key: cord- -v vv o authors: shen, mingwang; xiao, yanni; rong, libin title: modeling the effect of comprehensive interventions on ebola virus transmission date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: v vv o since the re-emergence of ebola in west africa in , comprehensive and stringent interventions have been implemented to decelerate the spread of the disease. the effectiveness of interventions still remains unclear. in this paper, we develop an epidemiological model that includes various controlling measures to systematically evaluate their effects on the disease transmission dynamics. by fitting the model to reported cumulative cases and deaths in guinea, sierra leone and liberia until march , , we estimate the basic reproduction number in these countries as . , . and . , respectively. model analysis shows that there exists a threshold of the effectiveness of isolation, below which increasing the fraction of latent individuals diagnosed prior to symptoms onset or shortening the duration between symptoms onset and isolation may lead to more ebola infection. this challenges an existing view. media coverage plays a substantial role in reducing the final epidemic size. the response to reported cumulative infected cases and deaths may have a different effect on the epidemic spread in different countries. among all the interventions, we find that shortening the duration between death and burial and improving the effectiveness of isolation are two effective interventions for controlling the outbreak of ebola virus infection. the ebola virus disease (evd) affecting multiple countries in west africa in has an unprecedented magnitude, which is far larger than all previous evd outbreaks combined. by march , , a total of , cases (including , confirmed, , probable, and , suspected cases) of evd, as well as , deaths from the infection, have been reported in guinea, sierra leone, liberia . ebola virus spreads primarily via contact with body fluids of infectious people, and with those dead but not buried who can still transmit the virus in traditional west african funeral practices. because of lack of licensed therapeutic treatment regimens and vaccines , major control measures are a combination of early diagnosis, case isolation, contact precaution, awareness campaigns, and sanitary burial practices. identifying infected people quickly by the polymerase chain reaction (pcr) assay and isolating them to break chains of evd transmission may be effective to control the outbreak. the effect of reducing the time between symptoms onset and diagnosis with rapid testing has also been investigated in the literature [ ] [ ] [ ] . chowell et al. used a mathematical model to study the effect of early diagnosis of pre-symptomatic individuals and found that the basic reproduction number always decreases as the fraction of early diagnosed individuals increases. the result may be affected by the effectiveness of isolation. although the isolated class has a lower transmission rate than the non-isolated class, isolated individuals may live longer due to supportive clinical care and have more chance to infect others. whether the relationship between the fraction of early isolation and the effectiveness of isolation affects the ebola epidemic has not been explored. in this paper, we will use a model to systematically evaluate the effect of a number of factors, including isolation of pre-symptomatic individuals and the time between symptoms onset and isolation, on both the basic reproduction number and the final epidemic size in the above three african countries. media report on the numbers of infected cases and deaths can greatly affect social behavior and play an important role in defining health issues in the evd epidemic [ ] [ ] [ ] [ ] . mathematical models have been used to explore the effect of mass media on infectious disease outbreaks such as sars in [ ] [ ] [ ] and h n in [ ] [ ] [ ] [ ] [ ] , assuming that media coverage depends on the number of infected individuals and reduces the incidence rate. very few models have considered the impact of media coverage on the transmission dynamics of evd. recently, the first large-scale trials that aim to assess the safety and efficacy of two ebola vaccines (cad -eboz and vsv-zebov) were initiated in liberia on february , . the effectiveness of these vaccines still remain unclear. we will include the effect of vaccination in our model. the objective of this paper is to assess the effect of all possible intervention strategies (isolation, media impact, safe burial, and vaccination) on controlling the spread of ebola virus in guinea, sierra leone, liberia. we develop a mathematical model on the basis of the model in ref. and fit to epidemiological data of reported cumulative numbers of infected cases and deaths . using the model, we evaluate the potential effect of increasing the fraction of latent individuals prior to symptoms onset, shortening the duration between symptoms onset and isolation, improving media coverage, following restrict burial procedures, and administrating timely vaccine on the epidemic of ebola infection. we develop a mathematical model (eq. ) to study the impact of comprehensive interventions including isolation, media impact, safe burial and vaccination. the population is divided into eight classes: s (susceptible individuals who can be infected by ebola virus following a contact with infectious cases), v (vaccinated individuals), e (latent undetectable individuals), e (latent detectable individuals), i (infectious individuals with symptoms), j (isolated individuals), d (individuals who are dead but have not been buried; they can still transmit the disease during funerals), and r (recovered individuals). let n denote the total population size, i.e., n = s + v + e + e + i + j + r. susceptible individuals become infected through contact with infectious individuals and enter into the latent class at rate where β is the mean human-to-human transmission rate per day,   ( ≤ ≤ ) quantifies the relative transmissibility of isolated individuals compared to infectious symptomatic patients who are not isolated. therefore,  ( − ) × % (reduction in transmissibility) provides a measure of the effectiveness of isolation. β d is the transmission rate during funerals. in the model, , where c i and c d are the cumulative infected cases and deaths, respectively. m and m are non-negative parameters that measure the effect of media reported cumulative numbers of infected cases and deaths on the contact transmission. when m i (i = , ) is or relatively small, the transmission rate β is equal or close to the constant β . for m i > , the public is more aware of the disease so that the transmission rate could be decreased to β (< β ) as the number of accumulated infected cases c i or deaths c d increases. similarly, the transmission rate β d is assumed to be less than β d during funerals. we assume that susceptible individuals receive the vaccine at a rate ξ. the effectiveness of immunization is assumed to be η where ≤ η ≤ with η = meaning that the vaccine is perfectly effective and η = meaning that the vaccine has no effect. latent undetectable individuals (e ) progress to the latent detectable class e at a rate k , and subsequently move to the infectious symptomatic class i at a rate k . let f t be the rate at which latent detectable individuals are detected and isolated. thus, θ = f t / (f t + k ) represents the fraction of isolated patients among latent detectable individuals exiting the class. infectious individuals are isolated at a rate α, or removed from the class at a rate γ due to recovery (with probability − δ) or disease-induced death (with probability δ). similarly, isolated individuals are removed at a rate γ r because of recovery (with probability − δ) or disease-induced death (with probability δ). a flow diagram of the model (eq. ) is shown in fig. and parameters are described in table . the dynamical model is as follows: figure . a schematic flow diagram of ebola infection with isolation, media impact, post-death transmission and vaccination. the description of parameters can be found in table . it should be noted that we keep track of the cumulative number of infected cases c i and deaths c d (c i and c d are not epidemiological states) by applying the solutions of eq. ( ) to the following equations in the absence of effective vaccine (i.e. ξ = η = ), we obtain the basic reproduction number r which is the spectral radius of the matrix ∼ −  fv by using the next generation matrix approach given in where ρ denotes the spectral radius and the matrices  f and ∼ v are note that each term of the aforementioned expression for r has clear epidemiological interpretation. − θ = k /(f t + k ) is the fraction of latent detectable individuals becoming symptomatic among those exiting the e class. /(α + γ) is the mean infectious period of symptomatic cases (not isolated). α/(α + γ) is the fraction of symptomatic cases that are isolated among those exiting the i class. /γ r is the mean infectious period of isolated cases and /γ d is the mean duration of the infectious period between death and burial. r i , r j and r d reflect the contribution to new infection from the symptomatic class (i), the isolated class (j), and the dead class (d), respectively. when vaccine is included, i.e., ξ > , η > , the reproduction number can be calculated as r v = ηr , where r is given by ( ). data fitting and parameter estimation. we obtained data of the total cumulative cases and deaths (as the sum of confirmed, probable and suspected cases) for guinea, sierra leone, liberia from the world health organization (who) . we used t (see table ) as the starting date for each country when the first infectious case was reported . we compiled publicly available time series of reported cases and deaths from who beginning from march , may , and june for guinea, sierra leone, liberia, respectively , . the data as of march were used to fit the model equation ( ) to estimate the unknown parameters. the latest data from march to may were used to assess how well our forecasts match additional data points. based on previous studies, the mean incubation time for evd is days ( /k + /k = ) with days from the latent undetectable class to latent detectable class ( /k = ) and days from the latent detectable class to symptomatic class ( /k = ) , . the mean time from symptoms onset to isolation is days ( /α = ). the mean time that infectious individuals are removed from the class after recovery or disease-induced death is days ( /γ = ) , . the average duration from death to burial is chosen as days ( /γ d = ) . as for the total population size n, we used the number of for guinea, for sierra leone, and for liberia, provided by the world bank . the other parameters were estimated by fitting the equation ( ) to the data on cumulative cases and deaths of each country and using an adaptive metropolis-hastings (m-h) algorithm to carry out the bayesian markov chain monte carlo (mcmc) procedure implemented by matlab. the estimated parameter values and their % confidence intervals are listed in table . model prediction and comparison with data. we first consider the situation without vaccination because of the unavailability of vaccine before february , , and then explore the effect of vaccination in liberia where the first large-scale vaccine trials were implemented since then . figure shows that the model provides a very good fit to the reported data of both infected cases and deaths in all three countries. using our parameter estimates (table ) , we calculated the basic reproduction number r in guinea, sierra leone and liberia as . , . and . , respectively. they are all within the range of estimated values in the literature (see table ). in guinea, the symptomatic component of we plotted the simulated newly infected individuals in fig. (red solid lines) and compared with the reported weekly confirmed cases in the three countries. most of the reported cases in guinea were confirmed ( / ), while only / and / (the numerator and denominator denote the confirmed and total cases on march , respectively) of cases were confirmed in sierra leone and liberia, respectively. there is a small difference between the simulated new infection and reported data in guinea ( fig. (a) ), while the simulated results are higher than confirmed cases in sierra leone and liberia ( fig. (b,c) ) due to a lower confirmation rate in these two countries. moreover, the simulated trend of evd outbreak and predicted peak time are in good agreement with what the who data and other references [ ] [ ] [ ] [ ] indicated. the solution of model ( ) is shown in fig. . the number of infected individuals is decreasing and tends to vanish gradually at the end of ( fig. (a-c) ). effect of isolation on r and the final epidemic size. to explore the effect of early and broad diagnosis on the basic reproduction number r , we calculate the derivative of r with respect to the parameter θ (i.e. the fraction of latent individuals diagnosed prior to symptoms onset) as follows the derivative is greater than when  > for guinea, sierra leone and liberia respectively (see table blue lines in fig. ) , respectively, which are all less than their respective threshold. thus, increasing the fraction θ of detecting pre-symptomatic cases results in a decline in the basic reproduction number. if the effectiveness of isolation increases to %, i.e., the relative transmissibility  of isolated individuals decreases to % (magenta lines in fig. ), then about %, %, % of pre-symptomatic patients need to be detected in guinea, sierra leone and liberia to control the disease. if the isolation is perfect  ( = ) , then the disease could be controlled easily (see yellow lines in fig. ). next, we study the effect of the fraction θ of latent individuals diagnosed prior to symptoms onset on the final epidemic size, which is defined as z = c i (t ) = c d (t ) + r(t ) . the time t is min{t > ,e (t) + e (t) + i(t) + j(t) + d(t) = }. although the explicit formula between the final size z and θ cannot be obtained, there exists a threshold which decides whether the final epidemic size increases as θ increases. the relationship between z and θ is similar to that between r and θ. for example, it follows from fig. (a) that if the effectiveness of isolation is lower than . %, i.e., the relative transmissibility  of isolated class is greater than . %, then the final epidemic size increases as θ increases. this suggests that strict measures must be taken to limit the contact of isolated individuals. otherwise, low effectiveness of isolation would lead to more infected individuals because isolated people live longer due to improved medical care and have more chances to infect other people. the benefit of having a reduced transmission rate would be counteracted by a longer infectious period of the isolated class. fig. (d-f) show the situation in which the final epidemic size decreases as θ increases. using the estimated value of θ (see table , θ = . , . , . in guinea, sierra leone and liberia, respectively), the simulated final size is , and in these three countries respectively (blue lines in fig. ). this simulated result in liberia where the outbreak of evd was considered ended by the who on may is very close to the actual final size with a relative error . %. similarly, to investigate the effect of shortening the time ( /α) between symptom onset and isolation (i.e, increasing the isolation rate α of infectious symptomatic individuals) on the basic reproduction number r , we plot fig. , which shows a similar relationship to that between r and θ when the the relative transmissibility  of isolated class varies. this is not surprising because the derivative of r with respect to α is it has the same threshold  = γ γ ⁎ r that decides whether the basic reproduction number increases with an increasing α. if the relative transmissibility  of isolated individuals decreases to % (see magenta lines in fig. ) , then the disease will be under control if the isolation rate α exceeds . (i.e., the time between symptom onset and isolation is less than days) in sierra leone and liberia. in this case  ( = %) , evd will always be controlled no matter how long the time ( /α) is in guinea. we study the effect of media coverage m (response to the reported cumulative number of infected cases) and m (response to the reported cumulative deaths) on the final epidemic size. let q ( ≤ q ≤ ) be the percentage of increase of m and m from its baseline estimate (table ) . we examine how the final epidemic size changes with an increasing media impact q in fig. . for example, increasing m (or m ) by % from its baseline value (while keeping other parameters fixed) can reduce the final epidemic size by . % (or . %) in guinea, table . scientific reports | : | doi: . /srep . % (or . %) in sierra leone, and . % (or . %) in liberia, respectively. this indicates that the response to the reported cumulative deaths (m ) may be stronger than the response to reported cumulative cases (m ) in guinea and liberia. a possible explanation of this result is that the case fatality rate in these two countries is relative high (see table ). thus, people had increased attention to evd-induced deaths. in sierra leone, an opposite scenario was observed. in conclusion, the analysis of media impact indicates that massive news coverage on cumulative cases and deaths would greatly curb the spread of the ebola disease. effect of post-death transmission on r . to assess the effect of reducing post-death transmission on the basic reproduction number r , we plot the variation in r with the efficacy of intervention, z d , at funerals (i.e., the post-death transmission rate β d is decreased to β d ( − z d )) for various durations of the traditional burial /γ d in fig. . it shows that increasing the efficacy of intervention leads to a decline in r in all three countries. in particular, the epidemic in guinea could be controlled by following very restrict burial procedures (a large z d ). for example, when /γ d = (blue line in fig. (a) ) and the efficacy of interventions z d exceeds about %, the basic reproduction number r will become less than . however, reducing transmission during funerals is insufficient to control the disease in sierra leone or liberia no matter how large z d is ( fig. (b-c) ). this is because the burial-related transmission contributes less to the spread of the disease in sierra leone (r d /r = . %) and liberia (r d /r = . %) than in guinea (r d /r = . %). figure also demonstrates that r decreases as the duration of funeral decreases. this suggests that the duration of funeral should be as short as possible for the control of the disease. sensitivity analysis. in order to identify which parameters the basic reproductive number r and the final epidemic size are most sensitive to, we perform sensitivity analysis using the latin hypercube sampling (lhs) technique and calculate the partial rank correlation coefficients (prccs) , in fig. . the magnitude of these prccs shows the sensitivity of these parameters and the sign of the prccs indicates a positive or negative correlation between the inputs (i.e. parameters) and outputs (i.e. r and table . the final epidemic size). it follows from fig. (a-c) that the two parameters with the most significant impact on r are the relative transmissibility  of isolated classes and duration of the burial /γ d . a smaller relative transmissibility  of isolated classes or a shorter duration of burial results in a smaller r , which is consistent with the results shown in figs and . these two parameters  ( and /γ d ) also have the most significant impact on the final epidemic size, as shown in fig. (d-f) . these results suggest that increasing the effectiveness of isolation (decreasing ) and shortening the duration of funerals ( /γ d ) are of crucial importance to reduce the evd infection. the effect of media (m or m ) has no effect on r (see the formula of r in ( )), which is in agreement with the findings in refs. - , that the media table . table . impact does not affect the epidemic threshold. however, the media impact leads to a decline in the final epidemic size, as shown in fig. . effective vaccination, if used before the epidemic peaks, would be projected to prevent tens of thousands of deaths. in liberia, the evd epidemic reached the peak around mid-september , , which is in good accordance with our simulated peak time, sep , as shown in fig. table ). after initiating the vaccine, the reproduction number is reduced from . to . (table ). using the estimated values of ξ and η, we plotted how the simulated cumulative cases would vary with different timing of vaccination. it shows that the earlier the vaccination is initiated, the lower the cumulative cases ( fig. (a) ). it follows from fig. (b) that the final epidemic size grows with delayed vaccination. there would be more cases if vaccination had started one week later ( fig. (b) ). this indicates that the timing of vaccine administration does not have a big effect on the final size when the epidemic declines. in this study, we developed a mathematical model to study the transmission dynamics of ebola virus. the model includes the effect of case isolation, media impact, post-death transmission and vaccination. by fitting the model to the who reported data of infected cases and deaths (fig. ) , we obtained reasonable estimates of the parameters ( table ). the basic reproduction number in guinea, sierra leone and liberia is estimated as . , . and . , respectively. they are all in good agreement with previous estimates ( table ). the simulated results indicate that the outbreak in the above countries reaches the peak on oct , oct , sep , respectively (see fig. ), which also agrees with the observations in sierra leone , , and in liberia [ ] [ ] [ ] . we found that isolation does not always contribute to the control of the evd transmission. whether this intervention is beneficial depends on the effectiveness of isolation (or the relative transmissibility  of isolated individuals). if the infectious period of isolated individuals is less than that of non-isolated, i.e., < γ γ , then isolation is always beneficial to disease control because of a lower transmission rate and a shorter infectious period of isolated individuals. on the contrary, if the infectious period of isolated individuals is longer than that of non-isolated, then when the isolation effectiveness is relatively low (e.g.  > ) γ γ r , there will be more infected cases as the fraction θ of latent individuals prior to symptoms onset increases (figs and (a-c) ) or the duration /α between symptoms onset and isolation table . scientific reports | : | doi: . /srep decreases (fig. ) . when the isolation effectiveness is high (e.g.  < ) γ γ r , then enhancing isolation can greatly reduce new infection (figs , (d-f), and ). these results suggest that isolation may not always have a positive effect on disease control as shown in refs. - . sensitivity analysis also shows that the relative transmissibility  of isolated classes has the most significant impact on both the basic reproduction number and the final epidemic size, which further explains why the isolation effectiveness determines whether the disease can be controlled successfully. therefore, strict measures should be taken to limit the contact with isolated individuals. a few reasons may explain why the isolated class contributes more to the basic reproductive number r than the symptomatic class and the dead class. one is that the infectious period of isolated individuals (i.e. /γ r ) is longer than that for non-isolated individuals because of improved clinical care. this is consistent with the parameter estimates shown in table . the other reason is that a majority of patients (θ) are isolated but the isolation may not be effective in reducing the transmission of the disease. ebola virus is very contagious and the transmission is also rapid, which makes isolation, as a containment strategy, usually inefficient . this agrees with the estimate of  in table , which is not small, implying that isolated individuals still have a high capacity to transmit the virus. in our model, we did not discriminate isolated individuals from the latent detectable class (e ) and from the infectious class with symptoms (i). isolated cases from the e class may have a different duration ( /γ r ) staying in the isolated class from the isolated cases coming from the i class. however, the above conclusion that the isolated class contributes more to r should still be valid as long as the infectious period of isolated individuals is longer than that of their corresponding preceding class. this is usually true in view of the improved health care received for isolated individuals. we also found that the estimate of the case fatality rate δ in guinea ( . ) is almost as twice as that in sierra leone ( . , see table ). one reason is a low level of preparedness, as well as poor availability and quality of medical care in guinea . another important reason is that the outbreak in guinea was caused by the zaire strain , which induces an average fatality rate of %, the highest death rate of the five known ebola strains . media impact can significantly affect the ebola infection, as shown in fig. . however, the responses to the reported cumulative deaths and cumulative cases may have different effect on the infection. we find that the response to the reported cumulative deaths (m ) is more sensitive than the response to the reported cumulative cases (m ) in guinea and liberia. it can be explained by a higher case fatality rate in these two countries (table ) . post-death transmission is an important factor that induces more infection and hence can not be ignored during evd outbreaks , . our simulation shows that increasing the efficacy of intervention at funerals can control the disease in guinea. however, this measure is insufficient to eliminate the disease in sierra leone and liberia (shown in fig. ) because the burial-related transmission does not contribute much to the spread of the disease in these two countries. sensitive analysis indicates that the duration of the burial /γ d is the second parameter which most affects r and the final epidemic size. thus, shortening the duration between death and burial and reducing transmission before burial would effectively reduce the infection. because the interventions we considered mainly address isolation of infected people within evd treatment centres, one limitation of our model is that we do not explicitly account for hospital bed capacity, which plays an important role in affecting both the outbreak dynamics and the intervention efforts . additionally, the conventional homogeneous mixing assumptions used in our model may be over-simplified, especially in countries where infection always occurs in households due to the structure of the community . taking into consideration the effect of social networks structure on ebola infection would also be interesting for future research. in summary, we used an epidemiological model to study the effect of various measures on evd transmission dynamics. depending on the effectiveness of isolation, early and massive diagnosis of pre-symptomatic individuals with rapid testing may remain beneficial to reduce the transmission of the disease. shortening the duration between death and burial and improving the effectiveness of isolation are two effective interventions for controlling the evd outbreak. world health organization, ebola situation report- ebola vaccination: if not now, when? modelling the effect of early detection of ebola ebola control: rapid diagnostic testing transmission dynamics and control of ebola virus disease (evd): a review sample nlpde and nlode social-media modeling of information transmission for infectious diseases: case study ebola ebola 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modelling analysis m.s. and y.x. designed the study and carried out the analysis. m.s. performed numerical simulations. m.s., y.x. and l.r. contributed to writing the paper. competing financial interests: the authors declare no competing financial interests. key: cord- -pdxm iiw authors: xiong, siping; tang, qi; liang, xudong; zhou, tingting; yang, jin; liu, peng; chen, ya; wang, changjun; feng, zhenqing; zhu, jin title: a novel chimeric anti-pa neutralizing antibody for postexposure prophylaxis and treatment of anthrax date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: pdxm iiw anthrax is a highly lethal infectious disease caused by the bacterium bacillus anthracis, and the associated shock is closely related to the lethal toxin (letx) produced by the bacterium. the central role played by the kda protective antigen (pa ) region of letx in the pathophysiology of anthrax makes it an excellent therapeutic target. in the present study, a human/murine chimeric igg mab, hmpa , was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmpa expressed in f cells could neutralize letx both in vitro and in vivo. at a dose of . mg/kg, it could protect all tested rats from a lethal dose of letx. even administration of . mg/kg hmpa h before letx challenge protected all tested rats. the results indicate that hmpa is a potential candidate for clinical application in anthrax treatment. the bacterium bacillus anthracis, which primarily affects livestock but can also infect humans, is the causative agent of anthrax, a zoonotic disease and bioterrorism threat , . b. anthracis spores can be used as bioterror agents in biological warfare. this threat has spurred significant efforts toward the development of countermeasures for anthrax, including anthrax vaccines and therapeutics . however, vaccines are effective only for prevention . currently, therapeutic antibodies that target the anthrax toxin are under development and are designed to protect against the disease. b. anthracis secretes a tripartite toxin comprising a protective antigen (pa), lethal factor (lf), and edema factor (ef) . this is an a-b (or "binary") bacterial toxin. pa is the "b" subunit, which is responsible for cell surface binding, while lf and ef are a subunits responsible for the enzymatic activity of the toxin , . pa combined with lf or ef constitutes the lethal toxin (letx) or edema toxin (edtx), respectively . the first step in cellular intoxication involves binding of an kd form of pa (pa ) to specific cell surface receptors (antxr and antxr ). following receptor binding, pa is cleaved after the arg-lys-lys-arg sequence at amino acid position by a furin-family protease. this results in a kda form (pa ) that spontaneously oligomerizes to either a heptamer or an octamer [ ] [ ] [ ] . dissociation of the kda form (pa ) from pa allows pa to bind to either or both ef and lf. then, oligomeric pa -receptor complexes translocate lf or ef into the cytosol, where they promote intoxication . previous studies have shown that pa inserts stably and irreversibly into lipid bilayers to form ion-permeable channels , . other research has shown that the protease cleavage site deletion or mutation in pa prevents ef and lf binding , , and that for cells treated with lysosomotropic agents, the ability of pa to mediate the actions of ef or lf is blocked . nasal immunization of mice with a mixture of pa , lf, and a poly-γ -d-glutamic acid conjugate have been shown to exhibit strong antibody responses against all three antigens . thus, pa seems to be an ideal target fragment for antibody generation and selection. since passive immunization with protective antibodies can provide immediate and extensive protection independent of the host response, it is an attractive option to enhance the current postexposure treatment of anthrax. especially with regard to biodefense, it is considered the primary available therapeutic measure . during the past years, extensive research has focused on development of therapeutic antibodies to target the main virulence factors of anthrax, namely, pa, lf, ef, and capsule [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among these, pa plays a central role in the pathophysiology of anthrax and is an excellent therapeutic target. further, pa is the most important part of pa. in the present study, we developed murine igg neutralizing antibodies that directly target pa . then, we selected an ideal antibody from among these and genetically recombined it to form human/murine chimeric igg (coded "hmpa "). hmpa could specifically bind to pa and protect j a. cells against letx challenge in vitro. further, it protected fischer rats (f ) from letx after challenge. elisa was performed to test the binding sensitivity of hmpa to pa . hmpa recognized rpa in a dose-dependent manner, and the graph of hmpa concentration and absorbance at nm was a hyperbolic curve (fig. ). western blot analysis showed that hmpa could specifically recognize rpa (fig. ) . no reaction was seen with the negative control. immunoprecipitation. immunoprecipitation was performed using pa , which could be split to active pa using trypsin. a protein of about kda was detected on sds-page, and its sequence matched that of the b. anthracis protective antigen in the swiss-prot database (fig. a,c,d) . a kda membrane protein was also detected using a commercial anti-pa antibody (fig. b) , and this protein did not reaction with any other antibodies. kinetics of binding. the equilibrium dissociation constant (kd) for hmpa was determined by biacorex analysis. the rate constants kon and koff were evaluated directly from the biacorex sensogram. the kd was also determined using the biacorex . one striking feature of hmpa is its very slow off rate, which may explain its high affinity of . × − m (fig. ) . in vitro letx neutralization assay. the ability of hmpa to protect against letx was assessed in j a. cells. hmpa , pa , and different concentrations of lf were simultaneously added to cells. cell viability test results indicated that hmpa could completely neutralize letx. at μ g/ml lf and . μ g/ml pa , > % of the hmpa -treated cells remained viable, while only % of the control igg antibody-treated cells remained viable. at . μ g/ml lf and . μ g/ml pa , % of the hmpa -treated cells were viable, while only % (fig. ) of the control cells were. protection of f rats. f rats were injected hmpa antibody via the tail vein either before or after letx injection. the survival time of group iii was significantly (p < . ) longer than that in groups i and ii. until the last observation, all group iv rats were alive (fig. a ). rats injected hmpa min before letx showed similar results. a dose of μ g antibody protected the rates from death ( fig. b) , although some symptoms, such as accelerated breathing and lethargy, were observed in one rat. hmpa injection before or after letx administration protected all rats from developing anthrax (fig. c) . the prophylactic function of hmpa was tested by injection of the antibody at different times before letx injection. in the groups that received prophylaxis min to h before letx injection, rats remained alive (fig. d ). when hmpa was injected before pa, it protected rats from anthrax death regardless of lf injection time (fig. e ). tissue pathology and immunohistochemical analysis. the lung of the rats were pathologically and immunohistochemically examined. h&e staining showed that the local tissue of rats injected only letx showed greater alveolar exudation (fig. c ) than untreated control rats (fig. a) . however, the group that received letx + μ g hmpa (fig. b) showed no significant differences from untreated control rats. since anthrax receptors were expressed in some cells including alveolar epithelial cells, cell binding to pa could be positive. when rats were injected with letx, the cells could dectect pa. further, the positive staining was mainly localized to the membrane. a strong positive reaction was found in the group injected only letx (fig. f) , while a weak positive reaction was found in the group injected letx + μ g hmpa (fig. e) . the untreated control group showed a negative reaction (fig. d ). this study revealed two major findings: first, the human/murine chimeric antibody hmpa can neutralize letx in vivo and can be used for prohylaxis before letx is released in the blood. second, since immunization with pa produces neutralizing antibody, it can be used to immunize animals. in the present study, we developed four murine mabs that could well neutralize letx in vitro, and one clone was selected to form a human/mouse chimeric antibody known as hmpa . this antibody showed excellent neutralization both in vitro and in vivo. the current status of therapeutic mabs was directed against the major virulence factors: pa, lf, ef and capsule. although lf could induce cell death, its effective result in vivo was unsatisfactory. pa was the critical factor which recognized cell membrane receptors. then pa was cleaved to active pa by furin which induced lf or ef into cells. our strategy was to get antibody against active pa . to immunize the mice, we used pa , which is formed by the dissociation of pa from pa , instead of pa , because although pa is a part of pa , these factors may have different structures and expose lf-or ef-binding sites. these differences may in turn lead to the production of completely different antibodies against anthrax in vivo. in the present study, we showed that pa could induce neutralizing antibodies, as proven through in vitro and vivo experiments. on the other hand, it was difficult to obtain active pa based on present methods. we screened out positive clones by active pa in a different way. based on traditional elisa, we coated plates with alive j a. cells overninght at cell culture condition. then pa was added into wells incubated about h. the negative control was cells without pa adding. the rest of elisa procedures was as normal. in this screening system, the active pa was more closed to in vivo state and could oligomerize to heptamer. in all, more diversity antibodies was generated in the immunization stage and more effective antibodies was obtained in the screening system. anthrax, whether resulting from natural or bioterrorist-associated exposure, is a constant threat to human health , and the low incidence of anthrax suggests that large-scale vaccination may not be the most efficient means of controlling this disease. passive immunization with protective antibodies is therefore considered the primary available biodefense measure , especially in bioterrorist-associated exposure. in the present study, hmpa was found to provide good protection in rats challenged with anthrax virulence factors. in the in vitro experiment, hmpa maintained % cell viability with . μ g/ml lf and . μ g/ml pa , while non-correlated igg maintained only % cell viability. further, with μ g/ml lf and . μ g/ml pa , hmpa maintained > % cell viability, while the control antibody maintained only %. moreover, in previous study, they often used f rats challenged with letx (table ) before tested with b. anthracis spores. therefore f rats was injected with letx vial tail vain. the hmpa with a kd of . nm protected all rats from death at a concentration of . mg/kg ( μ g per rat). however, in a previous study, . mg/kg raxibacumab (of human origin) with a kd of . nm was administered h before anthrax lethal toxin administration in f rats . we also tested an hmpa concentration of . mg/kg ( μ g per rat) administered h before letx injection; this dose also protected all rats. our findings indicate that hmpa may be used as a prophylactic. other humanized or chimeric mabs have been examined in other animal model, but the affinity found in the present study is the same as or better than those found previously , . murine mabs against pa have been developed, but our chimeric mab maintains a balance between the high-affinity murine component and the low-immunogenicity human component. other mabs of human origin are available but are difficult to produce and are expensive . the chimeric mab hmpa is produced in f cells, and the method of production is very convenient. in the in vivo test in the present study, irrespective of whether lf was injected before or after hmpa , the antibody protected the rats from death provided that it was administered before or simultaneously with pa. this finding indicated that hmpa could not prevent lf from binding pa . further, no morphological changes were observed in the rats of only injection letx, probably because the time between injection and sacrifice was very short (only about mins), and no histological changes occurred in this period. the ihc analysis showed a strong positive result in the group injected only letx. however, the group that received letx + μ g hmpa showed a weakly positive reaction. these findings indicate that hmpa prevents pa from binding to cell receptors. however, further experiments are required to validate this hypothesis. future studies should focus on detailed characterization of this mab (specificity, toxicity studies, autoantigen testing, etc.). second, epitope mapping and structure function analysis of hmpa should be performed. in the in vivo experiment in the present study, we demonstrated that hmpa could not interfere with lf binding to pa. further experimentation is needed to determine the exact mechanism by which hmpa neutralizes letx. lastly, more animal tests are required, for example, in which animals are challenged with anthrax spores. in summary, we reported a human/murine chimeric igg, namely, hmpa , which can specifically identify pa with high affinity, neutralize letx, and protect macrophages and f rats from anthrax-related death. we also showed that pa is a good immunogen. from our findings, we believe that once hmpa is further characterized, it can be used alone or in combination with other neutralizing mabs for treatment of anthrax. bl by using the pcoldii vector and purified by affinity chromatography. six balb/c mice aged weeks were intraperitoneally (ip) immunized with rpa and adjuvant as described previously . after immunization, the mouse spleen showing the best titer was removed, and splenocytes were extracted and fused with sp / myeloma cells using hybridoma technology . positive clones were screened by elisa using active pa -coated -well plates, and subcloning was conducted based on standard protocols. clonal expansion was conducted with hybridoma-sfm (gibco,usa). the cell supernatant was then removed and purified by affinity chromatography with protein g (ge, usa) according to the manufacturer's purification system. figure . in vivo letx neutralization assay in f rats. a. mean survival time. letx and the antibody were simultaneously injected via the tail vein. group i, μ g hmpa + μ g letx; group ii, μ g hmpa + μ g letx; group iii, μ g hmpa + μ g letx; group iv, μ g hmpa + μ g letx. ***p < . . b. different concentrations of the antibody were injected, and letx was injected min later via the tail vein. c. for each rat, μ g antibody was injected before (− min), after ( min), or simultaneously ( min) with letx. d. for each rat, μ g antibody was injected at different times before letx injection. e. group i, pa ( μ g) was injected min after lf ( μ g) + hmpa ( μ g); group ii, pa ( μ g) was injected min before lf ( μ g) + hmpa ( μ g). all experiments involving animals were performed in accordance with the protocols approved by the animal care and use committee of the national institute of allergy and infectious diseases, national institutes of health, usa. total rna was extracted from the pa hybridoma cells using the trizol reagent (invitrogen), and cdna was synthesized using reverse transcriptase superscript ii according to the manufacturer's instructions. eukaryotic vectors were constructed by separately cloning pa heavy and light variable regions into pth and ptl, which respectively include constant regions of igg heavy and light chains. murine variable regions of the heavy (v h ) and light chains (v l ) were first amplified by pcr using pa cdna as the template. to obtain v h and v l nucleotide sequences, these chains were cloned into the pmd- t vector. pcr primers were designed using the in-fusionr hd cloning kit (clontech), and v h and v l were amplified from right-sequenced pmd- t vectors by using these primers. finally, v h and v l were separately cloned into linearized pth and ptl vectors, respectively, by infusion pcr using the in-fusionr hd cloning kit. the recombinant elisa. ninety-six-well enzyme immunoassay plates were coated overnight at °c with μ l of rpa antigen ( μ g/ml) diluted in mm sodium carbonate buffer (ph . ). the plates were blocked and serial two-fold dilutions of hmpa were added to the wells ( wells for each concentration) as the primary antibody. the plates were incubated at °c for h and then washed times with μ l of pbs containing . % tween (pbst). subsequently, goat anti-human igg-hrp conjugate (sigma) was added as the secondary antibody and incubated at °c for min. after color development, the absorbance values of the wells were detected at nm. non-correlated igg was used as the control. the absorbance values at nm of hmpa were plotted using graphpad prism software version . (graphpad software, inc., la jolla, ca, usa). the cell lysates of rpa recombinant bacteria and e. coli bl were separately run on a % sds-page gel and then transferred onto a nitrocellulose membrane (bio-rad). the membrane was blocked with pbs containing % dry milk at °c overnight and then incubated for h at rt with : diluted hmpa from mg/ml stock. after it was washed times with pbst, the membrane was incubated with a : diluted secondary hrp-conjugated goat anti-human antibody (sigma) for an additional min at rt. following the same washing procedure, the signal was detected using ecl western blot substrate (pierce) according to the manufacturer's instructions. (ph . ) and mm nacl] with . μ g/ml trypsin (sigma) for min at °c, followed by addition of μ g/ml soybean trypsin inhibitor (sigma) . then, the mixture was incubated with μ g of hmpa at °c and rotated for h. next, μ l protein-a sepharose (invitrogen, usa) was added and incubated at °c. the immune complexes that formed were washed times with pbst. subsequently, μ l elution buffer was added to separate these antibody-antigen complexes from protein-a sepharose. as a negative control, another anti-tlr chimeric antibody (generated by our lab) was created using the same protocol. the protein complexes were isolated by running two % sds-page gels; one was transferred onto a nitrocellulose membrane, and the other was stained with coomassie blue. the nitrocellulose membrane was blocked at °c overnight, incubated with : diluted rabbit polyclonal anti-pa antibody (pierce, usa) for h at rt, washed with pbst times, and reacted with : diluted goat anti-rabbit igg-hrp conjugate (sigma) for an additional min at rt. the membrane was washed times with pbst, and the hybridization signal was detected using ecl western blot substrate. the target bands on sds-page gel were subjected to mass spectra identification with an abi proteomics analyzer and maldi-tof/tof mass spectrometer (applied biosystems, framingham, ma). the mass spectra were then searched within the swiss-prot database using the mascot search engine (http://www.matrix science.com; matrix science, uk). affinity and kinetic assay of antibody. the biacore x system (ge, usa) was used to analyze the affinity and kinetics of the hmpa antibody. pa was diluted to μ g/ml with acetate buffer ( mm naac, ph . ) and immobilized on the surface of a cm sensor chip (ge, usa) to capture purified mab, which was diluted in running buffer ( mm hepes, mm nacl, mm edta-na , . % p ; ph . ) to achieve different concentrations ranging from to nmol/l. the association time was set up at s and the dissociation time, at s, followed by regeneration with mm glycine-hcl (ph . ). sensograms were evaluated using the biacore x evaluation software. in vitro letx neutralization assay. the in vitro letx neutralization assay was performed as described previously injected via the tail vein with a mixture of pa + lf (letx) and different amounts of hmpa antibody prepared in sterile pbs. each rat was administered μ l of the mixture. further, the rats were also treated with different concentrations of the antibody min before exposure to letx. for this experiment, they were injected intravenously with pbs or , , or μ g of the antibody before receiving an intravenous injection of letx ( μ g pa + μ g lf). additionally, double the complete protection dose of antibody ( μ g) was injected to test its prophylactic ability. the rats were inoculated with μ g antibody followed by letx administration after different times, from min to h. two additional experiments were conducted with f rats. one group received pa ( μ g) injection min after lf + hmpa ( μ g + μ g, respectively), while the other received μ g pa min before lf + hmpa (at the same doses). after injection of letx, signs of malaise and death were checked for every min for the first h and then at h and h, followed by twice-daily checks for week. tissue pathology and immunohistochemical examination. the lungs of the f rats were embedded in paraffin wax at the department of pathology, nanjing medical university (jiangsu, china), using routine methods. sections ( μ m) were deparaffinized with xylene and then dehydrated in decreasing concentrations of alcohol. some sections were treated with h&e staining and examined by light microscopy to determine the pathological features of the lung tissues. for the remaining sections, endogenous peroxidase activity was blocked by incubation with % hydrogen peroxidase in tris-buffered saline. some of these tissue sections were then incubated with rabbit polyclonal anti-pa primary antibody (pierce, usa), followed by the envision hrp complex (dako, carpinteria, ca). they were then counterstained with hematoxylin qs (vector laboratories, burlingame, ca). the results were analyzed according to the ihc score (ihs) as described previously . briefly, the ihs was determined by evaluation of both staining density and intensity. multiplication of the intensity and percentage scores yielded the final ihs. samples with ihs ≤ were considered weakly positive, while those with ihs ≥ were considered strongly positive. the ihc results were evaluated by two independent investigators blinded to the rat groups. in cases of conflict, a pathologist reviewed the cases, and a consensus was reached. survival data were analyzed using the graphpad prism version statistical analysis software (san diego, ca). a t-test was used to compare the mean survival time between groups. a two-tailed log rank test was used to determine the statistical significance of differences between groups. a p value of < . was considered statistically significant. a human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro mouse monoclonal antibodies to anthrax edema factor protect against infection analysis of defined combinations of monoclonal antibodies in anthrax toxin neutralization assays and their synergistic action an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus anthrax-toxin-mediated delivery of a kda antigen of mycobacterium tuberculosis into the cytosol of mammalian cells binary bacterial toxins: biochemistry, biology, and applications of common clostridium and bacillus proteins antibodies against anthrax: mechanisms of action and clinical applications consequences and utility of the zinc-dependent metalloprotease activity of anthrax lethal toxin identification of the cellular receptor for anthrax toxin human capillary morphogenesis protein functions as an anthrax toxin receptor a receptor-based switch that regulates anthrax toxin pore formation structure and function of anthrax toxin anthrax toxin anthrax protective antigen: prepore-to-pore conversion anthrax toxin protective antigen: low-ph-induced hydrophobicity and channel formation in liposomes ph-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin a deleted variant of bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vivo cleavage of structural proteins during the assembly of the head of bacteriophage t nasal immunization with the mixture of pa , lf, and a pga conjugate induced strong antibody responses against all three antigens passive antibody administration (immediate immunity) as a specific defense against biological weapons human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against bacillus anthracis infection and enhance endogenous immunity to anthrax efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen raxibacumab for the treatment of inhalational anthrax human monoclonal antibody avp- d to protective antigen reduces dissemination of the bacillus anthracis ames strain from the lungs in a rabbit model prophylaxis and therapy of inhalational anthrax by a novel monoclonal antibody to protective antigen that mimics vaccine-induced immunity a high-affinity monoclonal antibody to anthrax protective antigen passively protects rabbits before and after aerosolized bacillus anthracis spore challenge in vitro and in vivo characterization of anthrax anti-protective antigen and anti-lethal factor monoclonal antibodies after passive transfer in a mouse lethal toxin challenge model to define correlates of immunity neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model an anthrax lethal factor-neutralizing monoclonal antibody protects rats before and after challenge with anthrax toxin protective and immunochemical activities of monoclonal antibodies reactive with the bacillus anthracis polypeptide capsule monoclonal antibody therapies against anthrax protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity a high-affinity human/mouse cross-reactive monoclonal antibody, specific for vegfr- linear and conformational epitopes derivation of specific antibody-producing tissue culture and tumor lines by cell fusion a novel lmp antibody synergizes with mitomycin c to inhibit nasopharyngeal carcinoma growth in vivo through inducing apoptosis and downregulating vascular endothelial growth factor production and characterization of monoclonal antibodies against the lethal factor component of bacillus anthracis lethal toxin novel chimpanzee/human monoclonal antibodies that neutralize anthrax lethal factor, and evidence for possible synergy with anti-protective antigen antibody this work is supported by the national natural science foundation (no. ).and science fund of jiangsu province, china (no. bk and no. cws j ) s.x., j.z., z.f. and q.t. wrote the main manuscript text and s.x. and x.l. prepared figures - and q.t. and s.x. prepared figures - . all authors reviewed the manuscript. competing financial interests: dr. zhu, dr. feng, qi tang and siping xiong have a common patent a issued in china. all other authors report no conflicts. key: cord- -a sbdhhn authors: xiaokaiti, yilixiati; wu, haoming; chen, ya; yang, haopeng; duan, jianhui; li, xin; pan, yan; tie, lu; zhang, liangren; li, xuejun title: egcg reverses human neutrophil elastase-induced migration in a cells by directly binding to hne and by regulating α -at date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: a sbdhhn lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. egcg has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. in this study, we demonstrated a novel mechanism by which egcg reverses the neutrophil elastase-induced migration of a cells. we found that neutrophil elastase directly triggered human adenocarcinoma a cell migration and that egcg suppressed the elevation of tumor cell migration induced by neutrophil elastase. we observed that egcg directly binds to neutrophil elastase and inhibits its enzymatic activity based on the cdocker algorithm, md stimulation by gromacs, spr assay and elastase enzymatic activity assay. as the natural inhibitor of neutrophil elastase, α -antitrypsin is synthesized in tumor cells. we further demonstrated that the expression of α -antitrypsin was up-regulated after egcg treatment in neutrophil elastase-treated a cells. we preliminarily discovered that the egcg-mediated induction of α -antitrypsin expression might be correlated with the regulatory effect of egcg on the pi k/akt pathway. overall, our results suggest that egcg ameliorates the neutrophil elastase-induced migration of a cells. the mechanism underlying this effect may include two processes: egcg directly binds to neutrophil elastase and inhibits its enzymatic activity; egcg enhances the expression of α -antitrypsin by regulating the pi k/akt pathway. signaling pathways, such as the activator protein (ap), nuclear factor-κ b (nf-κ b), and phosphatidylinostitol- -oh kinase pathways (pi k/akt) [ ] [ ] [ ] . however, the molecular mechanisms underlying the anti-tumor metastatic properties of egcg in the specific context of inflammation remain elusive. in this study, we demonstrated additional contributions of inflammation to the progression of lung cancer metastasis and a novel molecular target of egcg, human neutrophil elastase, which induces lung cancer cell migration. neutrophils, as a component of the tumor microenvironment, have only recently been thought to play an important role in tumor growth and invasiveness . the presence of increased infiltrated neutrophils within tumors was significantly associated with a poorer clinical outcome in patients with bronchioloalveolar carcinoma . neutrophil elastase, which is released by activated neutrophils, is the most potent neutrophil protease. the potential substrates of this protease include cytokines and cytokine receptors, indicating that neutrophil elastase may regulate the inflammatory process . recent reports have shown that neutrophil elastase directly induces uncontrolled tumor proliferation in a lung adenocarcinoma mouse model and in lung epithelial tumor cells. alpha- antitrypsin (α -at, aat) is a serum glycoprotein that contains three potential glycosylation sites. as an acute-phase protein, aat is thought to play an important role in limiting host tissue injury triggered by proteases, particularly human neutrophil elastase (hne). the clinical relevance of aat is demonstrated in individuals with an inherited deficiency in circulating aat, who exhibit increased susceptibility to early-onset pulmonary emphysema and liver and pancreatic diseases . neutrophil elastase and aat are a protease and protease inhibitor counterpart pair . it has been assumed that in aat-deficiency, the protease/ anti-protease balance is shifted toward hne, which leads to extensive tissue damage, particularly by causing emphysema. xu et al. demonstrated that the natural polyphenol product curcumin inhibits tumor proliferation induced by neutrophil elastase via the upregulation of aat in lung cancer. altering the balance between aat and hne may represent an innovative form of lung cancer treatment. cell culture and treatment. the human lung adenocarcinoma cell line a was used for in vitro experiments. the cells were maintained in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) containing % fetal bovine serum (fbs), u/ml penicillin and μ g/ml streptomycin in a humidified incubator containing % co in air at °c. the cells were treated with different concentrations of neutrophil elastase (merck group, darmstadt, germany) and/or egcg (sigma-aldrich co., st. louis, mo, usa) for h and then evaluated as described below. cell viability assay. cell viability was determined using a cell titer ® aqueous one solution cell proliferation assay (mts assay, promega, madison, wi, usa). a cells were seeded on -well plates at a density of cells/well and were allowed to adhere for h. after incubation in neutrophil elastase and/or egcg for h, μ l of the mts solution was added to each well, followed by incubation for h at °c. the resulting color was assayed at nm using a microplate reader (bio-rad laboratories, inc., hercules, ca, usa). in vitro wound healing assay. the wound healing assay was performed as previously described (liang et al., ) to assess the capacity for cell migration. briefly, the a cells were seeded on a -well plate. when the cells reached - % confluence, scratches were applied using a sterile -μ l pipette tip. the cells were washed three times with phosphate buffered saline (pbs), and the initial wounds were examined under an olympus microscope. then, the cells were incubated in medium containing neutrophil elastase and/or egcg for h, and the wounds were photographed again. the rate of cell migration was evaluated based on the rate of wound closure. transwell migration assay. cell migration was further investigated using the transwell migration assay. a cells were suspended in serum-free dmem ( × cells) and placed in the upper chamber. the plate was incubated at °c for h, followed by fixation of the cells in % formaldehyde. the upper chamber was gently wiped with a cotton swab to remove the non-migrated cells, and the migrated cells on the underside of the polycarbonate filter were stained with crystal violet and counted under an olympus microscope (× magnification). immunofluorescence assay. a cells were seeded on glass coverslips in an environment containing % co in air at °c and then incubated in nm neutrophil elastase and μ m egcg for an additional h. the coverslips were gently washed in pbs, fixed in . % paraformaldehyde for min, permeabilized in pbs containing . % triton x- for min and blocked in pbs containing % bsa for min. the fixed cells were incubated in an anti-human zinc finger e-box-binding protein- (zeb- ) antibody (cell signaling technology, beverly, ma, usa) overnight at °c in a humidified chamber. then, the cells were washed and incubated in the corresponding dylight -conjugated secondary antibody (jackson immunoresearch laboratories, west grove, pa, usa) for h at °c. after washing in pbs, the cells were incubated in hoechst ( μ g/ml, sigma-aldrich) for min at room scientific reports | : | doi: . /srep temperature. after several washes, the immunostained specimens were observed under a tcs-sp laser scanning confocal microscope (leica microsystems, wetzlar, germany). . (accelrys software, inc., san diego, ca) using the charmm engine was used in this study to evaluate the potential molecular binding mode between egcg and hne. the program was run using a dell optiplex server (round rock, tx). the cdocker algorithm employed a strategy involving the generation of the initial ligand orientation in the active site of the target protein followed by molecular dynamics (md)-based simulated annealing and final refinement via energy minimization. the crystal structure of hne (pdb id: z f) was obtained from the protein data bank (http://www. rcsb.org/pdb/). the water molecules in the protein were removed, the protein was refined based on the root mean square deviation (rmsd), and egcg was docked to the protein according to the appropriate parameters. beginning from the egcg configuration, a set of different orientations was randomly generated. the co-crystalized ligand sei was used as the positive control ligand, and the binding site was defined based on the binding of sei and hne. the interaction energy was calculated to analyze the interaction between the ligand and the receptor. once the ligand was docked to the active site, a molecular dynamics (md) simulation was performed using the gromacs . . program. the gromos a force field and the spc/e water condition were selected during the topology process. water was position-restrained using a force constant (kpr) of kj mol − ·nm − . the protein was placed in the box at least . nm from the box edge, and the box was defined as a cube. during the genion process, cl − were added to the system for charge equilibration. a -ns md simulation was performed. the gromacs program was used to constrain the bonds involving hydrogen atoms, allowing for an integration interval of fs. the particle mesh ewald method using a threshold of nm was employed to account for the electrostatic interactions. a threshold of nm was used for the van der waals interactions. the non-bonded pair lists were updated every . ps. the coordinates were saved every ps. accuracy testing was performed by calculating the rmsd after re-docking the internal ligand to the protein using the algorithm. binding free energy calculations. the trajectories obtained from molecular dynamics were used for binding free energy calculations. the calculations were performed using gromacs molecular mechanics poisson boltzmann surface area (g_mmpbsa) method, implemented in gromacs . . the binding free energy for a protein-ligand complex is given as: where, △g binding is an estimate of absolute free energy of binding and g is the average free energy of complex, receptor and ligand respectively. the average free energy on the other hand is defined as: where, ts is the solute entropic contribution to the system at temperature t. e mm is the molecular mechanical energy obtained from bonded and non-bonded interactions within the system and can be represented by following equation: g solv is the average solvation free energy and is equal to the sum of electrostatic and non-polar terms and represented as: g pb gb is the electrostatic contributions to solvation free energy which is evaluated by finite differential poisson boltzmann (fdpb) in case of pbsa method. − g non polar is the hydrophobic non polar contributions to solvation free energy, calculated as: where sasa is the solvent accessible surface area and, γ and b are constants. for the g_mmpbsa calculations the molecular dynamic trajectories were split into "snapshots". a total , snapshots were extracted from , frames obtained in ns of production run. this allows not only to sample the flexibility of the binding site, but also to obtain a more reliable free energy estimate of binding than compared to a single snapshot calculation. surface plasmon resonance (spr) biosensor analysis. the binding affinity of egcg to hne in vitro was assayed using the spr-based biacore instrument (biacore ab, uppsala, sweden) as previously reported , . recombinant hne protein (molecular mass, kda; pl . in pbs) with a purity of greater than % was purchased from merck group (darmstadt, germany). the manufacturer indicated that this material could be used for in vitro binding assays. the hne protein was dissolved in coupling buffer ( μ g/ml, in mm sodium acetate, ph . ), and response units (ru) of the hne protein, which was used as the ligand, were immobilized on a cm sensor chip using n-ethyl-n-( -dimethylaminopropyl) carbodiimide and n-hydroxysuccinimide according to the standard primary amine-coupling procedures; hbs-ep ( mm hepes, nm nacl, mm edta, and . % (v/v) surfactant p , ph . ) was used as the running buffer. equilibration of the baseline was performed by continuously flowing hbs-ep across the chip surface for - h. the biacore data were collected at °c using hbs-ep as the running buffer at a constant flow rate of μ l/min. egcg was serially diluted into the running buffer to create a series of concentrations. the samples were injected into the channels at a flow rate of μ l/min, followed by washing with running buffer. the binding responses were continuously recorded in ru at a frequency of hz as sensorgrams and were expressed as a function of time. the association (k on ) and dissociation (k off ) rate constants and the equilibrium dissociation constant (k d = k off ) were calculated as concentrations of egcg using bia evaluation software version . (biacore) and the : langmuir binding fitting model. the curve-fitting efficiency was evaluated according to the statistical parameter χ . western blot analysis. total protein was extracted using ripa lysis buffer, and equal amounts of proteins were subjected to % sds-page and then transferred to polyvinylidene difluoride membranes (millipore corp., ma, u.s.a). the membranes were blocked and then incubated in primary antibodies against vimentin, β -catenin, aat), insulin receptor substrate- (irs- ) ( : , cell signaling technology, beverly, ma), pakt and akt ( : , cell signaling technology), ppi k and pi k ( : , cell signaling technology), and gapdh ( : , sigma-aldrich) overnight at °c with gentle agitation, followed by incubation in the anti-rabbit ir-dye -or cw-labeled secondary antibody for h at room temperature. two -min washes in tbst were performed after secondary antibody labeling; then, the membranes were placed in tbst. the membranes were imaged using a licor odyssey scanner. boxes were manually placed surrounding each band of interest to measure the raw near-infrared fluorescence intensity values, and the intra-lane background intensity was subtracted using odyssey . analytical software (licor, lincoln, ne, usa). hne activity assay. the assessment of hne activity was performed according to löser the reaction was terminated via the addition of μ l of soybean trypsin inhibitor solution ( . mg/ ml in tris-hcl buffer, ph . ). the samples were then vortexed, and the absorbance was measured at nm. the remaining activity of hne (as a % of the control) was calculated relative the control in the absence of the inhibitor, considering the influence of the buffer, the substrate, the solvent and the extract. the inhibitory effect of the selective hne inhibitor sivelestat sodium ( μ mol/l), which was previously established using the same assay, was considered as a positive control. to evaluate the anti-metastatic effect of egcg on lung cancer with neutrophil elastase involvement in vitro, we first treated the metastatic a cells with various concentrations of neutrophil elastase ( - nm), egcg (up to μ m) or egcg (up to μ m) and neutrophil elastase ( nm). the results from the mts proliferation assay showed that neutrophil elastase at a concentration of at least nm enhanced tumor cell viability; among all of the concentrations tested, nm neutrophil elastase displayed the strongest effect (fig. b) . thus, we selected nm neutrophil elastase as the intervention condition. the mts proliferation assay results showed similar cell viability between the control group and the groups treated with various concentrations of egcg (fig. c ) and also showed a non-significant change in cell viability between the group treated with neutrophil elastase ( nm) and the groups treated with egcg and neutrophil elastase ( nm). however, in the wound healing assays, the cells treated with or μ m egcg migrated at a much slower rate than the control cells and the cells treated with neutrophil elastase ( nm) ( fig. a,b) , suggesting that egcg displays anti-migratory properties. to confirm this result, we performed further cell migration assays using the transwell chamber model. as shown in fig. c ,d, the group treated with nm neutrophil elastase displayed significantly more migrating a cells than the control group. alternatively, treatment with either or μ m egcg (after exposure to nm neutrophil elastase), resulted in dramatically fewer migrating a cells than the control treatment and treatment with neutrophil elastase ( nm) alone. these results indicated that treatment with egcg at a concentration between and μ m induces a substantial anti-migratory effect without affecting the proliferation of a cells exposed to neutrophil elastase. to determine whether egcg regulates the hne-induced emt process, the expression of emt marker scientific reports | : | doi: . /srep proteins was detected via western blot assays and confocal microscopy. the results showed that vimentin expression was significantly altered after hne treatment. however, as the specific inhibitor of hne sivelestat sodium significantly up-regulated the expression of vimentin. nevertheless, the vimentin protein levels were clearly down-regulated in response to egcg treatment in the presence or absence of hne (fig. b,c) . the expression of β -catenin was increased after hne induction but was significantly decreased after treatment with μ mol/l egcg (fig. b,c) . we also detected the expression and sub-cellular localization of zeb- because a recent study suggested that zeb- plays a role in lung cancer invasiveness and metastasis development. the results showed that zeb- expression was significantly increased after treatment with hne, and this effect was ameliorated after the addition of egcg (fig. a) . additionally, the sub-cellular translocation of zeb- from the cytoplasm to the nucleus was detected after treatment with hne. the expression of zeb- in the nucleus was decreased after treatment with egcg in the neutrophil elastase-treated a cells. we analyzed the binding pattern between egcg and hne. first, a crystal structure of hne bound to a selective inhibitor (pdb id: z f) was selected. the interaction energy between egcg and hne is - . kj/mol, which is similar to the interaction energy (− . kj/mol) between hne and its endogenous inhibitor sei . the active binding site between egcg and hne (pdb id: z f) was defined as the ligand-binding site with a -Å radius. as anticipated, egcg stably docked to the ligand-binding domain (lbd) (fig. b) . at least residues in the lbd were involved in the interaction between egcg and the hne protein. egcg potentially formed hydrogen bonds with ser , thr , arg and arg . moreover, egcg possessed a potential aromatic interaction with arg (fig. d) . the binding mode of egcg in the lbd of hne provided detailed structural insight into the interaction between this compound and the hne protein. furthermore, we performed a -ns md process using gromacs . . software. the time-averaged normalized ratio of the gyration radius, the rmsd, hydrogen bonds and the gromacs energy were analyzed to reflect the distribution of egcg molecules surrounding hne. the gyration radius and the rmsd of the ligand egcg and the receptor hne trended toward stability as time progressed (fig. a,b) . the interacting h-bond number was shown to change with in a stable range as time progressed (fig. c) . additionally, the gromacs energy did not fluctuate as time progressed changed (fig. d ). g_mmpbsa method calculated results showed that ∆g binding = − . ± . .kj/mol ( table ). to verify the prediction from the cdocker-based analysis that egcg directly binds to the hne protein, the binding affinity of egcg to hne was determined using spr biosensor technology. the ability of egcg to bind to hne was reflected by the ru values that were directly recorded from the biacore instrument. as shown in fig. a , the ru increased with increasing egcg concentration, indicating that egcg bound to hne in a concentration-dependent manner. the association (k on ), dissociation (k off ), and equilibrium dissociation constants (k d ) of egcg binding to hne were . × m − ·s − , . × − s − , and . × − m, respectively. the curve-fitting efficiency was evaluated according to the Χ , a statistical parameter in the spr assay. the Χ value was calculated to be . . the results indicated that egcg displayed specific binding affinity for hne (fig. a) . we first tested the enzyme activity of hne by adding the indicated concentrations of hne to the substrate solution system. the results showed that hne reacted with the substrate in a concentration-dependent manner (fig. b) . after establishing the steady enzyme-substrate reaction system, we tested the inhibitory effect of egcg at the indicated concentrations. sivelestat sodium was used as the positive control to evaluate the inhibitory effect of egcg. images of zeb- in a cells exposed to egcg, sivelestat sodium and neutrophil elastase. a cells were grown to % confluence and then incubated in μ m egcg, μ m sivelestat sodium and/or nm neutrophil elastase for h. the cells were labeled with an anti-human zeb- antibody, a dylight -conjugated secondary antibody (green) and the hoechst stain (blue). (b, c) the protein levels of vimentin were clearly down-regulated in response to egcg treatment with or without hne. (b, d) the expression of β -catenin was increased after hne induction but was significantly decreased after treatment with μ mol/l egcg. the gels were electrophoresed using the identical experimental conditions. the results showed that egcg inhibited hne activity in a concentration-dependent manner; the ic of egcg was . μ m (fig. c) , which was higher than that of sivelestat sodium (ic = . μ m) (fig. d) . aat is a serine protease inhibitor (serpin) and is the natural inhibitor of hne. the imbalance between aat and hne plays an important role in lung cancer progression . based on this understanding, we predominantly focused on the regulation of aat by egcg to investigate the mechanisms by which egcg inhibits hne-induced cell proliferation. western blot assays were used to assess whether the aat protein levels were altered by egcg in a cells. when co-incubated with neutrophil elastase ( nm), egcg ( μ m) enhanced aat expression in a cells (fig. a,b) . neutrophil elastase directly induced lung tumor cell proliferation by degrading irs- , which is an adapter protein of pi k, and subsequently activating the pi k pathway in the tumor cells . upon neutrophil elastase exposure, the protein level of irs- was decreased, and the phosphorylation of akt (thr ) and pi k were elevated. egcg ( μ m) enhanced the protein levels of irs- (fig. a,c) and reduced the phosphorylation of akt and pi k in the neutrophil elastase-stimulated a cells (fig. a,d-f ). in recent years, egcg has been reported to inhibit tumor proliferation and metastasis and to induce the apoptosis of lung cancer cells in vitro and in vivo . egcg suppresses proinflammatory cytokines and chemokines induced by toll-like receptor agonists in prostate cancer cells . in addition, egcg suppresses lung cancer cell growth via ras-gtpase-activating protein sh domain-binding protein- . many molecules are involved in the anti-tumor activity of egcg, including jak/stat, mapk, pi k/akt, wnt, notch, nf-κ b and ap- . because cancer progression is a complex process, its pathogenic mechanism remains somewhat elusive. egcg is considered as a natural multi-targeting chemopreventive agent. thus, the possibility that egcg inhibits tumor development via alternative mechanisms cannot be excluded. the purpose of this study was to investigate the novel inflammation-related mechanisms by which egcg mediates anti-cancer migratory activity in lung cancer. recent studies suggested that chronic inflammatory pulmonary diseases such as emphysema are highly associated with an increased risk of lung cancer, independent of smoking . hne, among the most potent proteinases released by neutrophils, is considered to be responsible for the elastolytic damage in emphysema . to some extent, neutrophil elastase may represent the link between emphysema and lung cancer. recent reports showed that modest levels of neutrophil elastase led to the uncontrolled proliferation of a lung epithelial tumor cells . we found that hne treatment ( nm) enhanced the , the pakt (thy )/ akt ratio (d), the pakt (ser )/akt ratio (e) and the ppi k/pi k ratio (f) in a cells treated with neutrophil elastase, egcg and sivelestat sodium. the data are presented as the means ± sem; n = . **p < . ; ***p < . . the images for irs- , aat and gapdh were cropped from the same gel. the images for p-pi k, p-akt (t ) and gapdh were cropped from the same gel. the images for pi k and p-akt (s ) were cropped from the same gel. the images for akt and gapdh were cropped from the same gel. all gels were electrophoresed using the identical experimental conditions. scientific reports | : | doi: . /srep models of breast cancer . therefore, the role of hne, which is generally considered as the major effector of human neutrophil function, particularly merits investigation. thus, we provide the first evidence for the proliferation-promoting effect of hne on a cells. next, we explored whether egcg directly binds to hne using the cdocker algorithm. cdocker is an efficient technique used to predict interactions between small molecules and proteins by modeling and rmsd analysis. the binding site was defined based on the definition of the positive control ligand (sei ) binding site. the analysis showed that arg a: and thr a: formed hydrogen bonds with the hydrogens on the phenolic hydroxyl groups of the small molecule egcg. additionally, arg a: formed a pi bond with egcg. applying minimization and md to egcg and hne enabled us to perform a ligand-protein interaction analysis, as depicted in fig. a-d. to determine the specific binding site, we directly transformed the cdocker binding results in the pdb file to an md preparation gro file. this analysis showed that the interaction between egcg and hne may not involve instantaneous binding but rather may be a long term interaction. based on the results of md simulation, we further calculated the binding free energy. according to study of sulea et al. , we verified that egcg could bind with hne protein. to verify these results concerning the interaction between egcg and hne, we further evaluated the binding affinity of egcg to hne using spr biosensor analysis. the results confirmed the computer modeling prediction. we are the first to discover this novel target of the multi-targeting molecule egcg. interestingly, the inhibitor of hne sivelestat sodium displayed a lower affinity to hne than egcg. we found that the low water solubility of sivelestat sodium led to the appearance of the lipid solvent as an interfering factor. because of equipment limitations, we could not resolve this issue in the present study. to confirm the inhibitory effect of the interaction between egcg and neutrophil elastase on the activity of this enzyme, we measured the enzymatic activity of neutrophil elastase after treatment with egcg or sivelestat sodium. alasbahi had verified the method of the elastase enzymatic activity assay. egcg exerted a concentration-dependent inhibitory effect on the enzymatic activity of neutrophil elastase. as the positive control, sivelestat sodium exerted a greater inhibitory effect than egcg. as aat is the specific and primary inhibitor of neutrophil elastase, we assumed that aat might mediate the inhibitory effect of egcg on neutrophil elastase-induced cell migration in vitro. aat is predominantly synthesized in liver but is also expressed in extra-hepatic tissues and cells, including carcinoma cells . the role of aat in cancer development has been supported by some laboratory data; however, the molecular mechanism underlying the role of aat in tumor cell migration is poorly understood. the primary function of aat is the inhibition of neutrophil elastase. our results indicated that the protein levels of aat in a cells were clearly increased after treatment with egcg. however, the contribution of aat to the tumor development is somewhat controversial. in contrast to its effects against tumor growth, aat has been shown to be correlated with poor prognosis in lung adenocarcinoma and with the inhibitory effect of polymorphonuclear neutrophils (pmns) on the proliferation and invasiveness of lung cancer hcc cells . upon binding to neutrophil elastase, aat activates cleavage within the reactive site loop (rsl) in a suicidal action . evidence has indicated that the aat levels are significantly elevated in the skin of ne −/− mice compared with ne +/+ mice in a murine model of the autoimmune disease bullous pemphigoid . it has been reported that the deficiency of circulating aat is highly associated with lung inflammation and, especially, the early onset of pulmonary emphysema . additionally, clinical findings have indicated that aat, which is elevated in the serum of cancer patients, is a serum biomarker for the diagnosis of lung cancer and prostate cancer . previous studies showed that egcg inhibits pi k/akt/mtor signaling in various tumor cells , . our previous data demonstrated that the inhibition of neutrophil elastase-induced cell proliferation using curcumin (a type of polyphenol similar to egcg) is also dependent on the pi k pathway in a cells . recently, it was shown that neutrophil elastase released by activated neutrophils within the lung is absorbed by adjacent epithelial tumor cells and degrades irs- , which is a binding partner of the p regulatory subunit of pi k, thereby inducing the hyperactivity of the pi k pathway, which leads to uncontrolled tumor cell proliferation . our results showed that the neutrophil elastase-induced decrease in irs- expression was significantly inhibited by egcg in a cells. the suppression of akt and erk / has been thought to mediate the anti-tumor migration property of egcg . therefore, we explored whether egcg treatment remarkably suppressed neutrophil elastase-induced phosphorylation and activation of pi k/akt. our results showed that the expression of ser -phosphorylated akt was increased in neutrophil elastase-induced a cells and was down-regulated after co-treatment with egcg and neutrophil elastase. moreover, we observed the phosphorylation of akt at thy . compared with the regulation of ser phosphorylation, the phosphorylation state of thy was not significantly changed after treatment with neutrophil elastase. these results indicated that the migration-promoting effect of neutrophil elastase was not mediated by akt phosphorylation at thy but might be mediated by the regulation of p -akt. in the positive control group, neither p -akt nor p -akt expression was significantly changed after treatment with the inhibitor of neutrophil elastase sivelestat sodium. we speculated that sivelestat sodium, as a selective neutrophil elastase inhibitor, cannot suppress the metastasis of lung cancer, in contrast to the multi-targeting natural product egcg, via the pi k/akt signaling pathway. taken together, these data suggest that the direct binding of egcg to neutrophil elastase and the stimulation of aat by egcg are crucial for the inhibition of the neutrophil elastase-induced migration of a cells by egcg. however, the molecules and signaling pathways that modulate the expression of aat remain unclear, and it is important to explore the mechanisms that regulate aat, which is a serpin that is highly related to lung inflammation and lung cancer. additionally, the role of egcg in the neutrophil elastase-induced induction of tumor metastasis and growth in other xenograft murine models of lung cancer remains to be investigated. this study demonstrates that egcg, an inhibitor of neutrophil elastase, modulates the migration of nsclc a cells and blocks the neutrophil elastase-induced migration of a cells by up-regulating aat expression. these results provide a novel inflammation-related mechanism by which egcg prevents tumor metastasis and suggests the potential of egcg for the treatment of other inflammation-related diseases in the lung, such as emphysema (fig. ) . global cancer statistics cancer-related inflammation green tea catechin, epigallocatechin- -gallate, attenuates the cell viability of human non-small-cell lung cancer a cells via reducing bcl-xl expression pi k/akt/mtor signaling is involved in (− )-epigallocatechin- -gallate-induced apoptosis of human pancreatic carcinoma cells inhibition of carcinogenesis by tea cancer chemoprevention with dietary phytochemicals targeting multiple signaling pathways by green tea polyphenol (-)-epigallocatechin- -gallate epigallocatechin- -gallate inhibits epidermal growth factor receptor signaling pathway. evidence for direct inhibition of erk / and akt kinases tumor-associated neutrophils: new targets for cancer therapy neutrophil alveolitis in bronchioloalveolar carcinoma: induction by tumor-derived interleukin- and relation to clinical outcome the dual role of neutrophil elastase in lung destruction and repair alpha -antitrypsin deficiency. : epidemiology of alpha -antitrypsin deficiency role of imbalance between neutrophil elastase and alpha -antitrypsin in cancer development and progression curcumin inhibits tumor proliferation induced by neutrophil elastase through the upregulation of alpha -antitrypsin in lung cancer open source drug discovery, c. & lynn, a. g_mmpbsa--a gromacs tool for high-throughput mm-pbsa calculations cinanserin is an inhibitor of the c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro severe acute respiratory syndrome coronavirus c-like proteinase n terminus is indispensable for proteolytic activity but not for enzyme dimerization. biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations inhibition of neutrophil elastase activity by cinnamic acid derivatives from cimicifuga racemosa neutrophil elastase-mediated degradation of irs- accelerates lung tumor growth the green tea polyphenol egcg potentiates the antiproliferative activity of c-met and epidermal growth factor receptor inhibitors in non-small cell lung cancer cells epigallocatechin- -gallate suppresses proinflammatory cytokines and chemokines induced by toll-like receptor agonists in prostate cancer cells epigallocatechin gallate suppresses lung cancer cell growth through ras-gtpase-activating protein sh domainbinding protein the role of the green tea component egcg in chemoprevention chronic obstructive pulmonary disease and altered risk of lung cancer in a population-based case-control study inflammatory proteinase slips into tumor cells tumor entrained neutrophils inhibit seeding in the premetastatic lung solvated interaction energy (sie) for scoring protein-ligand binding affinities. . benchmark in the csar- scoring exercise screening of some yemeni medicinal plants for inhibitory activity against peptidases effects of native and cleaved forms of alpha -antitrypsin on me tumor cell functional activity an evaluation of the prognostic significance of alpha- -antitrypsin expression in adenocarcinomas of the lung: an immunohistochemical analysis s. alpha -antitrypsin and its c-terminal fragment attenuate effects of degranulated neutrophilconditioned medium on lung cancer hcc cells, in vitro inhibitory mechanism of serpins. mobility of the c-terminal region of the reactivesite loop the serpin alpha -proteinase inhibitor is a critical substrate for gelatinase b/mmp- in vivo pulmonary emphysema and alpha -antitrypsin deficiency the importance of alpha- antitrypsin (alpha -at) and neopterin serum levels in the evaluation of non-small cell lung and prostate cancer patients green tea catechin, epigallocatechin- -gallate (egcg): mechanisms, perspectives and clinical applications epigallocatechin gallate inhibits growth and epithelial-to-mesenchymal transition in human thyroid carcinoma cell lines this work was supported by grants from the national natural science foundation of china (no. , , , , and ) and the research fund from the ministry of education of china ( project, no. b ). the funders played no role in the study design, data collection or analysis, the decision to publish, or the preparation of the manuscript. key: cord- - nzr authors: sugiyama, michael g.; armstrong, susan m.; wang, changsen; hwang, david; leong-poi, howard; advani, andrew; advani, suzanne; zhang, haibo; szaszi, katalin; tabuchi, arata; kuebler, wolfgang m.; van slyke, paul; dumont, dan j.; lee, warren l. title: the tie -agonist vasculotide rescues mice from influenza virus infection date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: nzr seasonal influenza virus infections cause hundreds of thousands of deaths annually while viral mutation raises the threat of a novel pandemic strain. antiviral drugs exhibit limited efficacy unless administered early and may induce viral resistance. thus, targeting the host response directly has been proposed as a novel therapeutic strategy with the added potential benefit of not eliciting viral resistance. severe influenza virus infections are complicated by respiratory failure due to the development of lung microvascular leak and acute lung injury. we hypothesized that enhancing lung endothelial barrier integrity could improve the outcome. here we demonstrate that the tie -agonist tetrameric peptide vasculotide improves survival in murine models of severe influenza, even if administered as late as hours after infection; the benefit was observed using three strains of the virus and two strains of mice. the effect required tie , was independent of viral replication and did not impair lung neutrophil recruitment. administration of the drug decreased lung edema, arterial hypoxemia and lung endothelial apoptosis; importantly, vasculotide is inexpensive to produce, is chemically stable and is unrelated to any tie ligands. thus, vasculotide may represent a novel and practical therapy for severe infections with influenza. whether as an agent of pandemics or as a seasonal pathogen, the influenza virus exacts a heavy toll on global public health. despite vaccination programs and antiviral drugs, seasonal influenza alone causes millions of cases of severe illness and hundreds of thousands of deaths annually , . there are concerns that ongoing mutation will lead to a novel strain of the virus that is both highly transmissible and highly virulent, as occurred in the pandemic leading to the deaths of million people . existing treatments for the virus are inadequate: antiviral drugs are not completely effective at reducing mortality , and exhibit declining efficacy unless given at the time of infection, a problematic limitation since the time of infection is usually unknown , . there is also the problem of antiviral resistance; of the two classes of drugs approved for use against influenza, one is largely ineffective due to widespread resistance, while sporadic cases of resistance to the other have been reported , . thus, there is a need for novel therapies; those that target the host rather than the pathogen may be ideal as they should be less susceptible to viral resistance. most deaths from influenza virus infection occur due to pulmonary complications, in particular the development of acute respiratory distress syndrome (ards) , , a potentially fatal syndrome of pulmonary edema that occurs due to increased permeability of the lung microvasculature , . blood vessels in the lung are lined by a continuous layer of endothelium; thus, loss of endothelial barrier integrity is a prerequisite for ards. the fact that mortality persists despite antiviral therapy suggests that elements of the host response may be maladaptive , . given the prominence of ards in severe infections, we hypothesized that enhancement of lung endothelial barrier function might improve survival. we tested the novel tie -agonist tetrameric peptide, vasculotide; this peptide has no sequence homology with any endogenous ligands, is easy to produce and is chemically stable . here we report that administration of vasculotide significantly improves survival from influenza virus infection, even when started several days post-infection. we first infected c bl/ mice with a lethal dose of influenza virus (hkx ;h n ) followed by daily intraperitoneal injection of vasculotide ( ng, see schematic, fig. a ). while no infected mice survived longer than days, treatment with vasculotide markedly improved survival even if started as late as hours post-infection ( % survival if started at hours after infection; % survival if at hours, p < . ); treatment starting hours after infection improved survival although this did not achieve protocol used to test vt in mice infected with influenza virus. arrows indicate the time after infection (hours) at which daily administration of vt was begun. c bl/ j mice were infected with mouseadapted influenza, strain x and received placebo or ng vt per day starting at the indicated times. (b) survival was monitored daily; numbers in parentheses indicate the number of mice per group. ***p < . vs. flu-alone mice. (c) arterial oxygen saturation on day after infection, ***p < . . (d) lung edema was measured by wet-to-dry ratio in mice sacrificed days after infection. (e) left ventricular ejection fraction on day after infection was measured by echocardiography. (f) c bl/ j mice were infected with mouseadapted influenza, strain pr and received placebo or ng vt daily starting at hours after infection, *p < . . scientific reports | : | doi: . /srep statistical significance (p = . ; fig. b ); treatment with a lower dose of vasculotide ( ng) did not appear to be as effective (supplemental figure ). prior to receiving vasculotide, mice displayed typical signs of progressive illness such as arterial hypoxemia, hypothermia, and declining body weight; these features are apparent as early as - hours after infection. vasculotide treatment started at hours post-infection is able to rescue a significant subset of infected mice (supplemental figures , ) . furthermore, virus-induced hypoxemia and lung edema were greatly ameliorated with the drug (fig. c-d) . echocardiography revealed that lung edema was not due to impairment of left ventricular systolic function by the virus, consistent with an increase in vascular leak from lung injury ; analogously, the reduction in lung edema by vasculotide was not from an improvement in left ventricular systolic function (fig. e) . of note, the drug was similarly effective against a lethal dose of pr (h n ), a second strain of influenza virus that is highly virulent in mice (fig. f) . next, we infected mice with the swine-origin pandemic h n influenza virus. although the mortality rate in these mice was only %, all of the vasculotide-treated mice survived (supplemental figure ) . thus, vasculotide appears to be effective against lung injury caused by multiple strains of influenza. in a clinical setting, an agent like vasculotide is most likely to be administered in combination with an antiviral drug. we therefore treated influenza virus-infected mice with amantadine alone or in combination with vasculotide. treatment with amantadine modestly but significantly improved survival (from to %, fig. a ). adjuvant vasculotide markedly increased survival even when started as long as hours after infection ( % survival) ( fig. a and supplemental figure ). this was in association with significant improvements in arterial hypoxemia (fig. b ), hypothermia and weight loss ( fig. c-d) . these findings are especially important because mortality from influenza virus persists despite antiviral therapy and because the efficacy of such therapy declines rapidly over time , , . to prove that the benefit of vasculotide is mediated through the tie receptor, and because homozygous deletion of tie is embryonically lethal , we obtained haploinsufficient tie +/− mice bred onto a cd background. these mice express significantly less tie on the lung endothelium . infection of cd wild-type mice with influenza virus (hkx ;h n ) resulted in significant lethality (only % survival) that was greatly improved by vasculotide ( % survival), even if started hours after infection (fig. a) . conversely, infection of haploinsufficient tie +/− mice resulted in a similar degree of mortality ( % survival) that was not significantly changed by the drug ( % survival), although there was a trend towards an intermediate phenotype (fig. b) . furthermore, in contrast to wild-type mice, vasculotide had no effect on influenza virus-induced hypoxemia or hypothermia in the haploinsufficient mice ( fig. c-d) . thus, the benefit of vasculotide is dependent on tie expression. while tie is widely thought to be almost exclusively expressed on endothelial cells , we considered the possibility that its benefit might reflect unanticipated activity on non-endothelial tissues or on the virus itself. we first established that vasculotide has no antiviral activity in vitro and in vivo (fig. a) . second, vasculotide did not affect chemotaxis of human and murine monocytes and neutrophils in vitro (fig. b-c and data not shown). to our surprise, despite significantly improved arterial oxygen saturation and body weight ( fig. d -e), vasculotide-treated mice exhibited no difference in the severity of lung injury determined by blinded quantification of lung histology at days post-infection ( fig. g ) (this timepoint was chosen as infected mice die soon afterwards). concordantly, while infection induced a marked increase in alveolar neutrophil recruitment, this was unaffected by treatment with vasculotide ( fig. f ); alveolar and serum cytokine levels on day post-infection were also unaffected (supplemental figure ). thus, the mechanism of benefit of vasculotide appears downstream of innate immune responses and instead involves the lung endothelial barrier. as expected, vasculotide induced tie phosphorylation and significantly attenuated thrombin-induced lung endothelial leak, as measured by electrical resistance (supplemental figure a -b). while the drug had no effect on human lung endothelial proliferation (supplemental figure ) , it significantly attenuated lung endothelial apoptosis in vitro in response to influenza virus, as assessed by cleavage of caspase- (supplemental figure c ); we observed a similar reduction in cleaved caspase- in lungs from infected mice who received vasculotide (supplemental figure d ). although the apoptotic cells in the lungs of infected mice undoubtedly reflect contributions from epithelium and leukocytes, we observed colocalization of cleaved caspase- with the endothelial-specific marker ve-cadherin, consistent with our in vitro data. finally, while influenza infection caused loss of tie from the lungs of infected mice, this was significantly attenuated by treatment with vasculotide. vasculotide treatment also markedly increased levels of phospho-akt in the lung, an event that is known to be downstream of tie activation and that acts as an endothelial pro-survival signal (supplemental figure ). our findings have a number of important implications. first, these data strongly implicate failure of the lung endothelial barrier as the cause of death in murine models of severe influenza, as vasculotide conferred a significant survival benefit against multiple strains of the virus in two strains of mice. the effect of the drug is tie -dependent, appears to be downstream of innate immune activation and does not require inhibition of viral replication. second, the enhancement of lung endothelial barrier integrity did not impair leukocyte recruitment to the lung, supporting the notion that vascular leak and leukocyte transmigration may be regulated independently . accordingly, administration of vasculotide at the time of infection (i.e. even before leukocyte infiltration could occur) was not harmful and still conferred great benefit (fig. b) . lastly, administration of vasculotide even - days after infection significantly improved survival, an observation with practical implications given that the moment of infection in patients is usually uncertain. while other ligands for tie have been studied as therapies for vascular leak (as reviewed in ), none have been reported in severe influenza virus infection and almost all required administration to be prophylactic or simultaneous with the time of infection , , limiting their clinical utility. the potential clinical use of recombinant angiopoietin , the endogenous ligand for tie , is limited by its tendency to bind to the extracellular matrix and to form undesirable aggregates ; furthermore, modified forms of the protein are reportedly difficult to synthesize and are unstable . in contrast, vasculotide was invented based on phage display experiments in which over billion unique peptides were screened to identify those capable of binding with high affinity to tie , . vasculotide bears no sequence homology to angiopoietin- and exhibits no activity against or binding to some cellular receptors (unpublished data); its specificity for tie is also strongly supported by our data using tie haploinsufficient mice. given the existing literature on ang and other putative barrier-enhancing agents , , , the ability of delayed administration of vasculotide to rescue mice dying of influenza (supplemental figure ) is unexpected and novel. this finding, combined with its relatively low cost of production and its chemical stability, suggests that the drug might constitute a practical therapy. our data indicate that vasculotide increases endothelial barrier integrity and reduces lung endothelial apoptosis, consistent with its agonism of tie . further work will determine whether it affects other mediators of vascular instability including angiopoietin- and ve-ptp . as its mechanism of action is independent of viral replication and instead involves modulation of the host response, it is possible that vasculotide may not engender viral resistance, but this hypothesis will require direct testing. since influenza virus infections are often complicated by secondary bacterial pneumonia , novel agents that impair the innate immune response may not be without risk. the fact that vasculotide does not impede neutrophil recruitment to the lung is therefore reassuring. finally, whether the impressive benefit of vasculotide in the therapy of the influenza virus carries implications for other infectious agents or non-infectious causes of ards will require further investigation. experimental design. fourteen-week old c bl/ j mice were purchased from jackson laboratories for all experiments, mice were sedated with % isoflurane and infected intranasally with influenza virus (see virus section, below) diluted in pbs to a final volume of μ l. after infection, mice were separated into weight-matched control and treatment groups; unless otherwise indicated, we used ten mice per group and mice were infected with hau of influenza virus, which caused greater than % mortality by day . in the first experiment, we included the following groups (see fig. b ): mice that received the influenza virus alone (flu) and infected mice that also received vasculotide ( ng in . ml pbs by intraperitoneal injection, the dose based on previous work ); of the latter, mice that received vasculotide were divided a priori into those who received it at the time of infection (vt ) and then once daily; those that in other experiments to test vt as an adjuvant therapy ( fig. and supplemental figure ), we divided mice into the following six groups: influenza-virus infected mice (flu); infected mice that received a dose of amantadine by oral gavage twice daily at a concentration of mg/kg/day (flu/amant); and infected mice that received amantadine and a daily intraperitoneal injection of vt starting , , to test the requirement for tie (fig. ) , cd- wildtype and tie +/− mice bred onto the cd- background were obtained from sunnybrook research institute (toronto, ontario); tie +/− mice were backcrossed onto the cd- background for a minimum of generations. these mice were divided into the following four groups, depicted in fig. : cd- wildtype mice infected with influenza virus (flu, fig. a) ; cd- wildtype mice that were infected and given vt daily starting hours after infection (flu/vt , fig. a) ; tie +/− mice infected with influenza virus (flu, fig. b) ; tie +/− mice that were infected and given vt daily starting hours after infection (flu/vt , fig. b) . in all experiments, mice were monitored times daily for the duration of the experiment ( - days after infection) and were scored for weight loss, hypothermia, hypoxemia, spontaneous activity (scored from (moribund) to (normal), as described in ) , and other clinical features of influenza infection. mice were euthanized if two or more of the following occurred: weight loss exceeded % of initial weight; temperature fell below °c; the animal appeared moribund. in some experiments, mice were sacrificed on the indicated day for collection of blood and tissue samples. . in vitro measurement of viral replication was also measured by qpcr as previously described . pulse oximetry measurements. arterial oxygen saturation was measured using the mouse ox plus device and software (starr life sciences, oakmont, pa) on awake (non-anesthetized) mice using either the small or medium collar clips for c bl/ j and cd- and tie +/− mice, respectively. for c bl/ j mice, a chemical depilatory cream was used to remove hair around the base of the neck several days before infection. for so measurements, mice were allowed to acclimate to the collar clip for several minutes in their home cage before recording the maximal so measurement. mice treated with or without vt starting hours after infection were sedated and sacrificed days after infection by cardiac puncture. this timepoint was chosen as infected mice die soon afterwards. after sacrifice, the trachea was exposed and the lungs were inflated with . ml cold pbs with . mm edta. after s, . ml balf was recovered and centrifuged at x g to pellet the cells. supernatant was collected and immediately frozen for analysis of balf cytokines. the balf pellet was resuspended in μ l pbs. neutrophils were stained with . measurement of edema. c bl/ j mice were infected with hkx and vt ( ng) or pbs was injected by ip injection once daily for days. on day , mice were sacrificed by cardiac puncture of the right ventricle. the circulatory system was flushed with ml cold pbs by instillation through the left ventricle. the entire right lung was removed, patted dry, and then weighed for determination of wet weight. lung tissues were dried for hours and then weighed for the dry weight. degree of lung edema is expressed as the wet-to-dry ratio of lung weights. echocardiography. c bl/ j mice were infected with hkx influenza and vt ( ng daily) or pbs treatment was started hours after infection. on day , mice were lightly sedated and a depilatory cream was used to remove hair from the ventral surface of the thorax and abdomen. on day , mice were lightly sedated and left ventricular function was measured by echocardiography as previously reported . histology and immunohistochemistry. hkx -infected c bl/ j mice (n = per group and scientific reports | : | doi: . /srep and eosin; a lung pathologist (d.h.) blinded to the experimental design scored acute lung injury based on consensus criteria, yielding a composite score between (no injury) to (highest degree of lung injury) . degree of lung injury was determined by scoring randomly selected high power fields ( x magnification) per mouse. for detection of cleaved caspase- , immunofluorescence microscopy was performed on formalin-fixed paraffin embedded lung tissue as previously described . sections were dual-stained with a goat polyclonal antibody directed against ve-cadherin (santa cruz biotechnology) and a rabbit polyclonal antibody directed against cleaved caspase- (cell signaling technology). slides were washed and subsequently incubated with alexa fluor donkey anti-goat and alexa fluor donkey anti-rabbit secondary antibodies, both from life technologies inc. (burlington, on, canada) . blinded quantification of cleaved caspase- was performed on randomly selected lung fields per mouse acquired by spinning disc confocal microscopy (zeiss axiovert m microscope, settings kept constant between conditions) and analyzed using imagej (nih). cell culture and reagents. primary human lung microvascular endothelial cells (hpmecs) obtained from promocell (heidelberg, germany) were cultured in ebm- media (lonza, basel, switzerland) with the recommended supplements and used in passages - . the thp- human monocytic cell line was a gift from amira klip (toronto). human neutrophils were obtained from healthy volunteers and were isolated from whole blood by density gradient separation . vasculotide, a pegylated tetrameric peptide agonist of the tie receptor, was synthesized as previously described and prepared to a concentration of ng/μ l in sterile phosphate-buffered saline (pbs) without calcium and magnesium. a working solution of vt was prepared by diluting the stock ten-fold in pbs. immunoblot. hpmec-l were grown to % confluency on -well plates and treated with x influenza or x influenza plus ng/nl vt for hours. lysates were prepared with lysis buffer ( . mm tris-hcl ph . , % sds, % glycerol, mm dtt). sds page was performed on a % polyacrylamide gel. immunoblotting was performed as previously described with the exception of the blocking step, which used % bovine serum albumin in tbs instead of % milk. in some experiments, lung homogenates were generated from infected mice as follows: after euthanasia, the circulation was flushed with ml ice-cold pbs. the lungs were homogenized in buffer containing mm nacl, mm edta, % glycerol, % triton x- , mm tris-hcl, mm sodium orthovanadate, and complete protease inhibitor cocktail tablets and separated using % polyacrylamide gels. proteins were transferred to nitrocellulose membranes, blocked for hour in % milk in tbs, and probed overnight with primary antibody at °c. after washing, blots were incubated with hrp-conjugated secondary antibodies for hour, washed, and then visualized by chemiluminescence (thermoscientific). band intensity was quantified using imagej (nih) and normalized to the loading control after background correction. the anti-phospho-akt (ser ) antibody was from cell signaling; the anti-tie (c ) antibody was from santa cruz; all other antibodies were from santa cruz biotechnology. chemotaxis assay. × neutrophils (isolated from healthy human volunteers) in μ l of dmem with low-glucose or × thp- cells in μ l of rpmi with % fbs and % penicillin/streptomycin containing ng/ml or ng/ml vt or pbs were seeded into the upper part of a transwell chamber ( -μ m pore size, . mm in diameter; corning life sciences). the lower chamber of the transwells was filled with μ l of dmem containing vehicle (dmso) or μ m fmlp for neutrophil chemotaxis or with μ l of rpmi , % fbs, % penicillin/streptomycin containing μ m atp for thp- chemotaxis. after h of incubation at °c, the migrated neutrophils or thp- cells were recovered from the lower chamber and counted using a hemocytometer. electric cell substrate impedance sensing (ecis). an ecis ztheta system (applied biophysics, troy, ny) was used to follow barrier function. hpmecs were seeded on applied biophysics ecis culture arrays pretreated with mm cysteine at . × /well and grown for h to allow the establishment of a confluent layer. next, the arrays were placed into the ecis system and impedance and capacitance (c) data were collected (and r values calculated by the software) continuously using the frequency scan mode. after obtaining a baseline, the measurement was paused and thrombin was added to the arrays in μ l medium to induce endothelial permeability, at a final concentration of unit/ml. some wells received vt alone or in combination with thrombin at a concentration of ng/ml. controls received μ l medium without thrombin. for each condition measurements were performed in duplicate. curves were normalized to the resistance level prior to thrombin administration using the ecis software. the ecis trace represents the data collected at hz and is representative of independent experiments. mtt and scratch assay. hpmecs were seeded on well tissue culture plates at a density of . × cells/ml ( wells per condition). cells were incubated for four hours to allow cells to attach. after hours (time ), the media was changed to egm- (control) or egm- with ng/ml vt. for baseline and endpoint measurements, mtt (sigma) was added to the cells at time and hours after treatment followed by a -hour incubation. mtt and media were aspirated and the cells were solubilized in ul dmso and shaken for minutes. absorbance was measured at nm. for the scratch assay, hpmecs were grown to confluency on -well tissue culture plates marked on the bottom with an open square to define the scratch area. a single scratch was made through the scratch area using a sterile p pipette tip. the initial scratch area was imaged by phase-contrast microscopy. cells were washed once with media and then treated with egm- or egm- with ng/ ml vt for hours. after hours the cells were imaged again to assess the degree of wound healing. phase-contrast images were exported to imagej for quantification of baseline and endpoint wound areas. wound healing is expressed as a fraction of the endpoint area occupied by cells over the baseline scratch area. quantification of wound healing was performed in a blinded fashion. statistics. all statistics were performed using graphpad prism (la jolla, ca) software. comparisons between two groups were made using unpaired, two-tailed student's t-tests with statistical significance set at p < . . comparisons of more than groups were made using one-way anova with post-hoc analyses applying bonferroni's adjustment for comparisons between selected groups. for comparing kaplan-meier survival curves, the log-rank test was used with p < . . statistics for ecis experiments were 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neutrophil isolation protocol co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and rac this work was supported by grants from the canadian institutes of health research (ocn and mop ), the physicians' services incorporated foundation ( ) ( ) ( ) ( ) ( ) and an early researcher award (government of ontario), all to wll. the authors thank chris spring, pamela plant, xiaofeng lu, caterina di ciano-oliveira and judy trogadis from the research core facility at the keenan research centre for technical assistance; johnny su for assistance with densitometry, julie khang for assistance with luminex and qinghong dan for assistance with ecis. key: cord- - yvyiiuy authors: nikas, jason b. title: inflammation and immune system activation in aging: a mathematical approach date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: yvyiiuy memory and learning declines are consequences of normal aging. since those functions are associated with the hippocampus, i analyzed the global gene expression data from post-mortem hippocampal tissue of old (age ≥ yrs) and young (age ≤ yrs) cognitively intact human subjects. by employing a rigorous, multi-method bioinformatic approach, i identified genes that were the most significant in terms of differential expression; and by employing mathematical modeling, i demonstrated that of the genes were able to discriminate between the old and young subjects with high accuracy. remarkably, % of the known genes from those most significant genes are associated with either inflammation or immune system activation. this suggests that chronic inflammation and immune system over-activity may underlie the aging process of the human brain, and that potential anti-inflammatory treatments targeting those genes may slow down this process and alleviate its symptoms. i n the absence of any neurodegenerative disease, the aging process of the human brain is inevitably and quintessentially characterized by memory and learning impairments. unlike in the case of a neurodegenerative disease, normal aging has not been associated with neuronal loss [ ] [ ] [ ] . rather, it has been observed that the impairments induced by normal aging are associated with synaptic remodeling, and that they are more likely to affect functions that are associated with the hippocampus, i.e. several areas of memory and learning , , . in order to study the process of human normal aging, this study focuses on the most vulnerable target of that process, namely, the hippocampus. given the long, gradual course of the normal aging process, i arbitrarily defined the boundaries of the two groups as follows: ) old subjects (o) with age $ years and ) young subjects (y) with age # years. this -yr age gap, i theorized, would accentuate the contrast between the two groups in connection with this otherwise continuous and overlapping process. i analyzed the global gene expression data from post-mortem hippocampal tissue (harvested from the body of the hippocampus at the level of the lateral geniculate nucleus) of old and young cognitively intact human subjects, posted at the gene expression omnibus (gse ) . demographical information pertaining to all subjects is shown in supplementary table . having employed three different and independent methods of statistical significance, namely, roc curve analysis, fold change, and p-value, i was able to identify genes that were the most significant in terms of differential expression. fig. b depicts the results of k-means clustering analysis based on the expression of the top most significant genes. all k-means clustering analysis results (with respect to both the housekeeping genes and the most significant genes) are shown in supplementary table . as can be seen in both fig. b and supplementary table , there is a clear separation of the two groups. fig. depicts the heat map that resulted by plotting the expression of those genes for all subjects ( young and old). as can be seen by the relative intensities, all of the most significant genes are over-expressed (red color) in the case of the old subjects as compared with the case of the young subjects (blue color). the direction of the differential expression of those genes also appears in table . moreover, fig. provides a d representation of the differential expression of those genes between the two groups in a surface-contour plot. classify the subjects with a high accuracy. such a model would be valuable in future studies of global gene expression analysis of post-mortem hippocampal tissue investigating biological and chronological aging. to that end, i randomly selected approximately % of the subjects [ / young subjects and / old subjects] for the development of the function (henceforward referred to as super variable), and i used the remaining subjects ( young and old) solely for the purpose of validating the super variable. employing a general methodology that i have previously introduced , , i was able to generate a super variable (function) that, based on the input of genes from the most significant genes, was able to identify/classify accurately all but one of the old subjects {subject # [ yrs (f)]} [sensitivity ( / ) . ] and all of the young subjects [specificity ( / ) . ]. those overall results of the performance of the f super variable were obtained by combining the results from the development and the validation phases. according to the rank that appears in table , the seven genes that provide the input to the f super variable are: c a (c b), adora , ms a , bcl , cd , c ar , and hla-drb . all of those seven genes are, in terms of biological function, either genes of inflammation or genes of immune system activation. supplementary fig. shows the f super variable function in relation to its input gene variables. fig. table show the overall results of the f super variable, i.e. the f scores of all subjects used in this study, as well as their respective classification. fig. and supplementary table were created by combining the results from the development phase (the f scores of all subjects that were randomly selected and used exclusively for the development of the model) with the results from the validation phase (the f scores of all subjects that were randomly selected and used exclusively for testing purposes). the results of the f super variable in the development phase are shown in supplementary fig. and supplementary table , whereas the results in the validation phase are shown in supplementary fig. and supplementary table . it is interesting to note here that, assessing and comparing the performance of the f super variable (supplementary table ) with that of the supervised k-means clustering (supplementary table ), one can see that the former yielded one misclassification as opposed to four yielded by the latter. finally, it should also be noted here that, owing to the constraints of this study, namely, the paucity of healthy, normal human brain tissue samples and respective available data, the f super variable needs to be further validated with a larger, independent cohort. biovariability of aging. it has long been observed empirically that aging is not a steady-state, uniformly continuous process; that it is characterized by a relatively wide biovariability; and that biological age may not necessarily coincide with chronological age. the results of my study corroborate those observations. looking at the expression of the most significant hippocampal genes of all subjects table ). moreover, the aforementioned observations about the biovariability of the aging process were also supported by the results of hierarchical clustering analysis performed on the f scores of all subjects (supplementary fig. ) . young subjects (y) with respect to the house-keeping genes (a), and with respect to the most significant genes (b). in (a), in connection with the house-keeping genes, the two groups are inseparable and indistinguishable; whereas in (b), in connection with the most significant genes, the two groups are separated and are clearly distinguishable. d is subject distance from the centroid of cluster , and d is subject distance from the centroid of cluster . inflammation and immune system activation in aging. remarkably, of the known genes out of the most significant genes, were -in terms of function -either genes of inflammation or genes of immune system activation (table ). this suggests that -to a large extent, and insofar as it pertains to the hippocampal area of the brain -the dual process of a chronic inflammation and the elicited chronic immune-system response and activity can differentiate between old and young brains with a high accuracy. this is further supported by the fact that the aforementioned seven genes employed by the f super variable, all of which are genes of inflammation or genes of immune system activation, can discriminate between old and young brains with almost a perfect accuracy [sensitivity ( / ) . and specificity ( / ) . ]. the seven genes [c a (c b), adora , ms a , bcl , cd , c ar , and hla-drb ], which are the constituent input variables of the model (f super variable), and all of which are -in terms of function -inflammation or immune system activation genes (table ) , were all found to be over-expressed in the old subjects compared with the young subjects (table ) . c a (c b) has been observed to be over-expressed in patients with huntington disease and alzheimer disease , in mice with rheumatoid arthritis , etc. adora has been found to be over-expressed in the hippocampus of patients with parkinson disease , in patients with astrocytomas , etc. ms a has been observed to be over-expressed in mice with rheumatoid arthritis . bcl has been observed to be over-expressed in patients with huntington disease , with ischemic stroke , with rheumatoid arthritis , with b-cell lymphoma , etc. cd has been found to be over-expressed in patients with systemic lupus erythematosus , with immune thrombocytopenia , with schwannomas , with huntington disease , and numerous other diseases and conditions. over-expression of c ar has been observed in patients with severe acute respiratory syndrome , with asthma , etc., while overexpression of hla-drb has been observed in patients with multiple sclerosis , with rheumatoid arthritis , with duchenne muscular dystrophy , etc. previous studies using animal models have observed associations between aging and inflammation in connection with the hippocampus, the neocortex, and the cerebellum , . using animal models or human subjects with early-stage neurodegenerative diseases, such as alzheimer, other studies have observed a link between neuroinflammation and deficits in synaptic plasticity, especially long-term potentiation (ltp) in the hippocampus, which is associated with longterm memory consolidation [ ] [ ] [ ] [ ] [ ] [ ] . the fact that definitive causality cannot be established here notwithstanding -in other words, whether it is the normal aging process that induces inflammation/ immune-system-overactivity, or whether the vice versa occurs, or whether another, hitherto unspecified, process engenders the normal aging process, which in turn induces inflammation/immune-systemoveractivity, or whether that unspecified process engenders inflammation/immune-system-overactivity, which in turn induces the normal aging process -the results of my study support a direct causal link between the normal aging process and the process of inflammation/immune-system-overactivity. when considered collectively, therefore, the results of my study and all of the above observations from the other aforementioned studies point to a plausible theory on the normal aging process. at some point in time, chronic, low-level inflammation establishes itself and elicits a corresponding chronic immune response and activity. these two conjugate processes ultimately are responsible for a gradual loss of synaptic plasticity, particularly ltp in the hippocampus, accompanied with a minimal neuronal loss , , [ ] [ ] [ ] . it is this loss of synaptic plasticity -at least in the hippocampus part of the brain -that is associated with the phenotypical changes of normal aging. the results of my study, in addition to providing evidence for this dual process of chronic, low-level neuroinflammation/immune-system-activation in connection with normal aging, suggest a means of a potential treatment. regardless of the exact causal sequence of the events, administration of anti-inflammatory drugs/chemicals that can normalize the expression of the aforementioned genes of inflammation/immune-system-activation may decelerate the onset of the aging process, as well as the aging process itself, and mitigate its symptoms by restoring synaptic plasticity throughout the hippocampus and possibly throughout the rest of the brain. supplementary table lists all those most significant genes as possible targets for the development of such an anti-inflammatory treatment, along with potential candidate drugs/chemicals that are known (via ingenuity pathway analysis) to interact with those genes. it is worth noting here that various anti-inflammatory drugs have been used in an effort to slow down the progression of neurodegenerative diseases, such as alzheimer, with various degrees of success , . the magnitude of the neuroinflammatory processes in the case of alzheimer disease or other neurodegenerative diseases, however, cannot be compared to that of the neuroinflammation in the normal aging process; and by virtue of the same argument, the task of . the f uses of the most significant genes as its input variables. using the expression value of those genes for a particular subject, the f yields the f score of that subject; and, based on the determined cut-off score of . , the f classifies that subject as young if the f score is , . or as old if the f score is $ . . as can be seen by the overall performance, the f classified correctly all subjects except one old one [sensitivity ( / ) . and specificity ( / ) . ]. the mean f score of the y subjects was . (top of the blue bar) and their standard deviation (whiskers above or below the top of the blue bar) was . . the mean f score of the o subjects was . (top of the red bar) and their standard deviation (whiskers above or below the top of the red bar) was . . the significance level was set at a . (two-tailed), and the probability of significance for the f was p . (independent t-test with t-value . ). the f is parametrically distributed with respect to both groups. the f scores of all subjects are shown in supplementary table . www.nature.com/scientificreports scientific reports | : | doi: . /srep halting neuronal cell loss during the course of a neurodegenerative disease cannot be compared to that of restoring synaptic plasticity during the course of the normal aging process. it would stand to reason, therefore, that anti-inflammatory treatment strategies may be more successful and efficacious than those employed against the progression of neurodegenerative diseases. finally, i should point out that, based on increased evidence over the last twenty years or so, neuroinflammation seems to be the common denominator of normal aging and neurodegenerative diseases, such as alzheimer. understanding the causal circumstances under which a chronic, low-level neuroinflammatory process can transition to a major neuroinflammation conducive to neuronal degeneration and death would be of paramount importance. according to the latest evidence , even a single substitution mutation on a single inflammation gene might suffice to trigger that transition in a small percentage of the population. data acquisition. i downloaded the raw intensity microarray data (cel files) of old subjects (age $ years) and young subjects (age # years) posted at the gene expression omnibus (accession number: gse ) . data processing. i processed the original raw intensity data (cel files) using the expression console software by affymetrix with the library for the hg-u plus . microarray chip, and choosing the rma algorithm ( (k) fda approved) with the standard settings. statistical methods. i first assessed the quality of the data by examining the expression of all housekeeping genes by all subjects ( old and young). with regard to the housekeeping genes, there was no statistically significant differential expression between the two groups (supplementary table ). as can be seen by the results of k-means clustering analysis in fig. a , the two groups cannot be discriminated based on the expression of the housekeeping genes. having, thus, established the quality of the data, i investigated for any differential expression among all gene variables using three different and independent methods. ) using a methodology that i have developed and introduced previously - , i performed roc curve analysis on all gene variables in order to assess their discriminating power with respect to the two groups (old vs. young), and with respect to this method, i set statistical significance at roc auc $ . . table . in order to minimize the number of false negatives in the case of the third method , , for the final selection of significant variables, i imposed the condition that if a given gene variable met the significance criteria of all three methods, or those of the first method and those of only one of the other two methods, it would be deemed significant. excluding multiplicities (different transcripts that corresponded to the same genes), thirty six genes made up the final list of the most significantly differentially expressed genes between the two groups, as assessed by the aforementioned three different and independent methods of statistical significance (table ) . in greater detail, to assess statistical significance, i used to assess statistical significance, i used the following three different and independent methods. ) roc curve analysis. i performed roc curve analysis on all gene variables in order to assess their discriminating power with respect to the two groups (old vs. young), and with respect to this method, i set statistical significance at roc auc $ . . ) fold change. for all gene variables, i defined fold change (fc) as the mean expression value of the old subjects over the mean expression value of the young subjects, and i set statistical y) . ) p-value. i used the independent t-test for parametric gene variables (both normality and homogeneity of variance conditions were met); the aspin-welch unequal-variance test (aw) for gene variables that met the normality condition but not the homogeneity of variance condition; and the mann-whitney u test (mw) for the non-parametric gene variables, i.e., for those variables that i) the normality condition was not met or ii) the normality and the homogeneity of variance conditions were not met. taking into account that there are , probe sets (including those of the housekeeping genes) in the affymetrix hg-u plus . chip, and using the bonferroni correction, i set the significance level for the entire study at a . . therefore, in order for any variable to be deemed significant according to the p-value method, the following condition must be met: p , a. regarding the mann-whitney u test (mw), since none of the non-parametric variables had any sets of ties (a subject from one group having the same expression value as a subject from the other group), i used the exact probability for all mw tests. incorporating the three aforementioned independent methods of statistical significance assessment, and in order to minimize the number of false negatives in the case of the third method , , i set the overall significance criterion as follows: in order for any variable to be included in the final list of the most significant variables, it would have to meet the significance criteria of the first method (roc auc $ . ) and those of at least one of the other two methods [fc $ . (or fc # . ) and/or p , . ]. mathematical modeling. utilizing the final most significant genes, i wanted to explore the possibility of developing -via mathematical modeling -a function that could identify as correctly as possible the age status (o or y) of an unknown subject based on the expression of any combination of those most significant genes. to that end, i randomly selected approximately % of the subjects [ / young subjects and / old subjects] that could be used only for the development phase of such function. in other words, a function could be developed only by the exclusive use of those subjects. the remaining subjects ( young and old ones) were designated unknown (test) subjects and were used solely for the purpose of validating any promising function generated in the development phase. this split into two fixed sets, whereby one is used only for training and the other only for validation, represents the simplest implementation of k-fold cross validation , . a function was deemed promising in the development phase only if it exhibited a sensitivity $ . and a specificity $ . in connection with the subjects of the development phase. pertaining to the validation phase, and in connection with the unknown subjects, a promising function would have to exhibit the same minimum classification accuracy (a sensitivity $ . and a specificity $ . ) in order to be accepted. i was able to generate one such function (f -henceforward also referred to as super variable) that fulfilled all of the aforementioned criteria. supplementary fig. shows the equation of f as a function of genes. the cut-off score of the f was determined by taking into account the results of the following two analyses: ) calculation of the optimal point on the roc curve based on the f scores of the subjects used in the development phase [optimal point is defined as the point with the highest sensitivity and the lowest false positive rate ( specificity)] and ) calculation of the . % confidence intervals for the mean f scores of the two groups (o and y) of those subjects and their respective standard deviations. the . % confidence intervals were calculated based on a bootstrap sample size of , . taking into account the aforementioned roc optimal point, as well as the relative overlap of mo and my [mo llo -sdo and my uly sdy] (llo: the . % confidence lower limit for the mean of the o group; sdo: standard deviation of the o group; uly: the . % confidence upper limit for the mean of the y group; sdy: standard deviation of the y group), the cut-off score of the f super variable was determined to be . . if a subject's f score is , . , then that subject is classified as y (young); otherwise, if the f score is $ . , then that subject is classified as o (old). it should be pointed out here that, based on the equation of the f ( supplementary fig. ), a given f score is just a numerical value and does not signify age or number of years. in addition to the main validation method explained above, and in order to further assess the performance of the f super variable, i employed two other and different cross validation methods: ) a -fold cross validation and ) a leave-one-out cross validation . both of those methods yielded a misclassification rate of . and a mean-squared error of . in connection with the f super variable. the results of those methods, along with the confusion matrices generated by them, are shown in supplementary table . as can be seen in supplementary table , each one of those two and different validation methods resulted in a correct classification of all of the young subjects and in a correct classification of all but two of the old subjects. computer software. all analyses in this study were carried out with custom software written in matlab r b. all computer programs in connection with the model were also created using matlab r b. the ageing cortical synapse: hallmarks and implications for cognitive decline neural mechanisms of ageing and cognitive decline aging and the human neocortex genomic and proteomic strategies to identify novel targets potentially involved in learning and memory gene expression changes in the course of normal brain aging are sexually dimorphic mathematical prognostic biomarker models for treatment response and survival in epithelial ovarian cancer prognosis of treatment response (pathological complete response) in breast cancer regional and cellular gene expression changes in human huntington's disease brain complement activation by beta-amyloid in alzheimer disease identification of arthritis-related gene clusters by microarray analysis of two independent mouse models for rheumatoid arthritis therapeutic potential of adenosine a( a) receptor antagonists in parkinson's disease a adenosine receptors in human astrocytoma cells: agonist-mediated desensitization, internalization, and down-regulation gene expression in huntington's disease skeletal muscle: a potential biomarker the future of genomic profiling of neurological diseases using blood inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of rheumatoid arthritis primary mediastinal b-cell lymphoma: high frequency of bcl- mutations and consistent expression of the transcription factors oct- , bob. , and pu. in the absence of immunoglobulins phosphorylated erm is responsible for increased t cell polarization, adhesion, and migration in patients with systemic lupus erythematosus microrna regulate immunological pathways in t-cells in immune thrombocytopenia (itp) cd expression is aberrant in benign schwann cell tumors possessing mutations in the neurofibromatosis type , but not type , gene expression profile of immune response genes in patients with severe acute respiratory syndrome complement regulates inhalation tolerance at the dendritic cell/t cell interface genome, epigenome and rna sequences of monozygotic twins discordant for multiple sclerosis autoimmune diseases cdna microarray analysis of individual duchenne muscular dystrophy patients immune response gene expression increases in the aging murine hippocampus gene expression profiling of aging using dna microarrays synaptic correlates of increased cognitive vulnerability with aging: peripheral immune challenge and aging interact to disrupt theta-burst late-phase long-term potentiation in hippocampal area ca age-related neuroinflammatory changes negatively impact on neuronal function neuroinflammation and synaptic loss circuitspecific alterations in hippocampal synaptophysin immunoreactivity predict spatial learning impairment in aged rats gene regulation and dna damage in the ageing human brain gene-expression profile of the ageing brain in mice preserved neuron number in the hippocampus of aged rats with spatial learning deficits aging, spatial learning, and total synapse number in the rat ca stratum radiatum protective effects of nsaids on the development of alzheimer disease relation of nsaids to incident ad, change in cognitive function, and ad pathology variant of trem associated with the risk of alzheimer's disease comparison of analytical mathematical approaches for identifying key nuclear magnetic resonance spectroscopy biomarkers in the diagnosis and assessment of clinical change of diseases roc-supervised principal component analysis in connection with the diagnosis of diseases application of clustering analyses to the diagnosis of huntington disease in mice and other diseases with well-defined group boundaries linear discriminant functions in connection with the micro-rna diagnosis of colon cancer apobec b is an enzymatic source of mutation in breast cancer what's wrong with bonferroni adjustments do multiple outcome measures require p-value adjustment? an introduction to the bootstrap asymptotic statistical theory of overtraining and cross-validation the elements of statistical learning this study was supported by the dept. of neurosurgery and the masonic cancer center at the university of minnesota. the author would like to thank douglas yee and walter low for their support. j.b.n. conceived, designed, and carried out all aspects of this study and wrote and edited the manuscript. key: cord- -ulq xqma authors: li, jing; rao, yuhan; sun, qinglan; wu, xiaoxu; jin, jiao; bi, yuhai; chen, jin; lei, fumin; liu, qiyong; duan, ziyuan; ma, juncai; gao, george f.; liu, di; liu, wenjun title: identification of climate factors related to human infection with avian influenza a h n and h n viruses in china date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ulq xqma human influenza infections display a strongly seasonal pattern. however, whether h n and h n infections correlate with climate factors has not been examined. here, we analyzed cases of h n infection and cases of h n infection. the spatial characteristics of these cases revealed that h n infections mainly occurred in the south, middle, and northwest of china, while the occurrence of h n was concentrated in coastal areas of east and south of china. aside from spatial-temporal characteristics, the most adaptive meteorological conditions for the occurrence of human infections by these two viral subtypes were different. we found that h n infections correlate with climate factors, especially temperature (tem) and relative humidity (rhu), while h n infections correlate with tem and atmospheric pressure (prs). hence, we propose a risky window (tem – °c and rhu – %) for h n infection and (tem – °c and prs - kpa) for h n infection. our results represent the first step in determining the effects of climate factors on two different virus infections in china and provide warning guidelines for the future when provinces fall into the risky windows. these findings revealed integrated predictive meteorological factors rooted in statistic data that enable the establishment of preventive actions and precautionary measures against future outbreaks. influenza viruses with high infectivity and widespread outbreak patterns, these two aivs often cause sporadic and small outbreaks, despite the lack of human-to-human transmission ability, so other more complex reasons were involved. because these two viruses are associated with both wild and domestic bird populations, it is likely that environmental factors play a significant role in the spread of the viruses. generally, wild birds may act as the primary spreading agent for the outbreak of the influenza virus . after the movement of the virus from wild birds to poultry, the virus may spread from flock to flock and farm to farm through several ways . these include wild bird migration, poultry-wild bird interaction, and human proximity to poultry. for example, poultry trade areas that are in proximity to wild waterfowl habitats, such as wetlands or lakes, can facilitate virus exchange between wild birds and poultry , . animal model data shows that the transmission of iav is more likely at low temperatures and relative humidity conditions . however, the prevention of secondary spread can be achieved through restricting bird movement and proper biosecurity procedures. given the mobility of wild birds and the challenges of sustained control, it is of great importance to identify which environment factors correlate with the occurrence of these two viruses. both the h n and h n influenza viruses are of avian origin and have similar infectivity to humans, but their transmission region, outbreak timing, and host range are diverse. outbreaks of h n in wild birds are concentrated in the central part of europe, while outbreaks in poultry are mainly found in eastern europe, with human infections mainly in southeast asia and eastern europe . human infections with h n are found in china, corresponding to its high prevalence in certain lpms (live poultry markets) , while the occurrence of h n in wild birds is rare . previously, data have indicated that environmental factors affect the prevalence of h n and h n , with the infection and spread of the two viruses being closely correlated with bird habitats, migration, and local climate factors (e.g., temperature and relative humidity) . however, we found that both viruses have species-specific manifestations; the outbreak patterns of these two viruses in humans are not coincident with infections in wild birds or waterfowl. the reasons for this discrepancy could be due to the prevalence of the viruses in wild birds and waterfowl, the proximity of humans to these animals, and/or characteristics of the viruses themselves. the susceptibility of humans to the viruses due to host immune function and inhibition of mucociliary clearance by the inhalation of cold, dry air could also be a factor. in the present study, we compared the physical environmental factors and anthropogenic environmental factors that were present during h n and h n aiv outbreaks, as these viruses have similar endemic areas, host ranges, and are the main concerns to public health in recent years in china. understanding the outbreak patterns of these viruses is important to identify high-risk populations and areas, as well as to establish preventive actions and precautionary measures against future outbreaks. spatial characteristics of h n cases and h n cases. as the temperature varied heterogeneously in gradients in different provinces, we classified the affected provinces into the three commonly used geographical sectors that have similar meteorological characteristics: south (south area of the yangtze river), central (the yellow river to the south and the yangtze river to the north), and north china (north area of the yellow river). south china is comprised of guangxi, guangdong, and fujian provinces, central china is comprised of sichuan, guizhou, hubei, hunan, jiangxi, anhui, jiangsu, shanghai, and zhejiang provinces, and north china is comprised of shaanxi, shanxi, henan, hebei, shandong, and liaoning provinces, as well as beijing and tianjin (municipal cities). as shown in table , the h n influenza infection cases mainly occurred in south, central, and northwest china, with the most (eight cases) in hunan province. in contrast to h n , the occurrence of h n in mainland china was concentrated in the coastal areas of south china. climatic characteristics of h n cases and h n cases. to understand the discrepancies in climate factors between h n and h n infections, the null hypothesis was tested using the t-test. as shown in table , the p-values for rhu.mean, prs.mean, and win.mean were < . . these results imply that there were significant differences between h n and h n infections in terms of humidity, pressure, and wind speed. h n infections occurred with the highest frequency in a humidity range of - %, while h n infections occurred in the - % rage (fig. b) . in terms of atmospheric pressure, the cases of h n were concentrated at - kpa, while the occurrences of h n infection were found at - kpa (fig. c ). in addition, as the composition of meteorological factors, the wind speed during h n cases was concentrated at - m/s, while the occurrences of h n infection were found at - m/s wind speed (fig. f ). temperature and specific humidity were the best individual influence factors for influenza infection. according to null hypothesis , both the mean value of the air temperature and the mean value of the ground surface temperature displayed no significant difference comparing h n and h n infections. h n influenza occurred with the highest frequency in a temperature range of - °c, with a second peak in the range of - °c. h n influenza occurred with the highest frequency in a temperature range of - °c, with a second peak in the interval of - °c. consistent with the tem value, the h n influenza infection cases were relatively more prevalent when the ground surface temperature was - °c, with a second peak at - °c; h n influenza infection cases peaked at - °c and - °c, respectively (fig. a,d) . to determine the correlation between climate factors and influenza infection, we established a null hypothesis that states that h n /h n infection is independent of climate factors. the chi-squared test was used to test this hypothesis. as shown in table , we found that the p-value for all of climate is < . . these results demonstrate that there were significant relationships between climate factors and h /h influenza infection. to further understand the relationships among these factors with h /h infection, we conducted principal component analysis (pca) for all climate factors employed in our study. as shown in table , fig. a and supplementary video s , the results from the h n influenza cases indicate that the first principle component, whose dominant contributors are temperature variables (i.e., tem.mean, tem.high, tem.low, and gst.mean) explain . % of the total variance; the second principle component, which is dominated by relative humidity variables (i.e., rhu.mean and rhu.low), explains . % of the total variance. the third principle component was dominated by wind speed (win.mean) and other variables and only explains . % of the total variance. the total contribution of these three sets of climatic variables accounted for . % of the total contribution. similar to the h n influenza cases, the first h n principle component was led by temperature variables (i.e., tem.mean, tem.high, tem.low, and gst.mean) and explains almost half of the total variance ( . %, see table , fig. c and supplementary video s ). in contrast to h n , the second principle component of h n was dominated by pressure (i.e., prs.mean), which explains . % of the total variance, and the third principle component included humidity and other variables, explaining . % of the total variance. the total contribution of these three sets of climatic variables accounted for . % of the total contribution. from the above analysis, we can see that among all of the climatic variables, temperature and humidity were the key factors for h n infection. for h n infection, temperature and atmospheric pressure were the key factors, though humidity also had significant effects. a heat map was generated to evaluate the factors conducive to the spread of h n (fig. b ). based on the heat map, we created a climate high-risk window that we consider represents a high risk for the spread of h n (temperature - °c and rhu - %) and encompassed h n infections ( . %), as well as a broader climate moderate-risk window that we consider represents a moderate risk for the spread of h n (temperature - °c and rhu - %), encompassing infections ( . %). according to these criteria, weeks - in north china, - and - in central china, and - and - in south china commonly satisfy the criteria of the climate high-risk window. furthermore, in weeks furthermore, based on the mean temperature-humidity and temperature-atmospheric pressure during - , the predictions of city distribution and month distribution were calculated ( supplementary fig. s , table s and s ). we found that the occurrence of h n influenza virus was concentrated in march, april, october, and november, while h n influenza peaked in january and february. darker color in the figure corresponds to a higher occurrence ratio of influenza in a specific province. to determine whether the risky windows were statistically meaningful, we used the t-test to examine the differences in case numbers within and outside risky windows. for each province i, we counted the cases that met the criteria of the risky windows (i.e., n r,i ) and did not meet these criteria (i.e., n n,i ), respectively. then, t-tests were performed to examine the mean value difference between n r,i and n n,i across all provinces. the null hypothesis is that the means value of n r,i and n n,i are the same, indicating that the risky window is not statistically meaningful. the alternate hypothesis is that the mean values are different, suggesting that the risky window is statistically significant. the t-test was performed on both h n and h n , yielding p-values of . and . , respectively. this suggests that case numbers within and outside risky windows are significantly different for both h n and h n . both avian h n and the newly emerged h n influenza viruses are highly pathogenic aiv subtypes that can directly infect humans and cause severe diseases with high mortality. unlike seasonal influenza, human infection with these two subtypes is sporadic, and little is known about the factors underlying the occurrence of human cases and the environmental factors favoring the occurrence of human infections. herein, we identified that temperature, humidity, and atmospheric pressure are potential warning meteorological factors for the incidence of both h n and h n subtype infections. the dependence of influenza virus transmission on environmental factors, including temperature, humidity, and atmospheric pressure, has been documented by many previous studies [ ] [ ] [ ] [ ] . however, we found that the most adaptive meteorological conditions for the occurrence of the human cases of these two aiv subtypes are different. for example, most h n cases appeared when the atmospheric humidity was - %, whereas h n cases predominantly occurred when the humidity was between - %. in addition, differential atmosphere pressure and wind speed favor the occurrence of h n and h n cases, respectively. however, what underlies these differences is unknown and deserves further investigation. several lines of evidence indicate that the high incidence of the featured seasonal pattern of h n virus infection in poultry in china , , consistent with the human cases of h n influenza virus in our study. furthering the understanding of the correlation between the survival ability of the viruses relative to climatic factors should be considered. aside from meteorological factors, many other factors also impact the occurrence of human cases of h n and h n infection , . the migration of wild birds and live poultry transactions play an important role in spreading h n and h n viruses. the occurrence of human h n cases has been suggested to overlap with wild bird flyways . although it is also reported that h n virus is found only in chickens, ducks, and pigeons at live poultry markets and that no migratory birds have tested positive for the virus, migratory birds are implicated in the transmission of the virus. the regions with high incidences of h n and h n are places located along migratory bird flyways and also have surrounding poultry farms serving the densely populated areas that have many live poultry markets . to assess the different roles that various climatic factors play in h n and h n infections, we applied pca to patient-specific meteorological data, and the results suggest that the first two principle components (pc and pc ) can be used to estimate correlations. temperature factors and relative humidity are the dominant variables for pc and pc (respectively) for h n , while temperature and pressure are the key factors for h n cases. these results suggest the possibility of using temperature and relative humidity to approximate the effects that the climate factors have upon human h n infection. just as compared with highly pathogenic aiv (hpaiv), the h n virus shows differential environmental factors. although climatic factors exhibit a strong correlation with h n or h n infection, other crucial factors doubtlessly include the density of the pathogen virus, human behavior, and population density. several previous studies depict risk maps in terms of h n /h n infection in birds , , [ ] [ ] [ ] . there are similar spatial distributions with h n infections in humans but differential spatial distribution with h n infections in humans. the risk areas for human h n infection were more concentrated in south and central china in our analysis. hpaiv viruses are generally thought to arise in poultry after domestic birds become infected by lpai h and h viruses from the wild-bird reservoir . the probable long co-existence of multiple viruses in multiple hosts provides the driving force for the virus to adapt to infect humans. furthermore, it is difficult to identify humans, birds, or pigs infected with subclinical h n /h n viruses from clinical signs, and it is extremely difficult to conduct large-scale surveillance to identify virus infections in humans, birds, or pigs for an extended period. therefore, we must pay more attention to overlap areas. the lpms act as a melting pot for a variety of viruses at a range of densities, and market networks with numerous trade connections should permanently be closed down to avoid future avian influenza virus infections. however, there is vast number of small-scale poultry producers, supplying a strong demand for live poultry in china. consequently, permanently closing lpms would likely prove unpopular, and therefore, their periodic closure may represent a more viable compromise. the climate risk windows delineated in this study should be considered as important references for the guidance of any such solution. this strategy of metrological factor-based prediction can avoid large-scale live poultry culling and blindly closing live poultry markets to save a large amount of social resources. outcome data. we collected the h n and h n influenza data reported on the chinese mainland from the climate dataset contained the air temperature, ground surface temperature, relative humidity, wind speed, and atmospheric pressure of all meteorological stations in the reported provinces at the reported dates for each record. there is more than one meteorological station in each province, and the site-level climate datasets were aggregated into the province-level (average for the climate factors), which are able to represent the weather situation in the study areas. furthermore, several indices were derived from the province-level climate data for further analysis, i.e., the maximum, minimum, and mean value of the air temperature (tem.mean, tem.high, and tem.low), the minimum and mean value of the relative humidity (rhu.mean and rhu.low), the mean value of the ground surface temperature (gst.mean), the pressure (prs.mean), and the wind speed (win.mean) on each reported date. briefly, the average daily data from each weather station in the province, observed four times on a daily basis ( : , : , : , and : ), were used after removing outliers. according to province-level climate data, the daily mean data were selected to average total weather stations of each province from january through december . the monthly mean data were calculated from the daily reports of each province. first, we employed eda to summarize the data distribution and statistical characteristics of our datasets for both h n and h n infection cases. we analyzed the distribution of all potential meteorological factors for the h n and h n datasets using histograms, respectively. to further determine whether the climatic factors differ between the h n events and h n events, a series of two-sample t-tests were employed in our research due to the unequal sample sizes of the two groups. all hypothesis tests had a similar null hypothesis that the mean values of specific factors (x) are equal. then, a t statistic was obtained by the equation below, where x i , s i , and n i represent the mean value, variance, and sample size of a specific factor of the ith group (i.e., group : h n ; group : h n ), respectively. statistically, this statistic denotes a t distribution with the degree of freedom of df, which was calculated as below, given the significance level of a p-value (i.e., . in this study), the null hypothesis should be rejected, which indicates that the mean values of the specific factors differ between the h n events and h n events, if the calculated t statistic meets the following critical region, where t −a/ ,df is the critical value of the t distribution with the degree of freedom of df. pearson's chi-squared test. in addition to the t-tests, pearson's chi-squared tests were used to examine the dependence of h n events and h n events on the occurring month and climatic factors. the null hypothesis of the pearson's chi-squared test is that a specific factor is statistically independent from the occurrence of h n and h n . considering that the pearson's chi-squared test could only be applied to the categorical variables, all continuous variables, including age, and all climate factors were converted into categorical variables by dividing observations into several groups using pre-defined thresholds. specifically, all observations of h n and h n were divided into: ) three groups for occurring month, (group : january to march; group : april to june; and group : july to december); ) nine groups for air temperature, from - to °c with a step of °c; ) eight groups for relative humidity, from % to % with a step of %; and ) eight groups for ground surface temperature, from - to °c with a step of °c, separately. after the grouping procedure, the counts of all observations in each group could be exhibited as in the table , accordingly, a χ statistic was calculated based on the following equations, where e i,j is the expectation value of each group at the ith row and jth column, and n is the total sample size of these two datasets. statistically, this calculated χ statistic denotes a chi-squared distribution with the degree of freedom (df) equal to r- . therefore, if the calculated χ meets the following critical region, we rejected the null hypothesis, which indicates that this factor is dependent on the occurrence of h n and h n , where χ -a/ ,df is the critical value of the chi-squared distribution, a is the significance level (i.e., . in this study), and df is the degree of freedom. pca. in addition to the descriptive analysis applied for our datasets, we also deployed pca for both datasets to determine relative contributions of each 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influenza a (h n ) in china genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia dynamic patterns of avian and human influenza in east and southeast asia epochal evolution shapes the phylodynamics of interpandemic influenza a (h n ) in humans synchrony, waves, and spatial hierarchies in the spread of influenza bird migration and risk for h n transmission into qinghai lake the emergence of influenza a h n in human beings years after influenza a h n : a tale of two cities environmental factors contributing to the spread of h n avian influenza in mainland china predicting the risk of avian influenza a h n infection in live-poultry markets across asia weather variability and influenza a (h n ) transmission in shanghai, china: a bayesian spatial analysis airborne transmission of influenza a/h n virus between ferrets predicting unprecedented dengue outbreak using imported cases and climatic factors in guangzhou j.l. analyzed data and wrote the paper. y.r., q.s., j.j. and x.w. collected data. j.c., y.b., f.l., d.l. and g.f.g. modified the paper. q.l., z.d. and j.m. analyzed data. w.l. designed the experiments. key: cord- -c vwr t authors: xiao, qiuyan; ren, luo; zheng, shouyan; wang, lili; xie, xiaohong; deng, yu; zhao, yao; zhao, xiaodong; luo, zhengxiu; fu, zhou; huang, ailong; liu, enmei title: prevalence and molecular characterizations of enterovirus d among children with acute respiratory infection in china between and date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: c vwr t ev-d is associated with respiratory tract infections (rtis). since its first isolation, ev-d has been detected sporadically. however, the us and canada have experienced outbreaks of ev-d infections between august and december . this study aimed to investigate the molecular epidemiology and clinical characteristics of ev-d in chongqing, southwestern china. from january to november , nasopharyngeal aspirate specimens (npas) were collected from hospitalized children with rtis. among the npas, ev-d was detected in samples ( . %, / ). of these, samples were detected in september and october ( . %, / ). phylogenetic analysis showed that all strains detected in the chongqing had high homology with the main strains of the us outbreak. among the children with ev-d infection, ( %) had a history of recurrent wheezing. a total of children had a discharge diagnosis of asthma. of these, children were diagnosed with acute asthma exacerbation. ev-d was the predominant pathogen that evoked asthma exacerbation in september and october . in conclusion, our results found that a history of recurrent wheezing may be a risk factor for the detection of ev-d and viral-induced asthma exacerbation may be a clinical feature of ev-d infection. have been reported in approximately % to % of ev-d cases . more critically, messacar k et al. found that the outbreak of ev-d was geographically and temporally associated with acute flaccid paralysis (afp) and cranial nerve dysfunction in children . there was a case report of afp following ev-d infection in europe in which ev-d was isolated from nasopharyngeal aspirate, stool and bronchoalveolar fluid specimens of the child . in a previous hospital-based viral surveillance study of rti cases in chongqing, china, from to , the detection rate of ev-d was . % ( / ) . however, the specific clinical characteristics and gene structural features of ev-d in china remain unclear. therefore, the current study aimed to investigate the molecular epidemiology and clinical characteristics of ev-d in chongqing, china. a total of npas were collected from children with rtis between january and november ( from , from , from ). children enrolled in this study were of ages ranging from month to years and months (median, months). the overall male-to-female ratio was : , with a . bias towards males. among all samples, ( . %) were positive for at least one viral agent. rsv was the most common viral agent (detected in children [ . %] fig. ). the frequency of ev-d detection was . % ( / ) from january through august and . % ( / ) from september through october . moreover, the detection rate of ev-d in september-october was higher than in september-october ( . %, / vs. %, / , p = . ) and in september-october ( . %, / vs. . %, / , p = . ). sequence analysis of vp revealed that the strains in chongqing could be grouped into two clades (clade a and clade b) that were the main epidemic genotypes in chongqing from to ( fig. ) . thirteen strains of chongqing detected, seven strains from the us outbreak, and most strains from the netherlands detected were grouped into clade b. however, only two chongqing strains from and could be grouped into clade b. the remaining four chongqing strains were grouped into clade a (two in , and two in ). unfortunately, only thirteen chongqing strains were nearly full-length nucleotide sequences, including ten strains from , two strains from , and one strain from . sequence analysis of nearly full-length nucleotide sequences found that the clinical isolates detected in the current study had nucleotide sequence similarities of . - . % with each other and similarities of . - . % with the fermon strain. strains of clade b demonstrated similarities ranging between . - . % (nucleotides). a phylogenetic tree constructed with nearly full-length nucleotide sequences revealed that the division of ev-d was consistent with fig. (fig. ) . kaida et al. first reported that ev-d osaka strains have two deletions at nt - and - in contrast to the fermon strain in the ′ utr . these ev-d osaka strains were grouped into clade c according to phylogenetic analysis (fig. ). in the current analysis, fifteen chongqing strains and seven strains from the us outbreak that were grouped into clade b also had two blocks of deletions at nt - and - in contrast with the fermon strain (fig. ) , which differed from clade c at nt and . tokarz et al. found that clade a only had a deletion at nt - . in the current analysis, four chongqing strains (cq , cq , cq , and cq ) and one strain from the us outbreak (us/ky/ - ) that were grouped in clade a had a deletion at nt - , which contrasted with the fermon strain by a nucleic acid substitution at position (fig. ) . the complete structural viral protein (vp -vp ) amino acid sequences were also analyzed. clade b had clearly different amino acid signatures relative to the prototype fermon strain and had different signatures relative to clades a and c ( figure s ). however, all clade b amino acid residue substitutions have been reported in previous studies [ ] [ ] [ ] [ ] [ ] [ ] . the clinical and epidemiological characteristics of children positive for ev-d are shown in table and . the children with ev-d ranged in age from month to years and months (median, months). the male-to-female ratio was : . the most common signs and symptoms exhibited by the children were cough ( %) and wheezing ( %), with only five ( %) children having fever. thirteen ( %) children had a history of recurrent wheezing ( table ) . thirteen children had a discharge diagnosis of asthma, in which twelve children dually diagnosed with pneumonia. among these thirteen children, eleven were diagnosed with acute asthma exacerbation. moreover, three children were diagnosed with pneumonia, one child was diagnosed with bronchiolitis, one child was diagnosed with mycoplasma pneumonia, and one child was diagnosed with bronchitis (table ) . ev-d was detected as a sole pathogen in / cases (with the exception of other common respiratory viruses, bacteria, and mycoplasma). children diagnosed with asthma were more commonly associated with cases of ev-d single detection than co-detection ( % vs. %, p = . , table ). the clinical isolates of ev-d in were all in clade b according to phylogenetic analysis. comparisons were made between clades b and a ( table ) . children infected with clade b viruses were significantly older than those infected with clade a viruses (median of months old vs. . months old, p = . ). the duration of hospitalization was shorter for clade b-infected children than for clade a-infected children (median of days vs. . days, p = . ). diagnoses of asthma and acute asthma exacerbation were more common among children infected with clade b viruses than those with clade a ( % vs. %, p = . ; % vs. %, p = . ). moreover, among the thirteen clinical isolates of ev-d in , eleven cases were among children diagnosed with acute asthma exacerbation. of these, five and six cases were among children diagnosed with severe and moderate acute asthma exacerbations, respectively (table ) . among the npas samples, were obtained from children admitted to the hospital with acute asthma exacerbation. the pathogen spectrum among the children with acute asthma exacerbation is shown in table with hrv and rsv predominating. hrv and rsv were the predominant pathogens associated with acute asthma exacerbation in september and october of and . ev-d was the predominant pathogen in september and october of following the decline of hrv. combined with the results of the previous study in chongqing, thirteen cases of ev-d were detected during the period - : two in , four in , four in , and three in . ev-d infection was substantially higher in september and october than in previous years. recent studies have shown that ev-d infection presents from mild respiratory illness to severe acute lower respiratory tract infection and even death , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . additionally, increasing evidence supports the relationship between ev-d infection and afp , , , . hasegawa et al. found a strong association between ev-d infection and exacerbation of asthma . in the ev-d outbreak in the us, the percentage of children who had a history of asthma or wheezing in kansas city and chicago was % and %, respectively, and most of these children were admitted to the pediatric intensive care unit . at the st. louis children's hospital in st. louis, missouri, ev-d was detected in seven of ten samples from patients with severe disease . in our study, most children infected with ev-d had a history of recurrent wheezing or asthma and a diagnosis of acute asthma exacerbation. in ev-d single detection, the phenomenon was more obvious. moreover, ev-d became the predominant pathogen associated with asthma exacerbation in september and october of . our study found that a history of recurrent wheezing or asthma may be a risk factor for the detection of ev-d and viral-induced acute asthma exacerbation may be a clinical feature of ev-d infection in chongqing area. the current study found that clade b viruses had a stronger association with asthma exacerbation than clade a, but children infected with clade a viruses were too young to be diagnosed with asthma . therefore, the difference between clade b-and clade a-induced asthma exacerbations could not be inferred. in addition, the duration of hospitalization was longer in clade a-infected children. however, the number of cases was too small to estimate differences in disease severity between clade b and clade a. further studies are thus needed to explore additional relationships between different clades. the ′ utr contains the internal ribosome entry site (ires) responsible for cap-independent initiation of translation as well as other secondary structural elements responsible for genome replication . studies have found that the enterovirus ires is associated with translational efficiency and virulence of the enterovirus , , . the ′ utr spacer region of ev-d ranged from the end of the ires and the polyprotein open reading frame (orf) . the role of the spacer region in viral fitness remains unclear . deletions in the spacer region might be correlated with the worldwide increase in ev-d , . our study showed different deletions among clades a, b and c. clade a only had one deletion (nt - ) , clade c had two deletions (nt - and - ) , and clade b had two deletions (nt - and - ) . the ′ utr was considered a conserved region but the spacer region likely had more mutations. therefore, more sequences would need to be collected to explore the nucleic acid differences of ′ utr sequences among different clades. more studies are needed to expound the relationship between the deletions within the ′ utr of ev-d and efficiency of translation, or virulence. the vp contains a serotype specific neutralization site, the bc and de loops, associated with viral antigenicity . previous studies found some distinctively different amino acid substitutions among the three clades, particularly in the bc and de loops of the vp protein - , - . such changes might modify the immune response and help to explain the biological basis for the increase in ev-d incidence , . however, an association between the substitutions and the severity of infection has not been established although the main ev-d epidemic clade in chongqing, the us, and the netherlands was clade b, it was different from the epidemic cluster observed in the philippines in , and no prior increase was reported. moreover, the ev-d strains in certain areas had regional features as evidenced by the fact that the ev-d epidemic strains in chongqing and us belonged to a different cluster than the netherlands strains. this phenomenon may be linked to the rapid evolution of enteroviruses . our study has several limitations. first, as this study was limited to inpatients, we could not analyze the clinical characteristics in outpatients and asymptomatic children. viral surveillance was performed in a tertiary referral hospital where diseases seen were often more severe, thus more ev-d might have been detected. second, because we only collected the npas of children with rtis, no children positive for ev-d in our study had muscle weakness or paralysis. we could not find any relationship between afp and ev-d . in summary, we found that clade b of ev-d increased in september and october in chongqing, china. a history of recurrent wheezing or asthma appeared to be a risk factor for the detection of ev-d and viral-induced acute asthma exacerbation might be a clinical feature of ev-d infection. our study provides additional epidemiological and clinical data on ev-d infection in china and a resource of genome sequences for genomic comparison of ev-d . at present, ev-d is not included in the viral surveillance of artis in china, but it has emerged as a considerable global public health threat. additional surveillance of ev-d is needed. ethics statement. this study was approved by the ethics committee of the children's hospital of chongqing medical university. the study was conducted in compliance with the principles of the declaration of helsinki. informed consent was obtained from each parent or guardian prior to enrollment. medicine at the children's hospital of chongqing medical university (chcmu) in chongqing, china. chongqing is located in a subtropical region, and chcmu is the largest children's hospital in southeastern china. nasopharyngeal aspirates (npas) were collected from children with rtis . a standardized questionnaire was designed to obtain the patients' information from medical records, including clinical assessments. we defined four clinical variables related to wheezing: ( ) a history of asthma, which was determined by asking the parents whether their child had ever been given a definitive diagnosis of asthma by clinicians or by documentation of a previous diagnosis of asthma in the medical record; ( ) a history of recurrent wheezing, which was determined by asking the parents whether their child had ever had the symptom of wheezing at least three times; ( ) a discharge diagnosis of asthma, which was defined as any discharge diagnosis of asthma following the guidelines of the global initiative for asthma (gina); and ( ) hospitalization for acute asthma exacerbation using criteria in accordance with previous and current published international guidelines . the degrees of acute asthma exacerbations were divided into mild, moderate and severe . molecular analysis. viral dna and rna were extracted from -μ l aliquots of the npa samples by a qiaampminelute virus spin kit (qiagen, hilden, germany). rna was applied as the template for cdna synthesis using the superscript iii first-strand synthesis system (invitrogen, california, usa). all experimental methods were carried out according to the manufacturers' instructions. all samples were screened for influenza viruses (iva-c), human parainfluenza viruses (piv - ), respiratory syncytial virus (rsv-a and rsv-b), human coronaviruses (hcov- e and hcov-oc ), human metapneumovirus (hmpv), adenovirus (adv) and human bocavirus (hbov) using molecular methods, as described previously , . a primer pair targeting bp of the ′ untranslated regions ( ′ utr) of the human enterovirus (hev) and human rhinovirus (hrv) genome was used to distinguish between hev and hrv . to determine ev-d sequences, we synthesized cdna using specific primer pairs (table ) , . the sequenced regions were all structural viral proteins (vp -vp ), partial ′ utr regions, and nonstructural viral proteins ( a- d). pcr was performed using a takara ex taq pcr kit (takara biotechnology, dalian, china), and all pcr products were sequenced by shanghai majorbio bio-pharm technology. detection of bacteria and yeasts. qualitative and semiquantitative cultures for bacteria and yeasts were performed using standard microbiological methods. for all samples, macroscopically distinct colonies were isolated from pure culture, and standard methods for identification, typing and establishing antibiotic sensitivity patterns were used . sequence analysis. sequence data for each clinical strain were formatted and assembled by the seqman program of dnastar software (v . ). the sequences of ev-d strains that were newly obtained in chongqing have been deposited in genbank under the accession numbers kt -kt . to study the distribution and diversity of ev-d identified in this study compared to other ev-d strains, multiple sequence alignments were made using the clustalw function of the bioedit program (v . . ). phylogenetic trees were constructed using the mega neighbor-joining algorithm ( . ). the statistical significances of the tree topologies were tested via bootstrapping ( replicates). only bootstrap values > % are shown in each tree. a phylogenetic tree that was constructed with bp of vp gene nucleotide sequences showed that modern ev-d strains were divided into three primary clades (a, b and c) according to the classification previously described by tokarz (fig. ) . nearly full-length nucleotide sequences that correspond to nt - of the ev-d prototype strain (genbank accession no. ay ) were obtained from strains of ev-d in the current study. a phylogenetic tree was constructed based on this region of nucleotide sequences with the strains from chongqing (ten strains from , two strains from , and one strain from ), seven strains from the us outbreak and available ev-d sequences that were long enough (fig. ). statistical analysis. data were analyzed using the spss . software package. categorical variables were compared using chi-square or fisher's exact tests, and continuous variables were compared using the nonparametric mann-whitney u-test. two-sided p-values of < . were considered statistically significant. a probable new human pi-cornavirus 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and receptor binding properties of enterovirus emergence and epidemic occurrence of enterovirus respiratory infections in the netherlands in molecular epidemiology and evolution of human enterovirus serotype in thailand enterovirus among children with severe acute respiratory infection, the philippines upsurge of human enterovirus infections in patients with severe respiratory tract infections clusters of acute respiratory illness associated with human enterovirus enterovirus infection in children with asthma attacks: virus-induced asthma in japanese children genome sequence of enterovirus d from st global strategy for asthma management and prevention: gina executive summary new insights into internal ribosome entry site elements relevant for viral gene expression internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants molecular analysis of virulent determinants of enterovirus first enterrovirus d (ev-d ) cases detected in hospitalized patients in a tertiary care university hospital in spain longitudinal molecular microbial analysis of influenza-like illness recombination among human non-polio enteroviruses: implications for epidemiology and evolution european respiratory society statement: asthma control andexacerbations: standardizing endpoints for clinical asthma trials and clinical practice development and implementation of a molecular diagnostic platform for daily rapid detection of respiratory viruses correlation between nucleotide mutation and viral loads of human bocavirus in hospitalized children with respiratory tract infection impact of bacterial colonization on the severity, and accompanying airway inflammation, of virus-induced wheezing in children the authors acknowledge the assistance of the patients and their caregivers involved in the study, the q.x. carried out the initial analyses and drafted the initial manuscript. q.x., s.z. and l.w. collected the nasopharyngeal aspirates and clinical informations, finished the detection of virus. l.r., x.x., y.d. and y.z. advised on data analyses. x.z., z.l., z.f. and a.h. reviewed and revised the manuscript. e.l. conceptualized and designed the study, reviewed and revised the manuscript. all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. key: cord- -rwnbxmt authors: lu, yi-fan; mauger, david m.; goldstein, david b.; urban, thomas j.; weeks, kevin m.; bradrick, shelton s. title: ifnl mrna structure is remodeled by a functional non-coding polymorphism associated with hepatitis c virus clearance date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: rwnbxmt polymorphisms near the interferon lambda (ifnl ) gene strongly predict clearance of hepatitis c virus (hcv) infection. we analyzed a variant (rs g/t) located within the ifnl mrna ′ untranslated region (utr); the g allele (protective allele) is associated with elevated therapeutic hcv clearance. we show that the ifnl ′ utr represses mrna translation and the rs allele modulates the extent of translational regulation. we analyzed the structures of ifnl variant mrnas at nucleotide resolution by shape-map. the rs g allele mrna forms well-defined ′ utr structure while the t allele mrna is more dynamic. the observed differences between alleles are among the largest possible rna structural alterations that can be induced by a single nucleotide change and transform the utr from a single well-defined conformation to one with multiple dynamic interconverting structures. these data illustrate that non-coding genetic variants can have significant functional effects by impacting rna structure. human genetic variation has a large influence on an individual's susceptibility to infectious diseases. studies on the hepatitis c virus (hcv), a major cause of liver disease , have revealed a striking example of how human genetics affects the outcome of infection. genome-wide association studies performed on diverse patient populations have identified polymorphisms near the interferon-λ (ifnl ; formerly il b) gene that predict the efficacy of interferon-based therapy for chronic infection. these polymorphisms are also strongly predictive of spontaneous hcv clearance during the acute phase of infection [ ] [ ] [ ] [ ] . ifnl is a secreted cytokine that binds a specific cell surface receptor complex expressed on epithelial cells, leading to jak-stat signal transduction and expression of interferon-stimulated genes [ ] [ ] [ ] . ifnls have been implicated in control of viral infections of epithelial-derived tissues, such as gut, lung, and liver [ ] [ ] [ ] [ ] . ifnls inhibit replication of multiple viruses in vitro [ ] [ ] [ ] and ifnl genotype has been linked to liver interferon-stimulated gene mrna expression in chronic hcv patients and primary human hepatocytes [ ] [ ] [ ] . the mechanisms by which ifnl genetic variants function to control hcv infection are not clear. steady-state levels of ifnl mrna in liver tissue and peripheral blood mononuclear cells may be linked to hcv clearance because the protective alleles (rs c and rs t) are correlated with a modest (< -fold) increase in ifnl mrna levels , , , . however, other studies on different patient scientific reports | : | doi: . /srep populations and cell types failed to detect correlations between differences in ifnl mrna expression and genotype in liver tissue , or plasmacytoid dendritic cells . thus, the propensity for hcv clearance appears to be related to differences beyond ifnl mrna levels. although primary hepatocyte cultures have been shown to express ifnl upon hcv infection , , evidence is emerging to support a role for dendritic cell subtypes in the production of ifnl [ ] [ ] [ ] . in addition, levels of ifnl protein were found to be strongly correlated with ifnl genotype in serum samples from hcv-infected patients , . one study also suggested that serum ifnl levels are a stronger predictor of viral clearance than ifnl genotype . these observations, taken together, suggest that ifnl abundance may be regulated by differential efficiency of mrna translation as a function of ifnl genotype. here we report the genetic association and functional consequences of a snp (rs ) located in the ′ untranslated region (utr) of ifnl mrna. this snp was previously reported to regulate ifnl expression by changing seed base pairing with hcv-induced micrornas . we genotyped a large number of patients with chronic hepatitis c from the ideal cohort to evaluate associations of this variant with treatment phenotypes. molecular analyses suggested that rs is functional in regulation of mrna translation efficiency. rna structure probing by shape provided a physical explanation for these functional differences and revealed that ifnl mrna structure is markedly altered by this common snp. together, these findings support a model in which ifnl mrna translation efficiency, governed by allele-specific mrna structures, modulates clearance of hcv infection. the rs discovery snp and rs are highly correlated and exhibit indistinguishable associations with clinical phenotypes. the most strongly associated single nucleotide polymorphisms (snps) found in genome-wide association studies (gwas) were rs in european-americans and african-americans, and rs in east asians. these snps likely co-segregate with polymorphisms that directly affect biological functions related to ifnl expression or activity. to identify candidate functional variants in the ifnl gene region, we searched for variants in high linkage disequilibrium with rs (hereafter referred to as the 'discovery snp') by analysis of available human genome sequence data ( genomes.org). we identified a snp (rs ) in the ′ utr of ifnl that is highly correlated (best tagging snp according to genomes project) to the discovery snp [ ] [ ] [ ] [ ] (fig. a) . the rs g allele is correlated with the protective c allele of the discovery snp. this variant was an intriguing candidate as the source of viral infection-related phenotypes because ′ utrs often contain cis-acting rna elements that control mrna translation and decay. rs is also flanked by au-rich elements (ares; fig. b ), sequence motifs that have been linked to post-transcriptional gene regulation . therefore, we conducted a large-scale genetic association study examining rs and hcv clearance in a cohort of chronically-infected patients. we genotyped and analyzed these variants in the ideal cohort , a large sample of chronic hcv patients treated with pegylated ifn-α and ribavirin combination therapy (n = european-american; n = african-american). the sample sizes of both populations are sufficient to achieve greater than . in statistical power ( . in european-american and . in african-american under the significance level of . ). linkage disequilibrium analysis in hcv patients revealed that rs is strongly associated with rs in european-american patients (r > . ; table ) , with somewhat lower linkage disequilibrium in african-americans (r = . ). we performed association tests of rs with clinical phenotypes using logistic regression. the association of rs with sustained virological response (defined as absence of detectable hcv rna in serum at least weeks after discontinuation of treatment) was extremely significant (p = . × − ) in the european-american population ( table ). the discovery snp has been previously associated with viral burden such that patients with the protective allele (c) exhibit a slightly higher baseline viral load . we found that rs exhibited comparable association with pre-treatment baseline viral load by linear regression in both european-and african-americans compared with the discovery snp (table ) . next, we performed a multiple logistic regression analysis to examine whether rs showed independent association with patient phenotypes in excess of that attributed to the discovery snp. this analysis revealed that rs did not show significant association with svr when we adjusted for rs genotype, in either european-or african-americans (table ) . thus, although the ideal cohort is one of the largest and most data-rich clinical trial cohorts available for genetic studies of patient response to ifn-based therapy, the extremely high linkage disequilibrium between variants in this region precluded independent statistical association of rs with svr in this sample size. recently, a functional dinucleotide snp rs that can influence the expression of ifnl protein was discovered upstream of the ifnl locus and was implicated in the clearance of hcv . in addition, the putatively favorable rs g allele was proposed to have an unexpected negative effect on the decrease of hcv rna level after treatment in the presence of the unfavorable rs Δ g allele , suggesting that rs may modify the effect of the rs variant associated with ifnl . here, we also performed the same multiple logistic regression or multiple linear regression approaches to assess whether rs or rs showed stronger association with patient phenotypes. we found that, despite the large sample size of the ideal cohort, this analysis did not reveal significant independent association of either snp after correcting for the effect of each other (table and table ), although we scientific reports | : | doi: . /srep note an interesting opposite trend of odds ratios (or) for svr after mutual adjustment ( . and . for rs g and rs tt, respectively) in african american patients (table ) . overall, rs demonstrated similar levels of association with patient phenotypes as did the discovery snp or a nearby functional snp rs , suggesting that rs is an interesting candidate variant and may be responsible for clinically important phenotypic differences. rs impacts ifnl mrna translation efficiency. although rs did not independently predict svr in patients, based on association evidence it is a good candidate to investigate. furthermore, its presence in the ifnl mrna ′ utr prompted us to test functionality. the ifnl ′ utr is nucleotides (nt) in length, is composed of % a and u nucleotides, and harbors several functional ares , including two (are and are ) flanking rs (fig. b) . comparison of utrs from primate ifnl and ifnl genes indicates that the region around rs is highly conserved and that the t variant is the ancestral allele (supplementary figure s ) . we first evaluated the effects of the variant ifnl ′ utrs on steady-state mrna and protein levels using a reporter assay. we took advantage of a stable hela cell line that harbors a genomic flippase recognition target site to create isogenic cell lines with distinct renilla luciferase (rluc) reporter constructs controlled by a cmv promoter that is transcriptionally regulated by tetracycline repressor protein. this system allows characterization of gene expression in cells containing a single copy of the reporter gene. three cell lines were established containing either rluc alone or rluc fused to each of the two ifnl ′ utr variants (fig. c) . the presence of either ifnl ′ utr caused modest but statistically significant (p = . for g allele, p = . for t allele, unpaired t-test) reductions in the levels of ifnl chimeric mrnas compared to rluc alone (fig. d) . interestingly, levels of the rluc protein were reduced by a much larger amount, -and -fold for the rs g and t reporters, respectively, compared to triple asterisks indicate that differences are significant at the p < . level. all data are shown as mean values ± s.d. the reporter lacking an ifnl ' utr ( fig. e ). the differential effects on mrna versus protein levels strongly suggest that the ifnl ′ utr regulates gene expression by repressing the efficiency of mrna translation rather than mrna abundance in hela cells. importantly, the ′ utr snp affected the magnitude of this repression: the rs t allele produced approximately % less rluc than did the corresponding g allele mrna ( fig. e ; p = . , unpaired t-test). induction of reporter mrna expression levels by tetracycline treatment reduced rluc differences between the cell lines, suggesting that reporter mrna over-expression led to escape from endogenous regulatory factor(s) present in hela cells (supplementary figure s a ,b). we also tested the ifnl reporter constructs in an immortalized human hepatocyte cell line (ph ch ), a human hepatoma cell line (lh ), and hela cells by transient plasmid transfection (supplementary figure s c ). in each cell line, the patterns of reporter expression mirrored that in the stable hela cell lines (fig. e ). we next directly tested the hypothesis that rs regulated efficiency of mrna translation. we used polysome profiling to examine whether the variant ifnl reporter mrnas were differentially associated with translating ribosomes in stable hela cell lines. the extent of association with multiple ribosomes is an indication of mrna translation efficiency. ifnl variant reporter cell lines, grown to equal densities, were harvested, lysed and subjected to sucrose density gradient centrifugation to separate non-translating and ribosome-associated mrna. gradients were subsequently fractionated and rluc reporter and gapdh mrna levels were measured by rt-qpcr. ribosomal rna sedimentation r * or** p p (after correcting for rs ) r * or** p p (after correcting for rs ) table . association of rs with the pre-treatment viral load. association tests were performed by linear regression or multiple linear regression (additive genetic model). patterns from the ifnl two reporter cell lines were nearly identical ( fig. a) , indicating that bulk mrna translation rates did not differ between cell lines. in contrast, the rs g and t ifnl reporter mrnas showed distinct profiles across the gradient. across three biological replicates, the g allele mrna was more highly represented in denser gradient fractions, reflecting incorporation into polysomes, than the t allele mrna (fig. b ,c and supplementary figure s ). we quantified the areas underneath the lines for both g and t alleles and found that the allelic difference in area between polysome fractions (fractions - ) and non-polysome (fractions - ) fractions were significantly different (p = . ) (fig. c ). this result indicates that the g allele mrna was more abundant in polysome fractions compared to the t allele mrna. correspondingly, levels of non-translating mrna (sedimenting between the s ribosomal subunit and the s monosome) were higher for the rs t mrna. figures b and s were quantified for both polysome fractions (fractions - ) and non-polysome fractions (fractions - ). the plot shows the differences in area between the g and t alleles (g minus t). unpaired t-test (two-tailed) was used to calculate the p value from the three independent experiments (p = . ). error bar represents the sem. we did note that a significant amount ( %) of the t-allele reporter mrna was found in dense gradient fractions ( ) ( ) ( ) , suggesting that a portion of the t allele reporter mrna escapes repression and is efficiently translated. combined with the discrepant luciferase protein and reporter mrna measurements shown in fig. , these data strongly suggest that the rs snp regulates efficiency of ifnl reporter mrna translation in hela cells. rs alters structural conformation of the ifnl ′ utr. there is extensive literature linking ′ utrs with translational control [see reference for a recent review]. a growing body of evidence indicates that ′ utr structure regulates mrna translation and decay by modulating interactions with trans-acting factors . moreover, genetic variants that alter mrna structure and function, termed ribosnitches, have been described . we directly examined whether the rs snp physically modulates the structure of endogenously expressed ifnl mrna using selective ′-hydroxyl acylation analyzed by primer extension (shape) . we examined the structure of ifnl mrna under non-denaturing conditions in the context of total purified cellular rna. shape analysis was performed on deproteinized rna as this approach allows for direct assessment of mrna structure independently of trans-acting proteins or rnas. the structure of the ifnl mrna was probed using the fast-reacting shape reagent -methyl- -nitroisatoic anhydride ( m ) and products were analyzed by massively parallel sequencing using the shape-map strategy (supplementary figure s ) . we used human a lung adenocarcinoma cells for these experiments because they express ifnl mrna upon transfection with poly(i:c) (supplementary figure s a) . importantly, a cells are heterozygous at rs and levels of mrna from each allele were directly correlated with the gene dosage (supplementary figure s b ). using the map approach, sequencing reads derived from ifnl were sorted by snp identity at rs ; this made it possible to examine the structures of both alleles under identical conditions in the same experiment (supplementary figure s ) . shape data were collected on a nt long region of the ifnl mrna containing the last nt of the coding sequence (including the stop codon) and the ′ utr, excluding the terminal nt and the poly(a) tail. shape-map reactivities report a model-free measurement of the degree of rna structure , . the rs g allele had lower shape reactivity, and thus more stable structure, near the region close to rs than did the t allele (fig. a) . we next calculated shannon entropy values for each rs allele. shannon entropies are derived from a shape-directed partition function and report a measure of whether an rna region is likely to form a single well-defined structure [ ] [ ] [ ] . the median shannon entropies across the ifnl ′ utr were much lower for the g allele, especially near the rs site, than for the t variant ( fig. b and supplementary figure s ) . we next performed a correlation analysis of shape reactivities between the g and t alleles by calculating r across the mrna regions to address the overall structural similarity between the two alleles (fig. c) . the shape reactivities in the open reading frame (orf) are highly correlated between alleles, and moderately correlated at the very end of ′ utr. in contrast, a very low or zero correlation was observed in the region of rs . this indicates that a drastic local change in structure is induced by the snp. in sum, the g allele ′ utr adopts an overall well-defined, highly structured conformation; whereas, nucleotide-resolution shape probing indicates that the t allele adopts multiple conformations. in addition, structural differences between the two ′ utr alleles are centered on the snp region. shape data can be used to develop accurate models for large, complexly structured rnas with well-defined structures , . shape-directed rna structure models for each variant over the ′ portion of the open reading frame and ′ utr are summarized in arc plots that display predicted short-and long-range base pairing (fig. d ). given its low shannon entropy, the structure of the g allele could be modeled with a high degree of confidence, as evidenced by the preponderance of highly probable helices (fig. d, bottom) . in contrast, the ′ utr for the t allele is predicted to adopt multiple conformations, comprised of helices with base pairs of lower individual probability (fig. d, top) . the combined shape reactivity and entropy differences indicate that the t-allele mrna has a more variable structure than the g-allele mrna. the shape-directed rna secondary structure model indicated that the rs g nucleotide forms a canonical base pair within a stable stem-loop ( fig. a and supplementary figure s ) that contains portions of are and are . in contrast, the u variant is not predicted to stably adopt this stem-loop structure (fig. a) . we calculated the free energy change of ′ utr rna folding for each allele and found that the rs g to u change alters relative free energy by approximately kcal/mol. this difference in relative free energy is close to the largest possible increment achievable by altering a single base pair . we calculated the expected free energy changes for all possible nucleotide changes in the context of the ifnl ′ utr ( sequence variants) and in the context of the intact mrna ( sequence variants). the increase in folding free energy due to the change from g to u at rs ranked the highest among all possible substitutions in ′ utr, and the third among all possible substitutions in the intact mrna, indicating that this position has a strong influence on rna folding (fig. b) . direct shape experimental interrogation and free energy increment calculations thus suggest that the rs snp induces close to the largest possible change in rna structure. we performed mutagenesis to further test whether functional differences for rs reflect changes in rna structure. three mutant versions of the ifnl reporter construct were established that contained mutations at the rs position (nt of the ′ utr) and/or at the nt position predicted to base-pair with rs (nt of the ′ utr) (fig. a) . changing rs from g to c resulted in reduced expression, similar to the rs t reporter construct (fig. b) . restoring the predicted base pair in this mutant by introducing a g at nt increased expression, consistent with rna structure opposing repression. interestingly, introduction of g at nt (c g) in the context rs g, which is predicted to interrupt base-pairing, had increased expression which was opposite of the effect on expression initially expected. however, this mutant resulted in a free energy of rna folding similar to the rs g ′ utr (fig. b) by inducing a register shift in base pairing and ultimately preserved the stem loop associated with escape from repression (supplementary figure s ) . for each of the constructs analyzed, the expression levels were inversely correlated with folding free energy. together, the mutagenesis data further reinforce the model that the t-allele has a more variable structure than the g-allele mrna and that stable structure is associated with enhanced hcv clearance. multiple genome-wide association studies have identified polymorphisms near the ifnl gene that predict efficacy of therapy and spontaneous hcv clearance [ ] [ ] [ ] [ ] . although rs is not independently associated with patient phenotypes in the cohort we analyzed, this snp occurs in the ′ utr of ifnl and showed clear functional effects on reporter gene expression, suggesting a role for this variant in control of hcv infection. rs influences regulation of mrna translation efficiency by the ifnl ′ utr in hela cells, perhaps through altering functionality of ares. although cytokine ′ utr ares are commonly associated with enhanced mrna decay, they have also been implicated as translational regulators [ ] [ ] [ ] [ ] and mrna decay and translation are closely associated cytoplasmic processes , . due to gene regulation at the level of mrna translation, the modest differences in ifnl mrna levels, as a function of genotype, reported in some studies may underestimate actual differences in ifnl protein levels, as we observed here for the ifnl rs snp (fig. ) . this is consistent with a recent report of ~ -fold differences in ifnl / serum levels between hcv patients who are homozygous at the discovery snp . consistent with this work, a recent independent study identified a distinction between cc versus non-cc genotype (discovery snp) for ifnl protein plasma levels . however, we acknowledge that our results do not formally establish a causal relationship between rs and ifnl levels in patients. to examine and define the physical basis for the mechanism of ifnl translational regulation, we used the shape-map strategy, which made it possible to resolve structural conformations and differences for the rs g and u variant mrnas in a single heterozygous cell line, in a single experiment. we discovered that the nucleotide at rs influenced the global structure of ifnl mrna. the g allele was much more highly structured and the free energy difference between the g-and u-containing mrnas was predicted to be close to the upper limit in structural stabilities achievable by a single-nucleotide sequence change. our work strongly suggests that rs is a ribosnitch that alters gene expression by inducing changes in rna structure. recent genome-wide analyses suggest that ribosnitches are widespread in the human transcriptome . the experimental and computational strategies developed here can be applied to other candidate functional variants to advance our understanding of how post-transcriptional gene regulation is affected by non-coding genetic polymorphisms. these approaches should be broadly useful for identifying human genome sequence variants in non-coding regions that exert functional effects by altering rna structure. the observed change in protein expression is likely the result of differential binding of trans-acting factors, induced by the differential folding of the ′ utr variants. the shape-derived secondary structures show that rs t reduces structure of a region overlapping are and (fig. a) , suggesting that unidentified trans-acting are-binding proteins may access these elements more efficiently in the t allele mrna. recently, mcfarland et al. reported that the ifnl mrna is regulated by au-rich elements and also targeted by muscle-specific micro(mi)rnas (mir- b and mir- a- p) whose expression is triggered by hcv replication. these authors found that rs was functional using ifnl reporter mrnas. however, the snp did not confer regulation when are or are were mutated, in agreement with our observation that rs alters structural conformation of regions encompassing are and are . with respect to the mechanism involving action of mirnas, the rs t to g allele would disrupt mirna-mrna seed base-pairing . thus, the dramatic effects of rs on ifnl mrna structure that we report here may regulate interactions with are-binding proteins and mirnas. however, we note that, when the snp position was mutated to a c, we observed a level of reporter expression similar to that of the t-allele (fig. b) . the rs c mutation abolishes seed-region base-pairing to the proposed regulatory mirnas, similar to the g-allele. this suggests that the regulation we characterized in hela cells, which may lack expression of the implicated mirnas, is mediated primarily by effects of rna structure on interaction with trans-acting factor(s) other than mir- b or mir- a- p. further study is needed to define the complement of relevant trans-acting factors that may differentially regulate variant ifnl mrna transcripts through repression of translation and/ or induction of mrna decay. recently, rna sequencing analysis of primary human hepatocytes (phh) revealed transcriptional activity upstream of the ifnl gene that is coincidentally induced by treatment with poly i:c . moreover, a dinucleotide variant (rs tt/Δ g) in this region is associated with hcv clearance and may alter translation of a specific mrna isoform encoding the novel ifnl . ifnl is antiviral against hcv in vitro yet is poorly secreted and does not appear to be strongly expressed in phhs , . in the rs Δ g genotype, which is predicted to express ifnl and is in high linkage disequilibrium (ld) with the non-protective (t) rs allele, ifnl production may exert effects that would be unexpectedly deleterious to hcv clearance . rs was recently reported to be more strongly associated with some patient phenotypes than rs in different patient cohorts, and thus may be dominant in determining hcv clearance in patients . further study is needed to understand the potentially maladaptive effects of ifnl expression and dissect possible interactions between rs and ss variants in control of hcv infection. in summary, we demonstrate a general strategy involving genetics, molecular biology, and rna chemistry for understanding gwas associations with non-coding genetic variants that may alter rna structure and post-transcriptional gene regulation. nucleotide-resolution shape probing revealed that the ′ utr of the rs g allele ifnl mrna, which is associated with hcv clearance, forms a well-defined structure whereas that of the t allele mrna is dynamic. the large alteration induced by a single nucleotide change illustrates the extent to which non-coding genetic variants can have significant functional effects by impacting rna structure. genotyping and statistical analysis. dna from the ideal cohort was used for genotyping. the genotyping of rs was performed by quantitative pcr (bioline) using, forward primer ′-acctg agatt ttatt tataa attag ccact tg(g/t)- ′, and reverse primer ′-ctttt cctca ttgtt tattt caaca aggat ttc- ′. data were clustered by ct value for genotype calling. rs was genotyped by the taqman genotyping assay (life technologies) according to the protocol recommended by the manufacturer, forward primer ′-tgggt cctgt gcacg gtgat- ′, reverse primer ′-tccct cagcg ccttg gca- ′, and probe ′-cgcag (aa/c)ggcc ccccg g- ′ . statistical analysis of phenotypic data was performed in stata (statacorp) and r (www.r-project.org). sustained virological response (svr) was used as the binary dependent variable, and genetic polymorphisms were considered independent variables in the logistic regression model (the assumption of logistic regression requires the dependent variable to be binary). both discovery snp and rs were used as independent variables in multiple logistic regression to distinguish its independent association after accounting for the effect of each other. viral loads (iu/ml) were transformed by box-cox method and considered as the dependent variable in linear regression to meet the multivariate normality assumption. sample size calculation was performed by the pwr package in r and the significance level was set at p = . under the multiple regression setting. all the p values reported in this paper are based on the two-sided statistical tests. cell culture, cloning and rt-qpcr. ph ch , lh , a and stable hela-frt cells expressing tetr were cultured in high glucose dmem supplemented with % heat-inactivated fbs and non-essential amino acids. stable hela-frt cells (a gift from elena dobrikova, duke university) harboring rluc reporter trans-genes were established as described by co-transfection of a flp recombinase plasmid (pog , life technologies) with rluc reporter plasmids cloned into pcdna /frt/to (life technologies, see below). stable cell lines were selected with blasticidin ( . μ g/ml) and hygromycin ( μ g/ml). analysis of rluc levels (renilla luciferase assay system, promega) in hela cell lines was conducted in the absence or presence of tetracycline ( μ g/ml) induction. stable reporter cell lines were independently derived and analyzed three times. reporter constructs were generated by pcr amplification of the rluc open reading frame using ′-gaggt accat gactt cgaaa gttta tgatc c- ′ and ′-gagat atctt attgt tcatt tttga gaact cgc- ′ and ligation into pcdna / frt/to (life technologies) using kpni and ecorv. the resulting clone was used for insertion of ifnl ′ utr sequences pcr amplified from a genomic dna using ecorv and noti (primers: ′-gagat atccc cttcc gccag tcatg caacc tgag- ′ and ′-gagcg gccgc cgcac acaca gtccc acgtc atggg t- ′ ). mutant constructs were established by cloning custom synthesized dna fragments (integrated dna technologies). all clones were verified by sanger sequencing. for rt-qpcr, total rna samples were obtained by rneasy column purification using on-column dnase treatment (qiagen). rna samples were converted to cdna (life technologies) and quantified by qpcr (power sybr master mix, life technologies; applied biosystems step one plus instrument). a minus rt control was included to control for contaminating genomic dna. primer sequences for qpcr were: gapdh_f: ′-agcca catcg ctcag acac- ′, gapdh_r: ′-gccca atacg accaa atcc- ′, rluc_f: ′-cagtg gtggg ccaga tgtaa acaa- ′, rluc_r: ′-taaga agagg ccgcg ttacc atgt- ′. cell transfections. transient transfections of ph ch , lh , or hela cells were performed using the pcdna /frt/to reporter plasmids described above. cells were plated onto -well plates at a density of . × cells/well hours prior to co-transfection with ng (lh , hela) or ng scientific reports | : | doi: . /srep (ph ch ) of rluc reporter plasmids and ng of pgl firefly luciferase plasmid (promega) using . μ l lipofectamine (life technologies). cells were lysed hours after transfection and luciferase measurements were made using by dual luciferase assay (promega). all reporter plasmids were analyzed in triplicate and p values were calculated by unpaired t-test. a transfections for rt-pcr analysis of ifnl expression were performed with . × cells plated hours prior to transfection into -well plates. cells were transfected with ng pcdna / frt/to plasmid, poly i:c (sigma), or purified hcv jfh strain rna produced by in vitro transcription with t rna polymerase (ambion). four hours after transfection, total rna samples were generated using trizol reagent (life technologies) and used for standard rt-pcr detection of ifnl mrna using primers designed to amplify the open reading frame. polysome profiling. hela cell lines were cultured on cm dishes and processed essentially as described . briefly, cells were washed, scraped and pelleted in ice-cold pbs containing μ m cycloheximide. cell pellets were resuspended in . ml lysis buffer [ mm koac, mm k-hepes (ph . ), mm mg(oac) , mm dtt, μ m cycloheximide] and incubated on ice for minutes before centrifugation for minutes at , × g to remove insoluble material. supernatants were layered onto ml - % sucrose density gradients containing mm koac, mm k-hepes (ph . ) and mm mg(oac) and then centrifuged for hours at , rpm using an sw rotor. gradients were fractionated (isco) and fractions were collected for rna extraction using trizol ls (life technologies). polysome analysis was conducted with single replicate per cell line in three biological replicates conducted on different days. adherent a cells were plated in a -well culture dish and grown overnight to % confluency. ifnl mrna was induced by transfecting cells (using lipofectamine ) with μ g of poly i:c (sigma) and incubated for hours before washing with pbs (ph . ). for shape modification of total deproteinized mrna, rna was purified using a modified trizol (invitrogen) extraction protocol. the cells were lysed with . ml of trizol for min at room temperature. the cell lysate was placed in a microfuge tube and . ml of chloroform was added followed seconds of shaking vigorously by hand. the sample was incubated for min at room temperature, and the sample was spun in the centrifuge at , × g for min. the deproteinized total rna in the aqueous layer was removed and immediately equilibrated into native folding buffer [ mm hepes (ph . ), mm mgcl , mm potassium acetate] using a pre-equilibrated g- spin column (ge health sciences). deproteinized rnas were incubated at o c for minutes in native folding buffer in order to allow folding state to come to equilibrium and then treated with . volume of dmso, or mm -methyl- -nitroisatoic anhydride ( m ) in dmso at o c for min. edta was added to mm final concentration; reactions were chilled on ice; and the rna was precipitated with isopropanol. for the parallel denaturing modification control, deproteinized total rna was equilibrated into x denaturing buffer [ mm hepes (ph ), mm edta, % formamide] using a pre-equilibrated g- spin column; heated to o c for min; modified with . volume of mm m in dmso; chilled on ice for min; and recovered by precipitation with isopropanol. shape-map library construction. dna libraries for massively parallel sequencing were prepared as described . briefly, targeted shape-map reverse transcription reactions ( μ l) contained μ m of ifnl rt primer ( ′-gtctt ttcct cattg tttat ttc- ′ ) in × shape-map buffer [ mm tris-hcl (ph ), mm kcl, mm mncl ] with μ l reverse transcriptase (superscript ii, invitrogen). reactions were incubated at °c for hours, and cdna products were purified (g- microspin column; ge lifesciences). purified cdnas were amplified for cycles using q dna polymerase (neb) with illumina adapted ifnl targeted primers (forward ′-gactg gagtt cagac gtgtg ctctt ccgat cnnnn ncctc cacca ttggc tgc- ′, reverse: ′-cccta cacga cgctc ttccg atctn nnnnn nnctc attgt ttatt tcaac aagga tttc- ′ ). the forward primer was designed with a ′ mismatch to the ifnl mrna to limit amplification of this similar transcript. pcr primers were designed to amplify sequences spanning different exons, so analyzed pcr product could only be generated from a spliced mrna and not contaminating genomic dna. pcr products were purified using a purelink pcr purification kit (invitrogen), and libraries were amplified by a second pcr reaction using q dna polymerase (neb) and illumina truseq pcr primers. pcr libraries were quantified by fluorescence (qubit fluorometer; life technologies), analyzed with a bioanalyzer dna kit (agilent), and sequenced on a miseq instrument (illumina). shape-map data analysis. shape-map reactivities for each nucleotide within the ifnl ′ utr were generated from the raw fastq files. raw shape reactivity data are shown in table s . a custom analysis pipeline identified all of the sequencing reads from ifnl rs g, ifnl rs t, and ifnl mrnas. ifnl reads were determined by analyzing six nucleotide positions that vary between ifnl and ifnl . reads with three or more nucleotide matches to ifnl ( . % of total reads) were removed prior to rna structure analysis. reads from the two ifnl alleles were sorted into separate fastq files and then analyzed using shapemapper . shannon entropies and minimum-free energy genome secondary structure models were generated from shape reactivities using a custom rna scientific reports | : | doi: . /srep folding pipeline . shannon entropy values were calculated to quantify the well-determinedness of structural states for a given rna region. the pipeline interfaces with rnastructure v . in order to computationally model rna secondary structure. the rna secondary structure models were diagramed using varna . global control of hepatitis c: where challenge meets opportunity genetic variation in il b predicts hepatitis c treatment-induced viral clearance il b is associated with response to chronic hepatitis c interferon-alpha and ribavirin therapy genome-wide association of il b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c genetic variation in il b and spontaneous clearance of hepatitis c virus ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il- , il- and their class ii cytokine receptor il- r ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo il- is the dominant type iii interferon produced by hepatocytes during acute hepatitis c virus infection lambda interferon is the predominant interferon induced by influenza a virus infection in vivo lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo interferons alpha and lambda inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics lambda interferon inhibits hepatitis b and c virus replication interferon-lambda is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden hepatic isg expression is associated with genetic variation in interleukin b and the outcome of ifn therapy for chronic hepatitis c il b genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis c interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness variants in il b in liver recipients and donors correlate with response to peg-interferon and ribavirin therapy for recurrent hepatitis c interferon-induced gene expression is a stronger predictor of treatment response than il b genotype in patients with hepatitis c hepatitis c virus pathogen associated molecular pattern (pamp) triggers production of lambda-interferons by human plasmacytoid dendritic cells hepatitis c virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures dendritic cells in hepatitis c virus infection: key players in the ifnl -genotype response human blood dendritic cell antigen (bdca )(+ ) dendritic cells are a potent producer of interferon-lambda in response to hepatitis c virus human type myeloid dendritic cells produce interferon-lambda and amplify interferonalpha in response to hepatitis c virus infection interferon-lambda serum levels in hepatitis c impact of il b gene polymorphisms on interferon-lambda plasma levels during pegylated interferon-alpha/ ribavirin therapy for chronic hepatitis c in patients coinfected with hiv ex vivo induction of ifn-lambda by a tlr agonist determines response to peg-ifn/ribavirin therapy in chronic hepatitis c patients the favorable ifnl genotype escapes mrna decay mediated by au-rich elements and hepatitis c virusinduced micrornas peginterferon alfa- b or alfa- a with ribavirin for treatment of hepatitis c infection deciphering the interleukin b variants that better predict response to pegylated interferon-alpha and ribavirin therapy in hcv/hiv- coinfected patients estimating the net contribution of interleukin- b variation to spontaneous hepatitis c virus clearance genome-wide association study of spontaneous resolution of hepatitis c virus infection: data from multiple cohorts analysis of il b variants in an egyptian population defines the kilobases minimal region involved in spontaneous clearance of hepatitis c virus au-rich elements and associated factors: are there unifying principles? a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus comparison of functional variants in ifnl and ifnl for association with hcv clearance activation of cap-independent translation by variant eukaryotic initiation factor g in vivo translational control by ′-utr-binding proteins a systematic analysis of disease-associated variants in the ′ regulatory regions of human protein-coding genes i: general principles and overview disease-associated mutations that alter the rna structural ensemble exploring rna structural codes with shape chemistry rna motif discovery by shape and mutational profiling (shape-map) assessing the reliability of rna folding using statistical mechanics using an rna secondary structure partition function to determine confidence in base pairs predicted by free energy minimization accurate shape-directed rna secondary structure modeling, including pseudoknots accurate shape-directed rna structure determination mfold web server for nucleic acid folding and hybridization prediction nndb: the nearest neighbor parameter database for predicting stability of nucleic acid secondary structure translational blockade imposed by cytokine-derived ua-rich sequences the ′ untranslated region of the human interferon-beta mrna has an inhibitory effect on translation endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level au-rich-element-dependent translation repression requires the cooperation of tristetraprolin and rck/p interrelationships of the pathways of mrna decay and translation in eukaryotic cells p bodies and the control of mrna translation and degradation landscape and variation of rna secondary structure across the human transcriptome interferon lambda signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses human hepatocyte clonal cell lines that support persistent replication of hepatitis c virus hepatitis c virus triggers apoptosis of a newly developed hepatoma cell line through antiviral defense system production of infectious hepatitis c virus in tissue culture from a cloned viral genome mrna translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation rna motif discovery by shape and mutational profiling (shape-map) varna: interactive drawing and editing of the rna secondary structure we thank merck research laboratories for allowing the use of patient specimens and data from the ideal trial. we thank bryan cullen and stacia philips y.l. study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript; d.m.m. study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript; d.b.g. critical revision of the manuscript for important intellectual content, study supervision; t.j.u. study concept and design, analysis and interpretation of data, critical revision of the manuscript for important intellectual content; k.m.w. study concept and design, analysis and interpretation of data, critical revision of the manuscript for important intellectual content; ssb: study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript, study supervision. supplementary information accompanies this paper at http://www.nature.com/srep key: cord- -a byk authors: saisawang, chonticha; saitornuang, sawanan; sillapee, pornpan; ubol, sukathida; smith, duncan r.; ketterman, albert j. title: chikungunya nsp protease is not a papain-like cysteine protease and the catalytic dyad cysteine is interchangeable with a proximal serine date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: a byk chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. in the last decade millions of cases have been reported around the world from africa to asia to the americas. the alphavirus nsp protein is multifunctional and is considered to be pivotal to viral replication, as the nsp protease activity is critical for proteolytic processing of the viral polyprotein during replication. classically the alphavirus nsp protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. we performed structure-function studies on the chikungunya nsp protease and show that the enzyme is not papain-like. characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. the enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. this switchable dyad residue has not been previously reported for alphavirus nsp proteases and would have a major impact on the nsp protease as an anti-viral target. and is proteolytically cleaved into individual mature proteins by the virus-encoded nsp protease in a specific manner while the structural proteins are subsequently translated from a subgenomic mrna . the alphavirus nsp protein is a multifunctional enzyme with the n-terminus of the protein comprising of rna helicase, nucleoside triphosphatase (ntpase) and rna-dependent ′ -triphosphatase activities [ ] [ ] [ ] ; whereas, the c-terminus of nsp contains the protease domain , . nsp is considered to be an essential protein as it is responsible for viral replication and propagation with its proteolytic and other activities . in addition nsp is translocated to the nucleus where it shuts down antiviral gene expression , . the proteolytic activity of nsp has been characterized in other alphaviruses, such as sindbis virus (sinv), semliki forest virus (sfv) and venezuelan equine encephalitis virus (veev), as a papain-like cysteine protease with a cysteine-histidine catalytic dyad in the active site , , [ ] [ ] [ ] [ ] . this led to the suggestion that the cysteine-histidine catalytic dyad possesses a nucleophilic cysteine residue that catalyzes the peptide bond cleavage with a histidine residue serving as a general base in the reaction. cysteine proteases or thiol proteases are comprised of different families each of which cluster into clans based on sequence identities, similarities and d-structure . the alphavirus nsp protease is classified into the togavirus cysteine endopeptidase family (c ) which belongs to clan cn . to date there is no vaccine against chikv and as the multifunctional nsp protein is critical for viral replication it makes an attractive potential anti-chikv drug target. to this end we have begun characterization of the protease active site with the initial focus on the catalytic dyad. although the dyad residues have been identified previously in other alphavirus nsp proteins; in sinv, sfv and veev , , [ ] [ ] [ ] , the chikv nsp protease active site has not been experimentally characterized. in performing this study a structural comparison showed that chikv nsp protease is not papain-like, and we have found what appears to be a unique feature of chikv nsp protease, in which the cysteine dyad residue can be catalytically replaced by a vicinal serine. previously the alphavirus nsp protease enzyme has been defined as a papain-like cysteine protease , - . as it appeared that the nsp was a cysteine-histidine dyad protease similar to papain, the papain protease has been used as a model for which structure was available for several decades. currently, there are available alphavirus nsp protease structures, for venezuelan equine encephalitis virus (veev; pdb id: hwk), sindbis virus (sinv; pdb id: gua) and chikungunya virus (chikv; pdb id: trk). structural superposition with papain and these nsp structures cannot be performed beyond atom pairs which supports that these alphavirus protein motifs are not related to papain (fig. ) . however, structural superposition of chikv nsp protease domain to the veev protease ( atom pairs) gives an rmsd of . Å and to sinv protease ( atom pairs) an rmsd of . Å. the superposition of the three alphavirus nsp proteases demonstrates a highly conserved tertiary structure for the nsp proteases despite the amino acid sequence variations, for example, chikv nsp pro aligned with sinv and veev nsp pro shows % and % amino acid identity, respectively. of interest in this report is the sequence difference as this is what makes the chikv nsp different from the other studied alphavirus nsp proteases. several studies have been reported characterizing the sfv, sinv, veev and chikv truncated nsp protease domain alone , , [ ] [ ] [ ] . moreover, the sfv full length nsp and protease domain have been stated to be similar . it was reported for sfv nsp that "the high solubility and specific activity of pro suggest that this fragment represents a structurally compact protease domain of nsp " . the requirement for the n-terminus of nsp has only been demonstrated for sfv and in the same study nsp protease from sinv processed the sfv polyprotein but the sfv nsp could not process the sinv polyprotein . these reports as well as the amino acid identity differences mentioned above demonstrate fundamental differences in the alphavirus nsp proteases. we have previously demonstrated that the full length chikv nsp and the truncated protease domain are not significantly different for protease activity . in our previous report we demonstrated that the chikv nsp protease (truncated and full length) could recognize small peptide substrates, which has not been reported for sindbis virus (sinv) or semliki forest virus (sfv) nsp protease. moreover, the chikv nsp protease recognition of small substrates also has been shown previously by another group . therefore an additional difference noted for chikv nsp is the length of the substrates that can be cleaved by the protease activity . the substrates employed in the present study are the biological cleavage site sequences of the chikv protease with fret tags; which is unlike previous reports for sfv and sinv nsp that use large gfp and thioredoxin fusion tagged sequences , , , . the sfv nsp showed no cleavage of / sequence unless amino acids of the n-terminus of nsp was part of the substrate . although even the egfp- nsp substrate showed incomplete cleavage suggesting the lack of activity for / cleavage could be due to steric hindrance by the presence of the thioredoxin fusion protein and, in any case, demonstrating specificity/activity differences compared to the other two sfv cleavage sites. in addition, chikv nsp -pro compared with the same region of sfv and sinv shows only % and % amino acid identity, respectively . the amino acid identity differences in the nsp proteins would by itself logically suggest that the nsp proteases will be different. moreover, the sfv cleavage sequences are actually different from the chikv sequences, for example the important sfv p residue of the / site, is tyr; but, in chikv it is asp. in fact, in the literature no data is presented concerning the testing of small peptides as substrates as all activity shown is with large fusion constructs. verification of our in vitro findings in an in vivo context is the logical scientific reports | : | doi: . /srep next study. the current study clearly demonstrates properties that the protease domain possesses which implies that these properties may also be expressed in vivo and this is the obvious justification for all recombinant protein characterization studies. several initial studies have shown cysteine and histidine residue involvement with catalysis, which gave nsp the papain-like designation , . as papain has been extensively studied (for example [ ] [ ] [ ] ) and the alphavirus nsp has been characterized as papain-like there appear to have been few studies to characterize the kinetic mechanism for the nsp protease. this is unfortunate as now available structures and the data in this report demonstrate that alphavirus nsp is not papain-like. for example, a tryptophan residue in papain has been shown to make an important contribution to the catalytic mechanism , . in the early literature for alphavirus, a tryptophan residue was also found to cause nsp enzyme activity loss, similar to what had been shown for the papain catalytic mechanism . later, the tryptophan residue was suggested to be involved with nsp protease substrate recognition of the glycine residue in the p position . now, the availability of the nsp protease structures shows that the tryptophan residue is actually in the wrong orientation to participate in catalysis. the tryptophan residue appears to be in a pocket surrounded by hydrophobic residues and van der waals contacts ( . to . Å in the chikv nsp , pdb id: trk); therefore, it is most likely involved in structural stability of the loop that contains the catalytic histidine residue (figs and ). to confirm this role we replaced the tryptophan residue with alanine and phenylalanine and performed characterization studies with the two proteins. the kinetic parameters obtained for the tryptophan position mutants show differences for the two engineered proteins for the substrates (table ). in contrast to the previous reports for the other alphavirus nsp enzymes neither mutant showed a total loss of activity for any substrate. perhaps surprisingly, the trp ala enzyme actually showed a -fold increase in activity for the agc substrate ( table ) . the alanine or phenylalanine residues affected the kinetic parameters to a different extent, and these effects also appeared to be substrate dependent. for example, both mutants showed decreased activity for aga but also an increase in affinity as shown by a decreased k m value such that the catalytic efficiency ratio (k cat /k m ) for both enzymes increased compared to the wild type enzyme (table ) . but both enzymes showed quite different effects on kinetic parameters with the agc substrate; the trp ala enzyme had increased activity, the trp phe had decreased activity and both had similar k m 's compared to wild type enzyme parameters (table ) . both tryptophan position mutants, for the agg substrate, showed similar activity as the wild type but displayed affinity (k m ) differences with trp phe similar to wild type but trp ala showing decreased affinity with an increased k m (table ) . we used several classical protease inhibitors as well as metal salts to characterize the nsp enzymes. although the alphavirus nsp has been classified as a cysteine protease there are many studies that show bacterial and viral cysteine proteases behave differently to papain-like cysteine proteases, such that the enzymes show little to no inhibition to e- , chymostatin, pmsf or leupeptin [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore to further characterize the tryptophan mutants we used several protease inhibitors and metal ions to observe the effects on the activity with the substrates (tables and ). both tryptophan mutants appeared to behave similar to the wild type enzyme in the presence of the protease inhibitors ( table ). the metal ion effects on the activity of the tryptophan mutants for the substrates suggested more complex conditions existed (tables and ). perhaps cobalt and zinc effects illustrate this complexity best. cobalt showed different effects depending on the substrate employed, with enhanced activity for aga, inhibition for distances are in angströms in green. alpha helices are shown in red and beta strands in purple. molecular graphics and analyses were performed with the ucsf chimera package. chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p -gm ) . agc and moderate increased activity for agg ( table ). the tryptophan residue mutants responded differently to each other as well as compared to the wild type suggesting topological changes in the active site upon protein interaction with the metal. with zinc these interactions allowed the tryptophan residue mutants to retain greater activity for all substrates (table ) . the modest effects of the tryptophan residue mutations on the kinetic parameters support the idea that the residue position does not play a direct role in catalysis. we suggest that the tryptophan residue position is involved in protein dynamics through anchoring of the loop that contains the catalytic dyad histidine residue. the residue changes in this position affected the conformational ensembles of the proteins, which indirectly affected the enzyme catalysis and affinity of interaction with the substrates. initial experiments to confirm the catalytic dyad cysteine residue were performed with the residue replaced with alanine. surprisingly the engineered enzyme did not lose all activity as had been reported for sinv and sfv alphavirus nsp proteases , . analysis of the available chikv nsp protease structure (pdb id: trk) suggested that a serine residue one helical turn away from the cysteine residue may be substituting in the catalytic role (fig. ) . in the chikv structure this serine is . Å from the dyad histidine residue (ser og to his ne ) which seems too far. however, examination of the veev (pdb id: hwk) and sinv (pdb id: gua) nsp protease structures show that the distances between the atoms of the dyad cysteine (sg) and histidine (ne ) residues range from . to . Å. this suggests dynamic movement of the static crystal structure conformations must occur to position the catalytic residues correctly and these types of movements could also account for placing the chikv serine residue an equivalent distance into a catalytic position. to test the hypothesis of serine catalysis we also generated ser ala mutant enzyme as well as the double mutant cys ala/ser ala. although the double mutant protein had no detectable activity with any substrate, both single mutation enzymes, cys ala and ser ala, had activity for all three substrates (table ). both enzymes, cys ala and ser ala, had a similar k cat to the wild type enzyme for the agc and agg substrates (table ) ; however, both enzymes had significantly less activity than wild type for the aga substrate ( table ). the binding affinity (k m ) for agc and agg was similar to wild type for both enzymes but was significantly increased (lower k m value) for the aga substrate. use of the aga substrate appears to allow discrimination of the kinetic parameters for the enzymes, cys ala, ser ala and wild type. this suggests that the conformational dynamics of the proteins are different with the changes in k cat of -and -fold as well as the changes in k m of -and -fold also demonstrating that other residues (in addition to the 'dyad' residues) are significantly involved in the binding and catalysis. previously a similar nsp protease from the alphavirus sfv was characterized and shown to be completely resistant to e- , pmsf and leupeptin as well as exhibiting metal ion effects , . in our study the protease inhibitors leupeptin and e- showed little to no effect on wild type, cys ala and ser ala enzyme activities for all substrates (table ) . chymostatin showed mild inhibition of the wild type for all substrates; but for cys ala and ser ala enzymes the inhibition appeared to be substrate dependent with even a slight enhancement for ser ala activity with aga (table ) . pmsf showed the greatest effect on cys ala activity for agg but little effect on aga activity of wild type, cys ala and ser ala enzymes ( table ). the five metal salts studied gave varying effects depending on the metal, the substrate and the enzyme being considered (table ) . for example, cobalt significantly enhanced aga activity for wild type and cys ala enzymes but not ser ala, but inhibited agc activity of all enzymes to a similar extent and gave a mild increase in agg activity for all enzymes (table ). using substrate equivalent to our agg (the cleavage site of nsp /nsp ) the sfv nsp showed approximately % inhibition with cobalt, whereas for chikv we observed a slight enhancement in activity. previously the sfv nsp was reported to be completely inhibited by copper and zinc and this was also reported for a chikv nsp protease ser ala enzyme using the substrate aga in the presence of zinc ( table ) . as previously observed, we suggest that the differences observed for the chikv nsp proteases originate with the different substrates used, as the different amino acid lengths around the scissile site as well as total substrate length appear to impact on the properties of the enzyme . a thermal stability study was performed to determine if the residue changes introduced structural properties that could be detected by variations in protein stability. the stability data showed the wild type enzyme and all the engineered mutants in this study behaved in a similar fashion ( table ). the similarities of the physical properties of the proteins were also corroborated by similar behaviors during expression and purification of the recombinant proteins. the enzyme differences observed in this report would therefore appear to be due to conformational dynamics and the varying ensembles available to each enzyme. of specific interest in this report is that the chikv nsp protease appears able to interchangeably employ either the canonical dyad cysteine residue or a proximal serine residue for catalysis (fig. ) . the enzyme retained activity with either a cys ala or a ser ala mutation but lost all detectable activity with the double mutation cys ala/ser ala. the data suggests that rather than an induced fit mechanism the substrate interaction employs conformational selection as either the cysteine or serine residue can be utilized for cleavage of all substrates. some conformations appear to yield greater activity and thereby display a slight residue preference for activity, such as cysteine residue for the aga substrate cleavage. the cysteine residue appears to have a structural influence as the cys ala mutant shows large experimental variation in the data. actually, both cysteine and serine residue positions seem to impact on structure to the extent of influencing changes in binding affinity such as for the aga substrate. the conformational dynamics also were impacted by the metal ions as demonstrated by specificity changes for wild type versus the cysteine and serine residue mutants for the substrates. several reports show viral protease enzyme activity is affected by metal ions , , , , , . the mechanism is unclear at this time but may be due to the metal interaction with the protein's amino acid residues (histidine, cysteine, aspartate, glutamate, tyrosine, tryptophan, methionine, serine, threonine, asparagine, glutamine or the main-chain amino and carbonyl groups ) impacting on the enzyme's conformational dynamics which then could yield an increase or decrease in enzyme activity. in our study, for example, cobalt significantly increases activity for aga substrate for the wild type and cys ala but not ser ala, while inhibiting all protease activities for agc substrate. these cobalt effects for aga substrate also support the concept that the wild type enzyme uses serine or cysteine residues interchangeably for catalysis. however, some of the inhibitor data, such as chymostatin, where the wild type activity is greater than either cysteine or serine mutant suggests that the catalytic mechanism is much more complex. indeed, taken together the data suggests that the available conformation ensembles are impacted by small molecules such as metal ions or inhibitors, as well as the residues present in the active site. these different ensembles present varying structures for the conformational selection process of the substrate binding which then yield the alterations in activity observed. the alphavirus nsp protease kinetic mechanism would seem to be more complex than the early literature has suggested. the chikv enzyme appears to be unique with the interchangeable cysteine/serine dyad residue; however, characterization of other alphavirus nsp 's with a similar serine -cysteine arrangement (for example sfv nsp ) needs to be performed. in addition, in vivo macromolecular assembly has also been suggested to play an important role in the functionality of nsp during viral replication . obviously, further characterization studies will be necessary if the nsp protease will be an anti-viral target. recombinant nsp protease characterization. the recombinant nsp proteases were constructed, expressed and purified as previously described . the engineered mutants were expressed and purified by the same protocol as the wild type protein. enzyme characterization was performed as previously described . briefly, the characterization was performed with synthetic fluorescent substrates corresponding to the cleavage sites of the chikungunya viral non-structural polyprotein. these substrates were designated nsp /nsp (aga), nsp /nsp (agc) and nsp /nsp (agg) with the amino acids nsp pro agg metal and protease inhibitor characterization. the effects on nsp protease activity of metal ions and protease inhibitors were characterized as previously described . briefly, a single concentration of mm metal ion was employed to test cobalt (co + ), magnesium (mg + ), zinc (zn + ), nickel (ni + ) and copper (cu + ) effects on protease activity. different final concentrations of four protease inhibitors were used; μ m chymostatin (inhibits chymotrypsin-like proteases), μ m e- (a selective cysteine protease inhibitor), μ m leupeptin (inhibits serine and thiol proteases) and mm phenylmethanesulfonyl fluoride (pmsf; a commonly used serine protease inhibitor). the enzyme activity in the presence of the metal ions or inhibitors was measured with the fluorescent substrates. the initial reaction rate was analyzed using graphpad prism ® software, version . . percent remaining activity was calculated from the enzyme activity obtained in the presence of inhibitor versus the control activity in the absence of inhibitor. stability test. thermal stability of the nsp protease was determined as previously described . briefly, freshly purified enzyme was incubated at and °c for min then activity was immediately measured using the agg substrate at . μ m final concentration. the initial reaction rate was evaluated using graphpad prism ® software, version . . percent remaining activity was assessed by comparison with the control reaction at °c. table . substrate sequences of chikungunya virus nsp protease used in this study. the substrate sequences used in the present study have been previously reported , and this table is adapted from the previous report. briefly, the three fluorescent substrates designated as aga, agc and agg (as underlined) were synthesized corresponding to the scissile site sequences (shown in upper case bold text) of chikungunya virus non-structural polyprotein (nsp / , nsp / and nsp / ), respectively. a -(n-methylamino)benzoyl (nma) fluorophore group was attached at the amino terminus and a , -dinitrophenyl (dnp) group attached to the carboxyl terminus of an additional lysine (k) residue. chikungunya: a re-emerging virus chikungunya on the move chikungunya virus in the americas-what a vectorborne pathogen can do chikungunya in the americas origin, evolution, and phylogeography of recent epidemic chikv strains 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activity on natural and synthetic substrates in vitro papain-like protease (plp ) from severe acute respiratory syndrome coronavirus (sars-cov): expression, purification, characterization, and inhibition characterization of the zinc binding activity of the rubella virus nonstructural protease cysteine proteinases of microorganisms and viruses metal ions in biological systems program dynafit for the analysis of enzyme kinetic data: application to hiv proteinase ucsf chimera-a visualization system for exploratory research and analysis funding: this work was supported by the thailand research fund (irg ) and grants from the office of higher education commission and mahidol university under the national research universities initiative and mahidol university. c.s., s.s. and p.s. performed the experiments. c.s. and a.j.k. re-analysed the data. c.s., s.u., d.r.s. and a.j.k. contributed to the manuscript preparation. competing financial interests: the authors declare no competing financial interests. key: cord- - a rfi k authors: knibb, wayne; luu, giang; premachandra, h. k. a.; lu, ming-wei; nguyen, nguyen hong title: regional genetic diversity for nnv grouper viruses across the indo-asian region – implications for selecting virus resistance in farmed groupers date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: a rfi k grouper aquaculture around asia is impacted by the nervous necrosis virus (nnv) and, in response, host resistance to this infection is being considered as a trait for selection. however efficient selection may be confounded if there are different genetic strains of nnv within and between regions and over years. this study uses statistical approaches and assessment of “characteristic attributes” (i.e. nucleotide positions that discriminate among strains) to assess whether published and new nnv rna cds sequences show genetic differentiation over geography, host species and years. rather clear evidence was found for regional strains of nnv. interestingly, most of the geographic defining “characteristic attributes” were in codon position three, and not translated into differences for the protein capsid (i.e. they were synonymous variations), suggesting that while nnv strains were geographically isolated and had diverged in different regions for rna sequences, selection had largely conserved the protein sequences among regions. the apparent selection constraint on the capsid protein may mitigate the risk that despite geographic subdivision, nnv strain variability will confound genetic selection for host resistance. the existence of regional asian nnv strains may suggest that hatcheries are at risk from nnv not only from imported material but also from endemic reservoirs. recently new genetic selection and domestication programs have commenced for large marine fish . various groupers (subfamily epinephelinae of the family serranidae) including giant groupers (epinephelus lanceolatus), tiger groupers (e. fuscoguttatus) and humpback groupers (cromileptes altivelis), compose one important new group of large marine species for domestication and selection. however, a viral disease, "viral nervous necrosis (vnn)" has been reported across grouper production centers in taiwan, malaysia, indonesia and elsewhere around asia , . the causative agent of vnn in groupers and other marine fish is the nervous necrosis virus (nnv) of the genus betanodavirus, within the family nodaviridae (international committee on taxonomy of viruses) (harikrishnan et al. many authors believe nnv is a serious threat to future marine fish aquaculture (including groupers). pakingking et al. concluded "catastrophic mortalities inflicted by piscine betanodaviruses remain as a major deterrent in the sustainable aquaculture of several species of groupers reared in ponds and floating net-cages in the open sea". in a similar vein, harikrishnan et al. concluded "viral nervous necrosis (vnn) caused by nervous necrosis virus (nnv) is one of the most important viral diseases that cause mass mortality in more than marine fish species in families". likewise munday et al. concluded "in the last decade betanodavirus infections have emerged as major constraints on the culture of marine fish in all parts of the world". several recent reviews recognize betanodavirus as a significant problem for marine fish farming almost world wide [ ] [ ] [ ] . fish host nnv resistance could be a key trait for future selection and the estimated heritability for nnv resistance in atlantic cod appears to be high [ ] [ ] [ ] . moreover qtls for resistance to nnv in asian sea bass were reported . however, notwithstanding such promising genetic selection opportunities, there are substantial knowledge gaps about the genetic diversity of nnv among asian grouper hosts: are there many strains within and between regions, perhaps with different virulence so that each region or nnv strain may then require different selection programs, or not? moreover, is there evidence that nnv strains vary over time? genetic programs are long term and expensive undertakings so selecting for resistance to the appropriate strain/s is an important consideration, but one with a relative absence of knowledge for asian grouper nnv. without some clarity, we may select for inappropriate nnv strains, or see new nnvs transported from a different region for which the grouper have no resistance. on a global scale, and considering many fish host species, previous phylogenetic analyses have resolved certain geographic and other patterns for nnv. for example there appears to be clustering of vnn genotypes into four main groups that include the so called barfin flounder nervous necrosis virus (bfnnv), the tiger puffer nervous necrosis virus (tpnnv), the striped jack nervous necrosis virus (sjnnv) and the red spotted grouper nervous necrosis virus (rgnnv) based on molecular phylogenetic analysis and percent nucleotide identity . these four clusters are somewhat associated with water temperatures (and also hosts), for example there is a tendency for bfnnv and tpnnv genotypes to occur in temperatures up to °c, for sjnnv to occur from to °c and for rgnnv for occur from up to °c . within the red spotted grouper nervous necrosis virus (rgnnv) strain, to which all asian grouper nnv belong, however, no one so far has reported evidence of genetic subgrouping by region, species or year in a formal statistical manner, especially when we restrict hosts just to asian grouper. available genbank data show all asian grouper nnv rna rna sequences, from japan to indonesia, are very closely related, varying by just one or two percent; this closeness means there are challenges to determine phylogenetic relationships especially when using traditional dna or rna distance based methods. however, lowenstein et al. have found for tuna that particular polymorphic nucleotide positions (characteristic attributes) may categorically discriminate groups/ species previous not previous distinguished using traditional analyses based on percent rna or dna similarity. ruan et al. reported certain polymorphic nucleotide positions could discriminate among strains of the sars virus while zou et al. reported that "character based" bar coding methods outperformed other approaches in discriminating closely related sea snails. within aquaculture, these approaches of identifying nucleotides that are present in one group but not others have yet to be considered to a large degree, although our group have applied these ideas of characteristic attributes to recently resolve a very long standing taxonomic issue in oysters, whether the very closely related crassostrea gigas and c. angulata are one or two species, as a prelude to conducting selection on the now identified c. angulata in vietnam . the goal of this report was to collate the most comprehensive data set to date on nnv rna sequences for warm water asian marine finfish, whether published and/or lodged in genbank over the last years, including some sequence data produced by our group for vietnamese and taiwanese grouper, to statistically test the data for evidence of nnv strain variation that associates with geography, host species and year and also to determine whether there are "characteristic attributes" that indicate regional (or host, year) specific differences among the strains. this knowledge will help guide future selection criteria/nnv strains to consider in future genetic selection programs. new samples from vietnam and taiwan. as a prelude to sequencing, a large number of grouper samples (nearly ) were collected as part of routine veterinary surveillance, from different species and hybrids from the national broodstock centre for mariculture species, nbcfms in cat ba, haiphong, northern vietnam in september and from qieding, kaohsiung, taiwan in july ( supplementary fig. s ) and tested for the presence of nnv using primers from nishizawa et al. . samples were taken, typically, at a time there were general mortalities of larvae, but other than being alive or dead, no other distinctive behaviours were noted; i.e. larval fish in this study looked normal. note: the lack of symptoms for larval fish infected with nnv was also reported . rna extraction and sequencing. total rna was isolated from the tissue samples using trisure reagent according to the manufacturer's instructions (bioline, aus). cdna synthesis was conducted from the total genomic rna using the quantitect reverse transcription kit (qiagen) following manufacturer's instructions. briefly, a genomic dna elimination reaction was performed by mixing a solution of µl of dissolved template rna ( ng/µl) with µl g dna wipeout buffer x and µl rnase-free water with an incubation at °c for minutes, then placed immediately on ice. the reverse transcription reaction was carried out for minutes at °c and then incubated at °c for minutes with the reaction mixture with final volume of µl: µl quantiscript rt buffer, µl quantiscript reverse transcriptase, µl rt primer mix and µl genomic dna elimination. the rna segment of nervous necrosis virus was amplified using the set of primers described in ransangan and manin . the forward and reverse pcr reactions were conducted separately in a μl total volume consisting of μl mytaq ™ red mix (bioline, aus), μl rnase-free water, μl each primer ( µm) (forward and reverse) and μl cdna. the amplifications were performed using an eppendorf mastercycler pro thermal cycler with an initial denaturation at °c for minutes, followed by collation of published sequences. genbank (the national centre for biotechnology information (ncbi) (http://www.ncbi.nlm.nih.gov/)) was searched in various ways for nnv sequences. first, the complete coding rna sequences for the nnv rna gene from a giant grouper host (gb accession number ay ) was blasted against all sequences in genbank, and over sequences recovered with at least % similarity. all complete coding rna sequences from all groupers in the indo-asian region were retained along with those from any other species that had similarity (to ay ) at least equal to the most distant nnv from a grouper host, these non grouper species included mostly barramundi (lates calcarifer). second, genbank was searched for "grouper" and "nnv" and "rna ", and all nnv complete coding rna sequences from the indo-asian region collated. a total of sequences were collated. to this we added (details following) a further four unpublished sequences reported for the first time here from vietnamese humpback grouper (c. altivelis) and three from taiwanese giant x tiger hybrids (e. lanceolatus ♂ x e. fuscoguttatus ♀) (supplementary table s ). only the protein encoding region of the sequences was used for analyses. full protein capsid encoding sequences of all sequences, published and new, is given in supplementary table s . analyses of sequences. alignment of nucleotide sequences was performed using clustal w . in the bp orf encoding the coat protein there were variable nucleotide sites which were considered, and referred to, as "loci" for the following tests. analysis of molecular variance (genalex ) assessed the probabilities for differences among regions over all loci, and for each locus separately. each region group needed at least two cases for statistical testing, so for example, the testing for region differences would have had regions considering the samples ( fig. ) . a region typically corresponds to a country, such as japan or taiwan, but for large countries where sites were separated by significant geography, such as north vs south china and east (borneo) vs west (peninsular) malaysia, regions within countries were considered. to address the possibility of lack of independence of some samples arising from a short term spread and propagation of a new infection of one viral genotype in a given hatchery, the data were also analysed at each of various culling steps that attempt to remove potentially non independent samples within hatcheries. first, samples that had the exact same sequence (genotype) as another case from the same location, same year and same species were removed; samples were available after this culling. the second culling included given samples that came from the same location, year and host species as another sample (leaving only one sample per species in a given place and time); samples were available after this round of culling. the intention was to remove cases that may represent propagation in a hatchery, so an exception to the rule is that wild fish samples even if from the same species and location were not culled at this stage. last, the third culling was for samples that came from the same site and same year as other samples; samples remained after this round of culling. at this stage, even different wild fish were culled if they came from the same site at the same year. similar culling procedures applied when testing for "species" grouping, and for "year" grouping. when a group contained only one case after culling, the single case was pooled with a neighbouring group (for region and year) or included in a group termed "other" for the species analyses. construction of phylogenetic trees based on rna nucleotide and amino acid sequences were conducted by mega using the maximum likelihood method. statistical anova model. the analysis of variance (anova) model assessing jointly effects of location, species and year on "percent rna sequence relatedness" to a consensus sequence take the following form: where y ijklmn = observations, i.e. "percent rna sequence relatedness" to a consensus sequence µ = mean analyses were conducted using spss software. relative synonymous codon usage analysis. relative synonymous codon counts were calculated for each of the main host fish species groups, namely, grouper and barramundi, using mega and counts were compared for differences between groups using a chi square test. bayesian tip-association significance testing (bats). phylogeny-trait (geographic location) associations were tested including sequences ( australian samples were removed since they were appeared to be outliers) using bayesian tip-association significance testing (bats) software . posterior sample of trees (pst) were obtained through bayesian markov-chain monte carlo (mcmc) analysis using beast software . beast was run for million mcmc chain lengths (sampled every generations; burn-in %) with the hasegawa-kishino-yano + gamma (hky + g) model, assuming coalescent constant population priors with relaxed lognormal clock . the convergence for all parameters (effective sample size > ) was assessed using tracer v . (http://beast.bio.ed.ac.uk/tracer). the resulting trees were used to test whether each region was a monophyletic clade (i.e. assessed for phylogeny-trait statistics considering geographic regions -india, malaysia , malaysia , singapore, vietnam, taiwan, china south, china north, korea and japan) using bats software . , japan = japan. † different groups that were culled to reduce impact of pseudo-replication, see details in following text. abbreviations for column : f = fish, fw = fish wild, fy = fish wild yearling, c = cell culture. additional tests for regional differences were conducted using bayesian tip-association significance (bats) tests; the association index and the parsimony score statistic assessing regional differences among all samples were both statistically significant (p < . ) as were tests of monophyletic clade (mc) sizes for each region, except for japan and south china. supplementary fig. s ) , with an average similarity between pairs of . %, and the most distant pair had . % identity. this maximum distance involved australia bass which has a rather atypical sequence from all other sequences. removal of this one outlier showed the average similarity between pairs was . %, and the most distant pair had . % identity; comparable similarity percentages restricting the data set just to groupers (n = ) were . % and . % respectively. there were nucleotide positions of the that varied over the cds, and most analyses focused on these variable nucleotide positions (supplementary table s ). characteristic attributes. regional grouping. regional differences were evident for a large number of nucleotides positions. analysis of molecular variance tests (amova ) recorded, out of polymorphic sites, nucleotide positions statistically significantly from zero considering region at p ≤ . ; an additional at p ≤ . ; and the probability when testing all loci together was ≤ . (data showing significance levels for each nucleotide position of fig. are in supplementary fig. s ). while there were nucleotides in the complete coding sequences in each of the samples considered, only contained two or more nucleotide variants at a given nucleotide position and would qualify as candidates for statistical testing. however, of the , just, contained three or more different alleles; every case of p ≤ . was restricted to nucleotide positions where there were three or more different bases at a given nucleotide position; this probably reflected the power of the statistical test and relationship with level of polymorphism. thus we can say that of moderately polymorphic nucleotide sites (with at least three variable nucleotides), more than half recorded statistically significant differences among regions. every indonesian and east malaysian (borneo) sample invariably had the same (less common) nucleotides at positions , , and (although a smattering of samples from other regions also had the same nucleotides as some of these positions). these characteristic attributes, hereafter referred to as "cats", were present in two different grouper species as well as barramundi, were present in multiple sites within borneo and evident in all (and different) years and collection times ( to ). considered separately, each of the "cats" in highly statistically significant ( fig. and supplementary fig. s ) , and together, with a single exception (ay ), they comprise a diagnostic genotype unique for borneo and indonesia irrespective of host fish species. "cats" were also evident for vietnam, and all samples share the same nucleotides at positions , , , , , , , . together they comprise a diagnostic genotype almost unique to vietnam but interestingly all samples from norther china share, with one exception ( ), the same genotype for these "cats", and the chinese samples come from three different species (flounder, sole, grouper) and from three different years ( , , ) . while four of the five vietnamese samples came from the same species, same location and same year (humpback grouper, north vietnam, ), the fifth, with the same "cat" genotype, came from a different species and different year (orange spotted grouper, ). together with the data from northern china, this described genotype would indicate these samples are not randomly drawn from a general population, nor are they solely outcomes of propagation in a given hatchery (although the four humpback grouper samples may be). other "cats", where only one nucleotide is found in all samples in a given region, are highlighted in darker grey in fig. ; "cats" where a given country mostly has one nucleotide at a given position is highlighted in medium grey, and signature genotypes, that characterise a region, composed of a number of "cats" are given in light grey (see accession numbers hq , hq , hq , hq ). culling to reduce impact of "pseudo-replication". removing all samples that had identical sequences to other same species samples from the same site and year (cases designated by a " ", column , fig. ) left remaining sequences for analysis (cases designated by a " ", " " or " ", column , fig. ); this made little change to the probabilities for regional differences recorded using all samples (supplementary fig. s ) . likewise, removing a further hatchery samples which came from the same location, year and host species as other samples (cases designated by a " ", column , fig. ), left samples for analysis (cases designated by a " " or " ", column , fig. ), but did not substantially alter the pattern of significant cases among regions ( supplementary fig. s ). wild fish were except from culling at this step as the intention was to remove cases of pseudo-replication in the hatcheries. the last culling was for a further samples that came from the same site and same year as other samples (cases designated by a " ", column , fig. ), leaving samples for analysis (cases designated by a " ", fig. ). even wild fish meeting these criteria were culled at this point. with these samples, the incidence of significant cases was reduced somewhat, but even in this most extreme vetting, amova analyses showed there were still a total of eight cases (nucleotide positions) with statistically significant differences among regions at p ≤ . , a total of eight cases (nucleotide positions) with statistically significant differences among regions at p ≤ . and the test over all loci (all nucleotide positions) was significant (p ≤ . ) (supplementary fig. s ). after culling, there were only nucleotide (allele) positions with three or more different nucleotides (the rest of the were now monomorphic or had one to two nucleotide differences among the samples). so we can say that of moderately polymorphic nucleotide sites (with at least three variable nucleotides of the nnv samples), more than % (i.e. eight) recorded highly statistically significant nucleotide differences among regions even under extreme culling (which is about a ten fold higher rate of highly significant cases than that expected by chance alone). species grouping. species differences at given nucleotide positions were also detected considering all samples, albeit at about half the incidence as that detected above for regional differences (amova recorded nucleotide positions statistically significantly from zero, p ≤ . ; with p ≤ . and the overall probability when testing all loci together was p ≤ . (supplementary fig. s ). there appeared to be a high degree of coincidence in the probability levels for nucleotides considering regions and considering species (r = . , p < . , considering all nucleotide positions). "cats" that were predominately restricted for one fish host species were not typical, with some rare exceptions (e.g. nucleotide ); within countries, "cats" are shared across species, e.g. barramundi and grouper share common "cats" in east malaysia and indonesia (fig. ) . on the other hand, with one exception (column , fig. ), different species did not have completely identical rna sequences. the various culling steps to reduce "pseudo-replication" culminated in the most extreme culling with samples remaining (cases designated by a " ", fig. ) and relative few cases of statistically significant differences among species, namely five cases of statistically significant nucleotide differences among species, only one at p < . from among moderately polymorphic nucleotide sites (i.e. those with at least three variable nucleotides). such an incidence of statistically significant results could be accounted for by chance or type i error. the test over all loci was not statistically significant (p > . ). year grouping. yearly differences were also detected considering all samples, again at about half the incidence as that detected above for regional differences (amova recorded statistically significantly from zero, p ≤ . ; with p ≤ . and the overall probability when testing all loci together was p ≤ . ; supplementary fig. s ) . again, there appeared to be a high degree of coincidence in the probability levels for nucleotides considering regions and considering years (r = . , p < . , considering all nucleotide positions). the various culling steps to reduce "pseudo-replication" culminated in the most extreme culling with samples remaining (cases designated by a " ", fig. ) and then there was only one case of a statistically significant difference among species, namely one at p < . from among moderately polymorphic nucleotide sites (i.e. those with at least three variable nucleotides). the test over all loci was not statistically significant (p > . ). for some regions, the same "cats" were recorded over years, for example, the "cats" at nucleotides position for singapore were evident from through to , and several "cats" for north china (nucleotide positions , , , , etc.) were evident from to . analogous results were evident for vietnam and western malaysia (borneo) but not for india. on the other hand, with one exception (column , fig. ), different years did not have completely identical rna sequences. sequence for all the polymorphic nucleotide sequences was generated (supplementary table s ), then "percent rna similarity" to the consensus was calculated for each sample (supplementary fig. s ); this variate ranged from . % identity (for australian bass) to . % for orange spot grouper. the bass seemed to be an outlier as the next most divergent sample was malaysian barramundi at . % similarity, noting all these percentages refer to just the polymorphic nucleotide data, and not the whole coding region sequences. on one hand, the metric of "percent rna similarity" contains less information than the preceding data on variable nucleotide positions, but on the other hand, "percent rna similarity", as a continuous variate, permits simultaneous partitioning of variance among the predictor variates (location, species, and year). an anova (section . . materials and methods), using all the samples and the three predictor variates (region, species, year), showed that all predictor factors accounted for a significant part of the variation in "percent rna similarity" (for region, anova f , = . , p < . ; for species, anova f , = . , p < . ; for year, anova f , = . , p < . ). repeating these analyses, but after the most extreme culling (removing all samples that come from the same year and site as another) still indicated highly statistically significant differences only for region (for region, anova f , = . , p < . ; for species, anova f , = . , p < . ; for year, anova f , = . , p > . ). maximum likelihood tree. the maximum likelihood tree (fig. ) shows some concordance with the regionality of nnv strains detected by "cats" (fig. ) , however the tree analysis revealed far fewer groupings that were statistically significantly different (branch numbers or greater) than previous revealed using individual nucleotides. confidence of the tree branches (fig. ) can be inferred from the number labelled on branches which indicate the proportion of times out of trials the same node was formed (using mega ). considering all samples (fig. ) there were samples taken from fish tissue that were sequenced, and samples from cell lines with viruses originally derived from fish, and samples of unknown origin. because propagation in cell tissue culture may represent new selective environment/s and/or foster the retention of new mutations and cause greater genetic variance, samples from fish were compared with those from cell lines. analyses of molecular variance showed there was only four percent variation among groups which was not statistically significant. moreover, similar results were evident in the preceding analyses whether data from cell cultures was included or not. the average pair wise rna sequence percent similarity among all the cell culture lines was . % and the most dissimilar pair had . % identity; respective values after removing the anomalous single australian bass sample were . % and . %; respective values for the grouper only samples were . % and . % (see supplementary fig. s ) , i.e. after discounting the anomalous australian bass sample, there was a greater range in sequence variation among the fish samples than among the tissue culture samples considering the whole data set. sequence diversity within fish and in "outbreaks". considering the new rna sequences generated in this report, each of the rna sequences from three humpback grouper in vietnam, sampled at the same time from the same hatchery, were all % identical. similarly, all three taiwanese giant grouper samples sampled at the same time and from the same hatchery were % identical (supplementary table s ). if there had been another strain, say at . frequency in both vietnam and taiwan, then chance of failing to detect it is less than % (even if another strain had been present in both stocks at a frequency of only %, the chance of failing to detect in among the six samples is about %). thus it appears unlikely there were different nnv sequences in the infections at moderate frequencies. apparently similar results, of identical rna genotypes were reported in most other grouper data sets where there were multiple rna sequences for the same species in the same hatchery at the same time, that is, these samples tended to have identical rna genotypes and examples included humpback grouper in indonesia, tiger grouper in indonesia, tiger grouper in east malaysia and in west malaysia. barramundi, however, tended to show slightly different genotypes in the same time/location (e.g. india, malaysia). translated protein sequence. of the polymorphic rna sites tabulated in fig. , were in codon position (rna polymorphic sites in codon or are in bold, top row, fig. ). the average divergence among pairs of samples for protein sequences, considering all samples listed in fig. was just % (supplementary fig. s ) , and there was little evidence for the pronounced protein "cats", as seen for the rna, that associated with region, species or year. the maximum likelihood tree for the protein sequence (fig. ) , notwithstanding some regional clustering, did not have significant nodes save for one exception. codon-based test of purifying selection (mega ) indicated statistically significant (p < . ) excess of synonymous substitutions over nonsynonymous substitutions per site when considering all sequences, just those for grouper or just those for barramundi (average numbers of synonymous sites per codon for all samples, grouper and barramundi, respectively, were . , . , . , and respective values for nonsynonymous sites were . , . , . ). on the other hand, relative synonymous codon counts for nnv were not statistically significantly different between two major host fish species groups, namely, grouper and barramundi. high level of rna and protein similarity across large regions. from japan to india, and considering various marine fish hosts, the average sample pair difference for nnv rna cds was just % for percent rna sequence similarity and % for the translated protein sequence. at face value, one could infer rather heavy evolutionary constraints on the virus throughout the region and among species, or perhaps co-transport with hosts among regions for commercial production is homogenizing the virus groups. these first impressions are reflected in the literature where previously no formal statistical analysis revealed genetic diversity of asian rgnnv that significantly associated with region, host or time. characteristic attributes identify regional strains. however, our analyses using "characteristic attributes" and statistical approaches such as amova and n-way anova, and bayesian tip-association testing (bats) and using the largest data set so far compiled for asian grouper (and other marine asian species), show clear regionality in rgnnv strains. this is the first clear evidence of regionality for rgnnv in the indo asian region, but interestingly a large survey in southern europe by panzarin et al. also detected regionality (geographic subdivision) of mostly rgnnv strains. this understanding, of regionality for rgnnv strains, is a completely novel one for asian marine species, and possibly has some implications for selective breeding programs for host resistance in different regions, and also possibly for general management and prevention of this disease. far more cases of statistically significant regional differences were detected using "characteristic attributes" than using maximum likelihood tree analyses, lending support to the postulate that characteristic attributes can better discriminate closely related species than traditional phylogenetic analyses , . our evidence for species specific and year specific "cats" is somewhat less than for regional differences. actually, there is evidence of sharing of "cat" genotypes among species, such as grouper and barramundi in eastern malaysia and indonesia, and flounder, grouper and sole in north china (fig. ) which questions whether nnv has yet evolved specificity for different marine finfish in asia. panzarin et al. , in their large survey from southern europe, considered many local host species (sea bass, mullet, sea bream etc.) and observed no "species-specific" mutations in the rna region. our evidence for temporal stability of nnv strains over years is mixed, on one hand, the same "cats" are evident over six or so years in northern china, and in eastern malaysia over two years, in vietnam over eight years but not in india over six years (fig. ) ; on balance we cannot conclude that each year brings a new wave of very different genotypes to a particular region, on the contrary, the overall picture is one of genotype stability over years in given regions. there is a possibility that the levels of rna variation would be artificially increased after passage in cell cultures, because of the different selective pressures in tissue culture than those found in the fish host. actually, we found no evidence of such a hypothetical effect, and indeed there was slightly more variation in sequences from fish hosts than from cell cultures; moreover amova tests did not show significant differences between the sequences from fish hosts and from cell lines. accordingly, there does not appear to be any evidence based reason not to include the rna sequences from cell cultures in our analyses on the grounds that they are intrinsically more variable, or intrinsically more different, than sequences from fish. another potentially confounding matter was that of substantial diversity of nnv sequences within hatcheries. we did not find evidence of much if any diversity for the same species, same hatchery and same sampling time for grouper species, although some barramundi samples from india showed some slight differences from the same collection time and place; in any case such slight differences were clearly, ipso facto, insufficient to confound the analyses that assessed regional differences (i.e. regional differences were statistically significant). rna genetic diversity is greater than the capsid protein diversity. the rna sequence considered here was from the rna gene, which encodes the virus protein capsid protein. interestingly, the translated rna sequences showed even less variation than the rna with less evidence of region specific amino acid "cats"; indeed most rna "cats" were found at codon position three which did not lead to changes in the capsid protein. statistical tests indicated a very substantial excess of synonymous substitutions over nonsynonymous sites, indicating "purifying" selection. every node in the maximum likelihood tree for the protein sequences was not significant for clusters save one. a parsimonious model to account for this interesting discrepancy between the rna sequence and the protein is that strains to some degree are geographically isolated and free to diverge especially at rna triplet positions not tightly constrained by selection (i. e. most codon positions), but those sites (rna codon positions one and two) where changes can lead to changes in the capsid protein, are more tightly constrained by selection. balancing all the evidence, the null hypothesis is that the regional asian strains of nnv are functionally equivalent which may give encouragement that selection for resistance in one region will lead to fish capable of resisting nnv in another. indeed various have reported that all rgnnv strains that were tested in asian and europe were serologically related (within each region) and had the same serotype despite different rna genotypes, further supporting the hypothesis that selection for one rgnnv rna genotype will give rise to resistance for all within a region , . if this hypothesis is confirmed, then variation evident in the rna sequences and "cats" may become less relevant to the issue of genetic selection, but still may inform about the epidemiology of the pathogen (see following). implications for disease management. munday et al. stated "lack of knowledge of the epidemiology of the diseases caused by nodaviruses, except for vertical transmission of the pathogen in some species, has impeded the development of control measures". by showing the existence of regional strains of rgnnv in asian marine fish for the first time, our study points to the existence of regional, endemic sources of nnv that may recurrently infect local hatcheries. this understanding is consistent with reports of finding nnv in wild fish . so in addition to taking precautions not to import nnv along with imported broodstock, grouper hatcheries may also now consider the possibility that nnv can enter the hatcheries from local reservoirs from local wild fish carrying nnv. • rna sequence variation shows the existence of regional asian rgnn strains • translated protein differences showed less regionality than the rna data • while gene flow appears largely restricted among regions, selection apparently constrains variability of capsid protein sequence • the apparent selection constraint mitigates the risk that despite geographic subdivision, nnv strain variability will confound genetic selection for host resistance • regional asian rgnn strains may suggest hatcheries are at risk from nnv not only from imported material but also from endemic reservoirs. comparison of lines shows selection response in kingfish (seriola lalandi) characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus genome analysis of betanodavirus from cultured 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in asian seabass genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene cloning of the fish cell line ssn- for piscine nodaviruses the real maccoyii: identifying tuna sushi with dna barcodes -contrasting characteristic attributes and genetic distances comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection comparing the usefulness of distance, monophyly and character-based dna barcoding methods in species identification: a case study of neogastropoda resolution of the controversial relationship between pacific and portuguese oysters internationally and in vietnam polymerase chain reaction (pcr) amplification of rna of striped jack nervous necrosis virus (sjnnv) current knowledge on viral nervous necrosis (vnn) and its causative betanodaviruses clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice genalex : genetic analysis in excel. population genetic software for teaching and research molecular evolutionary genetics analysis version . for bigger datasets correlating viral phenotypes with phylogeny: accounting for phylogenetic uncertainty beast : a software platform for bayesian evolutionary analysis relaxed phylogenetics and dating with confidence molecular epidemiology and evolutionary dynamics of betanodavirus in southern europe serological relationships among genotypic variants of betanodavirus molecular basis for antigenic diversity of genus betanodavirus pcr-based detection of betanodaviruses from cultured and wild marine fish with no clinical signs this study was supported by the genecology research centre, the university of the sunshine coast, queensland, australia. we gratefully acknowledge financial support from the john allwright fellowship. we highly appreciate national broodstock center for mariculture species in northern vietnam for providing the larvae samples from vietnam. w.k. conducted the genbank data collation, statistical analyses and wrote the draft ms. g.l. did the laboratory work including the sequencing and pcr testing; g.l. also conducted preliminary data collation, statistical analyses and draft writing. m.l. provided the samples and information from taiwan. n.h.h. assisted with editing the manuscript. h.p. conducted the "beast" analyses. supplementary information accompanies this paper at doi: . /s - - - competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -jyk f hz authors: branton, w. g.; lu, j. q.; surette, m. g.; holt, r. a.; lind, j.; laman, j. d.; power, c. title: brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: jyk f hz microbial communities reside in healthy tissues but are often disrupted during disease. bacterial genomes and proteins are detected in brains from humans, nonhuman primates, rodents and other species in the absence of neurological disease. we investigated the composition and abundance of microbiota in frozen and fixed autopsied brain samples from patients with multiple sclerosis (ms) and age- and sex-matched nonms patients as controls, using neuropathological, molecular and bioinformatics tools. s rrna sequencing revealed proteobacteria to be the dominant phylum with restricted diversity in cerebral white matter (wm) from ms compared to nonms patients. both clinical groups displayed , – , bacterial genomes/cm( ) and low bacterial rrna:rdna ratios in wm. rnaseq analyses showed a predominance of proteobacteria in progressive ms patients’ wm, associated with increased inflammatory gene expression, relative to a broader range of bacterial phyla in relapsing-remitting ms patients’ wm. although bacterial peptidoglycan (pgn) and rna polymerase beta subunit immunoreactivities were observed in all patients, pgn immunodetection was correlated with demyelination and neuroinflammation in ms brains. principal component analysis revealed that demyelination, pgn and inflammatory gene expression accounted for % of the observed variance. thus, inflammatory demyelination is linked to an organ-specific dysbiosis in ms that could contribute to underlying disease mechanisms. scientific reports | : | doi: . /srep bacterial proteins, in brains from humans (in the presence or absence of neurological disease), nonhuman primates , rodents and other species . herein, we examined bacterial quantity and genetic diversity in brains from patients with ms and other diseases. bacterial abundance and molecular diversity were associated with both neuropathology and proinflammatory gene expression in patients with ms, revealing disturbances in human brain microbiota in a disease context. quantitation and conventional sequencing of brain-derived bacterial s and groel amplicons. bacterial s ribosomal rdna and rrna v -v sequences (genbank: kx -kx ) were amplified from cerebral white matter of all tested ms (n = ) and nonms (n = ) patients (table ) with greater expression of rrna than rdna ( - fold) in all patients (fig. b) . quantitation of bacterial genomic (groel) dna ranged from , - genomes per cm (tissue) based on single gene copy detection per bacterium in both ms and nonms white matter (fig. c ). bacterial rrna sequence analyses of cloned amplicons derived from nonms white matter displayed greater molecular diversity within multiple clones per sample compared to ms white matter (p < . ) (fig. c ). phylogenetic assessment of bacterial sequences encoding the ribosomal s rrna v -v domain cloned from a subset of samples was performed, which revealed alignment of brain-derived bacterial sequences from ms (n = ) and nonms (n = ) patients with diverse bacterial species' sequences ( fig. a) . proteobacteria represented the most abundant phylum in both clinical groups although more cloned bacterial sequences were obtained from ms samples. these findings highlighted the preponderance of bacterial sequences resembling proteobacterial species in brains with reduced bacterial genomic molecular diversity in ms patients' white matter. massively parallel (deep) sequencing (rnaseq) of total rna permitted analysis of all rna sequences in ms (n = ) and nonms (n = ) white matter samples, revealing that bacterial rna (ribosomal and non-ribosomal) sequences were detected in all nonms and ms brain specimens, including ms patients with relapsing-remitting disease (receiving disease modifying therapy) (rr-ms, n = ) and progressive (untreated) ms (p-ms; n = ) ( fig. a) . greater than % of bacterial sequences were identified as unambiguously complementary to proteobacteria sequences. while actinobacteria was the second most abundant phylum in all cases, there was an enrichment of this phylum in rr-ms (p < . ) relative to nonms white matter with a marked reduction of actinobacteria sequence proportions in p-ms brains ( fig. a) . sequences matching bacteriophages with proteobacteria hosts predominated in both clinical groups but were overrepresented in nonms samples (fig. b) . the apparent contraction of bacterial molecular diversity within p-ms brains was associated with increased expression of immune genes in p-ms white matter (e.g., cd ε, hla-dra, il- , cd c) (fig. c ). proteobacteria sequence abundance was also examined in relation to host gene expression from the same sequencing analyses revealing a positive correlation with multiple groups of genes including nfkb-signaling, secondary metabolism, and energy metabolism ( supplementary fig. a) . conversely, expression of genes related to nervous system regulation, gene silencing, and cell proliferation were negatively correlated with proteobacteria rna tag abundance ( supplementary fig. b) . these observations highlighted the predominance of protobacteria-encoded rna sequences in brains from ms and nonms patients but also displayed variation in bacterial molecular diversity in relation to host immune responses. histopathology, immunohistochemistry and in situ hybridization. as ms is a heterogenous disease in terms of clinical features as well as site and type of lesions, we used premortem mris to guide the selection of tissue samples when possible (fig. a,b) for neuropathological studies. from brains of ms (n = ) and nonms (n = ) patients, serial brain sections were investigated based on the presence of lesions on mri that showed gadolinium-enhancement (t ) (fig. a ) and/or evident on t images (fig. a ) . strong lfb staining, indicative of intact myelin, was evident in nonms white matter ( fig. b ) but was reduced in demyelinating ms lesions (fig. b ). cd ε -immunolabeled t cells were occasionally detected in nonms tissue sections ( supplementary fig. a ) but cd ε -immunopositive cells were evident in ms lesions ( supplementary fig. a ). cd -immunopositive brain macrophages were minimally detected in nonms white matter ( supplementary fig. b ) but were abundant in demyelinating ms lesions ( supplementary fig. b ). bacterial peptidoglycan (pgn) immunostaining was particulate, detected in sections from nonms (fig. c ) and ms (fig. c ) brains but appeared to be more concentrated in ms lesions. this was in contrast to the lack of immunostaining apparent with the isotype control ( supplementary fig. c ). pgn immunodetection was co-localized with immunoreactivity to the astrocytic protein, glial fibrillary acidic protein (gfap in both ms and nonms cases (fig. c inset, fig. e ,e , supplementary fig. b ,b ) non-cns disease (cancer, sepsis, myocardial infarction, hiv/aids, drug overdose (n = ), amyotrophic lateral sclerosis (n = ), stroke (n = ), multi-system atrophy (n = ), huntington's disease (n = ), parkinson's disease (n = ) edss . - . n/a supplementary fig a , a , respectively) with approximately % of each cell type associated with pgn immunopositive structures ( supplementary fig. c,d) . bacterial rna polymerase beta subunit immunoreactivity was also evident in both nonms (fig. d ) and ms (fig. d ) brains together with in situ hybridization detection of bacterial s rdna (insets in fig. d ,d ). to verify these findings, neuropathological features were scored by a neuropathologist (jql), unaware of the slide identity, in the same brain sections from ms and nonms which showed significantly increased cd ε and cd immunodetection with reduced lfb staining in ms sections compared to nonms brains. concurrent semi-quantitative scoring of pgn immunoreactivity within each entire tissue section did not differ between clinical groups (supplementary table ). these findings implied that in nonms and ms brain tissues, bacterial genomes and proteins were detectable, recapitulating and extending earlier studies of bacterial detection in human brains with and without neurological disease. despite obvious neuropathological differences, total pgn abundance per brain section was similar in ms and nonms brains suggesting that overall bacterial burden did not distinguish ms from nonms patients. the current study shows the presence of bacterial rna and dna sequences and proteins in human brain which are disrupted in conjunction with inflammatory demyelination in patients with ms. proteobacteria represented the chief bacterial phylum detected in human brain with restricted molecular diversity in ms brains despite the increased density of bacterial glycoproteins within demyelinating lesions. evidence for bacterial presence in human brains was verified by using several monoclonal antibodies that recognized specific bacterial proteins, in situ hybridization, pcr amplification and cloning of bacterial rna and dna sequences together with massively parallel sequencing of brain-derived rna. the presence and type of bacteria in brain was associated with host immune gene expression, which were apparent using different methods. these findings indicated a strong interaction between bacterial presence and host responses involving nfκ b-related signaling in demyelinating lesions, which is a pivotal pathway in neuroinflammation and ms pathogenesis . as contamination of tissue specimens and experimental tools was a paramount concern in the present study, extensive precautions were implemented to mitigate this concern, given the relative low levels of bacterial burden observed in the present studies. phylogenetic analyses of pcr-derived sequences displayed similarities to propionibacterium, a common cutaneous and nasopharyngeal bacterium in a single ms patient which might reflect contamination during the autopsy process or alternatively in vivo transport from the nasopharynx via the cribriform plate, as reported for other bacteria . however, the correlations between peptidoglycan abundance and host neuroimmune responses, using both nanostring and deep sequencing, argues against contamination, particularly the consistent preponderance of nfκ b-associated transcriptional observations (figs and , supplementary fig. a) . moreover, the presence of viruses in the brain inducing local immune responses was unlikely here because of the relative paucity of detectable viral genomes (supplementary table ). reduced molecular diversity among ms brain-derived s v -v rrna sequences as well as the increased density of pgn immunoreactivity associated with demyelination are also at odds with contamination as the sole explanation for the present observations. the present studies revealed the ratio of bacterium-encoded s rdna to rrna in matched brain samples to be ~ : in both white matter (and cortex, data not shown) with bacterial numbers of - genomes/cm suggesting both bacterial burden and replication were low compared to active pathogenic infections in other tissues. the low bacterial rdna:rrna ratio and burden are not surprising given the sensitivity of the brain to bacterial molecules (e.g., endotoxin), which at high levels can lead to adverse effects on brain tissue. the reduced molecular diversity of bacterial rna sequences in ms brains implies an overgrowth in select bacteria, perhaps proteobacteria, as indicated by the current sequencing data (fig. ) from progressive ms patients, which was diversified in relapsing-remitting ms patients receiving disease modifying therapies. these results recapitulate changes in microbial populations in other diseased organs and are reminiscent of a dysbiosis that can be resolved by increasing microbial diversity. the differential distribution of individual bacterial phyla consistently associated with clinical phenotype and immune activation, observed across multiple lots of reagents, collectively support the specificity of the current findings. the detection of bacterial dna and rna sequences, proteins and cell wall components in human brains raises the question of how bacteria might enter the brain. the bacterial sequences detected in the present studies of human brain resemble those of environmental (soil-derived) bacteria , largely without human disease associations. phagocytes including macrophages, neutrophils and dendritic cells can engulf live microbes or microbial compounds at different mucosal sites . polymicrobial species have been detected in human blood, particularly in leucocytes including neutrophils and macrophages . in rats with experimental autoimmune encephalomyelitis, leukocytes enter the brain following activation in the lung , suggesting that lung epithelium also provides an interface for microbial translocation. given that the present bacterial species' sequences are similar to environmental bacteria, inhalation and phagocytosis with ensuing trafficking to the brain is a plausible route of cns entry. the presence of bacteria or their components (e.g., pgn), even in a quiescent state and in low copy numbers in the brain, could exert effects on neurocognitive functions, inflammatory gene expression, and perhaps on neural cell (e.g., oligodendrocyte) survival. potential consequences of resident bacteria in brain include altered myelin viability and repair as well as activation of host inflammatory genes with pathogenic or protective effects . these effects could be consequences of colonization by viable bacteria or the presence of bacterial pamps from non-viable bacteria. our previous study in which we successfully transmitted human brain-derived bacteria to immunocompromised mice suggests that some of these organisms are viable as the persistence of residual rna is not sufficient to explain the finding given the duration of those studies. identifying the individual bacterial species and their impact on inflammatory demyelination might yield insights into ms prevention and treatment. indeed, recent studies of minocycline treatment of ms (clinicaltrials.gov number nct ) could also be interpreted within the context of the current study. similarly the putative neurotoxic effects of antibiotics might be related to their actions on microbiota located in the brain (or gut) with potential adverse consequences for gut-brain interactions , . further delineation of the individual bacterial species in brain and other organs that contribute to neurological disease (or health) are warranted because they might offer new therapeutic approaches or targets for inflammatory degenerative neurological diseases. ethics statement. the use of autopsied brain tissues with associated clinical data (age, sex, ms phenotype, edss, duration of disease) was approved by the university of alberta human research ethics board (biomedical, protocol number ). written informed consents were signed before or at the collection time. the protocols for obtaining post-mortem brain samples were performed in accordance with the canadian association of pathologists policy statement and guidelines for the ethical use of human tissue in research and all federal and institutional guidelines with special respect for the confidentiality of the donor's identity. all frozen tissues were stored at − °c at the time of autopsy. brain tissues were obtained from ms patients including relapsing-remitting (rr-ms) and progressive (primary and secondary) (p-ms) or disease controls (nonms) ( table ) - . specificity and control measures. in view of the study's overall aim, explicit attention was paid to issues of contamination and specificity. brain tissues from ms and nonms patients was obtained by aseptic collection at autopsy into sterile vessels with immediate flash freezing. patient brains were collected at separate sites in canada (winnipeg, calgary, edmonton and vancouver) reducing the probability of shared contaminating microorganisms. all post-collection tissue manipulation was performed in decontaminated biosafety hoods with autoclaved or chemically decontaminated tools and no sampling was performed from the exposed surface of tissues. all assays including rna and dna extractions contained water controls that were carried forward through all subsequent steps and each new step added an additional water control including cloning and sanger sequencing. all data in which any steps showed evidence of reagent contamination were excluded and the previous steps were performed with new lots of reagents. all amplification steps were set up in areas and with equipment treated with dnase and rnase inhibitor (molecular bioproducts, san diego ca, usa). all post-amplification steps were performed in a discrete space with separate equipment. immunohistochemistry, histochemistry and quantitation. formalin-fixed paraffin-embedded brain was processed and brain sections (two regions from ms case , , , - and two from nonms cases - ) ( µ m) were stained with luxol fast blue (lfb) to visualize myelin. in addition, serial brain sections were immuno-labelled with antibodies to bacterial and host proteins. immunocytochemistry was performed with two mouse anti-pgn antibodies (mab , generated against streptococcus mutans peptidoglycan, chemicon, temecula, ca; mab e , mouse igg , generated against a human gut pgn preparation at erasmus mc rotterdam, netherlands ), and mouse anti-rna polymerase beta subunit, mouse igg , generated against e. coli recombinant protein (neoclone, madison, wi), rabbit anti-glial fibrillary acidic protein (gfap) (dako, carpenteria ca) and rabbit anti-iba- (wako pure chemical industries ltd., osaka japan), microglia/macrophages were detected with a rabbit polyclonal anti-cd and t-cells by anti-cd ε which was quantified as previously reported together with appropriate secondary antibodies for single or double immunolabeling - . immunofluorescence. slides were deparaffinized by incubation for hour at degrees followed by one minute and five minute incubations in xylene baths through decreasing concentrations of ethanol to distilled water. antigen retrieval was performed by boiling in mm sodium citrate ( ph . ) hr. slides were blocked with hhfh buffer ( mm hepes buffer, % (v/v) horse serum, % (v/v) fbs, . % (w/v) sodium azide in hank's balanced salt solution (hbss)) for hours at room temperature. slides were incubated with a cocktail of rabbit anti-gfap or anti-iba- ( : ), mab against peptidoglycan ( : ) overnight at four degrees primary antibody was removed by three min pbs washes and slides were incubated for three minutes in . micron filtered % (w/v) sudan black in % ethanol and washed an additional times in pbs. a cocktail of : alexa goat anti rabbit igg, alexa goat anti mouse igg for two hours, washed three times in pbs stained with dapi scientific reports | : | doi: . /srep for minutes, washed times in pbs and mounted with prolong gold antifade reagent. slides were imaged with wave fx spinning disc confocal microscope (zeiss). total gfap and iba- positive cells were counted in ten fields per case at x and the number of cells with attached peptidoglycan immune positive particles and internalized particles were counted. semi-quantitative scoring of neuropathology. neuropathological scoring was performed as modified from past studies , . scoring of lfb ( . %) staining, cd (dako, carpenteria, ca), cd (dako, carpenteria, ca) and pgn (mab ) immunoreactivity in serial brain sections was performed as follows (modified from , ) with differences in staining or immunoreactivity defined relative to normal appearing white matter in terms of area and/or intensity ( x magnification). decreased density of lfb staining for myelin was scored in (original magnification × ) as follows: , normal to minimal decrease; , identifiable to % of decrease; , more than % of decrease to little preservation; , complete loss. decrease in lfb staining was defined by reduction of tissue reactivity for lfb, compared to normal appearing white matter (nawm), in terms of its size and/or intensity. the scores of every consecutive fields were summed, and then - (depending on the tissue section sizes) sets were averaged for analysis. cd + and cd ε + cells were assessed by scoring the frequency of positive cells in a × (original magnification) field: , none; , sparse; , scattered; , frequent. positive cells were identified as those with visible immunoreactivity within the cytoplasm. the scores of every consecutive fields were summed, and then summed sets were averaged for analysis. pgn immunostaining was scored based on the following criteria: , none; , weak and focal; , strong but focal ( % or less of the entire tissue section); , strong and diffuse (more than % of the entire tissue section), overall sparse to scattered; strong and diffuse (more than % of the entire tissue section), overall frequent. to quantify lfb and peptidoglycan immunodetection simultaneously images from lfb/anti-peptidoglycan double-labelled slides were divided into three fields. lfb staining was quantified using image j fiji and pgn immunolabelled particles (brown) in pixels or greater in size (magnification x ) and separated by two or more pixels of another color were counted as immunopositive signals in coincident fields. in situ hybridization. ish performed as previous . in brief, slides were de-paraffinized, rehydrated then treated with mg/ml hen egg lysozyme (sigma, oakville, on, canada) for minutes followed by treatment with µ g/ml proteinase k in buffer for minutes at °c then washing and dehydration. µ l of pre-warmed ng/µ l double dig labeled eub (gctgcctcccgtaggagt) probe targeting bacterial s rdna or scrambled probe in hybridization buffer ( mm tris-hcl, mm nacl, . % sds ph ) was applied to each sample and incubated at °c for minutes (sigma, oakville, on, canada) . slides were then washed in six rapid changes of °c wash buffer ( mm tris, ph . , mm edta). the samples were then blocked first for min. with levamisole, then for hr with odyssey blocking buffer (licor, lincoln, ne, usa). a ∶ dilution of ap (alkaline phosphatase) conjugated sheep anti-dig fab' fragments (roche, mannheim, germany) was applied to the slides and incubated o/n at °c. the slides were washed times in pbs and incubated for hours in the dark at °c with ap substrate (roche, mannheim, germany), then washed times in pbs, mounted and imaged. dna and rna preparation. total rna was extracted using the qiagen rneasy and dna was extracted using the qiagen dneasy blood and tissue kit from frozen normal appearing white matter in parallel with ultrapure rnase-and dnase-free water (life technologies, - ) to monitor for reagent contamination according to the manufacturer's protocol (qiagen, toronto, on) (see supplemental material and methods). a subset of rna (ms - , - nonms , , - ) selected based on maximum rna quality, was subjected to massively parallel sequencing as described previously . cdna synthesis and bacterial rna and dna quantitation. first strand cdna synthesis and rt-pcr was performed as previously described . semi-quantitative rt-pcr was performed for several host response genes and viruses using primers described in supplementary table . the quantity of bacterially-encoded s rrna, rdna and groel was determined using the primers described in supplementary table on samples from ms cases - and nonms cases - by comparison to standard curves generated from pgem-t clones of the e. coli s rdna and groel, respectively. the number of bacterial genomes was estimated based on the quantity of groel-encoding dna sequences ) detected per mass (g) of tissue from which the gdna was extracted, assuming a single groel copy per genome. s rrna clones were generated from ms cases - and nonms cases - using the pgem t-easy vector system (promega, madison, wi) and sequenced in both directions with t and m r primers using big dye terminator cycle sequencing kit (thermo fisher, foster city, ca) and analyzed . nanostring analyses. rna was extracted from serial µ m brain sections (per case) and analyzed with the ncounter human inflammation panel containing genes as per the manufacturer's instructions (nanostring technologies, seattle, wa). bioinformatics. gene ontology analysis was performed using david bioinformatic resources . functional annotation , (http://david.abcc.ncifcrf.gov/). gene network analysis of transcripts identified by nanostring shown to be correlated with pgn immunostaining was performed using the network tool in ingenuity pathway analysis (ingenuity systems, www.ingenuity.com). alignment of rnaseq-derived bacterial sequences was performed (novoalign) . statistics. univariate and correlation analyses were performed using spss using the student t and tukey-kramer tests ( -tailed) and spearman correlation. principal components analysis (pca) was performed to investigate multivariate correlations within the data including pgn immunoreactivity, nanostring, lfb staining within individual patient groups (r core team, , http://www.r-project.org). widespread colonization of the lung by tropheryma whipplei in hiv infection a comprehensive repertoire of prokaryotic species identified in human beings spatial variation in the healthy 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proteinase-activated receptor modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis neuroinflammation and endoplasmic reticulum stress are coregulated by crocin to prevent demyelination and neurodegeneration neuroinflammation and demyelination in multiple sclerosis after allogeneic hematopoietic stem cell transplantation the regulation of reactive changes around multiple sclerosis lesions by phosphorylated signal transducer and activator of transcription assessment, diagnosis, and treatment of hiv-associated neurocognitive disorder: a consensus report of the mind exchange program fiji: an open-source platform for biological-image analysis in situ identification of micro-organisms by whole cell hybridization with rrna-targeted nucleic acid probes intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases improved template representation in cpn polymerase chain reaction (pcr) product libraries generated from complex templates by application of a specific mixture of pcr primers systematic and integrative analysis of large gene lists using david bioinformatics resources bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists cp and ms hold canada research chairs (tier ) in neurological infection & immunity and microbiology, respectively. the authors thank drs. vijay ramaswamy, farshid noorbakhsh and jens walter for helpful discussions and grace boldireff for assistance with manuscript preparation. w.b. was involved in the design of the study, acquisition of data, analysis of data including univariate statistical analysis and drafting, revising the manuscript. j.q.l. was involved in analysis and acquisition of data. m.g.s. was involved in study design and analysis and interpretation of the data as well as revising the manuscript for content. r.a.h. was involved in the acquisition and analysis of data as well as providing access to the tools for rnaseq. j.l. performed and interpreted analysis of the data including multivariate statistical analysis of the data. j.d.l. was involved in study design and analysis and interpretation of the data as well as revising the manuscript for content. c.p. was involved in the design of the study, analysis of data including and drafting and revising the manuscript. accession codes: genbank accession numbers kx -kx . key: cord- -pbb lp authors: chen, chin-pei; lin, meng-han; chan, ya-ting; chen, li-chyong; ma, che; fischer, wolfgang b. title: membrane protein assembly: two cytoplasmic phosphorylated serine sites of vpu from hiv- affect oligomerization date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: pbb lp viral protein u (vpu) encoded by human immunodeficiency virus type (hiv- ) is a short integral membrane protein which is known to self-assemble within the lipid membrane and associate with host factors during the hiv- infectivity cycle. in this study, full-length vpu (m group) from clone nl - was over-expressed in human cells and purified in an oligomeric state. various single and double mutations were constructed on its phosphorylation sites to mimic different degrees of phosphorylation. size exclusion chromatography of wild-type vpu and mutants indicated that the smallest assembly unit of vpu was a dimer and over time vpu formed higher oligomers. the rate of oligomerization increased when (i) the degree of phosphorylation at serines and was decreased and (ii) when the ionic strength was increased indicating that the cytoplasmic domain of vpu affects oligomerization. coarse-grained molecular dynamic simulations with models of wild-type and mutant vpu in a hydrated lipid bilayer supported the experimental data in demonstrating that, in addition to a previously known role in downregulation of host factors, the phosphorylation sites of vpu also modulate oligomerization. the generation of functional forms of membrane proteins comprises several steps: membrane insertion during the translation process via the translocon complex or other systems , and the proper assembly of the proteins into a quaternary structure, if necessary. it has been asserted that after insertion into the membrane, proteins undergo structural arrangements in the monomeric form. in an analogy with single protein folding, during synthesis proteins are thought to rapidly achieve an intermediate state referred to as the 'molten globule' or 'compact intermediate' state . since hardly any information is available about this state, at this point, how the final assembly is formed can only be speculated. viral channel forming proteins (vcps) encoded by the virus are a special type of membrane protein which are a dependant of the larger ion channels of the host - but smaller in size. since vcps are also known to interact with host proteins and initiate ion channel-independent functions, it can be hypothesized that they also need to 'exist' as monomers. in this respect, vcps can be used to explore the dynamics and structural features of membrane protein assembly within the lipid membrane [ ] [ ] [ ] [ ] . vpu of hiv- is one of vcps with amino acids in length and contains a single helical transmembrane domain (tmd) , followed by a cytoplasmic domain consisting of another two helices and further residues towards the c terminal side [ ] [ ] [ ] [ ] . the ion channel activity of vpu has been shown to be attributed solely to the tmd . a recent review has discussed speculations about the, as yet, unclear ion channel function of vpu in vivo . in addition, vpu is phosphorylated at two serines at positions and which are responsible for initiating downregulation of membrane proteins of the host, including cd , bst- , or ntb-a . this function of initiation of host factor degradation is independent of the function of altering electrochemical gradients via the formation of an ion channel , . the oligomeric state of vpu has not been univocally established. while gel permeation chromatography suggests that a maximum of five proteins are assembled . computational models which were based on nmr spectroscopic data show structural features of a tetrameric or pentameric form of the tmd of vpu . at present, the known architecture of ion channels based on crystallographic data suggests that hydrophilic residues face the lumen of a putative ion conducting pore (see for example ). in the case of the pentameric ligand gated ion channel of gloebacter violaceus (glic), the serines and threonines of the pore-lining helices m of each of the five subunits points into the lumen forming a hydrophilic ring . it was also speculated that the only hydrophilic residue in the transmembrane domain of vpu, ser- , should point into an ion conducting pore . however, in these computational models ser- is located at the helix-lipid interface leaving the putative pore as a pure hydrophobic stretch, they contradict the current notion of the putative pore architecture. consequently, there is a need for further refinement of the model of the formation of ion-conducting pore by assembled vpu. in addition, vpu is known to act against host factors for down-regulation. vpu was proposed to exist in a stable equilibrium between oligomeric and monomeric forms, which are inactive and active, respectively, for interacting with host proteins . however, how vpu is assembled and how it eventually reaches a pore-like formation remains to be characterized. in this study, we investigated the oligomeric behavior of vpu expressed in human hek cells and purified into detergents micelles to retain its tertiary folding. wild-type (wt) vpu and mutations at the sites of the phosphorylated serines at positions and were investigated to assess the role of phosphorylation in the dynamics of assembly. coarse grained molecular dynamics (cgmd) simulations of vpu proteins embedded in a planar lipid bilayer model were chosen to evaluate the oligomeric assembly under likely in vivo conditions such as an abundance of vpu proteins in a large lipid patch and simulated over a long time period. in addition, cgmd simulations proposed mechanical features of how individual domains of vpu, both transmembrane and cytoplasmic, contribute to the assembly process. protein dimers and higher oligomers in detergent micelles. vpu-wt and mutant vpu proteins were expressed in hek cells (fig. ). sds-page analysis from cells expressing vpu-wt revealed four bands (fig. a, lane ) . the sds-page analysis of the double mutants vpu-dd and vpu-nn, which lack phosphate groups at the serines, showed only a single band each on the sds-page at various molecular weights due to the decreased migration rate of the negative charged vpu-dd upon denaturation (fig. a , lanes and ) , . vpu- d and vpu- d each show two bands ( fig. a lanes and ) . taken together, these results indicate that the four bands of vpu-wt represent the following from high to low molecular weight, (i) phosphorylation of both of the serines, ser- and ser- , (ii) single phosphorylated serines at position and (iii) position , and (iv) fully non-phosphorylated serines. the ratio between phosphorylated and non-phosphorylated vpu remains the same as found in measurements directly from cell pellets using anti-strep-tag antibody for vpu in western blot (data not shown). the thrombin enzyme cleaves the strep-his fusion tag from vpu-wt and the mutants ( fig. a lanes , , and and supplementary fig. s ). the pattern mentioned for uncleaved vpu does not seem to be affected by thrombin treatment. the fusion tag-free vpu was further purified by size-exclusion chromatography and eluted with four peaks (fig. b) . vpu-wt showed two peaks, a smaller peak at . ml representing large protein/detergent complexes (p in fig. b ) and a larger peak representing smaller protein/detergent complexes (p in fig. b ) at . ml. mutant vpu- d shows a similar pattern. for vpu- d and vpu-dd the peak of the large complexes was not resolved. vpu-nn showed the peak of the large complexes being larger than that of the smaller complexes. sds-page analysis identified the two peaks representing vpu protein and its respective mutations (fig. c) . the third and fourth peaks correspond to thrombin and strep-his fusion tags, respectively. multi-angle light scattering analysis identified that p and p correspond to molecular weights of . ± . kda and . ± . kda, respectively ( table ). the respective averaged oligomeric state was calculated to be around . ± . for p and . ± . for p . thus, vpu is able to exist in two oligomeric states, which are most likely a dimer and higher oligomer. modulation of the dynamics of vpu-wt, vpu-nn and vpu-dd oligomerization by the two phosphorylation sites. after purification of the proteins from a stock solution, the peak ratio between the higher oligomer (referring to p ) and the dimer (referring to p ) for vpu-wt and vpu-dd was in favor of the dimer for all ionic strengths investigated, , and mm nacl (fig. a) . the peak of the higher oligomer is the largest at the highest ionic strength of mm nacl for the two proteins. the peak area of the higher oligomer was largest for vpu-nn at all ionic strengths (fig. b) . higher ionic strength screens the negative charges at the serine sites and even the partial charges of the amide group in asparagine indicating electrostatic type interaction modulates assembly. immediately after purification of vpu-wt from a stock solution, the peak of the dimer was larger than that of the higher oligomer (fig. a, top graph) . repeating the purification from the stock solution over a period of days revealed a gradual increase in the higher oligomer. a slower increase in the peak of the higher oligomer was observed for vpu-dd. purification after days showed just the beginning of a small peak for the dimer (fig. a , middle graph, green line). in the case of vpu-nn, the peak of the higher oligomer was larger than that of the dimer from the first day of the experiment and increased even more over three days, finally reaching a plateau over a longer period (fig. a , lower graph). the dynamics data was plotted as area of the peak of the higher oligomer (a p ), divided by the total area of a p and the peak area of the dimer (a p ), a p /(a p + a p ), over time for vpu-wt with a double logarithmic growth curve (fig. b , black and table ). vpu-dd (fig. b , red) and vpu-nn (fig. b , blue) can both be fitted with a single function (see also table ). vpu-nn (c = . day − ) and vpu-dd (c = . day − ) mark a fast and slow increase in the area of the higher oligomer, respectively. vpu-wt exhibited a fast increase (c = . day − ) first, followed by a slow increase (c = . day − ), similar to the afore-mentioned growth rates of vpu-nn and vpu-dd, respectively. as a result, vpu-dd, due to their negative charges at the two serine sites tended to assemble very slowly reaching a final assembly ratio of a = . , whilst vpu-nn, having charges removed at the site of the two serines, assembled very quickly reaching the largest assembly ratio of a = . . therefore, the fast increase in the peak of the higher oligomer of vpu-wt should be due to the assembly of non-phosphorylated vpu, whilst the slower increase of that peak should be due to the assembly of both, single and double phosphorylated vpu proteins. the single phosphorylated vpu proteins obscure the plot in as much they show 'mixed' assembly dynamics. the negative charges of the phosphorylated serine site slowdown or even prevent oligomerization. vpu dd in hydrated lipid bilayers. the computational model of vpu was generated by bending a helical motif of vpu - at the site of the eyr motif as reported previously (fig. a, left) . sequence alignment shows that the strains used for building the computational models and the one used in the experimental study share % sequence identity (data not shown). two copies of the kinked vpu - are run in a single lipid patch in an inverted orientation for ns (fig. s ). both of the helices remain in both of the structures. both of the structures (fig. s , black and red curves) show larger root mean square fluctuation (rmsf) values for residues glu- to ile- . the structure shown by the red lines in fig. s , named vpu - , was chosen for the next step, since the residues leu- to arg- of its second membrane-associated helix show lower rmsf values than those of the structure represented by the black curve. residues ile- to ala- of the second, membrane-associated helix of vpu - are overlapped with the n terminal side of the nmr-based structure of vpu to finally generate full-length vpu - with united atoms. md simulation of two copies of vpu - showed a leveling off of the root mean square deviation (rmsd) values after about ns (fig. s , upper left). one of the vpu - structures showed large fluctuations of the amino acids in the kink region (ile- to gln- , fig. s , upper right, red curve). these residues define the intermediate parts between the helices. based on these values and the leveling of the rmsd values this structure was considered further for cgmd simulations as vpu-wt (fig. a, left and fig. s ). in vpu-wt the serines are not phosphorylated. at this stage the cg mutant model vpu-dd was generated by replacing the two serines with two aspartic acids. a total of vpu-wt and vpu-dd are embedded in a hydrated lipid bilayer ( ns, fig. a , right) and simulated for μ s (fig. b) . the vpu-wt started to assemble into two large units consisting of and proteins (fig. b, left) . vpu-dd at the end of the simulation shows three units of , and proteins (fig. b, right) . after about μ s, vpu-wt reached an oligomerization ratio of nearly (a = . , table ), compared to vpu-dd which reached a value of about a = . (fig. a and table ). the oligomerization ratio of vpu-wt was due to large values of both tmd assembly (a = . ) and the cytoplasmic domain (a = . ) (fig. b) . for vpu-dd as well, the tmd assembly contributed the most (a = . ) to the overall oligomerization compared to the cytoplasmic domain (a < . ) (fig. c) . analysis of the growth curve showed that the growth rates c of the tmds are almost independent of the phosphorylation state. the higher growth rate of the cytoplasmic domains of vpu-dd compared to the rate of vpu-wt is due to an almost sudden assembly of a few proteins ( table ). in this state the growth rates were not compared with those of the experiments due to the different time scales. long lasting dimers of both vpu-wt and vpu-dd form close contact areas within the tmd along the line of valines (residues to ) of one monomer with the leucines and isoleucines of the other monomer . pore like structures with eventually serines (ser- of the tmd) pointing towards the center of a putative pore have not been observed. a striking feature is that assembly of vpu-wt is driven by an early assembly of the cytoplasmic domain within . μ s to an oligomerization ratio of ∼ . followed by an increasing rate of assembly due to the tmd within the table . fitting parameters using a logistic growth function ( = + ⋅ − y a b e ( ) cx ) where y is the rate over time, here x. the parameters are a = maximum oligomerization ratio, b = initial value at time t = , and c = growth rate (time − ). two terms of the logistic growth function are combined additively to fit the experimental data of vpu-wt. first micro second of up to ∼ . (fig. b) . for vpu-dd the sequence is reversed by assembly via tmds of up to ∼ . oligomerization followed by cytoplasmic assembly which remains a ratio of ∼ . (fig. c) . the oligomerization ratio of mixtures of vpu-wt and vpu-dd ( vpu-wt and vpu-dd, vpu-wt and vpu-dd as well as vpu-wt and vpu-dd) achieve maximum level at a later time step as for the 'pure' systems ( fig. d) . deriving the growth rate, c, from a fitting of the curves with a double logistic growth function indicates that in all the mixtures the first rate is faster than the second rate except for the mixture of vpu-wt and vpu-dd (supplementary table ). oligomerization of the tmds does not follow this trend due to internal reorientations within the patches (supplementary table and supplementary fig. s ) . rate of oligomerizaion is driven by the assembly of the tmd of vpu independent of negative charges due to phosphorylation of the two serines and , while maximum degree of oligomerization depends on the negative charges. in this study, full-length vpu from clone nl - was overexpressed in hek cells using an experimental protocol in which the protein never leaves the lipid or lipid-like environment. the precise oligomeric state of vpu has not yet been established. in many studies, synthetic peptides corresponding to the transmembrane domain of vpu or vpu expressed in escherichia coli and purified have been used (supplementary table s ). in these studies using electrophoresis, it was proposed that the tm of vpu exists in the tetramer to hexamer range. size-exclusion chromatography of full-length vpu by coupled transcription/translation systems suggests a pentameric structure . in the e. coli system, all forms of vpu, either the tm-containing segment or full-length vpu, were over-expressed into inclusion bodies and extracted into denaturating buffer. in a subsequent step the vpu protein is then refolded. in this study, full-length vpu is expressed in human cells and extracted into ldao micelles from cell membranes. ldao is a gentle and commonly-used detergent for membrane protein structure determination , . application of other detergents like cymal ( -cyclohexyl- -pentyl-ß-d-maltoside), also showed a similar pattern of two peaks in the size-exclusion chromatogram (data not shown). the expression system and purification steps in this protocol most likely keep the structure in the native states as much as was possible. in addition, sec-mals which measures molar mass directly is used as a tool to obtain the oligomeric states of vpu without relying on reference standards which are usually needed in conventional size-exclusion chromatography. from our results, the smallest oligomeric state of vpu and vpu with phosphate group is a dimer. experimental evidence about this has not been reported in previous studies. the dimer is assembling into larger assemblies of up to approximately proteins. mutating vpu-wt into vpu-nn is chosen as a way to remove phosphorylation sites in this protein since this technique is anticipated to maintain the overall structure of the protein - . comparison of computational and experimental data. the vpu model in respect to its cytoplasmic domain relies on nmr spectroscopic investigations in which the peptide is non-phosphorylated . the structural feature is of two helices connected by a loop, which harbors the two serine sites and . another study in which a much shorter peptide, vpu - , is used indicates that a short helical part towards the c terminal side disappears upon phosphorylation but the overall shape of a loop conformation remains , . thus, the cg models vpu-wt and vpu-dd reflect reliable structural features. the computational system is designed to represent an estimate of the in vivo system. the proteins are embedded within a planar lipid bilayer of a single type of lipid molecule. thus, the question of whether the vpu proteins would oligomerize in the same way and with the same dynamics when embedded in a lipid bilayer can be addressed. in this study, the computational models exhibit the same behavior as found experimentally. the dimer is smallest unit to assemble. the level and growth rate of oligomerization of vpu without the phosphate groups is bigger and faster than vpu with phosphate groups. in addition, structural features taken from the simulation data allowed specification of the interaction dependent on the cytoplasmic domain and tmd with the latter contributing mostly to the oligomerization ratio. the coarse-graining investigations of conformational dynamics are limited to emphasizing the diffusive aspects of the protein in the bilayer. the computational data represent a semi-quantitative analysis of protein diffusivity which matches the experimental findings. the number of vpu was chosen to be instead of the putative vpu molecules calculated from the experimental analysis. this is done due to the need to use a squared lipid patch with regularly positioned molecules of vpu to build the larger patch. in this paper the dynamics of oligomerization of the mer is segregated into contributions of the transmembrane and the cytoplasmic domain in a quantitative way to parallel the experimental data set in respect of growth rate and maximum oligomerization ratio. in an earlier computational study structural features of the assembly of two vpu proteins either as vpu-wt and vpu-dd are reported . the sequence of occurrence of individual oligomers of vpu-wt and vpu-dd during the simulation of lipid patches with up to mers and mers is explored on a qualitative level. the sequence of protein assembly. the computational models were built alongside a biological pathway . it is assumed that there is an equilibration of the monomeric unit of the membrane protein first, due to the distance between ribosomes (e.g., Å apart from each other) . the structure of the protein obtained in this state can be considered to be a 'molten globule' or 'compact intermediate' , an intermediate state before the formation of a fully functional channel . in a subsequent step larger assemblies are formed. a general feature is that the assembly of the host channels is in the minute to hour range . considerable time is dedicated to the folding of the subunits, a feature that is not explicitly considered in this study in as much cgmd simulation restrains the structure in its internal dynamics. the experimental part of this study verifies a "dimer" first step of oligomerization of vpu as simulated in an earlier study . this formation of a dimer is driven by the association of the tmds as indicated from computer simulations. in the dimer the two phosphorylation sites are the furthest apart due to electrostatic charge repulsion. during assembly into larger units the exposed negative charges of the phosphate groups have to be taken care of. whilst the cytoplasmic domain directs oligomerization, the tmds are responsible for holding the oligomer together. based on this study, how vpu is assembled and how it eventually reaches a pore-like structure is shown in the schema in fig. . some of the individual monomers assemble into dimers via association of tmds (fig. a,b) . within a larger assembly or patch (fig. c) , dimers and additional monomers are able to adopt conformations, which can either be channel-like (as marked by the red circles) or not channel-like (as marked by the dashed grey circles). conformational changes of the proteins will allow e.g., to the transformation of vpu proteins from the not channel-like region into the channel-like regions. it is always possible that the assemblies can be made out of the dimers or a mixture of both dimers and monomers. the generation of protein patches for more than - proteins may be restricted due to thermodynamic considerations taking into account protein binding affinities and protein dynamics due to the membrane environment. the phosphorylation sites are necessary for the role of vpu in initiating the ubiquinone-dependent downregulation of the proteins to which it attaches. according to this study, those sites seem to have another role in the regulation of the assembly of vpu itself. whether the interaction of vpu with host factors occurs with vpu as a monomeric or dimeric unit still needs to be investigated. it is also possible that vpu interacts with host proteins in its patch-like assembly. dimerization is generally an essential first step in the oligomerization of membrane proteins. specific sites within the protein, such as the two phosphorylation sites in the cytoplasmic domain of vpu, play a modulating role during the initial step of assembly whilst the tmd defines the stability of the oligomer. in the special case of vpu, the phosphorylated serines have an additional function. besides functioning in the initiation of the downregulation of an attached host protein it also regulates the oligomeric state of vpu. plasmids, cells and transfection. human codon optimized vpu genes derived from hiv- strain nl - (p : mqpiqiaiaa lvvaiiiaiv vwsiviieyr kilrqrkidr lidrlierae dsgnesegei salvemgvem ghhapwdidd l) were synthesized by multiple overlapping polymerase chain reaction (pcr) and cloned into the expression vector ptt-strep-his harboring a thrombin cleavage site for removing the tags. the cytoplasm domain mutants of single mutated vpu, vpu-s d and -s d, as well as double mutated vpu, vpu-s / d and vpu-s / n were generated by quick-change site-directed mutagenesis and overlapping pcr respectively, by standard methods using the phusion-ii polymerase (new england biolabs). for the single mutants, the second serine site is still available for phosphorylation during protein expression. vpu with the mutations was also expressed using the vector ptt-strep-his . all constructs were verified by sequencing analysis. the helix was bent around residues glu- to ile- so that the helical stretch from residues leu- to ser- aligned with the membrane surface as described earlier . asp- was pointing towards the bilayer surface and arg- was pointing into the aqueous phase, according to experimental findings supplementary fig. s ) was chosen to generate full-length vpu - . the first structure out of the structures of the models deposited in the pdb data bank (pdb id: k y, hv h , p ; residues to ) , gsidr to arg- , which adopt a helical motif, were merged with the helical motif of residues ile- to arg- of vpu - on the level of the cα atoms to generate full-length vpu, vpu - henceforth referred to as vpu-wt. . dsgnesegdq eelsalverg hlapwdvddl two of these structures ( atoms including united atoms) were embedded into a fully hydrated lipid bilayer as mentioned above. the last frame of the ns md simulation of vpu - (see red curves for root mean square deviation (rmsd) and root mean square fluctuation (rmsf) in supplementary fig. s ) was chosen to generate a coarse grained (cg) model vpu-wt . a computational model of mutant vpu-dd, was generated at the full-length structure prior to start the cgmd simulations by replacing ser- / . classical md simulations. md simulations on the systems reported in the present study were carried out with gromacs . . using gromos (ffg a ) force field with an integration step size of fs. the temperature of the protein, lipid, and the water molecules were separately coupled to a berendsen thermostat at k with a coupling time of . ps. a semi isotropic pressure coupling was applied with a coupling time of . ps and a compressibility of . e − bar − . long-range electrostatics were calculated using the particle-mesh ewald (pme) algorithm with grid dimensions of . nm and interpolation order . lennard-jones and short-range coulomb interactions were cut off at . and nm, respectively. vpu - and vpu - proteins were put on either side of the lipid bilayer consisting of / lipids ( / atoms) and hydrated with / water molecules ( / atoms) ( supplementary figs s and s ). lipids which overlapped with the peptide were removed. the system was then minimized ( steps of steepest descent and steps of conjugate gradient) and equilibrated for a total of . ns. equilibration was achieved by gradually increasing the temperature from k to k and then to k, whilst keeping the peptide fully restrained (k = kj mol − nm − ). the first two simulations (at k and k) were run for ps, the last simulation (at k) was run for . ns. it was verified that the space between helix and lipid membrane did not contain any water molecules, since the hydrophobic residues were pointing toward to the lipids. holding the system at k, the restraints, imposed by a force constant k on the peptide, were released in two steps (k = kj mol − nm − , k = kj mol − nm − ), running each of the steps for ps. the unconstrained systems were submitted to production runs of ns. the last frame of the ns md simulation of vpu - was used to replace the serines at site and into aspartic acid (vpu - -dd). coarse-grained md simulations. coarse-grained molecular dynamics (cgmd) simulations using the gromacs software were performed using the martini force field v . for water and v . for protein , . the martini script was used to convert vpu - and vpu - -dd into coarse-grained structural models. a default elastic network was used . the integration time step was Δ t = fs and periodic boundary conditions were applied. the non-bonded interaction had a cut off distance of . nm. the temperature of the protein, lipid, and the water molecules were separately coupled to a berendsen thermostat at k with a coupling time of . ps. a semi-isotropic pressure coupling was applied with a coupling time of . ps and a compressibility e- bar - . for the lipid bilayer, a pre-equilibrated lipid popc membrane hydrated by water molecules was used as a starting point. sixteen full-length vpu and vpu-dd mutants were embedded in a popc membrane ( supplementary fig. s ) with a protein: lipid ratio of : . na-ions were added to neutralize the system. the systems consisted of and beats for the vpu-wt and vpu-dd system, respectively. the simulations with mixtures of vpu-wt and vpu-dd were generated by replacing vpu proteins in a row by the other type of protein. the simulation systems were then neutralized with the respective number of na-ions. all the system were energy minimized ( steps of steepest decent) and equilibration with protein restrain (k = kj mol − nm − ) for a total of . ns. the unrestrained systems were submitted to production runs of μ s. oligomerization analysis. the oligomerization rate of the computational data was calculated with the concentration of trimer or higher oligomers divided by the concentration of total oligomer, in order to quantify the oligomerization level the maximum value was and the minimum was . the respective curves were fitted with a logarithmic growth function. cx with a = maximum oligomerization ratio, b = initial value at time t = , and c = growth rate (time − ). non-linear regression was performed by using non-linear curve fitting of originlab . . the initial values were set to a = , b = , and c = . . iteration was conducted until the difference between reduced χ values of two successive iterations was less than a specified tolerance value, here − by default. fitting the experimental vpu-wt data two logistic growth functions were combined additively. structures are considered as oligomers when the distance between pairs of cg-atoms of different vpu structures was below Å and observed continuously for more than times steps between the proteins. memrbane-protein integration and the role of the translocon channel structural basis of sec-independent membrane protein insertion by yidc ion-channel assembly viral channel forming proteins viral proteins function as ion channels mechanism of function of viral channel proteins and implications for drug development viroporins: structure and biological functions the minimalist architectures of viroporins and their therapeutic implications expression, purification, and activities of full-length and truncated versions of the integral membrane protein vpu from hiv- three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (vpu) from hiv- assembly of viral membrane proteins in silico investigations of possible routes of assembly of orf a from sars-cov correlation of the structural and functional domains in the membrane protein vpu from hiv- secondary structure and tertiary fold of the human immunodeficiency virus protein u (vpu) cytoplasmatic domain in solution solution structure and orientation of the transmembrane anchor domain of the hiv- -encoded virus protein u by high resolution and solid-state nmr spectroscopy nmr structural characterization of hiv- virus protein u cytoplasmic domain in the presence of dodecylphosphatidylcholine micelles the two biological activities of human immunodeficiency virus type vpu protein involve two separable structural domains hiv- vpu -an ion channel in search for a job putative a-helical structures in the human immunodeficiency virus type vpu protein and cd are involved in binding and degradation of the cd molecule tetherin inhibits retrovirus release and is antagonized by hiv- vpu the interferon-induced protein bst- restricts hiv- release and is downregulated from the cell surface by the viral vpu protein degranulation of natural killer cells following interaction with hiv- -infected cells is hindered by downmodulation of ntb-a by vpu ion channel activity of hiv- vpu is dispensable for counteraction of cd oligomerization of the human immunodeficiency virus type i (hiv- ) vpu protein -a genetic, biochemical and biophysical analysis structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel vpu from hiv- on an atomic scale: experiments and computer simulations assembling vrial channel forming proteins: vpu from hiv- detergent binding explains anomalous sds-page migration of membrane proteins host lipid and temperature as important screening variables for crystallizing integral membrane proteins in lipidic mesophases molecular dynamics simulations on the first two helices of vpu from hiv- patch formation of a viral channel forming protein within a lipid membrane -vpu of hiv- structure and dynamics of the hiv- vpu transmembrane domain revealed by solid-state nmr with magic-angle spinning membrane proteins, lipids and detergents: not just a soap opera crystal structure of the membrane-bound bifunctional transglycosylase pbp b from escherichia coli the human immunodeficiency virus type encoded vpu protein is phosphorylated by casein kinase- (ck- ) at positions ser and ser within a predicted a-helix-turn-a-helix-motif phosphorylation of both phosphoacceptor sites in the hiv- vpu cytoplasmic domain is essential for vpumediated er degradation of cd multilayered mechanism of cd downregulation by hiv- vpu involving distinct er retention and erad targeting steps deubiquitinases sharpen substrate discrimintation during membrane protein degradation fom the er hiv- encoded virus protein u (vpu) solution structure of the - hydrophilic region containing the phosphorylated sites ser and ser nmr studies of the phosphorylation motif of the hiv- protein vpu bound to the f-box protein beta-trcp protein folding in the cell glycans on influenza hemagglutinin affect receptor binding and immune response membrane interactions and alignment of structures within the hiv- vpu cytoplasmic domain: effect of phosphorylation of serines and the martini force field: coarse grained model for biomolecular simulations the martini coarse-grained force field: extension to proteins combining an elastic network with a coarse-grained molecular force field: structure, dynamics, and intermolecular recognition wbf. thanks the national science council (nsc- - -m- - -my ), taiwan, for financial support. cm thanks academia sinica, taiwan, for financial support. key: cord- -vi rth o authors: zhang, chao; yao, yao; zhu, juan-li; zhang, si-nong; zhang, shan-shan; wei, hua; hui, wen-li; cui, ya-li title: establishment and application of a real-time loop-mediated isothermal amplification system for the detection of cyp c polymorphisms date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: vi rth o single-nucleotide polymorphisms (snps) represent the most widespread type of genetic variation (approximately %) in the human genome, and the demand to overcome such variation has received more attention now than ever before. the capacity to rapidly assess snps that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. in this work, a rapid one-step snp detection method, real-time loop-mediated isothermal amplification (rt-lamp), was first applied for cyp c polymorphisms testing. the optimized method was established with specifically designed primers for target amplification by real-time detection in approximately min under isothermal conditions. rt-lamp amplified few copies of template to produce significant amounts of product and quantitatively detected human dna with compatible specificity and sensitivity. the success in the establishment of this rt-lamp protocol for cyp c polymorphism testing is significant for the extension of this technique for the detection of other snps, which will further facilitate the development of personalized medicine. clinical observations beginning in the s have suggested that individuals exhibit differences in their responses to drugs and that these variations could be inherited . the detection of dna sequence variations provides valuable insight into the diagnosis of genetic-related diseases and conditions, especially for early-stage treatment and response monitoring . thus, it is critically important to select a method with high sensitivity and specificity to detect single or small numbers of nucleotide polymorphisms , . current detection methods rely on sample amplification combined with meticulous control, including polymerase chain reaction (pcr), nucleic acid sequence-based amplification (nasba) , self-sustained sequence replication ( sr) , strand displacement amplification (sda) and direct sequencing. although these technologies are currently considered the gold standard for laboratory-based dna detection and diagnostics, these methods cannot meet the requirements of point-of-care testing (poct) strategies due to the high set up and operating expenses and the requirements for high-precision equipment. loop-mediated isothermal amplification (lamp) is an outstanding gene amplification procedure with high specificity, sensitivity and rapidity that was established by notomi et al. . the process amplifies nucleic acids under isothermal conditions and employs self-recurring strand-displacement synthesis primed by a specially designed set of target-specific primers , thus clearly distinguishing this technique from existing genetic tests . lamp is characterized by the use of six specifically designed primer regions to recognize eight regions on the target dna; thus, the specificity is extremely high . amplification and detection of a gene can be completed in a one step by incubating the mixture of sample, primers, dna polymerase with strand displacement activity and substrates under isothermal conditions between and °c. lamp has been used for the diagnosis of pathogens via the detection of gene segments, e.g., the diagnosis of infectious diseases, such as japanese encephalitis virus infection , rapid genotyping of carcinogenic human papillomavirus and herpesvirus , and the detection of middle east respiratory syndrome coronavirus [ ] [ ] [ ] . numerous investigations have demonstrated that this special identification system is more accurate than pcr-based methods, which use only two primers to recognize two regions. although detection of human dna polymorphisms using lamp is challenging, especially for single-nucleotide polymorphisms (snps) due to the complex nature of dna compared with microbes and viruses , snps represent the most widespread type of genetic variation (approximately %) in the human genome , and the capacity to rapidly test patients for snps that are correlated with disease predisposition, drug metabolism and disease development is a key step for the development of personalized medicine . thus, the wide application of this simple, rapid and low-cost genotyping lamp method in snp detection is imperative. numerous lines of evidence have strongly suggested that genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors and other drug targets are associated with inter-individual differences in drug treatment response . sequence variations in drug target proteins, drug-metabolizing enzymes, and drug transporters can alter drug efficacy, drug side effects, or both to cause variable drug responses in individual patients . for example, on march , , the us food and drug administration approved a black box warning regarding the diminished effectiveness of clopidogrel in patients who carry two loss-of-function alleles (poor metabolizers) , i.e., cyp c * (g a) and cyp c * (g a) alleles, which account for % and % of the nonfunctional alleles in whites and asians, respectively , . the warning addressed the need for polymorphism genotyping to identify altered clopidogrel metabolism in patients . we developed a rapid, one-step snp detection method (rt-lamp) that enables the detection of the cyp c allele in approximately min under isothermal conditions. the optimized rt-lamp technique is more suitable for point-of-care testing and will further facilitate on-site screening. the successful establishment of an inexpensive, rapid and real-time lamp protocol for cyp c * and cyp c * detection is significant for the extension of this technique for genotyping other snps. our results suggest applications for this rt-lamp assay system for both basic research and clinical diagnosis in pharmacogenomics. plasmid construction and identification. in this study, four plasmids were constructed by recombining the specific sequences of cyp c * g g, cyp c * a a, cyp c * g g and cyp c * a a. using the primer pairs * -seq-f/* -seq-r and * -seq-f/* -seq-r, listed in table , -bp cyp c * and bp cyp c * fragments were amplified and sequenced by beijing genomics institute (bgi; beijing, china), indicating the successful incorporation of the four plasmids (data not shown). based on the point mutations of the cyp c * (g a) and cyp c * (g a) genes, two sets of rt-lamp primers were designed to discriminate the snps. as shown in fig. , the basic principle of rt-lamp involves the use of specific primers, with a forward inner primer (fip) and backward inner primer (bip) that are designed to contain a snp nucleotide at the ′ terminus, each reaction including two common primers (f and b ) and two specific primers (fip-g and bip-g for g allele and fip-a and bip-a for a allele). the structures of the lamp primers and products based on this study are presented in fig. , and the information regarding the primer names and sequences are provided in table . the target snp was characterized using six different regions (f /f c-f /f c and b /b c-b /b c) specifically designed to recognize distinct regions on the target gene, which were designed to ensure that the primers would specifically amplify the g a and g a substitutions. the results depicted in fig. illustrate that the rt-lamp method could accurately detect and discriminate all possible homozygotes and heterozygotes of cyp c * (g a) and cyp c * (g a) snps. sensitivity of the rt-lamp assay. the sensitivities of the rt-lamp assay were tested using -fold serial dilutions of the four constructed plasmids. the detection limit of the rt-lamp assay was × copies of plasmid (the result for the cyp c g g plasmid is presented in fig. a ; the results for the other three plasmids are presented in supplementary fig. s ), indicating that the rt-lamp method was efficient and specific in snp detection under a constant temperature with greater than -fold increased sensitivity compared with conventional pcr , . moreover, as shown in fig. , a standard curve was generated using -fold dilutions of plasmids and calculated by regression analysis comparing the t peek with the copy number. the high correlation coefficient (r = . ) indicated that the rt-lamp assay could be applied in dna quantification. to test the reliability of the rt-lamp system optimized in this study, the accuracy of the rt-lamp assay was further verified using clinical samples. in addition, all of these samples were also assessed via conventional pcr (as-pcr) and sequenced by bgi. the detected genotypes together with their frequencies are presented in table . the observed allele frequencies were . %, . %, % and %, for * g, * a, * g and * a, respectively (calculated from: * g: f + f + f / + f + f / ; * a: f + f / + f / ; * g: f + f + f + f / + f / ; * a: f + f / + f / ). the frequencies of the cyp c * and cyp c * alleles were similar to those reported by chen et al. in a chinese population. the comparison of rt-lamp with as-pcr and direct sequencing revealed no discrepancies. in recent years, dna testing technology has been extensively used in the areas of diagnosis and disease detection. the lamp technique is a unique assay with high efficiency and high accuracy that was developed rapidly. as described in some reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the lamp assay has been widely used in the detection of pathogenic microorganisms. however, to the best of our knowledge, reports regarding the detection of mutations in human genomic dna, especially snps, using the lamp method are lacking. in this study, the successful establishment of an inexpensive, rapid and real-time lamp protocol for cyp c snp genotyping expanded the scope of application of this technique to human gene mutation detection. rt-lamp is a one-step method wherein the amplification itself is the signal for snp detection, whereas the difficulty in developing this technology involves the suppression of non-specific amplification . to help overcome this difficulty, the target snp is characterized using six different primer regions specifically designed to recognize eight distinct regions on the target gene , . in this work, by adding or subtracting a few nucleotides in the primer regions, the t m value and gc content were calculated until the six different primer regions were suitable for the lamp reaction. as a result, the primer regions were selected as noted in fig. (f -f and b -b ) . furthermore, as the arrangement and composition of human genomic dna is very complex, sequence alignment was necessary to avoid false recognition of the specific site. thus, primer-blast software (http://www.ncbi.nlm. nih.gov/tools/primer-blast) was used to ensure that the chosen primer regions were specific to the target snp, which helped avoid mismatches and locate the target snp as accurately as possible. the secondary structures of these primers may cause non-specific results in the lamp reaction given that the selectable sequence area for primer design is limited to less than four hundred nucleotides surrounding the target snp site . hence, the inner primers were designed (fig. ) to minimize the impact of the secondary structure given that the inner primers are the main component for dna strand extension. moreover, according to tomita et al., the formation of a starting structure is the key initiating step of lamp . specific nucleotides were added to the ′ termini ( ′ -term) of the fip and bip primers to establish a complete and effective starting structure, as shown in fig. . consequently the starting structure would be successfully established only when the ′ -term nucleotide was exactly matched with the target snp (fig. a) . otherwise, the starting structure was blocked, as shown in fig. c , and the amplification could not proceed. in conclusion, non-specific amplification was effectively suppressed in this work through special primer design and accurate target location. in addition, using real-time fluorescence detection equipment, amplification and detection can be performed in a closed tube, which could reduce the risk of contamination. thus, the rt-lamp method that was developed has an excellent sensitivity for detection of cyp c polymorphisms (as shown in fig. a ). similar results were observed by singh et al. and lee et al. with the same sensitivity of × copies. moreover, a standard curve with a high correlation coefficient was obtained in this study, as shown in fig. b , indicating that except for microorganism quantification [ ] [ ] [ ] , the rt-lamp assay can also be applied in human dna quantification. therefore, the rt-lamp method can be used for the determination of trace amounts of dna of interest among copious background dna, such as specific mutation detection in circulating tumour dna , , and can be applied for complex gene quantification, which is clinically meaningful , . in summary, as a rapid, feasible and cost-efficient point-of-care (poc) snp detection method, we demonstrated that rt-lamp could quantitatively detect human genomic dna with high specificity and sensitivity in a single step. moreover, the lamp method can amplify few copies of template to significant levels in min and can be used for both dna and rna targets . thus, this poc detection method should be helpful in basic research in a variety of fields, including medicine, pharmaceuticals, environmental hygiene, food security, and pharmacogenomics testing. peripheral blood and genomic dna extraction. peripheral blood samples were collected from unrelated chinese volunteers using edta-coated tubes at the shaanxi provincial people's hospital (xi'an, china) with informed consent. the study was approved by the ethics committee of the national engineering research center for miniaturized detection systems, xi'an, china. all methods were performed in accordance with these approved guidelines. the genomic dna from the volunteer was isolated from μ l of blood using a whole blood genomic dna isolation kit (xi'an goldmag nanobiotech co., ltd., xi'an, shaanxi, china), according to the manufacturer's instructions. the final dna quality and concentrations were measured using a nanodrop c/ uv-vis spectrophotometer (thermo fisher scientific, wilmington, de, usa), according to the manufacturer's instructions. primer design and synthesis. for each snp, six primers for rt-lamp were designed, including two outer primers (common primers, f and b ) and four inner primers (specific primers, fip-g, fip-a, bip-g and bip-a) that recognize distinct regions of the cyp c * and cyp c * alleles (rs and rs ). conventional pcr primers were designed based on the principle of as-pcr using the primer . software program (primer-e ltd., plymouth, uk). all oligonucleotide primers were synthesized by invitrogen biotechnology ltd. (shanghai, china). plasmids for cyp c * and cyp c * . plasmids - gg, - aa, - gg and - aa, which contain the cyp c * g g, cyp c * a a, cyp c * g g, and cyp c * a a genes, respectively, were constructed using the pmd tm optimization of rt-lamp reaction. the initial condition of the rt-lamp reaction was adopted from zhang et al. . the lamp reaction mixtures were incubated for min at , , , , , or °c to determine rt-lamp conventional pcr (n = ) total sequencing (n = ) total agreement (%) frequency (%) * /* * /* * /* * /* * /* * /* * /* * /* * /* * /* * /* * /* table . gene test results and frequency of clinical samples of cyp c alleles (type-specific concordance among rt-lamp, conventional pcr and direct sequencing). * /* : cyp c * g g type, cyp c * g g type; * /* : cyp c * a a type, cyp c * g g type; * /* : cyp c * g g type, cyp c * a a type, * /* : cyp c * g a type, cyp c * g g type; * /* : cyp c * g g type, cyp c * g a type; * /* : cyp c * g a type, cyp c * g a type. the optimal reaction temperature. then, the lamp reaction was performed at the optimal reaction temperature for , , , , and min to determine the optimal reaction time ) with the following parameters: one step of min at °c; cycles of s at °c, s at °c, s at °c; and one step of min at °c. all pcr products were detected by electrophoresis on a . % (w/v) agarose gel containing goldview nucleic acid stain (an alternative to ethidium bromide, xi'an heart biological technology co., ltd for the samples to be sequenced, a -bp fragment for cyp c * and a -bp fragment for cyp c * were amplified using sequencing primers (table ). the pcr products were sequenced by the beijing genomic institute sensitivity of rt-lamp assay. the sensitivities were assessed using the optimized rt-lamp assay with pharmacogenomics-drug disposition, drug targets, and side effects dna diagnostics: nanotechnology-enhanced electrochemical detection of nucleic acids array-based dna diagnostics: let the revolution begin a nanoliter fluidic 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endorsed by the society for cardiovascular angiography and interventions and the society of thoracic surgeons loop-mediated isothermal amplification targeting insect resistant and herbicide tolerant transgenes: monitoring for gm contamination in supply chain circulating tumor dna as a liquid biopsy for cancer genetic polymorphism analysis of cyp c in chinese han populations from different geographic areas of mainland china real-time loop-mediated isothermal amplification (realamp) for the species-specific identification of plasmodium vivax real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria real-time loop-mediated isothermal amplification (lamp) assay for group specific detection of important trichothecene producing fusarium species in wheat detection of middle east respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (rt-lamp) development of an electrochemical method for ochratoxin a detection based on aptamer and loop-mediated isothermal amplification clinical evaluation of a loop-mediated isothermal amplification (lamp) assay for rapid detection of neisseria meningitidis in cerebrospinal fluid loop-mediated isothermal amplification of dna (lamp): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review application of ethidium bromide monoazide for quantification of viable and dead cells of salmonella enterica by real-time loop-mediated isothermal amplification rapid real-time loop-mediated isothermal amplification combined with coated activated carbon for detection of low numbers of salmonella enterica from lettuce without enrichment clinical validation of the detection of kras and braf mutations from circulating tumor dna simultaneous quantification of mitochondrial dna copy number and deletion ratio: a multiplex real-time pcr assay comparison of four digital pcr platforms for accurate quantification of dna copy number of a certified plasmid dna reference material innate reverse transcriptase activity of dna polymerase for isothermal rna direct detection determination of abo blood group genotypes using the real-time loop-mediated isothermal amplification method this work was funded by the national science and technology major projects for "major new drugs innovation and development" of china (nos. zx - ) and northwest university graduate innovation and creativity funds (nos. yzz ). key: cord- -nmi n h authors: petriccione, milena; mastrobuoni, francesco; zampella, luigi; scortichini, marco title: reference gene selection for normalization of rt-qpcr gene expression data from actinidia deliciosa leaves infected with pseudomonas syringae pv. actinidiae date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: nmi n h normalization of data, by choosing the appropriate reference genes (rgs), is fundamental for obtaining reliable results in reverse transcription-quantitative pcr (rt-qpcr). in this study, we assessed actinidia deliciosa leaves inoculated with two doses of pseudomonas syringae pv. actinidiae during a period of days for the expression profile of nine candidate rgs. their expression stability was calculated using four algorithms: genorm, normfinder, bestkeeper and the deltact method. glyceraldehyde- -phosphate dehydrogenase (gapdh) and protein phosphatase a (pp a) were the most stable genes, while β-tubulin and s-globulin were the less stable. expression analysis of three target genes, chosen for rgs validation, encoding the reactive oxygen species scavenging enzymes ascorbate peroxidase (apx), superoxide dismutase (sod) and catalase (cat) indicated that a combination of stable rgs, such as gapdh and pp a, can lead to an accurate quantification of the expression levels of such target genes. the apx level varied during the experiment time course and according to the inoculum doses, whereas both sod and cat resulted down-regulated during the first four days, and up-regulated afterwards, irrespective of inoculum dose. these results can be useful for better elucidating the molecular interaction in the a. deliciosa/p. s. pv. actinidiae pathosystem and for rgs selection in bacteria-plant pathosystems. multiplication and growth of p. s. pv. actinidiae in a. deliciosa leaf. during the time course of the experiment, the multiplication and growth of psa cra-fru . inoculated at - × cfu/ml and - × cfu/ml into a. deliciosa cv. hayward leaves was assessed. when inoculated at the lower dose, the growth of the pathogen within the leaf never exceeded cfu/ml and showed a peak of - × cfu/ml at nine dpi; no symptoms were observed in the inoculated leaves. by contrast, when the pathogen was inoculated at - × cfu/ml, it incited the appearance of tiny necrotic spots on many of the inoculated leaves of nine dpi (see supplementary fig. s online). in this case, only the green tissue was precisely removed to prepare the samples. selection of candidate reference genes, amplification specificity and efficiency. nine rgs, commonly used as internal controls for expression studies in other pathosystem, were screened in a. deliciosa leaves inoculated with the pandemic psa strain cra-fru . . to determine the specificity of the primer pairs used in this study, melting curve analysis and agarose gel electrophoresis were performed following the rt-qpcr experiment. a single peak in the obtained melting curve confirmed the specificity of the amplicon, and no signal was detected in the negative controls for all of the tested rgs (see supplementary fig. s online) . in addition, a single band with the expected size was detected in a single pcr product (see supplementary fig. s online) . the standard curve method using a pool of all of the cdna samples was performed to calculate the pcr efficiency (e) and the correlation coefficient (r ) of each primer pair. average e values ranged from . to . %, with r varying from . to . (table ). the results showed that all of the primer pairs were suitable for rt-qpcr analysis. expression levels of the reference genes. rt-qpcr was used to quantify the mrna levels of nine candidate rgs, and the expression stability was investigated. to determine the expression levels of the candidate rgs, the raw quantification cycle (cq) values were determined. the nine candidate rgs displayed a wide expression range, with cq ranging from . to . , across all of the tested samples, with mean cq values between . ± . and . ± . (fig. ). all of the tested rgs showed a normal distribution in cq values according to the kolmogorov and smirnov method. these genes were clearly distributed into different expression level categories. the results showed that cyp was the most expressed gene with the lowest mean cq ( . ). on the other hand, glo a was the least expressed gene with the highest mean cq value ( . ) . tub showed the most variation in expression level among the evaluated rgs by the larger whisker taps and boxes compared to the other genes, suggesting its low stability. most of the candidate rgs were highly expressed, with average cq values between and cycles, except sand and glo a, which showed average cq values at intermediate expression levels (fig. ). primer sequence ( ′- ′) bestkeeper and the deltact method) were used to evaluate the stability of expression of selected rgs. the analyses were performed for three comparison groups considering both low-and high-dose bacterial inocula in the leaves and their combined dataset. in each comparison group, the nine rgs were ranked from the most stable to the least stable. the data obtained from biological replicates were analysed separately to verify that the variation was not due to the treatment, but was intrinsic to the gene itself , . genorm analysis. nine rgs were ranked in three comparison groups based on their average expression stability (m-value), as shown in tables , and . all of the tested rgs showed an overall limited variance, with m-values lower than . , which was the default limit (m≤ . ), indicating a high stability level of the analysed genes in our experimental conditions. gapdh, pp a and ubc were the three most stable genes in this pathosystem, with slight differences in ranking for three comparison groups. in a. deliciosa leaves inoculated with a low dose of bacterial inoculum, gapdh was the most stable gene (table ) , while in leaves inoculated with a high dose of bacterial inoculum and when all of the sample sets were analysed together, pp a was the most stable gene (tables and ). tub was the least stable gene in three comparison groups (tables , and ). in this study we used the genorm algorithm to find the optimal number of suitable rgs required for proper normalization. in three comparison groups, genorm analysis revealed that by step wise calculation the pairwise variation value v / was lower than the threshold value ( . ), suggesting that two rgs could be used for normalization under these conditions (fig. ). this suggested that the optimal number of rgs for normalization was two and that the addition of the third rgs showed no significant effect on the normalization of gene expression. finally, gapdh and pp a were identified as the best rgs and selected for normalization by genorm. normfinder analysis. normfinder ranks the rgs according to their stability values under the tested conditions. the results of normfinder analysis were slightly different from those of genorm. however, in the three comparison groups, gapdh emerged as the most stably expressed gene with the lowest tub . table . average stability values (sv) of the nine candidate reference genes are shown for leaves inoculated with high dose of pseudomonas syringae pv. actinidiae inoculum. stability value. gapdh and pp a still occupied the next two top positions for higher stability when we considered the total dataset (table ) or in a. deliciosa leaves inoculated with a high dose of bacterial inoculum (table ) , while in a. deliciosa leaves inoculated with a low dose of bacterial inoculum, gapdh and act were the most stable rgs ( table ). the normfinder results indicated that tub was the least stable rg in the total dataset, confirming our genorm results. tables , and . in the total dataset, bestkeeper analysis highlighted six rgs characterized by the least overall variation, with sd < ; sand and eef- a were the most stable genes, with sd values of . and . , respectively (p < . ) ( table ). in a. deliciosa leaves with a low dose of bacterial inoculum, sand ( . ) was the most stable gene, followed by eef- a and glo a, with sd values of . and . , respectively (table ). in kiwifruit leaves with a high dose of bacterial inoculum, bestkeeper revealed that only the expression of tub overcame the stability threshold; cyp and gapdh were considered to be the most stable genes, with sd values of . and . , respectively (table ). the results of the deltact method were reported in tables , and . gapdh was the most stable gene for the three comparison groups. for the entire dataset, the results were similar to normfinder and genorm analysis, with gapdh and pp a as the top two ranked rgs, with a slight difference in the ranking (table ) . tub was the least stable gene in three comparison groups, as demonstrated by other statistical algorithms. in this study, to determine the consistency of the ranks of candidate rgs produced by genorm, normfinder, bestkeeper and the deltact method, the pearson correlation coefficient was employed ( table ). the pearson correlations achieved from the calculations were positive and significant for all methods, except bestkeeper. the most significant correlation of the rank of all rgs ranked by two methods was genorm and deltact in a. deliciosa leaves inoculated with a high dose of bacterial inoculum (r = . ), followed by normfinder vs. deltact in a. deliciosa leaves inoculated with a low dose of bacterial inoculum (r = . ) ( table ) . for the overall final ranking obtained by the four algorithms, the two top rgs for the total dataset were gapdh and pp a, while the least stable were glo a and tub. expression analysis of the target genes for reference gene validation. the expression of three genes encoding the reactive oxygen species (ros) scavenging enzymes ascorbate peroxidase (apx), superoxide dismutase (sod) and catalase (cat), induced during the systemic infection of kiwifruit leaves with psa, were chosen to further validate the reliability of the selected rgs for the normalization of rt-qpcr data. in this study, we followed two normalization strategies to determine the expression of these target genes. the first used the best two rgs (gapdh and pp a) given by ranking from four methods (genorm, bestkeeper, normfinder and deltact), and the second used the least stable rgs (tub and glo a). in a. deliciosa leaves inoculated with a high dose of bacterial inoculum, an up-regulation in apx mrna expression was observed during the time course of the experiment with . -and . -fold changes after and dpi, respectively. instead, when we used a low dose of bacterial inoculum, we observed an accumulation of the apx transcript after dpi with a . -fold-change and a gradual decrease from to dpi (fig. a) a down-regulation in cat mrna expression during the first dpi was observed, and subsequently, we registered a gradual up-regulation in cat mrna expression in a. deliciosa leaves inoculated with a low-and high-dose of bacterial inocula. the maximum level of the transcript was reached after dpi, with a . -and . -fold change in infected leaves with high-and low-dose bacterial inocula, respectively (fig. b) . similarly, we observed in the accumulation of the sod transcript, that the maximum average value after dpi was a . -and . -fold change, with low and high bacterial inocula, respectively (fig. c) . our results confirm that the transcriptional levels of apx, cat and sod are subjected to complex regulation in psa-infected kiwifruit leaves. this information is distorted when we normalize against the least stable genes, upon which the expression levels of apx, cat and sod were inaccurate and altered transcriptional profiles were displayed (fig. ). in research of plant molecular pathology, studies on gene expression patterns are important for understanding the biological process involved in host-plant interactions. presently, several methods can be applied to study gene expression levels, but rt-qpcr has become the primary quantitative method for the high-throughput and accurate expressing profiling of target genes. for rt-qpcr analysis, the requirement of a normalization method against rgs is important to achieve reliable results. as suggesting by the "minimum information for publication of quantitative real-time pcr experiments" (miqe) guidelines , the use of rgs as internal controls is the most appropriate normalization strategy . ideal rgs should be stably expressed in all cells or tissues and remain stable under different experimental conditions . several studies highlighted that there is neither a universal rg nor a defined number of genes to use, but the choice and an optimal number of rgs should be experimentally determined , . many reliable rgs have been determined in plant cells and across different plant species, developmental stages, and biotic and abiotic stresses . however, to the best of our knowledge, few studies have been carried out to assess rgs in bacteria-plant pathosystems . here, we assessed nine rgs for their use as internal controls in gene expression studies of the a. deliciosa response to infection by psa upon leaf infiltration using two different doses of bacterial inoculum. to identify the best rgs, four different statistical algorithms were used. combined use of genorm, normfinder, bestkeeper and the deltact method to select and validate the best rgs generated substantial discrepancies in the final ranking due to different mathematical models associated with each algorithm, as confirmed by other studies , , . as reported in other studies, the most discrepant results in gene stability ranking were obtained with bestkeeper . in the total dataset, pp a, gapdh and ubc were identified as the top three rgs using genorm, while gapdh, pp a and act were suggested as the most stable rgs by normfinder and the deltact method. according to bestkeeper, act, gapdh, pp a and ubc were ranked fifth to eighth, respectively. among all of the tested rgs, tub was ranked as the least stable gene in the four statistical algorithms, and its use as a rg should be avoided in rt-qpcr experiments in this pathosystem. to overcome differences in the ranking of rgs, we adopted the geometric mean of all four algorithms to obtain a final ranking . as suggested by several studies, the accuracy of rt-qpcr can improve by using more than one rg . the optimal number of candidate rgs for normalization of rt-qpcr data has been evaluated by genorm software. our results showed a pairwise variation v / value below . , which indicates that combination of two-rgs was sufficient for optimal normalization in the three comparison groups. the final ranking showed that the two top rgs for the total dataset were gapdh and pp a and can be used as rgs for rt-qpcr normalization in this pathosystem. gapdh was indicated to be a stable rg in a tomato-virus interaction , in virus-infected mammalian cells and in wheat infected with barley yellow dwarf virus (bydv) , but was the least stable rg in coffea spp. hypocotyls inoculated with colletrichum kahawae . pp a was a stable rg in virus-infected leaf tissues of nicotiana benthiamiana and in virus-infected arabidopsis thaliana . in our study, ubc was among the four most stable rgs, as demonstrated in coffea arabica leaves inoculated with hemileia vastarix , but was considered to be the least stable rg in common bean inoculated with colletotrichum lindemuthianum . tub was not confirmed as a stable normalization factor in our conditions, confirming our previous proteomic study that showed the variability of this protein in a. chinensis shoot during systemic infection with psa ; however, in other pathosystems, such as puccinia graminis f sp. tritici-infected wheat, tub was one of the most stable rgs . furthermore, this rg showed highly variable expression levels in closely related cereals, such as wheat, barley and oat infected with bydv; tub was unstable in wheat and reasonably stable in two other species . the sand transcript was ranked lower among rgs in our pathosystem than was identified in nicotiana benthiamiana and lycopersicum esculentum plants inoculated with viruses , . these variations in the expression profiles of rgs in different pathosystems confirm the need for validation for rgs under each specific condition. some rgs can be involved in different metabolic pathways and influenced in a plant tissue-dependent manner during plant-pathogen interactions . the suitability of the selected rgs has been evaluated analysing the expression levels in three target genes (apx, cat and sod) that encode for proteins that are directly involved in ros detoxification, protecting cells from oxidative bursts induced as responses to pathogen invasion . sod catalyses the dismutation of o to h o , cat dismutates h o to oxygen and water, and apx reduces h o to water by utilizing ascorbate as a specific electron donor . the balance between sod and apx or cat activities in cells is crucial for determining the steady-state level of o and h o . in our study, the accumulation of apx, cat and sod gene transcripts was strongly influenced by the dose of bacterial inoculum used. indeed, these genes involved in ros detoxification and the oxidative-stress response have a key role for bacteria survival and pathogenesis . the apx up-regulation during a relatively long time course of infection (i.e., days) was observed upon the twig inoculation with the same high dose of psa cra-fru . also in the case of a. chinensis "soreli" . in the same study, however, neither cat nor sod were found differentially expressed days after the twig inoculation. irrespective of the inoculum doses, both cat and sod resulted up-regulated during the first four days of infection, and, subsequently, their level in the leaf tissues declined. interestingly, a similar trend was observed for sod in the phaseolus vulgaris/p. s. pv. phaseolicola pathosystem after the inoculation of bean leaves with the same high dose of bacterial inoculum used in the present study (i.e., × cfu/ml) . in this study, however, the sod level in the bean primary leaves and into the apoplastic fluid starts to decrease and hours after the artificial inoculation. furthermore, in this study, we demonstrated that to correctly quantify apx, cat and sod, it was necessary to choose the rgs that had transcript levels that were not influenced by bacterial infections and that the use of inappropriate rgs can markedly change the expression pattern of a given target gene, leading to incorrect results. this is the first study in which a set of candidate rgs was analysed in terms of their expression stability in a. deliciosa leaves infected with psa. four different statistical algorithms showed slight differences in the final ranking of rgs, but by combining and analysing the data together, we demonstrated that two genes, gapdh and pp a, are the most stably expressed transcripts in all infected kiwifruit leaves. the validation of rgs in our study provides new information that will be useful for a better understanding of the molecular mechanisms implicated in the expression profiles of target genes in the a. deliciosa/p.s. pv actinidiae pathosystem. it should be considered that ideal rgs can vary with the pathosystem under investigation, and therefore, these genes should be carefully selected for each study conforming to the miqe guidelines. plant material, p. syringae pv. actinidiae inoculations and experimental design. two-yearold, self-rooted, pot-cultivated a. deliciosa "hayward" plants and the pandemic psa strain cra-fru . were used in this study . this bacterial strain was originally isolated from a. chinensis leaf spot and further characterized , . plants were maintained in an aseptic room with % relative humidity with natural light and no further fertilization after their transfer from the nursery. they were watered regularly. inoculation took place in spring (i.e., may). the strain was grown for h on nutrient agar (oxoid) with % sucrose added (nsa) at ± °c. subsequently, a low ( - × cfu/ml) and high ( - × cfu/ ml) dose of bacterial inoculum, determined using spectrophotometry, were prepared in sterile, distilled water. to avoid wounding, the inoculation occurred by gently spraying the suspensions on the abaxial surface of fully expanded, healthy, young leaves, until the appearance of homogenous water-soaked areas on the whole leaf lamina. twenty plants per dose were inoculated. artificial inoculations were performed separately, according to the dose. control plants were treated in the same way with sterile, distilled water. after inoculation, plants were maintained separately and were kept for h in a moist chamber ( % humidity), which was required for optimal infection. during the experiment, the multiplication and growth of the pathogen was assessed as previously described . leaves were collected after one day post-inoculation and at intervals of three days for days, immediately frozen in liquid nitrogen and stored at − °c until rna isolation. in the same treatment group (inoculated and mock inoculated), each biological replicate was obtained by pooling three leaves from different plants harvested at random. three independent biological replicates were performed for each sample with three technical replicates each. total rna extraction and cdna synthesis. total rna was isolated from a. deliciosa leaves inoculated with psa as well as from control leaves as described by rubio-piña and zapata-perez . residual genomic dna was digested by rnase-free dnase (invitrogen life technologies, carlsbad, ca, usa) according to the manufacturer's instructions. the rna concentration was quantified by measuring the absorbance at nm using a jasco v- uv/vis spectrophotometer (tokyo, japan). the purity of all of the rna samples was assessed at an absorbance ratio of od / and od / , while its structural integrity was checked by agarose gel electrophoresis. only high-quality rna with od / and od / > was used for subsequent steps. single-stranded cdna was synthesized from μ g of total scientific reports | : | doi: . /srep rna using an iscript ™ select cdna synthesis kit and oligo(dt) primers (bio-rad, milan, italy), according to the manufacturer's instructions. test. for this study, special attention was paid to a select set of nine candidate rgs (act, eef- a, pp a, ubc , sand, tub, glo a, cyp and gapdh) to investigate their robustness as internal controls for rt-qpcr in a. deliciosa. these genes belong to different functional and abundance classes to significantly reduce the chance that they are co-regulated. apx, cat and sod were selected as genes of interest. gene-specific primers, such as sand, tub, glo a, cyp, gapdh, apx, cat and sod, were designed in our laboratory using primer expression software version ( table ). the amplification efficiency of each candidate/target gene was determined using a pool representing all of the cdna samples. first, all of the primers were examined by end-point pcr, all of the chosen candidates/target were expressed, and specific amplification was confirmed by a single band of appropriate size in a % agarose gel after electrophoresis (see supplementary fig. s online) . in a second step, the pool was used to generate a five-point standard curve based on a ten-fold dilution series. the amplification efficiency (e) and correlation coefficient (r ) of the primers were calculated from the slope of the standard curve according to the equation : quantitative real-time pcr (qpcr). quantitative real-time-pcr was performed using a cfx connect real-time pcr detection system (bio-rad) to analyse the specific expression of each reference/ target gene. cdna was amplified in -well plates using the ssoadvanced ™ sybr ® green supermix (bio-rad), ng of cdna and nm specific sense and anti-sense primers in a final volume of μ l for each well. thermal cycling was performed, starting with an initial step at °c for s, followed by cycles of denaturation at °c for s and primer-dependent annealing (table ) for s. each run was completed with a melting curve analysis to confirm the specificity of amplification and lack of primer dimers. determination of reference gene expression stability. data analyses were performed on three groups: a) infected plants with a low dose of bacterial inoculum compared to the mock-inoculated plants dataset (ldi), b) infected plants with a high dose of bacterial inoculum compared to the mock-inoculated plants dataset (hdi), and c) the entire dataset (total). the stability of candidate rgs for several comparison groups was analysed with the following four applets: genorm , normfinder , bestkeeper and the deltact method . the raw cq values were converted into relative quantities and imported into the genorm and normfinder software programs; no transformed cq values are required for bestkeeper and the deltact method. genorm calculates an expression stability value (m) for each rg and then determines the pairwise variation (v) of each rg with all of the other genes. at the end of analysis, by stepwise exclusion of the gene with the highest m-value (less stable), this tool allows for the ranking of the tested rgs according to their expression stability. the optimal number of rgs required for normalization was determined by pairwise variation v n /v n + ( . recommended threshold). normfinder calculates the expression stability value (sv) for each gene, taking into account intraand inter-group variations of the samples set . a low sv-value indicates the high expression stability of this gene. bestkeeper is an excel-based software tool that selects best-suited rgs by performing a statistical analysis based on pearson correlation coefficient (r), standard deviation (sd) a coefficient of variance (cv). only genes with a high r value and a low sd are combined into bestkeeper index (bki) value using the geometric mean of their cq values. finally, this tool determines the correlation coefficient of each candidate rg with the bki value, along with the probability (p) value. the rg with the highest coefficient of correlation with the bki is considered to be the most stable. the deltact (dct) method compares relative expression of pairs of rgs within each sample to identify stable rgs . a ranking of the rgs using the four algorithms together was obtained as suggested by velada et al. . correlations among the stability values of rgs obtained with different software were analysed using pearson's correlations (p < . and p < . ). all statistical analyses were performed using the spss v. . . to confirm the reliability of the rgs, the relative expression profiles of apx, cat and sod genes were determined and normalized with the most stable and less stable genes. relative fold changes in gene expression was calculated using the comparative −ΔΔct method and normalized to the corresponding rgs levels , . statistical analysis. data are displays as mean ± standard deviation. cq values were tested for normality (kolmogorov-smirnov test) prior to analysis. statistical analysis of data was performed by one-way anova followed by lsd post-hoc test. calculation were performed using the spss v. . . transcript profiling in host-pathogen interactions relative quantification in real-time pcr proteomics approach combined with biochemical attributes to elucidate compatible and incompatible plant-virus interactions between vigna mungo and mungbean yellow mosaic india virus the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments normalization of qrt-pcr data: the necessity of adopting a systematic, experimental conditions-specific, validation of references genome-wide identification and testing of superior reference genes for transcript normalization in arabidopsis real-time rt-pcr normalisation; strategies and considerations evaluation of reference genes for accurate normalization of gene expression for real time-quantitative pcr in pyrus pyrifolia using different tissue samples and seasonal conditions tracking the best reference genes for rt-qpcr data normalization in filamentous fungi standardization of real-time pcr gene expression data from independent biological replicates transcript profiling of a conifer pathosystem: response of pinus sylvestris root tissues to pathogen (heterobasidion annosum) invasion transcript profiling of poplar leaves upon infection with compatible and incompatible strains of the foliar rust melampsora larici-populina validation of reference genes for normalization of qpcr gene expression data from coffea spp. hypocotyls inoculated with colletotrichum kahawae detection of prune dwarf virus by one-step rt-pcr and its quantitation by real-time pcr evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant vitis vinifera cultivars selection and validation of reference genes for gene expression studies by reverse transcription quantitative pcr in xanthomonas citri subsp. citri during infection of citrus sinensis reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions evaluation of reference genes for real-time rt-pcr expression studies in the plant pathogen pectobacterium atrosepticum pseudomonas syringae pv. actinidiae: a re-emerging, multi-faceted, pandemic pathogen pseudomonas syringae pv. actinidiae draft genomes comparison reveal strain-specific features involved in adaptation and virulence to actinidia species pseudomonas syringaepv. actinidiae (psa) isolates from recent bacterial canker of kiwifruit outbreaks belong to the same genetic lineage genomic analysis of the kiwifruit pathogen pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease redefining the global populations of pseudomonas syringae pv. actinidiae based on pathogenic, molecular and phenotypic characteristics proteomic changes in actinidia chinensis shoot during systemic infection with a pandemic pseudomonas syringae pv. actinidiae strain proteomic analysis of the actinidia deliciosa leaf apoplast during biotrophic colonization by pseudomonas syringae pv. actinidiae accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes normalization of real-time quantitative reverse transcription-pcr data: a modelbased variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper -excel-based tool using pair-wise correlations selection of housekeeping genes for gene expression studies in human reticulocytes using real-time pcr a rapid transcriptional activation is induced by the dormancy-breaking chemical hydrogen cyanamide in kiwifruit (actinidia deliciosa) buds modified carotenoid cleavage dioxygenase expression correlates with altered branching in kiwifruit (actinidia chinensis) metabolic analysis of kiwifruit (actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism characterization of two alcohol acyltransferases from kiwifruit (actinidia spp.) reveals distinct substrate preferences identification of suitable reference genes for real-time rt-pcr normalization in the grapevine-downy mildew pathosystem quantitative rt-pcr analysis of differentially expressed genes in quercus suber in response to phytophthora cinnamomi infection technical advance: transcript profiling in rice (oryza sativa l.) seedlings using serial analysis of gene expression (sage) selection and validation of reference genes for quantitative gene expression studies by real-time pcr in eggplant (solanum melongena l) reference gene validation for quantitative rt-pcr during biotic and abiotic stresses in vitis vinifera selection of reference genes for expression studies in cicer arietinum l.: analysis of cyp e gene expression against ascochyta rabiei normalisation of real-time rt-pcr gene expression measurements in arabidopsis thaliana exposed to increased metal concentrations analysis of qpcr reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (scophthalmus maximus) gonad dataset identification and validation of reference genes for normalization of transcripts from virus-infected arabidopsis thaliana reference gene selection for quantitative real-time pcr normalization in caragana intermedia under different abiotic stress conditions the choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses identification of a novel reference gene for apple transcriptional profiling under postharvest conditions reference genes selection and normalization of oxidative stress responsive genes upon different temperature stress conditions in hypericum perforatum l reference gene selection for quantitative real-time pcr analysis in virus infected cells: sars corona virus,yellow fever virus, human herpesvirus- , camelpox virus and cytomegalovirus infections biphasic haustorial differentiation of coffee rust (hemileia vastatrix race ii) associated with defence responses in resistant and susceptible coffee cultivars validation of reference genes for rt-qpcr normalization in common bean during biotic and abiotic stresses reference gene selection for qpcr gene expression analysis of rust-infected wheat validation of reference genes for gene expression studies in virus-infected nicotiana benthamiana using quantitative real-time pcr assessment of reference gene stability influenced by extremely divergent disease symptoms in solanum lycopersicum l reactive oxygen and oxidative stress tolerance in plant pathogenic pseudomonas the antioxidant systems vis à vis reactive oxygen species during plant-pathogen interaction selected reactive oxygen species and antioxidant enzymes in common bean after pseudomonas syringae pv. phaseolicola and botrytis cinerea infection identification of pseudomonas syringae pv. actinidiae as causal agent of bacterial canker of yellow kiwifruit (actinidia chinensis planchon) in central italy molecular and phenotypic features of pseudomonas syringae pv. actinidiae isolated during recent epidemics of bacterial canker of yellow kiwifruit (actinidia chinensis) in central italy isolation of total rna from tissues rich in polyphenols and polysaccharides of mangrove plants guideline to reference gene selection for quantitative real-time pcr analysis of relative gene expression data using real-time quantitative pcr and the −ΔΔct method this work was financed by the regione campania programme under the grant agreement ur.co. fi. (unità di coordinamento e potenziamento delle attività di sorveglianza, ricerca, sperimentazione, monitoraggio e formazione in campo fitosanitario), decreto dirigenziale n° del giugno . key: cord- - plcxv authors: xu, zhiwei; hu, wenbiao; zhang, yewu; wang, xiaofeng; zhou, maigeng; su, hong; huang, cunrui; tong, shilu; guo, qing title: exploration of diarrhoea seasonality and its drivers in china date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: plcxv this study investigated the diarrhoea seasonality and its potential drivers as well as potential opportunities for future diarrhoea control and prevention in china. data on weekly infectious diarrhoea cases in provinces of china from to , and data on demographic and geographic characteristics, as well as climatic factors, were complied. a cosinor function combined with a poisson regression was used to calculate the three seasonal parameters of diarrhoea in different provinces. regression tree analysis was used to identify the predictors of diarrhoea seasonality. diarrhoea cases in china showed a bimodal distribution. diarrhoea in children < years was more likely to peak in fall-winter seasons, while diarrhoea in persons > = years peaked in summer. latitude was significantly associated with spatial pattern of diarrhoea seasonality, with peak and trough times occurring earlier at high latitudes (northern areas), and later at low latitudes (southern areas). the annual amplitudes of diarrhoea in persons > = years increased with latitude (r = . , p< . ). latitude . ° n and . ° n were the latitudinal thresholds for diarrhoea seasonality in china. regional-specific diarrhoea control and prevention strategies may be optimal for china. more attention should be paid to diarrhoea in children < years during fall-winter seasons. d espite remarkable progress in its control, diarrhoea remains a leading infectious cause of morbidity and mortality in low-income and middle-income countries, especially for children , years [ ] [ ] [ ] [ ] . it is estimated that in , worldwide, there were more than . billion episodes of diarrhoea in children , years, resulting in , deaths in . china, together with other countries, shoulder the heaviest burden of diarrhoea, accounting for % of total diarrhoea episodes and % of severe diarrhoea episodes in children aged , years . exploring the seasonality of diarrhoea could reflect the relative predominance of its aetiological agents and shed new light on future vaccination programs. further, understanding the determinants of seasonality could benefit the development of early warning systems. existing research puts emphasis on exploring the seasonality of rotavirus diarrhoea and suggests that it varies with region and climate [ ] [ ] [ ] [ ] . china is an economically, geographically and climatologically diverse country, in which the diversity of diarrhoea seasonality is not well unveiled. after the severe acute respiratory syndrome (sars) outbreak, chinese government has strengthened its public health disease system. the web-based real-time disease surveillance system-china information system for disease control and prevention (cisdcp), was built in to detect and respond to infectious disease outbreak . infectious diarrhoeal diseases, which have been reported to chinese center for disease control and prevention (china cdc) through cisdcp in the past decade, were grouped into three classes: class a (cholera); class b (bacillary dysentery, typhoid and paratyphoid); and class c (other infectious diarrhoea). the seasonality of classes a and b, which may largely be determined by their predominant aetiological agents (bacterium vibrio cholera, shigellosis and salmonella), has been extensively documented , . however, the seasonality of ''other infectious diarrhoea'' (class c) remains unknown. in this study, we reviewed the data on ''other infectious diarrhoea'' in china from - obtained from cisdcp, aiming to characterize the seasonality of diarrhoea in china and identify its potential drivers, as well as explore potential opportunities for future diarrhoea control and prevention. patterns of seasonality. there was a distinct seasonality in diarrhoea occurrence in the total population ( figure ) and in each age group (figures s a-s c (supplementary material)), and two peaks were observed every year. specifically, diarrhoea in children , years peaked in fall-winter seasons, and diarrhoea in persons . years peaked in summer ( figure ). further, we found that the peak times in children (, years) varied greatly over time, while the peak times in adults (. years) was consistent across years (figures s a-s f (supplementary material)). figure shows the seasonality of diarrhoea in different regions (listed by latitude), revealing that in both children , years and persons . years, diarrhoea at higher latitudes were more likely to be peaking in summer, and diarrhoea at lower latitudes were more likely to be peaking in fall-winter seasons ( figures a-c) . analysis by season ( figures s a- c (supplementary material) ) reveals this pattern more clearly. in terms of the amplitude distribution by age and region (seasonal amplitude refers to the relative fluctuation of diarrhoea within a certain period of time, and it provides pivotal information on the possibility of diarrhoea outbreaks), we found that adults (. years) had much greater diarrhoea amplitudes than children (, years) (figure ), but the amplitude in persons . years declined progressively with increasing age, reaching its valley in elderly . years (figure ) , and this decreasing trend was remarkably consistent across years ( figure s (supplementary material)). figures a-c depicts the spatial pattern of diarrhoea amplitude, indicating that, in persons . years, diarrhoea amplitudes at higher latitudes were greater than lower latitudes. drivers of seasonality. to better understand whether there was a statistically significant relationship between latitude and diarrhoea seasonal parameters (peak time, trough time and amplitude), we calculated the spearman correlations between them, and found that diarrhoea peak time and trough time were negatively correlated with latitude ( figures a-d) . no significant correlation was observed between amplitude in children , years and latitude, but amplitude in persons . years was positively correlated with latitude (r . , p, . ) ( figures e-f) . to identify the putative predictors of diarrhoea seasonality, we conducted regression tree analysis. table shows the summary statistics for the demographic, economic, and geographic characteristics, climatic factors, and diarrhoea seasonal parameters of the pro- epidemiological transmission zones. after identifying the latitudinal threshold for diarrhoea peak and trough times, we plotted the heat maps accordingly ( figure a -c), and found that: at latitudes. the national surveillance data allows us to do this first comprehensive study concerning diarrhoea seasonality by age and geography in china. diarrhoea in children , years normally peaked in fall-winter seasons, while diarrhoea in persons . years were more likely to peak in summer. three epidemiological regions characterized by distinct diarrhoea seasonality were identified in this study: northern provinces (latitudes. . un), intermediate provinces (latitudes. . un & , . un) and southern provinces (latitudes, . un). regression analysis indicated that mean temperature was predictive of diarrhoea vertical line: week, from week to week ; colour palette: ''high'' refers to relatively higher number of diarrhoeal cases within each age group, and ''low'' refers to relatively lower number of diarrhoeal cases within each age group. www.nature.com/scientificreports scientific reports | : | doi: . /srep peak time in persons . years, and relative humidity was linked to diarrhoea amplitude in children , years. the core of seasonality in diarrhoea is related to temporal oscillations in the pathogenic agents and host susceptibility (fluctuations of neuroendocrine function and immune response ). the most intriguing finding of this study is the remarkable difference in the diarrhoea seasonality between children , years and persons . years. we found that diarrhoea in children , years appeared to peak in fall-winter seasons, which is in accord with findings in brazil , and this may partially be attributable to the fact that rotavirus is the predominant aetiology of diarrhoea in infants and young children , and rotavirus favours low temperature , , , . compared with infants and young children, persons . years in china are more likely to be attacked by bacterial pathogens , possibly resulting in their summer peak time of diarrhoea . interestingly, we found diarrhoea seasonality (peak time, trough time and amplitude) in children , years varied over time, indicating a yearly-specific diarrhoea control and prevention strategy in children , years in china. further, the consistent diarrhoea seasonality in adults . years across years we observed in this study may facilitate the development of future early warning systems focusing on diarrhoea control in adults. in this study, we detected greater amplitudes of diarrhoea seasonality at higher latitudes among persons . years, implying that reduced winter sunlight and its potential effect on vitamin d deficiency might play a role in diarrhoea seasonality in persons . years. at northern latitudes, vitamin d deficiency is more common due to reduced ultraviolet light exposure . at latitudes . un, even with adequate sun exposure, the dermal generation of vitamin d is negligible , , which may render the greater amplitudes of diarrhoea seasonality in persons . years by altering their immune response . this finding should guide clinical and public health practice in those regions with high latitudes, and clinicians in those regions should be particularly made aware of the adverse impact of vitamin d insufficiency on diarrhoea and consider supplementation of vitamin d in persons with higher risk for diarrhoea. moreover, we found amplitudes of diarrhoea seasonality in persons . years were much higher than children , years. this is striking because children shoulder majority of diarrhoea burden. existing literature offers limited information on the mechanism explaining this finding, and we speculated that it may be because persons . years have had relatively recent diarrhoea compared with children , years . as reported in prior studies, though temperature, rainfall and relative humidity were associated with the occurrence of diarrhoea [ ] [ ] [ ] [ ] , no climatic factor alone can fully capture the complexity of diarrhoea seasonality. however, in this study, we found climatic factors helped distinguish the peak time, trough time and amplitude between regions, which is pivotal for future diarrhoea control. specifically, we found that . uc was the temperature threshold for diarrhoea peak time in persons . years, with diarrhoea in those provinces with mean temperature below . uc peaking earlier. relative humidity was associated with seasonal amplitude of diarrhoea in children , years, and provinces with relative humidity below . % had a greater seasonal amplitude of diarrhoea in children , years. it was also observed that . mm rainfall was the threshold for both trough time in children , years and amplitude in persons . years. for those regions with latitude . . un and monthly average rainfall , . mm, diarrhoea in children , years peaked the earliest, highlighting that public health sectors in these regions should take earlier precautionary measures to prevent infants and young children from being attacked by diarrhoea. further, for the similar regions (latitude . . un and monthly average rainfall , . mm), the seasonal amplitude of diarrhoea in persons . years was the greatest. the identification of climatic factors associated with diarrhoea seasonality in this study will shed new light on the possible role of climate variation in the occurrence of diarrhoea. our large-scale analysis of diarrhoea seasonality identified three epidemiological regions: northern china where diarrhoea in children , years and persons . years both peaked in summer, midlatitudes where diarrhoea in children , years peaked in fall-winter seasons and diarrhoea in persons . years peaked in summer, and southern china where diarrhoea in children , years peaked in fallwinter seasons and diarrhoea in persons . years peaked yearround. the three epidemiological zones indicate that diarrhoea seasonality in china may result from the interaction between climatic factors, behaviours likely to mix feces with food and water, and population immunity , . the seasonal patterns of diarrhoea at low latitudes (, . un) in children , years and persons . years we figure a implies that latitude is the primary predictor of diarrhoea peak time in children , years). the numbers on the tree refer to the thresholds of the predictors (e.g., figure a suggests that, for the regions with latitude , . un, diarrhoea in children , years are more likely to peak around week ). found were not consistent with prior studies [ ] [ ] [ ] . previous research mainly focused on rotavirus seasonality in children , years and found that rotavirus persists year-round in tropical areas (, un) and peaks from autumn to spring (cold and dry seasons) in temperate areas . information on diarrhoea seasonality in adults is scarce in existing literature, and one study looking at the rotavirus diarrhoea in japan did not find a significant seasonality . the motivation of this study is to assist on-going diarrhoea surveillance and the epidemiological regions we identified have important implications for development of early warning system. specifically, in northern china, diarrhoea preventive measures should be taken earlier (before summer); in intermediate latitudes, more attention should be paid to diarrhoea in children , years during fall-winter seasons, and diarrhoea control may focus more on persons. years in summer, and in southern china, diarrhoea control should also focus on children , years during fall-winter seasons while diarrhoea surveillance in persons . years should be strengthened, given its nondistinct seasonality. this is the first study to explore the seasonality of diarrhoea in china at the national level. the remarkably different diarrhoea seasonality in children , years and persons . years we found will be useful for designing and implementing future diarrhoea control and prevention strategies. the three epidemiological zones we identified will guide the development of targeted early warning systems and implementation of future risk management efforts. further, the potential drivers of diarrhoea seasonality we identified will facilitate future studies assessing the impact of social-environmental change on diarrhoea in china. several limitations should also be acknowledged. first, these diarrhoea cases were not all lab-confirmed. second, most diarrhoea cases may be undetected as people with mild symptoms may not seek medical care. third, access to health care may vary across provinces, and we cannot rule out surveillance bias in some areas such as tibet and xinjiang. finally, we did not have data on the pathogenic agents, which restricts us to examine the seasonality of specific pathogens and give more evidence-based implications for future vaccination programs. in conclusion, our study unfolds the striking difference in diarrhoea seasonality between children , years and persons . years. the distinct geographic patterns of diarrhoea, which may be impacted by climatic factors, were also unveiled. future research should focus more on elucidating the impact of social-environmental changes on diarrhoea in different epidemiological zones and mechanisms why diarrhoea seasonality differs greatly across different age groups. our work has practical implications for the development of early warning systems targeting different population in different regions. data collection. the territory of china lies between latitudes u and un, and longitudes u and ue. china's climate is mainly dominated by dry seasons and wet monsoons, and it is diverse from region to region due to the complex topography of china. table shows the summary statistics for the demographic, economic, and geographic characteristics as well as climatic factors of chinese provinces. infectious diarrhoea data from st january to st december were collected from cisdcp. cisdcp is the reporting system of nationally notifiable infectious diseases and public health emergencies throughout china . diarrhoea cases were those who have three or more loose or liquid stools per day diagnosed in the hospital or the local cdc. in this study, we used the weekly reports of diarrhoea cases from cisdcp. information on the age and residential address of the diarrhoea patients was obtained. ethical approval was obtained from the research institutional review board of public health of shandong university (china) prior to the data being collected. patient information was de-identified and thus no written informed consent was obtained. demographic, economic, geographic and climate information in all provinces from to , including population, per capita gross regional product (pgrp), latitude, longitude, monthly average mean temperature, monthly average relative humidity, and monthly average rainfall, were collected from china statistical yearbook to identify the putative drivers of diarrhoea seasonality. data analysis. a seasonal decomposition analysis was conducted to assess whether there was a distinct seasonality in diarrhoea occurrence . in this analysis, diarrhoea time-series was decomposed into seasonality, long-term trend, and irregular factors . heat maps were created to present the peak and trough times of diarrhoea in each age group and province. a cosinor function combined with poisson regression was used to quantify the seasonal parameters of diarrhoea (ie., peak time, trough time and annual amplitude) . the annual amplitude was expressed as a proportion of mean diarrhoea cases to facilitate the comparisons between different age groups and different regions. to identify the putative drivers of diarrhoea seasonality, a classification and regression tree (cart) analysis was conducted . cart model is based on simple splits of the data and does not require the assumptions such as the existence of linear regression among and variables and homoscedasticity in variance. cart model was chosen in this study mainly because the potential drivers of diarrhoea seasonality (e.g., latitude and temperature) are highly correlated with each other and cart can potentially better accommodate complex interactions between variables since they avoid some of the assumptions associated with linear regression . more importantly, cart model can identify the thresholds for the independent variables in the regression. visual maps for the spatial distributions of diarrhoea incidence and annual amplitude were created using arcgis version . (esri inc., redlands, ca, usa). all the remaining analysis was conducted using the r statistical environment (version . . ), with the ''season'' package (version . - ) to conduct the cosinor analysis and ''rpart'' package to conduct the regression tree analysis. diarrhoea morbidity and mortality in older children, adolescents, and adults global and regional mortality from causes of death for age groups in and : a systematic analysis for the global burden of disease study global burden of childhood pneumonia and diarrhoea setting research priorities to reduce global mortality from childhood diarrhoea by global seasonality of rotavirus infections seasonality of rotavirus disease in the tropics: a systematic review and meta-analysis global seasonality of rotavirus disease seasonality of rotavirus in south asia: a meta-analysis approach assessing associations with temperature, precipitation, and vegetation index a nationwide web-based automated system for early outbreak detection and rapid response in china trend and disease burden of bacillary dysentery in china cholera in bangladesh: climatic components of seasonal variation seasonal variation in host susceptibility and cycles of certain infectious diseases modeling diarrhea disease in children less than years old burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study analysis of reported infectious diarrhea (other than cholera, dysentery, typhoid and paratyphoid) in china in climatic factors associated with hospitalizations for rotavirus diarrhoea in children under years of age analysis of the aetiology of diarrhoea in outpatients in impact of ambient temperature on children's health: a systematic review seasonal variance of -(oh) vitamin d in the general population of estonia, a northern european country vitamin d: a millenium perspective prevalence of vitamin d insufficiency in canada and the united states: importance to health status and efficacy of current food fortification and dietary supplement use vitamin d deficiency associated with increased incidence of gastrointestinal and ear infections in school-age children seasonality of tuberculosis in the united states the global burden of diarrhoeal disease winter seasonality and rotavirus diarrhoea in adults emergence and control of infectious diseases in china national census in china in extreme temperatures and paediatric emergency department admissions chapter three: consinor] analysing seasonal health data temperature, air pollution and total mortality during summers in sydney classification and regression trees the authors thank the national basic research program of china ( program) (grant no. cb ). z.x., w.h., q.g. and s.t. designed the study and collected the data, z.x. and w.h. analysed the data and drafted the manuscript, w.h., y.z., x.w., m.z., h.s., c.h., s.t. and q.g. revised the manuscript. supplementary information accompanies this paper at http://www.nature.com/ scientificreportscompeting financial interests: the authors declare no competing financial interests. key: cord- -qbh ec authors: bi, yuhai; liu, jingyuan; xiong, haofeng; zhang, yue; liu, di; liu, yingxia; gao, george f.; wang, beibei title: a new reassortment of influenza a (h n ) virus causing human infection in beijing, date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: qbh ec a -year-old man was confirmed to have an influenza a (h n ) virus infection, and the causative agent a/beijing/ / (h n ) virus was isolated. genetic and phylogenetic analyses revealed that the virus belonged to a novel genotype, which probably emerged and further reassorted with other h or h viruses in poultry before transmitting to humans. this virus caused a severe infection with high levels of cytokines and neutralizing antibodies. eventually, the patient was cured after serially combined treatments. taken together, our findings indicated that this novel genotype of the human h n virus did not evolve directly from the first beijing isolate a/beijing/ / (h n ), suggesting that the h n virus has not obtained the ability for human-to-human transmissibility and the virus only evolves in poultry and then infects human by direct contact. hence, the major measures to prevent human h n virus infection are still to control and standardize the live poultry trade. early antiviral treatment with combination therapies, including mechanical ventilation, nutrition support and symptomatic treatment, are effective for h n infection. yellow-white phlegm and feeling fatigued on january . because the detection of the influenza a virus universal antigen was negative on the throat-swab by means of the immune colloidal gold technique and the radiologic findings revealed bronchitis, the patient was treated with anti-infective therapy by an intravenous injection of moxifloxacin. however, that treatment did not take effect, and the symptoms gradually worsened. on february , the patient appeared with hyperpyrexia (maximum temperature . °c), coughing with bloody sputum and dyspnoea with a low oxygen saturation ( . %). the h n viral rna was positive in the oropharynx swab confirmed by the real-time rt-pcr method according to the protocol of the chinese cdc . the patient was transferred into the intensive care unit (icu) of beijing ditan hospital, capital medical university. the case was diagnosed as a laboratory-confirmed case of influenza a (h n ) infection with severe pneumonia combined with the complications of acute respiratory failure, septic shock, stress ulcer and acute renal failure. antiviral treatment (oseltamivir) with combination of antibiotics (sulperazon), a gastric acid secretion inhibitor (omeprazole), mechanical ventilation, continuous renal replacement, supportive nutrition therapy and symptomatic treatment were given. on february , the h n viral nucleic acid was negative when detecting the tracheal aspirate specimens by real-time rt-pcr. on march , the infection symptoms and the respiratory function improved, and the circulation situation tended to be stable. after approximately four months of treatment, the patient recovered and was discharged from hospital on june (table ) . a h n virus, a/beijing/ / (h n ) (abbreviated as bj thereafter), was isolated and identified after one passage propagation in eggs. to further study the gene evolution of the h n virus, the whole genome was amplified and sequenced. genetic alignments showed that the ha, na, pb , pa, np and ns genes possessed the highest genetic similarities ( . - . %) with other h n virus genes. however, the pb and m genes possessed the highest nucleotide similarities ( . % and . %, respectively) with h n isolates (table ) . furthermore, the genetic homology between bj and bj displayed some diversity because there are . % and . % nucleotide similarities, respectively, of the pb and m genes ( table ) . these nucleotide identities suggested that bj might not be evolved directly from bj . the phylogenetic analyses showed that the ha and na gene sequences of bj clustered with previously identified human-infecting h n viruses (fig. a,b) . the phylogenies of the pb and m genes documented that bj and bj fell into different clades, and this finding implied that different evolutionary pathways might behind these two viruses (fig. c,d) . according to the genotypic assignment in the previous study (table ) . however, the pb and m genes of bj clustered together with other avian-source h n viruses (fig. c ,d). all of these findings suggested that this novel genotype virus should emerge and evolve in poultry prior to transmission to humans. neutralizing antibody and cytokine evaluation. the level of serum neutralizing antibody (nab) and cytokines in flu patients play pivotal roles in disease progression and recovery [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for this reason, the kinetic . the mn titre of the h n patient was higher than that of the contemporaneous h n patient on d.a.o. (fig. b ). the evaluation of serum cytokines in the h n patient revealed that the levels of il- , il- p , mcp- and ip- were significantly increased on , and d.a.o. compared with the normal ranges, whereas the ifn-γ , ifn-α and il- a levels did not notably increase (fig. c) . the levels of il- , il- p , and ip- displayed an increase-decrease-increase tendency, and the highest levels were on d.a.o., then decreased to the lowest points on d.a.o., and subsequently showed increasing levels on d.a.o. additionally, most of the detected cytokines in the h n case showed higher levels than in the contemporaneous severe h n case on d.a.o. (fig. d) , which suggested that the h n virus might cause higher cytokine secretions than the h n virus, although the case number in our study was limited. the present data documented that the novel genotype g . of the h n virus (bj ) could infect and cause severe disease in humans. in addition to the g . virus (bj ), there are at least two genotypes of h n virus existing in beijing. to date, only one g . and one g . virus from humans have been isolated; and the other g . strain (sc ) was first isolated from chickens. although high homology is shared between bj and sc (table ), the pb and m genes of the bj virus clustered with other avian-source h n or h n viruses (fig. c,d) . these data indicate that bj likely evolved from a novel genotype found in chickens (sc ) that then reassorted with other h or h virus in birds before being transmitted to humans. this finding suggested that the reassortment with other subtype viruses in avians is still the major evolutionary path of the h n virus; then, sensitive people would be infected after exposure to the mutated viruses. hence, diverse h n genotypes or reassortants were consecutively isolated in humans. to standardize or close the live poultry trade would be the most efficient way to prevent and control the human-avian influenza virus infection disease. to date, a total of genotypes of h n viruses, including bj (g . ) and bj (g . ), have been identified according to our classification and nomenclature system . however, there are only genotype viruses isolated and identified in humans . this finding suggested that diverse genotype viruses should possess heterogenic host-specific preference and pathogenicity for humans. the bj virus only caused the patient to have a mild to moderate respiratory syndrome , whereas the bj virus tended to cause a severe disease. the underlying mechanisms need to be further clarified. in addition, the oseltamivir resistance mutation (r k) in the na protein of bj could not be found after days of oseltamivir usage; however, the mutation emerged in the a/shanghai/ / (h n ) strain just after one day treatment with oseltamivir , . this finding further suggested that diverse biologic characteristics might appear in different h n viruses. in general, viral and host factors contribute to disease severity and outcomes. a previous multivariate analysis revealed that the presence of coexisting medical conditions (such as chronic heart disease and chronic obstructive pulmonary disease) were the only independent risk factors for severe illness with h n infection . recent studies found that the presence of host genetic factors might be closely related to h n influenza disease susceptibility and/or severity , . the ifn-induced transmembrane protein- (ifitm ) c/c genotype was reported to be associated with severe clinical outcomes, as reflected by a higher viral load, more rapid progression to ards, higher cytokine/chemokine levels, and an increased mortality rate after h n infection . recently, increasing data showed that elevated concentrations of inflammatory cytokines/chemokines (especially il- , il- , mip- β , ip- , mif, scf, mcp- , and hgf) in the infected lung and plasma (hypercytokinaemia) are highly positively linked to disease severity in h n infected patients [ ] [ ] [ ] [ ] . cytokine production is closely related to the severity of host lesions due to influenza virus infection [ ] [ ] [ ] . proper cytokine expression is necessary for disease recovery, defending against virus infection, and recruiting inflammatory cells to the sites of infection through a delicate balance between pro-and anti-inflammatory mediators . however, cytokine over-expression breaks the immunologic balance, which causes systemic inflammation, acute organ dysfunction and even death , , . h n -infected patients present the manifestations of acute respiratory disease syndrome (ards) and hypercytokinaemia , , which also were observed in the present case. in the present study, the h n patient displayed the highest levels of ip- , il- , il- p and mcp- secretion in the serum obtained during the acute phase of the disease ( d.a.o.) . notably, the elevation of the chemokine ip- was the most robust among all of the detected cytokines and chemokines. because ip- is a critical player in the induction of lung injury, its upregulation in h n infection might be positively linked to disease severity, and this molecule could be a sensitive outcome predictor . although the case number was limited in our study to compare the serum cytokine levels between h n and h n infected patients, a similar profile of mediator secretion patterns in h n and h n infected patients has been seen by other groups with more influenza a patients , . hence, the higher level of cytokine secretion might explain the more severe syndromes caused by h n virus infection than by the contemporaneous h n virus. it should be mentioned that there are also some different changes between our study and other studies in the cytokines for the h n infections , [ ] [ ] [ ] [ ] . this finding might be related to the individual differences of host immune responses and the virus pathogenic characteristics, which should be further determined with more sample cases in the future. although this was a severe h n -infected case with cytokine storm-like appearances and multiple organ failure, the patient was eventually cured after combination therapy with antivirals, mechanical ventilation, supportive nutrition and symptomatic treatment. in our study, the -year-old patient was not prescribed oseltamivir until day d.a.o. because the h n virus was not identified at the onset of his illness. according to the clinical finding reports of the h n -infected patients in , the median time of the initiated antiviral therapy was days after the onset of illness . although it is difficult to identify, diagnose and initiate antiviral therapy within d.a.o., neuraminidase inhibitors should be employed as soon as possible (ideally, within hours following symptom onset) to maximise the therapeutic benefits and reduce the incidence of severe illness. to achieve this purpose, we need to further optimize the diagnostic tools for influenza-infection detection. a previous study showed that the early and rapid induction of nab was correlated significantly with better clinical outcomes , . as shown in our study, the seroconversion of nab in this h n case appeared on day d.a.o., and the mn titres increased rapidly until reaching a plateau at ~ d.a.o. with a significantly high level ( : ~ : ). hence, the quickly increased nab level probably contributed to the virus clearance and the patient's recovery. the h n influenza virus is spreading, evolving and becoming widespread among chickens in china . fortunately, the virus has not completely obtained the ability for human-like receptor binding and human-to-human transmission. moreover, the h n virus still mainly originates and evolves in avian species; therefore, it is possible to intercept the interspecies transmission by controlling or standardizing live poultry trade. additionally, we should persistent in monitoring the gene evolution of the h n virus isolated from humans and avian species and should optimise diagnostic tools, develop antiviral drugs and spread effective combination therapies for h n infections. clinical samples and ethical approval. a suspected case of h n influenza virus infection was confirmed by a real-time rt-pcr assay in the beijing centers for disease control and prevention (cdc). the epidemiologic and clinical data were collected. a confirmed case was defined as evidence of pneumonia with h n viral rna or isolation of h n virus from respiratory specimens. informed consent was obtained from all participating individuals. this experimental protocol was approved by the local ethics committee of beijing ditan hospital, capital medical university. the methods were carried out in accordance with the approved guidelines. virology analyses. an oropharynx swab, collected on day after the onset of flu-like symptoms, was used for virus isolation. the virus was propagated in -day-old specific pathogen free (spf) embryonated chicken eggs and mdck cells for to hours at °c, respectively. for genetic analysis, the complete gene segments were amplified using improved primers (table s ) based on previous reports , and were sequenced by an abi xl automatic dna analyser (applied biosystems, foster city, ca, united states). genetic identification and homology of the isolate were performed using the blast method in ncbi. a phylogenetic analysis was constructed using the maximum likelihood method with mega (http://www.megasoftware.net). the genotype analysis of the h n virus was classified by the criteria previously described . neutralizing antibody and cytokine evaluation. sera from the h n patient were consecutively collected on , and d.a.o. for kinetic evaluations of the neutralizing antibodies and cytokine levels. a pandemic h n severe case, onset on february , was used as a control, and the serum was collected on d.a.o. the neutralizing antibodies (nabs) were detected by microneutralization assays with a/anhui/ / (h n ) and a/california/ / (h n ) viruses in mdck cells, according to the previously described method . the levels of cytokines in the h n patient's serum were detected by the bio-plex human cytokine panel (bio-rad laboratories, inc.) according to the instructions. clinical findings in cases of influenza a (h n ) virus infection mapping spread and risk of avian influenza a (h n ) in china dynamic reassortments and genetic heterogeneity of the human-infecting influenza a (h n ) virus genetic changes of reemerged influenza a(h n ) viruses sequential reassortments underlie diverse influenza h n genotypes in china dissemination, divergence and establishment of h n influenza viruses in china detection of mild to moderate influenza a/h n infection by china's national sentinel surveillance system for influenza-like illness: case series human infection with avian influenza a(h n ) virus re-emerges in china in winter surveillance of the first case of human avian influenza a (h n ) virus in beijing human infection with a novel avian-origin influenza a (h n ) virus fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia biological features of novel avian influenza a (h n ) virus the role of alpha/beta and gamma interferons in development of immunity to influenza a virus in mice kinetics of serological responses in influenza a(h n )-infected patients correlate with clinical outcome in china into the eye of the cytokine storm amino acid substitutions in polymerase basic protein gene contribute to the pathogenicity of the novel a/h n influenza virus in mammalian hosts human h n and h n influenza viruses differ in induction of cytokines and tissue tropism a single mutation in the pb -f of h n (hk/ ) and influenza a viruses contributes to increased virulence tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells computational assay of h n influenza neuraminidase reveals r k mutation reduces drug binding affinity editorial commentary: host and viral factors in emergent influenza virus infections early hypercytokinemia is associated with interferon-induced transmembrane protein- dysfunction and predictive of fatal h n infection clinical, virological and immunological features from patients infected with re-emergent avian-origin human h n influenza disease of varying severity in guangdong province the serum profile of hypercytokinemia factors identified in h n -infected patients can predict fatal outcomes a(h n ) virus results in early induction of proinflammatory cytokine responses in both human lung epithelial and endothelial cells and shows increased human adaptation compared with avian h n virus a broadly neutralizing human monoclonal antibody is effective against h n h n and pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice assessment of the internal genes of influenza a (h n ) virus contributing to high pathogenicity in mice characterizing and controlling the inflammatory network during influenza a virus infection immunoparalysis and adverse outcomes from critical illness correlation between serum tnf alpha and il levels and severity of group a streptococcal infections the cytokine storm and factors determining the sequence and severity of organ dysfunction in multiple organ dysfunction syndrome human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome cytokine and chemokine levels in patients infected with the novel avian influenza a (h n ) virus in china kinetic analysis of the immunity in a pregnant patient infected with avian influenza h n immune derangement occurs in patients with h n avian influenza avian-origin influenza a(h n ) infection in influenza a(h n )-affected areas of china: a serological study universal primer set for the full-length amplification of all influenza a viruses reliable universal rt-pcr assays for studying influenza polymerase subunit gene sequences from all haemagglutinin subtypes risk perceptions for avian influenza virus infection among poultry workers this study was supported by grants from the national natural science foundation of china (nos and gfg is a leading principal investigator of the nsfc innovative research group ( ). we would like to thank pro. hui zeng and xingwang li for their professional comments to the study. we are also grateful for the contributions of dr. linghang wang from beijing ditan hospital, capital medical university for his comments on sample processing and clinical data collection. this study was conceived and designed by y.b. and b.w., who lead the research groups on the pathogenesis of pathogenic microbes. y.b. and d.l. performed the virus isolation, phylogenesis, antibody and cytokine analyses. j.l., h.x. and y.z. were in charge of the case recruitment, sample and clinical data collection. y.b., d.l. and b.w. performed the data analyses, prepared the manuscript, and completed the revisions. g.f.g. and y.l. provided helpful suggestions regarding the study. all authors read and approved the final manuscript. key: cord- -kw psrmz authors: beniac, daniel r.; booth, timothy f. title: structure of the ebola virus glycoprotein spike within the virion envelope at Å resolution date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: kw psrmz we present the structure of the surface ebola virus (ebov) trimeric glycoprotein (gp) spike at Å resolution, in situ within the viral plasma membrane of purified virus particles. gp functions in cellular attachment, endosomal entry, and membrane fusion to initiate infection, and is a key therapeutic target. nevertheless, only about half of the gp molecule has yet been solved to atomic resolution, excluding the mucin-like and transmembrane domains, and some of the glycans. fitting of the atomic resolution x-ray data from expressed, truncated deletion constructs within our Å structure of the entire molecule demonstrates the relationship between the gp -gp domains, the mucin-like and transmembrane domains, and the bilaminar lipid envelope. we show that the mucin-like domain covers the glycan cap and partially occludes the receptor binding sites prior to proteolytic cleavage. our structure is also consistent with key antibody neutralisation sites on gp being accessible prior to proteolysis. based on the findings of us and others, gp-mediated binding may create an angle of degrees between the planes of viral and endosomal membranes. truncated mutants without a transmembrane domain, or as smaller sub-domains of the gp molecule, or as artificial virus-like particles (vlps) , , , , [ ] [ ] [ ] [ ] . the structure presented in the current investigation is based solely on data from the entire glycosylated gp on the surface of ebov, using virions purified from ebov infection in cell culture, and not recombinant expressed versions of the gp spike. to establish a more definitive structure for the native spike within the ebov particle, we analysed purified bona-fide ebov in order to image the gp spike within the virion envelope (fig. a,c) . these gp spike images were analysed using the single particle method only (fig. s ,s ) , as a comparison to structures previously obtained by us and others using tomographic methods. discrepancies had been observed between the structures of the entire, untruncated ebov gp determined using material produced with differing heterologous expression systems, and between structures obtained using alternative tomographic or single-particle three-dimensional image processing methods , . due to safety concerns, the virus preparation was treated using paraformaldehyde crosslinking (after centrifugation) in a protocol that has previously been shown to preserve protein and lipid structures , . ebola virions are flexible, and viral filaments are frequently curved when prepared in the frozen-hydrated state for cryo-electron microscopy (fig. b) . therefore, regions of virions that were as straight as possible were selected for image processing (fig. c) . our data included , individual spike images for single particle analysis. in addition, , images were selected for reference-free analysis of the half-diameter of ebov to investigate the spatial distribution of the gp spikes, as well as the periodicity and symmetrical relationships between gp and the matrix protein vp in the envelope, and the underlying nucleocapsid layer (figs c and s ) . a -d structure for the gp spike trimer in situ, within the viral envelope, with a resolution of Å was calculated (figs and , supplementary fig. s ) using the projection-matching procedure on masked images , . we were able to dock the atomic structure of the ebov gp ( jq ) containing the full-length gp and gp domains, but with the mucin-like domains truncated (fig. b) . this clearly demonstrates a number of features of the viral-derived spike, including the structure and arrangement of the mucin-like domains with respect to the gp -gp structure. in particular, the base of the spike, consisting of the gp fusion domains with the heptad repeat motifs, fits extremely well: the atomic resolution structure jq fills the volume of our -d cryo-em structure (fig. a) . the closeness of fit, especially in the stalk or "neck" area of the gp trimer, is apparent when the spike structure is displayed at a density where the nm thick bilayer nature of the virion envelope is clearly visible (figs a,c and a) and also when the surface is truncated to a level where the volume approximates to a molecular weight of kda (figs and b) . the alpha-helices of gp are visible at the base of the spike, (fig. a) and when the reconstruction is displayed at a slightly higher contour level, densities that cross the interior of the membrane are visible (fig. c) . since the n-terminal ends of the docked alpha- gp domains ( ebo ) appear to line up with these transmembrane densities, the latter may be an indication of the putative hydrophobic alpha-helical transmembrane regions of gp (fig. c ). using the predicted mass of the gp (as measured by gel electrophoresis of virion-derived gp ) we adjusted the volume of the -d structure in fig. , using a value of . da/Å as an approximate density for protein . since the structure jq is kda , the truncated mucin-like and transmembrane domains, including glycans, represent ~ - kda, approximately half the mass of the spike. when viewed from above, the gp spike looks like a three-bladed propeller (figs a and b) . the length of the three "blades" encompasses a circle that is nm in diameter. when viewed from the side, the gp spike has a stalk region adjacent to the viral envelope, which then spreads out to the upper part of the gp spike. the toremifene-binding "pocket" or "tunnel" identified in jq is near the surface of the -d em structure, on the side of the stalk at the base of the propellers (figs a and b) . at the base of the stalk in fig. a , the three heptad repeat helices at the base of gp fit neatly into three strands visible in our em -d structure, that appear to penetrate the virion membrane (figs a and a) . also, when viewed from the top, each blade has a smaller nub closer to the -fold axis that protrudes distally (figs a and b ). using the kda contour level, these nubs correspond closely with the receptor binding site, covering most of the residues known to be involved in binding, as well as protruding adjacent to the glycan cap regions. each propeller blade, which is known to contain the mucin-like domain , completely covers the site known to bind to the npc- receptor (fig. b) . thus approximately half of the mass in our structure, external to the envelope, are the mucin-like domains. when the density map is contoured to a level that removes the lipid bilayer from display, the stalk still conforms closely to the surface of the atomic resolution structure, whereas there distal ends of the blades and the glycan cap "nubs" are slightly truncated. this indicates that these regions of the structure probably have a lower density, consistent with their being highly glycosylated, as predicted by the amino acid sequence of the mucin-like domain.we also docked the jnx structure , which includes part of the cleaved glycoprotein (gpcl) in complex with full-length human npc , and and jnx (npc -gp, magenta) were docked within the ebola gp using chimera (gp trimer subunits coloured in green, blue and yellow; npc -gp in magenta). the cryo-em reconstruction is presented at a threshold to show the viral envelope, and using the same colour code as in fig. . (b) the gp spike reconstruction is presented at a threshold equivalent to its molecular mass to illustrate the tight fit of jq . the inset shows the location of toremifene (red), and the residues of the receptor-binding site are coloured red. (c) the atomic resolution structures of the neutralizing antibodies kz fab ( csy, beige), mr fab, ( x d, pink) were docked within the ebola gp using chimera. overlaid the atomic resolution structure onto one monomer using the program module "fit in map" in ucsf chimera (fig. a) . this indicates that the mucin-like domain, (all of the unoccupied density remaining in the -d em structure when gp -gp are fitted) completely covers the glycan cap, with the nub at the side of the propeller covering most, if not all, of the receptor binding site (fig. a) , indicating that removal or cleavage of the mucin-like domain (probably including the nub), as well as the glycan cap, may be prerequisites for receptor binding to be achieved. the docked jnx structure also includes the transmembrane region of npc , and thus we aligned the approximate plane of the plasma membrane when the gp spike docks with the receptor npc , shown in transparent blue in fig. a . in the absence of any bending of endosomal membrane to accommodate the contour of the gp trimer and npc complex, the plane of the plasma membrane would make an angle of about degrees from the viral membrane when the spike is perpendicular to the viral envelope (fig. a) . this is consistent with the suggestion by gong et al. that perhaps only one npc -receptor binding site out of three on each trimer can be occupied at once, and that binding to more than one receptor might be precluded due to stearic interference. the possible line of cleavage delineating the mucin-like domain and glycan cap from the rest of the trimer is shown in fig. a and b. our structure suggests that the mucin-like domain and the glycan cap might be cleaved at the same time, since the latter is entirely within the density of the putative mucin-like domain, and is consistent with a molecular weight for the mucin-like domain of about kda, which is similar to that predicted by the sequence as well as gel electrophoresis, although the length of the sugar side-chains is still unknown. in addition, we fitted the atomic-resolution fab antibody-gp structures that bind to each of the two major ebov gp neutralising epitopes that have been previously investigated, mr ( x d ) and kz ( csy ): (fig. c) . this indicates that, within the limitations of the resolution achieved in our -d structure of the spike in situ in the envelope, the putative mucin-like domain (likely to consist of the propeller blade and possibly the nub features described here) is unlikely to interfere or stearically hinder either of these antibody neutralisation sites, consistent with neutralisation being possible prior to cleavage of the mucin-like domain in the endosome. it is clear that our structure differs from the -d em structures of the heterologously-expressed ebola gp spike observed in situ in the plasma membrane previously published by us and others (fig. ) . both of these structures included engineered expressed material using mutated or partially sequence-truncated proteins made in virus-like particles. the spike structure of beniac et al imaged within the virus-like particles was somehow clipped, since this should have included the mucin-like domains: in addition, tomography was combined with single-particle analysis, which may have distorted the results . the structure that we report here is based entirely on the well-accepted method of single-particle analysis using projection matching, and is broadly similar to that published of expressed gp in virus-like particles using tomography , : there are noticeable differences in shape and size of the spike, as well as in resolution (fig. ) . the previously determined atomic resolution structures of gp appear to fit well within the cryo-em structure we report here (figs and ) and the resolution, although modest ( Å), is an improvement compared to ~ Å for the structures generated from tomography alone that appear to have a slightly different shape (fig. ). while our structure shows a spike length of nm and a stalk length of nm and width of . nm, the tomographic structures show a spike length of nm, with a more pinched, shorter and more narrow stalk region of . nm in length and nm width, while the virion envelope at the base also appears to be sloped away from the gp stalk (fig. ) . our structure clearly delineates the nm bilayer of the virion envelope (that has an internal spacing of about Å, fig. a ) as well as the alpha helices of the heptad repeat domains (that have a diameter of about Å) and the nub feature at the base of the propellers, whereas these features were not discernable in the previous structures , (fig. b) . these inconsistencies could be due to the differences between the expressed and viral materials, the latter of which was used in the present report. differences in the methods used could also be significant, and a factor in the improved resolution was the use of an optimally sized t-shaped mask, and the selection of straight regions of the virion membrane for analysis (supplementary figure s ) . this allowed accurate alignment and selection of images taken at right-angles to the three-fold axis of the spikes, while getting an excellent coverage of side views, since the spikes are randomly distributed on their -fold axes, using euler angles of - degrees of rotation around the z-axis (since it is a trimer). thus, we avoided the "missing wedge" of information associated with tomography, where the constraint of tilt angle limits the angular distribution of views. our analyses did not reveal any longitudinal symmetry or well-ordered periodicity of the gp spikes along the axis of the virion filament, showing that the spike arrangement of virions may not follow a rigid symmetry. nor was any longitudinal symmetry apparent in the virion vp matrix layer (although a nm lattice spacing in the vp layer has been observed in images showing a perpendicular view of the vlp membrane ). we have shown that the gp spikes of ebov virions likely have an inconsistent or variable spacing, similar to that previously shown for ebov vlps (supplementary fig s ) . we also confirmed previous results showing that the nucleocapsid layer of virions maintains a consistent ordered longitudinal spacing of about nm (supplementary fig s ) , despite the curves and bends of the filament. thus any contacts between gp and vp and/or the nucleocapsid proteins may be variable and non-equivalent: or if a preferred alignment and stoichiometry exists, it was not detectable by cryo-em of whole virions with our current data. in conclusion, the current structure is consistent with the mucin-like domain occluding the glycan cap and receptor-binding region, such that cleavage is required for the functioning of the latter in the endosome to reveal the receptor binding site on gp , , [ ] [ ] [ ] . it is likely that the cleavage of the glycan cap also includes removal of both the "propeller" and "nub" structures described here. it is clear that the density in our structure partially covers the receptor binding domain, and would likely inhibit npc- binding until cleavage of the mucin-like domain occurs. further high resolution studies of the viral-derived gp structure and virion particles are needed to answer these questions, and for progressing knowledge of ebov morphogenesis. in future studies, analyses of the spatial arrangement of the spikes in the membrane, the structural arrangement of the transmembrane and cytoplasmic domains, and analysis of flexible, quasi-equivalent connections between the envelope matrix protein vp and the nucleocapsid, will all be important to further our understanding of how these viral components function in the replication cycle of ebov. scientific reports | : | doi: . /srep methods cells and viruses. zaire ebola virus (mayinga strain) was produced in vero (e ) culture and purified and concentrated by centrifugation . viral preparations were characterised using polyacryalamide gel electropheresis and western blotting. samples were inactivated by using paraformaldehyde fixation ( %) followed by dialysis against pbs to remove the excess. dialysis was carried out using a . ml slide-a-lyzer g cassette ( , mwco: thermo scientific, usa). all virus culture and purification, and handling of infectious materials was carried out at the national microbiology laboratory within the biosafety level laboratories in winnipeg, canada. virus specimens ( ul) were plunge-frozen on glow-discharged quantifoil grids ( um holes, at um intervals: quantifoil, germany). as a focussing aid, a bsa-colloidal gold suspension ( nm particles: aurion, the netherlands) was added to virus preparations at a rate of per cent by volume. freezing was carried out using liquid ethane as a cryogen with a vitrobot (mark iv: fei company, usa). images were taken at kv with a tecnai g electron microscope (fei) using a gatan ct tr single tilt rotation specimen holder at − °c. data was recorded with an eagle k ccd camera (fei company, usa) at , x magnification at - um defocus, with a dose of electrons/Å . this gave a pixel size of . Å at the ccd chip. image recording used the automated low-dose tia software (fei company, usa). xplore d data acquisition software (fei company, usa) was used to automate eucentric height and focus. image processing. particle selection was carried out using eman, and correction for contrast transfer function (ctf) was made using eman . image processing and -d structures used the spider and web programs . analyses were carried out on a mac pro -core computer (apple inc, with . ghzintel xeon nehalem processors, gb ram, running os x . . ) and on a dell -core power edge (r , -bit xeon x . ghz cpus, gb ram, running on linux os . ). correction of images for the ctf was carried out with the "e ctf.py" module in eman this estimated defocus, and used phase-flipping to correct for the ctf. spike images (n = , ), and half-width images of the virus (n = , ) were selected for image analysis. in all subsequent sections, spider software was used for image processing except where stated otherwise. methods are described and illustrated in supplementary figs s -s . resolution of the final cryo-em structure was estimated using the fourier shell correlation . criterion (supplementary fig. s ). single particle image analysis: half width of virus images. specific image regions were analysed for single particle image processing. two-dimensional masks were generated in the canvas x software package (acd systems, seattle, washington, usa). masks were imported as tiff files for image processing in spider. reference-free classification, to bring all images into register, was carried out with the eman software package. the class average was then used as reference to bring all the images into rotational and translational register. these pre-aligned images were then used with a series of masks for various regions of interest, both including the lipid bilayer, and with this region masked. eman was again used to perform a reference free classification, and the averages generated were processed in spider to perform a multi-reference alignment. the results of these analyses were then applied to both masked and unmasked images to investigate effect of the masking procedure and periodicity of the different layers including the spikes, viral envelope and nucleocapsid. single particle image analysis: gp spike analysis. the masking described for -d image analysis was adapted to -d projection matching of ebov spike images ( supplementary figs s -s ) . for -d processing, images were pre-aligned to a global average by reference-free classification in eman, followed by data alignment to this average in spider. pre-aligned images were then masked with an upside down "t"-shaped mask, thus selecting image regions containing both the lipid bilayer and a single spike (supplementary fig s ) . a dual set images was then sub-filed, one masked with the "t"-shaped mask, and the other unmasked. the d reconstruction of the gp spike was then generated by projection matching, using methods similar to those previously used with other viral spikes within their envelopes , . the main difference was the use of "t"-masked images for two-dimensional analysis and alignment, and unmasked images to apply the alignment parameters to generate new image averages. in addition a cylindrical mask was used on the -d reference volume. in both cases, the -d and -d masks selected data from the envelope and spike, and suppressed noise from adjacent spikes, as illustrated in supplementary figs s and s . structure visualisation and docking atomic resolution data. the d cryo-em data, including docked atomic resolution structures csy, x d, and ebo were visualised with the chimera software package (computer graphics laboratory, university of california, san francisco, supported by nih p rr- ). data and materials availability. the -d electron microscopy structure of the gp spike has been deposited into the electron microscopy data bank, www.emdatabank.org (accession number emd- ). marburg and ebola 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glycoprotein-dependent manner cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection toremifene interacts with and destabilizes the ebola virus glycoprotein structural basis for marburg virus neutralization by a cross-reactive human antibody structures of protective antibodies reveal sites of vulnerability on ebola virus biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity structure of the ebola virus glycoprotein bound to an antibody from a human survivor ebola virus entry requires the host-programmed recognition of an intracellular receptor role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein endosomal proteolysis of the ebola virus glycoprotein is necessary for infection the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding role of ext and glycosaminoglycans in 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determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases the potential and limitations of neutrons, electrons and x-rays for atomic resolution microscopy of unstained biological molecules structure of glycosylated npc luminal domain c reveals insights into npc and ebola virus interactions structural insights into the niemann-pick c (npc )-mediated cholesterol transfer and ebola infection replication-deficient ebolavirus as a vaccine candidate eman: semiautomated software for high-resolution single-particle reconstructions spider and web: processing and visualization of images in d electron microscopy and related fields architecture of the sars coronavirus prefusion spike ucsf chimera-a visualization system for exploratory research and analysis we would like to thank the canadian government genomics research and development initiative for funding, dr. steven jones (national microbiology laboratory, canada) for providing us with ebov and shannon hiebert for excellent technical assistance. key: cord- - s hpakk authors: kwok, hoi-hin; poon, po-ying; fok, siu-ping; ying-kit yue, patrick; mak, nai-ki; chan, michael chi-wai; peiris, joseph sriyal malik; wong, ricky ngok-shun title: anti-inflammatory effects of indirubin derivatives on influenza a virus-infected human pulmonary microvascular endothelial cells date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: s hpakk influenza a virus (iav) poses global threats to human health. acute respiratory distress syndrome and multi-organ dysfunction are major complications in patients with severe influenza infection. this may be explained by the recent studies which highlighted the role of the pulmonary endothelium as the center of innate immune cells recruitment and excessive pro-inflammatory cytokines production. in this report, we examined the potential immunomodulatory effects of two indirubin derivatives, indirubin- ′-( , -dihydroxypropyl)-oximether (e ) and indirubin- ′-oxime (e ), on iav (h n ) infected-human pulmonary microvascular endothelial cells (hpmecs). infection of h n on hpmecs induced a high level of chemokines and cytokines production including ip- , rantes, il- , ifn-β and ifn-γ . post-treatment of e or e could significantly suppress the production of these cytokines. h n infection rapidly triggered the activation of innate immunity through phosphorylation of signaling molecules including mitogen-activated protein kinases (mapks) and signal transducer and activator of transcription (stat) proteins. using specific inhibitors or small-interfering rna, we confirmed that indirubin derivatives can suppress h n -induced cytokines production through mapks and stat signaling pathways. these results underscore the immunomodulatory effects of indirubin derivatives on pulmonary endothelium and its therapeutic potential on iav-infection. scientific reports | : | doi: . /srep manufacturer's instruction. complementary dna was synthesized from dnase-treated total rna using superscript ii first-strand synthesis system (invitrogen). the relative expression of target gene was quantified by real-time rt-pcr using kapa sybr fast abi prism qpcr kit (kapa biosystems, woburn, ma, usa) and detected by a steponeplus real-time pcr system (applied biosystems, foster city, ca, usa). the relative expression of target gene was normalized by the level of glyceraldehyde- -phosphate dehydrogenase (gapdh), and then calculated by the comparative ct method. plaque assay. the virus titers were determined by standard plaque assay on madin-darby canine kidney (mdck) cells. in brief, mdck cells were grown in mem and seeded onto -well plates. diluted cell culture medium from influenza virus-infected hpmecs were added to the confluent mdck cells monolayers for h. then, the inoculum was removed, and a mixture of agarose ( %, w/v) containing l-(tosylamido- -phenyl) ethyl chloromethyl ketone (tpck) ( μ g/ml) was added onto the mdck cells monolayers. after h of incubation, the plate was fixed by formaldehyde ( %, v/v) overnight and then the agarose was discarded. the plaques were counted after staining with crystal violet ( . %, w/v). were prepared by ne-per nuclear and cytoplasmic extraction reagents (thermo scientific, rockford, il, usa) according to the manufacturer's protocol. for extraction of whole-cell lysate, cells were lysed by cytobuster tm protein extraction reagent (novagen, madison, wi, usa) containing protease ( . %, v/v) and phosphatase inhibitor cocktails ( . %, v/v) (calbiochem, san diego, ca, usa). the cell lysate was collected after centrifugation. protein concentration of the sample was determined by the detergent-compatible protein assay (bio-rad, hercules, ca, usa). equal amounts of protein were loaded and separated by % sds-page followed by electroblotting onto nitrocellulose membrane. the membrane was soaked in blocking buffer ( % non-fat milk in tbs-t, w/v) and then incubated with specific primary antibodies overnight at °c and secondary antibody for h at room temperature. immunoreactive bands were visualized using supersignal west pico kit (thermo scientific). in vitro mitogen-activated protein kinases assay. to detect the activity of individual mapks after treatment with iav and indirubin derivatives, the non-radioactive in vitro protein kinase assay kit from cell signaling technology was used. in brief, the sepharose bead-immobilized antibody was used to immunoprecipitate active mapks from an equal amount of total cell lysate ( μ g) overnight. the immunoprecipitate was washed twice with cell lysis buffer and kinase reaction buffer. the immunoprecipitate were then incubated with indirubin derivatives e or e ( μ m) for min before addition of atp. subsequently, kinase reactions using corresponding protein substrate were performed at °c for min. the kinase reaction was stopped with sds loading buffer. phosphorylation of protein substrate was detected by immunoblotting with specific antibody. immunofluorescence microscopy. hpmecs at a density × were seeded onto a glass coverslip in a -well plate. after treatment for the indicated time, cells were fixed with % paraformaldehyde for min at room temperature. cells were incubated with primary antibody ( : dilution rabbit anti-phospho-stat (tyr ) antibody) overnight at °c. the coverslip was washed and then incubated with fitc-conjugated goat anti-rabbit secondary antibody ( : dilution) (invitrogen) for h at room temperature. nuclei were visualized by staining with dapi ( . μ g/ml). the coverslip was washed and mounted on a slide using dako fluorescence mounting medium (carpinteria, ca, usa). fluorescence image was captured by the olympus fluoview fv confocal laser-scanning microscope (tokyo, japan). small interfering rna (sirna) transfection. transfection of sirna was performed using lipofectamine rnaimax (invitrogen). non-targeting-sirna ( nm) was used in parallel with stat -specific sirna ( nm) (ambion, austin, tx, usa). cells plated at % confluence were transfected in opti-mem medium (gibco brl, grand island, ny, usa) for h. after transfection, cells were rinsed with opti-mem prior to further treatment. all results were expressed as mean ± standard derivation (s.d.) of at least independent experiments. statistical significance between groups was determined by one-way anova with tukey's post hoc test. p < . was considered to be statistically significant. influenza a virus h n is a potent inducer of cytokines production in pulmonary endothelial cells. recent studies suggested that lung endothelium is the central regulator of cytokine amplification during influenza a virus infection, while dysregulation of cytokines production may result in systemic inflammation . in this study, we found that the infection of influenza a virus subtype a/quali/hong kong/g / (h n ) on hpmecs induced excessive production of various pro-inflammatory cytokines and chemokines, including ip- ( to examine the immunomodulatory effects of indirubin derivatives, hpmecs were infected with h n for h followed by incubation with indirubin derivatives e or e for another h. we have tested the cytotoxicities of indirubin derivatives in hpmecs prior to the elisa. as shown in fig. was observed at or below μ m in hpmces. next, indirubin derivatives were found to suppress h n -induced ip- ( fig. a) , rantes (fig. b ) and il- ( fig. c ) expression in a concentration-related manner. e significantly inhibited cytokines expression at μ m, a similar inhibitory effect was observed when a higher concentration of e ( μ m) was used. both indirubin derivatives slightly induced and inhibited the basal level of rantes and il- respectively. it may be due to the regulatory effects of indirubins on innate immunity . for . similar to the results of direct virus infection, viral rna stimulated cytokines expressions, and the inhibitory effects of mapks inhibitors were very similar to the experiments in direct infection. this confirmed that the suppression effects of mapks inhibitors on cytokines expression were not due to their potential effects on viral load. as a result, the anti-inflammatory effects of indirubin derivatives are mainly due to its inhibitory effects on different kinases. in contrast, stat -specific sirna had no effects on ip- , rantes and il- production induced by h n infection in hpmecs (fig. d-f) . time-course experiments showed that h n rapidly induced p and jnk phosphorylation in min after addition of the virus, then a second wave of p and jnk phosphorylation were induced at h.p.i. and h.p.i., respectively (fig. a,b) . no activation of erk was found after h n infection. similar to the early phosphorylation of stress-related mapks, stat was phosphorylated early in min after addition of the virus. however, the second wave of stat phosphorylation was found at h.p.i. taken together, these results suggested that h n infection on hpmecs could activate p , jnk and stat signaling pathways rapidly, and the expression of cytokines including ip- , rantes and il- were mainly due to the activation of p and jnk. indirubin derivatives suppress h n -induced cytokines expression through direct inhibition of p and jnk activity. to study the underlying mechanism of the anti-inflammatory effects of indirubin derivatives, hpmecs were treated with indirubin derivatives e or e after h n infection. as shown in fig. , e can significantly reduce h n -induced phosphorylation of p (fig. a) and jnk (fig. b ) at and h.p.i., respectively, and e demonstrated a more potent effect than e . it has been suggested that indirubin and its derivatives are potent inhibitors of various kinases, including mapks. in vitro kinase assay on p and jnk showed that e but not e inhibited p and jnk kinases activity. this action was reflected by the reduced phosphorylation of their direct downstream substrates atf (fig. c ) and c-jun (fig. d) respectively. indirubin derivatives prevent h n -induced ifn-β expression through inhibition of stat phosphorylation and nuclear translocation. though we found no relationship between stat and h n -induced ip- , rantes and il- expressions (fig. d-f) , stat signaling pathway is indispensable for the induction of interferons. we showed that knockdown of stat strongly inhibited h n -induced ifn-β (fig. b ) mrna expression. to elucidate the inhibitory effects of e on stat , western blot analysis was performed. treatment with e or e inhibited h n -induced stat tyrosine phosphorylation (fig. c ). upon activation, stat forms homo-or heterodimers that translocate to the nucleus. hpmecs fractionation of nucleus and cytoplasm was obtained by means of subcellular fractionation followed by western blot analysis. the results showed that h n infection increased phosphorylated stat in the nuclear fraction, while treatment with e significantly reduces the nuclear translocation (fig. d) . similar to the result of western blot analysis, the confocal image also showed that increased fluorescence signal was found in the nucleus after h n infection in hpmecs (fig. e) . treatment with e reduced stat fluorescence signal in the nucleus. the emergence of iav poses a serious global threat to human health. besides regular epidemic outbreaks, severe pandemics like the spanish flu and the more recent swine flu had caused enormous social and economic burden. current treatment of iav infection by m -ion channel inhibitors or na inhibitors emerged high frequency of resistance, and the efficacy and effectiveness of these antiviral drugs are limited by disappointing success rate , so alternative or complementary therapies that modulate the signaling pathways utilized by iav came into focus. in this report, we demonstrated that indirubin derivatives, particularly e is a potent immunomodulatory compound for iav-infection in vitro by inhibiting intracellular signaling pathways in pulmonary endothelial cells. during the early stage of iav infection, innate immune cells are recruited to the site of infection and are associated with an overwhelming production of pro-inflammatory cytokines and chemokines. endothelial cells in the pulmonary vasculature form a barrier between the blood and interstitium. this strategic position suggests that pulmonary endothelial cells are prone to be affected by the cytokines and viral particles released from the iav-infected epithelial cells. a recent study by teijaro et al. identified endothelial cells as the central orchestrator which contribute to the aberrant pro-inflammatory cytokine and chemokine production during early iav infections . concomitant with our in vitro data, we showed that h n virus can efficiently infect hpmecs (fig. a,b) and induce a significant amount of ip- , rantes, il- , ifn-β and ifn-γ . ip- and rantes are the chemoattractants for leukocytes including t cells, nk cells, and granulocytes, while production of il- by endothelial cells initiates infiltration of neutrophils in the early phase of infection . these cytokines have been found histopathologically in the lungs (including epithelial and endothelial cells) of h n infected patients, who showed acute respiratory distress syndrome (ards) , and ards can be characterized by progressive pulmonary endothelial damage. it has been suggested that treatment with antibodies against ip- in h n infected mice can improve the survival rate and reduce acute lung injury . furthermore, suppression of early innate cytokine and chemokine production in the pulmonary endothelium can significantly improve survival of mice infected with lethal h n swine iav . these studies suggested that inhibition of cytokines production of the pulmonary endothelium is an attractive therapeutic strategy against iav-induced cytokine storm. since severe infection of the influenza virus triggers the activation of the innate immune response and sometimes results in the induction to a cytokine storm. in this study, we demonstrated the immunomodulatory effects of indirubin derivatives, particularly e on iav-infected pulmonary endothelial cells. over the past two decades, many studies have identified that indirubin derivatives are potent inhibitors of various kinases, including mapks , , src kinase , glycogen synthase kinase- β (gsk- β ) and cyclin-dependent kinases (cdks) . based on these findings, potential functions of indirubin derivatives have been proposed, including anti-inflammation , [ ] [ ] [ ] , anti-leukemia , antiviral , and angiosuppression , . crystal structure analysis revealed that indirubin can form three hydrogen bonds with the atp-binding pocket of cdks, thereby competitively inhibiting atp binding in the catalytic domain of cdks . the results from our in vitro kinase assay also demonstrated that e is a potent inhibitor of p and jnk (fig. c,d) . the cytokine elisa data also suggest that h n -induced ip- expression of hpmecs was dependent on p and jnk activation, while rantes and il- were controlled by jnk and p , respectively ( fig. a-c) . however, the western blot analysis showed that e could also inhibit phosphorylation of p (fig. a) and jnk (fig. b) , which mean upstream kinases of p and jnk may also be inhibited by e . mapks are important mediators of influenza-induced cytokine expression. in fact, p has been shown to regulate the stability of il- mrna . meanwhile, another study also indicated the critical function of p on ip- during viral infection . furthermore, inhibition of p by specific inhibitor sb in vivo can greatly diminish h n lethal infection . however, the role of jnk in iav infection has not been fully examined. nacken et al. elucidated that influenza viral rna induces jnk phosphorylation in an rig-i dependent manner, but the ns of iav has also an intrinsic jnk activating property . taken together, the potent inhibitory effect of p and jnk signaling pathways by e strongly correlates with its anti-inflammatory function. ifns are pro-inflammatory cytokines crucial for antiviral responses to iav infection. stat and stat are predominant and essential transcription factors of type i and ii interferons signaling pathway, but the role of stat activation after ifn binding remains controversial. undeniably, stat is indispensable for downstream signaling pathway of many other cytokines like il- , vegf or egf . our data showed that h n infection on hpmecs can significantly induce ifn-β and ifn-γ expression. interestingly, stat -specific sirna has no effect on h n -induced il- ( fig. f ) and ifn-γ (fig. b ), but strongly inhibit ifn-β expression level, indicating the involvement of stat in ifn-β induction. the western blot data showed that iav-infection could activate stat in early stage ( min after addition of virus) followed by another activation at h.p.i. (fig. ) . h n was found to upregulate tlr- and myd , which is critical to the induction of ifn-β . in line with this finding, the early activation of stat may function together with the downstream signaling molecules of tlr- , possibly the interferon regulatory factors (irfs), to induce rapid expression of ifn-β mrna at h.p.i. (fig. h) . and then the autocrine effect of ifn-β induces a further amplification of ifn-β expression and result in a cytokine storm. a previous study suggested that e could inhibit stat dimerization and subsequent dna binding . our translocation experiments clearly indicated that e could inhibit stat phosphorylation and nuclear translocation. since the induction of many pro-inflammatory cytokines requires type i interferon signaling, inhibition of ifn-β production by e may blunt the early induction of these cytokines. though ifn-β is a well-known antiviral cytokine, it is also involved in the pathogenesis of influenza infection. in vivo studies focusing on the s p receptor and p pathway also suggested that, even the iav-induced ifns were suppressed by an s p receptor agonist or p inhibitor , the survival rate of the infected mice could still be significantly improved if those strongly induced cytokines were suppressed. in many studies, increased viral load were concomitant of reduced interferons expressions. however, in the present study, viral titer did not further increased in indirubin derivatives treated cells (fig. b ) even ifnβ and ifn-γ were suppressed, the results indicated that suppression of cytokines produced by the infected pulmonary endothelium could reduce iav pathogenicity independent of the viral clearance. in fact, many kinases including cdk and mapks, which are suggested being involved in the influenza replication process are also the target of indirubin derivatives, this might explain the partial antiviral effect of indirubin derivatives in this model. meanwhile the phosphorylation and nuclear translocation of stat leaded to induction of ifn-β . indirubin derivatives particularly e is a potent inhibitor of p and jnk signaling pathways. e could also reduce the phosphorylation and nuclear translocation of stat . by inhibition of these signaling pathways, e could significantly suppress h n -induced cytokine burst in hpmecs. scientific reports | : | doi: . /srep combination therapies coupling with antiviral and immunomodulatory drugs have been investigated intensively . the encouraging results from in vitro and pre-clinical studies have led to an increased interest on this topic. however, to achieve the best clinical outcome, antiviral and immunomodulatory drugs should be administrated at the appropriate time during an infection. further understanding of the immune dynamic could allow us to design an optimum therapy strategy. in this report, we demonstrated for the first time, the potent immunomodulatory effects of indirubin derivatives on pulmonary endothelial cells and their therapeutic potential for iav-infection (fig. ) . as a result, the combinational effects of indirubin derivatives and antiviral drug in animal model warrant further investigation. perspectives on influenza evolution and the role of research structure and assembly of the influenza a virus ribonucleoprotein complex pathogenesis of influenza virus infections: 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cyclin-dependent kinase inhibitors an indirubin derivative, e , exhibits potent angiosuppressive activity automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish the p map kinase pathway signals for cytokine-induced mrna stabilization via map kinase-activated protein kinase and an au-rich region-targeted mechanism innate immune response to h n and h n influenza virus infection in a human lung organ culture model activation of c-jun n-terminal kinase upon influenza a virus (iav) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of iav ns stats in cancer inflammation and immunity: a leading role for stat human intestinal epithelial cells are susceptible to influenza virus subtype h n this work was supported by the area of excellence scheme of the university grants committee, hong kong sar government (aoe/m- / ) and dr. gilbert hung ginseng laboratory fund. key: cord- -d hnox authors: opuu, vaitea; silvert, martin; simonson, thomas title: computational design of fully overlapping coding schemes for protein pairs and triplets date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: d hnox gene pairs that overlap in their coding regions are rare except in viruses. they may occur transiently in gene creation and are of biotechnological interest. we have examined the possibility to encode an arbitrary pair of protein domains as a dual gene, with the shorter coding sequence completely embedded in the longer one. for × domain pairs (x, y), we computationally designed homologous pairs (x′, y′) coded this way, using an algorithm that provably maximizes the sequence similarity between (x′, y′) and (x, y). three schemes were considered, with x′ and y′ coded on the same or complementary strands. for % of the pairs, an overlapping coding exists where the level of homology of x′, y′ to the natural proteins represents an e-value of (− ) or better. thus, for an arbitrary domain pair, it is surprisingly easy to design homologous sequences that can be encoded as a fully-overlapping gene pair. the algorithm is general and was used to design triple genes, with three proteins encoded by the same dna segment. the ease of design suggests overlapping genes may have occurred frequently in evolution and could be readily used to compress or constrain artificial genomes. of a hypothetical antisense polypeptide. a special case of this hypothesis is that peptide ligands might have arisen for some proteins from the antisense strand complementary to their coding region , . in addition to their biological importance, overlapping genes could be of significant interest in biotechnology, to compress or constrain artificial genomes. in particular, a synthetic organism where several genes overlap would have fewer available neutral mutations, since each mutation in the overlapping region would affect more than one protein. thus, the organism would undergo reduced genetic drift and remain closer to its original, designed genotype. an example of a designed overlapping gene was produced recently, where each dna strand coded for a simplified but functional aminoacyl-trna synthetase "urzyme" [ ] [ ] [ ] [ ] , with the two enzymes being homologous to the two modern synthetase classes. we have developed a general method to design overlapping genes, with two main objectives: ( ) to expand our ability to engineer artificial genes and genomes and ( ) to help evaluate the importance of overlapping genes in evolution. indeed, to evaluate their role in evolution, we should have a precise idea of the difficulty to create them. the rarity of contemporary examples and the stringent overlap constraints suggest that it is very difficult. to quantify in a general way the difficulty to create overlapping genes, we have examined the possibility of encoding an arbitrary pair of protein domains in a single dna segment, as a fully-overlapping dual gene. we considered three possible reading frames for the second protein, relative to the first: two on the antisense strand and one on the sense strand. indeed, sense and antisense overlap schemes have different implications for the biological hypotheses above and different capabilities to code a variety of amino acid types , . one sense:antisense overlap scheme placed the third, wobble base in each codon opposite the central nucleotide of another codon (fig. ) . given the structure of the genetic code, this is expected to be the most favorable scheme, as confirmed below. another scheme placed the central bases of the sense and antisense codons opposite each other (f = scheme, fig. ). this is less favorable, since each central base constrains the other. the two sense:sense overlap schemes are similar and we chose one of them. we considered protein domains from the pfam database , - amino acids long, and all , corresponding domain pairs (x, y). of the proteins ( %) were viral proteins. for each (x, y) pair, we searched for two homologs (x′, y′) that can be encoded in a fully overlapping manner. we required that the coding sequence of the smaller domain be completely embedded within that of the larger one. for each pair, we explored three possible overlap schemes, where x′ and y′ are encoded either on the same dna strand in two different reading frames or on opposite strands. the search used a dynamic programming scheme , presented here, that provably maximizes a total x/x′ and y/y′ similarity score. the method's cost is proportional to the length of the shorter protein, allowing a large-scale study. considering all three reading frames, we found that over % of the (x, y) pairs have homologous pairs (x′, y′) that can be encoded in a fully overlapping manner, with a level of x/x′ and y/y′ homology corresponding to blast e-values of − or less and a match length of at least % of the total sequence length. the success rate was % for pairs of non-viral proteins and % for pairs of viral proteins. none of the viral pairs were natural pairs occurring within the same virus. thus, it appears that many pairs of protein domains have close homologs that can be encoded by a fully-overlapping dual gene and the tendency is especially high for viral proteins. this is in striking contrast to naive expectation. differences between the proteins that do or do not allow overlapping schemes are analyzed and discussed. given the level of success and the generality of our algorithm, we also designed triple genes, with three proteins encoded by the same dna segment using both strands and three different reading frames. all the designed proteins had e-values better than − against their natural homologs. the designs included triplets of bacterial proteins and triplets of viral proteins. such triple overlaps have been found in viruses , , , but the corresponding protein sequences may be unstructured, in contrast to our designed sequences. it has also been proposed that triple overlaps may have existed in ancestral ribosome sequences . the ease of design revealed here of both double and triple genes is consistent with the overprinting mechanism for gene creation and suggests that overlapping genes could have occurred frequently in evolution. to facilitate their use in artificial genomes, our design code is provided online (see github). designing overlapping homologs: goal of the algorithm. we start from a pair of protein domains, whose amino acid sequences we denote x and y. the goal is to determine homologous amino acid sequences x′, y′ such that the coding sequence of the smaller protein is entirely embedded within that of the larger protein, being coded either on the same dna strand in a different reading frame or on the opposite strand. the five choices of reading frames for y are shown in fig. . we choose a reading frame f = − , − , , , or for y, and a starting point for the smaller of the two proteins, such that its coding sequence will be entirely embedded within that of the larger protein. applying a sequence optimization algorithm described below, we obtain the protein sequences (x′, y′), which are as similar as possible to x, y and whose coding sequences completely overlap. the similarity is measured by a blosum similarity score, summed over the length of both proteins: is the i th amino acid of x (resp., y, x′, y′), and p i , q j are position-dependent weights that reflect sequence conservation among natural homologs of x and y. they are chosen so that positions in x, y that are highly conserved (respectively, variable) have high (low) weights (see below). designing overlapping homologs: an exact algorithm. we now describe the optimization scheme, starting with the case f = . in this reading frame, y′ is coded on the antisense strand in register with x′: each codon of y′ overlaps with a single x′ codon on the opposite strand. to maximize the similarity score (eq. ( )), we simply choose the optimal nucleotides for each pair of base-paired codons, by comparing the possible choices and picking the one that gives the largest contribution to s(x′, y′). in all the other reading frames, each codon of x′ and y′ overlaps with two other codons and another approach is needed. we consider the f = − case first. the dna sequence to be optimized is the region of x′, y′ overlap. we number the x′ codons in this region in the ′ → ′ direction (the direction of the polypeptide sequence). for each x′ codon c x (k), we denote c y (k) the y′ codon on the opposite strand that has two overlapping nucleotides. the two codons c x (k) and c y (k) define a quartet of nucleotides on the x′ strand, which we denote q k , shown in fig. a . the following quartet q k+ shares its first nucleotide with q k : q k+ ( ) = q k ( ), so that the sequences of any two consecutive quartets are linked . the key step is to express the dna sequence, not as a series of codons but as a series of quartets, whose sequences will be varied subject to the linkage constraint. this representation of the dna sequence is shown in fig. b . with this representation, the problem of maximizing s(x′, y′) is closely analogous to sequence alignment and can be solved by a similar recursive method, schematized in fig. . let n be the number of x′ codons, which is also the number of quartets. for any position k in the sequence, there are = possible quartets q, which can be subdivided into four groups of , depending on their last nucleotide: q( ) = a, c, g, or t. this nucleotide defines the "state" of the quartet: . the first step of our algorithm consists in filling a -by-n table of optimal scores, where each entry corresponds to a position in the sequence of quartets and a quartet state. we fill the table from left to right, one column at a time. to fill in column k, we assume column k − contains, for each state , the score of the optimal sequence that terminates in this state, say − m k ( ; )  . for column k, for any quartet q, an optimal sequence terminating with q is necessarily obtained by adding q to a sequence whose k − quartet has the state  = q( ). let s(q) be the contribution to s(x′, y′) of the two codons contained within a quartet q (and its complementary strand). we have the recursion: the maximum is taken over the quartets q j that have  as their last nucleotide (q j ∈ ). their optimal character is tested by adding s(q j ) to the score m k q the first nucleotide of each x codon appears twice in the sequence of quartets. only the x strand is shown, for simplicity. as we fill in entry m k ( ; )  in the table, we also tabulate the "optimal" quartet, called q k , that led to its score. initialization of the leftmost column is done by adding a column " " to the left of the table, filling it with zeroes, and applying ( ) to column k = . once the table is filled, we perform a traceback operation, analogous to sequence alignment. it consists in concatenating the optimal quartets backwards, with the first nucleotide q k ( ) of the each optimal quartet serving as a pointer to the state  we should move to in the previous column. the cost of the whole procedure is proportional to the length n of the overlapping sequences. for the reading frames other than f = − , the same method applies; only the positions of the x′ and y′ codons within each quartet q change, changing slightly the mechanics for calculating the score s(q). this method leads to the sequences x′, y′ that globally maximize the score s(x′, y′). the method also applies, with straightforward adjustments, to cases where each nucleotide quartet contains three or more overlapping codons; for example, a reference x codon, a second y codon on the opposite strand (f = − frame) and a third z codon on the same strand (f = frame). only the quartet scoring mechanics (eq. ( )) change slightly, with each codon in the quartet contributing a term to the score s(x′, y′, z′). the method can thus be used to design three, four, or even six overlapping genes. to design five or six overlapping genes (using all six reading frames at once), the coding sequence should be represented as a series of nucleotide quintets, instead of quartets. it is also straightforward to extend the algorithm to situations where one uses more than four nucleotide types, or codons that are four nucleotides long, instead of three , which could be of interest in synthetic biology, or still longer codons, which could be of interest for other types of information storage. below, we present the successful design of triple genes as an illustration. test data and calculations. the algorithm was tested on pfam protein domains, - amino acids long, from pfam families. within each family, the first protein was chosen. each domain was assigned a conservation pattern based on the pfam seed alignment of its family. specifically, we assigned to each position i of x a weight p i , inversely proportional to the exponential of the sequence entropy s i of the corresponding column . the entropy was computed not from the amino acid types, but from a simplified classification into six groups: the search algorithm was applied to all × domain pairs, in three possible reading frames. for the frames f = − and f = , we did two calculations per pair, with the beginning of the shorter protein x′ aligned with either end of y′. for the frame f = , we also did two calculations per pair; the beginning of the two proteins were aligned, and either y or x was in the f = frame (with the other protein in the f = frame). this led to a total of , optimization runs. the nucleotide sequences do not include a stop codon; we assume one can be added (to the shorter protein) by manually changing a few nucleotides. to evaluate the similarity between the homologs x′, y′ and the target proteins x, y, we did a blast search of the swissprot database using the homolog sequence x′ or y′ as the query, and retrieved the best e-value corresponding to the target protein family. this was not always the target protein x or y, but sometimes a natural homolog from the same pfam family. the corresponding e-values for x′ and y′ were compared, and the largest one, denoted e p (x′, y′), was taken as the homology metric. for the longer protein, say y, the non-overlapping part of the protein sequence was not included in the blast query; only the overlapping sequence was employed. we only considered a blast result if the match length was at least % of the length of the overlapping sequence region; all shorter blast matches were discarded. to evaluate the similarity further, we submitted each homolog to the superfamily library of hidden markov models , which each correspond to one family in the scop classification of protein domains , to determine if ( , )  of the best sequence that terminates in that state. to annotate the a state in column k, we consider the quartets on the right and select the one that leads to the maximal score, m k a ( , )  = . q j ( ) is the first nucleotide of quartet j. the pointer in each box (grey arrow) indicates by its slope which is the previous state; for example the pointer in the state g box on the left comes from state a at the very top of the k − column; the pointer in the state t box comes from the g state one line above in the k − column. the submitted sequence belongs to the correct scop family. only the shorter protein of the pair was tested. of the natural pfam domains, had a scop match according to superfamily; the superfamily tests were limited to their predicted homologs. for all , pairs of pfam proteins (x, y) and all three reading frames considered here, we used our search algorithm to find homologous sequences (x′, y′) that are coded by two fully overlapping dna sequences, with the shorter protein's coding sequence completely embedded in the longer one's. the similarity of the computed homologs to natural sequences from the same family as x or y was characterized by their blast e-values versus the swissprot sequence collection. for each pair and reading frame, we made two predictions, corresponding to two different positions of the x′ ′-terminus relative to y′, for a total of , predictions. blast hits were only counted if the match lengths represented at least % of the total sequence length. the fractions of pairs with low e-values are reported in table . histograms of e p values are shown in fig. . summing over all three reading frames, we obtained an e p (x′, y′) value of − or better for , pairs, representing . % of all pairs. for , pairs ( . %) the e p (x′, y′) value was − or better. for pairs of non-viral proteins, the success rate (e p (x′, y′) value of − or better) was . %. for pairs of viral proteins, it was especially high: . %. none of the viral pairs were naturally-existing pairs. for viral/non-viral pairs, it was %. the success rate was best in the f = − frame and poorest in the f = frame ( . % of pairs with −log e p > ), which both correspond to sense/antisense overlap. this was expected, given the structure of the genetic code and the relative importance of the central and wobble codon bases (see below). for the f = sense:sense overlap scheme, the success rate was intermediate: . % of pairs had −log e p > . similar results were obtained from superfamily searches. of the pfam domains considered here, were recognized by superfamily as similar to a scop family. we considered the protein pairs (x, y) where the shorter of the two, say x, is among these domains, and where the predicted homolog pair (x′, y′) has a low e p value. we submitted each homolog x′ to superfamily. notice that we only considered the shorter protein, since the longer protein y has non-overlapping amino acids that make it easier to recognize. thus, x′ represents a more stringent test. for all three reading frames, over % of the homologs x′ were correctly recognized by superfamily, further supporting their similarity to natural sequences. predictions corresponded to e p values better than − . three of the pairs were (x, x) "self " pairs involving three bacterial proteins (pfam families pf , pf , pf ). three were (x, y) pairs involving bacterial −log(e p ) range pair type a total number of pairs percentage of successful pairs overlap scheme the f = − sense:antisense overlap scheme was the most favorable above, although f = and f = also gave many hits. in prokaryotic genomes, a slight preference for f = − was noted in surviving overlapping genes. indeed, the coding properties of this scheme are especially favorable, and we analyze them briefly here. in this frame, each x′ codon has its middle nucleotide in register with a y′ wobble nucleotide, and vice versa. since the wobble nucleotide has little effect on the coded amino acid, this allows the middle nucleotide of each x′ and y′ codon to be chosen almost freely. figure shows two sense/antisense codons that share their first bases. we denote them  and . we denote the sense nucleotides , , and the antisense nucleotides ′, ′, ′. the possible amino acid choices for  and  are listed in table . if we classify the amino acid types into four classes: Π = (large, polar), Φ = (large, nonpolar), π = (small, polar), ϕ = (small, nonpolar), with four choices of class on each strand, there are possible / s a combinations. despite the base-pairing constraint, of them can be encoded freely in this overlap scheme. only the combination (π, π) cannot be encoded unambiguously. in contrast, the f = frame (central bases opposite) can only encode of × possible pairs of amino acid types. to identify sequence properties that favor gene overlap, we characterized the pfam proteins that were most "successful," participating most frequently in high scoring dual genes in the f = − frame (ones where the designed homologs had e p < − ), as well as the most successful viral proteins. we compared them to "unsuccessful" pfam proteins that gave no hits (all e p values > − ). we repeated the analysis for the f = and f = frames. the f = − and f = "successful" sets shared proteins out of , so we only present the f = − and f = sets. tables and and fig. summarize the results. the "successful" proteins are slightly longer on average and the corresponding pfam families are slightly less diverse, especially for the viral set (f = − scheme), with a mean entropy of . , vs. . for the unsuccessful set ( table ). the mean codon degeneracy and differences in codon usage are more striking, especially when comparing the most successful viral proteins (f = − scheme) to the least successful pfam proteins (fig. ) . in contrast, in the f = scheme (which gave fewer successful designs), the successful/unsuccessful differences are much smaller, with only six codon frequencies shifted by just over . . in the overall set of f = − successes amino acid classes produced by sense:antisense nucleotides nucleotide or ′ sense:antisense nucleotides needed to produce pairs of aa classes sense class antisense ( ′ ′ ′) aa class s, p = π ambiguous → a g :u ′ c ′ table . sense/antisense amino acid (aa) types for overlapping codons, f = − reading frame. the relation between the sense/antisense overlapping codons and the nucleotide numbers are defined in fig. . ( fig. , right), many of the viral trends are confirmed, even though this set contains just viral proteins out of . thus, there are codon choices that are more favorable for dual genes, and the corresponding trends are especially visible in the most successful viral proteins. finally, given the high success rate for dual gene design and the generality of our algorithm, we used it (with slight adjustments) to design triple genes, with three proteins x, y, z coded in a fully-overlapping manner. taking x as the reference, we chose to code y on the antisense strand in the f = − frame and z on the sense strand in the f = frame. we tested , triplets where the corresponding pairs gave high-scoring dual genes. this led to a high success rate: more than % of the triplets gave −log e p > . we obtained triplets where all three e-values (x′, y′, z' vs. x, y, z, respectively) were better than − . of these, were triplets of non-viral proteins, were triplets of viral proteins, and involved one viral and two non-viral proteins. the dna and protein sequences for one triplet are shown in fig. . x is bovine, y is viral, and z is a bovine tick protein. the e-values vs. swissprot for the predicted homologs x′, y′, and z' were, respectively, − , − , and − . we have shown that for a significant fraction of protein domain pairs, at least %, homologs with fully-overlapping coding sequences can be produced rather easily. this contrasts sharply with naive expectations based on the stringent overlap constraint. the fraction of successes obtained is only a lower bound, since we only considered one representative from each pfam family and only two positions for the x′ ′-terminus relative to y′. we also observed that while our algorithm provably maximizes the similarity score s(x′, y′), a heuristic search that does not reach the maximum can produce additional hits (in other words, maximizing s(x′, y′) does not always minimize e p (x′, y′)). however, our focus here was not to be exhaustive but to demonstrate that overlapping genes could be produced for close homologs of thousands of domain pairs. since the calculation was done for , unique pairs, we expect the results are general. to identify properties that favor overlapping genes, we analyzed the most successful and the least successful of our pfam proteins. the most striking differences were in codon usage, with codons out of significantly enriched or depleted within the most successful viral proteins, compared to the least successful proteins (with the f = − overlap scheme; fig. ). this may be the result of ancestral selective pressure for gene overlap, even though the modern viral proteins in our dataset do not overlap with each other. similar trends were found in our larger set of successful proteins, with both the f = − and f = overlap schemes. the f = − coding properties can be considered in the light of various hypotheses regarding simpler, ancestral genetic codes that would have contained fewer amino acid types or classes [ ] [ ] [ ] . if we express the sequences of the proteins x and y in the simplified alphabet {Φ, ϕ, Π, π}, the simplified sequences can be exactly expressed by a dual gene in the f = − frame, with the exception of the (π, π) pairs, which would sometimes be replaced by (π, Π) pairs. thus, in an ancestral world with a simpler {Φ, φ, Π, π} amino acid alphabet, arbitrary overlapping genes could be encoded almost perfectly thanks to the properties of the genetic code , exploited by the f = − reading frame. this would presumably have been favorable for genome compaction and consequently for viruses, which evolve essentially at fixed capsid volume. the ease of overlap coding also has implications for mechanisms of gene creation by "overprinting", in viruses or otherwise , . the success rate for designing overlapping coding schemes for the viral proteins considered here was remarkably high: almost % of the considered pairs gave designs with e-values better than − vs. the natural proteins. it could be of interest to test whether the modern genetic code is especially tolerant of overlapping genes, compared to ancestral codes (including competing codes that did not survive), or artificial codes (including codes that use larger or smaller amino acid alphabets or codons of a different length). this could be tested by repeating our calculations using one or more alternative codes and comparing the level of success obtained to the values reported above. such comparisons could help determine whether a high tolerance for overlapping coding schemes played a role in the selection of the genetic code. another hypothesis related to gene creation is the rodin-ohno hypothesis for the appearance of the two aminoacyl-trna synthetase classes, proposed to have occurred on the sense:antisence strands of an overlapping gene , . statistical evidence for such an ancestral dual gene was found by analyzing the dna sequences of modern aminoacyl-trna synthetases . a hypothetical dual gene that exhibits a weak similarity to both synthetase classes was discovered in the mold achlya klebsiana and several other organisms , . the recent design of two overlapping synthetase genes [ ] [ ] [ ] [ ] also provides some support for this hypothesis. our method should allow overlapping genes to be designed and produced readily for applications. this could be of technological interest, for example to produce a very compact plasmid or to prevent the fixation of mutations and genetic drift in a plasmid or a synthetic organism. given the surprising ease with which a dual gene can be designed, we also considered and verified the possibility of creating triple genes that use not two, but three of the six possible overlapping reading frames. with additional effort, other triple genes with even better e-values could be obtained. this could be a way to reduce genetic drift even more drastically, constraining an artifical organism to stay close to its original genotype. purifying and 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strands? is there a relationship between dna sequences encoding peptide ligands and their receptors? a minimal trprs catalytic domain supports sense/antisense ancestry of class i and ii aminoacyl-trna synthetases histidyl-trna urzymes class i and ii aminoacylt-trna urzymes have comparable catalytic activities for cognate amino acid activation aminoacylating urzymes challenge the rna world hypothesis functional class i and ii amino acid-activating enzymes can be coded by opposite strands of the same gene the combinatorics of overlapping genes the pfam protein families database biological sequence analysis sequencing of coronavirus ibv genomic rna: three open reading frames in the ′ "unique" region of mrna d the p gene of bovine parainfluenza virus expresses all three reading frames from a single mrna editing site the ribosome as a missing link in prebiotic evolution ii: ribosomes encode ribosomal proteins that bind to common regions of their own mrnas and rrnas natural mitochondrial proteolysis confirms transcription systematically exchanging/deleting nucleotides, peptides coded by expanded codons the superfamily database in : families and functions scop database in : refinements integrate structure and sequence family data an asymmetric underlying rule in the assignment of codons: possible clue to a quick early evolution of the genetic code via successive binary choices emergence of a code in the polymerization of amino acids along rna templates trna acceptor stem and anticodon bases form independent codes related to protein folding the origin of a novel gene through overprinting in escherichia coli two types of aminoacyl-trna synthetase could be originally encoded by complementary strands of the same nucleic acid the rodin-ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed statistical evaluation of the rodin-ohno hypothesis: sense/antisense coding of ancestral class i and ii aminoacyl-trna synthetases did trna synthetase classes arise on opposite strands of the same gene? molec no rosetta stone for a sense/antisense origin of aminoacyl-trna synthetase classes discussions with pierre plateau and sylvain blanquet were very helpful. t.s. conceived the project and calculations, derived algorithms, analyzed results, wrote manuscript. v.o. and m.s. derived and implemented algorithms, performed calculations, analyzed results, reviewed manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -shk n f authors: fahmy, ahmed m.; labonté, patrick title: the autophagy elongation complex (atg - / l ) positively regulates hcv replication and is required for wild-type membranous web formation date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: shk n f hepatitis c virus (hcv) infection induces intracellular membrane rearrangements, thus forming a membranous web (mw) in which hcv replication and assembly occur. the hcv-induced mw is primarily composed of double membrane vesicles (dmvs) transfused by multi-membrane vesicles. the autophagy machinery has been proposed to participate in the formation of such vesicles. however, no clear evidence has been found linking autophagy to the formation of these dmvs. in this study, we evaluated the role of the autophagy elongation complex (atg - / l ) in hcv replication and mw formation. using a dominant negative form of atg and an sirna approach, we demonstrated that the atg - conjugate, but not lc -ii formation, is crucial for efficient viral replication. furthermore, purification of hcv mw revealed the presence of atg - and atg l along with hcv nonstructural proteins. interestingly, lc was not recruited along with the elongation complex to the site of viral replication. finally, inhibition of the elongation complex, but not lc , greatly impaired the formation of the wild-type mw phenotype. to our knowledge, this study provides the first evidence of the involvement of autophagy proteins in the formation of wild-type mws. hepatitis c virus (hcv) infection is a leading cause of liver diseases, including cirrhosis and hepatocellular carcinoma. hcv, a member of the flaviviridae family, is a hepacivirus with a positive-strand rna genome . the virus replicates exclusively in the cytoplasm of the host cell. after cell entry, the . kb hcv genome is released and translated at the rough endoplasmic reticulum (rer) into a single polyprotein. this translated polyprotein is then proteolytically processed by cellular and viral proteases into distinct proteins consisting of structural (core, e , and e ) and nonstructural (p , ns , ns , ns a, ns b, ns a, and ns b) proteins . the expression of hcv proteins results in the induction of a major rearrangement of host cell membranes, thus leading to the formation of a complex membranous compartment termed the membranous web (mw), which favors viral rna replication and assembly , . this massive remodeling of the host cell membrane network is associated with all positive-strand rna viruses and is typically characterized by the generation of either convoluted membranes or double membrane vesicles (dmvs) [ ] [ ] [ ] [ ] . importantly, the hcv-induced mw is primarily composed of dmvs thus suggesting that autophagy plays a role in the construction of the hcv replication scaffold , . macroautophagy, referred to hereafter as autophagy, is a catabolic pathway that degrades proteins and organelles, thereby maintaining cell homeostasis and directing cell fate. during cellular stress such as amino acid starvation, autophagy is triggered, thereby forming an organelle called the autophagosome. the de novo formation of the autophagosome begins by initiation of the growth of a double-membraned phagophore that, by closing, sequesters cytoplasmic contents. the autophagosome then fuses with the lysosome, thus allowing the degradation of the intra-autophagosomal cargo by the action of lysosomal enzymes and the release of free amino acids and other products. this process is orchestrated by more than autophagy-related gene (atg) proteins and other autophagy-linked proteins . hcv does not perturb formation of the atg - / l complex. atg forms a conjugate with atg , but the monomeric forms of these two proteins have been shown to be nearly undetectable under normal conditions . we first tested whether this conjugation occurs in hcv-infected cells. the assessment of the atg protein by western blotting showed that monomeric atg ( kda) was undetectable in both infected and uninfected cells. atg was detected only in the atg - -conjugated form ( kda) (fig. a) . infection with hcv jfh strain was confirmed by detecting hcv ns protein using anti-ns antibody (fig. a) . the difficulty in detecting unconjugated atg suggested that the majority of the atg is readily conjugated to atg in huh cells, as has previously been reported for other cell types . in addition, hcv infection did not inhibit this conjugation. furthermore, hcv infection induced lc -ii accumulation (fig. a) . this result confirms the capability of hcv to modulate autophagy, as has previously been reported by several groups , , . after atg is conjugated to atg , it forms a multimeric complex by associating with atg l . to test whether this complex forms in hcv-infected cells, we overexpressed atg -flag in infected and uninfected cells. using co-immunoprecipitation with an anti-flag monoclonal antibody, we detected the atg - / l complex under both conditions (fig. b) . whereas atg - associates spontaneously with atg l , , our results indicated that hcv infection did not disturb the formation of the atg - / l complex. to test whether the decoupling of the atg - conjugate influences hcv replication, we overexpressed the dominant-negative form of atg (atg dn) in hcv-infected huh cells. this mutated form of the atg protein lacks the c-terminal glycine, which is crucial for conjugation with atg . interestingly, the overexpression of this conjugation-defective mutant had an adverse effect on hcv lifecycle, as indicated by a decrease in the ns protein (fig. c) , as well as the viral rna level (fig. d ), as compared with the levels in mock-treated cells. the specificity of the effect of atg dn overexpression was assessed by trans-complementation with wild-type atg . as expected, trans-complementation with atg restored the normal level of replication (fig. c,d) . altogether, these results suggest that the atg - conjugate, rather than the monomeric form of the atg and atg proteins, acts as an hcv proviral factor. the atg - conjugate is involved in the hcv lifecycle at a post-translational step. the conjugation of atg to lysine of atg is mediated by atg , an e -like enzyme, thus allowing the formation of the atg - / l complex. a fraction of the atg - / l complex localizes to the isolation membrane, where it facilitates lc lipidation. another role of atg , along with atg , an e -like enzyme, is to activate lc after its processing at the c-terminus by atg b, thus allowing its conjugation to the amino group of phosphatidylethanolamine (pe) and formation of the membrane-associated lc -ii, which assists in the expansion and closure of the autophagosome , . therefore, silencing of atg allows the inhibition of lc -ii and atg - conjugation (fig. b) . thus, by silencing lc , atg or atg , we were able to analyze the independent contributions of the two autophagy conjugation systems in the hcv lifecycle. for this purpose, we first determined the efficiency of the selected sirna to knock down their respective targets ( fig. a ,b,c). we then analyzed the effects of the silencing of atg , lc or atg on hcv entry, viral rna translation/replication and virion maturation and secretion. as shown in fig. d , huh infection by hcvpp was not altered in cells treated with sirna against lc , atg or atg , thus suggesting that neither lc nor the atg - conjugate is involved in viral entry. previously, dreux and colleagues have reported that hcv rna translation is affected by inhibition of lc conjugation using sirna against atg b. in that report, the authors followed the luciferase activity expressed from a replication-defective subgenomic replicon rluc/sgr harboring an inactivation mutation (gdd to gnd) at the active site of the hcv polymerase ns b (rluc/sgr-gnd) . using a similar approach, we evaluated the effects of lc , atg and atg silencing on viral rna translation and/or replication. we used full-length hcv jfh rna with a firefly luciferase reporter containing the gnd mutation in the active site of ns b (jfh /fluc-gnd) and then analyzed the fluc activity expressed from the hcv internal ribosomal entry site (ires). a significant decrease in luciferase activity (> %) was observed in jfh /fluc-gnd-transfected cells pretreated with silc ( fig. e) . silencing of atg , which inhibits lc -ii formation as well as atg - conjugation, decreased viral translation by %. this effect probably occurred through the inhibition of lc -ii conjugation, because silencing of atg expression was much less efficient than lc silencing at inhibiting viral rna translation (fig. e ). in contrast, silencing of atg severely affected the luciferase activity of a replication competent jfh /fluc wild-type virus. the effect on replication was not due to the toxicity of sirna treatment (fig. s ), thus suggesting that the atg - conjugate is involved in an hcv lifecycle step(s) beyond entry and rna translation (fig. e ), whereas lc expression and/or conjugation is primarily important for viral translation, as previously suggested . assessed by western blotting in mock (ui) or huh cells infected at more than % with jfh using specific anti-atg antibody. hcv infection and lc lipidation were detected using anti-ns and anti-lc antibodies, respectively. β -actin represents loading control. (b) uninfected or jfh -infected huh cells at more than % were transiently transfected with a plasmid encoding for flag-atg protein. two days later, cells were lysed and atg was immunoprecipitated using anti-flag antibody or igg as a control followed by western blot analysis using anti-flag and anti-atg l . (c) huh cells were infected with jfh (> % infected) before being transfected with control (mock), flag-atg , gfp-atg , flag-atg dn or flag-atg and flag-atg dn encoding plasmids. cell lysates were analyzed for hcv ns , atg and atg protein expressions at h post-transfection by western blotting. (d) jfh -infected cells at more than % were transfected with control plasmid (mock), flag-atg dn or flag-atg and flag-atg dn. two days later, intracellular hcv rna was quantified by rt-qpcr. data were collected from three independent experiments (n = ). (**p = . , ns, none-significant. statistical analysis was performed by using one-way anova). scientific reports | : | doi: . /srep the atg - conjugate positively regulates hcv rna replication in an lc -independent manner. to determine whether lc or the atg - conjugate modulates hcv rna replication, we analyzed the effects of silencing these autophagy genes on viral rna replication in huh cells stably expressing the jfh subgenomic replicon (sgr). using these specific cells, which are capable of only intracellular hcv rna replication and lacked the capacity to produce infectious viral particles, allowed us to study hcv rna replication independently of viral entry and egress. again, silencing of atg but not lc efficiently inhibited rna replication, thus supporting the role of the atg - conjugate in viral replication and ruling out the possibility that lc participates in viral replication (fig. a,b) . because silencing of lc expression led to a clear inhibition of hcv rna translation after electroporation of the viral rna but did not significantly affect replication in jfh -sgr cells, we sought to compare the effect of sirna treatment before and after infection with hcvcc jfh (fig. c) . the results clearly demonstrated that silc inhibited hcv only when it was transfected before infection, whereas siatg was effective when it was transfected before or after infection. these results suggest that lc is important early in infection and primarily for initial hcv rna translation, as has previously been reported . finally, we evaluated the effects of sirna treatment on intracellular and extracellular hcv infectious particle production in jfh -infected cells (fig. d) . these results suggested that hcv maturation and secretion was not significantly affected by sirna treatment. although silencing atg led to a significant decrease in hcv particle formation, this effect was attributed to a severe reduction in viral replication. collectively, atg silencing and to a lesser extent atg but not lc , impaired viral replication. atg - and atg l are associated with purified mw extract. in a previous study from our lab, we have shown that atg interacts with the hcv polymerase (ns b) and co-localizes with the mw associated protein ns b . the major limitation in our ability to investigate the composition of the mw has recently been resolved by dr. ralf bartenschlager's group, which has developed a method to purify the hcv mw by using hcv replicon cells harboring an ha-tag ns b (ns b-ha) . this method allowed us to evaluate the presence of the autophagy elongation complex proteins in purified mw extract. through this protocol, after membrane enrichment from a discontinuous sucrose gradient via ultracentrifugation, we pooled fractions that were rich in viral nonstructural proteins (fraction to ) but mostly devoid of soluble proteins (gapdh or lc i, fractions - ) (fig. a) . the mw vesicles were then pulled down from pooled fractions by using a specific antibody against the ha-tag of the hcv ns b protein. subsequently, the mw-enriched extract was used for either western blot analysis or vesicle visualization by transmission electron microscopy (tem). as expected, hcv ns b ha , ns and ns a were readily detectable in the purified extract from ns b ha replicon cells but not that from control untagged ns b replicon cells (fig. b) . the autophagy elongation complex proteins (atg - and atg l ) were also detected in the purified mw from ns b ha replicon cells, but not in the control extract, thus indicating that the elongation complex is indeed present at the hcv replication site. in contrast, we were unable to detect lc ii in the purified mw (fig. b) , thereby suggesting that lc is not recruited with the autophagy elongation complex to the mw. we then examined the morphology of purified membranes and compared them with er membranes purified from a cell line expressing ha-tagged calnexin, as previously described . the results showed that almost % of the ns b ha purified membranes were spherical vesicles as compared with clnxn ha purified material, in which the majority of membranes were composed of partially collapsed large membranes (fig. a,b) . our results are in agreement with those of paul and colleagues, who have demonstrated that most of the purified er membranes are composed of elongated structures, as opposed to the spherical vesicles found in mw extracts . finally, the specificity of the pull-down using ha-beads was confirmed by using extracts from untagged sgr cells (fig. c) . altogether, these results indicate that at least a fraction of the autophagy elongation complex is localized in the virus-induced mw compartments. silencing of atg or atg , but not lc , alters the phenotype of the mw. to evaluate the putative role of host cell proteins in mw formation, reiss and colleagues have established a t -polymerase-based specific antibodies were used to detect ns , ns b ha , ns a, lc and gapdh as indicated on the left. fractions containing membrane-associated proteins (boxed in red) were pooled for affinity capture immunoprecipitation. the density of the different fractions is shown in the lower panel. tcl, total cell lysate. (b) ha-specific affinitycaptured protein content from pooled fraction in (a) was analyzed by western blotting to detect viral ns , ns b, ns a and autophagy elongation complex atg - / l by using specific antibodies. pooled fractions from sgr cell lysate was used to demonstrate pull-down specificity. hcv rna synthesis system in which continuous production of hcv polyproteins persists even when hcv replication is abrogated . this system is particularly useful to evaluate the formation of the mw while targeting host cell proteins in the absence of hcv rna replication. with this system, it has been shown that alterations in mw formation result in a clustered phenotype of hcv nonstructural proteins , . thus, using the same system (obtained from dr. volker lohmann), we analyzed mw formation indirectly by monitoring viral protein localization after treatment with silc , siatg and siatg . under normal conditions, the ns and ns a cellular distribution appeared as small punctate structures that appeared to be membrane associated (fig. a,b) . treatment with silc did not alter the cellular distribution of the viral proteins, as observed by confocal microscopy ( fig. a-c) . strikingly, silencing of atg or atg resulted in the formation of larger protein clusters in most of the transfected cells ( fig. a-c) . this effect was not due to decreased hcv protein expression level (fig. d) , thus suggesting that the atg - conjugate, but not lc , is required to obtain a wild-type mw phenotype. silencing of atg or atg , but not lc , modifies mw morphology. next, using tem, we analyzed mw morphology after treatment with silc , siatg or siatg . expression of ptm-ns - b in cells treated with sictl induced heterogeneous membrane alterations composed of dmvs of an average size of nm interspersed by multi-membrane vesicles (mmvs) that were distributed throughout the cytoplasm (fig. a,b) and that were not seen in negative cells (fig. e) . silencing of atg resulted in more homogenous dmvs with a markedly decreased average size ( nm) and led to the disappearance of mmvs (fig. a,b,d) . silencing of atg led to a similar effect but with a much lower abundance of dmvs (fig. c) . however, silencing of lc had no effect on the dmv size (average diameter nm) (fig. b) or on the vesicle types in which both dmvs and mmvs coexist (fig. c,d) . thus, a similar morphology of membrane alterations was observed in sictl-treated cells (fig. a,b) . these results strongly suggest that the atg - conjugate is crucial for the formation of a typical hcv-induced mw architecture. in the present study, we demonstrated the requirement of the atg - / l complex for the completion of the hcv lifecycle. hcv infection does not hamper atg conjugation to atg or the formation of the multimeric complex atg - / l (fig. a,b) . in contrast, the conjugation of atg to atg is crucial for the hcv lifecycle. more specifically, our study suggests a role of the atg - / l complex in hcv genome replication and the formation of the mw. the involvement of the autophagy elongation complex in the hcv replication step was investigated by using sirna targeting of atg , atg or lc . because the silencing of atg is known to inhibit the conjugation of both lc and atg , we were able to address the importance of these two conjugation systems in hcv replication. indeed, the atg - conjugate acted as a proviral factor at a step beyond entry and rna translation but before virion maturation and secretion, as depicted in figs and . we also observed that silencing of lc interfered with hcv rna translation after electroporation of replication-defective replicon (fig. e) . these results are consistent with those of dreux and colleagues, who have found a defect in viral rna translation after silencing of beclin- or atg b, thus leading to inhibition of lc -ii formation . silencing of atg had little effect on replication-deficient virus but was detrimental to the replication of the jfh /fluc virus, thus indicating that its primary target is beyond the translation step (fig. e) . this result was further confirmed in cells stably expressing the jfh subgenomic replicon, in which silencing of atg or atg , but not lc , significantly inhibited hcv replication (fig. a,b) . silencing of lc impeded hcv only when performed before infection, thus suggesting that the atg - conjugate, but not lc , is important in viral replication after the establishment of infection (fig. c ). in addition, the co-purification of the elongation complex proteins with the mw suggested that the atg -ns b interaction previously described by our laboratory might actually participate in targeting of the elongation complex to the mw and/or in supplying of autophagic isolation membranes for the formation of the virus-induced vesicles. in canonical autophagy, the atg - / l complex is recruited to the isolation membrane prior to lc and is released just before the completion of autophagosomes. the absence of lc in the purified mw suggests that hcv either hijacks atg - / l -positive lc -negative isolation membranes or initiates the de novo formation of the isolation membrane at the mw rather than utilizing lc -positive autophagosomes for the formation of dmvs within the mw (fig. ) . interestingly, the recruitment of the elongation complex to the mw was not accompanied by lc lipidation or its relocation at that site. recently, it has been demonstrated that the atg - / l complex has a membrane-tethering activity that is independent of lc , . this finding highlights the possibility that in hcv infected cells the major role of the elongation complex is to tether vesicles during mw formation. concomitantly, it has been reported that some atg proteins, including atg l , can traffic in lc -free vesicle-like structures to the site where they probably act to generate de novo isolation membranes . this finding also raises the possibility that hcv may recruit similar structures that aid in the formation of the mw. recently, reiss and colleagues have developed a system to evaluate the importance of host factors in membranous web formation . using this system, we demonstrated that atg as well as atg expression, but not lc , are important to obtain a wild-type mw phenotype, as observed using confocal microscopy (fig. ) . furthermore, the morphology of the hcv-induced vesicles was severely altered after silencing of atg or atg , but not lc . notably, knocking down atg decreased the size and the number of dmvs, whereas silencing of atg mainly affected their size (fig. ) . at the moment, it remains unknown whether the altered mw is hcv-replication competent. however, the importance of the atg - conjugate in hcv rna replication suggests that the autophagy elongation complex inhibits hcv replication through destabilization of the viral replication factories present within the mw. in summary, recruitment of the autophagy elongation complex to the mw, which is normally involved in dmv formation, promotes viral replication and maintains proper formation of the wild type mw. cell culture and reagents. huh and huh -lunet cells stably expressing calnexin or ns b-ha replicon were obtained from dr ralf bartenschlager. huh -lunet cells stably expressing the t polymerase (huh -lunet-t ) was obtained from dr. volker lohmann, and the huh . cell line was obtained from dr. charles rice. all huh -derived cell lines were cultured in dulbecco's modified eagle's medium (dmem; gibco) supplemented with % v/v fetal bovine serum (fbs) (multicell), u/ml penicillin, μ g/ml streptomycin, and mm l-glutamine (gibco) at °c, % co , in a humidified incubator. cell lines harboring the wild-type replicon or ns b ha were maintained in medium supplemented with g (gibco) at a final concentration of μ g/ml. huh -lunet-t and huh -lunet cells stably expressing calnexin were cultured in the presence of μ g/ml blasticidin (in vivo gen). plasmids and antibodies. the hatg and hatg l sequences were cloned into the pegfp-c plasmid (clontech), thus forming pgfp-atg and pgfp-atg l , respectively. the flag-tagged atg (patg ) and its dominant-negative derivative patg Δ g (atg dn) constructs were kindly provided by dr. adi kimchi . the ptm vector for the expression of hcv nonstructural proteins ns to b (ptm-ns - b) was kindly provided by dr. volker lohmann. rabbit polyclonal anti-lc , rabbit polyclonal anti-atg , mouse monoclonal anti-flag, and mouse monoclonal anti-β -actin antibodies were purchased from sigma aldrich. rabbit polyclonal anti-atg and anti-atg were purchased from cell signaling. rabbit polyclonal anti-atg l antibody was purchased from mbl. mouse monoclonal anti-ha was purchased from roche. mouse monoclonal anti-ns and anti-ns a antibodies were purchased from biofront. rabbit polyclonal anti-ns and ns a were obtained from dr. olivier nicolas. rabbit polyclonal anti-ns b and anti-ns b antibodies were kindly provided by drs. kouacou konan and takaji wakita, respectively. mouse monoclonal anti-gapdh was purchased from santa cruz. the cell culture-derived hcv (hcvcc) jfh virus was generated in huh cells by transfection of in vitro-transcribed full-length jfh rna (megascript, ambion). viral stocks were produced by infection of huh cells at a multiplicity of infection (moi) of . , as described previously . a replicative bicistronic jfh -based full-genome construct expressing firefly luciferase (pjfh /fluc) and a clone with a mutation in the viral polymerase (gdd-to-gnd) (pjfh /fluc-gnd) were generated as previously described . to reach % infected cells, huh cells were infected at an moi of . , passaged for days and then analyzed by immunofluorescence using an anti-ns a antibody. western blot analysis. cells were lysed in μ l of lysis buffer [ mm tris-hcl, mm nacl, mm edta, % np , complete protease inhibitor (roche)]. the lysates were normalized for total protein content using the bca protein assay kit (pierce). the proteins were then resolved by sds-page, transferred to polyvinylidene fluoride (pvdf) membranes (bio-rad), blocked for min at room temperature (rt) with pbs- % milk, and then incubated overnight at °c with primary antibody in pbs- % bsa. after being washed with . % tween in pbs (pbst), the membranes were incubated for h at rt with a goat-anti-rabbit or goat-anti-mouse igg conjugated to horseradish peroxidase in pbs- % milk. protein bands were visualized with either the clarity western ecl (bio-rad) or femto chemiluminescence substrates (pierce). purification of hcv-induced mw. hcv-remodeled membrane purification was performed using a method adopted from a previously described protocol . briefly, . × huh -lunet cells harboring either wild-type or ha-tagged ns b replicons or control cells stably overexpressing canx ha were washed, scraped and then resuspended in μ l of hypotonic buffer and incubated on ice for min. the cells were lysed with strokes with a dounce homogenizer. the lysates were centrifuged at × g for min at °c. supernatants were collected and layered on top of a discontinuous sucrose gradient ( % to %) and centrifuged at , × g for h at °c using an sw i rotor (beckman coulter). ten fractions were collected from the bottom ( μ l each) and analyzed for protein content. for ha affinity capture, fractions to were pooled, and then an equal amount of protein contained in pooled fractions was equilibrated to mm nacl. incubation with ha-agarose beads (sigma-aldrich) was performed as previously described . membrane visualization by transmission electron microscopy. to examine purified membranes, μ l of eluted material was centrifuged at p.s.i. on a copper grid for min at rt in an airfuge (beckman). structures were negatively stained using % aqueous uranyl acetate for sec and examined with an h- (hitachi) transmission electron microscope. quantification of hcv rna by rt-qpcr. isolated rna was reverse transcribed with m-mlv (invitrogen). the generated cdna was used for qpcr using taqman probes, as previously described . results were analyzed using the comparative Δ ct method. small interfering rna (sirna) transfection. huh cells were reverse transfected in a -well plate with sirna ( nm final concentration) using lipofectamine rnaimax reagent (life technologies) according to the manufacturer's protocol. huh cells were transfected with sirna to gfp, lc b sirna (uaccuguauacguuagugaaauu) or with an on-targetplus human atg sirna-smart pool (catalog no. l- - - ). to study the onset of replication, huh . cells were reverse transfected with sirna, as described above, in -well plates. forty-eight hours later, the cells were trypsinized, washed twice with cold pbs, resuspended in μ l of cold ingenio electroporation solution (mirus) and then electroporated with μg of in vitro-transcribed viral rna (jfh /fluc or jfh /fluc-gnd) in mm gap electroporation cuvettes by using a btx harvard apparatus with the following settings: v, μ s, pulses, . s interval. the cells were then resuspended in dmem- % fbs and seeded in -well plates and further cultured for h. the cells were then lysed in μ l luciferase lysis buffer (rlb) and stored at − °c until measurement of luciferase activity. for the determination of intra-and extracellular virus titers, jfh -infected huh cells were reverse transfected with sirna in -well plates. two days later, the cells were washed three times with pbs and supplemented with fresh dmem. after h, cells and supernatants were harvested. the cells were washed twice with pbs, trypsinized, resuspended in ml culture medium and subjected to rapid freeze-thaw cycles in a dry ice/ethanol bath and °c water bath, respectively. cell debris was removed by centrifugation at , rpm for minutes. samples were analyzed using a limiting dilution assay. production of hcvpp and cell entry assay. viral pseudotyped particles harboring the hcv glycoproteins (hcvpp) were produced by transfection in hek- t cells of vectors encoding viral glycoproteins, packaging proteins and a luciferase marker. after h, viral pseudoparticle supernatants were harvested and filtered through -μ m filters to remove the cell debris. for the entry assay, huh . cells were reverse transfected with sirna in -well plates. after h, the cells were infected with μ l hcvpp containing supernatant. forty-eight hours post-infection, the cells were washed three times with pbs, lysed in μ l luciferase lysis buffer (rlb) and stored at − °c until measurement of luciferase activity. luciferase assay. cell lysates were prepared with reporter lysis buffer (rlb) (promega), and luciferase activity was measured with a luciferase assay system (promega), per the manufacturer's protocol. for the immunofluorescence experiment, huh -lunet-t cells were reverse transfected with sirna as previously described . forty-eight hours later, a second round of transfection with sirna was performed. after h, cells were transfected with ptm-ns - b using lipofectamine (invitrogen). the coverslips were then fixed with % formaldehyde in pbs for min, washed in pbs and incubated in blocking buffer (pbs, % bovine serum albumin, % fbs, . % triton x- ) for min at rt. after being washed three times with pbs, the coverslips were incubated with primary antibody in blocking buffer for h at rt. then, the coverslips were washed with pbs and incubated with either alexa fluor ™ -( or ) goat anti-mouse igg or alexa fluor ™ -( or ) goat anti-rabbit igg (invitrogen) for h at rt. after being washed, the coverslips were mounted on glass slides with prolong ™ . antifade (invitrogen) and examined with a laser scanning confocal zeiss lsm . for tem analysis, a similar setup was used, except that after transfection with ptm-ns - b, the cells were trypsinized and seeded into lab-tek chamber slides (thermo fisher). after h, the monolayer of cells was washed with pbs, fixed with . % glutaraldehyde (electron microscopy science) and incubated overnight at °c. the cells were then washed in . m cacodylate (electron microscopy science) and incubated in % osmium tetroxide (mecalab) for h at °c. the cells were dehydrated in a graded series of ethanol/deionized water solutions (from % to %). the cells were then infiltrated with a : and : epon for h for embedding and polymerized overnight in an oven at °c. the polymerized blocks were trimmed, and nm ultrathin sections cut with an ultracut e ultramicrotome (reichert jung) and transferred onto -mesh copper grids (electron microscopy science) with formvar support film. the sections were stained with % uranyl acetate (electron microscopy science) for min, then with lead citrate for min (fisher scientific). the cells were imaged with an fei tecnai transmission electron microscope (fei company) operating at an accelerating voltage of kv and equipped with an amt xr c ccd camera. vesicle size was measured using image j (nih). cell viability assay. cells were reverse transfected with different sirnas used in this study in a -well plate for h. cell viability was then assayed using the celltiter ® aqueous non-radioactive cell proliferation assay reagent (promega). the results shown represent the mean of at least three independent experiments. student's-t-test and one-way anova with dunnett's post-test (as indicated in the figure legends) were performed using graphpad prism . p-values below . were considered statistically significant. development of novel treatments for hepatitis c replication of hepatitis c virus identification of the hepatitis c virus rna replication complex in huh- cells harboring subgenomic replicons expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus replication of hepatitis c virus rna on autophagosomal membranes autophagy in immunity and inflammation the atg l complex specifies the site of lc lipidation for membrane biogenesis in autophagy subversion of cellular autophagosomal machinery by rna viruses autophagy is involved in the early step of japanese encephalitis virus infection flavivirus ns a-induced autophagy protects cells against death and enhances virus replication dengue virus-induced autophagy regulates lipid metabolism autophagy sustains the replication of porcine reproductive and respiratory virus in host cells hcv induces the expression of rubicon and uvrag to temporally regulate the maturation of autophagosomes and viral replication hepatitis c virus inhibits akt-tuberous sclerosis complex (tsc), the mechanistic target of rapamycin (mtor) pathway, through endoplasmic reticulum stress to induce autophagy hepatitis c virus genotype a growth and induction of autophagy persistent expression of hepatitis c virus non-structural proteins leads to increased autophagy and mitochondrial injury in human hepatoma cells changes in autophagic response in patients with chronic hepatitis c virus infection hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro hcv infection selectively impairs type i but not type iii ifn signaling hepatitis c virus core protein activates autophagy through eif ak and atf upr pathway-mediated map lc b and atg expression hepatitis c virus-induced autophagy is independent of the unfolded protein response irgm is a common target of rna viruses that subvert the autophagy network knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles dissection of autophagosome formation using apg -deficient mouse embryonic stem cells autophagy protein atg interacts transiently with the hepatitis c virus rna polymerase (ns b) early during infection mouse apg l, a novel wd-repeat protein, targets to the autophagic isolation membrane with the apg -apg conjugate generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size molecular mechanism of autophagic membrane-scaffold assembly and disassembly formation of the approximately -kda apg -apg .apg multimeric complex, mediated by apg oligomerization, is essential for autophagy in yeast the autophagy protein atg associates with antiapoptotic bcl- family members to promote mitochondrial apoptosis methods in mammalian autophagy research characterization of autophagosome formation site by a hierarchical analysis of mammalian atg proteins morphological and biochemical characterization of the membranous hepatitis c virus replication compartment recruitment and activation of a lipid kinase by hepatitis c virus ns a is essential for integrity of the membranous replication compartment the lipid kinase phosphatidylinositol- kinase iii alpha regulates the phosphorylation status of hepatitis c virus ns a dissecting the role of the atg -atg -atg complex during autophagosome formation mechanism and functions of membrane binding by the atg -atg /atg complex during autophagosome formation structures containing atg a and the ulk complex independently target depolarized mitochondria at initial stages of parkin-mediated mitophagy novel hcv replication mouse model using human hepatocellular carcinoma xenografts ski- /s p inhibitor pf- impairs the onset of hcv infection ski- /s p inhibition: a promising surrogate to statins to block hepatitis c virus replication we thank dr. takaji wakita for providing the hcv jfh- and the anti-ns b antibody. the huh -lunet, ns b hareplication and canx ha cells were generously provided by dr. ralf bartenschlager. the huh -lunet-t cells and the ptm-ns - b vector were provided by dr. volker lohmann. we are also grateful to dr. kouacou konan for providing the anti-ns b antibody and dr. adi kimchi for providing the atg and atg Δ g plasmids. we also thank dr. hojatollah vali and his group for performing tem with af. in addition, we thank david paul, jessy tremblay and micheline letarte for technical assistance. this work was supported by a research grant from the nserc of canada. af received fellowships from the ncrtp-hepc. a.f. and p.l. designed the study. a.f. performed the experiments. a.f. and p.l. analyzed the data and wrote the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep key: cord- -rueg gsj authors: huang, ao; li, weiwei; shi, shuo; yao, tianming title: quantitative fluorescence quenching on antibody-conjugated graphene oxide as a platform for protein sensing date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: rueg gsj we created an immunosensing platform for the detection of proteins in a buffer solution. our sensing platform relies on graphene oxide (go) nanosheets conjugated with antibodies to provide quantitative binding sites for analyte proteins. when analyte proteins and standard fluorescein-labelled proteins are competing for the binding sites, the assay exhibits quantitative fluorescence quenching by go for the fluorescein-labelled proteins as determined by the analyte protein concentration. because of this mechanism, measured fluorescence intensity from unquenched fluorescein-labelled protein was shown to increase with an increasing analyte protein concentration. as an alternative to the conventional enzyme-linked immunosorbent assay (elisa), our method does not require an enzyme-linked second antibody for protein recognition and the enzyme for optical signal measurement. thus, it is beneficial with its low cost and fewer systematic errors caused by the series of antigen-antibody recognition steps in elisa. immune globulin g (igg) was introduced as a model protein to test our method and our results showed that the limit of detection for igg was . pmol ml(− ) in the buffer solution. this sensing mechanism could be developed into a promising biosensor for the detection of proteins, which would broaden the spectrum of go applications in both analytical biochemistry and clinical diagnosis. conventional enzyme-linked immunosorbent assay (elisa) has been widely used in analytical biochemistry and clinical diagnosis. however, the accuracy of elisa is affected by a few shortcomings intrinsic to the technique. for example, the uncertainty of the recognition between antibody and antigens could cause false signals - the elisa plates made of polystyrene lack definitive chemical surface properties to provide chemical binding of the protein ; and intricate procedures such as "plate cleaning" increase opportunities for systemic and human errors. in recent years, many research groups have tried to improve the accuracy of elisa by synthesizing new elisa polymer substitutes , introducing nanomaterials , , employing more sensitive biomarkers , and applying monoclonal antibodies . all of these studies were restricted by the framework of traditional elisa, which still relies on elisa plates, and the problems mentioned above remain unsolved. obviously, new immunosorbent assay techniques call for quantitative surface sites for antibody binding and fewer steps or reagents in the whole process to avoid the above-mentioned drawbacks. herein we present a sensing platform based on graphene oxide (go) with definitive surface binding sites as an alternative sensing platform to that of elisa plates. the mechanism is illustrated in fig. . human igg was chosen as a model protein to test our design. the antibody-conjugated go surface was able to specifically bind with both a regular igg protein (analyte) and a fluorescein isothiocyanate (fitc)-labelled igg protein (standard, mentioned as igg-fitc below), thus exhibiting competitive binding of the types of igg proteins. the amount of analyte igg protein controls the adsorption of igg-fitc as well as the degree of its fluorescence quenching by go. as shown in fig. , when analyte igg proteins are present to compete for the available binding sites, few igg-fitcs adsorb on the remaining binding sites, resulting in fluorescence signals from unquenched free igg-fitcs. thus, the fluorescence intensity increases with an increasing amount of analyte igg protein, given the sensing principle of our method. in the following, we briefly outline the experimental methods and then present the results in detail, followed by a discussion. first, go was chemically modified with rabbit anti-human igg antibody by forming of a peptide bond between the carboxyl of go and the amino group of the antibody. this surface provides antibody mediate binding sites that are superior to elisa plate surfaces made of polystyrene, which rely on physical adsorption. then, analyte igg proteins in increasing concentrations were added to a set of buffer solutions containing antibody-conjugated go. after adsorption of analyte igg proteins on part of the binding sites, fitc-labelled igg proteins with a certain concentration were also added to the above set of solutions. in this case, the go surface not only played a role as a binding platform but also as an energy acceptor [ ] [ ] [ ] [ ] to quench the fluorescence of igg-fitc , . since quenching occurs in close proximity , only the igg-fitcs that were adsorbed on antibody-conjugated go would be quenched [ ] [ ] [ ] [ ] [ ] . as the number density of the binding sites was limited on the modified go, more adsorbed analyte igg proteins resulted in a stronger fluorescence signal from free igg-fitcs in the solutions. in essence, the analyte igg proteins control the fluorescence quenching and determine the fluorescence intensity of the whole system. within a certain concentration range, one can generate a linear plot correlating the igg-fitc fluorescence intensity with the analyte igg concentration. according to our results, we achieved a limit of detection (lod) of . pmol ml − . we first present the experimental evidence for go surface morphology, which changes upon various stages of surface modification. we then present the quenching effect of igg-fitc on bare go, antibody-conjugated go, and bovine serum albumin (bsa)-blocked go surfaces to characterize and optimize assay parameters. finally, we show how analyte igg may be detected in a wide range of concentrations based on the fluorescence signal change of igg-fitc. in addition, we provide the limit of detection and selectivity of the assay. atomic force microscopy (afm) was introduced to measure the various stages of the surface modification of go. according to the works of lee et al. and hosseini et al. , the height of go would obviously increase after being activated by edc-nhs and further upon conjugation by antibodies. these studies served as reference points for us to use afm to characterize the surface morphologies of bare go, edc-nhs-activated go, and antibody-conjugated go to provide evidence of the edc-nhs coupling reaction and the presence of antibodies on the go surface. in fig. (a) , bare go has a thin and flat appearance with a height of approximately nm on mica, which may correspond to the monolayer state of go. the corresponding line scan and height profile of the sample are shown in fig. (d). after activation by edc-nhs, go exhibits an increased thickness of about nm, similar to what was reported before , . the line scan (arrow) gives a corresponding cross-sectional height profile in fig. (d) , showing a uniform surface of the go. because the antibody is very large (with a molecular weight of over kda), one can correctly expect that the antibody-conjugated go should be much thicker. as shown in fig. (c), we observed that the height of antibody-conjugated go increased dramatically, with a typical thickness of about nm, as suggested in the height profile in fig. (d). some bright peaks were observed on this sample, which was also similar to what hosseini et al. had observed . the bright peaks are likely due to the accumulation of antibodies on the go surface. all of the surface morphology results implied that the antibody was successfully retained on the go surface. the key design of our immunoassay is the adsorption of igg on antibody-conjugated go, which controls the adsorption of igg-fitcs and leaves some free igg-fitcs in the solution to generate fluorescence signals. due to the fact that binding sites on the antibody-conjugated go were limited, when more analyte igg proteins adsorb on antibody-conjugated go, fewer binding sites remain for igg-fitcs, so that more free igg-fitcs will be in the solution and produce more fluorescence signals. therefore, there is a positive and quantitative correlation between the analyte igg concentration and the fluorescence intensity of free igg-fitcs. according to this principle, our assay relies on the effectiveness of go as an energy acceptor to provide efficient fluorescence quenching. the quenching efficiency of igg-fitc by bare go, antibody-conjugated go, and bsa-blocked go was investigated by adding a variety of these types of go to standard samples containing μ g ml − of igg-fitc, respectively. as we expected, the fluorescence of the free igg-fitcs decreased when the igg-fitcs bound to the graphene surfaces. in fig. (a) , a rapid decrease of fluorescence intensity was observed with the increasing amount of bare go, exhibiting a quenching efficiency of about % with μ g ml − of go. the signal change versus go concentration is plotted in fig. (d) . the quenching efficiency of antibody-conjugated go may be lower than bare go, because the modification of go with antibodies changed the electronic property of go, changing go to a semiconductor and making the transfer of energy from the excited state of fitc to bare go easier than that to antibody-conjugated go. in fig. (b) , the fluorescence intensity also decreased with the increasing amount of antibody-conjugated go, and the quenching efficiency indeed dropped to about % at μ g ml − , showing antibody-modified go could still be a quencher. with μ g ml − of antibody-conjugated go, about % of igg-fitc initial fluorescence had been quenched, as can be seen in fig. (d) . therefore, with analyte igg in the solution, the fluorescence intensity change of igg-fitc will be between to % with respect to initial fluorescence with no analyte. one last characterization of the assay was to determine whether the diminishing of fluorescence intensity could originate from other principles. if the fluorescence of igg-fitcs will be quenched no matter if it is free or attached to the go surface, the design is meaningless. by blocking the go surface with % bsa in the buffer solution, we found that the fluorescence intensity change remained almost unchanged with increasing amount of bsa-blocked go. only the igg-fitcs adsorbed on the surface of the go could be quenched. according to our results, the appropriate concentration of antibody-conjugated go was μ g ml − (~ % quenching). although more go might quench the fluorescence even further, a higher antibody-conjugated go concentration could be less sensitive for sensing the analyte igg with a very small concentration. therefore, there is a delicate balance between the concentration of go and the change of fluorescence signal. in the following experiment, antibody-conjugated go always had a concentration of μ g ml − . based on the above results, we carried out quantitative sensing of analyte igg. various amounts of igg ranging from to μ g ml − was prepared in the buffer solution and tested. in our experiment, the "blank" sample consisted of antibody-conjugated go and igg-fitc, without analyte igg. in order to show the difference in fluorescence quenching with the addition of the analyte, we always compared the fluorescence intensity of the blank without the analyte versus that when the analyte was present. as mentioned above, fluorescence quenching efficiency is determined by the energy coupling between the go surface and the adsorbed igg-fitc. surface modification and interfacial environment largely affect quenching efficiency. with μ g ml − of conjugated go and μ g ml − of igg-fitc, our measured fluorescence intensity was ~ at nm as obtained by spectrometer. the addition of analyte igg induced an increase of the signal, starting from the relatively large background signal. in order to make the results in direct proportion to the analyte concentration, we used the change of intensity for plotting the assay response curve in fig. (a) . the value of Δ i was calculated by subtracting the final fluorescence intensity at nm and the "blank" sample without analyte igg. the experiment was repeated times, and the average of the results was plotted in fig. (a) , from which we can see the assay's fluorescence signal increased readily with the increasing analyte igg concentration. there are main features that can be observed from the plot. first, the signal change Δ i showed a rapid increase with analyte igg up to ~ μ g ml − , after which the change became less steep (the initial rise of the fluorescence signal was very rapid with a nearly linear region at concentrations below about μ g ml − ). second, the signal change reached about half of the maximum value with μ g ml − of the analyte concentration (versus that of μ g ml − ). according to our design, since binding sites on antibody-conjugated go are limited, the more analyte igg that was added, the fewer binding sites remained for igg-fitcs, resulting in more free igg-fitcs in the buffer solution. thus, we observed that increasing fluorescence intensity is quantitatively controlled by the analyte igg concentration. the result indicates that the principle of our platform is feasible, and the analyte igg proteins could control the fluorescence quenching and determine the fluorescence intensity. within the concentrations of to μ g ml − , we generated a linear plot relating to the igg-fitc fluorescence intensity with the analyte igg concentration. this is plotted in fig. (b) . we performed the experiment under the same conditions times and the data points in fig. (b) fit well with the linear function. the fitting function of the curve is Δ i = . c (concentration of igg) with an r of . . we then repeated the blank experiment times in order to calculate the standard deviation (sd). we took times the value of the sd and divided it by the slope of the calibration curve, and got the lod of . pmol ml − . up to now, we have assumed that igg-fitc proteins are all uniformly labelled with fluorescent fitcs. because the quality of antibodies and percentage of fitc labelling on igg may vary in different batches and from different manufacturers , the lod could in principle be improved with well-controlled and uniform labelling of igg by fitc. however, this is beyond the scope of this paper. one major assumption we held is that igg proteins bind on antibody-conjugated go via the antibody-antigen type of specific bonding. this discriminative binding process means that analyte igg will be adsorbed by antibody-conjugated go, preventing the fluorescein-labelled igg-fitc from binding to and being quenching by the surface. in order to know whether other biological molecules might interfere with the sensing process and give false positive signals, we performed a series of experiments to determine more about the specificity of our assay. some interference molecules, such as human serum albumin (hsa), bsa, and igg, were introduced to test the selectivity. in this set of parallel testing, the signals from the solutions containing the above interference substances, analyte igg, and blank sample without analyte igg were compared. the results in fig. clearly show that the possible interfering biomolecules gave no detectable signal change, as compared with the blank sample. only the analyte igg protein showed a significant positive signal as the fluorescence intensity of the other proteins was close to the blank sample. this indicates that our assay design is highly selective for igg protein. we established an immunosensor platform based on quantitative fluorescence quenching between fluorescein-labelled antigens and antibody-conjugated go nanosheets, a process controlled quantitatively by the concentration of analyte proteins. human igg, a widely used model protein, was chosen to test the feasibility of our design. with the competitive binding of analyte igg and standard igg-fitc the surface of go's limited binding sites, the process conveniently paves a way to control free igg-fitc in the solution by analyte igg adsorption on the binding sites. the increase of fluorescence signal from igg-fitc correlates directly with an increasing analyte igg concentration. the assay based on antibody-conjugated go does not require any phase separation steps or wash steps as with commercial elisa procedures, which are used to remove unadsorbed antibodies. thus, our assay is beneficial since it uses fewer reagents, has a lower cost, and there are fewer opportunities for systematic and human errors. the lod was . pmol ml − , which still leaves room for further improvement. the sensing mechanism in this study could become a viable immunosensor platform for the detection of proteins, which will broaden the spectrum of graphene oxide applications in both analytical biochemistry and clinical diagnosis. materials. bsa was purchased from solarbio (beijing); igg, igg-fitc, and rabbit anti-human igg antibody were obtained from sangon biotech co. ltd. (shanghai); graphene oxide was bought from xfnano (nanjing); figure . selectivity. the concentrations of hsa and bsa were both μ g ml − , the igg-fitc was μ g ml − , and the antibody-conjugated go was μ g ml − for each experiment. the blank sample did not contain analyte igg or an interfering molecule. and n-( -dimethylaminopropyl)-n-ethylcarbodiimde hydrochloride (edc) and n-hydroxysuccinimide (nhs) were purchased from tokyo chemical industry co. ltd. (japan). they were all of analytical reagent grade and used without further purification. characterization methods. the fluorescence intensity of each sample was measured under the excitation wavelength of nm, with the slit width of nm, and voltage of v using a f- fluorescence spectrophotometer (hitachi, japan). the emission peak of fitc was centered at nm, and corresponding filters were introduced to obtain the analyte spectral region. for afm measurements, sample solutions containing bare go or modified go were dripped on freshly cleaved mica using a pipette and then dried in ambient air. surface topographic features were scanned in contact mode using a commercial afm (cspm , benyuan, china) equipped with a silicon cantilever. antibody-conjugated go was synthesized by a classic two-step edc-nhs ( -ethyl- -( -dimethylaminopropyl) carbodiimide and n-hydroxysuccinimide) method [ ] [ ] [ ] . briefly, mg of go was ultrasonically dispersed in mmol of l − -(n-morpholino) ethanesulfonic acid (mes) buffer (ph = . ). then a mes buffer solution containing mg ml − of edc and mg ml − of nhs was added into the go-dispersed mes solution to activate the go. the mixture was first stirred for min at °c, then centrifuged and washed with mmol l − of phosphate buffer solution (pbs, ph = . ) times to remove unreacted coupling reagents. the activated go was redispersed in pbs to react with μ g of rabbit anti-human igg so as to modify the go with the antibody. the samples were mixed on an electronic shaker at °c for h. remaining active sites of go were blocked with % bsa in mmol l − of pbs buffer solution for min. the solution was then centrifuged at , rcf for min to remove any unbound biomolecules in the supernatant. process for igg protein assaying. for a typical procedure of analyte igg sensing, a series of concentrations of human igg as the analyte ranging from - μ g were separately added into these tubes and reacted for h at °c. afterward, human igg-fitc standard was added to each sample to reach a concentration of μg ml − and allowed to react for another hour at the same temperature. when the reaction ended, the fluorescence intensity of each sample was measured. the response curve of the assay was obtained by plotting the igg-fitc's fluorescence intensity change as a function of the analyte igg concentration. methods for determining the lod and selectivity of the assay were essentially the same as the above processes. recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus development of an enzyme immunoassay using a monoclonal antibody against the psychoactive diterpenoid salvinorin a hapten synthesis, monoclonal antibody production and development of a competitive indirect enzyme-linked immunosorbent assay for erythromycin in milk synthesis and processing of elisa polymer substitute: the influence of surface chemistry and morphology on detection sensitivity nanomaterials for sensing and destroying pesticides novel optical nanoprobes for bioanalysis development of a lipovitellin-based sandwich elisa for quantification of vitellogenin in surface mucus and plasma of goldfish (carassius auratus) interfacial immobilization of monoclonal antibody and detection of human prostate-specific antigen simultaneous determination of human enterovirus and coxsackievirus b by dual-color quantum dots and homogeneous immunoassay hemin-graphene hybrid nanosheets with intrinsic peroxidase-like activity for label-free colorimetric detection of single-nucleotide polymorphism flexible graphene films via the filtration of water-soluble noncovalent functionalized graphene sheets immunosensor for the detection of cancer biomarker based on percolated graphene thin film applications of spectrophotometry biosensing platform based on fluorescence resonance energy transfer from upconverting nanocrystals to graphene oxide a graphene oxide-peptide fluorescence sensor tailor-made for simple and sensitive detection of matrix metalloproteinase the rise of graphene hapten-grafted graphene as a transducer for homogeneous competitive immunoassay of small molecules the photoluminescent graphene oxide serves as an acceptor rather than a donor in the fluorescence resonance energy transfer pair of cy . -graphene oxide graphene-based chemiluminescence resonance energy transfer for homogeneous immunoassay finding the right antibody for the job bioconjugate techniques one-step homogeneous magnetic nanoparticle immunoassay for biomarker detection directly in blood plasma utilization of kinetically enhanced monovalent binding affinity by immunoassays based on multivalent nanoparticle-antibody bioconjugates this study was supported by grants from the national nature science foundation of china. (no. and ). we thank letpub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. a.h. and t.y. conceived the experiments. a.h. and w.l. performed most of the experiments. s.s. and t.y. commented on the manuscript. a.h., w.l. and s.s. wrote the manuscript. competing financial interests: the authors declare no competing financial interests. key: cord- -mmpijsqb authors: philp, lisa k.; day, tanya k.; butler, miriam s.; laven-law, geraldine; jindal, shalini; hickey, theresa e.; scher, howard i.; butler, lisa m.; tilley, wayne d. title: small glutamine-rich tetratricopeptide repeat-containing protein alpha (sgta) ablation limits offspring viability and growth in mice date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: mmpijsqb small glutamine-rich tetratricopeptide repeat-containing protein α (sgta) has been implicated as a co-chaperone and regulator of androgen and growth hormone receptor (ar, ghr) signalling. we investigated the functional consequences of partial and full sgta ablation in vivo using cre-lox sgta-null mice. sgta(+/−) breeders generated viable sgta(−/−) offspring, but at less than mendelian expectancy. sgta(−/−) breeders were subfertile with small litters and higher neonatal death (p < . ). body size was significantly and proportionately smaller in male and female sgta(−/−) (vs wt, sgta(+/−) p < . ) from d . serum igf- levels were genotype- and sex-dependent. food intake, muscle and bone mass and adiposity were unchanged in sgta(−/−). vital and sex organs had normal relative weight, morphology and histology, although certain androgen-sensitive measures such as penis and preputial size, and testis descent, were greater in sgta(−/−). expression of ar and its targets remained largely unchanged, although ar localisation was genotype- and tissue-dependent. generally expression of other tpr-containing proteins was unchanged. in conclusion, this thorough investigation of sgta-null mutation reports a mild phenotype of reduced body size. the model’s full potential likely will be realised by genetic crosses with other models to interrogate the role of sgta in the many diseases in which it has been implicated. full, but not partial, sgta deficiency elicits a phenotype of reduced body size. from weaning through to adulthood ( - wks) , and irrespective of gender, sgta −/− mice weighed significantly less than their sgta +/− and wt littermates (p < . ; fig. a ). body length was reduced in sgta −/− adult males and females as compared to sgta +/− and wt littermates (p < . ), confirming that sgta −/− mice were proportionately smaller (fig. b ). reduced adiposity did not contribute to lower body mass in sgta −/− adults, as neither the absolute nor relative mass of pooled adipose tissue depots were affected by genotype (fig. b ). adipose tissue cellularity was also unaffected at the microscopic level, as assessed by a pathologist (suppl fig. ). changes in lean and skeletal mass are logical contributors for this size difference. however, pooled absolute hind limb muscle mass, whilst significantly reduced in sgta −/− adult mice vs sgta +/− and wt (p < . ), was unchanged when expressed relative to body sgta −/− x sgta −/− sgta +/− x sgta +/− intact comparator number of breeding pairs number of litters average litter size (± sem) . ( ± . statistics: # p ≤ . , trend † p < . , vs intact; * p ≤ . , trend ‡ p < . , vs sgta +/− . (fig. b) . skeletal muscle was also histologically normal across genotypes (suppl fig. ) . likewise, absolute femur weight, whilst significantly reduced in sgta −/− vs wt adults (p < . ), when corrected for body weight, was unchanged (fig. b ). therefore changes in lean and skeletal mass are unlikely to account for smaller body mass. we next aimed to determine the timing and cause of body weight divergence as a result of sgta ablation. sgta-null neonates appeared macroscopically indistinguishable from wt, displaying no signs of disadvantage (fig. c ). measuring pup weight from birth to weaning (fig. c ) revealed a genotype* age interaction (p < . ). from postnatal d - , sgta −/− pups exhibited similar body weight to sgta +/− and wt littermates. while body weights were monitored from birth, it was not until day onwards that sgta −/− pup weight diverged significantly from sgta +/− and wt animals (p < . ; fig. c inset) . feed intake did not account for smaller body mass as feed efficiency, measured from postnatal d - was unchanged by genotype ( fig. a ) and during early neonatal life, milk bands were clearly visible (fig. c inset) . serum growth hormone (gh) levels were highly variable and did not significantly differ by genotype (fig. b ), but serum levels of another growth mediator, igf- , were genotype-and sex-dependent (genotype* sex: p < . ). in males, serum igf- levels were lower in sgta −/− than sgta +/− and wt (p < . ; fig. b ). in females, serum igf- was lower in sgta +/− (p < . ), but not sgta −/− mice, compared to wt (fig. b ). mrna expression of the receptors responsible for gh and igf- action, growth hormone receptor (ghr) and igf- receptor (igf- r), and for igf- transcript were assessed by qrt-pcr in tissues. in the ovary, ghr mrna was higher in sgta −/− than wt (p < . ), but levels were unchanged by genotype in the mammary, prostate or testis (table ) . igf- mrna levels were significantly lower in the mammary gland of sgta −/− females than sgta +/− (p < . ; table ). in testis, igf- mrna was unchanged with genotype, as were igf- r mrna levels in the ovary, mammary, prostate and testis (table ) . sgta −/− breeders bear offspring with reduced perinatal viability. as sgta-null mice were viable, male and female sgta −/− mice were paired to assess fecundity. subfertility and reduced pup viability were observed in this cohort (table ) . litter size trended smaller when from sgta −/− breeding pairs, vs litters from sgta +/− (p < . ) breeders. delayed time to litter with decreasing sgta 'dose' (sgta-wt > sgta +/− > sgta −/− ) failed to reach significance (p = . ). offspring were no more susceptible to stillbirth, but were more prone to neonatal death between postnatal d - and markedly less mice survived to weaning in sgta −/− vs sgta-intact (p < . ) and sgta +/− (p < . ). sex ratios in sgta −/− litters did not deviate from normal ( . m: . f vs expected : , p = . ). effect of sgta ablation on reproductive organ development and ar signalling. sgta has been implicated as a putative co-chaperone for the ar and is believed to limit translocation of ar to the nucleus until the ar complex is ligand-bound . androgen and ar signalling pathways are involved in the development of the male-specific phenotype during early development and in spermatogenesis, sexual behaviour and fertility in male adult life, but are also important in female sex organ development, including uterus and breast, and normal reproductive physiology, such as ovarian folliculogenesis and embryonic implantation, in females. chemical or genetic disruption of androgen/ar signalling perturbs normal male and female reproductive development or function , . we therefore assessed agd (testis descent) and penis and preputial size as surrogates for androgen/ar signalling during early prenatal, prepubertal , and pubertal , development, respectively (fig. a,b) . in general, the phenotypic effects of sgta deficiency on the male reproductive system were subtle. the most significant effect was on testis descent, as agd was significantly increased in sgta −/− males compared to other males (wt and sgta +/− ; p < . ; fig. a ). penis length was increased in sgta −/− vs wt males (p < . ; fig. a ), and penis weight was greater in sgta +/− (p < . ), and trended higher in sgta −/− (p = . ), vs wt (fig. b) . similarly, preputial gland weight was greater in sgta +/− vs wt males (p < . ; fig. b ). however, other male reproductive organs were unchanged as a result of homozygous sgta ablation in their mass (suppl fig. a ), macroscopic appearance (suppl fig. b ) and pathology (suppl fig. ) when compared to wt. sgta −/− males also exhibited sperm in both the testes, the site of spermatogenesis , and epididymis, the site of sperm maturation and storage . similarly, female reproductive organs were unchanged as a result of homozygous sgta ablation in their mass (suppl fig. c ), morphology (suppl fig. d ) or histology (suppl fig. ) when compared to wt. the ovaries, which depend on androgens for normal function and early follicular development , were phenotypically normal at the macro-(suppl fig. c ), and microscopic levels in sgta −/− females as assessed by a pathologist (suppl fig. ). ovaries also exhibited follicles at all stages of follicular development , including primordial, primary, secondary and antral follicles, and the corpus luteum. sgta +/− mice (l, ladder; bp, base pairs; h o, water control; m, male; f, female; n.b. ladder's two brightest bands at (upper) and (lower) bp). (d) quantitative real time pcr confirming homozygous sgta mutation ablated sgta mrna compared to sgta +/− and wt, while heterozygous sgta mutation significantly diminished sgta mrna compared to wt. analyses were conducted in brain, mammary, ovary, prostate, and testis tissues with primers spanning in either sgta exon - and sgta exon - . means (n = /group) with different letters are significantly different, p < . . (e) western blots (brain) and immunohistochemistry (prostate, ovary) confirming loss of sgta protein in homozygous sgta-null (− /− ) tissue and lowered sgta protein expression in heterozygous sgta-null (+ /− ) tissue (lc, loading control; n-sgta, probed with 'n-terminal' sgta antibody; f-sgta, probed with 'full length' sgta antibody; h- , histone h- ; act, actin). for western blots, representative cropped blots are depicted, with all blots run under the same experimental conditions. scale bars represent μ m. scientific reports | : | doi: . /srep as some, but not all, androgen-regulated organs displayed potential signs of hyperandrogenisation during development in sgta +/− and sgta −/− mutants, we measured ar mrna expression in androgen-regulated tissues, the brain, and the prostate and testis in males, and the mammary and ovary in females (fig. c) . ar mrna was unchanged by genotype in the prostate, testis, ovary, mammary and brain. to determine if sgta ablation altered the localisation and distribution of ar protein, we undertook dual-labelled immunofluorescence staining of prostate and testis sections (fig. d ). in the prostate, sgta protein resided chiefly in the cytoplasm of wt and sgta +/− epithelial cells, with weaker intensity staining in sgta +/− than wt, and no staining in sgta −/− animals, as expected (fig. d) . conversely, ar protein was principally nuclear in wt and sgta +/− prostate epithelial cells, with weaker intensity staining in sgta +/− vs wt. however, in sgta −/− prostates, ar immunostaining was equally as intense as in the nuclei of wt prostate, but exhibited greater immunoreactivity in the cytoplasm than wt and sgta +/− . in the testis, sgta was also localised predominantly in the cytoplasm of cells and was detected in the leydig, sertoli, spermatid and spermatocyte cells of wt testis (fig. d ). ar immunostaining was detected predominantly within the leydig cells, with intense nuclear and distinct cytoplasmic immunoreactivity in wt testis. partial sgta ablation increased ar immunoreactivity, especially in the cytoplasm of leydig cells, although distinct nuclear immunostaining was also detected. and finally, full sgta ablation was associated with a visual reduction of ar immunoreactivity, especially in the cytoplasm, although some nuclear and cytoplasmic staining was still observed (fig. d) . hence, some tissue-specific effects of sgta dosage were evident. in order to determine if these changes were associated with changes in ar signalling, the mrna content of known androgen-regulated target genes was assessed in the testis, prostate, ovary and mammary ( table ) . none of the ar-dependent genes measured were altered as a result of full or partial sgta ablation. aging was associated with no genotype-specific abnormalities. as the majority of organs tended to exhibit lighter absolute weight in sgta −/− , presumably due to their smaller body size, we assessed the effect of genotype on organ weights relative to body weight. this allowed direct comparison between genotype and gender. other than an increased heart weight in sgta +/− vs wt (p < . ), brain weight in sgta −/− vs wt (p < . ), and intestine weight in sgta −/− than wt (p < . ), and a reduced relative stomach weight in sgta +/− vs sgta −/− (p < . ) and wt (p < . ), the mass of most vital organs relative to body weight was unchanged by genotype (suppl fig. e ,f). all organs were also phenotypically normal in terms of morphology and pathology (suppl fig. ). aging is considered to be a stressor and is associated with the accumulation of misfolded proteins . given the key role of sgta in protein processing and folding , we were interested to determine if aging resulted in a more females pronounced phenotype in sgta-null animals. however, the relative weights of vital and sex organs, adipose tissue, muscle and bone were not affected by a genotype* age interaction, suggesting that sgta-null mice are not any more vulnerable to age-related stress than wt. sgta has also been implicated in neuro-synaptic transmission and in responses to amyloid-associated toxicity , however in this study sgta-null animals showed no overt symptoms of neurodegeneration in either adulthood or aging. other tpr-containing proteins are largely unchanged in sgta −/− mice. given the mild phenotype observed in sgta −/− animals, we hypothesised that one or multiple tpr-containing proteins could possibly recent studies from our laboratory , , and others , , have linked altered sgta expression to tumour cell proliferation and/or cancer prognosis. this is not surprising, given that sgta has been implicated in cell cycle and apoptosis , , and in the molecular co-chaperoning of numerous protein clients to ensure their correct folding, compartmentalisation and/or trafficking . each of these processes, if defective, has the potential to contribute to tumorigenesis. aside from a role in the pathophysiology of several disease-states , , [ ] [ ] [ ] [ ] [ ] [ ] , little is known about the normal physiological role of sgta. in vitro, sgta knockdown in kidney (nbe) , cervical cancer (hela) and prostate cancer (c - b) cells reduces proliferation and viability due to impaired mitosis , , in association with defective cytokinesis and failed completion of chromosomal alignment . because of this, and the fact that sgta is ubiquitously expressed and a wide range of protein interactions with sgta are documented , we hypothesised that sgta would be vital for life and that its ablation in mice would prove lethal. on the contrary, however, we demonstrated here that viable sgta −/− animals were produced from breeding sgta-deficient mice, as is the case in the knockout/down of conserved sgta orthologs , in c. elegans, d. melanogaster and s. cerevisiae , , . the tendency we observed for reduced numbers of sgta −/− offspring in comparison to expected mendelian inheritance is consistent with that of mutants for similar tpr-containing protein, fkbp (fkbp ) , . given considerable evidence that sgta is involved in tail-anchored (ta) protein insertion , , , and as ta biogenesis is essential for early development and genetic ablation disrupting this process has been linked to embryonic lethality , , it is possible that aberrant ta protein processing may contribute to the reduced yield of mutant animals. the cause of prenatal mortality remains undetermined, however, in the case of fkbp −/− , genetic background did contribute to the penetrance of fkbp action. that is, independent generation of fkbp −/− mice on c bl/ j and svev backgrounds yielded lethality at % and % of embryos from heterozygous matings, respectively. moreover, backcrossing fkbp - svev mutants to the c bl/ strain amplified lethality to almost % ; highlighting the caveat that the underlying genetics of a mutant model influence the phenotype presented. whether a more pronounced phenotype of sgta −/− would have been observed if it been characterised on a different background strain of mice, remains to be determined. in contrast to fkbp −/− , deletion of the similar protein, fkbp (fkbp ), had no effect on early survival and elicited no apparent altered phenotype in mice , although fkbp knockout (c bl/ j background) in combination with fkpb deletion ( svev background) caused complete lethality in early embryonic life . this highlights that redundancy exists in the co-chaperoning system. consistent with this notion, fkbp −/− males developed normal testes, but displayed penile hypospadias . increased fkbp mrna and protein expression in fkbp −/− testis, but not in penis, may actively prevent testis dysgenesis. in the context of homozygous sgta ablation, it is possible that a structurally similar tpr-containing protein(s) intervenes, fulfilling the role of sgta in its absence. increased dnajc (tpr ) mrna in the ovary may serve this purpose, however the mrna expression of tpr-containing proteins was unchanged in the prostate, testis and mammary gland. whether changes in protein expression and/or localisation of tpr-containing protein(s) compensate for loss of sgta in normal physiology merits further investigation and could explain why only a mild sgta-null phenotype was observed in the current study. a more overt phenotype may be induced by applying a stressor to sgta-null mutants, as co-chaperones are essential for optimal folding/refolding and trafficking of stress-affected client proteins . in yeast null for sgta ortholog, sgt- , two distinct cellular stressors reduced yeast viability , growth and colony formation compared to wild-type yeast. this further supports that sgta is essential under certain cellular conditions and it is likely that its loss is compensated for to prevent dysfunction when these conditions arise. in sgta-null mice, reduced body size comprising a proportionate decrease in body weight and length was the most overt phenotypic change observed and was limited to homozygous mutants. inadequate nutrition could result in stunted sgta −/− growth, especially as weight differences coincided with a phase where dependence on nursing is lessened. this appears not to be the case, as feed efficiency was unchanged in sgta −/− mice, albeit their ability to absorb and utilise nutrients from food consumed remains unknown. whether sgta ablation prompted changes in the signalling of hormonal growth mediators warranted investigation, especially as myostatin and the ghr are sgta client proteins. defective development and altered mass of skeletal muscle would be predicted if sgta deficiency influenced the maturation and signalling of myostatin, a negative regulator of muscle mass ; however neither muscle pathology nor mass were affected. gh, acting via ghr, is essential for somatic growth, cellular differentiation , and paracrine production of igf- . aberrant ghr signalling, and gh resistance, causes cessation of igf- production and growth stunting. despite both gh and ghr normally being expressed in early embryogenesis, growth changes due to altered gh signalling are only observed from postnatal d - in mice , . dwarfing at weeks in ghr-null mice is associated with heightened gh and lowered levels of igf- in the circulation , . likewise, decreasing functional igf- r in mice (igf- rneo) leads to growth stunting from - weeks of age, corresponding to a plateau in size, which is maintained . analogous to ghr/ igf- r deficiency , , sgta-null mutants emerged as being physically smaller than wt at ~ weeks and plateaued at ~ weeks, at which time females were %, and males %, of the weight of wt animals. similar to gh/ghr, sgta is detected prenatally in the embryo, but its functionality during early development is not known. given its interaction with the ubiquitin-dependent endocytosis motif of precursor and mature ghr, sgta has been speculated to prevent ghr interaction with ubiquitin machinery whilst being trafficked from the endoplasmic reticulum, where precursor ghr resides, to the cell membrane, where mature ghr functions . aberrant ghr/ igf- r trafficking and potentially ubiquitin-mediated ghr degradation could be triggered by sgta ablation. we hypothesise that this may contribute to the growth stunting observed in sgta −/− mice, providing the loss of sgta is not compensated by another co-chaperone. lowered igf- serum and tissue mrna levels in sgta −/− animals may reflect defective ghr signalling, whereas raised serum gh and elevated ghr in the ovary in sgta −/− may indicate compensation for deficient gh/igf- signalling; however, the latter requires confirmation at the protein level. unlike homozygous fkbp deletion which rendered mice sterile , sgta deficiency elicited only mild subfertility in this study. dysgenesis in sex organs dependent on androgen/ar-driven development were the leading cause of defective fkbp −/− male reproduction , while failure of progesterone receptor-dependent uterine implantation conferred sterility in fkbp −/− females . increased surrogate measures (agd, penis and preputial gland size) for androgen signalling in utero, both prepubertally and at puberty, in full or partial sgta deficiency are suggestive of altered ar signalling and/or hyperandrogenisation. however, several other androgen-dependent organs were unaffected by sgta ablation in males and females, as was the mrna expression of ar and ar-responsive genes. regardless, an effect of sgta ablation on sex steroid production cannot be excluded. in addition to ar regulation, sgta also exhibits regulatory specificity for progesterone and glucocorticoid receptors , ; thus the influence of sgta ablation on biological processes controlled by progesterone and glucocorticoid signaling also warrant future investigation. reduced nuclear ar immunoreactivity without a concomitant increase in the cytoplasm in the sgta +/− prostate, is suggestive of sgta, like other tpr-containing proteins tpr /dnajc and chip/stub , playing a role in ar stabilisation and/or degradation . this appears to be the case for ghr , where its stabilisation is not compensated for in an environment of partial sgta deficiency. in summary, this study has provided an intriguing first glimpse into the multi-faceted role of sgta in vivo. full, but not partial, ablation of sgta conferred subfertility and limited the viability and growth of offspring. the complex interplay of molecular co-chaperones and the importance of redundancy in their physiological roles, particularly in hormone receptor maturation and signalling, is highlighted by the current findings. this sgta-null model is amenable to further study of several clinical disorders, including hormone-dependent and β -amyloid diseases, where a role for sgta has been implicated. heterozygous sgta knockout mice were generated by ozgene pty ltd (bentley, aus.). the gene encoding m. musculus sgta is located on chromosome and has exons, of which are protein coding. structurally, sgta exhibits a central tandem array of tpr motifs, a glutamine-rich c-terminal domain and an n-terminal domain containing a potential short coiled coil motif (as reviewed in ). our strategy was to delete the coding region of exons and of m. musculus sgta. this approach would result in either: ( ) cryptic splicing between exons - , generating a stable mrna coding for a truncated protein lacking the key functional domains, tpr and tpr , for a functional knockout, or ( ) a transcript lacking exons and , potentially rendering it unstable and leading to a complete knockout. a targeting vector was designed to introduce a conditional mutation into the mouse sgta gene (ensembl gene id: ensmusg ; ncbi nucleotide accession number: nm_ ) (suppl fig. c) , employing mutant loxp sites to enable deletion of the loxp flanked ("floxed") sequence under cre recombinase-expressing conditions. loxp sites were inserted into the introns flanking exons and , incorporating tpr completely and part of tpr of m. musculus sgta. the loxp site flanking exon was inserted upstream and the loxp site flanking exon was inserted downstream of a neomycin selection cassette (phosphoglycerate kinase (pgk)-neo). the selection cassette was flanked with flippase recognition target (frt) sites to enable removal by flpe-mediated recombination. the targeting vector was inserted into a plasmid backbone peller (ozgene) containing the pgk-neo cassette (suppl fig. c ), linearised and electroporated into c bl/ -derived embryonic stem cells (ozgene). correctly targeted stem cell clones were microinjected into recipient murine blastocysts, which were transferred into pseudo-pregnant foster mothers. offspring were crossed with c bl/ mice to generate embryonic stem cell-derived targeted mutant progeny (heterozygous sgta wt/flox), as confirmed by genotyping. these mice were crossed with ozcre mice, which express cre knocked into the ubiquitously expressed gt(rosa) sor locus. global tissue expression of cre initiated loxp-mediated deletion of exons and of sgta in early embryonic development and generation of sgta +/− + cre mice. to minimise the impact of cre-related toxicity , cre was eradicated by back-crossing sgta +/− cre mice to c bl/ and it was only cre-negative (Δ cre) mice that were used subsequently and described herein. mice were housed under specific pathogen-free conditions on a -hr light/dark cycle at the university of adelaide medical school animal house (adelaide, aus). mice were group-housed (unless specified below) in individually ventilated cages in a temperature-controlled environment and were provided autoclaved standard rodent chow (meat free rat and mouse diet, specialty feeds, glen forrest, aus; . mj/kg ( % and % energy from protein and fat, respectively, pre-autoclave)) and water ad libitum. pcr-based genotyping. confirmation of removal of the "floxed" sgta sequence in mice was determined by pcr-based genotyping. extraction of genomic dna from tail or ear clippings was performed using the redextract-n-amp tissue pcr kit (sigma-aldrich, st louis, us) according to manufacturer's instructions. a portion of this dna was applied to pcr-based genotyping for the determination of the presence of ( ) sgta alleles (wt vs knockout) and ( ) cre (positive or negative). ( ) genotyping for the sgta alleles employed primer sets, referred to as 'sgta knockout' or 'sgta intact' primer pairs. the 'sgta knockout' primer pair was designed to yield a bp product in response to a deleted sgta allele (+ /− , − /− ) and a higher molecular weight product ( bp) was observed in the presence of wt sgta alleles. the 'sgta intact' primer pair was designed to yield a bp product in response to an intact sgta allele (wt, + /− ) and the absence of a product in the presence of homozygous pcr conditions were an initial denaturation at °c for min; cycles of °c for min, °c for min and °c for min; followed by final elongation at °c for min. the distribution of sgta mrna in normal human tissue samples was assessed from publically available data through the oncomine database (https://www.oncomine.org/). sgta mrna content (log median-centred intensity) was measured using the human genome u plus . array on and normal human tissue samples for the roth normal and roth normal cohorts, respectively . a heat map was generated using conditional formatting and a colour scale based on the value in the cell and represented graphically, and categorised into tissues of endodermal, ectodermal and mesodermal embryonic origin (suppl fig. a) . mouse sgta gene expression during embryonic development and in gametes was assessed using public data in gene expression barcode . (http://barcode.luhs.org/). assessment of fertility, breeding efficiency and offspring viability. all sgta +/− and sgta −/− breeding pairs were monitored for time to litter (date paired to date litter born) and pup yield was assessed for litter size, live born, stillborn (birth/d ) and neonatal death (postnatal d - ). offspring were reared by their mother until wks-of-age, at which point they were removed from the breeding cage, weaned, sexed, tail-tipped (for genotyping), and placed in a cage with littermates of the same sex. genotype distribution of litters was monitored for normal mendelian inheritance in the case of sgta +/− breeders. sex distributions were determined for all litters. from wks, body weight was determined once-weekly. a cohort of offspring from sgta +/− and sgta −/− breeders were analysed for birth weight (d ), neonatal weight (d - ) and for food intake or for body weight post-weaning (d - ). to assess birth weight, a cohort of female breeders were left paired with males and checked daily - hrs after lights-on ( am), and any newborn pups were weighed. pups were weighed daily and any dead pups collected and genotyped for sgta status and sex. upon weaning (strictly at d ), pups were individually housed and food intake assessed. during food intake assessment, food and water were provided ad libitum. from d - , feed was first weighed, then placed in the cage for ~ hrs whereupon the remaining feed and crumbs were weighed. feed efficiency was calculated as weight gained from d - , relative to cumulative feed consumption for this time period (g weight gained/g feed consumed* ). a cohort of offspring from sgta +/− breeding pairs were assessed for phenotypic differences at adult ( wks-of-age) and aged ( wks-of-age) time points. from weaning ( wks), body weight was measured weekly and on the day culled. at or -wks-of-age, food was removed between - am and mice were fasted for hrs, with continued access to water. post-fast, mice were humanely culled by carbon dioxide asphyxiation. body weight and length (nose-to-tail base), and anogenital distance (agd), were measured. blood was drawn by cardiac puncture and serum separated using minicollect tubes (greiner bio-one international, kremsmünster, austria). the following organs were rapidly dissected and weighed: in males, the whole genitourinary (gu) tract and subsequently dissected testes, vas deferens, epididymis, seminal vesicles and prostatic lobes (anterior, dorsolateral and ventral), penis and preputial gland; in females, the left and right abdominal mammary glands, whole gu tract and subsequently dissected uterus, ovaries and fallopian tubes; and in both males and females, the thymus, heart, lungs, pancreas, liver, spleen, stomach, intestine, colon, kidneys, brain and femur; adipose tissue depots (visceral mesenteric, pooled perirenal and retroperitoneal, gonadal (epididymal -males and periovariac -females) and interscapular brown); and skeletal muscles (gastrocnemius, tibialis, extensor digitorum longus and soleus). male penis length was also assessed. in the case of paired organs, one of the pair was placed into rnalater (ambion inc, austin, us) and the other in % neutral buffered formalin (nbf). singular organs were halved and placed in rnalater or % nbf. following incubation ( °c, ~ hrs), organs in rnalater were stored at − °c and those fixed in % nbf underwent overnight processing and were embedded in paraffin wax. serum aliquots were stored at − °c. tissue pathology, immunohistochemistry and immunofluorescence. formalin-fixed paraffin-embedded tissue sections ( μ m) from adult and aged mice were stained with haematoxylin and eosin and assessed for tissue morphology by a pathologist (sj). for immunohistochemical analyses, adult mouse tissue sections were incubated in . % hydrogen peroxide ( min) and antigen retrieval was performed by decloaker ( mm citrate buffer (ph . ); min at °c and sec at °c). cooled sections were subjected to successive min blocking incubations in avidin/biotin block (invitrogen, mulgrave, aus); rodent block m (biocare medical, concord, us); and % goat serum. sgta primary antibody-treated sections were incubated overnight ( °c). sgta immunoreactivity in normal wt mouse tissues was assessed using 'full length sgta' proteintech antibody ( - -ap rabbit polyclonal; chicago, us) and in sgta +/− offspring tissues by 'n-terminal sgta' santa cruz antibody (sc- mouse monoclonal; dallas, us). primary antibody omission was employed as a negative control. immunogenic sgta was visualised by a standard immunoperoxidase reaction using biotinylated anti-rabbit or anti-mouse antibody ( : , dako aus pty ltd, north sydney, aus), streptavidin-horseradish peroxidase complex ( : , dako) and diaminobenzidine tetrahydrochloride. all sections were scanned using the nanozoomer digital pathology image scanner (hamamatsu photonics, hamamatsu city, japan). for immunofluorescent analyses, tissue sections from adult mice were subjected to blocking as above, except % goat serum was employed, and then incubated overnight ( °c) in a solution containing sgta (sc- ) and ar (santa cruz, sc- ) primary antibodies. primary antibody omission served as a negative control. visualisation of immunogenic sgta and ar were achieved using red alexa fluor goat anti-mouse- and green alexa fluor goat anti-rabbit- (invitrogen), respectively. sections were mounted (prolong gold anti-fade reagent with dapi (invitrogen) blue) and visualised by a zeiss confocal microscope, 'zen' software and zeiss ldm with lumen x-cite series pc camera. western blot analysis. protein was extracted from homogenised adult mouse brain (~ mg brain; μ l ripa buffer containing protease inhibitor cocktail (roche diagnostics, indiana, us)) by centrifugation. protein yield was determined by bradford assay (bio-rad laboratories, hercules, us; bovine serum albumin (bsa) standard). protein ( μ g) was subjected to gel electrophoresis utilising criterion xt . mm gels (bio-rad) and transferred to nitrocellulose membrane (hybond-c extra . μ m; amersham biosciences, rydalmere, aus). membranes blocked in % bsa-tbs/t (tris buffered saline (tbs)/ . % tween- (t)) overnight ( °c) were probed for sgta using 'full length sgta' (proteintech), 'c-terminal sgta' (aviva biosciences, san diego, us) or 'n-terminal sgta' (santa cruz) antibodies diluted at : , (full, c-terminal) or : (n-terminal) in % bsa-tbs/t ( min, room temperature). immunoreactivity was detected using polyclonal goat anti-rabbit ( : , ) or rabbit anti-mouse igg/hrp ( : ) antibodies (dako) followed by chemiluminescent visualisation (super signal west dura extended duration, thermo fisher scientific, scoresby, aus) and chemidoc mp imaging (bio-rad). reference proteins used were actin (santa cruz biotechnology, sc- , : , ) or histone h- (abcam, ab , : ). quantitative real time (qrt-) pcr. total rna was extracted from adult brain, ovary, mammary, anterior prostate and testis tissue. tissues were homogenised in buffer rlt (qiagen, germantown, us) and -mercaptoethanol (sigma-aldrich) using a precellys- homogeniser then passed through a qiashredder column (qiagen). homogenate was then subjected to an rneasy spin column workflow, as per qiagen instructions. eluted rna quality and quantity were assessed (nanodrop, thermo fisher scientific), following which rna ( μ g) was dnase treated (ambion rna turbo dna-free kit) and reverse transcribed (iscript first-strand cdna synthesis kit, bio-rad; ng total rna used as template). qrt-pcr primers for target genes of interest were designed using vector nti (life technologies) and synthesised by sigma-aldrich pty ltd (sydney, aus) (see suppl table ). triplicate qrt-pcr reactions were carried out using iq sybr green supermix and the cfx real-time pcr detection system (bio-rad). in each reaction, μ l cdna, . μ l each of fwd and rev primers ( pm/μ l) and μ l sybr green mastermix (applied biosystems) were added to a final volume of μ l. pcr cycling conditions were °c for min, followed by cycles of: °c for sec, annealing temperature (as described in suppl table ) for sec, and °c for sec; following which steps at °c for min, °c for min and - °c for sec ( -step amplification and melt curve) were performed. in parallel, mrna expression of a candidate reference gene panel was determined and the most stable (determined by genorm analysis) were used to normalise target gene mrna expression using the delta-delta ct method. pcr product specificity was validated with the use of a melt curve and by gelred agarose gel electrophoresis. reference genes utilised for normalisation were: ovary and anterior prostate -actb and tbp; mammary -tbp and hprt ; testis and brain -actb and tfrc. statistical analyses. all data presented as mean ± sem of parameters. shapiro-wilk tests of normality and levenes statistic tests of homogeneity of variance were performed and q-q plots assessed for all parameters. one-way anova, with pairwise comparisons (tukey or dunnett's t post-hoc), was used to determine the effect of genotype (wt, sgta +/− , sgta −/− ) on mrna expression. the effect of genotype, age ( , wks) and sex (male, female) on body weight were assessed using a linear mixed model. two-way anova, with pairwise comparisons (sidak or dunnett's t post-hoc), was used to determine the effect of genotype, age, sex and their interaction on the remaining parameters. if tests of normality failed, non-parametric median tests were employed. likewise, if data did not display homogeneity of variance, post-hoc tests in which equal variances are not assumed (dunnett's t ) were employed. observed vs expected frequencies of genotype and sex were assessed by chi-square analyses using graphpad prism ver. . and linear regression analyses to determine relationships between variables (pearson score (r) and p value) were performed using sigmaplot ver. ; all other aforementioned statistics were performed using statistical package for social scientists ver. . p < . was considered statistically significant. sgta: a new player in the molecular co-chaperone game the tetratricopeptide repeat: a structural motif mediating protein-protein interactions tpr proteins: the versatile helix identification of a novel cellular tpr-containing protein, sgt, that interacts with the nonstructural protein ns of parvovirus h- human sgt interacts with bag- /bat- /scythe and cells with reduced levels of either protein display persistence of few misaligned chromosomes and mitotic arrest functional interaction of human immunodeficiency virus type vpu and gag with a novel member of the tetratricopeptide repeat protein family structural and functional characterization of human sgt and its interaction with vpu of the human immunodeficiency virus type association of vpu-binding protein with microtubules and vpudependent redistribution of hiv- gag protein severe acute respiratory syndrome coronavirus protein a interacts with hsgt control of androgen receptor signaling in prostate cancer by the cochaperone small glutamine rich tetratricopeptide repeat containing protein alpha subdomain structure of the co-chaperone sgta and activity of its androgen receptor client small glutamine-rich tetratricopeptide repeat-containing protein (sgt) interacts with the ubiquitin-dependent endocytosis (ube) motif of the growth hormone receptor a trimeric protein complex functions as a synaptic chaperone machine cloning, purification and characterization of the caenorhabditis elegans small glutamine-rich tetratricopeptide repeat-containing protein small glutamine-rich protein/viral protein u-binding protein is a novel cochaperone that affects heat shock protein activity cooperative and independent activities of sgt and get in the targeting of tail-anchored proteins small glutamine-rich tetratricopeptide repeat-containing protein is composed of three structural units with distinct functions interaction of intracellular β -amyloid peptide with chaperone proteins crystal structure of get -get complex and its interactions with sgt , get , and ydj sgt and mdy interact with molecular chaperone ydj in saccharomyces cerevisiae a structural model of the sgt protein and its interactions with chaperones and the get /get complex a chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum hsgt interacts with the n-terminal region of myostatin identification of a novel cellular tpr-containing protein, sgt, that interacts with the nonstructural protein ns of parvovirus h- a brain-specific isoform of small glutamine-rich tetratricopeptide repeat-containing protein binds to hsc and the cysteine string protein knockdown of the cochaperone sgta results in the suppression of androgen and pi k/akt signaling and inhibition of prostate cancer cell proliferation androgen receptor protein levels are significantly reduced in serous ovarian carcinomas compared with benign or borderline disease but are not altered by cancer stage or metastatic progression expression of sgta correlates with prognosis and tumor cell proliferation in human hepatocellular carcinoma high expression of sgta in esophageal squamous cell carcinoma correlates with proliferation and poor prognosis small glutamine-rich tetratricopeptide repeat-containing protein alpha (sgta), a candidate gene for polycystic ovary syndrome regulation of chaperone effects on a yeast prion by cochaperone sgt isolation and characterization of human sgt and identification of homologues in saccharomyces cerevisiae and caenorhabditis elegans cysteine-string protein's neuroprotective role the human small glutamine-rich tpr-containing protein is required for progress through cell division gene expression analyses reveal molecular relationships among regions of the human cns insulin-like growth factor (igf- ): a growth hormone relative importance of prenatal and postnatal androgen action in determining growth of the penis and anogenital distance in the rat before, during and after puberty androgen receptor (ar) physiological roles in male and female reproductive systems: lessons learned from ar-knockout mice lacking ar in selective cells response of preputial and adrenal glands of the rat to sex hormones identification of testosterone-dependent volatile compounds and proteins in the preputial gland of rat rattus norvegicus duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium the role of the human epididymis in sperm maturation and sperm storage as reflected in the consequences of epididymovasostomy an analysis of follicle development and ovum maturation protein misfolding and cellular stress in disease and aging sgt, a hsp β binding partner, is accumulated in the nucleus during cell apoptosis overexpression of small glutamine-rich tpr-containing protein promotes apoptosis in cells physiological role for the cochaperone fkbp in androgen receptor signaling fk -binding protein is essential to uterine reproductive physiology controlled by the progesterone receptor a isoform the association of bag with sgta and tail-anchored proteins targeted disruption of the mouse asna gene results in embryonic lethality caml is required for efficient egf receptor recycling susceptibility to diet-induced hepatic steatosis and glucocorticoid resistance in fk -binding protein -deficient mice essential role for co-chaperone fkbp but not fkbp in androgen receptor-mediated signaling and physiology regulation of steroid hormone receptor function by the -kda fk -binding protein (fkbp ). current opinion in pharmacology chl is a selective organizer of the presynaptic machinery chaperoning the snare complex a deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle molecular mechanism of growth hormone action a mammalian model for laron syndrome produced by targeted disruption of the mouse growth hormone receptor/ binding protein gene (the laron mouse) a targeted partial invalidation of the insulin-like growth factor i receptor gene in mice causes a the cochaperone sgta (small glutamine-rich tetratricopeptide repeat-containing protein alpha) demonstrates regulatory specificity for the androgen, glucocorticoid, and progesterone receptors versatile tpr domains accommodate different modes of target protein recognition and function growth inhibition and dna damage induced by cre recombinase in mammalian cells pitfalls of pcr-based strategy for genotyping cre-loxp mice key: cord- -o azey m authors: chan, louisa l. y.; bui, christine t. h.; mok, chris k. p.; ng, mandy m. t.; nicholls, john m.; peiris, j. s. malik; chan, michael c. w.; chan, renee w. y. title: evaluation of the human adaptation of influenza a/h n virus in pb protein using human and swine respiratory tract explant cultures date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: o azey m novel avian h n virus emerged in china in resulting in a case fatality rate of around % and continues to pose zoonotic and pandemic risk. amino acid substitutions in pb protein were shown to influence the pathogenicity and transmissibility of h n following experimental infection of ferrets and mice. in this study, we evaluated the role of amino acid substitution pb - k or compensatory changes at pb - k and pb - n, on the tropism and replication competence of h n viruses for human and swine respiratory tracts using ex vivo organ explant cultures. recombinant viruses of a/shanghai/ / (rgh n ) and its mutants with pb -k e, pb -k e + q k and pb -k e + d n were generated by plasmid-based reverse genetics. pb -e k was essential for efficient replication of rgh n in ex vivo cultures of human and swine respiratory tracts. mutant rgpb -k e + d n replicated better than rgpb -k e in human lung but not as well as rgh n virus. the rgpb -k e mutant failed to replicate in human type i-like pneumocytes (ati) and peripheral blood monocyte-derived macrophages (pmϕ) at °c while the compensatory mutant rgpb -k e + q k and rgpb -k e + d n had partly restored replication competence in pmϕ. our results demonstrate that pb -e k was important for efficient replication of influenza h n in both human and swine respiratory tracts. in march , a novel avian-origin h n virus emerged in china. as of th july , a total of laboratory confirmed human infections and deaths were reported from provinces and municipalities in mainland china, hong kong, macau, taiwan, malaysia and canada. h n viruses have spread from eastern china in the first wave of the outbreak in early , to southern china in the second wave, and has now become enzootic in multiple provinces in china. since infection in poultry is asymptomatic, h n virus is likely to spill over borders and spread across the region in a pattern similar to that observed with h n and h n influenza viruses previously , . human h n infections can lead to a rapidly progressing viral pneumonia, acute respiratory distress syndrome (ards) and multi-organ failure , especially in older patients and in those with underlying co-morbidities. most zoonotic h n disease is associated with exposure to poultry within live poultry markets , with no evidence of sustained human-to-human transmission. active surveillance of chickens in live poultry markets in five provinces in china showed an average isolation rate of . % . phylogenetic analysis indicated that the novel h n virus originated through reassortant of avian influenza viruses from wild aquatic birds and poultry; the hemagglutinin gene and the neuraminidase gene respectively, being derived from h n and h n viruses in the replication kinetics of the rgh n and its pb mutants in an avian chicken fibroblast cell line (df- ) and a mammalian cell line (mdck) at °c and °c were compared (fig. ). in the avian df- cells, rgpb -k e demonstrated its temperature sensitive phenotype and replicated poorly at °c (fig. a) while the mutant viruses with the human compensatory markers, rgpb -k e + q k and rgpb -k e + d n were able to replicate to similar titers as rgh n which has pb - k (fig. a) . at °c, all h n viruses achieved similar viral titers by hpi indicating their replication competence in avian cells, though rgpb -k e virus had a delayed replication kinetics with a lower viral titer at hpi (fig. b) . in the mammalian mdck cells, the replication kinetics of rgh n (pb - k) was similar at both °c and °c with greater replication efficiency than all three mutants with pb - e (fig. c,d) . the mutant virus rgpb -k e failed to replicate at °c and yielded limited virus progeny at °c at hpi. pb -k e mutants with compensatory human adaptation markers, pb -q k or d n, had partially restored the replication competence in mdck cells at both temperatures, but they failed to replicate as efficiently as rgh n which possesses pb - k. replication competence of rgh n and pb mutants in ex vivo cultures of human bronchus and lung at °c. the rgh n virus replicated to significantly higher titers than each of the pb mutants in the human bronchus ( fig. a) . it also had a trend for a more efficient replication than each of the pb mutants in the lung (fig. b) at all time-points. the rgpb -k e mutant virus failed to replicate in either human bronchus or lung. the introduction of compensatory human markers, pb -q k and d n, partly rescued the replication competence of the rgpb -k e virus in lung. the mutant virus with rgpb -k e + d n showed partly restored replication competence in bronchus but its counterpart virus, rgpb -k e + q k did not. tissue tropism of the rgh n and pb mutants in ex vivo cultures of human bronchus and lung. immunohistochemical staining of the infected tissues indicated that rgh n virus possessing pb - k infected the human bronchial epithelium and the alveoli more extensively than its pb mutants (fig. c,d) . the rgpb -k e mutant virus failed to replicate in the ex vivo cultures of human respiratory epithelia (fig. c,d) . moreover, the extent of infection correlated well with the viral replication titers. there were very limited influenza antigen positive cells in the rgpb -k e + q k and rgpb -k e + d n inoculated ex vivo culture of human bronchus (fig. c ) while more infected cells were shown in these mutant viruses infected lung tissues (fig. d ). replication competence and tissue tropism of rgh n and pb mutants in ex vivo cultures of swine trachea, bronchus and lung. rgh n virus failed to replicate in ex vivo cultures of swine trachea but replicated in bronchus and lung ( fig. a-c) . the mutant viruses rgpb -k e + q k and rgpb -k e + d n also replicated in ex vivo lung cultures (fig. c ) while minimal or no viral replication was observed in swine trachea and bronchus (fig. a,b) . rgh n and all pb mutants replicated to lower titers than h n pdm did, especially in the swine lung ( supplementary fig. a) . in ex vivo culture of the swine respiratory tract, the epithelial cells in the terminal bronchioles were the main target cell types. the most extensive influenza nucleoprotein antigen positive cells were seen with the rgh n virus, followed by rgpb -k e + q k mutant virus-infected tissues (fig. f ). limited numbers of infected alveolar macrophages and type ii pneumocytes were seen in the virus inoculated swine alveolar ex vivo cultures (fig. g) . none of the viruses were able to infect the ex vivo cultures of swine trachea and bronchus, with the exception of some positive cells found in the interstitial tissue within the trachea (fig. d ) and bronchus (fig. e ). h n pdm also failed to infect the swine trachea and bronchus, the extent of infection in the terminal bronchioles and alveoli in the swine lung was similar to that of rgh n (supplementary fig. b -e). with a moi of . infection, rgh n replicated to titers significantly higher than all its pb mutants at all the time points examined in both type i-like pneumocytes (ati) and peripheral blood monocyte-derived macrophages (pmφ ) (fig. a,b) . rgpb -k e failed to replicate in both primary cells, while compensatory human markers pb -q k and d n partially restored the viral replication efficiency. therefore, we investigated if the lack of replication was due to the lack of infectivity or the incompetence in producing new progeny viruses. we compared the infection rate of these viruses in ati at hpi ( supplementary fig. ) and pmφ at hpi ( supplementary fig. ). with a moi of infection, there were no statistical significant differences in the infection rate among the rgh n virus to its mutants, ranging from - % in ati ( supplementary fig. e ) and - % in pmφ . cytokine and chemokine gene expression in the h n virus infected human ati and pmφ. the cytokine and chemokine gene expression was compared among the rgh n and its mutants in ati and pmΦ at a moi of . in ati, rgpb -k e + q k and rgpb -k e + d n induced significantly more il- gene expression than the rgh n (fig. e ) while mutant rgpb -k e induced significantly lower ifn-β and ccl than rgh n (fig. c ,i) at hpi. in pmφ , though the cytokine and chemokine expression induced by the pb mutants was comparable with rgh n , rgpb -k e + d n upregulated the expression of il- compared to the other h n viruses (fig. f ). in accordance with our previous findings , h n virus represented by rgh n in this study, were found to be moderate inducers of pro-inflammatory cytokine and chemokine with h n being in this study, we evaluated the effects of pb -e k mutation as well as two mammalian adaptation markers, pb -q k and d n on h n virus replication competence in ex vivo cultures of human and swine respiratory tracts and in vitro cultures of human primary ati and pmφ . while avian h n viruses invariably have pb - e, a/shanghai/ / (sh ) (h n ) virus which was isolated from a patient with severe disease had acquired the mammalian adaptation mutation pb -e k, and this is reflected in recombinant rgh n virus used in this study. tissue tropism and virus replication competence of rgh n virus and its pb mutants were compared. recombinant h n virus with pb - k replicated to similar titers with h n pdm in ex vivo cultures of human bronchus and lung as we previously described . we demonstrated that h n possessing pb - e failed to infect and replicate in the ex vivo cultures of human bronchus, lung, in vitro cultures of human ati and pmφ and in ex vivo cultures of swine trachea and bronchus suggesting that the avian virus is not adapted for replication in the human or swine respiratory tract. the compensatory mammalian adaptation mutations in pb , q k or d n could partially rescued the virus replication in human and swine cells and tissues though it was not reaching the full competence of the rgh n virus with the pb - k. this emphasized the key role of pb -e k in contributing to the efficient viral replication of h n in the human respiratory tract. these findings are in agreement with others who have found that lysine in position in pb was found to be essential for mammalian adaptation in terms of the enhancement of polymerase activity, viral growth kinetics and virulence in mice when compared with the mutants of sh or a/anhui/ / (h n ) having pb - e , . from our observation, the viral replication efficiency of h n and the pb mutants in ex vivo cultures of human respiratory tract was greater than in swine respiratory tract for approximately -fold difference in viral titer. this might contribute to the differential polymerase activities in human cells and porcine cells , the relatively lower polymerase activity detected in porcine cells may be responsible for the overall low viral titers resulted from h n virus infection in the ex vivo cultures of swine respiratory tissues [ ] [ ] [ ] . the profiles of sialic acid distribution in human and swine respiratory tract might play a crucial role as well though the distribution pattern of sialic acid receptors in swine and human respiratory tracts are similar , , , but higher galα - gal expression and comparatively rare extended sialylated lacnac repeats can be observed in swine . several previous studies have investigated the replication and infection potential of h n using swine as experimental model, both in vivo and ex vivo. here, we isolated the ex vivo cultures of swine trachea, bronchus and lung from intact respiratory organs of -month old pigs (sus scrofa domestica). these pigs were farm-raised, exposed to field conditions and represent the natural population of animals . jones et al., in contrast, used the explant cultures of tracheal and lung explants of one-week-old laboratory piglets to examine the infectivity of h n in swine. they found that three human h n viruses including a/anhui/ / , a/shanghai/ / and a/shanghai/ / replicated efficiently in trachea and lung . in our case, although no replication and positive staining were found in the swine trachea after virus inoculation, the influenza nucleoprotein antigen positive cells were also found in terminal bronchiolar epithelial cells in the lung. the lectin binding and glycan array profiles of the respiratory tracts of adult domestic pigs are different from infant pigs, the binding of sambucus nigra agglutinin (sna, a lectin binds α - , glycans) was strong for the upper respiratory tract of both infant and adult pigs but binding to maackia amurensis agglutinin-i (maa-i, a lectin binds α - , n-glycans) and maa-ii (a lectin binds α - , o-glycans) was only observed infant pigs. there was a strong binding of sna lectin to the alveoli of infant pigs but weak to that of adult pigs , . therefore, it is possible that age of pigs may affect the our data in swine ex vivo cultures is compatible with previous data from pigs experimentally infected with h n where the viral rna load in the trachea and bronchus was low . they found that the wild type and e viruses replicated well in the lung and trachea and was found transmissible between direct contact animals . our observations suggest that h n with either pb - k or e is able to infect and replicate the ex vivo cultures of swine lung and thus providing a virus reservoir for possible transmissions, however, the lack of efficient replication of h n in swine conducting airways, the trachea and bronchus, would imply that transmission could be inefficient. the tropism observations presented in this paper partly addressed the reason why there is not yet a report on the isolation of h n from pigs in the field [ ] [ ] [ ] . apart from the study in pig models, the effect of amino acid substitution in pb position of h n were also evaluated in chickens and ferrets. a recent report suggested that an avian h n virus isolated from pooled oropharyngeal swabs of silkie chickens (a/sck/hk/ / ) with pb - e transmitted efficiently among chickens via direct contact. the transmission efficiency of this isolate was comparable to that found in a human h n isolate with pb - v/e/k polymorphism (a/hk/ / ) . when a chicken-to-ferret transmission experiment was performed using this silkie chicken strain, a rapid increase in the proportion of k over e was observed in the nasal washes of transmitted ferrets. the e k adaption in pb gene was therefore shown to be associated with mammalian adaptation in ferrets , , . besides, we showed that in contrast to the tropism data found in the tissue and cells of human and swine, rgpb -k e could still replicate in the chicken fibroblast df- cells at °c, albeit less efficiently than rgh n or the mutants rgpb -k e + q k or rgpb -k e + d n. the rgpb -k e virus replicated poorly if at all in df- or mdck cells at °c, exhibiting its temperature sensitive phenotype , . apart from the determination of tissue tropism, the effect of pb mutations in h n virus was also evaluated. our results demonstrated that, with a similar percentage of infection, the proinflammatory cytokines and chemokines induced by the rgh n and its mutants in ati and pmφ was generally similar, except il- gene was upregulated by rgpb -k e + q k and rgpb -k e + d n in ati and by rgpb -k e + d n in pmΦ compared to rgh n at hpi. the reduced mrna expression levels of ifn-β and ccl in ati infected with rgpb -k e in comparison to rgh n with pb - k followed the similar story in a mouse study that h n with pb - k would induce a significantly higher level of proinflammatory in the lungs of mice than those infected with rgpb - e . in general, the overall profile of cytokine and chemokine induction by h n virus, follow the previous findings that h n induced a lower cytokine response when compared to hpai h n as shown in human primary cultures and mice . although rgpb -k e viruses failed to replicate in human ati and pmφ cells in the low moi experiment, these cells were infected at similar rate and cytokine responses were elicited in the infection with a moi of . influenza viruses are detected through the recognition of toll-like receptor (tlr) expressed in the endosome and on the surface of human respiratory epithelial cells [ ] [ ] [ ] and in the phagosome of macrophages [ ] [ ] [ ] . thus abortive infection may be sufficient to trigger cytokine responses. infected cells are sensed by tlr with the presence of double-stranded rna (dsrna) and leads to the production of pro-inflammatory cytokines dependent on the expression of nuclear factor-κ b (nf-κ b), type i interferon and ifn-stimulated genes (isgs) , , . macrophages are active innate immune cells able to patrol around to detect potential pathogens by amoeboid movement. one of the characteristic differences between macrophages and lung epithelial cells is the function of phagocytosis for the removal of infected or dying cells , . although rgpb -k e was not replicating in pmφ , it might be possible that pmφ phagocytoses the influenza virus-infected and apoptotic cells therefore activates the induction of pro-inflammatory cytokines since pmφ is capable of secreting cytokines once they exposed to any inflammatory stimuli , , . to conclude, rgh n possessing pb -k e failed to replicate and infect in both ex vivo cultures of human bronchus and lung and in in vitro model of human ati and pmφ . thus, pb -e k, or the compensatory adaptations such as pb -q k or d n would be essential to at least partly rescue the replication competence of virus possessing pb - e in the mammalian system. the current ex vivo cultures of human and swine lung, and in vitro culture of primary human peripheral blood derived macrophages would be a good risk assessing model for the virus competence in replication and host innate immune response induction. our experimental findings explain why many h n virus isolates from humans have one or other of these mammalian adaptation mutations in pb . viruses. table listed the viruses used in this study. all the recombinant h n viruses were generated by plasmid based reverse genetics of influenza virus a/shanghai/ / . virus stocks used for these experiments were propagated and titrated in the madin-darby canine kidney (mdck) cells to determine the tissue culture infection dose % (tcid ). mdck cells were cultured in eagle's minimal essential medium (mem) containing mm hepes, % fetal calf serum (fbs) and u/ml penicillin and μ g/ml streptomycin. chicken fibroblast df- cells were cultured in dulbecco's modified eagle's medium (dmem) containing %fbs and %ps. tcid assay. sub-confluent mdck -well tissue culture plates were prepared one day before the virus titration assay. cells were washed once with warm phosphate-buffered saline (pbs) and replenished with serum-free mem with %ps and μ g/ml of tosylsulfonyl phenylalanylchloromethyl ketone (tpck)-treated trypsin. serial dilutions of culture supernatants from experiments, ranging from . log to log, were performed before adding scientific reports | : | doi: . /srep the virus dilutions onto the plates in quadruplicate and cytopathic effect (cpe) was monitored daily. the end point of viral dilution leading to cpe in % of inoculated wells was estimated using the karber method. ex vivo organ cultures of human respiratory tract. fresh biopsies of human normal bronchus and lung tissues were obtained from patients undergoing surgical resection of bronchus and lung tissues at queen mary hospital. informed consent has been obtained from all subjects. this study was approved by the institutional review board of the university of hong kong and hospital authority hong kong west cluster (uw - ) and all methods involving human tissues were performed in accordance with relevant guidelines and regulations. tissue fragments of bronchial tissue were placed on a sterile surgical sponge for air-liquid interface (ali), while lung tissue were cut into thin slices and cultured with f k medium in -well plate as previously described , . these ex vivo cultures were maintained at °c for culture and infection. ex vivo organ cultures of swine respiratory tract. intact respiratory organs were obtained from freshly slaughtered pigs in hong kong sheung shui slaughterhouse and delivered within hours in a sterile plastic box at °c. a tracheal swab was taken to exclude current influenza infection in the animal. it was tested for influenza m gene by real-time qpcr and data generated from any swine tissues positive for influenza a virus in the swab sample were excluded. tracheobronchial epithelium (tbe) was prepared by removing the mucosa and submucosa from the cartilage with a pair of fine forceps and a sharp scalpel. lung slices were prepared by perfusing the lung with cold transport medium and then % agarose in pbs using a clear tracheal tube . mm through the bronchiole until the lobe was fully expanded. lung tissue was cut in cubes and embedded with % agarose. the agarose-embedded lung tissue was cut in slices, using a cryotome blade with < mm thickness. tbe epithelia and lung slices were cut into circular snippets with mm diameter using a disposable biopsy punch (miltex). these thin snippets were placed on surgical sponge in a -well culture plate. . ml corresponding medium was added into each well with the epithelial explants and the surgical sponge floating on the medium, as previously described . tracheal epithelium was cultured in a : mixture of rpmi and dmem high glucose with %ps, μ g/ml gentamicin, . mg/ml glutamine, bronchial epithelium was cultured in mem with %ps, μ g/ ml kanamycin, . mg/ml l-glutamine and mm hepes while the thin lung slices were cultured in dmem high glucose with . μ g/ml bovine insulin, . μ g/ml hydrocortisone, . μ g/ml vitamin a (sigma, usa) and . mg/ml gentamycin. culture medium of these explants was changed every hour within the first four hours and incubated in a °c water-jacketed incubator with % co . all experiments involving swine tissues were carried out in accordance with relevant guidelines and regulations. all experimental protocols were approved by the institutional review board of the university of hong kong. to examine the infectivity of the viruses, the control and infected respiratory tissues were collected at , and hpi and fixed in % neutral buffered formalin and processed for paraffin embedding. immunohistochemistry using a mouse anti-influenza nucleoprotein antibody (hb , evl laboratories, netherlands) was performed to examine the iav antigen as previously described . culture of human type i-like pneumocytes (ati). after the removal of visible bronchi, the lung tissue was minced into pieces of thickness < . mm with scissors. lung pieces were washed with hanks's balanced salt solution at ph . for three times to remove macrophages and blood cells. a combination of . % trypsin and u/ml elastase (worthington biochemical corporation, usa) was added to the lung pieces and incubated for min in a °c water bath with shaking for digestion and stopped with dmem/f medium with %fbs and dnase i ( u/ml) (sigma, usa). undigested lung fragments were separated with a μ m pore size disposable cell strainer and cell clumps were dispersed by repeated pipetting for min. single cell suspension in flow-through was centrifuged and resuspended in a : mixture of dmem/f medium and small airway growth medium (sagm) (lonza, usa) with % fbs and u/ml dnase i. resuspended cells were plated on a tissue culture flask (corning, usa) for min adhesion at °c. the non-adherent cells were centrifuged, pelleted and resuspended with sagm with supplements and %ps and plated on a new culture flask. the growth medium was changed daily starting from h after cell plating. at % confluence, the ati cell layer was trypsinised for seeding. human peripheral blood monocyte-derived macrophages (pmφ). buffy coats of healthy blood donors were obtained from the hong kong red cross blood transfusion service and peripheral blood leucocytes were separated by centrifugation on a ficoll-paque density gradient and purification of monocytes were done by adhering on plastic petri dishes as previously described . monocytes were seeded onto tissue culture plates in rpmi medium supplemented with % heat-inactivated autologous plasma. immunostaining for surface receptor cd (bd biosciences, usa) was done to ensure the purity of monocyte preparations. a -day differentiation was done and the culture medium was changed to macrophage serum free medium sfm (gibco, usa) a week before infection. quantification of pro-inflammatory cytokine and chemokine mrnas by quantitative rt-pcr. human ati and pmφ were lysed in μ l rlt buffer with beta-mercaptoethanol after the infection in moi of . rna extraction was performed using an rneasy minikit (qiagen, germany) following the manufacturer's protocol with dnase treatment followed by reverse transcription using primerscript rt reagent kit (takara, china). the gene expression level was quantified by real-time pcr amplification using viia ™ real-time pcr system (applied biosystem, usa). gene expression profiles of cytokines tumor necrosis factor alpha (tnf-α ), interleukin (il)- , interferon β (ifn-β ); chemokines cxcl , and ccl were normalized using housekeeping gene β -actin mrna at the time points stated above. absolute copy numbers of cytokine, chemokine and β -actin gene were determined from a standard curve generated from a standard plasmid with a known copy number which was simultaneously included in qpcr. the primers and methods used for these assays have been reported previously , , . immunofluorescence assay for influenza viral antigen. the infectivity of the influenza viruses in ati and pmφ was determined by the percentage of cells that expressed the influenza viral antigens, matrix (m) protein and nucleoprotein (np). cell monolayer seeded on coverslips were fixed with % pfa for at least one hour. the cell monolayers were washed once with pbs and permeabilized with . % triton-x- for min at rt. cells were washed with pbs once and stained with a mouse monoclonal antibody conjugated to fluorescein isothiocyanate (fitc) specific to m and np of influenza a virus (imagen, usa) for min incubation at °c. the stained cells were washed with pbs once and mounted on glass slides using a mounting medium with ' , -diamidino- -phenylindole, dihydrochloride (dapi) (vector laboratories inc, usa). fluorescence images were viewed and captured using a nikon eclispe ti-s inverted microscope system. the percentage of cells with positive influenza viral antigen expression was interpreted as the percentage of infection. control and statistical analysis. experiments were performed independently using ex vivo cultures from at least three human donors and pigs. ati and pmφ were isolated from three human donors. each virus used for all these experimental replicates was from same batch. mem inoculated tissue or cells served as mock infection and negative controls. results showed in figures were the arithmetic mean and standard error of mean (sem). the differences between log -transformed viral titers among different viruses at different times post-infection and the gene expression profiles of quantitative cytokine and chemokine of influenza virus-infected cells were compared using two-way anova with a post-hoc bonferroni multiple-comparison test. differences were considered significant at a p value of < . . statistical analysis was performed using graph-pad prism . as the aim of this part was to determine the effect of pb mutations in replication kinetics and cytokine and chemokine induction in human primary ati and pmφ , the result generated from the control viruses, h n and h n pdm, were only included as reference benchmark. biosafety. all the recombinant viruses with pb mutations were loss-of-function mutations. all experiments were carried out in a biosafety level facilities at the university of hong kong. dissemination, divergence and establishment of h n influenza viruses in china the genesis and evolution of h n influenza viruses in poultry from southern china clinical, virological, and histopathological manifestations of fatal human infections by avian influenza a(h n ) virus. clinical infectious diseases: an official publication of the infectious diseases society of america clinical findings in cases of influenza a (h n ) virus infection human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome genetic analysis of novel avian a(h n ) influenza viruses isolated from patients in china the genesis and source of the h n influenza viruses causing human infections in china biological features of novel avian influenza a (h n ) virus tropism and innate host responses of a novel avian influenza a h n virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract pb amino acid at position affects replicative efficiency, but not cell tropism, of hong kong h n influenza a viruses in mice the pb -e k mutation attenuates viruses containing the h n influenza pandemic polymerase the effect of the pb mutation k on highly pathogenic h n avian influenza virus is dependent on the virus lineage amino acid substitutions in polymerase basic protein gene contribute to the pathogenicity of the novel a/h n influenza virus in mammalian hosts amino acids substitutions in the pb protein of h n influenza a viruses are important for virulence in mammalian hosts the pb e k mutation contributes to the high polymerase activity and enhanced replication of h n influenza virus genomic signatures for avian h n viruses adapting to humans mutations to pb and np proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells the viral polymerase mediates adaptation of an avian influenza virus to a mammalian host introduction of virulence markers in pb of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission human h n influenza a viruses replicate in swine respiratory tissue explants molecular basis for the generation in pigs of influenza a viruses with pandemic potential the role of swine as "mixing vessel" for interspecies transmission of the influenza a subtype h n : a simultaneous bayesian inference of phylogeny and ancestral hosts long-term evolution and transmission dynamics of swine influenza a virus infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs investigation of influenza virus polymerase activity in pig cells potential for transmission of avian influenza viruses to pigs distribution of sialic acid receptors and influenza a virus of avian and swine origin in experimentally infected pigs efficient transmission of swine-adapted but not wholly avian influenza viruses among pigs and from pigs to ferrets comparative distribution of human and avian type sialic acid influenza receptors in the pig replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution infection of swine ex vivo tissues with avian viruses including h n and correlation with glycomic analysis analysis of recombinant h n wild-type and mutant viruses in pigs shows that the q l mutation in ha is important for transmission rapid adaptation of avian h n virus in pigs a novel neutralizing antibody against diverse clades of h n influenza virus and its mutants capable of airborne transmission emergence of avian influenza a(h n ) virus causing severe human illness -china transmission of h n influenza viruses with a polymorphism at pb residue in chickens and ferrets molecular determinants of adaptation of highly pathogenic avian influenza h n viruses to efficient replication in the human host emergence of the virulence-associated pb e k substitution in a fatal human case of highly pathogenic avian influenza virus a(h n ) infection as determined by illumina ultra-deep sequencing biochemical impact of the host adaptation-associated pb e k mutation on the temperature-dependent rna synthesis kinetics of influenza a virus polymerase complex residue of pb is a determinant of cold sensitivity in rna replication of avian influenza viruses human h n and h n influenza viruses differ in induction of cytokines and tissue tropism cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells involvement of toll-like receptor in the immune response of lung epithelial cells to double-stranded rna and influenza a virus toll-like receptor expression and induction of type i and type iii interferons in primary airway epithelial cells innate immunity to influenza virus infection sensing of viral infection and activation of innate immunity by toll-like receptor h n influenza virus-induced mediators upregulate rig-i in uninfected cells by paracrine effects contributing to amplified cytokine cascades detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia type i ifn triggers rig-i/tlr /nlrp -dependent inflammasome activation in influenza a virus infected cells evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice virus clearance through apoptosis-dependent phagocytosis of influenza a virusinfected cells by macrophages macrophage cytokines: involvement in immunity and infectious diseases phagocytosis and the inflammatory response tropism and innate host responses of the pandemic h n influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza h n and h n viruses tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures key: cord- -wvo jx authors: liu, cheng; he, tao; rong, yanxiao; du, fengjiao; ma, dongxing; wei, yujie; mei, zhiqin; wang, yuling; wang, haibin; zhu, yuehua; zhang, zongde; zheng, li; wu, xueqiong; liu, huiliang; ding, wenjun title: association of mannose-binding lectin polymorphisms with tuberculosis susceptibility among chinese date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: wvo jx tuberculosis (tb) is caused by infection of mycobacterium tuberculosis. host genetic variability is an important determinant of the risk of developing tb in humans. although the association between mbl polymorphisms and tb has been studied in various populations, the results are controversial. in this study four functional single-nucleotide polymorphisms (snps, h/l, x/y, p/q and a/b) across the mbl gene were genotyped by direct dna sequencing of pcr products in a case-control population of chinese han origin, consisting of , patients with pulmonary tb and , controls. we found that individuals carrying variant allele at a/b (namely bb or ab genotypes) was associated with increased susceptibility to tb (odds ratios [or] = . , % confidence interval [ci] . – . , p = . × (− )). additionally, lypb haplotype showed a significant association with increased risk of tb (or = . , % ci . – . , p = . × (− ); global haplotype association p = . × (− )). furthermore, individuals bearing low- or medium- mbl expression haplotype pairs had an increased risk of tb (or = . , % ci . – . , p = . × (− )). thus, the reduced expression of functional mbl secondary to having mbl variants may partially mediate the increased susceptibility to tb risk. scientific reports | : | doi: . /srep and phagocytosis or lysis of pathogens. in addition, mbl can regulate inflammatory responses and immune activation . mbl is encoded by the mbl gene, which is located on chromosome . six single-nucleotide polymorphisms (snps) in exon (codon a/d, codon a/b, and codon a/c) and in the promoter and ′ -untranslated regions (nt − h/l, nt − x/y, and nt + p/q) of the mbl gene are associated with serum levels and/ or functions of mbl. these genetic variations are associated with a wide variety of diseases, including respiratory tract infections , [ ] [ ] [ ] [ ] . however, in previously reported studies mbl polymorphisms have conflicting results showing protection from or susceptibility to tb . for example, low levels of mbl (associated with variant alleles at the promoter and exon regions of the mbl gene) were reported to protect against tuberculosis , , supporting the hypothesis that mbl binding can enhance the uptake of intracellular pathogens by phagocytes, and then promote infection , . other investigators instead claim that protection against the disease is associated with high levels of mbl (controlled by the wild mbl alleles) [ ] [ ] [ ] [ ] . in the chinese han population, no convincing evidence of association between mbl sequence variants and tb was observed [ ] [ ] [ ] [ ] [ ] . for example, in a meta-analysis totally including tb patients and controls of chinese origin, shi j and colleagues reported that variants at a/b were associated with increased susceptibility to tb . additionally, chen m and colleagues found that variants in y/x was associated with increased susceptibility to tb among chinese , . however, most recently wu l reported that variants in h/l, but not y/x, p/q or a/b, were associated with decreased susceptibility to tb among chinese . conflicting results are not unexpected in association studies for several reasons, including small sample size, marginal statistical significance, detection of genotypes, or ethnic heterogeneity. therefore, here we conducted a genetic association study in a large case-control population of chinese han origin (totally consisting of , patients with tb and , controls), to better define the association between the mbl snps and tb. population characteristics. the selected characteristics of patients with tb and control subjects in this study are shown in table . the study consists of , patients with tb and , control subjects. the male/female ratio of patients with tb was . , and the mean age was . years (± sd, . ) ( table ) . for the , controls, the male/female ratio was . , and the mean age was . years (± sd, . ). controls were comparable with cases with regard to mean age and gender (all p values > . ). the genotyping results are presented in table . the observed genotype frequencies for the four polymorphisms conformed to the hardy-weinberg equilibrium in controls (all p values > . ). consistent with the previous reports , , the codon (a/d) and (a/c) variants were not observed in our ethnic northern chinese individuals. the frequencies of variant alleles l, x, q and b in the controls were . %, . %, . % and . %, respectively, similar to those reported in other southern or northern chinese subpopulations , . polymorphisms and risk of tb in this study. on the basis of unconditional logistic regression analysis with adjustment for known confounding factors including age and gender, a statistically significant association with the susceptibility to tb was observed with the condon variant. subjects homozygous and heterozygous for variant b allele (i.e. b/b plus a/b genotype) had an increased susceptibility to tb compared to those homozygous for the wild-type a allele (or = . , % ci . - . , p = . × − ; simulation number = , , ; table ). the association was still significant even after correction for multiple comparisons. none of the remaining three variants (h/l, y/x and p/q) were found to be significantly associated with susceptibility to tb. logistic regression models including random effects obtained results similar to those obtained in models including fixed-effects (table ). in addition to single snp analysis, haplotype analysis was also performed for the mbl gene. we identified haplotypes (hypa, lypb, lxpa, lyqa and lypa) using the phase program, with the frequencies of these haplotypes greater than % in both cases and controls. the frequencies of these haplotypes in controls ranged from . % (hypa haplotype) to . % (lypa haplotype), similar to those reported in other asian populations of japanese and vietnamese . in haplotype analysis, only lypb showed a significant association with increased risk of tb (or = . , % ci . - . ; p = . × − ; global haplotype association p = . × − ; simulation number = , , ; cases are patients with tuberculosis. comparisons of gender and age distributions between cases and controls were performed by use of the one-sided χ test. differences of mean age between cases and controls were analyzed by use of an unpaired t test. table ). furthermore, we grouped cases and controls by harboring haplotypes associated with high, medium, or low mbl expression , to evaluate whether haplotype pairs associated with various levels of mbl expression were associated with susceptibility to tb risk. in , patients with tb, ( . %) were in the high expression group, ( . %) in the medium group, and ( . %) in the low group. in , controls, this distribution was ( . %), ( . %) and ( . %), respectively (or = . , % ci . - . , p = . × − , with adjustment for age and gender; simulation number = , , ; table ), implicating that the individuals bearing medium or low expression haplotype pairs had an increased risk of tb and that the mbl deficiency might play a potential role in susceptibility to tb. the associations between the four mbl polymorphisms and susceptibility to tb were further examined with stratification by age and gender, with no differences observed for mbl polymorphisms individually or in haplotype between stratums (all p values > . , test for homogeneity). recent studies on the genetic association between snps in the mbl gene and patients with tb of chinese origin have generated different and even contradictory results. these studies have small sample size, usually involving about or less ptb patients of chinese origin [ ] [ ] [ ] [ ] [ ] , which might lead to artificial associations. in addition, the four functional snps in the mbl gene is in linkage disequilibrium (ld) and more appropriate to be investigated together (in both haplotype and haplotype pairs). moreover, marginal statistical significance reported in these association studies means that poor reproducibility of their results is not unexpected, given the concern over the possible unreliability of case-control studies . in this study, we investigated all of the four functional snps in the mbl gene, individually or in haplotype or haplotype pairs, in a large case-control population of chinese han origin, totally consisting of , patients with tb and , controls. this is the first study to investigate the association between mbl polymorphisms with polymorphisms no. of cases a (n = , ) table . haplotypes for the four snps in mbl gene and their associations with tb risk in chinese. or, odds ratio; ci, confidence interval. the haplotype is in the order of h/l, y/x, p/q and a/b. a haplotype-specific scores and p values for each haplotype versus all others and the p value for global haplotype association are evaluated using the haplo.score-function. the global p value is . × − . b the ors ( % ci) are calculated for haplotypes lypb, lxpa, lyqa and lypa using the haplo.glm-function, with haplotype hypa as control. pulmonary tb susceptibility in northern han chinese. by genotyping all of the four functional snps in the mbl gene in the case-control population of relatively large sample size, we found that one snp (a/b) was associated with susceptibility to tb (p = . × − ). furthermore, we found that the lypb haplotype showed a significant association with increased risk of tb in northern han chinese (p = . × − ). in addition, individuals bearing haplotype pairs indicating medium or low mbl expression had an increased risk of tb (p = . × − ). we noted that wu et al. have reported that lypb haplotype was associated with tb in southern chinese . however, frequencies of mbl haplotypes in wu's study were remarkably different from those in chinese subjects observed in other studies. for example, frequencies of hypa, lypb, lxpa, lyqa and lypa were . %, . %, . %, . % and . % respectively in the controls in the present study, similar to the frequency distribution of these haplotypes in southern and northern chinese in the genomes project. however, in the controls in wu's study frequencies of hypa, lypb, lxpa, lyqa and lypa were . %, . %, . %, . % and . % respectively. based on the large sample size, haplotype pairs analyses and small p values, our results may be closer to the real situation of mbl polymorphisms in tb susceptibility. the mbl gene encodes a homotrimeric molecule harboring a carbohydrate recognition domain and a collagenous tail . the variant b allele can impair the formation of a triple helix in the collagenous tail, and therefore disrupts mbl polymerization and leads to enzymatic degradation and functional deficiency of this protein . the other snps, h/l, x/y, and p/q, also play roles in determining functional mbl concentration, among which the x allele shows the lowest transcriptional activity , , . when in haplotype, these snps form three defective haplotypes (lypb, lyqc and hypd, with the latter two absent among chinese), and five functional haplotypes with different expression levels (a low-producing lxpa haplotype, an intermediate-producing lypa haplotype, and two high-producing haplotypes lyqa and hypa) . the results that b allele, lypb haplotype, and medium-or low-expression haplotype pairs were associated with increased risk of developing tb (tables , and ) suggest that mbl deficiency possibly plays a role in susceptibility to tb. there is biological plausibility for our observed genetic associations, although the exact mechanism by which mbl deficiency is associated with increased susceptibility to tb requires further investigation. previous studies have revealed that mbl recognizes m. tuberculosis by direct interaction, resulting in lectin pathway activation, agglutination of bacteria and enhancement of phagocytosis , . additionally, the serum samples from carriers of haplotype pairs associated with high mbl levels (hya/hya) display significantly higher antibacterial activity of neutralization than did those associated with lower mbl levels, and the activity is mediated by mbl . therefore, one speculative interpretation of our results is that low serum concentrations of functional mbl caused by haplotype pairs indicating medium or low mbl expression might result in reduced neutralization and favor the survival of m. tuberculosis. there are several potential limitations in the present study. the first, the patients with tb were selected from the hospital and the controls were recruited from the community population, which means that inherent selection bias cannot be completely excluded. however, by matching on age and gender and by analyzing data with further stratification, potential confounding effect might have been minimized. the second, some of the control subjects used in this study may be asymptomatic with latent tb infection. therefore, we cannot directly specify which stage of tb, infection of mtb or development of active disease, was more affected by mbl . the third, although the highly significant association between mbl deficiency and increased susceptibility to tb derives from a biologically based a priori hypothesis, the initial findings presented here should be independently verified in other subpopulations of ethnic chinese origin (e.g. southern chinese) or of different ancestry. in summary, our results reveal an association between genetic mbl deficiency and increased susceptibility to tb among northern chinese, and provide supports for the importance of mbl in the pathogenesis of tb. knowledge of genetic factors contributing to the pathogenesis of tb revealed in this study could lead to improved treatment and prevention of this disorder. ethics statement. written informed consent was obtained from all participants involved in this case-control genetic association study, and the study was approved by the medical ethics committee of the pla hospital (beijing, china) and the fifth hospital of shijiazhuang (shijiazhuang city, hebei province, china). all the experiments were performed in accordance with the relevant guidelines and regulations. a total of , patients with pulmonary tb were recruited at the fifth hospital of shijiazhuang (shijiazhuang city, hebei province, china), between january and january . all patients with pulmonary tb were newly diagnosed based on smear or culture positive for m. tuberculosis and chest radiography. patients with confirmed diagnosis of extrapulmonary tb were excluded. the response rate for case patients was %. a total of , healthy adults frequency matched to the tb patients on the basis of age and gender were recruited as control subjects during the same time period as the tb patients were collected. the response rate for controls was %. all participants were unrelated northern han chinese, and had no clinical histories of diabetes mellitus, hiv infection, or receipt of immunosuppressive therapy. the authors had access to identifying information during and after data collection. extraction of genomic dna. about and + (p/q, rs ) and the structural polymorphism at nt + (codon a/b, rs ) of the mbl gene were selected for genotyping by use of pcr direct sequencing in the case-control population. the pcr primers were designed using the web-based software primer (available at: http://primer .ut.ee/). the forward primer ′ -atgggaggaggattcaagg- ′ and reverse primer ′ -ccttgtgacactgcgtgact- ′ were used for amplifying the target region containing the mbl h/l and y/x variants. the forward primer ′ -cggtcccatttgttctcact- ′ and reverse primer ′ -cacaaactgctgtgtggaat- ′ were used for amplifying the target region containing the mbl p/q and a/b variants. then the pcr products were sequenced by an abi sequencer. a nucleotide was identified as a candidate polymorphism by an observer using the polyphred program (available at: http://droog.gs.washington.edu/polyphred/) and confirmed by another observer independently. genotyping of all samples was performed in a blinded manner so that the case or control status was unknown. the call rates were % for all snps. the accuracy of genotyping data for each snp was validated by masking, choosing at random, and resequencing % of the samples from the opposite strand. statistical analyses. comparisons of gender and age between tb patients and controls were performed using the one-sided χ test (spss software, version . ). differences of mean age between tb patients and controls were analyzed using the unpaired t test (spss software, version . ). genotype frequencies for each snps were determined by gene counting. the significance of deviations from the hardy-weinberg equilibrium was tested using the online software snpstats (http://bioinfo.iconcologia.net/snpstats_web). unconditional logistic regressions under a dominant inheritance model were used due to the limited sample size, to calculate p values, odds ratios (ors), and % confidence intervals (cis) for assessing the association between snps and disease risk, adjusting for age and gender (fixed effects). in additional analyses, age and gender were treated as random effects in the logistic regression to deal with the uncertainties with more flexibility. the glm-function and glmer-function in the lme package (version . - ) of r (version . . ) were used to fit the models with fixed effects and random effects, respectively. to show the robustness of the conclusions, we performed resampling statistics as previously reported . the p values, ors, and % cis in single snp analyses were calculated by a bootstrapping test with the number of simulations was set to , , . as to multiple comparisons, the correction factor n(m- ) (in which there are n loci with m alleles) was used to correct the significance level. the haplotypes of the mbl gene of the case-control population were assigned by the phase program . p values of association between haplotypes and disease risk were evaluated using the haplo.score-function in the haplo.stats package (version . . ) of r, with the number of simulations was set to , , . ors and % cis were calculated for each haplotypes using the haplo.glm-function in the haplo.stats package. all p values and ors ( % ci) were calculated by adjusting for age and gender. analyses of haplotype pairs were similar to those of single snps. potential modification of the effect of the snps on tb risk was assessed for age and gender by the addition of interaction terms in the logistic model and by separate analyses of subgroups of subjects determined by these factors. a homogeneity test was used to detect the difference of ors within each subgroups. all tests are two-tailed unless otherwise indicated. p values less than . were considered statistically significant. genetics of susceptibility to human infectious disease polymorphisms in the interleukin receptor gene and tuberculosis susceptibility among chinese for better or for worse: the 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identification of high-quality cancer prognostic markers and metastasis network modules a new statistical method for haplotype reconstruction from population data score tests for association between traits and haplotypes when linkage phase is ambiguous estimation and tests of haplotype-environment interaction when linkage phase is ambiguous we thank everyone who provided blood samples and consent for genetic analysis. in addition, we thank all of the clinicians, nurses and study coordinators for their contributions to the work. the study was funded by grants from the army scientific research foundation (cws j ), collaborative innovation center of infectious diseases (pxm _ _ and pxm _ _ ), and beijing municipal administration of hospitals clinical medicine development of special funding support (zylx ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. l.z., x.w., h.l. and w.d. conceived and designed the study. c.l., t.h. and y.r. performed genotyping. y.z., z.z., d.m. and y.w. helped to analyze the data. z.m., y.w. and h.w. were responsible for recruitment of the casecontrol samples. f.d. helped to prepare samples. l.z., h.l., w.d., c.l., t.h., f.d. and y.r. conducted samples selection and data management, performed the statistical analyses, interpreted the results, and drafted the manuscript. all authors reviewed the manuscript. l.z., x.w., h.l. and w.d. approved the final version of the manuscript. competing financial interests: the authors declare no competing financial interests. key: cord- -bbtslrrt authors: almogy, gal; stone, lewi; bernevig, b. andrei; wolf, dana g.; dorozko, marina; moses, allon e.; nir-paz, ran title: analysis of influenza and rsv dynamics in the community using a ‘local transmission zone’ approach date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: bbtslrrt understanding the dynamics of pathogen spread within urban areas is critical for the effective prevention and containment of communicable diseases. at these relatively small geographic scales, short-distance interactions and tightly knit sub-networks dominate the dynamics of pathogen transmission; yet, the effective boundaries of these micro-scale groups are generally not known and often ignored. using clinical test results from hospital admitted patients we analyze the spatio-temporal distribution of influenza like illness (ili) in the city of jerusalem over a period of three winter seasons. we demonstrate that this urban area is not a single, perfectly mixed ecology, but is in fact comprised of a set of more basic, relatively independent pathogen transmission units, which we term here local transmission zones, ltzs. by identifying these ltzs, and using the dynamic pathogen-content information contained within them, we are able to differentiate between disease-causes at the individual patient level often with near-perfect predictive accuracy. ltzs are defined by the property that the transmission rate of an infectious disease within an ltz is significantly greater than the average transmission rate of the disease between the different ltzs. this means that the population within an ltz is in relative terms highly connected, and we suppose sufficiently connected to justify the assumption of 'random mixing, ' so that an invading pathogen could come into contact reasonably rapidly with most of members of the ltz. however, an invading pathogen may have difficulty in spreading beyond the confines of the relatively isolated ltz. extreme examples of ltzs have been documented in the literature and include confined military bases or cruise ships [ ] [ ] [ ] [ ] or other small isolated communities (e.g. religious or ethnic) through which diseases rapidly propagate, reaching most members of the population. to investigate the possibility of ltzs in a large regional area, we analyze clinical data from a healthcare medical center in the city of jerusalem, containing the clinical test results for influenza virus and respiratory syncytial virus (rsv) from patients presenting with ili symptoms. using a 'k-means clustering' algorithm, putative ltz groups are identified solely based on the physical distance between home locations of the patients. the methodology is akin to the procedure of 'community detection' applied in the study of complex networks and designed to locate highly connected clusters of nodes , . our analysis finds that while influenza and rsv incidences tend to overlap and show more or less equal number of cases over the whole region, individual ltzs show a far more homogeneous disease content at most given times, with some being dominated by rsv while others by influenza. we use these findings to arrive at a prediction algorithm that, applied to patients presenting at the hospital with ili, is capable of differentiating between cases of influenza and rsv, often with near-perfect accuracy. defining local transmission zones. the transmission dynamics of respiratory pathogens in a population are constrained by the physical distance between infected and susceptible individuals. an ltz for a given pathogen represents a group of individuals within the general population, such that the transmission probability of the pathogen within the group is greater than transmission probability between that group and any of the other groups. thus we suppose the population of a region can be subdivided into a set of k groups or ltzs, such that for any two ltzs i and j: here p(ltz i , ltz j ) is the average probability that an individual from ltz i infects an individual from ltz j . in our case we are given the geographic coordinates of a group of individuals, and we assume that the probability of transmission between two individuals is proportional to the euclidean distance between them. to divide the population into a set of k distinct ltzs we make use of an optimization technique known as k-means clustering that calculates the k different geographic zones while attempting to ensure that eq. holds in an optimal fashion . for any preassigned k we are able to divide the set of all patients home-locations into a set of k ltzs using the aforementioned clustering method, after determining all pair-wise euclidean distance between address locations of the full patient set (see also methods). thus the resulting ltzs represent k groups of patients, partitioned purely on the basis of the physical proximity between these patients' home locations , . note that when examined over the entire period , the spatial distributions of influenza and rsv are very similar and we have found that the ltzs obtained using only the influenza or rsv data for clustering purposes are very similar to those obtained when using the entire data (not shown). the clustering method used was effective at identifying geographically distinct areas as clusters, e.g. neighborhoods outside the jerusalem municipal boundary (fig. , highlighted area). here we chose k = ltz groups as a representative example because it guaranteed that the smallest ltz consisted of at least individuals. interestingly, the clustering algorithm was also able to make meaningful distinctions within the municipal boundaries, e.g. between the 'bet-safafa' and 'gilo' neighbourhoods (indicated on map as circles). hadassah-hebrew university medical centers between and is shown (fig. a) . the unusual dynamics in - may be the result of the unusual ili dynamics during this season, caused by the then emerging h n influenza pandemic strain (h n pdm, ref. ). in the - season, we note the occurrence of first an influenza epidemic followed by an rsv epidemic, with very small overlap between the two epidemics. as such there is a notable time-delay between the peaks of the epidemic curves of influenza and that of rsv. in contradistinction, over the - season, the influenza and rsv epidemics peak at almost the same time and overlap almost totally. ideally we expect to find that within a single well defined working ltz, the two disease signals show relatively small overlap. the underlying concept is that any pathogen arriving at a susceptible ltz is able to spread rapidly through the entire local population. this domination within an ltz is to be expected since disease transmission within an ltz is stronger than transmission between them. such a situation would be particularly favoured if only a limited number of infected individuals invade a susceptible but heterogeneous region. the pathogen dominating an ltz is likely to be the first successfully invading pathogen. in this extreme case, there would be zero overlap of the diseases in any ltz, because each ltz has only a single pathogen. here, our working assumption is that transmission between ltzs has relatively minor impact at these time-scales. hence, even if at the whole region scale (i.e. k = ) disease signal overlap is high, as say in - , in the individual ltzs within the region the disease signal overlap should be expected to be far smaller, ideally close to zero. this motivates us to develop a quantitative index for measuring disease signal overlap (so). to do so, we let the number of influenza and rsv cases at time t be represented by i(t) and r(t) respectively. let ρ i,r (τ) be the lagged cross-correlation between i(t) and r(t − τ), that is, the cross-correlation between the influenza and rsv time series when there is a time-delay τ between the signals. the signal overlap so i,r between the two time-series is then defined as (see also methods): , , here ρ i,r ( ) is precisely the usual pearson correlation between i(t) and r(t) and this is divided by the maximum such correlation possible when the time-series are delayed for a time τ, ranging from minus to plus weeks. the index is first used to examine the disease signal overlap when the whole region is considered a single ltz (i.e., k = ). the overlap for influenza and rsv ranges from a minimal value of so = . disease ratios. disease signal overlap is a useful index for studying and comparing the intersection of two diseases over a season. for shorter term dynamics we make use of the disease ratio index (dr) of the weekly incidence of the two pathogens. for a particular week, the disease ratio for influenza and rsv is defined here as (see also methods): where [i] and [r] are the number of new cases of influenza and rsv respectively, for that week. the disease ratio quantifies the degree to which one pathogen is dominant over the other during a period of one week. when only influenza is detected over a week, the disease ratio is dr = + , when only rsv is detected then dr = − , and hence the absolute value of the dr is maximal when only one pathogen is present (|dr| = ). if the pathogen incidences are equal over the week then dr = . we show the disease ratio for the whole region (k = ) colour-coded and plotted as a function of time in consecutive weeks (fig. a) . in the first half of the - season there are only cases of influenza and dr = + (red), whereas for the second half of the season there are only rsv cases (dr = − , blue). in contrast, during most of the - season dr = (green), as expected given the almost complete overlap of the influenza and rsv disease signals. in the - season, despite the high degree of overlap in the entire season, there is initially a period of rsv dominance, reflected in a negative dr (blue). this dominance erodes into a disordered, mixed pattern where neither pathogen achieves strong dominance for any contiguous period. it is informative to repeat the above qualitative examination of the whole region but in terms of its individual ltz groups. plotting the disease ratio for k = ltzs as a representative example (fig. b) , reveals substantial variation in the dr of different ltzs. in the first season ( - ), nearly all ltzs reflect the pattern observed for the k = analysis. namely, for the first part of the year dr = + (influenza dominance) while for the second half of the year dr = − (rsv dominance). note however, that the individual ltzs show a large degree of variability in terms of when the appearance of the first pathogen was detected in a patient, and when the dominance pattern changed from influenza to rsv. the - season shows a different phenomenon. most importantly, even when there are roughly equal number of influenza and rsv cases in the whole region, the dr in individual ltzs will often show a more binary pattern, with long periods where some ltzs contain only influenza (red), while others only rsv (blue). the inset in fig. b shows in detail a period of over months ( - season) where the single area dr for k = is near zero while of the highlighted ltzs contain (almost) only rsv, and the remaining ltz contains only influenza consistently over the entire season. we make these visual observations concrete by calculating the average per-season absolute disease ratio (|dr|) and making quantitative comparisons between analyses based on the whole area (k = ) and analyses based on an ltz approach (k > ). we are particularly interested in knowing when the ltz is dominated entirely with influenza or dominated entirely with rsv. for either case the absolute disease ratio attains the value |dr| = . dividing the whole region into ltz groups (k > ) led to a significant increase (p < . , n = , using -sided student t-test) in the absolute value of the disease ratios compared to the whole region (k = ) results for all k values (fig. c) . for the - season the ratio increased from |dr| = . in the whole region to |dr| = . using k > ltzs. for the - season, there was an increase from |dr| = . to |dr| = . for k > , and from |dr| = . to dr > . for k > in the - season. shortly, we use the concept behind this index to assess the likelihood of someone residing in that ltz to be infected by influenza or infected with rsv. frequency-based differentiation of influenza and rsv. the above results show that compared to the regional level (k = ), there is a reduced coexistence of influenza and rsv when examined at the ltz level (k > ), leading to lower signal overlap (fig. b) , and increased disease ratios within ltzs (fig. ) . together, these results support our hypothesis that patients from the same ltz will have a strong propensity to carry the same pathogen. this suggests that the pathogen incidence within an ltz may be better predicted by considering the data from that ltz, rather than the more abundant, yet less specific data collected at the whole region level (k = ). we now make use of the ltz concept to implement a test-algorithm designed to predict whether a patient has influenza or rsv given that a person arrives at hospital with ili symptoms, based solely on previous data kept in the hospital database. the test depends on determining the specific ltz that the patient resides in and recent information about the pathogens present in that ltz. if the ltz hypothesis is correct, ltz-based predictions should significantly outperform predictions based on the whole region data, where ltzs are ignored. if on the other hand, the ltz hypothesis is incorrect, then an attempt to predict a patient's disease by using ltz-specific pathogen content would be no better than predictions where the whole region is considered as a single unit. the test proceeds on a day-by-day basis beginning from the earliest time-point in the data (january ) and advances in chronological order (up to may ). predictions are made on each day t for all patients that arrive in the hospital with ili symptoms on that day. predictions for a newly presenting patient arriving on day t are made as follows: (i) the ltz associated with the patient is determined. it is to be expected that the per season accuracy of the predictions will improve with smaller signal overlap and increased disease ratio. the per season predictive accuracy for the three seasons as a function of the number of ltzs k are presented (fig. a) . during the first season, where signal overlap was minimal, the predictive accuracy was greater than a = % in all tests, ranging from a minimum of approximately a = % for the whole region, up to a = % for k = ltzs. this high accuracy, even for the whole region, reflects the low signal overlap between rsv and the pandemic influenza strain that emerged in . during the - season, the whole region (k = ) predictions were accurate only in about a = % of cases, whereas the ltz-based predictions (k > ) reached accuracy of over a = %. this is consistent with what might be expected given the great reduction in signal overlap during that season for larger k values (fig. b) . during the - season, where the signal overlap was almost unity, predictions based on the whole region were similar to predictions made at random, with an accuracy level of a = %. the ltz-based approach (k > ) preformed significantly better but only achieved an accuracy of a = % correct predictions (k = ). the increase in predictive accuracy when using the ltz-based approach was significant (p < . ) for all values of k in - and - , but only for k = , and during the - season (fig. a, significant changes marked with asterisk) . these results are consistent with the differences in the signal overlap between the whole region and ltzs groups, which we describe above (fig. b) . in summary, the increased predictive accuracy using ltz-data compared to the whole region data is sufficient to prove the ltz concept, and that home-location can provide a good basis for identifying ltzs. increasing the value of k did not always improve the accuracy of the predictions; e.g. k = performed better than k = in - and in - , and k = provided better accuracy than k = in - . however, higher |dr| (and lower so) values were associated with improved predictive accuracy over a given season (compare examination of the predictive performance of individual ltzs made clear that a small proportion of ltzs were far less accurate than others; by removing % of the ltzs that have the worst performance, the remaining ltzs, representing approx. % of patients, provide predictions that are nearly % accurate (fig. c) for the - and - seasons (k = , , ). while the majority (approx. %) of ltzs included in the top % (fig. c) were common to all seasons, we did not find any obvious characteristics that distinguish these ltzs from those included in the % worst performers. poor predictive accuracy in a specific ltz might be the result of limitations in the data itself, or difficulties in achieving perfect clustering by the k-means clustering method, i.e. it is quite possible that some ltzs were poorly identified or in fact 'misidentified' due to errors introduced by the k-means clustering . this study is the first of its kind in using a combination of clinical and geographical data to demonstrate that from a 'pathogen's perspective' even a geographic unit as small as one urban area is not a single, perfectly mixed ecology, but is in fact comprised of more basic transmission components, i.e. ltzs, which should be seen as tightly connected micro-community structures that allow for rapid pathogen transmission. specifically, we proposed the hypothesis that in terms of the spread of ili, the city of jerusalem and its surrounding areas are not a single disease transmission unit but may be more appropriately described by a set of multiple smaller-scale component ltzs. these ltzs, due to spatial fragmentation and isolation effects, will tend to have extended time-periods in which only a single disease dominates, often completely. to test this hypothesis we examined the geographic distribution of influenza and rsv cases in jerusalem over seasons, from - to - . analysis of the putative ltz groups showed unusually small signal overlap and large absolute disease ratio (figs and ) as compared to the whole region values (i.e. k = ). these results show that the spatial distribution of influenza and rsv incidences may be better differentiated in location-based groups of patients than it is in the entire area as a whole. this confirms our hypothesis that the jerusalem region is comprised of multiple ltzs, and is not by itself a single transmission unit. we also measured the performance of a simple algorithm for predicting whether incoming hospital patients with ili symptoms were infected with rsv or influenza. the prediction scheme made use of a frequency based approach and at the regional (k = ) level, the predictions simply matched the relative prevalence of the diseases. dividing the whole region into ltzs led to a substantial increase in predictive accuracy in all seasons (fig. a) and moreover, after taking into account that a proportion ( %) of the ltzs used may have been misidentified, when removed the predictions for two of the seasons attained routine prediction accuracy often of % in differentiating between cases identified as associated with influenza and those that were associated with rsv (fig. c ). an important part of the work preformed by hospitals and health organization consists of the diagnosis and surveillance of communicable disease, which may be carried out at the molecular and/or the symptomatic levels. the results presented here hint at the potential epidemiological importance in examining disease data at the ltz scale,and the possibility for generating 'maps' of the real-time distribution of pathogens at resolution levels sufficient to guide improved public health policies. furthermore, the improved predictive accuracy achieved by focusing the analysis at the patient-specific ltzs (fig. ) indicates a potential utility in data-driven diagnostics, where more sophisticated algorithms taking into account ltz information as well as other relevant population parameters, such as the patients' age, medical background etc. is expected to further increase the predictive accuracy. we hope to explore this further in future research. scientific reports | : | doi: . /srep our choice of ltzs was based entirely on patients' approximate home-locations, without any reference to urban motifs, e.g. socioeconomic divisions, public transportation etc. , and clearly provides only a partial picture of the complexity of urban transmission ecology. the social and spatial structures differ among cities, and jerusalem may not be representative of any 'generic' city; e.g. it might be that in some areas the population connectivity pattern is far more homogenous, and hence the ability to leverage ltz-based identification of a pathogen might be limited, or provide better results at other values of k. the "optimal value" of k will be the true number of ltzs in a given area, and for a given choice of pathogens, and as such is expected to vary. unfortunately, there is no simple way of determining this optimal k in advance of the analysis, which is a key problem with many clustering algorithms and "community detection" network algorithms in particular . it would be of great interest to test the methodology presented here in other geographical locations, on a more comprehensive list of pathogens, and include a wider dataset of human social activities , , , , which together could provide a finer, and more dynamic definition of area-and pathogen-specific real life ltzs. settings. the study was performed using data obtained from the computerized database of hadassah-hebrew university medical center, jerusalem, israel, a tertiary care medical institution serving a population of approximately million people from the entire jerusalem metropolitan. the demographics and microbiological data included in this study were collected from the institutional database between january and may . the dataset contained , positive and negative clinical test results for two causing agents: influenza virus (influenza) and rsv. the area considered in the analysis is a square shape of approximately sq. km. with vertices at: ( , ); ( . , ); ( . , . ); ( , . ). the whole jerusalem urban area population is well covered by this sample since the hospital provides care to over % of the referrals from the population in all parts of the city homogeneously. diagnosis of both influenza and rsv at the hadassah-hebrew university was done on routine basis for patients arriving either to the emergency room or being admitted. the diagnosis was based on direct immunofluorescence assay using commercial monoclonal antibodies (chemicon, temecula, california) until th march , and by in house rt-pcr assay after that date . the original data contained patient addresses in free text format collected from patients during admission (e.g. 'house number x, street y, town z'), which were converted into standard coordinate system using a process of reverse geocoding. in order to maintain patient anonymity the last digits of the obtained coordinates were omitted, resulting in a location resolution of on meters. the study was approved by the hadassah institutional ethics committee. clustering. patients were partitioned into k groups using the k-means clustering method , applied to the (approximate) home locations of the entire patient set. the minimal number of individuals in any ltz was reduced as k increased, however at the highest k value tested (k = ) there was still a minimum number of individuals in any ltz (with minimum numbers of , , , and individuals for k = , , , and , respectively). thus our schemes provides reasonably sufficient data for statistical estimates even for k-values as large as k = . the k values tested were arbitrarily set to multiples of twelve. results for the values k = and k = , or of other intermediate k-values we tested (e.g., k = or k = ) provided qualitatively similar results and are not presented in this study for clarity. note the number of meaningful clusters that may be found is limited by data type/availability. as k is increased, more data is required to accurately extract ltzs. this is because a minimum number of data-points is needed in an ltz before it can be identified accurately by the algorithm. adding a temporal element to the clustering process would be critical for detecting "pathogen hotspots" (using satscan, www.satscan.org, kulldorff, harvard medical school, boston, ma), typically characterized by a significantly elevated incidence of one disease/pathogen. there is no question that detection of disease hotspots is an important activity in modern disease surveillance where it may be used as a real-time warning alert of any atypical activity. our aims however are quite different, since here our goal is to determine if a city-area (i.e. jerusalem) is essentially a single, well-mixed ecology in terms of pathogen transmission, or if there are additional spatial partitions within this limited geographic scope. to address the question in the most direct way, we make use of the robust k-means clustering algorithm, based only on proximity between home locations addresses. disease-signal overlap. we define the general form of a disease signal (ds) for some causing agent v as a function: v ds v (a, t) is the number of positive cases of infection v in a monitored area a over a time period t. for our analysis below, v is a boolean variable, where denotes the influenza virus and denotes rsv. from the data available to us, this function can be immediately constructed for any chosen area a and time period t. the time period t is usually defined to be a week at a time, hence ds v (a, t) represents the total number of positive cases of disease v in area a during week t. we choose ∈ + t z . our data runs from january to may , and hence ∈ … t ( ). generically, any area analyzed may contain different disease signals at (or over) the same (period of) time, e.g. positive clinical test results for influenza and for rsv. in this case the signals from the v = , are termed 'overlapping' . we calculate the per season overlap so v ,v of two disease signals v and v using the following methodology: global epidemiology of influenza: past and present. annual review of medicine incidence of medically attended influenza during pandemic and post-pandemic seasons through the influenza incidence surveillance project emergency department syndromic surveillance providing early warning of seasonal respiratory activity in england demonstrating the use of high-volume electronic medical claims data to monitor local and regional influenza activity in the us ten-year performance of influenzanet: ili time series, risks, vaccine effects, and care-seeking behaviour infectious disease epidemiology infectious disease dynamics seasonality of viral infections: mechanisms and unknowns seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center syndromic surveillance for influenza in the emergency department-a systematic review clinical signs and symptoms predicting influenza infection the a (h n ) influenza virus pandemic: a review spatial transmission of pandemic influenza in the us unifying viral genetics and human transportation data to predict the global transmission dynamics of human influenza h n modeling human mobility responses to the large-scale spreading of infectious diseases transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination influenza a virus transmission via respiratory aerosols or droplets as it relates to pandemic potential community detection in graphs a high-resolution human contact network for infectious disease transmission understanding individual human mobility patterns emergence of new norovirus variants on spring cruise ships and prediction of winter epidemics norwalk-like virus infection in military forces: epidemic potential, sporadic disease, and the future direction of prevention and control efforts outbreak of acute gastroenteritis associated with norwalk-like viruses among british military personnel-afghanistan hepatitis e outbreak on cruise ship data clustering: years beyond k-means dynamics and control of diseases in networks with community structure find me if you can: improving geographical prediction with social and spatial proximity inferring social ties from geographic coincidences structure of urban movements: polycentric activity and entangled hierarchical flows interplay between telecommunications and face-to-face interactions: a study using mobile phone data association of human metapneumovirus with radiologically diagnosed community-acquired alveolar pneumonia in young children we would like to thank mor amitai, gideon almogy, nadav harel, amit gruber, amos cahan and nicolas regnault for their critical reading and helpful suggestions, and the packard foundation for funding. the support of the australian research commission grant dp is gratefully acknowledged. here ds a ( ) v is the average over the season of ds v (a, t), and where the maximization is done over a time-shift ∆ ∈ … t weeks, spanning all events over each entire season. this is also called the ratio of the cross correlation coefficients of the unnormalized vectors ds v (a, t) and ds v (a, t):, which is a quantity depending on the area in question (see eq. , main text). the maximization over the time-shift Δ t is done in absolute value, i.e. we are maximizing the absolute value of the time-shifted cross correlation. in the context of the current paper v and v represent influenza and rsv respectively. the resulting degree of signal overlap per a given period varies between − and , where indicates ds v and ds v completely overlap (same frequency) and zero time shift, and a − score indicates anticorrelation and zero time shift. the overlap is calculated per period, in the results presented here, per 'winter season' , defined here as august year x to may year x + , that is three seasons in total: - , - , and - . notice that our definition of the ratio in eq. [ ] is different from the usual cross correlation: a score of implies two possibilities: . that the data is perfectly correlated at zero time shift or . that the data is perfectly correlated and periodic with some period Δ t.by dividing the whole region into k = …k, we can compute the signal in eq. [ ] for each ltz. this provides a more local way to analyze the correlations between diseases. the total signal is then the weighted sum of the signal of the individual ltzs with weight factors w i equal to the percentage population size n i of the i' th ltz:we define the resulting overall signal overlap as a weighted sum over all ltzs that comprise the area analyzed. given some (number n of) ltzs … ∈ … i i k the signal overlap in each ltz, and consequently the overall ratio in the whole region are bound between negative one -anticorrelation) and one (complete overlap).disease signal ratios. we define the relative disease ratio dr of the incidence of two viruses using the following formula:the relative signal ratios are bound between a value of one (all cases are influenza) and negative one (all cases are rsv) and are specific per time-period, here calculated per one week time periods; when the incidence of influenza is equal to that of rsv the ratio is equal to zero.the per-season absolute |dr| values (presented in fig. c) were calculated as the average of the absolute values of the per-week |dr| values; when the per-week incidences of influenza and rsv are identical, the average value is minimal, i.e. zero, and when influenza and rsv do not coincide on any week the average |dr| is . when calculating the per week relative disease ratio over multiple ltzs, the overall ratio is the weighted average of the ratios in the ltzs. key: cord- - q g authors: imai, kenji; kotani, tomomi; tsuda, hiroyuki; nakano, tomoko; ushida, takafumi; iwase, akira; nagai, taku; toyokuni, shinya; suzumura, akio; kikkawa, fumitaka title: administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: q g the aim of the present study was to investigate long-term outcomes of the offspring in a lipopolysaccharide (lps)-induced maternal immune activation (mia) model and the effect of maternal molecular hydrogen (h( )) administration. we have previously demonstrated in the mia mouse model that maternal administration of h( ) attenuates oxidative damage and neuroinflammation, including induced pro-inflammatory cytokines and microglial activation, in the fetal brain. short-term memory, sociability and social novelty, and sensorimotor gating were evaluated using the y-maze, three-chamber, and prepulse inhibition (ppi) tests, respectively, at postnatal or weeks. the number of neurons and oligodendrocytes was also analyzed at postnatal weeks by immunohistochemical analysis. offspring of the lps-exposed dams showed deficits in short-term memory and social interaction, following neuronal and oligodendrocytic loss in the amygdala and cortex. maternal h( ) administration markedly attenuated these lps-induced abnormalities. moreover, we evaluated the effect of h( ) on lps-induced astrocytic activation, both in vivo and in vitro. the number of activated astrocytes with hypertrophic morphology was increased in lps-exposed offspring, but decreased in the offspring of h( )-administered dams. in primary cultured astrocytes, lps-induced pro-inflammatory cytokines were attenuated by h( ) administration. overall, these findings indicate that maternal h( ) administration exerts neuroprotective effects and ameliorates mia-induced neurodevelopmental deficits of offspring later in life. the association between maternal immune activation (mia) and subsequent neurodevelopmental disorders in offspring has become increasingly recognized, with both epidemiological evidence and research findings in various animal models [ ] [ ] [ ] [ ] . mia against viral or bacterial infections influences the developing fetal central nervous system (cns), and increases the risk of schizophrenia and autism spectrum disorder (asd) later in life [ ] [ ] [ ] . it has been suggested that mia has a strong impact on microglial development, and microglial disturbance disrupts neurogenesis, neuronal migration, and myelination, thus leading to consequent impairments in the brain function of the offspring . based on these findings, mia is believed to be a disease primer . it has also been reported that mia and mia-induced fetal neuroinflammation stimulate the generation of reactive oxygen species (ros) and disturbances in pro-inflammatory cytokine production in the fetal brain , . these changes are reported to maternal h administration attenuated the behavioral deficits induced by lps exposure. since mia has been considered to be a cause of behavioral abnormalities in offspring, including asd/ schizophrenia-like behavior, we subsequently evaluated the effect of lps and maternal administration of hw on short-term memory, sociability, social novelty, and sensorimotor gating. as shown in fig. a , spontaneous alternation was markedly reduced in the lps group than in the control group (p < . ) in the y-maze test, which is indicative of impaired short-term memory. treatment with h significantly attenuated the lps-induced impairment of short-term memory (p < . ). there was no difference in the total number of arm entries among the three groups (control, . ± . ; lps, . ± . ; hw + lps, . ± . ). in order to investigate social behaviors, we evaluated sociability and preference for social novelty in the three-chambered social test. during the habituation phase, the mice in each group spent equal amounts of time exploring both compartments, with no biased preference for either of the two empty cylinders (data not shown). during the sociability phase, the mice in each group demonstrated a preference for spending more time in the chamber containing an unfamiliar mouse (stranger ) relative to the opposite, empty chamber. however, the lps-treated offspring approached the chamber containing stranger significantly less frequently than the control and hw + lps groups (p < . and p < . , respectively; fig. c ). during the social novelty preference phase, the mice in each group showed a significant preference for the new, unfamiliar mouse (stranger ) over the previous, now familiar, mouse (stranger ). however, the lps-treated offspring approached the chamber containing the stranger significantly less frequently than the control group (p < . ; fig. d ). there was no difference between the lps and the hw + lps groups, although a trend toward attenuation of the lps-induced reduction in the preference for social novelty was detected in the hw + lps group. the prepulse inhibition (ppi) test was performed to evaluate sensorimotor integration by measuring the startle response to administered acoustic pulses, however, no significant differences were detected among the groups (fig. b ). maternal h administration attenuated the lps-induced neuronal loss. in our previous investigation, activated microglia was most accumulated in mainly temporal association area, h after the lps insult. neurons in this area were reported to be related to sociability deficits . in addition, the association between autism and amygdala, especially in lateral amygdala , , is well known. moreover, the amygdala interacts with the hippocampus in relation to memory . based on those findings, we subsequently performed nissl staining and evaluated the number of neurons in the amygdala, cerebral cortex, and hippocampus to investigate the brain regions related to the results of the behavioral deficits induced by lps exposure. the quantitative results revealed significant reductions in the number of neurons in the amygdala and cerebral cortex in the lps-treated offspring than in the control group (p < . and p < . , respectively; fig. a , panels a,b,d,e and b), but not in the hippocampus (fig. s ). the lps-induced neuronal loss in the amygdala and cerebral cortex was improved in the hw + lps group than in the lps group (p < . and p < . , respectively; fig. a , panels c,f and b). maternal h administration attenuated lps-induced oligodendrocytic loss and suppressed astrocytic activation. the pathophysiologic mechanisms of the brain injury due to mia are incompletely understood; however, under prenatal inflammation, activated astrocyte was reported to have a potential cause an adverse effect on neurons and oligodendrocyte, which would result in the abnormal behaviors in later life . the number of oligodendrocytes was evaluated by using olig , a transcription factor involved in the differentiation of cells in the oligodendroglial lineage, and a mature oligodendrocyte marker , . compared with the control group, the number of olig -positive cells was clearly reduced in the white matter, amygdala, and cerebral cortex in the lps group (p < . , p < . , and p < . , respectively; fig. a , panels a,b,d,e,g,h and c). h treatment completely blocked the lps-induced decrease of olig -positive cells in all three regions (p < . , p < . , and p < . , respectively; fig. a , panels c,f,i and c). the hallmarks of astrocytic activation include the development of a hypertrophic morphology with fewer processes and upregulation of intermediate filament proteins, particularly glial fibrillary acidic protein (gfap) , . in the control group, some gfap-positive cells were detected, most of which were in the resting state (fig. b , panels a,d). compared with the control group, a significantly increased number of activated astrocytes, characterized by a large soma and fewer processes (thus obtaining a more rounded shape), was observed in the white matter and amygdala of the lps-treated offspring (p < . and p < . , respectively; fig. b , panel b,e and d). to evaluate the hypertrophic soma, the cellular size of the astrocytes was also quantified by calculating the ratio of gfap immunostained area to the number of gfap-positive cells. larger soma were observed in the white matter of lps-exposed mouse brains (p < . ; fig. b , panel b and e). although the number of gfap-positive cells was unchanged following h treatment, the size of the gfap-positive cells was significantly reduced in the white matter (p < . ; fig. b , panel c and e), indicating a reduced number of reactive astrocytes by h treatment. in the amygdala, the number of gfap positive astrocytes in the hw + lps group was significantly decreased (p < . ; fig. b , panel f and d). h treatment suppressed lps-induced activation in primary cultured astrocyte. because the over-activation of immune cells, including astrocytes, could have an adverse effect on neurons with the overproduction of pro-inflammatory cytokines , we investigated the anti-inflammatory effect of h on astrocyte using primary cultured astrocyte. as shown in fig. , stimulation of astrocytes with lps for three hours led to marked increases in tumor necrosis factor α (tnf-α) (p < . ), interleukin (il)- β (p < . ), and il- (p < . ) mrna expression. these increases in expression of tnf-α (p < . ), il- β (p < . ) and il- (p < . ) were significantly attenuated by h treatment. altered gene expression patterns in primary cultured astrocytes from the control group, the lps group, and the hw + lps group. as we previously described the gene expression profile of microglia , differential gene expression patterns of astrocytes from newborn mice in the control group, the lps group, and the hw + lps group were also observed (tables s -s , supplementary information). these results suggest that h might also influence the function not only of microglia but also of astrocytes via changes in gene expression. for the first time, we have demonstrated that maternal h administration improved mia-induced neurological impairments of offspring, including short-term memory and social interaction at postnatal - weeks. previously, we reported that maternal h administration suppresses microglial activation in the same model. in the present study, h also suppressed the activation of astrocytes, both in vitro and in vivo. taken together, these results suggest that h exerts a protective role against mia-induced neuroinflammation through the suppression of microglia and astrocytes. as a result, mature oligodendrocytes were restored and neurons were protected from damage, thus leading to improved long-term neurological outcomes following maternal h administration. prenatal exposure to lps also resulted in a transient reduction in body weight of the offspring between p -p , which was attenuated by maternal h administration. this might be due to a change in the activity of the hypothalamic-pituitary axis . deficient post-neonatal growth has been reported to contribute to poor neurologic outcome , thus, the transient decrease of weight observed in this model might be related to the neurological impairments in the offspring. on the basis of the epidemiological evidence, several translational rodent models have been established to investigate a potential causal relationship between mia and asd-like behavioral abnormalities . following the discovery, in the s, of the increased risk of schizophrenia after maternal influenza infection , models of viral-like immune activation by polyriboinosinic-polyribocytidilic acid (poly(i:c)) have been widely studied , , . in addition, models of bacterial-like immune activation by lps are also well known to precipitate inflammatory responses and behavioral abnormalities in offspring, similar to the poly(i:c) models . however, with respect to neurotoxicity, lps exposure may have a stronger effect on the fetal brain than poly(i:c) exposure . thus, the type of pathogen is important for the pattern of neurodevelopmental abnormalities of the offspring, and the timing and dose of the pathogen's administration, in addition to the strain or species, may also have an influence. the present model of prenatal exposure to lps led to impairments in short-term memory and social interaction at to weeks after birth. alterations in short-term memory were demonstrated by the marked reduction in alternation behavior in the y-maze test, which is consistent with previous reports , and is a persistent finding in poly(i:c) models . the y-maze test is well known as a hippocampal-dependent short-term learning task. in our mia model, a remarkable loss of neurons was observed in the amygdala and cerebral cortex of the offspring, but not in the hippocampus. the formation of memory is encoded in a broad network of cortical/subcortical regions including the hippocampus, cingulate cortex, and amygdala . moreover, the amygdala is directly connected to and dynamically interacts with the hippocampus in relation to memory . therefore, a decreased number of neurons in the amygdala and cerebral cortex could consequently result in short-term memory impairments. in this model, increased activation of astrocytes was observed in the white matter and amygdala, which be related to impaired short-term memory , . previous reports have demonstrated a causal relationship between prenatal exposure to lps and deficits in social behavior and sensorimotor gating in offspring , [ ] [ ] [ ] [ ] . in this model, the social behavior including sociability and social novelty preference was impaired. the number of oligodendrocytes in the amygdala, cerebral cortex, and white matter in the offspring was decreased, which is consistent with previous findings ; and a reduced number of neurons in the amygdala and cortex was also observed. strong evidence suggests a crucial involvement of the amygdala in social processing and social cognition in humans [ ] [ ] [ ] , as well as in social behavior in animals [ ] [ ] [ ] . a recent report also suggested an association between the function of the cortex and the amygdala, and social behavior . therefore, the social behavior impairment observed in the present study is compatible with the neuronal and oligodendrocytic loss in the amygdala and cortex. a locomotor test was not performed in this study, although we observed no apparent differences in speed of movement among all groups in the three-chamber social test. interestingly, there were no remarkable differences in the ppi test in our mia model, and sensorimotor gating was also normal in comparison with the control group. a possible explanation for these results is that ppi abnormalities might occur following exposure to a pathogen at mid-gestation , while impairments in short-term memory and social behavior might be independent of the timing of exposure to a pathogen . thus, the negative results of the ppi test might be due to the fact that in the present model, lps exposure occurs at late gestation, on embryonic day (e ). impairments in short-term memory and social behavior are well described in schizophrenia and asd , . ppi impairments manifest primarily in adults with asd , and are not observed in children with asd . a recent study further suggested that sensorimotor gating is only impaired in certain asd subgroups , although it may be a globally common feature in schizophrenia . therefore, the present model may reflect asd-like rather than schizophrenia-like behavioral impairments. several characteristics that are reported in human asd brains were also observed in the present model, including neuronal loss in the amygdala and cerebral cortex , and astrocytic activation in the white matter . we and others have also previously demonstrated microglial activation, enhanced pro-inflammatory cytokine levels, including il- , and elevated oxidative damage in fetal brains , [ ] [ ] [ ] . in the mia model, microglia play a major role, along with astrocytes, in synaptic pruning and neural circuit formation . in addition, a single injection of il- is known to lead to the same behavioral abnormalities as those observed in the lps models (il- is thought to be a key cytokine in the link between mia and the behavioral deficits in the offspring ). in our previous report, maternal h administration reduced both the excessive microglial activation and the increased il- levels in the mia fetal brains , which could ameliorate the behavioral deficits later in life. previous studies in adult rodents reported that h treatment could mitigate the behavioral dysfunctions, including short-term memory deficits, cognitive impairments, and depressive-like behaviors, by regulating inflammation and ros production , , which is in line with our current results. moreover, the present study demonstrated that prenatal h administration restored the mia-induced neuronal and oligodendrocytic loss, and suppressed the excessive activation of astrocytes at postnatal weeks. it was also shown that h directly attenuated the lps-induced expression of pro-inflammatory cytokines, including tnf-α, il- β, and il- , in primary cultured astrocytes. these cytokines are known to induce apoptosis in oligodendrocytes and hypomyelination in the neonatal rodent . thus, the suppressive effect of h on both microglial and astrocytic activation would protect neurons and oligodendrocytes against lps exposure. the pathological mechanism by which mia causes psychiatric diseases in offspring is completely unknown, however we speculate that the following steps may be involved , . intrauterine inflammation activates fetal microglia during the acute phase, and subsequently, microglia also activate astrocytes. in the chronic phase, microglial activation is attenuated, while astrocytes are continuously activated . both microglia and astrocytes are important for normal neurodevelopment, however under prenatal inflammation, they are activated, their function is altered, and they have an adverse effect on neurons and axons, with the overproduction of pro-inflammatory cytokines . inflammation also prevents the maturation of oligodendroglial progenitor cells, followed by a reduction of the oligodendrocyte lineage , . this reduction leads to axonal loss. the neuronal and axonal damage caused by microglia or astrocytes in the developing brain would result in the abnormal behavioral features in later life. in our model, we have previously reported over-activation of fetal microglia h after the lps insult , and the present study demonstrated that activation of astrocytes continued even ~ weeks after the insult. moreover, the number of neurons and oligodendrocytes were reduced in the amygdala and white matter, respectively. we have previously reported that h has a suppressive effect on microglial activation , and the present study demonstrated that h had a similar effect on astrocytes, as described above. from these findings, we speculate that h would protect neurons and oligodendrocytes indirectly via suppression of the over-activation of immune cells, including microglia and astrocytes, which would result in a reduction of the behavioral abnormalities present in mia offspring. however, in this study, a direct effect of h on neurons and oligodendrocytes was not evaluated in vitro, thus, this possibility cannot be excluded. several limitations of this study should be noted. in the current mia model, additional core features of asd, including restricted and repetitive behavioral patterns, were not investigated . thus, the effect of maternal h administration on these symptoms remains unknown. the sensorimotor gating abnormality may be evaluated at an older age than was described in the present model, however, this might require changing other factors including the timing of exposure. since the mouse brain is not considered to be fully mature until weeks of age , the interpretation of the results of behavioral tests in the present study requires considerable caution. in addition, we could not avoid the detrimental effects of maternal separation required for the measurement of body weight on offspring development. however, the separation was performed similarly for the offspring in all three groups, thus any effect should be minimal, and consistent across groups. in humans, it is currently thought that the occurrence of multiple risk factors, including genetic mutations and postnatal exposure to inflammation or other environmental triggers, may be required for the onset of asd , . before advancing the clinical application of h , it should be investigated whether h could exert similar effects in so called 'multi-hit' models. finally, in the previous and present studies, maternal h administration was performed prior to the lps exposure; thus, the therapeutic potential of h remains unclear. therefore, it might be relevant to evaluate the therapeutic effects of h by administering h to dams after exposure to lps and in offspring. in conclusion, we have provided evidence that maternal h administration reduced deficits in short-term memory and social interaction in the lps-induced mia model, and also demonstrated an attenuation of the lps-induced neuronal and oligodendrocytic cell loss. remarkably, the neuroprotective effect of h appeared in the amygdala and cortex in this mouse model. the neuroprotection is thought to be due to the suppression of neuroinflammation, including excessive activation of microglia and astrocytes and the increased level of pro-inflammatory cytokines, as reported previously . although further investigation is required, our current and previous results point to the potential efficacy of maternal h administration on the long-term neurological outcomes of offspring exposed to inflammation in utero. reagents. lps (serotype o :b ) was obtained from sigma-aldrich (st. louis, mo, usa). hw and hydrogen medium (hm) were prepared by dissolving h gas as described previously , . both hw and hm had a concentration of > . mm h . animals and treatments. the experimental protocols in this study were approved by the animal experiment committee of nagoya university (approval number: ), and were carried out in accordance with the regulations on animal experiments in nagoya university. pregnant icr (cd- ) mice ( - weeks) were purchased from charles river laboratories (kanagawa, japan). all mice were allowed free access to food and water and were maintained on a -h light/dark cycle (lights on at : am). the pregnant mice were assigned randomly to three groups of five dams each: control group, lps group, and hw + lps group (fig. ) , as in our previous report . all pregnant mice, except for those in the control group, received an intraperitoneal injection (i.p.) of μg of lps dissolved in sterile saline on e . in the control group, the pregnant mice were injected with an equal volume of sterile saline. in the hw + lps group, the pregnant mice were administered hw, beginning h before lps injection and continuing until parturition. hw was aliquoted in glass drinking bottles to prevent h degassing as well as air refilling. offspring growth evaluation and behavioral testing. as shown in fig. , after birth, all offspring were housed with their mother until p , when they were weaned, as previously reported , . we only used male offspring in all of the experiments because of previously reported sex effects . all animals were left undisturbed except for measuring body weights and weekly cage changes. body weight was recorded at p , , , , , , and . the order of the behavioral tests was as follows: ( ) y-maze test at postnatal weeks ; and ( ) three-chamber sociability and social novelty test and ppi test at postnatal weeks, according to previous reports (fig. ). all behavioral tests were conducted between pm and pm. y-maze test. the y-maze test was carried out as described previously . briefly, the test was performed in a y-shaped maze with three opaque plastic arms situated at ° angles from each other. each mouse was placed individually in the center of the apparatus and allowed to explore the maze freely during an -min session. the series of arm entries were recorded visually. alternation was defined as successive entries into the three arms, on overlapping triplet sets. the percent alternation was calculated as the ratio of actual to possible alternations (defined as the total number of arm entries minus two) multiplied by . spontaneous alternation (%) was used to quantify short-term memory. three-chamber social test. the three-chamber social test was performed as described previously . briefly, the apparatus consisted of a black plexiglas rectangular box and two identical clear plexiglas cylinders. there were three interconnected chambers in the box. the light was conditioned at lux in an experimental room. during the habituation phase, empty cylinders were placed in each of the end chambers. a mouse was introduced to the center chamber and its behavioral approach to the end chambers was monitored for min. during the sociability test, an unfamiliar male icr (cd- ) mouse (stranger ) that had no prior contact with the test mouse was placed in one of the empty chambers, and the behavioral approach of the test mouse to the empty chamber and stranger was monitored for min. during the social novelty test, a new unfamiliar male icr (cd- ) mouse (stranger ) was placed in the third chamber, and the behavioral approach of the test mouse to stranger and stranger was monitored for min. the time spent in each zone was calculated using the ethovision automated tracking program (noldus, wageningen, the netherlands). prepulse inhibition test. the ppi test was carried out as described previously with the sr-lab system (san diego instruments, san diego, california). briefly, after habituation for min, the animals received startle trials, no-stimulus trials, and ppi trials. each startle trial consisted of a single db white noise burst lasting msec. the ppi trials consisted of a prepulse ( msec burst of white noise at , , , or db intensity) followed, msec later, by the startle stimulus ( db, msec white noise). the total session lasted min. the resulting movement of the animal in the startle chamber was measured for msec after the startle stimulus onset (sampling frequency khz), rectified, amplified, and input into a computer, which calculated the maximal response over the -msec period. the basal startle amplitude was determined as the mean amplitude of the startle trials. the ppi was calculated according to the following formula: × [ − (pp×/p )] %, in which ppx was the mean of the ppi trials (pp , pp , pp , or pp ) and p was the basal startle amplitude. after the behavioral analysis, offspring in each group were selected randomly and sacrificed by decapitation at postnatal weeks (fig. ) . the offspring were perfused via the heart with % paraformaldehyde (pfa) in pbs. the brains were then fixed in % pfa for at least h, embedded in paraffin, and cut in coronal planes (approximately . mm posterior to bregma). immunofluorescence staining was performed using the following primary antibodies: rabbit monoclonal anti-olig antibody (ab , : ; abcam, tokyo, japan) or rabbit polyclonal anti-gfap antibody ( - -ap, : ; proteintech, chicago, il). the sections were incubated with the primary antibodies at °c overnight and were further incubated with secondary antibodies (alexa fluor , : or alexa fluor , : ; invitrogen, carlsbad, ca) for min at room temperature. nissl staining was also performed in order to evaluate the number of neuronal cells. following deparaffinization, the tissue sections were incubated in . % cresyl violet (muto pure chemicals co.), and subsequently dehydrated in graded ethanol, and permanently mounted. images were acquired with a bz- microscope (keyence corporation, osaka, japan). the bz image measurement software program (keyence) was used to quantify the number of neuronal cells and olig -positive cells, as well as the number and the soma size of the gfap-positive cells. astrocytic cultures. mouse primary astrocytes were isolated from primary mixed glial-cell cultures of newborn icr mice, as described previously , . the purity of the astrocytes was > %, as determined by immunostaining with the anti-gfap antibody. astrocytes were plated at a density of × cells/well in -well dishes. cells were maintained in dulbecco's modified eagle's minimum essential medium (sigma-aldrich) supplemented with % fetal bovine serum, mg/ml bovine insulin, and . % glucose. to assess the expression of pro-inflammatory cytokines, confluent astrocytes were treated with μg/ml lps and/or hm. h treatment was performed by replacing the astroglial medium with hm, and dehydrogenated hm was used as a control medium. the time course of h concentration in hm was described previously . replacement of the astroglial medium with hm or control medium was performed h before the addition of lps. quantitative reverse transcription-polymerase chain reaction (rt-pcr). total rna from the astrocyte cultures was extracted using the rneasy mini kit (qiagen inc., tokyo, japan). the rt reaction with ng of total rna was carried out with a first strand cdna synthesis kit (revertra ace; toyobo co., ltd, osaka, japan). the expression levels of mrnas encoding tnf-α, il- β, and il- were evaluated by quantitative pcr (qpcr) using the thermal cycler dice (takara bio inc., tokyo, japan) and sybrii premix ex taq (takara bio inc.) reagents. primers for qrt-pcr are listed in table . statistical analysis. the data are presented as means ± standard error of the mean (sem). to analyze the neonatal body weights, two-way repeated measures analysis of variance (anova) was used, followed by a bonferroni post-hoc test. the mrna expression levels in astrocyte cultures were also analyzed by two-way anova, followed by a bonferroni post-hoc test. all other data were analyzed using one-way anova followed by tukey's test. the statistical analyses were performed using prism for windows (graphpad software, san diego, ca). values of p < . were considered to be statistically significant. tnf-α ′-gtagcccacgtcgtagcaaac- ′ ′-ctggcaccactagttggttgtc- ′ il- ′-acaaccacggccttccctac- ′ ′-tccacgatttcccagagaaca- ′ il- β ′-catccagcttcaaatctcgcag- ′ ′-cacacaccagcaggttatcatc- ′ β-actin ′-cgtgggccgccctaggcacca- ′ ′-acacgcagctcattgta- ′ table . list of primers for qrt-pcr. maternal immune 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injury preterm birth and the role of neuroprotection transcription factor mef c influences neural stem/progenitor cell differentiation and maturation in vivo molecular hydrogen ameliorates several characteristics of preeclampsia in the reduced uterine perfusion pressure (rupp) rat model axon-glia interactions and the domain organization of myelinated axons requires neurexin iv/caspr/paranodin impact of maternal cigarette smoke exposure on brain inflammation and oxidative stress in male mice offspring a transient insulin system dysfunction in newborn rat brain followed by neonatal intracerebroventricular administration of streptozotocin could be accompanied by a labile cognitive impairment seizure-dependent mtor activation in -ht neurons promotes autism-like behaviors in mice combined effect of neonatal immune activation and mutant disc on phenotypic changes in adulthood heterozygous disruption of autism susceptibility candidate causes impaired emotional control and cognitive memory excitatory amino acid transporter expression by astrocytes is neuroprotective against microglial excitotoxicity coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes the author would like to thank dr. j. kawanokuchi (institute of traditional chinese medicine, suzuka university of medical science) for technical support for primary cultured astrocytes. we would like to acknowledge mrs sachiko morisaki for her laboratory work. we would like to thank editage (www.editage.jp) for english language editing. this work was supported by jsps kakenhi grant number h and k and msd life science foundation, public interest incorporated foundation. the authors have no conflicts of interest to declare in association with this study. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -lrkscs authors: kurosaki, yohei; martins, danyelly bruneska gondim; kimura, mayuko; catena, andriu dos santos; borba, maria amélia carlos souto maior; mattos, sandra da silva; abe, haruka; yoshikawa, rokusuke; de lima filho, josé luiz; yasuda, jiro title: development and evaluation of a rapid molecular diagnostic test for zika virus infection by reverse transcription loop-mediated isothermal amplification date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: lrkscs the recent outbreak of zika virus (zikv) disease caused an enormous number of infections in central and south america, and the unusual increase in the number of infants born with microcephaly associated with zikv infection aroused global concern. here, we developed a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay using a portable device for the detection of zikv. the assay specifically detected zikv strains of both asian and african genotypes without cross-reactivity with other arboviruses, including dengue and chikungunya viruses. the assay detected viral rna at . tcid( )/ml in virus-spiked serum or urine samples within min, although it was slightly less sensitive than reference real time rt-pcr assay. we then evaluated the utility of this assay as a molecular diagnostic test using plasma or serum samples and urine samples collected from suspected cases of arbovirus infection in the states of paraíba and pernambuco, brazil in . the results of this assay were consistent with those of the reference rt-pcr test. this portable rt-lamp assay was highly specific for zikv, and enable rapid diagnosis of the virus infection. our results provide new insights into zikv molecular diagnostics and may improve preparedness for future outbreaks. zikv is a positive-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. zikv shares its vector, the aedes mosquito, with other flaviviruses, including denv, yellow fever virus (yfv), and chikv . zikv has been isolated from humans in east and west africa and in southeast asia and polynesian countries where the host mosquitoes, a. aegypti and a. albopictus, are found , . based on phylogenetic analyses, these isolates can be categorised into two genotypes, african and asian. epidemiological studies have revealed that the recent outbreak of zikv in brazil occurred via the introduction of a virus from french polynesia, where an outbreak of the disease occurred in . all of the viruses isolated in brazil and other countries on the american continent belong to the asian genotype . in patients with zikv infection, the virus can be detected in several sample types, including blood, urine, saliva, and other body fluids [ ] [ ] [ ] [ ] [ ] . the viral load in blood reaches a peak at to days after the onset of illness, but decreases rapidly thereafter. therefore, it is difficult to detect zikv in blood samples from patients after the acute phase of infection, even with sensitive molecular diagnostic methods, such as reverse transcription-polymerase chain reaction (rt-pcr) , , . the virus can be detected in urine samples for longer durations (> - days after the onset of symptoms) than in those for blood samples . currently, blood and urine samples are typically used for the molecular diagnosis of zikv. zikv infection is diagnosed in the laboratory by nucleic acid amplification tests (naats) to detect viral rna [ ] [ ] [ ] [ ] [ ] or by elisa to detect igm or igg antibodies , . the naats such as rt-pcr and other technologies (e.g. recombinase polymerase amplification) are highly accurate, and rt-pcr is considered the gold standard to confirm zikv infection , . rt-pcr, however, requires a step for viral rna extraction prior to the assay and the use of expensive equipment, such as thermal cycler, to conduct the test. moreover, there is a risk of reduced sample quality due to rna degradation during transportation to the laboratory. for elisa, serological cross-reaction between zikv and other circulating flaviviruses like denv makes accurate diagnosis with serology difficult , . therefore, novel diagnostic technologies that can be conducted at the point-of-care or in regional laboratories are greatly needed to control zikv infections. reverse transcription loop-mediated isothermal amplification (rt-lamp) is a rapid, sensitive rna detection method performed under isothermal conditions using four or six unique oligonucleotide primers , . since lamp reactions can be performed with simple inexpensive equipment, rt-lamp assays can be conducted in the field and by under-funded laboratories . we previously developed a rt-lamp assay using a portable isothermal amplification and detection device for ebola virus in response to the recent outbreak of ebola virus disease in west africa, and the assay has been deployed for field surveillance in guinea , . here, we developed a rt-lamp assay for the detection of zikv with a portable battery-powered device. then, we evaluated the utility of this assay for molecular diagnosis using clinical specimens collected from the recent zikv outbreak in brazil. sensitivity. we designed zikv genotype-specific lamp primers that targeted conserved sequences in the e protein-coding region (table ) . each genotype-specific primer recognised the same genomic position. to detect all known zikv strains, we used a mixture of primers specific for each genotype in a single reaction. first, we examined the sensitivity of the assay using serial -fold dilutions of in vitro synthesised standard rnas from strain uganda, which was isolated from a rhesus macaque in uganda, and strain prvabc , which was isolated at the centres for disease control and prevention (cdc) from a patient who travelled to puerto rico in . ten copies of the rna standards were detected from both strains in quadruplicate reactions (fig. a) . the times to obtain positive results (tp) for rna standards ranging from to copies were mostly less than min, and within this range, tp was correlated with the number of rna copies ( fig. b and c) . single copies of the standard rnas from the uganda and prvabc strains were detected with % and % positivity, respectively, and tp values were dispersed. these results suggested that the rt-lamp assay could be used as a rapid, sensitive diagnostic test for zikv, the tp value (i.e., less than min) can be used as an indicator of the number of rna copies in each reaction. we evaluated the specificity of each primer in silico using zikv strain sequences available in genbank as of november . twenty-seven sequences of african genotype isolates collected in - and sequences of asian genotype isolates collected in southeast asia and polynesia in - as well as from the current outbreak in the americas were used for this analysis. the lamp primers consist of nucleotides in total length and recognise eight separate sites on zikv genome (fig. a) . we determined the proportion of sequences that had identical nucleotides at each position for either the asian or african genotype primers (fig. b) . african genotype zikv sequences had identical residues at out of positions ( . %) in the african or asian genotype-specific primers. at positions in the primer recognition sites, more than % of african genotype zikv sequences had mismatched nucleotides. at six positions, scattered in the f , f , lf, and b sites in the asian and african genotype primers, more than % of the african genotype zikv sequences had nucleotide differences (fig. b, upper panel) . asian genotype sequences showed greater identity than african genotype sequences to the lamp primers. asian genotype sequences had identical residues at out of positions ( . %) in the asian or african genotype primers. for one residue at the ′ terminus of the f site of the fip primers, . % of the asian genotype sequences had nucleotide differences (fig. b, lower panel) . to assess the primer specificity for zikv strains, we synthesised rnas with the partial genome sequences of two african genotype zikv strains, -dak and ard , and two asian genotype zikv strains, p - and cpc- , which had more mismatched nucleotides against the primer sequences compared with the average for all strains. these rna sequences were also detected using the rt-lamp assay, in addition to the sequences in the uganda and prvabc strains (table ) . furthermore, no cross-reactions with other tested arboviruses, including denv, yfv, west nile virus (wnv), chikv, and rift valley fever virus (rvfv), and plasmodium falciparum were observed. these results suggested that the rt-lamp assay developed here was highly specific for detecting zikv strains of both african and asian genotypes. and/or urine is used, since viral rna can be detected in these clinical specimens during the acute phase of infection. the feasibility of using the rt-lamp assay for clinical specimens was evaluated using zikv-spiked human serum and urine samples. we prepared human serum and urine spiked with four-fold serially diluted zikv strain uganda, and obtained samples with titres of . - . tcid /ml. the sensitivity of the rt-lamp assay was compared to that of the real time rt-pcr (rrt-pcr) assay developed by the cdc . using the rt-lamp assay, we detected viral rna in both serum and urine samples at a titre of . tcid /ml in quadruplicate reactions. the ct values in the rrt-pcr were . ± . and . ± . for serum and urine samples, respectively, which corresponded to . and . genome equivalents (geq) per reaction, respectively (table ). using the rrt-pcr assay, we detected viruses in both serum and urine samples at a titre of . tcid /ml, which corresponded to . and . geq per reaction, respectively; however, the rt-lamp assay failed for these samples, suggesting that the rt-lamp assay was less sensitive than the cdc rrt-pcr assay for zikv detection. together with the results table ). the rt-lamp assay did not show any false-positive results, even for six confirmed denv samples (data not shown). the ct values of these eight zikv-positive samples were . - . , and the viral loads were estimated to be . × - . × geq/ml using the viral rna standards (table ) . these viral titres were higher than those reported in previous studies. to examine whether the assay can detect viral rna in samples with lower titres, we randomly selected two zikv-positive samples confirmed in this study, mrl and mrl , and conducted a dilution test (table ). while the rt-lamp failed to detect samples with the ct value > , however, it detected viral rna at the ct < , consistent with our earlier results obtained using the virus-spiked serum and urine samples (table ). these results show that the rrt-lamp assay had sufficient specificity for the detection of zikv as a molecular diagnostic test. the assay can be used to detect an amount of viral rna equivalent to that yielding ct values of - in the reference rrt-pcr test. we developed a rapid molecular detection assay for zikv in response to the recent outbreak in south america. lamp assays and modified diagnostic methods for zikv have been reported; however, these molecular techniques have never been evaluated for clinical use [ ] [ ] [ ] [ ] . this is the first evaluation of the clinical usage of a lamp assay for molecular diagnostic testing in the recent outbreak of zikv infections. since zikv shares a vector with denv and chikv, these viral diseases can occur simultaneously, and northeast brazil is an endemic area for dengue and chikungunya . numerous severe mosquito-borne diseases, including arbovirus infections as well as malaria, share clinical symptoms during the acute phase. however, zikv infection is generally associated with mild symptoms. a major concern with respect to molecular diagnostic testing for zikv is the potential for cross-reactivity with other flaviviruses, especially dnev, which have close antigenic relation with zikv , , , . in contrast, our assay showed no cross-reactions with other arboviruses or p. falciparum, and did not show false-positive results when applied to zikv-negative samples. these results indicated that the rt-lamp assay is specific for the detection of zikv and is a reliable molecular diagnostic test. another potential limitation of molecular diagnostic testing is that zikv-infected samples often have low titres after the acute or early phase of infection due to rapid clearance by the host immune system. this makes it difficult to identify zikv cases, even using rt-pcr-based tests. the limit of detection for this assay was copies table . detection of zikv by rt-lamp and rrt-pcr using diluted zikv-confirmed samples. . for both genotypes. the assay was slightly less sensitive than the cdc rrt-pcr test, which was commonly used to confirm zikv infection during the recent outbreak. zikv-infected clinical samples often show high ct values (> ) , . however, the zikv-positive samples detected in this evaluation showed ct values of less than . (more than . × geq/ml), which was a higher titre than that reported in other studies. to confirm its clinical utility, this assay should be tested using samples with lower titres or borderline zikv infections. it has been reported that viral rna can be detected for longer periods in urine than in blood , . therefore, we considered urine to be one of the best sample types for detecting zikv infections. recently, paz-bailey et al. reported contradictory results for the persistence of viral rna in blood samples of zikv patients; rna can be detected or weeks after the onset of illness . in some cases, viral rna can also be detected at higher titres in saliva than in blood, but persists for shorter periods , . it is necessary to determine the sample types suitable for the rt-lamp assay and to establish a standardised rna extraction protocol adjusted to each clinical specimen type in order to improve the sensitivity of this assay. owing to the sequence diversity among zikv isolates, we designed lamp primers specific for each genotype and used a mixture of these primers to detect all known isolates of both african and asian genotypes. as shown in fig. , we conducted an in silico evaluation of each primer using available zikv sequences. african genotype strains supposedly have a longer history of circulation in african mosquitos and humans than that of asian genotype strains , and african genotype sequences showed a lower identity at some positions in the lamp primers. the lamp primers designed here showed high identities at most positions against the sequences of strains involved in the recent outbreak on the american continent, as well as its ancestral southeast asian and polynesian isolates. during the outbreak of zikv in americas, confirmed or probable zikv-infected cases has been continuously reported in southeast asia . our assay will be useful for virus detection and may contribute to preparedness for future outbreaks in these zikv endemic countries as well as in asia and africa. however, the evolution of zikv sequences must be constantly monitored to guarantee primer specificity. using samples obtained from subjects with suspected arbovirus infection, we did not find any zikv-positive samples in paraíba in march or july by rrt-pcr or our rt-lamp test. these samples were collected from patients within or weeks after the onset of arbovirus infection-like symptoms as part of an education and follow-up campaign for cardiovascular diseases. many samples might have been collected after the acute or early stage of infection. in addition, when this campaign was conducted, the prevalence of zikv infection may have been low, since most cases were reported from november to march , which is closely linked to the ecology of the vector aedes mosquito. the main advantages of this assay are its speed (positive results can be obtained within min) and the use of a battery-operated portable device. since the device has a user-friendly interface, training is not necessary to conduct the assay and interpret the results. recently, freeze-dried reagents for lamp assays have been made available, making cold-chain-free lamp assays a possibility. our assay is suitable for use in field surveillance or remote areas where it is difficult to implement laboratory diagnostic tests. the assay should be evaluated in a prospective study to confirm its utility for molecular diagnostic testing, especially under limited resources and by field laboratories in zikv endemic countries. in this paper, we successfully developed a rt-lamp assay for the detection of zikv by designing asian and african genotype-specific primers. the assay showed results consistent with those of the reference rrt-pcr assay in diagnostic tests with suspected cases of zikv infection. our results provide a potential new molecular diagnostic test for zikv and may serve as a basis for the development of alternative rapid diagnostic techniques to prepare for potential outbreaks. and were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % penicillin/streptomycin and % foetal bovine serum (fbs). zikv strain uganda was kindly provided by dr. shigeru tajima (national institute of infectious diseases; niid). the virus was propagated in vero cells grown in dmem supplemented with % fbs. two days after infection, culture supernatants were harvested, clarified by low-speed centrifugation, and then stored as virus stock at − °c until use. the infectious titre of the virus stock was determined by the % tissue culture infective dose (tcid ) using vero cells; titres are expressed as tcid / ml. viral rna was extracted from μl of infected culture supernatant using the qiaamp viral rna mini kit according to the manufacturer's protocol. the rna was eluted in μl of elution buffer and stored at − °c until use. viral rna from zikv strain prabc was kindly provided by dr. shigeru tajima (niid). viral rnas from other arboviruses, including denv serotype - , yfv, wnv, chikv, and rvfv, as well as genomic dna from p.falciparum strain d were kindly provided by dr. kouichi morita and dr. osamu kaneko (institute of tropical medicine, nagasaki university). preparation of rna standards. rna standards, consisting of partial genome sequences of zikv strains uganda and prvab c , were amplified by rt-pcr using for ward ( ′-ggagtcaggatggtacttgtacc- ′) and reverse ( ′-aaaattggatattcaggaacc- ′) primers with the primescriptii high fidelity one step rt-pcr kit (takara bio, shiga, japan). the reactions were performed using the takara pcr thermal cycler dice with the following program: °c for min, °c for min, followed by cycles of °c for s, °c for s, and °c for s. amplified pcr fragments were cloned into the pcr . vector using the topo-ta-cloning kit (invitrogen, carlsbad, ca, usa). the plasmids were digested with bamhi, purified from the agarose gel slice using a column purification kit (qiagen, hilden, germany), and used as templates for rna synthesis. the partial genomic rnas of each zikv strain were synthesised in vitro using t rna polymerase (promega, madison, wi, usa) and purified using the rneasy mini kit (qiagen). the rna concentration was determined by measuring the optical density at nm (od ) with scientific reports | : | doi: . /s - - - a nanodrop (thermo fisher scientific, waltham, ma, usa), and the rnas were diluted in depc-treated water to achieve the desired concentrations. primer design. lamp primers for zikv detection were designed based on the coding sequences for the e protein. the zikv sequences available in genbank were aligned using clustalx to identify conserved regions. a consensus sequence for a region in the e gene was used to design lamp primers using lamp designer (optigene; http://www.optigene.co.uk/lamp-designer/). primers specific for asian genotype viruses were designed first, and then african genotype-specific primers were designed by adapting each position to the african genotype consensus sequence. the rt-lamp assay required a set of six primers, two outer primers (f and b ), a forward inner primer (fip), a reverse inner primer (bip), a forward loop primer (lf), and a reverse loop primer (lb). the fip consisted of the f c sequence, which was complementary to the f and f sequences. the bip consisted of the b c sequence, which was complementary to the b and b sequences . the lb primer was designed to detect both asian and african genotype sequences. the sequences and locations of the oligonucleotide primers are shown in table . rt-lamp was performed with isothermal master mix reagent (optigene, west sussex, uk) using the genelyzer fiii real-time fluorescence detection platform (toshiba medical systems, otawara, japan). the reaction mixture (total volume, µl) contained µl of isothermal master mix; µl of warmstart rtx reverse transcriptase ( u; new england biolabs, ipswich, ma, usa); µl of the lamp primer mix consisting of pmol f and b , pmol fip and bip, pmol lf and lb; and µl of rna sample (template). the assay was carried out using a mixture of primers specific for the asian and african genotypes. all primers were cartridge-purified oligonucleotides purchased from hokkaido system science (sapporo, japan). the reaction was performed at °c for min, followed by a dissociation analysis at °c- °c. depc-treated distilled water and rna synthesised from uganda or prvabc were used for the negative and positive controls, respectively. nonspecific amplification was excluded by comparing the melting temperature to that of the positive control . real time rt-pcr. real time rt-pcr for zikv was performed using the quantitect probe rt-pcr kit (qiagen) as reported previously . the reaction mixture (total volume, µl) contained . µl of × quantitect probe rt-pcr master mix, . µl of quantitect rt mix, pmol each of primers and c, and pmol fam-labelled probe for zikv. then, aliquots of the rna samples ( µl) were added to the -µl reaction mixtures. each reaction was performed using the real-time pcr system (applied biosystems, tokyo, japan) with a thermal cycle profile consisting of °c for min, °c for min, followed by cycles of °c for s and °c for min. cut-off values were set at ct . . to quantify viral rna, a standard curve, generated with -fold serial dilutions of synthesised standard rna from uganda or prvabc , was used. table except zikv was quantified by droplet digital pcr (ddpcr). the complementary dna (cdna) of each arbovirus rna was synthesised from an extracted rna stock using the superscript iii first-strand synthesis system (invitrogen) with forward primer for rvfv and reverse primers for denv, wnv, yfv, and chikv, respectively (supplementary table ). the primers used for ddpcr were designed using primer (supplementary table ). all -μl ddpcr mixtures contained × evagreen ddpcr supermix (bio-rad, hercules, ca, usa), . μm forward and reverse primers, and μl of cdna. each oil compartment of the droplet generator dg cartridge (bio-rad) was filled with μl of droplet generation oil for evagreen (bio-rad), and approximately , droplets were generated in each well by the qx droplet generator (bio-rad). the reactions were performed in a -μl droplet emulsion using a geneamp pcr system (applied biosystems) under the following thermal cycling conditions: °c for min, followed by cycles of °c for s and °c for min, with a final step at °c for min. controls without the template were used to monitor for signals from contamination or primer-dimer formation. the cycled droplets were read individually using the qx droplet reader (bio-rad) and analysed with quantasoft droplet reader software (bio-rad). clinical specimens. peripheral blood and urine samples were obtained from patients between and year old with suspected arbovirus infection, who presented with fever, rash, and/or arthralgia symptoms. venous whole blood samples were collected in one vacuette ® z serum separator clot activator and two vacuette ® edta tubes (greiner bio-one, kremsmünster, austria). to one edta tube, rnalater (thermo fisher scientific) was added at half the volume of the collected blood samples to prevent rna degradation during transport. in total, plasma/serum and urine samples from patients with suspected arbovirus infection, including paired samples from cases, were used in this study. the separated plasma or serum samples and urine samples were stored at − °c until use. rnas were extracted from sera and urine using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions. rna samples were eluted with µl of elution buffer and stored at − °c until use. ethical declaration. this study was approved by the ccs-ufpe ethical committee (caae: . . . ) and all patients gave informed consent. whole blood and urine samples were collected as part of an education and follow-up campaign for arboviruses and cardiovascular diseases conducted by lika in the states of paraíba and pernambuco, brazil in february-july . all experiments were 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anti-dengue immunoassays in patients with acute zika virus infection loop-mediated isothermal amplification of dna development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus loop-mediated isothermal amplification (lamp): principle, features, and future prospects deployment of a reverse transcription loop-mediated isothermal amplification test for ebola virus surveillance in remote areas in guinea development and evaluation of reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay coupled with a portable device for rapid diagnosis of ebola virus disease in guinea complete genome sequences of three historically important, spatiotemporally distinct, and genetically divergent strains of zika virus: mr- , p - , and prvabc- rapid and sensitive detection of zika virus by reverse transcription loop-mediated isothermal amplification attomolar zika virus oligonucleotide detection based on loop-mediated isothermal amplification and ac susceptometry instrument-free point-of-care molecular detection of zika virus simple and highly sensitive molecular diagnosis of zika virus by lateral flow assays investigation into an outbreak of dengue-like illness in pernambuco, brazil, revealed a cocirculation of zika, chikungunya, and dengue virus type zika virus infections imported to italy: clinical, immunological and virological findings, and public health implications zika virus in asia pan american health organization and world health organization. zika -epidemiological update the authors would like to thank sayaka okada, shota koyano and olamide k. oloniniyi for technical assistance with the experiments at nagasaki university, renato p. melo neto and carlos henrique m. castelletti for bioinformatics support at lika, and all members of the staff for their hospitality during the visit when the main results of this paper were obtained. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -mena g authors: chen, jiajia; wu, jie; hao, shaorui; yang, meifang; lu, xiaoqing; chen, xiaoxiao; li, lanjuan title: long term outcomes in survivors of epidemic influenza a (h n ) virus infection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: mena g patients who survive influenza a (h n ) virus infection are at risk of physical and psychological complications of lung injury and multi-organ dysfunction. however, there were no prospectively individualized assessments of physiological, functional and quality-of-life measures after hospital discharge. the current study aims to assess the main determinants of functional disability of these patients during the follow-up. fifty-six influenza a (h n ) survivors were investigated during the -year after discharge from the hospital. results show interstitial change and fibrosis on pulmonary imaging remained months after hospital discharge. both ventilation and diffusion dysfunction improved, but restrictive and obstructive patterns on ventilation function test persisted throughout the follow-up period. for patients with acute respiratory distress syndrome lung functions improved faster during the first six months. role-physical and role-emotional domains in the -item short-form health survey were worse than those of a sex- and age-matched general population group. the quality of life of survivors with ards was lower than those with no ards. our findings suggest that pulmonary function and imaging findings improved during the first months especially for those with ards, however long-term lung disability and psychological impairment in h n survivors persisted at years after discharge from the hospital. chest radiography. chest radiography indicated ground-glass opacities and consolidation at the onset of disease, with the exception of . % ( / ) that showed minor changes. radiologic changes included linear fibrosis, isolated areas of pleural thickening, and small bullous cysts on cct at months. at months after discharge from the hospital, all patients showed improvement on cct; however, no marked change was evident after months. at the -month follow-up, . % ( / ) of patients were proximally normal, . % ( / ) had fibrosis and . % ( / ) had parenchymal opacification including ground-glass opacities (ggo) and reticular patterns. imaging abnormalities including bronchiectasis (n = ; . %), pneumatocele (n = ; . %), small bullous cysts (n = ; . %), nodules (n = ; . %), and pleural thickening (n = ; . %) were also identified. the radiologic findings of a -year-old female patient with hypertension were monitored from admission until the -month follow-up visit (fig. ) . lung function. forty seven of them were included in the analysis of the index of lung function. of the patients, were diagnosed with ards. their first visit's clinical and laboratory features were compared and summarized in table . the proportion of female gender was similar between the patients with ards and those without ards. however, the patients with ards were significantly older than those without ards. similarly, patients with ards had higher reported acute physiology and chronic health evaluation ii scores (apach ii) than the patients without ards. ards patients tended to stay longer in the hospital than non-ards patients. overall, lung function at the -month visit was better in patients without ards than in those with ards. both ventilation and diffusion dysfunction persisted throughout the follow up. the percentage of ventilation dysfunction in patients decreased from the first visit to the -month follow-up visit. the influence of ards on lung function during follow-up. the mean and % ci of parameters of lung function over time are plotted in fig. . estimated longitudinal effects on lung function from the mixed-effects regression models are shown in table . we observed general increases in forced expiratory volume in one second (fev ), dlco and forced vital capacity (fvc) for patients regardless of ards status. however, patients without ards consistently achieved higher fev , dlco and fvc scores over the study period (fig. , table ). the ratio of forced expiratory volume in one second to forced vital capacity score (fev /fvc) declined over the follow-up period, and was higher in ards patients than patients without ards (fig. , table ). the estimated improvement in fev for ards patients was . (p = . ), . (p < . ), . (p < . ), and . (p < . ) at the -, -, -and -month follow-up assessments compared to -month follow-up, respectively. for non-ards patients, the estimated improvement in fev was smaller ( table ) . the results were similar for other measures, such as fvc, dlco, and fev /fev. quality of life. the scores for all domains of the sf- did not change significantly from to months after discharge from the hospital. because the patients were residents in and near hangzhou, so we chose the sf- results of the residents in hangzhou as the control surveyed by wang, li et al. . the scores for role-physical (rp) and role-emotional (re) domains were significantly lower than those of the control population during the first year . rp remained lower than that of the controls, but there was no difference in re at the -month follow-up. social functioning (sf) and body pain (bp) were both lower than those of the controls; a significant difference was detected in the former at the -month follow-up and in the latter at the -and -month follow-up visits ( table ). the mean and % ci of parameters of quality of life over time are plotted in fig. . generally, patients with no ards reported higher scores on all the domains of quality of life except for re, which were comparable between patients with ards and patients without ards across the study period. hospitalized patients with h n virus infection usually present with fever and cough, with early sputum production, and the illness progresses rapidly to severe pneumonia, moderate-to-severe ards, and shock. the development of refractory hypoxemia is the usual cause of death . however, there are no previous reports on the quality of life of h n patients after hospital discharge. our study found that more than half of the survivors of h n virus infection had respiratory tract manifestations after discharge from the hospital. most symptoms improved within month (data not shown). six months after discharge, more than % of patients had returned to work, and the percentage of abnormal dlco was lowest. psychological impairment persisted throughout the follow-up period. all survivors were found to have lung involvement on hrct images, possibly due to diffuse alveolar damage with proteinaceous exudates, occasional cytomegaly, and intra-alveolar hemorrhage . imaging showed improvement in inflammation over time, especially during the first months after hospital discharge. however, no further significant changes in interstitial fibrosis or ground-glass opacities were detected at the -and -month visits. an autopsy study of patients with h n infection suggested that lung histology varied according to the duration of illness. after acute diffuse alveolar damage, post-inflammatory changes such as pulmonary pneumocyte hyperplasia and parenchyma fibroproliferation occurred during the later course of the disease . we speculate that changes during the -month convalescence period are irreversible. absorption occurred slowly and was coincident with clinical symptoms. in survivors of h n virus infection, radiologic abnormalities including ground-glass opacities with a reticular pattern remained evident at the -month follow-up visit . moreover, in a study of the long-term outcomes of pandemic h n -associated severe ards, the patients also had abnormal imaging findings, with mildly distorted septal lines, parenchymal bands, pneumatocele and distal bronchiectasis, at year post-icu discharge . at the -month visit, ground-glass opacities were evident in . % of patients . these features are generally similar to those of survivors of h n infection in this study. fibrosis ( . %) and parenchymal pacifications ( . %), which paralleled lung dysfunction, were common at the -year visit. parenchymal pacifications were more sensitive than ct imaging in the evaluation of fibrotic changes . pulmonary function has been reported to be near normal, with the exception of decreased diffusion capacity, in h n patients . in our study, approximately half of the survivors had ventilation dysfunction at months. hybrid patterns and restrictive ventilation dysfunction accounted for most types of dysfunction, which may be caused by muscle weakness and fatigue . . % of patients exhibited decreased dlco levels at the -year follow-up visit, which was higher than reported previously [ ] [ ] [ ] [ ] . the overall pattern of lung function impairment suggests impairment in the small airways and the alveolar diffusion pathway. furthermore, patients with ards had larger lung function changes at each follow-up time. the improvement between month and months after discharge was larger than the improvement between months and months, as was previously reported for ards . for example, patients with ards achieved . units of improvement in fev within months, but have only . units of improvement in the next months. a study of the long-term outcomes of survivors with ards reported a mild restrictive pattern on lung-function testing, with a mild-to-moderate reduction in carbon monoxide diffusion capacity at months; the median dlco improved by % of the predicted value from to months . in our study, the median dlco of the patients with ards improved by . - . % of the predicted value, which is considerably higher than the rates reported previously. these survivors stayed a long period of time in the hospital or icu and suffered from lung injury physically. they also suffered from the fear of death. when they went back home, they not only lacked of activities, but also were isolated by their relatives and neighbors because h n attack made people fear of infection and death. thus survivors have significantly lower hrqol than that of the general population and are likely to have social functioning and mental health deficits . similarly, h n survivors experience persistent hrqol decrements after discharge. thus, the disease affected hrqol mainly in the rp, bp, sf, and re domains compared with normal controls. a meta-analysis showed that recovery in the hrqol of ards survivors occurred during the first months after discharge , but no significant improvement was evident at the -year follow-up in our study. these findings suggest that the quality of life of survivors with ards was lower than that of those without ards. the severity of the diseases may influence the quality of life the patients. to our knowledge, this is the first prospective study of the physical and psychological health status of patients with influenza a(h n ) pneumonia during the convalescent period. this study had several limitations. first, most h n infections occurred in china between and the present. this was a single-center study involving a limited number of patients over a -year period in zhejiang province, china. second, follow-up visits were offered to all patients discharged from the hospital, but some refused to attend and some did not complete follow up. the follow-up rates for lft were %, %, % and % and those for hrqol were %, %, % and % at , , and months, respectively. although many indices did not change significantly after year, the study population may not be representative of the entire population of h n survivors. third, this was a prospective study on the impact of h n on the physical and psychological health of survivors. however, no information on the baseline lung function and quality of life of these patients was available. although some patients may have underlying pulmonary diseases, most of them received the medicine without further examination. so we cannot compare the index of lung function before and post infection of h n . in particular, this group of patients had pre-existing conditions, which may also have affected the hrqol results. patients who had suffered acute pathologies reported significant decreases in quality of life, whereas other patients with pre-existing conditions reported significant improvements in terms of reduced bp and improved mh, vt and sf scores . in our study, h n survivors had significantly higher vt and mh scores than the population norms. thus, those scores may have been higher at baseline, i.e., prior to admission. finally, after discharge from the hospital, there was no significant improvement; however, whether improvements in physical and mental health would have been detected had the follow-up duration been longer is unknown. thus, further expanded research is needed. in summary, long-term lung disability and psychological impairment in h n survivors persisted at years after discharge from the hospital. pulmonary function and imaging findings improved during the first months especially for those with ards. most survivors returned to work, but at the -year follow-up, more than half of survivors still had ventilation and blood-gas diffusion dysfunction. the h n survivors had impaired hrqol scores that were lower than those of a sex-and age-matched control population, and ards substantially influenced these scores. follow-up protocol. patients were evaluated in clinics at , , , and months after their discharge from the hospital. at each visit, computed tomography of the chest (cct) and lung function tests (lft) were performed. the -item short-form health survey (sf- ) (chinese version) of the medical outcome study assessing health-related quality of life (hrqol) was completed. patients who declined the face-to-face interview were telephoned to obtain survival information. statistical analysis. patients' characteristics were summarized with means ± standard deviation (m ± sd) for continuous variables or with frequency and proportion for categorical variables. baseline differences in ards status were assessed using student's t tests, fisher's exact tests or chi-square test, whenever is applicable. we plotted the means of lung function and quality of life and the corresponding % confidence intervals (cis) over time to graphically examine the changes in outcomes over time. we estimated mixed-effect models to fit lung function with patients' ards status as the main effect, visit ( , -, -, -, or -month follow-up), and the ards status-by-visit interaction. the models also included a first-order autoregressive covariance structure to account for repeated measures within each patient. we also assessed the estimated difference in the outcome measures at the -, -, -, or -month follow-up visits compared to those at -month visit according to ards status through model contrast. the estimated change in lung function relative to -month visit was assessed. one sample t tests were used to compare sf- scores at the -, -, -, or -month follow-up visits with that of the control group. data availability. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. ethical approval and informed consent. the study design was approved by the human ethics committee of the first affiliated hospital, school of medicine, zhejiang university. the methods were carried out in accordance with the relevant guidelines and regulations. informed consent was obtained from each patient included in the study. human infection with a novel avian-origin influenza a (h n ) virus clinical findings in cases of influenza a (h n ) virus infection human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome assessing change in avian influenza a(h n ) virus infections during the fourth epidemic -china clinical features of pneumonia caused by influenza a(h n ) virus in beijing clinical features of human influenza a (h n ) infection in vietnam cumulative number of confirmed human cases for avian influenza a(h n ) reported to who development and psychometric tests of a chinese version of the sf- health survey scales the research on quality of life of civil residence in hangzhou with chinese version of the sf- questionaire. chinese journal of preventive medicine radiological features of lung changes caused by avian influenza subtype a h n virus: report of two severe adult cases with regular follow-up long-term outcomes of pandemic influenza a(h n )-associated severe ards follow-up study on pulmonary function and lung radiographic changes in rehabilitating severe acute respiratory syndrome patients after discharge one-year outcomes in survivors of the acute respiratory distress syndrome -year pulmonary function and health status in survivors of severe acute respiratory syndrome impact of severe acute respiratory syndrome (sars) on pulmonary function, functional capacity and quality of life in a cohort of survivors pulmonary function and exercise gas exchange in survivors of adult respiratory distress syndrome of survivors after the first outbreak of human infections with avian influenza a(h n ) virus in shanghai, china recovery of function in survivors of the acute respiratory distress syndrome health-related quality of life after acute lung injury quality of life after acute respiratory distress syndrome: a meta-analysis changes in quality of life after intensive care: comparison with normal data all authors contributed to the interpretation of results and approving the decision to submit the article for publication. l.j. li designed the study. j.j. chen, s.r. hao, m.f. yang were investigators in this study. x.q. lu, x.x. chen and j.j. chen collected the data. j.j. chen prepared the first draft of the article and j. wu completed the data analysis. all authors reviewed the manuscript. competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -yzan du authors: ren, rongrong; wu, shuxian; cai, jialin; yang, yuqin; ren, xiaonan; feng, yanling; chen, lixiang; qin, boyin; xu, chunhua; yang, hua; song, zhigang; tian, di; hu, yunwen; zhou, xiaohui; meng, guangxun title: the h n influenza a virus infection results in lethal inflammation in the mammalian host via the nlrp -caspase- inflammasome date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: yzan du the avian origin influenza a virus (iav) h n has caused a considerable number of human infections associated with high rates of death since its emergence in . as a vital component of the host innate immune system, the nucleotide-binding domain leucine-rich repeat containing receptor, pyrin domain containing (nlrp ) inflammasome plays a critical role against h n viral infection. however, the function of nlrp inflammasome in host immunological responses to the lethal h n virus is still obscure. here, we demonstrated that mice deficient for nlrp inflammasome components, including nlrp , caspase- , and apoptosis-associated speck-like protein containing a card (asc), were less susceptible to h n viral challenge than wild type (wt) controls. inflammasome deficiency in these animals led to significantly milder mortality and less pulmonary inflammation compared with wt mice. furthermore, il- receptor deficient mice also exhibited a higher survival rate than wt controls. thus, our study reveals that the nlrp inflammasome is deleterious for the host during h n infection in mice, which is due to an overwhelming inflammatory response via caspase- activation and associated il- signal. therefore, fine-tuning the activity of nlrp inflammasome or il- signaling may be beneficial for the host to control h n associated lethal pathogenesis. in march , the novel avian-origin influenza a virus (iav) h n was identified in china. h n exhibits low pathogenicity in birds, but has an ability to bind certain mammalian cell receptors, and replicates at temperature close to the normal body temperature of mammals [ ] [ ] [ ] . the infected patients often develop severe symptoms, including pneumonia and acute respiratory distress syndrome (ards) . the h n infection had caused confirmed human cases, with deaths by february [data from fao (food and agriculture organization of the united nations)]. the severe symptoms and high mortality associated with h n infection pose a big threat to the public health. although recent studies of h n have achieved great progress, clear definition of the host immunological responses to h n is still lacking , , . influenza virus infection mainly induces two types of innate immune responses . a rapid antiviral response, i.e. type i interferon production, to facilitate intracellular control of viral replication; and a proinflammatory response characterized by secretion of cytokines and chemokines, which promotes recruitment of various immune cells for viral clearance . as a key mediator of inflammation, the proinflammatory cytokine il- β plays a crucial role in the pathogenesis of various diseases . the maturation and secretion of il- β require a protein figure . h n rna activates the nlrp inflammasome in bmdcs. (a) bmdcs were primed with ng/ml lps and transfected with different dose of h n rna for hours, the supernatant were harvest and the levels of il- β and tnf-α were measured by elisa. (b) bmdcs from wild type and nlrp −/− , asc −/− , casp / −/− mice were primed as in (a) and transfected with . ug/ml h n rna for hours, supernatant were collected for elisa as indicated. (c) supernatants or cell lysates from h n rna-stimulated bmdcs were harvested and the expression of proteins was analysed by immunobloting. the upper lanes, marked as pellet, showed the complex called inflammasome, which activates caspase- to cleave proil- β to its mature form . more importantly, caspase- and the nlrp inflammasome have been found essential for protecting mice against h n iav infection [ ] [ ] [ ] . in a previous study, we have demonstrated that h n influenza virus isolated from human patients could establish successful infection of mice , . employing this murine model, in the current study we demonstrate a detrimental role for the nlrp -caspase- inflammasome and associated il- signal in h n infection of mice. our data suggest that dampening the nlrp inflammasome activity or inhibiting il- signaling should be beneficial for the host during a lethal h n challenge. h n rna induces nlrp inflammasome activation. it has been reported that h n rna plays a critical role in activating the nlrp inflammasome . to assess whether the nlrp inflammasome could be activated during h n challenge, we transfected lps-primed bmdcs (bone marrow derived dendritic cells) with h n rna and measured the production of il- β. as expected, robust il- β secretion was detected in the h n rna-transfected samples, while the production of an inflammasome-independent cytokine tnf-α was unaltered (fig. a) , suggesting that h n rna-induced il- β production was likely dependent on inflammasome activity. to ensure the role of inflammasome in h n rna-mediated induction of il- β, we generated bmdcs from nlrp −/− , asc −/− and casp / −/− mice. transfection of lps-primed bmdcs from such mice led to significantly compromised il- β secretion compared to wild type controls (fig. b) . and western blotting revealed that caspase- activation and asc oligomerization were also decreased in bmdcs from nlrp −/− and asc −/− mice (fig. c) , which might account for the compromised il- β production. thus, our results suggest that h n rna activates caspase- via the nlrp inflammasome in mouse dendritic cells. deficiency of nlrp or caspase- protects mice against h n infection associated morbidity and mortality. next, we investigated the function of nlrp inflammasome during h n infection in vivo. to this end, age and gender matched wt, nlrp −/− and casp / −/− mice were intranasally (i.n.) infected with h n virus as described . we found that the nlrp −/− mice survived significantly better than the wt controls (fig. a) . consistently, the mortality of casp / −/− mice was also significantly lower compared to wt controls days post infection (d.p.i) (fig. b) . in terms of body weight changes, nlrp −/− mice had less weight loss compared to wt controls (p < . at , , d.p.i) (fig. c) . moreover, wt mice lost significantly more body weights than casp / −/− mice from to d.p.i (p < . at d.p.i and p < . at other time points) (fig. d) . however, on d.p.i the survived wt mice began to recover quicker than casp / −/− mice. the gain of body weight started at d.p.i for wt but d.p.i. for casp / −/− mice. by d.p.i, wt mice returned to the initial level of body weight but the casp / −/− mice did not (fig. d) . notably, no significant difference for h n viral quantity in the lungs was observed between the infected nlrp −/− or casp / −/− vs. wt mice (fig. e ,f). deficiency of nlrp or caspase- decreases pulmonary inflammation during h n challenge. to study the pathological changes in the h n infected nlrp −/− , casp / −/− and wt mice, lung tissues were collected and h&e stained samples were examined by microscopy. on d.p.i, infiltration of inflammatory cells was observed in the infected lungs of all strains of mice. there was an obvious thickening of alveolar septum, destruction of partial alveolar structure, as well as pulmonary septal rupture ( fig. a and c) . however, infiltration of inflammatory cells to the lungs of wt mice was much more severe than that in nlrp −/− or casp / −/− mice, especially in the bronchus. in addition, the destruction of alveolar structure in the lungs of nlrp −/− and casp / −/− mice was weaker than that in wt mice ( fig. a and c) . on d.p.i, all strains of mice showed more severe infiltration of inflammatory cells to the lungs comparing to that on d.p.i ( fig. b and c) . nonetheless, the pulmonary pathology of nlrp −/− and casp / −/− mice was still milder than that in wt mice on d.p.i ( fig. b and c) . collectively, these results suggested a role for nlrp and caspase- in promoting pathological changes in the lungs of mice infected with h n . caspase- mediates the recruitment of proinflammatory leukocytes to the lungs during h n infection. caspase- is the critical executor of inflammasome activation mediating the cleavage of proil- β. therefore, we further studied the impact of caspase- deficiency on the infiltration of proinflammatory leukocytes to the lungs after h n infection. the single-cell suspensions, prepared from the bronchoalveolar lavage fluid (balf) of h n infected wt or casp / −/− mice on , and days post infection were stained with anti -cd b, -f / , -cd c, -ly c, -ly g, -cd and -b antibodies conjugated to different fluorescent conjugates, and analyzed through flow cytometry. in both wt and casp / −/− mice, the percentages of leukocytes expressing cd b, f / , cd c, ly c and ly g peaked around d.p.i, then declined ( fig. a-e) . the percentages of f / ( . % ± . %) and cd c ( . % ± . %) positive cells in the wt balf samples were significantly higher than that in casp / −/− balf samples (f / : . % ± . %; cd c: . % ± . %) on d.p.i. meanwhile, the mean fluorescence intensities (mfis) of cd b ( , ± , ), f / ( , ± , ) and cd c ( , ± , ) in wt balf cells were significantly increased compared with that of casp / −/− mice (cd b: . ± . ; f / : . ± . ; cd c: . ± . ) on d.p.i (fig. a-c) . for ly c and ly g, albeit there was no statistical difference between the wt and casp / −/− mice, the percentage was at least more than two folds higher in the wt mice than that in casp / −/− mice; and the differences of the mfis in those two oligomerization of asc. the right panel indicated the relative density of the immunoblotting. the full-length gels were displayed in supplementary figure . values represent the mean of triplicate samples ± sd. data are representative of three independent experiments. *p < . . scientific reports | : | doi: . /s - - - groups were even bigger on d.p.i ( fig. d and e). for lymphocytes, cd positive cells and b positive cells showed no significant differences between wt and casp / −/− mice on both d.p.i and d.p.i ( fig. f and g) . thus, deficiency of caspase- reduced the proinflammatory leukocytes recruitment to the lungs upon h n iav challenge. it is reported that the caspase- , activated by inflammasome, plays a critical role in murine proinflammatory responses to iav infection , , . to investigate the role of caspase- in h n infection related inflammation, cytokines and chemokines in the supernatants of lung homogenates were analyzed (fig. ). in casp / −/− lung tissues, the levels of il- β ( . ± . pg/g tissue) and il- ( . ± . ng/g tissue) were significantly lower than those in wt tissues (il- β: . ± . pg/g; il- : . ± . ng/g, respectively) on d.p.i (p < . ) (fig. a) . other cytokines such as il- / p , il- α, ifn-γ and ifn-β also exhibited decreased level in casp / −/− lung tissues compared with wt control on either d.p.i or d.p.i, although the differences were not significant (fig. a) . the level of il- fluctuates and was lower in the lungs from casp / −/− mice on d.p.i, but higher on d.p.i compared to wt counterparts, without significant differences in both cases (fig. a) . other cytokines such as il- , tnf-α and il- did not show significant differences between tissues from casp / −/− and wt mice (fig. a) . as for the detected chemokines, mip- α was significantly lower in casp / −/− lung tissues than that in wt control on d.p.i (p < . ). the levels of other chemokines, including mip- β, ip- , cxcl , ccl and gm-csf were just slightly lower in the lungs of casp / −/− mice than that in wt counterparts on either d.p.i or d.p.i. therefore, caspase- was involved for an optimal production of il- β, il- as well as mip- α in the lung in response to h n challenge. to note, the production of above mentioned cytokines and chemokines from nlrp −/− mice showed a similar pattern as that from casp / −/− mice (fig. b) , indicating that deficiency of nlrp inflammasome genes reduced the secretion of several critical proinflammatory mediators in the lung of mice upon h n iav challenge. in addition, the production of these cytokines and chemokines in the serum of nlrp −/− , casp / −/− and wt mice showed a similar trend with that from the lung homogenates ( supplementary fig. s ). asc and il- receptor mediated signal promoted mortality and weight loss of mice after h n infection. the adaptor protein asc plays a vital role in the nlrp inflammasome formation, which is required for caspase- activation and maturation of il- β. the mature il- β binds to il- receptor , the functional receptor of il- β, induces downstream signal transduction and executes inflammatory responses. thus, we set out to determine whether asc and il- r play a role similar as that of nlrp and caspase- during h n iav infection. to this end, we infected asc −/− and il r −/− mice with h n virus and monitored their weight change and mortality daily. our results demonstrated that the mortality of asc −/− and il r −/− mice was lower compared with that of the wt mice (fig. a) . the wt mice also suffered more weight loss than these knock-out animals (from to d.p.i) after h n challenge. of note, the difference for body weight loss between wt and asc −/− mice was statistically significant (p < . ) on and d.p.i. (fig. b ). to analyze the pulmonary inflammation of asc −/− and il r −/− mice, lung tissues of these mice were harvested for h&e staining and microscopy. on d.p.i, more inflammatory cells were recruited to the alveoli and blood vessels in the lungs of wt mice compared with that of the asc −/− or il r −/− mice, and more inflammatory cells infiltrated into the bronchus resulting in a local blockade (fig. a and c) . moreover, the pulmonary exudation was even milder in the il r −/− mice compared with wt mice (fig. a and c) . on d.p.i, all mice suffered more severe inflammation than that observed on d.p.i. furthermore, there were more inflammatory cells infiltrated in the alveoli of wt mice than in asc −/− or il r −/− mice (fig. b and c) . moreover, the damage in the lung of asc −/− mice was milder compared with that in wt mice, and there was less inflammatory exudation in il r −/− mice than in wt controls ( fig. b and c) . therefore, our results suggest that signals mediated by asc and il- receptor lead to more severe disease upon h n iav challenge. aberrant and excessive inflammation during h n viral infection is linked to severe morbidity and mortality in human patients . a recent study demonstrated the histological distribution of inflammation-related genes expression in the lungs of balb/c mice infected with h n influenza a virus . significantly higher expression levels of nlrp , rip , il- β and tnf-α in the lung of h n -infected mice were noted, indicating possible role of these molecules in driving lung pathogenesis . for example, the involvement of tnf-α activated signaling pathway during influenza virus infection was clearly demonstrated , . however, the exact roles of other molecules involved in inflammatory response during h n challenged remained elusive. specifically, the role of inflammasome signaling had not been defined before our present work. here, using the gene knockout mice we demonstrated that activation of the nlrp inflammasome was detrimental to the host upon h n iav infection. we noted that wt mice lost more body weight than casp / −/− , asc −/− and nlrp −/− mice and their overall survival rate was lower during h n infection (figs and ) . pathology analysis showed that there were inflammatory responses in the infected lungs of all strains of mice, both at d.p.i and at d.p.i; and the pulmonary inflammations at d.p.i. were more severe than that at d.p.i.; moreover, the pulmonary inflammations of the wt mice were stronger than casp / −/− , asc −/− and nlrp −/− mice (figs and ) . flow cytometric analysis showed that inflammatory cells has started to infiltrate into the alveolar spaces in the balf at d.p.i. it seemed that cd b + or f / + (high-expressed on microphages) or cd c + (high-expressed on dendritic cells) cells in the balf of wt mice were significantly more than those in casp / −/− mice at d.p.i. there were also ly c + (expressed on monocytes), ly g + (high-expressed on neutrophils) and cd + (expressed on t cells) in the balf of wt mice exhibited higher level, though it was not significant differences (fig. ) . however, except the cd b + cells were retained and b + (expressed on b lymphocytes) cells were increased, the percentage of the most infiltrated cells were decreased in the balf at d.p.i. (fig. ) , which seem to be "contrast" to the severer pulmonary inflammation pathology observation at d.p.i. (figs and ) . . a) , and macrophages/dendritic cells and a few other cells started to be recruited to the infected area by chemokine mip- α (figs a-c and a) . the production of il- β was still at low level at this stage (fig. a) . with the development of disease, at d.p.i., severe inflammation occurred in the infected lung tissue (fig. b) . with the migration of the lymphocytes, especially b cells, to the inflammatory area (fig. g) , the total number of infiltrated inflammatory cells were increased but the percentage of monocytes, macrophages, dendritic cells were decreased ( supplementary fig. s , fig. ) . the infiltrated inflammatory cells produced large amount of il- β (fig. ) , which accelerated the pulmonary inflammation. from d.p.i. to d.p.i, the body weight of survived wt mice recovered completely, whereas casp / −/− mice could not returned to the initial level ( d.p.i) (fig. d) . microscopic analyses revealed that on d.p.i, most tissues from wt mice exhibited complete recovery, while the casp / −/− tissues still manifested strong inflammation (data not shown). thus, wt mice suffered more due to the lung damage and inflammation in the early phase (before d.p.i) of h n infection and either died of organ failure, or recovered at the later phase. in contrast, casp / −/− mice did not develop as strong inflammation and lung damage as seen in wt counterparts in the early phase, consequently fewer of them died. however, casp / −/− mice failed to recover completely at the later phase. these results might suggest a potentially dual role for caspase- in h n infection, detrimental role to the host upon h n infection before d.p.i. and possibly necessary role for recovery from the lung inflammation after d.p.i. phase, likely due to a contribution to tissue regeneration. but it should be kept in mind that the comparison between % surviving wt and % surviving casp / −/− is not justified, and more stringent experiments are needed for further explorations on the role of caspase- to host recovery. studies on the role of nlrp -caspase- inflammasome during iav infection produced conflicting results. some studies have demonstrated that nlrp -caspase- inflammasome is protective against h n infection through viral clearance or virus induced pulmonary necrosis , . inflammation is essential to protect host from infection, but can also cause tissue damage or dysfunction of important organs and lead to death . dysregulation of the proinflammatory response may result in a "cytokine storm" that contributes to a severe viral pneumonia and serious complications in the lung . the highly pathogenic avian influenza virus h n causes hypercytokinemia and severe tissue damage . it is reported that production of proinflammatory cytokines and chemokines, including il- and ip- , is remarkably increased in h n infected patients , , and h n infection associated inflammation in the lungs resulted in rapidly progressive pneumonia and development of ards in the majority of hospitalized patients , . in addition, the h n iav infected patients who progressed severe lung injury have elevated levels of ccl , ip- , cxcl and il- compared with the seasonal h n infected patients . therefore, for the highly pathogenic influenza virus the hyperactivated proinflammatory responses may be detrimental for the host. moreover, a recent study demonstrates that nlrp inflammasome plays both a protective and detrimental role during h n and h n infection of mice depending on the disease phase . in our study, we found the inflammasome activation was deleterious in the case of h n infection, possibly due to overwhelming and fatal acute inflammation in the lungs following h n infection. pathology and infiltrating cellular analyses as well as cytokine and chemokine analyses all supported this possibility (figs - ) . pathology results revealed that on d.p.i, there were more inflammatory cells around the blood vessels and bronchus in wt mice compared with casp / −/− mice (fig. ) . the flow cytometric analyses further demonstrated the abundance of inflammatory cells in the balf of wt mice compared to casp / −/− counterparts (fig. ) , which were consistent with the observed pulmonary pathological changes. both casp / −/− and nlrp −/− mice produced relatively less level of ifn-β in the lung compared with control mice. although no significant difference was noted, these data indicated a possible role for the nlrp -caspase- inflammasome signaling in affecting the type i interferon production. theoretically, type i interferon response plays a role in limiting viral replication, however, we did not observe any significant difference in viral load between casp / −/− or nlrp −/− versus wt mice. it is likely that the h n could employ strategies to counteract the generation and function of type i interferon. chemokines are the driving factors promoting the recruitment of inflammatory cells. mip- α (ccl ) can activate granulocytes and lead to acute neutrophilic inflammation. it also induces the synthesis and release of il- β, il- and tnf-α from fibroblasts and macrophages . mip- α level is increased in h n and h n infected mice, but not in h n infected animals . we also noticed a clear increase of mip- α in wt mice upon h n infection (fig. ) . interestingly, this elevation was abrogated in the early phase ( d.p.i) in the absence of caspase- (fig. ) . we reasoned that the mip- α production may be, directly or indirectly, influenced by nlrp or caspase- -dependent il- β and/or il- secretion. il- β showed significant reductions in nlrp −/− and casp / −/− mice compared with wt on d.p.i (fig. ). il- β is a critical proinflammatory cytokine that is increased during infections with h n and h n influenza viruses . il- β also mediates the recruitment of monocytes and neutrophils into the lung . deficiency of il- β or its receptors manifests different responses to respiratory challenge with influenza virus [ ] [ ] [ ] . hence, the function of il- β in controlling iav infections remains obscure. our data showed that il r −/− mice exhibited attenuated tissue pathogenesis upon h n infection (figs and ) . during the preparation of this manuscript, we noticed that another group found the pb -f protein derived from h n virus also activates the nlrp inflammasome . probably, the viral particle of h n contains multiple factors such as rna and pb -f that can be detected by the host inflammasome. beside nlrp inflammasome, whether other inflammasomes, such as nlrp , ipaf, aim , could also be involved in h n infection needs to be investigated in further study. in summary, our present work demonstrates that the nlrp -caspase- inflammasome is deleterious for the mammalian host during h n influenza virus infection. therefore, fine-tuning the nlrp -caspase- activity or il- -mediated signaling should be beneficial to control h n -associated lethal pathogenesis. isolation of h n virus was described before and the viral titer was measured via tissue culture infective dose (tcid ) assay , . all experiments related to h n virus were performed in biosafety level laboratories in shanghai public health clinical center (sphcc), and followed the standard operating protocols approved by the institutional biosafety committee at sphcc, fudan university. mice. all mice used in our experiments are on a c bl/ genetic background and all experiments were carried out with age and gender matched mice ( - weeks old, female). c bl/ mice were purchased from the b&k universal group limited (shanghai, china). nlrp deficient mice (a gift from dr. warren strober) and asc deficient mice (a gift from dr. vishva m. dixit) had been described , . caspase- / and il- receptor deficient mice were purchased from the jackson laboratory (bar harbor, me, usa). herein, these mice were described as cells. bone marrow derived dendritic cells (bmdcs) were generated as described . briefly, bone marrow cells were harvest and lysed with rbc lysis buffer, and seeded in cm dish with ml rpmi with gm-csf ( ng/ml). the media were refreshed days later and the cells were harvested for experiments on day . antibodies. antibodies for immunoblotting include: rabbit anti-mouse asc (sc- -r; santa cruz, ca, usa), mouse anti-mouse caspase- (ag- b- ; adipogen, san diego, ca, usa), rabbit anti-mouse mature and pro-il- β (sc- ; santa-cruz, ca, usa) and mouse anti-mouse β-actin (sc- ; santa-cruz, ca, usa). asc oligomerization detection. asc oligomer was prepared as described . brifely, h n rna transfected bmdcs were lysed with lysis buffer [ mmol/l tris (ph . ), mmol/l nacl, % nonidet p- ], pelleted and washed with pbs, and cross-linked with disuccinimidylsuberate (dss) before subjected to immunobloting. mice infection. mice were infected with × tcid of h n virus in a volume of µl intranasally (i.n.) after sevoflurane (hearem, shanghai, china) inhalation anaesthesia, and monitored for clinical signs, body weight changes and physical survival during the days observation period. the infected mice were sacrificed on , or days post infection for collection of samples for analysis as reported . to monitor the body weight changes and survival rate, to mice were used in each group (n = ~ ). for the rest analysis of murine samples, more than mice were used in each group. ++ some blood vessels are cuffed by inflammatory cells ++++ most blood vessels are surrounded by a thin layer ( - cells thick) of inflammatory cells ++++++ most blood vessels are surrounded by a thick layer (> cells thick) of inflammatory cells ++ increased numbers of inflammatory cells in alveolar walls are evident ++++ as above, plus one to three foci per section showing alveolar exudate and atelectasis ++++++ as above, plus more than three foci per section showing alveolar exudate and atelectasis lung tissue inflammation ++ some blood vessels are cuffed by inflammatory cells ++++ a part of lung tissue shows consolidation and interstitial fibrous tissue proliferation ++++++ consolidation appears in most areas of the lung ++ some bronchus are cuffed by inflammatory cells ++++ some bronchus exhibit squamous metaplasia and are blocked by inflammatory exudates ++++++ most bronchus exhibit squamous metaplasia and are blocked by inflammatory exudates table . histology scoring criteria. the three levels of inflammatory response for each index, which was designated as ++, ++++ and ++++++, according to the criteria listed in the above table. the milder inflammation was designated with reduced number of "+" as indicated in respective figures. to warrant the objectivity of the histology scoring, two pathologists from different labs read each slide in a double-blinded way and an average value was taken as the final result. origin and diversity of novel avian influenza a h n viruses causing human infection: phylogenetic, structural, and coalescent analyses influenza: pathways to human adaptation amino acids substitutions in the pb protein of h n influenza a viruses are important 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pb -f derived from avian influenza a virus h n induces inflammation via activation of the nlrp inflammasome differential activation of the inflammasome by caspase- adaptors asc and ipaf nitric oxide suppresses nlrp inflammasome activation and protects against lps-induced septic shock internalized cryptococcus neoformans activates the canonical caspase- and the noncanonical caspase- inflammasomes histologic analysis of an immune response in the lung parenchyma of mice. angiopathy accompanies inflammatory cell influx this work was supported by grants from the national major projects for science and technology analysis of cytokines and chemokines. lung homogenate supernatants and serum were collected and stored at − °c before analysis. the levels of cytokines and chemokines in the collected samples were determined quantitatively using a procartaplex multiplex immunoassay kit (ebioscience, viennna, australia) according to the manufacturer's protocol.flow cytometry. bronchoalveolar lavage fluid (balf) was obtained via flushing the lungs three times with . ml/time of pbs. the single-cell suspensions from balf of h n infected mice were first treated with anti-mouse cd /cd antibody (ebioscience, vienna, australia) to block fc receptor, then surface stained with antibodies against b , cd , cd b, cd c, ly c, ly g and f / , and fixed with % paraformaldehyde before analysis with facs array (bd biosciences, franklin lakes, nj, usa). the final graphical output was generated through flowjo software (tree star, inc., ashland, or, usa).extraction of total rna and rt-pcr quantification. total rna was extracted from lung homogenates using a kit (qiagen, hilden, germany) according to the manufacturer's protocol. relative quantification of h n viral load via rt-pcr was conducted as reported previously . calculation was carried out by comparing h n hemagglutinin with gapdh, which served as an internal control gapdh (ΔΔct method).pathology analysis. excised mouse lung tissues were fixed in % paraformaldehyde followed by paraffin embedding and sectioning, then subjected to hematoxylin and eosin (h&e) staining. images were collected by leica dmi microscopy with indicated magnifications.histology scoring. four indexes were analyzed for scoring on each slide: ) blood vessel region inflammation, ) pulmonary alveoli inflammation, ) lung tissue inflammation, ) bronchial inflammation. the detailed criteria were listed in table , which was referred to a previous report supplementary information accompanies this paper at doi: . /s - - - competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - wjvykx authors: liu, chia-lin; hung, hui-chen; lo, shou-chen; chiang, ching-hui; chen, i-jung; hsu, john t.-a.; hou, ming-hon title: using mutagenesis to explore conserved residues in the rna-binding groove of influenza a virus nucleoprotein for antiviral drug development date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: wjvykx nucleoprotein (np) is the most abundant type of rna-binding viral protein in influenza a virus–infected cells and is necessary for viral rna transcription and replication. recent studies demonstrated that influenza np is a valid target for antiviral drug development. the surface of the groove, covered with numerous conserved residues between the head and body domains of influenza a np, plays a crucial role in rna binding. to explore the mechanism by which np binds rna, we performed a series of site-directed mutagenesis in the rna-binding groove, followed by surface plasmon resonance (spr), to characterize the interactions between rna and np. furthermore, a role of y in np stability and np-rna binding was evaluated. the aromatic residue of y was found to stack with a nucleotide base. by interrupting the stacking interaction between y and an rna base, we identified an influenza virus np inhibitor, (e, e)- , -bis( -hydroxy- -methoxyphenyl) - , -heptadiene- , -dione; this inhibitor reduced the np’s rna-binding affinity and hindered viral replication. our findings will be useful for the development of new drugs that disrupt the interaction between rna and viral np in the influenza virus. characterization of rna-binding activity of wild-type and mutant nucleoproteins from h n . the crystal structure of np revealed that the rna-binding groove was located between the head and body domains on the exterior surface of the np oligomers, which is exposed and highly accessible. to examine the effects of conserved positively charged residues and the y residue in the rna-binding groove of np, the amino acid residues y , r , r , r , r , r , k , r , r , r , r , r , and r were respectively replaced with alanine ( fig. ). each form of np was expressed and purified. subsequently, spr analysis was employed to determine the interactions between the full-length nps and rna. the repeated intergenic sequence of influenza virus, ′ -bio(uccaaac) - ′ , was used as a probe in the spr experiments. the dissociation constants, k d (k d /k a ), for the various np and rna complexes were obtained from kinetic analyses of spr experiments (fig. ). the k a values for rna binding to r a and r a were similar to that for wt, whereas y a, r a, r a, r a, r a, r a, k a, r a, r a, r a, and r a had smaller k a values than that of wt, suggesting these np residues are likely important for rna recognition and interaction. the k d values for rna binding to all mutants were larger than that of wt, and the y a mutant had the largest k d value ( fold that of wt), indicating the side chain of y increase rna residence times in np (table ). in addition to k a, r a, and r a, the dissociation constants for rna binding to y a, r a, r a, r a, r a, r a, r a, r a, r a, and r a ranged from . × - m to × - m and were much larger than those for wt ( fig. a) , implying that these residues contribute more to rna binding. previously, li et al. reported that influenza a virus with np mutations in y , r , r , r , r , r , r , r , r , and r were significantly attenuated . these biological properties of mutant viruses corroborate our observations at the biochemical level. one conserved amino acid, y , at the end of the rna-binding groove of the np was identified as an indispensable residue affecting the rna binding . to explore the role of y in the interactions between h n -np and rna, the y residue in the full-length np was replaced by amino acids with various characteristics (e.g., f, r, s, and a) via site-directed mutagenesis. spr analyses were then performed to measure the binding affinities between rna and the purified h n -np (wt and five mutants). traces from the spr experiments show the binding capacity of rna to the wt and mutant nps. comparison of the binding capacities of these proteins (fig. a) showed that the binding capacity was decreased in the following order: wt > y f > y s ~ y r > y a. these results emphasize the importance of the involvement of y in stacking interactions between the np and rna. the kinetic association and dissociation constants were obtained from analyses of the spr sensorgrams and the results are listed in table . as shown in fig. b , the k a values followed the order wt > y r > y f ~ y s > y a, with the k a of y a smaller than that of the wt by . %. on the other hand, the k d values increased in the following order: y a > y s y f > y r > wt (fig. c) with the k d of y a being . fold larger than that of wt. taken together, the k d values for the h n wt and five mutant nps decreased in the following order: y a > y s > y f > y r > wt (fig. d) , implying that the positive charge of y r was also involved in the interaction between the np and rna. circular dichroism spectral analysis of the thermal stability of wt and mutant nucleoproteins. the conformation of h n wt and mutant nps including y f, y s, y r, and y a with and without rna were monitored using cd spectroscopy. as shown in figure s , the cd spectra of these proteins were scanned from to nm at °c. the cd spectra of h n wt and mutant nps showed similar well-structured domains with α -helical and β -sheet secondary structures as well as two negative peaks at approximately and nm. upon addition of rna, the cd spectra of h n wt and mutant nps also showed similar intensities at approximately and nm, suggesting that they possessed a similar secondary structure composition upon rna binding, as compared to h n wt and mutant nps without rna. to examine the thermodynamic stabilities of h n wt and mutant nps including y f, y s, y r, and y a, we measured the melting temperatures (t m ) of the purified wt and mutant nps using cd in which the absorbance at nm was analyzed as a function of temperature (fig. a,b) . the wt np showed a thermal denaturation midpoint at . °c as determined by cd (table ) . however, as temperature increases, the mutant nps unfold more rapidly than the wt np does, as evidenced by a lower t m of approximately . to . °c (from . - . °c), indicating that y contributes significantly to the structural stability of the np. compared to wt and other mutant nps, y f had a lowest t m value. the results indicate that deletion of the tyrosine hydroxyl would significantly reduce the stability of np, suggesting tyrosine hydroxyl is required for proper np folding. we also characterized the stabilizing and structural effects of np bound to rna by cd melting analysis (fig. b ). the wt np-rna complex showed a higher t m value ( °c) than other mutant np-rna complexes ( table ). the t m values of the wt and y f nps increased by . and . °c, respectively, upon the addition of rna (table ) suggesting that a benzyl side chain has an important effect on the stability of the np-rna complex. however, the difference was reduced to . , . and . °c for y r, y s, and y a, respectively, upon addition of rna. according to previous studies , wt rnp complexes extracted from influenza virus were melt around °c in replicate experiments, compared to °c for protein heated in isolation. consistent with the early study, our finding show that rna binding enhance the stability of np within °c and our result would be meaningful. molecular docking studies to identify compounds that target y of h n nucleoprotein. previous studies have reported that the tyrosine residue in nucleocapsid protein is an attractive target for novel antiviral drug development , . thus, a virtual screening was performed to target the y of np. to identify compounds that might interact with the rna-binding site of np at y , we used the libdock docking program to evaluate the candidate ligands binding to np. in the current study, the potential hits with docking scores over were analyzed based on their docking results. we selected seven candidate ligands with high scores (h to h ) for the functional assay based on the energy calculated by molecular docking (table s ). they all contain an aromatic core to stack onto y of the np. in addition, the aromatic core contains hydrogen bondforming moieties to mediate the specific interactions with the np. more importantly, among the potential hits, these seven compounds were readily available. because our result reported that y is important for np stability and folding, the top hits were assayed in an in vitro drug-induced fluorescence change experiment. the purified recombinant np was treated with top hits from the in-house compound collection, then the change of tryptophan fluorescence caused by the compound was used to reflect the np-drug interaction . the four compounds that significantly decreased the np fluorescence by more than % were selected for further characterization (fig. a) . np possesses multiple rna binding sites. compound that targets y in the local area can't inhibit the binding affinity between rna and np absolutely. due to this reason, we determined the binding affinity in the presence of compounds under saturation conditions at protein: compound ratio of : . we further studied the effects of the four compounds (h , h , h , and h ) on the rna-binding capacity of np by spr experiments. only h decreased the rna-binding capacity of np by more than % (fig. b ). we also evaluated the kinetic association (k a ) and dissociation constants (k d ), respectively, and the dissociation rate constant (k d ) to determine the effects of these four compounds on the rna-binding affinity toward np. in the presence of compounds under saturation conditions, the affinity of np bound to rna (fig. a ) for h (k d = . nm) was higher than those for the other compounds. the np exhibited weaker rna binding in the presence of h , with a -fold increase in the dissociation constant. the k d values for h , h , and h were . , . , and . nm, respectively, which are at the same level as the k d without drug (table ). figure a shows that the np fluorescence decreased with increasing h concentration, suggesting that this decrease reflected the interaction of np molecules with h ; this result was derived by a hill plot analysis. assuming that the amount of quenched fluorescence corresponds to the fraction of np bound to h , fitting of the binding curve ( fig. b ) resulted in an unambiguous : stoichiometry for the interaction between h and np with a k d of . × - m. based on the docking results of np bound to h , the methoxyphenyl on the h participates in stacking interactions with the tyr side chains. hydrogen bonds were also formed between r and h (fig. c) . we have analyzed the proposed interactions using in y and r mutants. however, we observed that there is relatively low fl change in y and r mutants upon addition of h by fluorescence titration assay, compared to the wild type, suggesting that h inhibits the rna-binding activity of np by directly interacting with y and r ( figure s ). interacts with np of the influenza a virus, we assessed the effects of h after an early step involved in virus replication. np associates with viral rna to form a helical nucleocapsid after the first h. we performed a time-of-addition experiment to detect the viral rna and viral protein synthesis in virus-infected a cells. different concentrations of h were added to a cells after virus infection (t : h after infection), and culture supernatants were collected to determine the viral rna after one viral replication cycle. the matrix protein (m ) viral rna expression levels during replication were normalized based on the vehicle control (same volume of dmso added at t ). as shown in fig. a , upon h treatment at μm, the m viral rna synthesis in the influenza virus-infected cells was reduced by % at t . when the infected cells were treated with μ m h , a similar phenomenon was observed. h dose-dependently reduced the viral rna synthesis during the later steps of the viral cycle after t . this assay further demonstrated that h inhibited influenza virus replication, with an ic (inhibition concentration at which viral synthesis was reduced by %) of . μ m. we next investigated whether h inhibited viral rna synthesis in virus-infected a cells, which would also reduce viral protein synthesis. the expression level of np in the influenza virus-infected cells as compared to the viral control was reduced by % upon h treatment in the later steps of the viral cycle. as shown in fig. b , there was a > % decrease in np expression. m controls the viral rna levels in the later steps of the viral replication cycle . we also observed the protein expression level of m in the virus infected cells at t upon h treatment. the results suggested that h can slow viral protein synthesis after the early stages of viral replication in virus-infected cells. the cytotoxicity of h in this study was monitored by mts assay (fig. c) ; results indicate that the % cytotoxic concentration (cc ) value was > . μ m. overall, the time-of-addition experiment indicated that h exerts its antiviral effects through rna-binding capacity with np after the viral absorption stage. np is the most abundant rna-binding viral protein in influenza virus-infected cells and is responsible for recognizing rna and forming a filamentous nucleocapsid . it is necessary for viral rna transcription and replication. because influenza is a significant threat to both human and avian populations, understanding the molecular mechanisms governing rnp formation may facilitate better control of influenza virus infections . previous x-ray analyses revealed that folding of the np is essentially conserved across various influenza virus strains, having a crescent-shaped structure with a head domain, body domain, and tail loop region. the area between the body and head domains is rich in conserved basic residues providing a scaffold for rna binding, and the tail loop region is involved in the oligomerization of np . in this study, we investigated the mechanism of influenza virus np bound to single-stranded rna. using spr, we explored the effect of single amino acid substitution at hot spots on the surface of the groove between the head and body domains on the rna-associated properties of np, including the kinetic behavior of np bound to rna, by evaluating the spr association and dissociation phases. the association phase mainly reflects the entry of rna into the np groove, whereas the dissociation phase measures the hydrogen bonding environment within the groove that accommodates the rna molecules. the structure of the full-length n protein should be taken into consideration in our studies. moreover, in gel filtration we obtained and checked the uniformity of the purified construct np. in addition, in the spr experiment we designed the nt in length rna oligomer, and the length of rna oligomer can be bound by one protein molecule. during data analysis, we fit the spr sensorgram of the wild and mutant np bound to rna by using : langmuir model. we showed that the dissociation rate of np from the single-stranded rna increased in all mutants, while the presence of y a, r a, r a, r a, r a, r a, r a, r a, r a, and r a mutants significantly decreased the rates of association between np and rna, suggesting these residues play a crucial role in rna binding. interestingly, the k d values of wt, r a, r a, and r a were similar, a finding that is consistent with previous studies showing that the single amino acid substitutions at r a, r a, or r a had no effect on viral-genome replication and transcription . to characterize the interaction of y with rna, tyrosine was mutated to phenylalanine, serine, and arginine. the association rates (k a ) for rna binding to most of the mutants was decreased significantly, except that the y r mutant is similar to that for wt, suggesting that y r may participate in rna-binding of nps via electrostatic interactions. no structural data were available regarding influenza virus np binding to single-stranded rna. thus, to understand the structural interactions responsible for the rna recognition by influenza virus np, we modeled the structure of influenza virus np in an rna-bound state (fig. ) . this model indicates that the rna-binding groove of the np contains y , r , r , r , r , r , k , r , r , r , r , r , and r , which together clamp the rna into groove. y stacks against the first nucleotide of the bound rna to extend the quasi-helix. current antiviral drugs developed to treat influenza virus infections primarily target the viral m channel and neuraminidase. however, the use of m inhibitors, such as amantadine and rimantadine, has been limited by the propensity of these drugs to cause central nervous system side effects and the rapidly increased number of drug-resistant viral strains , . the neuraminidase inhibitors (zanamivir and oseltamivir) are commonly employed to treat influenza virus infections with minimal adverse effects . yet, recent studies have reported that the influenza virus has started to develop resistance to zanamivir and oseltamivir . therefore, novel antiviral strategies are required to combat the drug-resistant influenza viruses. the influenza virus np is a multifunctional rna-binding protein that is associated with genome and antigenome rna, and it is necessary for viral rna transcription and replication . recent studies suggest that np represents a potential anti-influenza drug target because of its many crucial functions during the viral life cycle. on the other hands, there are no np homologous proteins in the cell. antiviral inhibitors targeting np may not act nonspecifically on proteins in the cell, causing host cell toxicity and severe side effects. two strategies to inhibit oligomeric np function have been reported. the first strategy is to impair normal np function by interfering with monomer-oligomer equilibrium through either reported that some small molecules targeting e … r from virtual screening were shown to disrupt the formation of np trimers and inhibit replication of wt and nucleozin-resistant strains . the second strategy is to target the rna-binding site, which contains several conserved residues , . lejal et al. discovered naproxen, which interferes with the rna-binding activity of the influenza virus np and inhibits viral titers . mutation of conserved residues in the rna-binding groove of the influenza virus np led to a significant decrease in its rna-binding affinity and subsequent decrease in viral replication . according to the protein sequence alignment, the -aa np is highly conserved among all strains of influenza viruses. because the location of the np ligand-binding site is highly conserved among influenza viruses, the rna-binding groove of np would be a valid target for broad-spectrum antiviral drugs through interference with the rna-binding activity of the np. competitive binders of np may be employed to combat all strains of influenza virus, including h n , h n , and influenza b. according to our mutational analyses of the influenza virus np, the tyrosine residue on np (tyr ) appears to interact with rna bases via stacking and hydrogen-bonding interactions and to play a crucial role in protein stability. docking results suggest that several hits can bind to y of the np-ntd's groove using virtual screening. using fluorescence titration and an spr assay of the nps, we identified one potential natural compound, curcumin (h ), that targets y of influenza virus np and potently interferes with its rna-binding activity. we also found that h could inhibit influenza virus replication and the efficacy of h is relatively diverse. previous studies demonstrated that curcumin exist no or low cytotoxicity in normal cells . importantly, the safety, tolerability, and nontoxicity of curcumin at high doses have been well established by human clinical trials. therefore, h is an ideal point in designing a new class of inhibitors to interfere with rna-binding activity of np. here, we formulated two general guidelines for developing influenza virus np-y targeting agents. first, a polycyclic aromatic core is required to enable π -π stacking with the tyrosine residues in the np groove. second, introducing hydrogen bond-forming moieties to the aromatic core would mediate the specific interactions with the np. our findings will be useful for the development of new drugs to disrupt the interactions between rna and influenza viral nps. wild-type (wt) and mutant nps encoded by the a/tw/ / (h n ), np genes were amplified with the polymerase chain reaction (pcr) using various primers. the pcr products were digested with ndei and xhoi, and the dna fragments were cloned into pet b (novagen) using t ligase (new england biolabs). dh α competent cells that were transformed with the resultant plasmid were grown in culture. protein expression was induced by supplementing the culture media with mm iptg, followed by incubation at °c for h. after the bacteria were harvested by centrifugation ( × g for min at °c), the bacterial pellets were lysed with lysis buffer in the presence of rnase ( mm tris-buffered solution [ph . ], mm nacl, mm imidazole). the soluble proteins were isolated from the supernatant following centrifugation ( , rpm for min at °c) to remove the precipitate. the recombinant nps carrying an n-terminal × his-tag were purified using a ni-nta column (novagen) with an elution gradient that ranged from to mm imidazole. the pure fractions were collected and dialyzed against the buffer that lacked imidazole. purified nps (with greater than % purity) were analyzed by gel filtration with superdex (ge healthcare) and coomassie blue staining ( figure s a ). the protein concentrations were determined with the bradford method using bio-rad protein assay reagents. surface plasmon resonance binding experiments. the affinity, association, and dissociation between the nps and rna were measured using a biacore a surface plasmon resonance (spr) instrument (pharmacia, uppsala, sweden) equipped with a sa sensor chip from pharmacia. the repeated sequence, ′ -bio(uccaaac) - ′ , was used as a probe in our spr experiments. experiments were conducted by injecting np at different concentrations in mm tris (ph . ) with mm nacl ( figure s b ). we calculated the stoichiometric ratio (s m ) between np and rna, based on the equation, circular dichroism spectroscopy. the circular dichroism (cd) spectra were obtained using a jasco- cd spectropolarimeter. the temperature was controlled by circulating water at the desired temperature in the cell jacket. each protein was dissolved in mm tris-hc (ph . ) and mm nacl. the cd spectra were collected between and nm with a -nm bandwidth at -nm intervals. all of the spectra were obtained from an average of five scans. the photomultiplier absorbance did not exceed v during the analysis. the cd spectra were normalized by subtraction of a background scan with buffer alone or rna alone. the mean residue ellipticity, [θ ], was calculated based on the equation where mrw is the mean residue weight, θ λ is the measured ellipticity in millidegrees at wavelength λ , l is the cuvette path length ( . cm), and c is the protein concentration in g/ml. in addition, the t m was determined from the polynomial fitting of the observed curve and taken as the temperature corresponding to half denaturation of the np. the first derivative of the absorption with respect to temperature, da/dt, of the melting curve was computer generated and used to determine the t m . fluorescence spectroscopy and compounds. the small molecules tested were primarily obtained from an in-house collection of compounds and were included for testing of np inhibitors. in the drug-induced fluorescence change experiment, a final concentration of μ m np was added to a buffer that contained various concentrations of compound, and the samples were incubated at °c for various durations. the buffer consisted of mm tris (ph . ) and mm nacl. the tryptophan fluorescence was measured using a hitachi f- fluorescence spectrophotometer that was equipped with a cuvette with a -cm light path. the excitation wavelength was nm, and the emission data were collected between and nm. all static measurements were recorded in triplicate. the relative fluorescence titration intensity was determined using the following equation: where Δ f max is the saturating value of the fluorescence change, x is the drug concentration, k d is the dissociation constant, and n is the hill coefficient. to exclude the inner filter effects caused by ligand, a correction for the inner filter effect for the binding of h and np is employed by the following equation: l is the pathlength of the cuvette used, the a terms are the absorbance at the excitation and emission wavelengths, and f values are the corrected and observed fluorescence intensities. small-molecule screening against nucleoprotein. the libdock molecular docking software was used to screen for small molecules that may bind to a structure of the y of np. the molecules comprised more than , compounds from several drug databanks in the zinc database. the binding pocket of the np, which includes tyr and r , was represented by a set of spheres during the docking process. (atcc accession no. ccl- ), were grown in rpmi medium (gibco). madin-darby canine kidney (mdck) cells (atcc accession no. nbl- ) were grown in dulbecco's modified eagle medium (gibco). all media were supplemented with % fetal bovine serum (gibco), u/ml of penicillin and streptomycin, and mm l-glutamine. wsn viruses were provided by the clinical virology laboratory of chang gung memorial hospital (linkou, taiwan). viruses were amplified by using mdck cells. virus titer was determined by a plaque assay using mdck cells. in the time-of-addition experiment, a cells were infected with wsn (moi ) and incubated in e medium; at t time points after infection, h was added at different concentrations. the cells were rinsed with pbs to remove the drug and culture medium, and the cell lysates were applied to detect the viral rna and viral protein at h post-infection. a total of × a cells were seeded into six-well plates, allowed to reach confluence, and then challenged with virus (moi ). total rna was extracted from cells using the trizol reagent (invitrogen, carlsbad, ca, usa). following phenol-chloroform extraction and isopropanol precipitation, the rna pellet was washed, dried, and dissolved in ml of rnase-free water. the protocol of rt-pcr amplifications and q-pcr were as described by hsu et al . western blotting. the cell lysates were collected and lysed with lysis buffer ( mm nacl, % ca , mm tris-base, ph . ) for detecting the target proteins. proteins with sample buffer were subjected to sds-page and electroblotted onto hybond ecl membranes (amersham). blocking and incubation with antibodies were done using . % tween and in tris-buffered. western blot membranes were incubated with : dilution of anti-np rabbit polyclonal ab (kindly provided by dr. shin ru shih, research center for emerging viral infectious, chang gung university). cytotoxicity assay. cell cytotoxicity of inhibitors was determined by mts assay. a cells were grown ( cells/well) in -well plate for h. the medium was replaced with that containing serial diluted compound after virus infection (t : h after infection) and the cells were further incubated for h. the culture medium were removed and added μ l including mts and pms mixture solution, the plate was incubated for min. mts and pms are purchased from sigma and prepared in pbs (phosphate buffered saline). to identify a -well microtiter plate, ml reagent containing both mts and pms at the ratio of : was mixed immediately with ml serum-free dmem. each drug concentration was performed with four repeats. the optical density was measured at od nm in multi-well spectrophotometer (elisa reader). the crystal structure of the influenza a virus np (protein data bank code iqh) lacks a defined tertiary structure in the regions between amino acid residues - , - , and - . the swiss-model program was used to model the complete structure of the np. we used the complete structure of the np as a template to construct a plausible np-ssrna complex using the molecular modelling programs discovery studio . and haddock . the rna force field parameters of parkinson et al. were used . the quality of the model geometry was evaluated using the rms derivation of the bond length and bond angle. the quality of the modeled structure was tested with procheck the first influenza pandemic of the st century the first pandemic of the st century: a review of the pandemic variant influenza a (h n ) virus recent strategies in the search for new anti-influenza therapies strategies of development of antiviral agents directed against influenza virus replication the war against influenza: discovery and development of sialidase inhibitors advances in the structure-based design of the influenza a neuraminidase inhibitors antiviral strategies against influenza virus: towards new therapeutic approaches at the centre: influenza a virus ribonucleoproteins structure and assembly of the influenza a virus ribonucleoprotein complex structure of influenza virus 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viruses maintain fitness and transmissibility in ferrets nuclear traffic of influenza virus proteins and ribonucleoprotein complexes oligomerization of the carboxyl terminal domain of the human coronavirus e nucleocapsid protein high-throughput identification of compounds targeting influenza rna-dependent rna polymerase activity inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers e …r salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors therapeutic roles of curcumin: lessons learned from clinical trials correction of inner-filter effect in fluorescence excitation-emission matrix spectrometry using raman scatter identification of bpr p as an inhibitor of cap-snatching activities of influenza virus crystallography & nmr system: a new software suite for macromolecular structure determination new parameters for the refinement of nucleic acidcontaining structures aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr resonance, circular dichroism spectroscopy, and fluorescence spectroscopy experiments. s.c.l. analyzed the results and all author approval the final version of the manuscript. key: cord- -jv o authors: marcos-villar, laura; díaz-colunga, juan; sandoval, juan; zamarreño, noelia; landeras-bueno, sara; esteller, manel; falcón, ana; nieto, amelia title: epigenetic control of influenza virus: role of h k methylation in interferon-induced antiviral response date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: jv o influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. using an unbiased search, we analyzed the epigenetic changes at dna methylation and post-translational histone modification levels induced by the infection. dna methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. a particular increase in h k methylation was observed and the use of an inhibitor of the specific h k methylase, dot l enzyme, or its silencing, increased influenza virus replication. the antiviral response was reduced in conditions of dot l downregulation, since decreased nuclear translocation of nf-kb complex, and ifn-β, mx and isg expression was detected. the data suggested a control of antiviral signaling by methylation of h k and consequently, influenza virus replication was unaffected in ifn pathway-compromised, dot l-inhibited cells. h k methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. epigenetic methylation of h k might have an important role in controlling interferon-induced signaling against viral pathogens. chromatin is a dynamic structure that adapts itself to alter the spatial arrangement of genetic information and thus meet the changing demands of cell functions; it has a key role in the control of gene expression. epigenetic modifications of chromatin regulate heritable and reversible gene expression without altering the dna sequence, and can also modify chromatin accessibility. dna methylation and post-translational histone acetylation and methylation are major mechanisms responsible for epigenetic regulation of gene expression . dna methylation typically represses gene transcription when takes place in the promoters of regulated genes . histone acetylation opens the condensed chromatin structure by reducing dna affinity for histones; this enables the dna to uncoil from nucleosomes so that transcription factors and rna polymerase can access the dna and increase transcription . histone methylation can increase or decrease gene transcription, depending on which amino acids in the histones are modified and how many methyl groups are added to specific residues . influenza virus is an rna virus whose genome consists of eight single-stranded rna segments with negative polarity. the virus uses a very uncommon transcription mechanism. the heterotrimeric viral polymerase synthesizes capped, polyadenylated viral mrnas through an initiation process; it uses short-capped oligonucleotides as primers, which are scavenged by a viral endonuclease from newly synthesized host rna polymerase ii transcripts , . the viral transcription strategy thus requires continuous cellular transcription for viral mrna infected cells. to identify epigenetic changes induced by influenza virus infection in the cell transcription machinery, we first analyzed dna methylation levels. human a lung epithelial cells were mock-infected or infected at high multiplicity of infection (m.o.i.; pfu/ml) with a hypervirulent a/pr / / (pr hv) strain [ ] [ ] [ ] [ ] (fig. a) . at h post infection (hpi), dna was isolated and methylation profiles evaluated in triplicate using the k infinium dna methylation beadchip. correlation analysis of the , valid probes showed an extremely strong relationship between samples (pearson correlation coefficient from r = . to r = . ; fig. b ). to identify specific differentially methylated candidates, we performed a parametric analysis to compare average beta values from infected with mock-infected cells, selecting those with differences in methylation levels > (− . >delta> . ) and a standard deviation value < % (desvest< . ). we found no significant variations, even after analysis of selected cpg sites from infection-associated genes (not shown), which indicated that global methylation of dna does not change during influenza virus infection in this system. histone fractions were purified from the same samples used for the study of dna methylation and mass spectrometry (ms) analysis were performed. we used unbiased analysis with the nanolc-ms platform to identify and quantify influenza virus-induced epigenetic changes in histone post-translational modifications (ptm). three biological replicates were analyzed and quantitative analyses completed (see methods). we considered a mascot score threshold of as high-confidence peptide identifications, and all peptides methylated/acetylated on lysine (above or below this threshold) were validated manually to assure correct tandem mass spectra matches. only ptms that were present in at least two replicas are presented (fig. c) and it has to be pointed out that all modified and unmodified peptides were identified reliably more than once. results in infected cells showed a reduction in lysine acetylation of histone and . this decrease would impair host cell expression, in accordance with the role of acetylation in opening the condensed chromatin and activating transcription . non-methylated h k and non-acetylated h k levels increased moderately during infection; since h k methylation and h k acetylation are hallmarks of transcription elongation , , the increased levels of these unmodified residues would also contribute to transcriptional inactivation of host cells. histones have a large proportion of basic residues. this property impedes detection of specific residues, probably due to the presence of another lysine(s) that trypsin proteolysis would render as peptides too small to be identified reliably by ms-based techniques. lysines not detected by ms include h k and h k , whose ptm are recognized as histone marks that control cell transcription. h k trimethylation is a common modification in active chromatin , whereas h k methylation is linked to transcriptional repression , . decreased h k me levels have been observed during influenza virus infection ; here we evaluated possible changes in methylated h k and in acetylated h k , as the latter is a conventional histone marker of active chromatin . total extracts of a mock-infected or pr hv-infected cells at high m.o.i. were collected at various times post-infection and levels of acetylated h k and dimethylated h k were tested by western blot. the results indicated that at late times after infection, influenza virus triggers a decrease in h k ac and an increase in h k me (fig. d ), which could contribute to a general decrease in host cell transcription activity. in contrast to this general effect on histone modifications, which seems to impair transcription, we detected a clear increase in methylation of lysine of histone , especially monomethylation (fig. c ). this result was unanticipated, since studies in saccharomyces cerevisiae and in humans, h k methylation levels correlated clearly with transcriptional activity . as methylation of this lysine gave the highest score in ms analysis, we evaluated this increase by two additional methods. we purified histones from cells mock-or pr hv-infected at high m.o.i.; at hpi, h k me levels were evaluated by western blot analysis (fig. e ) and a quantitative colorimetric method based on anti-h k me antibody detection (epigentek) (fig. f ). both techniques verified the h k methylation increase observed in proteomic analysis. these results indicate that epigenetic modifications induced by influenza virus infection mainly target the histone component of host cell chromatin, with h k residue methylation the most frequently modified. that methylation of infected cell dna is unaffected indicates, that in these conditions, influenza virus does not permanently injure the host cell, but the epigenetic modifications may be transitory. methyltransferases modify different lysines, only one known methyltransferase, dot l, mono-, di-and trimethylates h k ; the inhibitor epz- (epz) specifically inhibits dot l. to confirm this ability to inhibit h k methylation in our system, we incubated a cells with the inhibitor and used western blot to analyze h k me accumulation at various times after epz addition. treatment with or μm epz substantially decreased h k methylation at - h post-epz addition, without affecting h k me levels (fig. s a ). the effect of dot l inhibition on cell viability was analyzed by mtt assay (see methods and ); at these doses, epz did not notably affect cell viability, even at h after treatment (fig. s b) . to determine whether h k methylation alters influenza virus infection, we inhibited dot l and analyzed its effect on production of infectious particles. a cells were plated alone or with dot l inhibitor ( h), then infected at low m.o.i. with the pr hv strain, with another laboratory-passaged influenza strain, a/wsn/ (wsn) ( fig. a ) or with one of two natural pandemic isolates, a/california/ / (cal ) and a/ california/ / (cal ) (fig. b ) and analyzed production of infectious particles in each condition. dot l inhibition caused an increase in viral replication, higher in cells infected with the natural isolates, which suggests a general role of h k methylation in control of the influenza virus life cycle. to confirm the effect of dot l activity on influenza virus replication, we tested the effect of dot l knock-down on production of infective particles. for rnai-mediated dot l silencing experiments, we used lentiviruses expressing shrna specific for dot l (shdot l , shdot l ) or a control that expresses irrelevant shrna (shtm) , . treatment of a cells with the dot l inhibitor or expression of the dot l-shrnas reduced h k me levels compared with shtm, measured by western blot analysis (fig. a ) and quantitative colorimetric method (fig. b ). dot l silencers decreased h k methylation in infected cells in a lesser extent than epz treatment. to analyze the relevance of dot l in virus replication, we transduced a cells with lentiviruses expressing the dot l silencers or control shrna ( days), followed by infection with the pr hv ( role of h k methylation in antiviral responses. the above results suggested that modification of lysine of histone may mediate the antiviral response; its demethylation could decrease the host response against the pathogen allowing higher production of infectious particles. in agreement with this proposal we did not find significant reduction of viral titer in cells infected at high m.o.i where ifn signaling does not play a major role, in the presence of the dot l inhibitor (data not shown). these data suggested that h k methylation would affect host response in conditions close to natural infections that take place in the epithelium of the respiratory tract at low multiplicity of infection and trigger an efficient ifn induction. nf-κb is a protein complex that controls dna transcription, cytokine production and cell survival. nf-κb is involved in cell responses to stimuli such as viral antigens and has a key role in regulating the innate immune response to infection . nf-κb has been shown to be activated upon accumulation of influenza virus rna . in response to specific stimuli, nf-κb translocates from cytoplasm to the nucleus, where it binds promoters of regulated genes, activates transcription of the interferon pathway , and stimulates isg transcription. type i interferon, which includes the ifnα and β subtypes, is induced during influenza virus infection and has an essential role in host defense against the virus by activating expression of a large number of isg . to analyze the effect of dot l methylase in nf-κb activation and ifn signaling, we evaluated nf-κb subcellular distribution in cells treated with tumor necrosis factor (tnf)-α ( ng/ml, min), or infected with influenza virus ( pfu/ml, hpi), alone or with dot l inhibitor ( h). after tnf-α binding to the receptor, the inhibitory protein iκbα, which normally binds nf-κb and inhibits its translocation, is phosphorylated; this is followed by ubiquitination and degradation, releasing nf-κb, which is then translocated to the nucleus . nf-κb distribution was monitored using antibodies to the p subunit of the nf-κb complex and anti-lamin a/c antibodies to identify the nuclear envelope; influenza np detection was used to monitor infection. we analyzed > cells to quantitate nuclear and cytosolic nf-κb distribution (fig. ) . tnf-α addition produced a significant increase in nuclear import of nf-κb, and dot l inhibitor treatment significantly decreased its nuclear translocation (fig. a) . influenza virus infection similarly induced nf-κb nuclear import, and inhibition of h k methylation reduced its nuclear translocation (fig. b) . subcellular distribution of nf-κb was additionally examined in cells previously transfected with lentiviruses that express the specific dot l silencers or the control ( days). using the conditions described above, the corresponding cells were treated with tnf-α, or infected with pr hv influenza virus. in agreement with the results obtained using the dot l inhibitor, the dot l silencers decreased the nuclear translocation of nf-κb both, in tnf-α treated cells (fig. a ) or in influenza virus infected cells (fig. b) , whereas the expression of the control silencer did not change the subcellular distribution of nf-κb. together these results indicate that methylation of h k modulates the pathway involved in the nuclear translocation of nk-κb. as dot l inhibition impaired nf-κb activation, we examined its effect on steps that follow its nuclear translocation, that is, ifn and isg rna production. a cells were left untreated (control), treated with u/ml ifnαβ (ifn) or were infected with pr hv strain ( pfu/cell) (pr hv), alone or with dot l inhibitor ( h). after h or h of ifn treatment or virus infection, rna was extracted and used for qpcr detection. at h, we found a weak increase on ifnβ, ifn-stimulated gene (isg ) and interferon-induced protein mx (mx ) rna levels after ifnαβ addition or influenza virus infection, and dot l inhibitor treatment did not significantly decreased their accumulation (fig. b,c) . at h, we observed an increase in ifnβ, isg and mx rna levels and a reduction of these rnas when cells were pretreated with the h k methylase inhibitor (fig. b,c) . the significant reduction in nf-κb nuclear translocation and decreased ifnβ, isg and mx rna expression after inhibition of dot l both, support a role for h k methylation in modulating the antiviral response. given the role of h k methylation in the control of ifn signaling, we analyzed the effect of dot l inhibitor on influenza virus replication in cells with normal or deficient ifn responses. we used pr hv and cal strains at low m.o.i. to infect mdck, mdck v or mdck npro cells, untreated or treated with dot l inhibitor. mdck v cells express v protein, which targets stat and for degradation , and mdck npro cells express npro protein, which targets irf- for polyubiquitination and subsequent degradation ; ifn, but not isg, is thus induced in mdck v cells, and neither ifn nor isg are induced in mdck npro cells. we analyzed viral titers at different hpi and observed that the increased infectious particle production in mdck cells treated with the inhibitor and infected with pr hv or cal (fig. a and b) was reduced or lost in 'ifn-compromised' cells treated with dot l inhibitor. the effect of influenza virus infection on h k methylation in mdck cells with normal and deficient response to ifn was checked by colorimetric assays. influenza virus infection increased h k methylation levels in normal and interferon deficient cells, likewise in a cells (fig. s a) . since h k methylation does not affect influenza virus replication in cells with impaired ifn signaling, we analyzed the effect of dot l inhibitor in subsequent stages of viral infection. we evaluated ifnβ, isg and mx rna amounts after influenza virus infection ( pfu/cell) in ifn-competent or -deficient cells, untreated or treated with dot l inhibitor. influenza virus infection increased ifnβ, isg and mx production in mdck control cells, and inhibitor treatment reduced their accumulation (fig. c, top) . infection of mdck v cells increased ifnβ production, which was reduced by dot l inhibitor, whereas accumulation of isg was not observed, in accordance with the need for stat signaling for their induction (fig. c, center) . infection of mdck npro cells did not increase ifnβ, isg or mx rnas, independently of the presence of dot l inhibitor (fig. c, bottom) , which coincided with the inability of these cells to produce ifn and isg. in addition, total extracts of mdck, mdck v and mdck npro cells infected with pr hv at m.o.i for h were used for western blot analysis to detect irf- , stat , mx and isg to confirm the differential expression of these proteins in normal or ifn-deficient cells (fig. s b) . these results indicate that the interferon antiviral response to influenza virus infection is at least partially regulated by h k methylation via canonical type i ifn signaling mediated by the stat pathway at the level of ifn activation and response. these results indicated the ability of dot l methylase to control interferon signaling pathways. to evaluate whether h k methylase has a broader role in the control of virus multiplication, we tested the effect of dot l inhibitor and the lentiviruses expressing the dot l silencers on the replication of two distinct rna viruses, respiratory syncytial virus (rsv) and vesicular stomatitis virus (vsv). like influenza virus, rsv is a respiratory virus, and is the most common cause of viral lower respiratory tract infections in infants and children . although ifnβ appears to restrict viral replication in lung epithelial cells, it is generally agreed that rsv is relatively resistant to ifnαβ, effects, and ifn has not been detected in natural infection . vsv can infect insects, cattle, horses and pigs and is a potent ifn inducer both in cell culture and animal models . human epithelial a cells, untreated or treated with dot l inhibitor ( h) (fig. a,c) , or transduced with lentiviruses expressing the dot l silencers or control shrna ( days) (fig. b,d) , were infected at low m.o.i. with rsv ( pfu/ml) (fig. a,b) or vsv ( − pfu/ml) (fig. c,d) ; at various times post-infection, cell supernatants were obtained for viral titration (see methods). no changes were observed in rsv replication in the presence of the inhibitor or the dot l silencers, whereas infectious particle production clearly increased in vsv-infected cells treated with dot l inhibitor or when dot l was down-regulated through the expression of the specific silencers. we also evaluated the expression of viral and isg proteins (isg and mxa) during infection with rsv (fig. b, right) , and vsv (fig. d, right) . the corresponding viral proteins were detected and we observed a lack of ifn-stimulated gene induction in rsv-infected cells, while infection with vsv efficiently induced ifn response in our system. these results support a role for h k methylation in the control of ifn signaling, and a potential general dot l methylase function in regulating pathogen infection controlled by the ifn pathway. viruses are obligate intracellular parasites that completely subordinate host cell metabolism. although influenza virus does not integrate into the genome of the infected cell, the virus efficiently switches off expression of host cell genes , a result of a complex interplay of virus-induced activities closely coordinated to reduce the host response to eliminate the viral infection. consequently, during infection there is a network of viral-and host-induced modifications of cellular gene expression with a close dependence of chromatin-based functions and therefore of chromatin dynamic. accordingly, specific interactions between chromatin remodelers of the chd family and influenza virus proteins have been described such as the association of chd with the non-structural protein ns or chd and chd with the viral polymerase complex , , . in spite of this association, little effort has been made to identify epigenetic changes in chromatin induced by the infection. few alterations in dna methylation have been reported during influenza virus infection [ ] [ ] [ ] . among them changes in promoter methylation levels of some proinflammatory cytokines and interleukine genes when using the high virulence h n strain have been described. infection of human respiratory epithelial cells with pr hv strain did not show variations on dna methylation (fig. b) , however it is possible that changes in promoter methylation of specific genes and their subsequent inactivation, take place in response to particular virulent influenza strains and/or in different systems. previous studies showed that influenza virus infection modulates ptm of several isg. chromatin immunoprecipitation assays and antibodies that recognize canonical marks of transcription activation such as h k me or of inactivation such as h k me showed addition or removal of these ptm in several isg; modifications depended on strain virulence when h n virus was compared with the h n pandemic strain . this study emphasized the importance of epigenetic control in the antiviral response elicited by influenza virus infection; nonetheless, they focused on the search for specific canonical transcription activation or repression marks of histones that correlate with up-or down-transcriptional regulation of the corresponding genes and do not provide an overview of all possible changes to chromatin in response to the viral infection. for a broad overview of chromatin modifications in influenza virus-infected cells, we used an unbiased search for global changes in chromatin epigenetics at the dna and histone levels. we observed a general decrease on histone acetylation in h and h lysine residues after infection, as well as increased levels of unmodified h k , h k and dimethylated h k (fig. c and d) . in addition, previous studies showed reduction of h k me ; one of the hallmark of active chromatin . histone acetylation has a key role opening the condensed chromatin, resulting in charge neutralization and a more relaxed, open, and transcriptionally active chromatin structure . decreased acetylated histones, trimethylated h k and increased non-methylated h k and dimethylated h k , all associate with transcriptional inactivation [ ] [ ] [ ] [ ] . these epigenetic changes would impair host cell expression, in accordance with the transcription inactivation of the host cell that occurs during infection and constitutes one of the major mechanisms triggered by the virus to inactivate host transcription machinery and consequently to decrease the antiviral response. unexpectedly, changes in methylation of lysine of histone were the most prominent. lysine is located within the globular domain of histone h and is mono-, di-, and trimethylated by dot l, which modifies this lysine exclusively . h k methylation is increased in actively transcribing genes and appears to have a role in cell cycle regulation and dna damage response . dot l has been studied particularly in the modulation of mixed-lineage leukemia (mll)-related leukemogenesis, as mll fusion target gene expression depends specifically on the functional dot l gene ; indeed, inhibition of dot l activity is currently in clinical trials. the role of h k methylation in viral infection, is poorly characterized. infection of human primary fibroblasts with human cytomegalovirus (hcmv), produces a marked increase in h k me levels and downregulation of dot l expression results in a decreased viral growth . the genome of hcmv is a double-stranded dna molecule that is maintained as episome during infection. the viral dna lacks histones when encapsidated in the virion however, upon infection the viral genome is transported to the nucleus, where it becomes associated with host cell histones. it has been speculated that dot l directly mediates replication of the hcv genome, in agreement with the chromatinization of its dna genome . the function of h k methylation modulating influenza virus replication, suggest a very different mechanism, since influenza virus is an rna virus that does not integrate in the host chromatin and its genome lacks histones. in physiological conditions where antiviral signaling is induced, decreased h k methylation clearly enhanced viral replication (figs and ) . moreover, nuclear translocation of nf-κb (figs and ) and accumulation of ifnβ and isgs (isg and mx ) decreased in influenza virus-infected cells with dot l downregulated (fig. ) . influenza virus and vesicular stomatitis virus induce a strong antiviral immune response characterized by robust production of antiviral type i interferons. during infection, double-stranded rna molecules are produced, which are recognized by the rig-i helicase . after activation, rig-i recruits various tnf receptor-associated factors, which trigger phosphorylation and activation of the iκκ complex, leading to iκbα phosphorylation and degradation to allow nfκb nuclear translocation . nf-κb and ifn regulatory factor direct expression of type i ifn, which promote transcription of a variety of genes that further limit viral replication , . we observed a marked increase on replication of influenza virus and vsv, both potent inducers of ifn, in cells with low levels of methylated h k (figs , and ). this effect is lost when cells deficient on ifn signaling are infected with influenza virus (fig. ) . together the data indicate that demethylation of h k would decrease antiviral ifn signaling and therefore, viruses may develop different strategies attempting to control epigenetic modification of h k that elicit the anti-pathogen response. methylation of lysine of histone might have a general role in controlling the host response of pathogens that are interferon inducers. epz was purchased from novagen, human tumor necrosis factor α from sigma-aldrich, and recombinant interferon αβ (pbl - universal type i ifn) was provided by s. guerra. lentiviral particle production and cell transduction. lentiviral particles were produced in hek t cells by cotransfection of plasmids pspax and pmd .g with each of the plko-based shrna vectors, as described , . supernatants were collected to h post-transfection, filtered through a . μm filter, and used to transduce the corresponding cells. as the lentiviral vectors confer puromycin resistance, the minimum amount of supernatant necessary to confer % resistance to puromycin ( μg/ml) was used. silencing was tested by western blotting or colorimetric assays, normally at days post-transduction. western blot was performed as described . to detect influenza virus proteins, the following antibodies were used: for pa, monoclonal antibodies (mab) and ( : ; ; for pb , mab ( : ; , and for pb , rabbit polyclonal antibody ( : ; ; for β-tubulin, mouse anti-β-tubulin mab ( : ; sigma). to analyze histones, we used h k ac ab ( : ; abcam) and h k me ( : ; active motif). polyclonal antibodies to h k me d e , h k me c d , h # and h d h ( : ) were from cell signaling. for vsv detection, a monoclonal antibody mix against g, n and m viral proteins provided by m. esteban was used. for rsv detection, a monoclonal antibody against np protein provided by j.a. melero was used. colorimetric determination of h k me . the iquik global di-methyl histone h k quantification kit (colorimetric) from epigentek was used for elisa-like measurement of total h k me amounts, following the supplier's protocol. proteomic analysis of histone modifications. enzymatic digestion and itraq- plex labeling. the enriched histone fraction ( μg) for each condition, prepared following the epigentek protocol, was precipitated by the methanol/chloroform method, reconstituted in m urea/ m thiourea/ mm teab buffer (triethylammonium bicarbonate, ph . ), reduced with mm tris( -carboxyethyl) phosphine (tcep, sciex), and alkylated with mm methyl methanethiosulfonate (mmts, pierce); this was followed by trypsin (sigma-aldrich) proteolysis at a : enzyme:protein ratio. the tryptic peptides were labeled using the itraq- plex isobaric mass tagging kit (sciex) according to manufacturer's instructions (mock, tag- ; pr lv, tag- , pr hv, tag- ). samples were pooled, dried and desalted on a sep-pak c cartridge (waters). peptide fractions were subjected to lc-ms/ms analysis using a nano liquid chromatography system (eksigent technologies nanolc ultra d plus) coupled to a high speed triple tof mass spectrometer (sciex) with a nanoelectrospray ion source. samples were injected on a c pepmap trap column ( μm, μm i.d. x cm; thermo scientific) at μl/min, in . % formic acid in water, and the trap column was switched on-line to a c nanoacquity beh analytical column ( . μm, Å, μm i.d. x cm, waters), equilibrated in mobile phase a ( . % formic acid in water), and peptide was eluted in a min linear gradient from - % b ( . % formic acid in acetonitrile) at nl/min. the mass spectrometer was operated in data-dependent acquisition mode. for tof scans, accumulation time was set to ms, and up to precursor ions were monitored per cycle. data analysis. ms and ms/ms data obtained were converted to mgf files, which were also searched against a homo sapiens protein database containing protein-coding genes (including reversed entries to calculate false discovery rate; fdr) using the mascot server v. . (matrix science). search parameters were set as follows: enzyme, trypsin; allowed missed cleavages, ; fixed modifications, beta-methylthiolation of cysteine and itraq- plex (n-term and k); variable modifications, oxidation of methionine, acetylation (k and n-term) and methylation and dimethylation (k). peptide mass tolerance was set to ± ppm for precursors and . da for fragment masses. the confidence interval for protein identification was set to ≥ % (p < . ) and only peptides with an individual ion score above the % fdr threshold were considered correctly identified. dna methylation. isolated dna was processed according to the manufacturer's protocols for illumina infinium assay, as described . data filtering. from the initial , cpg, we excluded all probes with detection p-values > . ( cpg removed). we eliminated cpg containing single-nucleotide polymorphisms (snp) located within bp of the target cpg (snp ) and internal controls (ch and rs), resulting in cpg removed. a total of , valid probes were included in the final analysis. methylation score was represented as β-values, average for each probe was calculated and β-value differences were used for analysis. confocal immunofluorescence microscopy. cells were fixed in % paraformaldehyde ( min, room temperature) and stored in pbs. for immunofluorescence, cells were permeabilized ( min) in pbs containing % triton x- and incubated with primary antibodies diluted in pbs/ % bsa (w/v) as follows: rat anti-np ( : ;, anti-p ab ( : ; abcam) and anti-lamin a/c ( )( : ; sc- , santa cruz) to label nuclear envelope. confocal microscopy was performed with a leica tcs sp laser scanning system. images of × pixels and an eight-bit grayscale depth were acquired sequentially every . - . μm using las af version . . software (leica) and analyzed using las af and metamorph premier version . . image analysis software (molecular devices). p nuclear translocation was quantified by counting at least cells/condition, and the ratio of relative p intensity in nucleus and cytoplasm of each cell was calculated. qrt-pcr analysis. for rna extraction, cell pellets were resuspended in ml trizol reagent (invitrogen) and rna was purified as recommended by the manufacturer. rna was digested with rnase-free dnase ( u/ mg; h, °c), extracted with phenol-chloroform-isoamyl alcohol and ethanol-precipitated. for reverse transcription, we used the high-capacity cdna rt kit (applied biosystems). pcr were performed in -well pcr plates using sybr green pcr master mix (applied biosystems). pcr were carried out in a prism sequence detection system (applied biosystems). the cycle threshold (ct) was determined with analytical software (sds; applied biosystems). serial dilutions of cdna were used to ensure amplification. chromatin remodeling, histone modifications, and dna methylation-how does it all fit together? cancer epigenomics: dna methylomes and histone-modification maps histone acetylation and chromatin remodeling histone modifications for human epigenome analysis the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit influenza virus transcription and replication influenza mrna translation revisited: is the eif e cap-binding factor required for viral mrna translation? pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses changes in methylation of genomic dna from chicken immune organs in response to h n influenza virus infection infection with influenza a viruses causes changes in promoter dna methylation of inflammatory genes interleukin- expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsrna treatment influenza a virus-induced caspase- cleaves the histone deacetylase in infected epithelial cells replication fitness determines high virulence of influenza a virus in mice carrying functional mx resistance gene attenuated strains of influenza a viruses do not induce degradation of rna polymerase ii specific residues of pb and pa influenza virus polymerase subunits confer the ability for rna polymerase ii degradation and virus pathogenicity in mice influenza virus and chromatin: role of the chd chromatin remodeler in the virus life cycle histone acetylation: a switch between repressive and permissive chromatin chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression hat , a golgi apparatus-anchored b-type histone acetyltransferase, acetylates free histone h and facilitates chromatin assembly profile of histone lysine methylation across transcribed mammalian chromatin pr-set -dependent methylation of histone h lys functions in repression of gene expression and is essential for mitosis 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interferon regulatory factor and targets it for proteasomal degradation activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental infections ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis polypeptide synthesis in influenza-virus infected cells chd facilitates vrnp nuclear export by interacting with nes of influenza a virus ns pa subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators chd chromatin remodeler is a negative modulator of influenza virus replication that relocates to inactive chromatin upon infection influenza virus infection causes specific degradation of the largest subunit of cellular rna polymerase ii dot l/kmt recruitment and h k methylation are ubiquitously coupled with gene transcription in mammalian cells mll-rearranged leukemia is dependent on aberrant h k methylation by dot l quantitative proteomic discovery of dynamic epigenome changes that control human cytomegalovirus (hcmv) infection temporal dynamics of cytomegalovirus chromatin assembly in productively infected human cells activation and regulation of pathogen sensor rig-i mavs recruits multiple ubiquitin e ligases to activate antiviral signaling cascades. elife , e emerging role of ubiquitination in antiviral rig-i signaling a third-generation lentivirus vector with a conditional packaging system lentivirus-delivered stable gene silencing by rnai in primary cells monoclonal antibodies against the influenza virus pb and np polypeptides interfere with the initiation step of viral mrna synthesis in vitro distinct regions of influenza virus pb polymerase subunit recognize vrna and crna templates molecular cloning. a laboratory manual genetic trans-complementation establishes a new model for influenza virus rna transcription and replication we are indebted to j. ortin, p. gastaminza and u. garaigorta for critique of the manuscript. the technical assistance of s. ciordia and r. navajas from the proteomics facility (cnb-csic, proteored isciii) is greatly appreciated. we thank c. mark for editorial assistance. lmv was supported by ciber de enfermedades respiratorias. this work was funded by the spanish ministry of economy and competitivness, plan nacional de investigacion científica, desarrollo e innovacion tecnologica (bfu - -r, (aei/feder)) and ciber de enfermedades respiratorias. js is a miguel servet researcher at isciii (ms / ). supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - e xrd y authors: watanabe, tokiko; iwatsuki-horimoto, kiyoko; kiso, maki; nakajima, noriko; takahashi, kenta; jose da silva lopes, tiago; ito, mutsumi; fukuyama, satoshi; hasegawa, hideki; kawaoka, yoshihiro title: experimental infection of cynomolgus macaques with highly pathogenic h n influenza virus through the aerosol route date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: e xrd y several animal models are used to study influenza viruses. intranasal inoculation of animals with a liquid inoculum is one of the main methods used to experimentally infect animals with influenza virus; however, this method does not reflect the natural infection with influenza virus by contact or aerosol route. aerosol inhalation methods have been established with several influenza viruses for mouse and ferret models, but few studies have evaluated inoculation routes in a nonhuman primates (nhp) model. here, we performed the experimental infection of nhps with a highly pathogenic h n influenza virus via the aerosol route and demonstrated that aerosol infection had no effect on clinical outcome, but caused broader infection throughout all of the lobes of the lung compared with a non-aerosolized approach. aerosol infection therefore represents an option for inoculation of nhps in future studies. influenza viruses cause annual epidemics and periodic pandemics. we have experienced three pandemics in the th century (i.e., spanish flu in - , asian influenza in , and hong kong influenza in ) and one in the st century (i.e., pandemic influenza a (h n ) in ). these influenza pandemics have a huge impact on public health and the global economy . additionally, recent sporadic human infections with h n and h n avian influenza viruses have raised the pandemic threat of these viruses [ ] [ ] [ ] [ ] . the continued circulation of h n viruses in birds provides opportunities for them to infect humans. indeed, human cases of h n infections have been reported in several countries, with a total of confirmed cases and fatalities as of oct , which is a case fatality rate of approximately % (http://www.who.int/influenza/human_animal_interface/ _ _ _tableh n .pdf?ua= ). therefore, concern over the pandemic potential of h n viruses is clearly justified. influenza virus can be transmitted from person to person through several transmission modes, including direct contact, indirect contact with a contaminated object (fomite), droplet transmission (droplets of > µm in diameter), and droplet nuclei transmission, also referred as airborne or aerosol transmission (droplet nuclei of < µm in diameter, which can remain suspended in the air for prolonged periods). experimental infection of animal models with influenza viruses is an essential component of the characterization of influenza viruses. mice, ferrets, and nonhuman primates (nhps) are commonly used to examine the pathogenicity, replicative ability, and transmissibility of influenza viruses, although each model has its advantages and limitations. nhps have been frequently used as a model to characterize the virulence of various influenza viruses, such as highly pathogenic h n , and the and pandemic viruses, because of their close genetic relationship to humans [ ] [ ] [ ] . division of virology, department of microbiology and immunology, institute of medical science, university of tokyo, tokyo, - , japan. department of pathology, national institute of infectious diseases, shinjuku-ku, tokyo, - , japan. intranasal and/or intratracheal inoculation with a liquid inoculum of influenza viruses has been the primary method used to infect nhps; however, a liquid inoculum does not reflect the natural infection. aerosol inhalation methods for inoculation of mice and ferrets have been established with several influenza viruses, including h n viruses [ ] [ ] [ ] [ ] [ ] . in the ferret model, these studies demonstrated that the inoculation of animals with highly pathogenic avian influenza h n virus via the aerosol route led to higher nasal wash virus titers, earlier onset of clinical signs, and/or a broader spectrum of disease compared with infection via intranasal inoculation despite no difference in lethality [ ] [ ] [ ] . in a murine model, belser et al. recently established aerosol infection with highly pathogenic h n influenza virus in the nhp model; however, they have not compared the outcomes in the nhps infected via the aerosol route with those in nhps infected via the conventional method of liquid virus inoculation. therefore, in this study, we compared the virus distribution, pathogenicity, and disease outcome in nhps infected via the aerosol route with these parameters in nhps infected through multiple routes (e.g., the intranasal and intratracheal routes). establishment of an h n virus infection system in nonhuman primates through an aerosol route by using a commercial nebulizer. a wide range of commercial nebulizers are available for clinical and research use. in this study, we used the ultrasonic nebulizer ne-u from omron healthcare co, ltd, japan. this was chosen because it is easy to use and could reduce the treatment time needed compared with other nebulizer devices , thereby making the procedure less stressful for the animals. the particle size is also critical for aerosol inhalation because aerosol transmission (also referred to as droplet nuclei transmission) occurs when small particles (< µm) are inhaled. a previous study demonstrated that the ne-u nebulizer generates a large fine particle fraction of aerosol mass < µm (the mean percentage of the fine particle fraction was %) . we used -to -year-old male cynomolgus macaques that weighed - kg each. four animals were inoculated with ml of a pfu/ml solution of the highly pathogenic h n avian influenza virus a/vietnam/ ut / strain (vn ) through the aerosol route by using the ultrasonic nebulizer ne-u . these animals were defined as 'the aerosol method group' . as a control, ml of the virus solution was administered through multiple routes by the conventional method, that is, by the intranasal ( . ml each to the left and right nostril), intraoral ( . ml each to the left and right tonsil), intraocular ( . ml per eye), and intratracheal ( . ml using a tracheal catheter) routes (defined as 'the conventional method group'). the virus dose of × pfu of influenza virus was chosen for inoculation because a similar virus dose has been widely used in previous studies to infect nhps to ensure the greatest likelihood of virus infection and replication in the inoculated animals , , [ ] [ ] [ ] . upon virus infection, none of the animals exhibited noticeable clinical signs (i.e., no appreciable body weight loss or fever was observed in any of the infected animals) except for a slight decrease in appetite among all of the infected animals. to examine virus shedding, nasal swab samples were collected from the infected animals on days and post-infection for virus titration. on day post-infection, vn virus was recovered from four animals in the conventional method group, whereas viruses were detected from three of four animals in the aerosol method group (table and fig. ). the mean virus titers were not significantly different between the conventional and the aerosol method groups [ . and . log (pfu/ml), respectively] (fig. ). on day post-infection, vn virus was recovered from nasal swabs of two and three animals in the conventional and aerosol method groups, respectively, and the mean virus titers were comparable between the two groups. to compare the distribution of h n virus in animals infected via the conventional method with that in animals infected via the aerosol method, animals from each group were euthanized on day post-infection and organ samples were collected for virus titration. relatively high virus titers were detected in the tonsils of all infected animals in both groups ( table ) . viruses were recovered from the nasal mucosa, trachea, and bronchus of some of the infected animals in both groups (table ). additionally, virus was recovered from the mediastinal lymph nodes of two and one of animal id virus titers (log pfu/ml) of animals infected with vn virus via the: aerosol method # - # - # - # - # - # - # - # - table . virus titers in nasal swabs from cynomolgus macaques infected with the h n virus via the conventional or aerosol method a . a cynomolgus macaques were inoculated with ml of a pfu/ml solution of a/vietnam/ut / virus (vn ) by the conventional method (through a combination of the intratracheal, intranasal, ocular, and oral routes) or via the aerosol method by using a nebulizer. b -, virus not detected (detection limit: . log pfu/ml). virus titers in respiratory swabs from infected cynomolgus macaques. cynomolgus macaques were inoculated with ml of a pfu/ml solution of the highly pathogenic h n avian influenza virus a/vietnam/ ut / strain (vn ) through the aerosol route by using the ultrasonic nebulizer ne-u (defined as "the aerosol method group"). as a control, ml of the virus solution was administered through multiple routes, which is the conventional method; by intranasal ( . ml each to the left and right nostril), intraoral ( . ml each to the left and right tonsil), intraocular ( . ml per eye), and intratracheal ( . ml using a tracheal catheter) routes (defined as "the conventional method group"). nasal swab samples were collected on days and postinfection for virus titration. red and blue horizontal bars show the mean titers for the aerosol and conventional method groups, respectively. virus titers (log pfu/g) of animals infected with vn virus via the: four animals in the conventional and aerosol method groups, respectively. also, virus was detected in the duodenum and brain of one of four animals in the conventional and aerosol method groups, respectively (table ) . interestingly, the virus distribution in the lower respiratory tract differed between the conventional and aerosol method groups. vn virus replicated efficiently in all of the lung lobes of the infected animals in the aerosol method group, whereas no virus was recovered from some of the lung lobes of some animals in the conventional method group ( table ). the mean virus titers in right upper, right middle, left upper, and left middle lobes of the lungs of the infected animals in the aerosol method group were significantly higher than those of the infected animals in the conventional method group [the mean virus titers in the aerosol method group were . , . , . , and . log (pfu/g), whereas those in the conventional method group were . , . , . , and . log (pfu/g), respectively] (fig. ) . in contrast, vn replicated well in the right-and left-lower lung lobes of the infected animals in the conventional method group [the virus mean titers were . and . log (pfu/g), respectively]. presumably, the introduction of virus as a liquid via the intratracheal and intranasal routes led to a large influx of virus solution into the right-and left-lower lobes. we assume that this also occurs in ferrets because we often observe similar macroscopic pathological changes in the lower lung lobes of infected ferrets (unpublished data). taken together, these findings demonstrate that a productive h n virus infection in nhps can be induced via the aerosol route by using the commercial ultrasonic nebulizer ne-u , and inoculation of nhps with aerosolized h n virus can result in the development of virus infection across a large area of the lower respiratory tract and the distribution of virus in all of the lung lobes of the infected animals, whereas inoculation via the conventional method can result in some lung lobes not being exposed to or infected with virus. histopathologic changes in the respiratory tract of animals infected with h n virus via the conventional and aerosol methods. next, we compared histopathological changes in the infected animals in the aerosol method group with those in the conventional method group. for this histopathologic analysis, we focused on two animals for each group in which virus was recovered from a variety of organs (i.e., # - and # - from the conventional method group, and # - and # - from the aerosol method group). nasal cavity, trachea, lung, epiglottis, heart, liver, spleen, kidney, small intestine, large intestine, and brain (i.e., olfactory bulb, cerebrum, cerebellum, and brainstem) were examined using hematoxylin-and-eosin-stained sections obtained from the selected animals. we also examined the distribution of influenza virus antigen by immunohistochemistry. we found no obvious changes and no viral antigens in most of the tissues tested, except for the tracheal and lung tissues. in the trachea, we observed mild inflammatory cell infiltration but no virus-antigen-positive cells. pulmonary edema and inflammatory cell infiltration into the alveolar spaces were observed in all of the infected figure . replication of h n virus (vn ) in the lung lobes of infected monkeys. cynomolgus macaques were inoculated with ml of a pfu/ml solution of vn through the aerosol route or conventional multisite routes (i.e., intranasal, intraoral, intraocular, and intratracheal routes) by using the aerosol or conventional method, respectively. four animals from each group were euthanized on day post-infection for virus titration. red and blue horizontal bars show the mean titers for the aerosol and conventional method groups, respectively. in the aerosol method group, the virus titers in some of the lung lobes were significantly higher than those in the conventional method group on day post-infection (p < . in the right-upper, right-middle, and left-middle lobes, and p < . in the left-upper lobe). detection limit = . log pfu/ml/tissue sample. scientific reports | ( ) : | doi: . /s - - - animals tested. the degree of inflammation, which had spread from around the bronchi, was different for each part of the lungs of each animal. the total pathologic scores appear to be higher in the animals infected by the conventional method than those by the aerosol method, although no statistical analysis was conducted because of the small number of animals used ( table ). the parts of the lung section with a pathologic score of are shown in fig. . neutrophils and mononuclear cell infiltration with fibrinous exudates were observed in the interstitial areas and alveolar spaces. edema and desquamation of alveolar epithelial cells into the alveolar spaces were also observed. these observations are identical to those reported in human patients who died as a result of h n infection [ ] [ ] [ ] [ ] . the conventional method group showed more severe inflammation in the lungs compared with the aerosol method group (table ) . all animals showed virus-antigen-positive signals in the bronchial and alveolar epithelium (table ). the use of animal models is essential for studying the biological properties of influenza viruses such as pathogenicity, replicative ability, and disease outcome. in most cases, intranasal administration with a liquid containing the virus is used to infect animals with influenza virus; however, a liquid inoculum does not reflect the natural influenza virus infection. recently the aerosol inhalation method has been tested in several mammalian models, including mice, ferrets, and nhps due to its resemblance to human exposure to influenza virus. here, we inoculated nhps with a highly pathogenic h n influenza virus via the aerosol route and showed that aerosol infection did not affect clinical outcome, but did cause more widespread infection throughout the lower respiratory tract compared with the conventional approach. the uniform infection in all of the lung lobes and the bronchial epithelium afforded by the aerosol infection system offers an advantage in some experimental settings. for example, when researchers take lung tissues for multiple purposes (e.g., virus titration, histopathology, and various omics analyses), they need to take samples from different parts of the lungs. therefore, infection via the aerosol route would minimize experimental variability. recently, wonderlich et al. demonstrated that aerosol infection of nhps with a highly pathogenic h n avian influenza virus caused fulminant pneumonia that rapidly progress to acute respiratory distress syndrome, and some of the infected animals died or were humanely sacrificed due to respiratory failure. the same phenomenon has also been observed in the ferret model [ ] [ ] [ ] ; however, findings by wonderlich et al. are not consistent with our findings here. the differences in disease outcomes between their study and ours may be caused by the different conditions used for the aerosol infection; that is, they used a head-only exposure chamber (into which the nhp's head was inserted), which allowed the animals to inhale the aerosolized h n virus with an exposure duration ranging from to min. in contrast, we used a standard inhalation mask, which is not a tight-fitting mask and allows the animals to breathe the aerosol mist through the nose and mouth spontaneously; the exposure duration was approximately min, potentially leading to virus infection with a lower dose compared to wonderlich's study. in addition, other parameters, such as origin, gender, and age of the macaques, as well as the conditions for virus stock generation (i.e., egg-grown virus vs. mdck-grown virus (our study), which could table . pathologic scores and detection of influenza virus antigen in animals infected with h n virus via the conventional or aerosol method a . a cynomolgus macaques were inoculated with ml of a pfu/ ml solution of vn virus by using the conventional method (through a combination of intratracheal, intranasal, ocular, and oral routes) or via the aerosol method by using a nebulizer. b pathologic severity scores for infected animals. to represent comprehensive histological changes, respiratory tissue samples were evaluated by scoring pathologic changes. pathologic scores were determined for each lung lobe in summary, here we established a system for aerosol infection of nhps with highly pathogenic h n influenza virus. we found that experimental infection with aerosolized h n virus led to a wider distribution of virus in the lower respiratory tract compared with liquid virus inoculation via the conventional method. however, no difference in clinical outcome was observed between the two inoculation methods. a highly pathogenic h n avian influenza virus, a/vietnam/ut / (h n ; vn ), was isolated in our laboratory from the same human specimen from which a/vietnam/ / (h n ; vn ) was isolated. there are no amino acid differences between vn and vn although there are two nucleotide differences, one in the na gene at position and the other in the m gene at position (in both cases, vn has adenine at these positions and vn has guanine). all experiments with vn virus was performed in enhanced biosafety level (bsl ) containment laboratories at the university of tokyo and kyoto university, japan, which are approved for such use by the ministry of agriculture, forestry, and fisheries, japan. cells. madin-darby canine kidney (mdck) cells were maintained in eagle's minimal essential medium (mem) containing % newborn calf serum. the genetic origin of the mdck cells was confirmed by dna fingerprinting (using random amplified polymorphic dna) conducted by takara bio japan. mdck cells were used for plaque assays to titrate viruses. experimental infection of nonhuman primates. two-to three-year-old male cynomolgus macaques, weighing . - . kg and serologically negative by neutralization assays for currently circulating human influenza viruses, were obtained from cambodia (obtained from japan laboratory animals, inc., tokyo, japan). all experiments with macaques were performed in accordance with the regulation on animal experimentation guidelines at kyoto university and were approved by the committee for experimental use of nonhuman primates in the institute for virus research, kyoto university. for the aerosol inoculation (aerosol method group), animals were anesthetized with ketamine via intramuscular injection and, by using the ultrasonic nebulizer ne-u (omron healthcare co, ltd, japan), ml of pfu/ figure . pathological findings in the lungs of infected monkeys. shown are representative pathological findings in the lungs of monkeys infected with vn by using the conventional (a,c; right-lower lobe of animal # - , whose pathologic score and score of detection of antigen-positive cells were and +, respectively) or aerosol (b,d; right-middle lobe of animal # - , whose pathologic score and score of detection of antigen-positive cells were and +, respectively) method at day post-infection with hematoxylin-eosin (he) staining (a,b) and immunohistochemistry for influenza viral antigen (np) (c,d). the animal id numbers correspond to those shown in tables , , and . scale bar is μm. scientific reports | ( ) : | doi: . /s - - - ml of virus was aerosolized and administered to each animal via an inhalation mask, which is not a tight-fitting mask and allows the individual to breathe the aerosol mist through the nose and mouth spontaneously. for virus inoculation through multiple routes, that is, the conventional method (the conventional method group), animals were anesthetized with ketamine via intramuscular injection and inoculated with a suspension containing a total of × pfu of virus through a combination of the intratracheal ( . ml), intranasal ( . ml per nostril), ocular ( . ml per eye) and oral ( . ml each to the left and right tonsil) routes. body temperature was monitored at , , , and days post-infection by use of a rectal thermometer. at the indicated timepoints post-infection, two or four macaques per group were euthanized for virologic and pathologic examinations. the virus titers in various organs and nasal swabs were determined by using plaque assays in mdck cells. pathologic examination. animal tissues were fixed in % phosphate-buffered formalin for pathologic examination. they were then processed for paraffin embedding and cut into -µm-thick serial sections. one section from each tissue sample was subjected to standard hematoxylin-and-eosin staining, while another was processed for immunohistochemistry with a mouse monoclonal antibody for type a influenza virus np antigen that reacts comparably with all of the test viruses. this antibody was produced in our laboratory and used for immunohistochemistry at : dilution. specific antigen-antibody reactions were visualized by use of , ′-diaminobenzidine tetrahydrochloride staining and a dako envision system (dako co. ltd., tokyo, japan). for statistical analyses, we used r (www.r-project.org) and lme to fit a linear mixed effects model to the virus titer data. different models were fitted for each biological question, namely, (i) the differences in virus titers between the groups over time (table ) , and (ii) infection of different organs (table ) , and infection of different lung parts (table and fig. ). in the first case, we used as fixed effects the different groups (i.e., the aerosol or conventional infection methods), and the time of the measurement. in the second case, we used as fixed effects the different groups (i.e., the aerosol or conventional infection methods), and the different organs in which the virus titers were measured. in both models, as random effects, we had intercepts for the different animals in the study. finally, we used the lsmeans package , to compare the groups for each model separately, and the p-values were adjusted using holm's method. p-values were considered significant if they were less than . . modified organisms of the university of tokyo and kyoto university, and, when required, by the competent minister of japan. all experiments were approved by the respective committees at the university of tokyo and kyoto university. all experiments with h n viruses were performed in enhanced biosafety level laboratories at the university of tokyo (tokyo, japan), and kyoto university (kyoto, japan), which are approved for such use by the ministry of agriculture, forestry and fisheries, japan. staff working in enhanced bsl- and bsl- ag wear disposable overalls and powered air-purifying respirators. the enhanced bsl- facilities at the university of tokyo and kyoto university include controlled access, effluent decontamination, negative air-pressure, double-door autoclaves, hepa-filtered supply and exhaust air, and airtight dampers on ductwork connected to the animal cage isolators and biosafety cabinets. the structure is pressure-decay tested regularly. all personnel complete biosafety and bsl- training before participating in bsl- -level experiments. human infection with a novel avian-origin influenza a (h n ) virus epidemiology of human infections with avian influenza a(h n ) virus in china a. h n influenza-continuing evolution and spread pandemic influenza as a current threat early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus pathogenesis of influenza a (h n ) virus infection in a primate model aberrant innate immune response in lethal infection of macaques with the influenza virus ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus a/vietnam/ / (h n ) influenza virus aerosol exposure and analytical system for ferrets comparison of the levels of infectious virus in respirable aerosols exhaled by ferrets infected with influenza viruses exhibiting diverse transmissibility phenotypes comparison of traditional intranasal and aerosol inhalation inoculation of mice with influenza a viruses experimental transmission of influenza virus infection in mice. i. the period of transmissibility influenza a virus challenge models in cynomolgus macaques using the authentic inhaled aerosol and intra-nasal routes of infection widespread virus replication in alveoli drives acute respiratory distress syndrome in aerosolized h n influenza infection of macaques identification and validation of nebulized aerosol devices for sputum induction lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes massive mobilization of dendritic cells during influenza a virus subtype h n infection of nonhuman primates experimental infection of macaques with a wild water bird-derived highly pathogenic avian influenza virus (h n ) molecular biology, and pathogenesis of avian influenza a (h n ) infection in humans the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h n -a review apoptosis and pathogenesis of avian influenza a (h n ) virus in humans h n infection of the respiratory tract and beyond: a molecular pathology study fitting linear mixed-effects models using lme least-squares means: the r package lsmeans we thank susan watson for scientific editing. we also thank naomi fujimoto, mikiko tanaka, kazue goto, yuko sato, hiromi sakawaki, and tomoyuki miura for technical assistance. competing interests: y.k. has received grant support from chugai pharmaceuticals, daiichi sankyo pharmaceutical, toyama chemical, and ttsumura co., ltd; royalties from medimmune; and is a co-founder of flugen.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -exbs tz authors: pumchan, ansaya; krobthong, sucheewin; roytrakul, sittiruk; sawatdichaikul, orathai; kondo, hidehiro; hirono, ikuo; areechon, nontawith; unajak, sasimanas title: novel chimeric multiepitope vaccine for streptococcosis disease in nile tilapia (oreochromis niloticus linn.) date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: exbs tz streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including nile tilapia (oreochromis niloticus linn.). vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. immunoproteomics analysis of s. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable rna and protein structure for protein expression. f and e were identified as the best candidates for a chimeric multiepitope vaccine. recombinant plasmids were constructed to produce a recombinant protein vaccine and dna vaccine system. overexpressed proteins were determined to be kda and kda in the e. coli and tk systems, respectively. the efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and dna vaccine was evaluated in nile tilapia, followed by s. agalactiae challenge at × ( ) cfu/ml. relative percentage survival (rps) and cumulative mortality were recorded at approximately – % and – %, respectively. these chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases. immunogenic protein characterization. proteins bound to a s. agalactiae antibody were eluted from protein a agarose and divided into two fractions. the first fraction was subjected to - % gradient sds-page to observe the protein features and compare the protein profile from serotypes ia and iii. the second fraction was subjected to lc-ms/ms mass spectrometry to identify the immunogenic proteins. the protein profile from the immunoprecipitation on d-sds-page demonstrated that the major protein (approximately kda) corresponded to rabbit immunoglobulin. however, several bacterial proteins could not be bound to rabbit immunoglobulin and were removed through the flow-through fraction (ft), whereas the protein that specifically bound to the anti-s. agalactiae antibody could be detected in the eluted fraction (fig. ) . comparative immunoproteomics analysis of s. agalactiae serotypes ia and iii was determined by lc-ms/ ms and assessed by a venn diagram ( supplementary fig. ). one hundred proteins were matched and identified between serotype ia and serotype iii via in-house protein databases, resulting in shared proteins between serotype ia and serotype iii. the protein expression levels of the common proteins were determined by hierarchical clustering (hcl). two groups of immunogenic proteins were demonstrated based on their abundance, and proteins were overexpressed in serotype iii, whereas there was a lower abundance of immunogenic proteins in serotype iii than in serotype ia (fig. ) . regarding specific antigen-antibody interactions, and proteins were uniquely identified in serotypes ia and iii, respectively (supplementary figs. , ) . linear β-cell epitope prediction and chimeric vaccine design. the epitopes of immunogenic proteins were predicted by the bcpreds server based on b cell epitopes to be used in chimeric multiepitope vaccine construction. in this study, not only immunogenic proteins from the immunoproteomics analysis were used but also other subunit vaccine candidates were subjected to epitope prediction and combined to produce a chimeric multiepitope vaccine. the amino acid sequences of the c-β protein (bac), surface protein rib (rib), lpxtg cell wall anchor domain-containing protein (spb ), surface immunogenic protein (sip), and cell surface protein heat map with hierarchical clustering (hcl) of normalized protein abundance reveals the differentially expressed immunogenic proteins. the expression value showed in the relative intensities ranges from the highest protein abundance (red) to the lowest protein abundance (green) expression value. alternatively throughout the backbone likely provides potential bioactivity . considering α/β fold structure, flavodoxin from escherichia coli [pdb accession code: chy] was utilized as a linker to combine the epitope fragments from five antigenic proteins. predicted epitopes were randomly displayed on the α-helix structure of flavodoxin, generating , designed models due to the variance of epitopes of bac and of epitopes of sip protein. after joining, protein conformation was examined by molecular modeling with , constructs. i-tasser and stereochemical qualitative allowance manifested from f and e showed appropriate potential tertiary structure with optimal c-scores between − and . f and e also demonstrated the highest score of the amino acid allowance region in the ramachandran plot. the f multiepitope model represented . %, . %, and . % of residues located in the most favored, allowed, and disallowed regions, respectively. meanwhile, the ramachandran plot regions for the e designed model comprised . %, . %, and . %, respectively, of the residues ( supplementary fig. ). the epitope arrangements in f and e were represented in a d structure of chimeric proteins, showing that all chosen epitopes were exposed to the protein surface. the five epitopes were displayed as α-helical layers surrounding chy linkers, which appeared as five-stranded parallel β-sheets at the structure's center, with the order (fig. ). codon optimization of chimeric multiepitope vaccines and plasmid construction. the ectopic expression of bacterial protein in the fish cells may not be achieved due to different codon utilization in the bacterial system. subsequently, codon optimization of the chimeric multiepitope vaccine was analyzed by geneart ™ 's gene optimization according to iso standards (registration no. ) to apply the codon bias of oreochromis niloticus. the region of an ideal gc content range-between % to %-was well optimized. moreover, negative cis-acting sites included internal tata-boxes, chi-sites and ribosomal sites; at-rich or gc-rich sequence stretches; rna instability motifs; repeat sequences; rna secondary structures; and splice donor and acceptor sites in higher eukaryotes, which were successfully removed from these chimeric multiepitope dna vaccine sequences. the best two predicted chimeric multiepitope vaccines were designated f and e . codon adaptation index (cai) presented f and e scores that matched in codon utilization with that of nile tilapia of . and . , respectively. the codon quality distribution index of f and e demonstrated that the codons within the dna sequence were distributed frequently in - positions at % and % ( supplementary fig. a-d) . the average gc content of both chimeric multiepitope vaccines was % ( supplementary fig. e ,f). single-stranded rna-folding prediction revealed the minimum free energy (mfe) secondary structure of f and e ( supplementary fig. the protparam server demonstrated a theoretical pi of . and a molecular mass of kda for f and e . the total number of negatively (asp and glu) and positively (arg and lys) charged amino acid residues of f was and residues, while for e , there were and residues, respectively. the estimated half-life of both chimeric multiepitope constructs was approximately h in mammalian reticulocytes (in vitro), more than h in yeast (in vivo), and over h in e. coli (in vitro). f showed aliphatic index and grand average of hydropathicity values of . and − . , respectively, whereas e showed values of . and − . , respectively. the f and e proteins were indicated to be stable proteins, as represented by instability indexes of . and . , respectively. antigenicity of the f and e chimeric multiepitope vaccines was predicted as . % and . % at a . % threshold for the bacterial model, consistent with antigenpro server prediction by representing . and . , respectively. these results indicate that both vaccine candidates have high potential antigenic www.nature.com/scientificreports www.nature.com/scientificreports/ properties. conformational b cell epitopes from the d protein structure computed by the discotope server demonstrated b cell epitope residues in both f and e at a − . threshold ( table ) . interestingly, the number of epitopes was reduced when computed at the − . and − . thresholds, with f showing and b cell epitope residue regions, respectively, while e contained only and b cell epitope residue regions, respectively (table ). recombinant plasmids harboring e and f were constructed, namely, pet a (+)_ e or _ f and pcdna . (+)_ e or _ f , which were used to determine the recombinant chimeric multiepitope vaccine expression (fig. ) . chimeric multiepitope protein expression was tested in a bacterial expression system and a fish cell (tk- ) culture expression system. these results demonstrated that both chimeric multiepitope proteins could be expressed in both systems, with the expression detectable within h in e. coli ( kda) and within days post-transfection in tk- cells ( kda) (fig. ). larger-sized chimeric multiepitope proteins in the e. coli expression system resulted from an additional tag at the n-terminus, which was contained in the pet expression vector. vaccine efficacy. after vaccination, fish were challenged with s. agalactiae, and infected fish showed clinical signs of streptococcosis disease, such as swirling swimming, opaque eye, exophthalmia and abscess. these moribund fish were collected, and bacteria were re-isolated, showing that they were infected with s. agalactiae serotype iii ( supplementary fig. s ) . www.nature.com/scientificreports www.nature.com/scientificreports/ dna vaccine efficacy testing showed that fish immunized with either f or e had cumulative mortality rates of . ± . % and . ± . %, respectively, which were not significantly different from those of the fkc-vaccinated fish (p > . ). however, in the control group [empty vector; pcdna . (+)], . ± . % mortality was observed at days post-challenge (fig. a ). the recombinant chimeric multiepitope protein vaccination showed that f and e produced cumulative mortality rates of . ± . % and . ± . %, respectively, which were significantly lower than those of the negative control group, at % (p < . ) (fig. a) . the f and e dna vaccines demonstrated similar patterns of rps, with . ± . % and . ± . %, respectively, which were not significantly different from those of the fkc-immunized fish ( . ± . %). however, they were significantly higher than those of the recombinant protein vaccines, which showed . ± . % and . ± . % for e and f , p < . , respectively (fig. b ). immune response. to determine the immune response, dot blot analysis of serum prepared from e -or f -vaccinated fish was used. it was demonstrated that the dna vaccine could gradually activate the production of fish antibodies from the st to the th week. the pattern of antibody response differed from that for the recombinant protein vaccine, with the highest activation of antibody production being significantly produced in the nd - rd week and suddenly dropping in the th week. the highest induction was observed in fkc-immunized fish (fig. ) . dot blot analysis of vaccinate fish sera against whole cell lysate of s. agalactiae serotype ia and iii demonstrated that fish vaccinated with recombinant protein vaccine e and f showed cross-reactivity to whole cell lysate of s. agalactiae serotype ia and iii ( supplementary fig. s ). for the reverse vaccinology approach, computational analysis using a variety of bioinformatics tools is robust and beneficial when identifying appropriate vaccine candidates . bacterial genomics and proteomics analysis indeed help researchers analyze proteins, short domains, and pathogenic epitopes that provide high immunogenicity and high antigenicity for multimeric vaccine development . therefore, immunoproteomics should be applied as a preliminary process to screen antigenic proteins and minimize potential candidates for vaccine development . several immunogenic proteins in this study were described previously, such as c a peptidase and laminin-binding surface protein (lmb), which are cell surface proteins that have an important function in www.nature.com/scientificreports www.nature.com/scientificreports/ chemoattractant activities and are proteins promoting invasion of group b streptococcus (gbs) , . however, the current immunoproteomics analysis from this study identified new immunoreactive proteins, such as bacteriocin transport accessory protein, dihydrofolate reductase, ssu ribosomal protein s p, transposase tnpa, , -alpha-glucan, cell wall surface anchor family protein, and the gtp-binding protein era. as expected, most of these are cell surface proteins, which are suggested to be associated with bacterial virulence , . subsequently, the identified immunoreactive proteins may be used in further vaccine development. multiepitope vaccines are an interesting issue since constructed vaccines designed by in silico analysis may elicit cellular immunity and provide effective responses , . it is known that immunodominant b cells could strongly induce both cellular and humoral immunity; thus, evaluation of b cell epitopes was performed to identify potent epitopes before integrating them to produce a multiepitope vaccine. moreover, this vaccine type is more efficient than whole antigens for controlling staphylococcus spp. infections , . from the present study, table . predicted conformational b-cell epitopes from d structure of designed chimeric multiepitope vaccines using discotope . server. www.nature.com/scientificreports www.nature.com/scientificreports/ linear b cell epitope prediction was assessed and identified potent epitopes from common immunogenic and virulence proteins that were present in serotypes ia and iii. the previous studies supported that one of the chosen proteins, sip, represented a highly conserved protein among gbs isolates and showed cross-protective immunity against gbs infections , , , . prediction of candidate antigenic proteins can be used to select the bacterial strains that carry antigenic genes, as well as to determine high expression levels in the target host and the accessibility of particular antigens in host organisms . therefore, these selected immunogenic proteins might be suitable for consideration in a rational vaccine design. rational chimeric multiepitope vaccine design was achieved by randomly combining epitopes from immunogenic proteins and conjugating with core structures of flavodoxin (β- - - chy) to produce a secondary structure with α/β folding. in addition to the α/β-type folding of flavodoxin, it was also useful to construct our chimeric multiepitope vaccine by forcing the chosen epitope segments to fit within α-helix loops and protrude out of the d-folded structure since that configuration benefits protein solubility by exposure to water molecules . additionally, this linker may promote the solubility of the constructed vaccine and help enhance the recognition of the vaccine by the host's immune system, which contributes to vaccine efficacy. f and e presented the most favored region of protein folding, with the stereochemical quality representing the disallowed region at only . %, which is acceptable since the minimum quality should be less than % . it is suggested that in silico analysis could design a chimeric multiepitope vaccine that could probably manifest effective properties , . to achieve a high level of protein expression in nile tilapia, codon optimization was conducted to improve the transcription and translation capability by removing all possible cis-acting sequence motifs, which may have a negative impact on protein expression. both proteins had a cai > . and a codon with frequent distribution (cfd) > %, which are acceptable for high expression in the target organism , . the gc content of f and e was optimized between - % and had a suitable thermodynamic ensemble free energy, which allowed rna folding and thermodynamic stability , . the overall points suggested that the modeled f chimeric multiepitope vaccine was clearly the best candidate vaccine. numerous effective single-serotype gbs vaccines have been reported, including vaccines for controlling streptococcosis in tilapia , , , , , , . however, it is known that single-serotype whole-cell inactivated vaccines have limitations during cross-prevention against different serotypes. for instance, a s. iniae vaccine (serotype i) could not protect atlantic salmon from infection by s. iniae (serotype ii) . meanwhile, mixed-serotype vaccines (serotypes iv and vii) could promote antiserum levels and enhance the survival rate of newborn pups against streptococcal infection . although formalin-killed vaccines generally provide highly protective effects compared with those of subunit vaccines and dna vaccines, the subunit and dna vaccines may replace the original formalin-killed vaccines or inactivated vaccines due to their promising efficacy, which are similar to those of inactivated vaccines, and longer shelf life , . evidence suggests that dna and subunit vaccines can efficiently trigger the immune system and promote protective efficacy, with an rps value greater than % , , , . nevertheless, these vaccines have limitations, such as their mass production costs, and they may require various optimizations to obtain the highest stable storage conditions , . regarding this idea, a chimeric multiepitope vaccine composed of different epitopes from different proteins common in both serotypes ia and iii was generated to achieve broad protection against different serotypes and increase their stability. interestingly, the designed chimeric multiepitope dna vaccine and protein vaccine exhibited effective prevention in nile tilapia against s. agalactiae, with efficacy similar to that of the whole-cell inactivated vaccine. this evidence supports the strategy of rational vaccine design through b cell recognition using in silico analysis. importantly, immunoproteomics analysis could assist the preliminary determination of suitable immunogenic proteins for vaccine development due to the distinct antigenic determinants that can mediate dissimilar immune responses. the criterion in immunogenic protein selection for vaccine development has focused on the ability of a particular protein to induce an immune response. among identified proteins shared in both serotypes, in addition to providing the highest bcpred scores (table ) , the proteins chosen were also reported as virulence proteins and used as vaccine candidates for streptococcosis disease prevention . for example, c-β protein (bac) can lead to antibody production through fc region binding of human iga . sip protein has been shown to mediate protection against streptococcal infection , , . additionally, the chosen immunogenic protein should be conserved among streptococcus spp., so it would be suitable for application in cross-reactive prevention among s. agalactiae serotypes , . moreover, it should be mentioned that peptide vaccines or epitopes with only amino acid residues may trigger immune responses through binding directly to mhc-i or mhc-ii molecules. these molecules localize to nonprofessional antigen-presenting cells. vaccines containing proteins with longer amino acid sequences can enhance the presentation of epitopes to dendritic cells due to t cell induction , , . herein, the comparative efficacy of both the f and e dna and recombinant protein vaccines indicated that the dna vaccine provided a higher efficacy than the recombinant protein vaccine. this result suggests that the dna vaccine can prolong the activation of the immune response by triggering both humoral and cellular immune responses , . moreover, the clearance rate of the recombinant protein vaccine in the host system may be faster than that of the dna vaccine. this difference implies that the dna vaccine can enter the host cell to produce chimeric multiepitope protein, with that protein existing in the host system for longer than the recombinant protein vaccine, thus enhancing its bioavailability. taken together, these data indicate that the antigen combination has shown promise for streptococcosis disease control in nile tilapia. this research demonstrated a novel platform for rational vaccine design based on chimeric vaccine development that used flavodoxin with a tim-barrel structure as a template. our chimeric protein backbone is suitable for presenting epitopes to be recognized by the host immune system. with epitopes, it could activate antibody production and demonstrated promising protection against bacterial disease similar to that of a whole-cell inactivated vaccine. this platform will promote the production of multivalent vaccines to control multiple diseases and for other applications in the future. experimental fish, bacterial strain and antibody. all male s. agalactiae-free nile tilapia (oreochromis niloticus linn.) were obtained from a commercial gap farm in thailand. the experiments were conducted in accordance with guidelines approved by the national research council of thailand. the experimental fish were anesthetized with clove oil to minimize stress during vaccination and challenge testing. s. agalactiae serotypes ia and iii were cultured as described previously . s. agalactiae serotype iii was used for polyclonal antibody (pab) production, which was kindly provided by prof. ikuo hirono, tumsat, japan. antibody against igm of nile tilapia was kindly provided by assist. prof. eakapol wangkahart. mahasarakham university, thailand. immunoproteomics analysis. s. agalactiae was grown in bhi broth at °c with agitation until reaching exponential phase. bacterial cells were collected by centrifugation, lysed in µl of lysis buffer [tris-buffered saline (tbs) with % tween- and . % lysozyme] and incubated at °c for min following sonication on ice. protein a agarose beads (cell signaling, usa) were added to the bacterial protein lysate, and nonspecific proteins were removed by min of centrifugation at , × g at °c. clarified supernatant was supplemented with % glycerol and then with a pab specific to s. agalactiae serotype iii ( : dilution). then, µl of protein a agarose beads were added to separate bound immunogenic proteins, and the bound proteins were separated by acetone precipitation [ : (v/v)]. precipitated proteins were solubilized in mm tris-hcl with . % sds, and a lowry assay was used to measure the protein concentration. the protein profile was assessed by fractionating µg of protein on a nupage - % bis-tris protein gel (thermofisher, usa). µg of immunogenic protein was mixed with a lysis buffer ( . % rapidgest sf in mm ammonium bicarbonate) and mm dtt in mm ammonium bicarbonate at °c for h. this step was followed by incubation with mm iodoacetamide (iaa) in mm ammonium bicarbonate at room temperature for min in the dark. the protein solution was cleaned up by a zeba spin desalting column before digestion with ng of sequencing-grade trypsin (promega, germany) at °c for h. tryptic peptides were dried at °c under a vacuum and then protonated with . % formic acid in lc water before injection into an lc-ms/ms. the tryptic peptides' immunoproteomics profiles were analyzed using an ultimate ™ nano/capillary lc system (dionex, uk) and hybrid quadrupole q-tof impact ii ™ (bruker daltonics gmbh, germany) equipped with a nano-captivespray ion source. first, nl of extracted peptide was subjected to a trapping column (thermo scientific, pepmap , c , μm i.d. × mm) through a full loop injection before being resolved in an analytical column (pepswift c nano column, μm × cm, i.d.) at °c. the linear gradient method was used to elute peptides with mobile phase a ( . % formic acid in water) and mobile phase b ( . % formic acid in % acetonitrile) at a . µl/min constant flow rate into the mass spectrometer. electrospray ionization was conducted at . kv using captivespray. mass spectra (ms) and ms/ms spectra were fully acquired in positive ion mode (compass . for otofseries software, bruker daltonics). mass accuracy was assessed using positive detection mode after internal calibration with sodium trifluoroacetate (na-tfa) within . ppm. raw lc-ms/ ms spectra were collected using compassxport version . . . (bruker daltonics gmbh, germany) to convert all spectra into the mzxml data format. the mzxml files of the lc-ms/ms datasets for label-free quantification of peptides were evaluated based on the ms profile by maxquant software. chimeric multiepitope vaccine design. the linear b cell epitope was predicted by bcpred . the scop and cath databases were used to design an appropriate chimeric multiepitope vaccine structure (an: wp_ ). a d structure was rendered by i-tasser (iterative threading assembly refinement) using the qualifying c-score value as a confidence score . to refine the tertiary structure, the derived i-tasser results in the pdb files were prepared using the galaxyrefine server, which performed a repeated structure perturbation, and the best structural relaxation candidates were chosen . moreover, to obtain the best chimeric multiepitope vaccine candidates, the residues were determined according to residue stereochemical quality for all the refined chimeric multiepitope models and validated by the procheck program v. . . to generate ramachandran plots . codon optimization. amino acid sequences were reverse-translated to nucleotide sequences using nile tilapia codon usage (oreochromis niloticus [gbvrt]: ). the codon adaptation index (cai) of the designed vaccine candidates' nucleotides was analyzed by an optimizer program (http://genomes.urv.es/optimizer/) and combined with geneart tm 's gene optimization process (thermo fisher scientific, usa). the secondary structure of the single-stranded rna folding and free energy of the thermodynamic ensemble were calculated by the rnafold web server . the optimized dna sequence was synthesized by geneart ® gene synthesis (thermo www.nature.com/scientificreports www.nature.com/scientificreports/ to verify the ectopic expression of the chimeric multiepitope dna vaccine, pcdna . (+)_ e or _ f was transfected into tk (tilapia kidney ) tilapia cells using effectene transfection reagent (qiagen, germany). the transfected fish cell cultures were maintained with leibovitz's l- media containing % fbs and penicillin-streptomycin at °c, and dna vaccine expression was determined after week. recombinant chimeric multiepitope protein was purified by ni-nta agarose beads (qiagen) with a gradient concentration buffer of imidazole ranging from mm to mm. subsequently, the gel filtration chromatography method was performed by fast protein liquid chromatography (fplc) incorporated with a hiprep / & / sephacryl s- high-resolution column (ge healthcare, usa) using a × pbs buffer with a ml/min flow rate. recombinant protein detection was confirmed by sds-page analysis and western blot analysis using an anti-his tag antibody (recombinant protein vaccine) or an anti-flag (rabbit igg) (dna vaccine) and anti-rabbit antibody conjugated to ap (alkaline phosphatase). vaccine efficacy analysis. to evaluate vaccine performance, nile tilapia (o. niloticus) were immunized with chimeric multiepitope vaccines (recombinant protein and dna vaccines), followed by bacterial challenge. a total of experimental groups, namely, ) the f recombinant protein vaccine, ) e recombinant protein vaccine, ) f dna vaccine, ) e dna vaccine, ) formalin-killed (fkc) s. agalactiae vaccine , and ) pcdna . (+) [empty vector], were conducted in triplicate. before vaccination, streptococcosis-free nile tilapia ( ± g) were transferred into glass aquarium tanks containing l of water for one week. after a week of acclimatization, fish were vaccinated according to above mentioned groups. all fish were maintained under running and aerated water at ± °c and fed with commercial pellet feed twice a day. for the chimeric multiepitope protein vaccination, purified f and e proteins were mixed with montanide isa (seppic, france) in a : ratio prior to intraperitoneal injection with µg of protein per fish. for the chimeric multiepitope dna vaccine, plasmid dna of f and e were purified by ultracentrifugation using a cscl gradient and dissolved in te buffer (ph . ) to obtain a concentration of . µg/µl. the dna vaccine was applied to the fish with µg of dna through intramuscular injection. fkc and pcdna . (+) were used as positive and negative controls, respectively. the schedule of vaccine efficacy analysis and immune response analysis was demonstrated in supplementary fig. . for the immune response analysis, blood was drawn from the caudal vein to separate serum for the immunoblotting assay, and those fish were transferred to another separate tank. the analysis was performed every week, using fish in each treatment from the st week to the th week. after one month of vaccination, vaccinated fish in each treatment group were taken from among the remaining fish for serum collection and anesthetized with eugenol before challenge with s. agalactiae (serotype iii) at × cfu/ml through ip administration. mortality and clinical signs of infected tilapia were recorded daily for weeks. the brain, head kidney, and liver were collected from moribund fish for bacterial isolation and identification . cumulative mortality and relative percentage survival (rps) were calculated . a one way analysis of variance (anova) was used for statistical analysis and p < . was considered significant. to detect the antibody response after immunization, antibody production was evaluated through dot blot analysis using the minifold ® i dot blot system (ge healthcare, germany). briefly, µl of purified e , f proteins, or a whole-cell lysate of s. agalactiae ( µg/ml) were spotted on a nitrocellulose membrane and blocked with blocking solution ( . % bsa in tbst) before adding µl of serum of the different treatment groups as above mentioned. then, the membrane was probed with a primary antibody (anti-igm at : , ) for . h, followed by washing times with tbst buffer and min of incubation with an anti-mouse igg hrp-linked ab ( : , ). subsequently, the signal was detected with a chemidoc ™ imaging system (bio-rad) after adding a substrate reagent (perkinelmer, usa). the integrated density of the dot blot was analyzed by imagej (version .x) . five different piscidins from nile tilapia, oreochromis niloticus: analysis of their expression and biological functions prevention and control of viral disease in aquaculture development of a quantitative pcr assay for monitoring streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia parasites and diseases streptococcus agalactiae serotype distribution and antimicrobial susceptibility in pregnant women in gabon microevolution of streptococcus agalactiae st- from australia indicates dissemination via imported tilapia and ongoing adaption to marine hosts or environment molecular serotyping, virulence gene profiling and pathogenicity of streptococcus agalactiae isolated from tilapia farms in thailand by multiplex pcr a microwave-irradiated s. agalactiae vaccine provides partial protection against experimental challenge in nile tilapia oreochromis niloticus efficacy of an experimentally inactivated s. agalactiae vaccine in nile tilapia (oreochromis niloticus) reared in brazil development of live attenuated streptococcus agalactiae vaccine for tilapia via continuous passage in vitro a recombinant truncated surface immunogenic protein (tsip) plus adjuvant fia confers active protection against group b streptococcus infection in tilapia development and efficacy of feed-based recombinant vaccine encoding the cell wall surface anchor family protein of s. agalactiae against streptococcosis in oreochromis sp safety and immunogenicity of an oral dna vaccine encoding sip of streptococcus agalactiae from nile tilapia oreochromis niloticus delivered by live attenuated salmonella typhimurium protective efficacy of cationic-plga microspheres loaded with dna vaccine encoding the sip gene of s. agalactiae in tilapia assembly and role of pili in group b streptococci dentification of universal group b streptococcus vaccine by multiple genome screen protection of nile tilapia (oreochromis niloticus l.) against streptococcus agalactiae following immunization with recombinant fbsa and alpha-enolase designing 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classification of proteins extended, integrating scop and astral data and classification of new structures aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr viennarna package . . algorithms for molecular biology dna synthesis and biological security prediction of n-glycosylation sites in human proteins precision mapping of the human o-galnac glycoproteome through simplecell technology protein identification and analysis tools on the vaxijen: a server for prediction of protective antigens, tumor antigens and subunit vaccines high-throughput prediction of protein antigenicity using protein microarray data first report of streptococcus agalactiae isolated from oreochromis niloticus in piura, peru: molecular identification and histopathological lesions dna extraction from . µm sterivex filters and cesium chloride density gradient centrifugation potency and efficacy test of a vaccine in addition with adjuvant against koi herpesvirus in koi (cypronus carpio) nih image to imagej: years of image analysis the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to s.u. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - l d ew authors: lv, yang; hu, guangyao; wang, chunyang; yuan, wenjie; wei, shanshan; gao, jiaoqi; wang, boyuan; song, fangchao title: actual measurement, hygrothermal response experiment and growth prediction analysis of microbial contamination of central air conditioning system in dalian, china date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: l d ew the microbial contamination of central air conditioning system is one of the important factors that affect the indoor air quality. actual measurement and analysis were carried out on microbial contamination in central air conditioning system at a venue in dalian, china. illumina miseq method was used and three fungal samples of two units were analysed by high throughput sequencing. results showed that the predominant fungus in air conditioning unit a and b were candida spp. and cladosporium spp., and two fungus were further used in the hygrothermal response experiment. based on the data of cladosporium in hygrothermal response experiment, this paper used the logistic equation and the gompertz equation to fit the growth predictive model of cladosporium genera in different temperature and relative humidity conditions, and the square root model was fitted based on the two environmental factors. in addition, the models were carried on the analysis to verify the accuracy and feasibility of the established model equation. with the large-scale use of central air conditioning system and the improvement of people's living standard, more and more attention has been paid to the increasingly serious problem of indoor air pollution. studies showed that air handing unit is an important source of microorganisms for indoor biological pollution, and some microorganisms tend to stay in the dust of air conditioning units with the appropriate temperature and humidity environment. the microorganisms grow and then enter the indoor space through the air, resulting in the destruction of indoor air quality [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . national institute for occupational safety and health (niosh) conducted a study of buildings, and the research results showed that among the sources of indoor air pollution, central air-conditioning system pollution accounted for % . summary of measurement by professor fanger showed that air-conditioning systems accounted for % in the indoor pollution sources . based on the supervision and inspection of air conditioning and ventilation system in the public places of china's provinces, municipalities and autonomous regions by china academy of building research and the chinese center for disease control and prevention, it was found that more than % of the central air conditioning systems could not meet china's national sanitary standard . thus, air conditioning system should eliminate negative impact caused by its own, on this basis, it may relate to positive effect of the ventilation. in recent years, h n , sars and other popular virus spread [ ] [ ] , and some researches showed that the hygienic, reasonable air conditioning systems were important to reduce damage [ ] [ ] . therefore, microbial pollution in central air conditioning system has become a critical topic in the field of indoor air pollution. studies showed that the filter, cooling coil, humidifier, and cooling tower in central air-conditioning system were easy to microbial breeding [ ] [ ] [ ] [ ] [ ] [ ] . in this study, a venue in dalian was selected as the research object. as the working condition of the air conditioning system was down, the environment parameters were measured, and microorganisms existing on the wind pipe, filtering net, surface cooler, and condensate water, on the floor and in the air were collected. besides, according to the tested microbial density and the identified genome sequence of collected microorganisms, the hygrothermal response experiment of dominant fungal was detected, and the fitting analysis was carried out based on the prediction model, followed by a series of statistical analysis. the aim of the present study was to clarify characteristics of the microorganisms in air conditioning systems, and the study would be helpful for policymakers and hvac engineers to develop the appropriate strategies and to conduct the bacteria and fungi characteristic assessments in hvac system. preliminary survey. the object of study is a venue in dalian, which covers a total area of m and building area is m . the aboveground part includes a swimming pool, ball training venues, gymnasium, the lounge room and the clinic. the underground part consists of a table tennis hall, air conditioning equipment rooms and reservoir area. the whole building is centralized-controlled by the central air conditioning room, which includes two air handling units. two measured units were all air system, which only had a coarse efficiency filter, and the unit is also provided with a heater, cooler and fan etc. both units are the primary return air system and the filters are removable types. the running time of the air conditioning system is from may to october, and the daily operation period is : - : . all components are cleaned every two years. when the measurement was carried on, the unit a and b were both cleaned a year ago. both units were closed during the sample collection. measurement method. the actual measurement is divided into two parts: the environment parameter measurement and air unit sampling. first, the temperature, humidity, and co concentration were automatically recorded by the temperature, humidity and co monitor (mch- sd, japan). second, the disinfected planktonic microorganism sampler (hkm series dw- , china) was installed where fresh air and return air mixed. once installing the sampler, we loaded medium in the sampler and set the parameter of air flow in sampler as l loaded medium. after the sample collection, the petri dishes must be sealed for preservation. finally, according to the hygienic specification of central air conditioning ventilation system in public buildings of china , we sampled the dust by using sterile non-woven ( mm* mm) on components of unit a and b, respectively, and each sampling point covered a cm* cm area at the sampling area. the non-woven fabrics were put into sterile water and stirred to make sure that organic substance on the non-woven was fully dissolved in sterile water. then, the samples of sterile water containing organic substances were prepared in times and times diluted concentration, respectively. there are sampling points in unit a and points in b, and two measuring point positions of the units are shown in fig. . the microorganisms collected in the air were directly cultured, and the samples in the dust were times and times diluted and μ l of the sample was inoculated into the two kinds of solid culture media. beef extract peptone medium was used for cultivating bacteria and potato dextrose agar was used for cultivating fungus , . each dilution was done in parallel samples to reduce the error, and final results showed the average value. the blank samples and test samples were set up for each of the cultures. if there is no colony detected on the blank sample, the test results are valid. both field test and laboratory measurements were performed in accordance with the hygienic specification of central air conditioning ventilation system in public buildings requirements . genome sequencing. only a small part of microorganisms are cultivable. therefore, the traditional cultivation method can not harvest all the species in ecological samples . fungal genome sequencing is an emerging method to identify the microbial genome, which could directly indicate related species information from environment samples . fungal amplicon sequencing analysis was used in this study, because the existing research showed that fungal spores have stronger vitality than other microorganisms in the air, and fungi dominated the microorganism in air conditioning systems. therefore, this method was mainly used to identify fungi in this study , - . environment parameters in air handling units. temperature, humidity and co concentration of unit a and b are shown in table . unit a is located in the ground floor (b ), and the unit b is located on the ground floor. compared to the unit b, the humidity of unit a is higher, and the temperature is lower. microbial colony analysis. the distribution density of bacteria and fungi in the unit a is obtained through statistics, as shown in fig. . the concentration of airborne fungus was cfu/m , and the concentration of airborne bacteria was cfu/m . the unit a showed the obvious microbial contamination status, though all components and airborne microorganism meet the hygienic specification of central air conditioning ventilation system in public buildings of china . the microbial distribution in filter net is central < edge < bottom and bacteria accounted for a larger proportion; the microbial distribution in surface cooler is center > against the wall > edge, and fungi accounted for a large. the fungal contamination in the air is more serious than the bacteria. the distribution density of bacteria and fungi in the unit b were obtained through statistics, as shown in fig. . the concentration of airborne fungus was cfu/m , and the concentration of airborne bacteria was cfu/m . parts of the measuring point in the unit b were polluted seriously. the bacterial colonies in the corner and the ground of the surface cooler were beyond the hygienic index (≤ cfu/cm ) in the hygienic specification of central air conditioning ventilation system in public buildings of china regulates . limited by unit placement, there were less measuring points in unit b, and we chose the same measuring points in both units for comparison (centre of surface cooler, surface cooler against wall, corner of surface, and ground of surface cooler). the comparison between unit a and b indicates that the bacterial density in unit a was less than that in the same sampling point in unit b, but the fungal density in unit a was more than that in the same sampling point in unit b. if the cleaning and disinfection is not enough before the air conditioning system running, it may make the fungus to enter the indoor environment, which results in make the pollution of indoor air. compared with cooling coil, the fungus contamination is worse in the floor dust and the air suspension. during the actual measurement, it is found that the unit internal is unprecedentedly narrow and low intensity of illumination in a closed state. according to the description by technicians, it is easy to trample damage to the underground pipes, which leads to the disinfection and cleaning work rarely in the unit. fungal genome sequencing analysis. in this study, we analysed the samples from the sampling points a , b , and b by amplicon sequencing information analysis, respectively named a a, b a, and b a. all collected samples in the air conditioner were transferred to the eppendorf tubes and processed with the extraction step. samples were resusponded in tens buffer with sds and proteinase k as described by vinod . after incubation at °c, phenol/chloroform/isoamyl alcohol was added to remove proteins, and the nucleic acid was precipitated with isopropanol and sodium acetate ( . m). total dna was dissolved in × te after washing with % ethanol. and then the quality and quantity tests were conducted by agarose gel electrophoresis, . for pcr product, the jagged ends of dna fragment would be converted into blunt ends by using t dna polymerase, klenow fragment and t polynucleotide kinase. then add an ' a' base to each ' end to make it easier to add adapters. after all that, fragments too short would be removed by ampure beads. for genomics dna, we use fusion primer with dual index and adapters for pcr, fragments too short would be removed by ampure beads too. in both cases, only the qualified library can be used for sequencing. the quality and quantity of libraries were assessed using the bioanaylzer (agilent technologies) and the steponeplus real-time pcr system (applied biosystems). the raw data generated by miseq and hiseq sequencers was processed to eliminate the adapter pollution and low quality to obtain clean reads. the qualified clean data was used for the further bioinformatics analysis. firstly, paired-end reads with overlap were merged to tags by software flash (v . . ) , and then tags were clustered to otu at % sequence similarity using usearch (v . . ) . secondly, taxonomic ranks were assigned to otu representative sequence using ribosomal database project (rdp) na, e bayesian classifier v. . . at last, alpha diversity, beta diversity and the different species screening were analyzed based on otu and taxonomic ranks by mothur (v . . ) . in order to fully understand the community structure of fungal sample and analyse fungus microbial diversity, while excluding errors that human operation brings, genome sequencing method in fields of molecular biology was employed in this study to obtain micro biological information. illumina company developed miseq method with higher flux and simple operation and lower cost for genome sequencing. besides, the synthesis of side edge sequencing method with higher reliability is more suitable for laboratory community structure. the high-throughput sequencing was found to be useful to characterize compositions and diversities of moulds. the gene sequence of the test samples from genome sequencing was dealed with, such as stitching and matching, and the sample had a total of high quality fungal sequences, with an average length of bp. the optimized sequence and the average length of the sample are shown in table . otu and abundance analysis. stitching and optimising the tags, in order to be the otu (operational taxonomic units) for species classification, gathering in the % similarity, and the statistics of each sample in the abundance of information in each otu were done , [ ] [ ] . rank otu curve is a form of species diversity in the sample, which can explain two aspects of sample diversity, that is, the richness and evenness of species in the sample. the richness of species in the samples represented by the horizontal length of the curve is wide, so that the sample species is more abundant. the uniformity of species in the samples from the curve reflects the longitudinal axis of the shape. that the curve is flat means that the sample has a higher composition of the species evenness. from fig. , the species composition of b a is the most abundant, and the uniformity is the highest. sample diversity analysis of observed species. alpha diversity is the analysis of species diversity in a single sample , including the species observed index, shannon index, etc. the greater the two indices are, the more abundant species is in the sample. the species observed index reflects the richness of the community in the sample, which also refers to the number of species in the community, without taking into account the abundance of each species in the community. shannon index reflects the diversity of the community, and the species richness and evenness of species in the sample community. in the case of the same species richness, the greater the evenness of the species is in the community, the greater the diversity of the community is. observed species exponential dilution curve. random sample in the processed sequence, draw the sequence number for the abscissa and the corresponding species number can be detected as the ordinate, so as to form a curve production dilution curve, shown in fig. (a) . with the increase of the sample quantity, the number of species increase and gradually become stabilized. when the curve reaches the plateau, it can be considered that the depth of the sequencing has been basically covered all the species in the sample. at the same time, the observed species index can reflect the richness of community in the sample, that is, the number of species in the community. it can be seen that the distribution of fungal species richness is b a > b a > a a. shannon exponential dilution curve. shannon index is affected not only by species richness in the sample community, but also by the evenness of species. in the case of the same species richness, the greater the evenness of the species is in the community, the more abundant diversity of the community is. it can be seen in the fig. (b) that the fungal species diversity of the unit b is significantly more complex than the unit a, and the similarity of species diversity in two sampling points of unit b was very high. composition of microbial samples. figure illustrates the species composition proportion of the three sampling points, and the proportion was redrew by removing the strains which were not detected in the sample. the results are shown in table . the species with the largest proportion is the dominant fungi. according to the fungal genome sequencing analysis results, fungal components in different units at the same sampling were different, and that in the same unit at different sampling points were roughly similar. they were caused by the different environmental conditions. on the center of air cooling coil in unit a, candida accounted for %; on the center and against the wall of the air cooling coil in unit b, cladosporium accounted for %, accompanied by alternaria, emericella and other fungus. cladosporium is usually rich in outdoor air, but they will also grow on the indoor surfaces when the humidity is high. existing research shows that the cladosporium spore is an extremely important allergen in the airborne transmission, which could cause asthma attacks or similar respiratory disease in patients with allergic reactions . some species of candida is a kind of conditional pathogenic fungi in the human body. growth prediction analysis of models. traditional microbial detection generally have the characteristics of hysteresis, which cannot play the role of prediction, but the use of mathematical models to predict the growth of microorganisms can timely and effectively predict the growth of microorganisms. therefore, it is very important to study the growth prediction model of the fungi in the air conditioning system. according to environmental conditions mentioned before, we established growth kinetics prediction model of cladosporium spp. to predict the rapid fungal growth in the experimental conditions, which can provide a theoretical basis for air microbial contamination prediction system and help evaluate the health risk inside buildings. the models were fitted by origin software (version ) and matlab r a, and the fitting conditions of logistic model and gompertz model were compared under different temperature and humidity conditions. the fitting effect between these two models and the fitting results of the two models were compared, and the corresponding model parameters were obtained. in addition, the square root model was fitted based on the two environmental factors. experimental study on the hygrothermal response of fungus. laboratory studies have revealed that fungal growth and reproduction are affected by water, temperature, nutrients, micro-elements, ph, light, carbon dioxide, and oxygen tension .the most relevant determinants of fungal proliferation in the building context are water/moisture and temperature, and to a certain extent those factors affect other environmental factors such as substrate ph, osmolarity, nutrient, material properties etc , .in order to lay the foundation for the fitting model, and to study the growth characteristics of fungi in different temperature and relative humidity, we set an experimental study on the hygrothermal response of fungus. from the results of fungal genome sequencing and literature research - , we selected cladosporium spp. and penicillium spp. as the research objects which are both common in air conditioning systems.this paper mainly studied the status of microbial contamination in air handling units so that the air temperature of each part of the air handling unit should be considered. the temperature gradient of °c − °c − °c and relative humidity gradient of %− %− %− % were selected as experimental hygrothermal conditions. the results of hygrothermal experiments are shown in figs , , . it can be known that growth rate of cladosporium spp. is faster than that of penicillium spp., in any experimental conditions, which is the essential characteristics of a strain, is hygrothermal response control method cannot change. these data indicated that low rh environments can reduce or even inhibit fungal growth. this observation agrees with findings by w. tang and pasanen , . growth prediction analysis based on logistic model. logistic model is a typical model of biological ecology, which has been widely used in the field of biological ecology . according to the actual research, the following formula equation ( ) was obtained after the appropriate change of the logistic equation. n was the colony growth diameter, cm; t was the microbial growth culture time, h; a , a , x , p as the model parameters. it can be seen from the table , the fitting curve of logistic model is similar to the experimental results. at °c and °c temperature conditions, the model's fitting effect is excellent, and r is greater than . ; at °c temperature conditions, the model fitting effect is not as good as other temperature conditions. predicting the growth of microorganisms. the pmp program developed by the ministry of agriculture to establish the model of pathogenic bacteria is the basic model for the study of gompertz equation. gompertz model has been widely used in the field of biology. gompertz model expression was as equation ( ): c n was the colony growth diameter, cm; t was the microbial growth culture time, h; a, a, k, x c as the model parameters. it can be seen from the table that the fitting curve of gompertz model is better fitted to the measured parameters. at the same temperature, with the increase of relative humidity, gompertz model fitting effect is better; the model is well fitted at the temperature of °c and the fitting effect is better than °c and °c temperature conditions. the fitting of logistic model to the growth of the fungus is better than that of the gompertz model. the two models are tested by the deviation factor b f and the accuracy factor a f in the mathematical model test. staphylococcus xylosus were studied by mcmeekin . they found that when t min is fixed, for each ϕ , the relationship between growth rate and temperature can be described by using the square root model. the combined effects of these two variables can be expressed by the modified equation ( ): in the formula, u is the growth rate of fungus, cm/h; b is the coefficient; t is the culture temperature, °c; t min is the most important parameter of square root equation, and it refers to the lowest temperature when the growth rate is zero, °c; ϕ is relative humidity of the cultivation, %. by using the logistic primary model, the predictive value of the growth rate of the cladosporium colony growth rate (instantaneous velocity) was obtained, as table shows. through the model fitting, the parameters of the square root model could be obtained, as table shows, and the model fitting of predicting growth of cladosporium was shown as fig. . the model equation of b f value was between . - . , indicating that the model used to predict the range of the experimental environment in cladosporium colony growth condition. at the same time, the a f value of the model was . that is closed to , which shows that the model has high accuracy. table . model fitting and model parameters of double factor square root. this study selected two central air conditioning systems at a venue in dalian as the objects. actual measurement and a series of studies were carried out on microbial pollution characteristic, and the results are shown as below: ( ) the bacterial colony forming units of the two measuring points in unit b were cfu/cm and cfu/cm , respectively, which exceeded the hygienic specification of central air conditioning ventilation system in public buildings of china (≤ cfu/cm ), and the rest of the test points met the relevant standards of china. the distribution of bacteria was more than fungi, and the concentration was higher. with the total characteristics of different distribution density, the area of dust associated microorganisms and the air pollution were more serious. ( ) alternaria spp., candida spp., cercospora spp. and cladosporium spp. existed in both units. the candida spp. accounted for % in unit a, and the cladosporium spp. occupied % in unit b. the composition of fungi in b was more complicated. two dominant fungi are both deleterious to health, so the timely maintenance and cleaning are required. it is suggested that the operating space should be reserved in the air conditioning room, so as to avoid incomplete cleaning and disinfection. ( ) within the experimental temperature and relative humidity, with the increase of relative humidity or temperature, the colony growth of the same strain showed an increasing trend. for the prediction model of the fungus growth, the study found that the overall fitting effect of logistic model is better, and r values were greater than . . logistic model for the cladosporium spp. growth was better than gompertz model. at the same time, considering the influence of temperature and relative humidity, the square root model can well predict the growth of cladosporium spp. it provides a theoretical basis for the growth of fungi in the air conditioning system under the hygrothermal environment conditions. why, when and how do hvac-systems pollute the indoor environment and what to do about it? the european airless project the control technology of microbial contamination in air conditioning system overview of biological pollution prevention methods in air conditioning system. heating 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database project: improved data processing and web-based tools naive bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy study on the relationship between dominant fungi in the air and the allergic asthma in children field guide for the determination of biological contaminants in environmental samples separate effects of moisture content and water activity on the hyphal extension of penicillium rubens on porous media investigation and review of microbial pollution in air conditioning systems of public buildings. heating ventilating and air conditioning microorganisms and particles in ahu systems: measurement and analysis the indoor fungus cladosporium halotolerans survives humidity dynamics markedly better than aspergillus niger and penicillium rubens despite less growth at lowered steady-state water activity effects of temperature, humidity and air flow on fungal growth rate on loaded ventilation filters fungal growth and survival in building materials under fluctuating moisture and temperature conditions mathematical modeling of growth of salmonella in raw ground beef under isothermal conditions from to °c model for combined effect of temperature and salt concentration/water activity on the growth rate of staphylococcus xylosus the study is supported by the national nature science foundation of china ( ), beijing key lab of heating, gas supply, ventilating and air conditioning engineering (nr k ), the fundamental research funds for the central universities (dut qy ) and the urban and rural housing construction science and technology plan project ( -k - ). key: cord- -yyfgl x authors: guo, jinyue; li, fei; qian, shaoju; bi, dingren; he, qigai; jin, hui; luo, rui; li, shaowen; meng, xianrong; li, zili title: tgev infection up-regulates fcrn expression via activation of nf-κb signaling date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: yyfgl x it has been well characterized that the neonatal fc receptor (fcrn) transports maternal igg to a fetus or newborn and protects igg from degradation. we previously reported that fcrn is expressed in a model of normal porcine intestinal epithelial cells (ipec-j ). transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (tgev). how porcine fcrn (pfcrn) expression is regulated by pathogenic infection remains unknown. our research shows that ipec-j cells infected with tgev had up-regulated pfcrn expression. in addition, the nf-κb signaling pathway was activated in ipec-j cells by tgev infection. furthermore, treatment of tgev-infected ipec-j cells with the nf-κb-specific inhibitor bay - resulted in down-regulation of pfcrn expression. transient transfection of pfcrn promoter luciferase report plasmids with overexpression of nf-κb p transcription factor enhanced the activation of the luciferase report plasmids. we identified four nf-κb transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. together, the data provide the first evidence that tgev infection up-regulates pfcrn expression via activation of nf-κb signaling. immunoglobulin g is a major ig isotype in mucosal secretions and is involved in host defense. it has now been years since the remarkable foresight by f.w.r. brambell, who described a saturable receptor that transports maternal igg to a fetus or newborn. a few years later, he put forth the hypothesis of the presence of a similar receptor that protected igg from degradation, eventually identified as the neonatal fc receptor (fcrn) . fcrn was originally isolated from the intestine of neonatal rodents and identified as the receptor responsible for the transmission of maternal antibodies from mother to pup [ ] [ ] [ ] [ ] . in recent decades, researchers have showed that fcrn is most closely structurally related to the major histocompatibility complex class i molecule and is composed of a heavy chain associated noncovalently with β -microglobulin (β m) , . fcrn was also shown to bind igg at the ch -ch interface in a ph-dependent way. binding occurs in acidic (ph ≤ . ) environments, and igg is released at neutral (ph ≥ . ) conditions , . fcrn is a transport receptor which mediated transfer of iggs across the human placental barrier or the rodents intestinal epithelial barrier to a fetus or newborn , , . fcrn, therefore, plays a major role in the passive acquisition of maternal immunity by newborn mammals. in addition, fcrn is capable of protecting igg from degradation and maintaining igg levels in the bloodstream . fcrn has been indicated to be expressed in a variety of mammalian species, including mouse, human, rat, sheep, cow, pig, possum and camel . the level of fcrn expression plays an important role in controlling igg levels in tissues and blood . some studies have shown that mice deficient in either β m or the heavy chain of fcrn fail to transport igg, meanwhile the serum half-life of igg is shortened , . more recently, several publications indicated that transgenic (tg) mice that over-express bovine fcrn in the mammary gland have increased igg levels in both milk and serum . meanwhile, some researchers have reported fcrn overexpression by tg modification in mice and rabbits not only prolongs the igg half-life but also enhances the humoral immune response of these animals [ ] [ ] [ ] . more specifically, these tg animals displayed significantly larger spleens containing a higher number of ag-specific b cells and plasma cells in response to immunization, increased antibody diversity and prolonged ag-specific igg half-life . this augmented immune response is also reflected in the ability of fcrn tg mice to produce high levels of ag-specific antibodies, b cells and plasma cells to weakly immunogenic targets or evade recognition by the immune system . nuclear factor-κ b (nf-κ b) is a family of transcription factors that mediates signal-induced expression of numerous genes involved in the innate and adaptive immune responses, inflammation, and autoimmune diseases . some articles have reported that nf-κ b signaling regulates functional expression and function of the human and bovine fcrn , . in the present study, we have analyzed the nf-κ b binding site in the promoter of pfcrn gene. transmissible gastroenteritis virus (tgev) is a member of the family coronaviridae in the order nidovirales . it replicates in the differentiated enterocytes covering the villi of the porcine small intestine and causes severe gastroenteritis in young tgev-seronegative pigs. diseased pigs often present with vomiting, dehydration, and severe diarrhea. consistent with in vivo pathological changes, tgev infection induces morphological and biochemical changes in host cells and some porcine cell lines in vitro. previous studies have reported that tgev infection induces apoptosis in pk- cells via activation of the p signaling pathway . quantitative proteomic analysis reveals that tgev infection activates the janus kinase signal transducer and activator of the transcription (jak-stat ) signaling pathway . several publications have also shown that tgev could infect ipec-j cells, and that it could also down-regulate proteins involved in tight and adherens junctions to alter the epithelial barrier integrity . these findings suggest that tgev infection induces a number of changes in host-cell biology that could influence cells function. in the present study, we investigated how tgev infection activated the nf-κ b pathway in vitro and up-regulated pfcrn expression in ipec-j cells. our studies showed that pfcrn expression can be triggered by tgev infection, through nf-κ b signal activation. an nf-κ b-specific inhibitor significantly down-regulated expression of pfcrn gene by tgev infection. furthermore, we identified the direct involvement of nf-κ b-specific binding sites by using several complementary strategies. these observations support the hypothesis that up-regulation of pfcrn expression following virus infection appears to be an innate immune response against invading pathogens that could help the host clear infection effectively. to determine whether tgev is capable of modulating pfcrn expression in ipec-j cells, infectious tgev or uv-inactivated virus was adsorbed to confluent monolayers of ipec-j cells at an moi of and pfcrn mrna was measured , , , , and h post-virus exposure by real-time rt-pcr. we found that the level of pfcrn mrna expression was -fold higher at , and h post-infection, compared to mock-infected cells (fig. a) . however uv-inactivated virus treatment only caused a . -fold up-regulation in pfcrn expression, compared to mock-infected cells (fig. a) . replicating virus had a more pronounced effect than uv-inactivated virus. to determine whether the increased mrna levels resulted in increased pfcrn protein production, ipec-j cells were cultured in the presence or absence of infectious virus at an moi of and pfcrn protein was measured in cell lysates by western blot (fig. b) . increased levels of pfcrn were detected by h and h tgev exposure (fig. b) . thus, the elevation in protein levels correlated with the increased mrna levels by h. tgev infection activates nf-κb signaling pathway. ipec-j cells were transiently transfected with p -egfp fusion expression plasmid and the subcellular localization of the fusion protein was analyzed using confocal laser microscopy. as shown in fig. a , p -egfp was located exclusively in the cytoplasm in mock-infected ipec-j cells (top panels), but it rapidly translocated to the nucleus when those cells were infected with tgev (bottom panels). cells were cotransfected with ng/well of nf-κ b luciferase reporter plasmid pnf-κ b-luc and ng/well of the renilla luciferase construct prl-tk which was served as an internal control. twenty-four hours later, cells were infected or control-infected with tgev. cells were harvested at the indicated times and luciferase activity was measured using a dual-luciferase assay system. data represent relative firefly luciferase activity normalized to renilla luciferase activity. nf-κ b-regulated luciferase expression was significantly enhanced during tgev infection at , , and h, compared to mock-infected samples (fig. b) . these two results demonstrated that the classical pathway of nf-κ b was activated in ipec-j cells by tgev infection. effect of nf-κb inhibition on pfcrn expression. inhibition of pfcrn induction by bay - , a small molecule that blocks the phosphorylation of iκ bα , suggested that the early proinflammatory response involved activation of the classical nf-κ b pathway. ipec-j cells were pretreated with bay - ( μ m) for min and subsequently infected by tgev. treatment with bay - significantly reduced tgev infected pfcrn mrna levels to that of the tgev infected cells without bay - treatment as assessed by real-time rt-pcr at h (fig. ). therefore inhibition of pfcrn induction by bay - suggested that early activation of the classical pathway of nf-κ b activation was critical for subsequent up-regulation of pfcrn mrna. software was used for searching putative transcription start sites in the ′ -flanking region of the pfcrn gene (minimum promoter score: . ). computational analysis revealed that the pfcrn gene contains two transcription start sites (fig. s ). transcription start site- has a higher score than the transcription start site- . furthermore, the alignment of pfcrn and human fcrn (hfcrn) upstream sequences of the start codon showed that regions near transcription start site- of pfcrn gene have a high homology with the transcription start site nucleotide sequence of hfcrn gene (fig. s ). in the study, the transcription start site- was considered as the transcription start site of pfcrn gene (fig. s ). scientific reports | : | doi: . /srep to study whether nf-κ b regulates pfcrn expression through a mechanism that involves direct binding to a putative regulatory nf-κ b binding sequence located in the pfcrn gene, we utilized luciferase reporter constructs of the pfcrn promoter that contained nf-κ b p binding sites. we created four luciferase reporter gene constructs with sequentially shortened fragments of the promoter region: pfcrn-luc- (− ~+ ), pfcrn-luc- (− ~+ ), pfcrn-luc- (− ~+ ) and pfcrn-luc- (− ~+ ). furthermore, transient transfection of the four luciferase reporter plasmids in ipec-j cells revealed that these plasmids significantly increased the basal promoter activity above that of the empty vector (fig. a ). next, we tested whether pfcrn promoter activity could be up-regulated by tgev infection. the results showed that luciferase activity was significantly enhanced using the pfcrn-luc- , pfcrn-luc- and pfcrn-luc- constructs during tgev infection compared with mock-infected ipec-j cells, while the luciferase reporter construct harboring the shortest fragment of pfcrn-luc- had not significant differences comparing with mock-infected ipec-j cells (fig. b ). to directly assess the involvement of nf-κ b in pfcrn expression, we investigated whether overexpression of nf-κ b p affects the promoter activity of pfcrn. we transiently transfected ipec-j cells with the p -tag b plasmid. we found that pfcrn-luc- , pfcrn-luc- and pfcrn-luc- constructs were induced in the presence of p , while pfcrn-luc- had not significant differences comparing with mock group (fig. c) . these results indicate that the overexpression of nf-κ b p is sufficient to up-regulate pfcrn expression. meanwhile the nf-κ b sensitive region is located in the sequence between − and − of the pfcrn promoter. screening for nf-κb binding sites adjacent to the pfcrn gene. the canonical nf-κ b dna binding sequence is a common -bp consensus dna element that has been identified as ′ -gggrnnyycc- ′ (where r is an a or g; n is any nucleotide; y is c or t) . we searched for putative nf-κ b-binding sequence(s) along this promoter region (− ~− ). computational inspection revealed that the promoter of the pfcrn gene contained sequences with a similarity to the nf-κ b consensus sequence (fig. a ). we used a chip assay to verify that these putative nf-κ b binding sequences had the capability to directly bind nf-κ b proteins in living cells. first, cross-linking the dna with bound nf-κ b proteins in situ in tgev infection verus mock-infected ipec-j cells, nf-κ b-dna complexes were co-incubated with p specific antibody. then, the purified dna fragments were measured by pcr. as shown in fig. b , pcr with specific primers (table ) produced a band from dna ) were separated by electrophoresis in a % sds-polyacrylamide gel, transferred to a pvdf membrane, and blotted with an pfcrn-specific (top panel) or a gapdh-specific ab (bottom panel). blots were then incubated with a hrpconjugated secondary ab and visualized using chemiluminescence. the bar graphs represented results of three independent experiments. intensities of proteins bands were calculated from the peak area of densitograms by using image software. coprecipitated with p (fig. b , − , − , − and − ), however the sequence (− ) failed to produced a band. in a negative control, immunoprecipitation with normal mouse igg did not generate any corresponding pcr products (fig. b, lane ) . the results suggest that p transcription factor interacted with the four nf-κ b binding sequences of pfcrn gene in ipec-j cells. emsa and supershift assays further confirmed the four nf-κ b binding sites that were identified from the chip assay. nuclear extracts co-incubated with oligonucleotides containing nf-κ b binding sequences (fig. a , − , − , − and − ). as shown in fig. a , a higher amount of complexes formed when bound to nuclear extracts from tgev-infected cells than when bound to nuclear extracts from mock-infected cells. these complexes were further shifted by anti-p monoclonal antibody, indicating that the complexes contained p transcription factor (fig. a, upper arrow, supershifted nf-κ b-specific complex, lane ) . to verify the binding specificity, a competition assay was performed. although the specific band could not be significantly inhibited by unlabeled oligonucleotides in − , − and − nf-κ b binding sequences (fig. a, lane ) . nonlabeled oligonucleotide was added to a -fold excess, the specific band was inhibited by unlabeled oligonucleotides in − , − and − nf-κ b binding sequences (fig. b, lanes , , ) . fcrn has more recently been shown to express in a variety of mammalian species . meanwhile, several studies have reported the distribution, function, and regulation of human and rodent fcrn expression , , , . our study demonstrated that pfcrn mediated bidirectional igg transport across polarized ipec-j cells that potentially provide the mucosal protection . some studies have showed that up-regulation of fcrn expression can augment the igg transport across the polarized epithelial cells . fcrn overexpression boosts humoral immune response in transgenic mice . tgev is an enveloped, non-segmented, single-stranded positive-sense rna virus . the ′ one-third of the genome encodes viral structural and accessory proteins, including the spike (s) glycoprotein, the membrane (m) glycoprotein, the small envelope (e) glycoprotein, the nucleocapsid (n) protein, and three accessory proteins a, b, and , meanwhile ′ -proximal two-thirds of the genome encode the viral replicase . our study is the first to show that tgev infection up-regulates pfcrn mrna and protein in ipec-j cells (fig. ) . levels of pfcrn mrna in uv-inactivated virus treatments are only . -fold lower compared to replicating virus infection. these results show that tgev-mediated fcrn up-regulation is mainly related to the replication of virus, meanwhile uv-inactivated tgev-mediated fcrn up-regulation maybe related to viral structural and accessory proteins. some researchers have shown that fasl-and mitochondria-mediated pathways were activated by tgev infection, which induces apoptosis in pk- cells . moreover, tgev infection promoted the activation of p signaling to induce cell apoptosis . our results show that tgev infection activates the nf-κ b signaling pathway, determined by a p nuclear translocation assay and nf-κ b luciferase report system assay (fig. ) . furthermore, pfcrn expression induced by tgev infection was strongly reduced by the nf-κ b-specific inhibitor bay - in ipec-j cells (fig. ) . this was corroborated by the overexpression of nf-κ b p , which up-regulated the activation of pfcrn promoter luciferase report plasmids. this complementary experiment lessens the concern of specificity or toxicity of the chemical inhibitor. the promoter regions for human and bovine fcrn have been analyzed, and the regulation of expression has been shown to be mediated with p transcription factors , . in this study, we analyzed the ′ -flanking region of the pfcrn gene by tess and tfsearch programs, and found there are five potential nf-κ b transcription factor p binding sites between position − and − . therefore, we constructed luciferase reporter plasmids with sequentially shortened fragments of the fcrn promoter region. transient transfection of the pfcrn promoter luciferase reporter plasmids revealed that pfcrn-luc-( - ) plasmids resulted in increased promoter activity in the presence of tgev infection (fig. b) , further demonstrating that tgev up-regulates pfcrn expression in ipec-j cells. pfcrn-luc-( - ) plasmids were also activated by overexpression of nf-κ b p plasmid (fig. c) . these results showed that the nf-κ b sensitive region of fcrn promoter is located in the sequence between − and − . purified dna fragments were subjected to pcr analysis using primer pairs (table ) . chip was performed at least three times. co-fcrn- ′ -atgtgccgtgggtgtggcccta- ′ ′ -aacttgcatccctgataaga- ′ co-fcrn- ′ -atggctgtggtataggctgata- ′ ′ -cctttttacagcaatacatgcc- ′ co-fcrn- ′ -tggttgatccagacaatagaat- ′ ′ -gccgcagcagtgatccca- ′ co-fcrn- ′ -ggcaccatattgtaggattcca- ′ ′ -caatcaccgatgtgtacgtt- ′ co-fcrn- ′ -catcggtgattgccagga- ′ ′ -tgccaccacggccacta- ′ we mapped four nf-κ b binding sequences in our chip experiment (fig. ). this result was further confirmed by emsa and supershift analysis (fig. ) . these results indicated strong and effective molecular interactions between nf-κ b p and the selected transcription binding sites of the pfcrn promoter. nf-κ b is a ubiquitous transcription factor that regulates the transcription of genes such as chemokines, cytokines, proinflammatory enzymes, adhesion molecules, proinflammatory transcription factors and so on. based on previous studies, nf-κ b is activated via two distinct kinase-dependent pathways, the classical nf-κ b pathway and the alternative nf-κ b pathway. while our research mainly concentrated on the classical nf-κ b pathway, its activity can be modulated significantly by additional factors that transfer into the nucleus and bind to nf-κ b binding sequences. in this regard, we analyzed the ′ -flanking region of the pfcrn gene. and found there are several other transcriptional factors in this region, such as ap- and sp . some studies have suggested that nf-κb dimers can act synergistically with ap- and sp transcriptional factors to influence gene regulation [ ] [ ] [ ] . these protein-protein interactions may be involved in mediating transcriptional regulation of the pfcrn gene in response to stimuli and can functionally cooperate to elicit maximal activation of the promoter. in summary, tgev infection up-regulates pfcrn expression in ipec-j cells, and activates the nf-κ b signaling pathway. furthermore, pfcrn expression was strongly reduced by inhibitor bay - treatment after tgev infection. this result deduced that the up-regulation of fcrn expression by tgev infection may be related to nf-κ b signaling pathway. we constructed fcrn promoter luciferase report plasmids by computational inspection. according to the analysis of the activation of pfcrn promoter luciferase report plasmids by tgev infection and overexpression p plasmid, the nf-κ b sensitive region of fcrn promoter is located in the sequence between − and − . there are four nf-κ b binding sites confirmed by chip and emsa experiments. in the study, upregulation of pfcrn expression following virus infection may play an important role in effectively protecting the mucosal surface from the pathogens invasion. and ) , and these complexes could be further shifted by p -specific abs (*supershifted κ b-specific complex, lane ). n.s, non-specific bands. (b) a competition assay was performed. nonlabeled oligonucleotide in − , − and − nf-κ b binding sequences was added to a -fold excess (fig. b, lanes , and ). distinct κ b-specific protein-dna complexes were detected using nuclear extracts (fig. b, lanes , and ). scientific reports | : | doi: . /srep cell lines, antibodies, virus and plasmids. ipec-j cells were cultured in dulbecco's modified eagle's media (dmem)/ham's f- ( : ) (hyclone, usa) with % fetal bovine serum (gibco, usa) and % penicillin/ streptomycin. all cells were grown in a humidified atmosphere of % co at °c. tgev strain wh- (genbank accession no. hq ), which was isolated in china, was propagated in pk- cells. affinity-purified rabbit anti-cytoplasmic tail of porcine fcrn (anti-pfcrn-ct) polyclonal antibody was prepared in our laboratory. horseradish peroxidase (hrp)-conjugated goat anti-rabbit and rabbit anti-mouse igg were purchased from thermo scientific pierce (usa). mouse anti-gapdh monoclonal antibody was purchased from boster (china). p -egfp fusion expression plasmid was prepared in our laboratory, p gene was pcr subcloned into pegfp-c vector. p -tag b plasmid encoding p was prepared in our laboratory, p gene was pcr subcloned into pcmv-tag b vector. quantitative real-time rt-pcr. confluent monolayers of ipec-j cells were inoculated with tgev at a multiplicity of infection (moi) of for , , , , , and h at °c. total rna was extracted from ipec-j cells with trizol reagent (invitrogen, usa). then, total rna was reverse-transcribed into cdna using reverse transcriptase (takara, china). real-time rt-pcr was performed in triplicate in three separate experiments using pfcrn primers ( ′ -ggcgacgagcaccactactg- ′ and ′ -agccgaccatgattccaacc- ′ ) and gapdh primers ( ′ -acatggcctccaaggagtaaga- ′ and ′ -gatcgagttggggctgtgact- ′ ) and the sybr premix ex taq ii (takara, china). all reactions were performed for cycles: s at °c and s at °c. pfcrn expression was calculated following normalization to gapdh levels by the comparative delta delta threshold cycle (Δ Δ c t ) method. the specific amplification reactions were confirmed by melt curve analysis. lysates from ipec-j cells were prepared as described previously . the total proteins were resolved on a % sds-polyacrylamide gels under reducing conditions and electrotransferred onto a polyvinylidene fluoride membrane (bio-rad, usa). affinity-purified rabbit anti-pfcrn-ct polyclonal antibody and mouse anti-gapdh monoclonal antibody were used as primary antibodies, then hrp-conjugated goat anti-rabbit igg or hrp-conjugated rabbit anti-mouse igg were used as secondary antibodies. western blot analysis was performed as described previously . confocal laser microscope assay. ipec-j cells were grown to approximately - % confluency on coverslips placed in -well plates. after transfection with . μ g of p -egfp fusion expression plasmid per well, cells were mock-infected or infected with tgev at a moi = . h after stimulation, cells were fixed with % paraformaldehyde, permeabilized with . % triton x- , and stained with dapi (invitrogen, usa) to detect nuclei. construction of reporter plasmids. four fragments of the ′ -flanking region of the pfcrn were pcr subcloned into the luciferase expression vector pgl (promega, usa) through sac i and hind iii digestion. pfcrn-luc- , containing sequences from − to + of the pfcrn gene promoter was amplified by the pcr primer pairs ( ′ -gcgagctcgctatag ctctgattcgacc- ′ and ′ -caagcttctgagcgggagacctgggg- ′ ); pfcrn-luc- , which contains the segment from − to+ of the pfcrn gene promoter was amplified using the reverse primers described above and a forward primer ( ′ -gcgagctcgcttcagctggacccgtagc- ′ ). plasmid pfcrn-luc- , which contains the segment from − to + of the pfcrn gene promoter, was generated using the reverse primers described above for pcr amplification and a forward primer ( ′ -gcgagctctacttaaaggggtacggggt- ′ ). plasmid pfcrn-luc- , which contains the segment from − to + of the pfcrn gene promoter and was generated using the reverse primers described above for pcr amplification and a forward primer ( ′ -gcgagctcgggggtgctgacgaggtaagaa- ′ ). lipofectamine (invitrogen, usa). cells were transfected with n g of pfcrn promoter luciferase report plasmids and n g of a renilla luciferase prl-tk control plasmid. twenty-four hours later, cells were infected with or without tgev (moi = ). cells were harvested at the indicated time and luciferase activity was measured using a dual-luciferase assay system (promega, usa). the values for firefly luciferase were normalized to the renilla luciferase activity and expressed as fold activation over the mock-infected group. in order to detect whether tgev infection activates nf-κ b signaling pathway, ipec-j cells were cotransfected with ng/well of nf-κ b luciferase reporter plasmid pnf-κ b-luc and ng/well of the renilla luciferase construct prl-tk. cells were infected or control-infected with tgev and were harvested at the indicated times and luciferase activity was measured using a dual-luciferase assay system. chromatin immunoprecipitation (chip). chip was performed according to the manufacturer's recommendations (epigentek, usa). in brief, ipec-j cells were infected with or without tgev (moi = ) for h. the cells were fixed with % formaldehyde. the nuclei were isolated and dna was sheared by sonication. chromatin was immunoprecipitated with μ g of ab specific for p or with μ g of normal igg as negative control for h at room temperature by an orbital shaker ( - rpm). the dna samples were amplified by pcr primers (table ) in optimized conditions. preparation of nuclear extracts, emsa and supershift assay. nuclear extracts were prepared using a nuclear and cytoplasmic extraction kit (cwbio, china). the single-strand oligonucleotides was labeled with biotin on the ′ end dna, annealed to form double-stranded oligonucleotides containing the tested nf-κ b sequences from the pfcrn promoter: pfcrn κ b- ( ′ -aaaaatggga gtttccatttccg- ′ ), pfcrn κ b- scientific reports | : | doi: . /srep ( ′ -gtagcctgggaacttccagat gcc- ′ ), pfcrn κ b- ( ′ -ccagaagaggcaaattcctagagac- ′ ) and pfcrn κ b- ( ′ -aaggggtacggggtctccttgggg- ′ ). for competition assays, a -fold excess of nonlabeled oligonucleotide was incubated during the preincubation time. for the supershift assays, μ g anti-p monoclonal antibody directed against nf-κ b p was preincubated with the nuclear extracts. the complexes were run on a % native polyacrylamide gel. emsa experiments were performed according to the lightshift chemiluminescent emsa kit (thermo scientific pierce, usa). data from three independent studies were analyzed using anova to identify significant changes between tgev-infected and mock-infected cells. all results are expressed as mean ± sem from three independent experiments. p values < . were considered significant (*p < . and **p < . ). interference by human and bovine serum and serum protein fractions with the absorption of antibodies by suckling rats and mice ph-dependent binding of immunoglobulins to intestinal cells of the neonatal rat the mechanism of intestinal uptake and transcellular transport of igg in the neonatal rat receptor-mediated transport of igg an fc receptor structurally related to mhc class i antigens crystal structure of the complex of rat neonatal fc receptor with fc isolation and characterization of an fc receptor from neonatal rat small intestine crystal structure and immunoglobulin g binding properties of the human major histocompatibility complex-related fc receptor(,) bidirectional fcrn-dependent igg transport in a polarized human intestinal epithelial cell line expression of functionally active fcrn and the differentiated bidirectional transport of igg in human placental endothelial cells neonatal fcr expression in bone marrow-derived cells functions to protect serum igg from catabolism chapter : multitasking by exploitation of intracellular transport functions the many faces of fcrn enhanced half-life of genetically engineered human igg antibodies in a humanized fcrn mouse model: potential application in humorally mediated autoimmune disease the major histocompatibility complex-related fc receptor for igg (fcrn) binds albumin and prolongs its lifespan the mhc class i-like igg receptor controls perinatal igg transport, igg homeostasis, and fate of igg-fccoupled drugs over-expression of the bovine fcrn in the mammary gland results in increased igg levels in both milk and serum of transgenic mice fcrn overexpression in transgenic mice results in augmented apc activity and robust immune response with increased diversity of induced antibodies characterization of the rabbit neonatal fc receptor (fcrn) and analyzing the immunophenotype of the transgenic rabbits that overexpresses fcrn transgenic expression of bovine neonatal fc receptor in mice boosts immune response and improves hybridoma production efficiency without any sign of autoimmunity neonatal fcr overexpression boosts humoral immune response in transgenic mice jankovics, i. & kacskovics, i. fcrn overexpression in mice results in potent humoral response against weakly immunogenic antigen nf-kappab regulation in the immune system nfkappab induces overexpression of bovine fcrn: a novel mechanism that further contributes to the enhanced immune response in genetically modified animals carrying extra copies of fcrn nf-kappab signaling regulates functional expression of the mhc class i-related neonatal fc receptor for igg via intronic binding sequences molecular biology of transmissible gastroenteritis virus transmissible gastroenteritis virus infection induces cell apoptosis via activation of p signalling quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat signaling pathway transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells distribution of rat neonatal fc receptor in the principal organs of neonatal and pubertal rats characterization of the rat intestinal fc receptor (fcrn) promoter: transcriptional regulation of fcrn gene by the sp family of transcription factors neonatal fc receptor-mediated igg transport across porcine intestinal epithelial cells: potentially provide the mucosal protection molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model transmissible gastroenteritis virus infection induces apoptosis through fasl-and mitochondria-mediated pathways tgev nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p signaling communication between nf-kappa b and sp controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein gene sp binding is critical for promoter assembly and activation of the mcp- gene by tumor necrosis factor cross-coupling of the nf-kappa b p and fos/jun transcription factors produces potentiated biological function transfer of igg in the female genital tract by mhc class i-related neonatal fc receptor (fcrn) confers protective immunity to vaginal infection this work was supported by national natural science foundation of china ( to z.l.) j.g. and z.l. conceived and initialed the study; j.g., f.l. and s.q. extracted the data set; j.g., d.b., q.h., h.j., r.l., s.l. and x.m. performed the analysis. j.g. and z.l. wrote the paper. key: cord- -m amd y authors: mathur, kalika; anand, abhishek; dubey, sunil kumar; sanan-mishra, neeti; bhatnagar, raj k.; sunil, sujatha title: analysis of chikungunya virus proteins reveals that non-structural proteins nsp and nsp exhibit rna interference (rnai) suppressor activity date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: m amd y rnai pathway is an antiviral defence mechanism employed by insects that result in degradation of viral rna thereby curbing infection. several viruses including flaviviruses encode viral suppressors of rnai (vsrs) to counteract the antiviral rnai pathway. till date, no vsr has been reported in alphaviruses. the present study was undertaken to evaluate chikungunya virus (chikv) proteins for rnai suppressor activity. we systematically analyzed all nine chikv proteins for rnai suppressor activity using sf rnai sensor cell line based assay. two non-structural proteins, namely, nsp and nsp were found to exhibit rnai suppressor activity. we further validated the findings in natural hosts, namely in aedes and in mammalian cell lines and further through emsa and agrobacterium infiltration in gfp silenced transgenic tobacco plants. domains responsible for maximum rnai suppressor activity were also identified within these proteins. rna binding motifs in these domains were identified and their participation in rnai suppression evaluated using site directed mutagenesis. sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. further validation of rnai suppressor activity of these proteins awaits establishment of specific virus infection models. are transmitted through arthropods, and display differential replication pattern in the two hosts. while invertebrate hosts like mosquitoes and ticks behave as maintenance hosts where the viruses persists in low levels , , vertebrate hosts get infected to high titers with efficient virus replication and this alteration between hosts has been shown to play an important role in evolution of arboviruses . until recently, it had been speculated that these arbovirus do not possess rnai suppressor activity unlike other insect-specific viruses like fhv and crpv , . however, recent studies have established that flaviviruses encode for vsrs , . alphaviruses are member of arboviruses containing recognised members with a very wide geographic distribution and several geographical variants on the basis of which they have been classified as old world and new world viruses . grouped into seven complexes based on serological cross-reactivity, they infect a variety of host including birds, fishes, mammals including humans and are maintained in natural cycles by transmission between susceptible vectors and vertebrate hosts . alphaviruses have a single plus-stranded rna genome encapsulated by capsid proteins. the approx. kb genome consists of two open reading frames that encodes four non-structural proteins (nsp to nsp ), three structural proteins (capsid, envelope glycoproteins e and e ) and two small cleavage products (e and k). while the structural proteins are involved in mediating entry and fusion of the virus into the hosts, the non-structural proteins (nsps) form the replication complex . the non-structural proteins that are initially translated as one or two polyprotein from the full-length genomic viral rna are processed exclusively by the virus-encoded protease, nsp . upon cleavage, each of the mature proteins as well as the intermediates actively participates in virus replication. as of date, some properties of these nsps are known : nsp is membrane-associated and possess both guanine- -methytransferase and guanyltransferase activity, nsp exhibit several enzymatic activities including helicase, protease activity, nsp possess phosphatase and rna-binding activity , and nsp is a rna-dependent rna polymerase (rdrp) . even though much information is available from the above and other studies, it is expected that these proteins have more novel roles in viral replication. the current study undertaken to identify viral encoded suppressors of rnai in chikungunya virus (chikv) has thrown light to possible rnai suppressor activity in two non-structural proteins of chikv. systematic analysis of all chikv proteins using a sf rnai sensor cell line based assay revealed that non-structural proteins nsp and nsp exhibited rnai suppressor activity. we further validated this finding in its natural hosts, namely, an aedes and mammalian cell line. after dissecting the domain responsible for suppressor activity, we evaluated the suppressor strength by mutating the rna binding motifs in these domains. aligning known sequences of nsp and nsp across all alphavirus species, we observed that these rna binding motifs are conserved across species thereby confirming the importance of these motifs in alphavirus-host interactions vis-à-vis rnai pathway. recombinant alphavirus with an endogenous rnai suppressor have shown an increase in alphavirus replication in aedes mosquitoes . while it was previously thought that flaviviruses do not possess any rnai suppressors, it was recently proved that these viruses do possess vsrs that are active in both insect and mammalian cells , . in case of alphaviruses, reports have suggested that rnai suppressors in this class of viruses may either be weak or undetected due to inappropriate assay conditions , . we undertook the present study to systematically evaluate chikv proteins (fig. a) for possible rnai suppressor activity using a sf rnai sensor cell line developed in our lab . using this rnai sensor cell line, in which gfpshrna is expressed constitutively to silence gfp in a gfp sf cell line; we checked for reversion of gfp due to rnai suppressor activity of the cloned chikv genes in pib vector using flow cytometry ( fig. b and c) . post h transfection, it was observed that nsp , nsp and nsp showed a reversion in gfp expression of %, % and % respectively (p values < . ). the other genes showed reversion similar to empty vector. denv rnai suppressor ns b and flock house virus protein b served as positive controls. previous studies have emphasised the importance of testing the efficiency of vsrs in relevant host systems . having checked suppressor activity of all chikv genes using an established cell line based assay, we next checked for their suppressor activity in its natural hosts, namely a mosquito and mammalian system. for this purpose, we utilised an aedes cell line, aag and the mammalian cell line, hek t. using transient transfection protocols in which gfpshrna in pizt/v -his and pgfp vrs plasmid were co-transfected with nsp /nsp pib/ pcdna . + plasmids in aag and hek t respectively (fig. a) , we evaluated suppressor activity of all nine genes. overall, it was seen that transfection worked better in hek as compared to aag owing to better transfection efficiency in the mammalian system. with respect to gfp reversions, it was observed that nsp and nsp showed significant reversion (p value < . ) in both aedes and hek cell lines ( fig. b and c) . in case of nsp , however, rnai suppressor activity was not evident in both the natural hosts and only around % gfp reversions were seen in the cells upon transfections with nsp . based on these results, we identified chikv_nsp and chikv_nsp to possess rnai suppressor activity and further proceeded to validate these findings. nsp and nsp are suppressors of rnai. having established that nsp and nsp show rnai suppressor activity, we proceeded to detect the level of gfp protein due to effect of the putative vsrs. western analysis also revealed increase in gfp expression in nsp and nsp transfected cells as compared to sf rnai sensor cells (fig. a) . in addition to the above mentioned in vitro assays, we utilised transgenic nicotiana leaf based assay to validate rnai suppressor activity of nsp and nsp under in vivo conditions . the putative vsrs were cloned into the plant binary vector pbi and reversal of silencing by agro-infiltration-mediated transient ectopic expression of chikv nsp and chikv nsp was assessed in transgenic tobacco leaf tissues in which gfp is silenced. the infiltrated leaves showed reversal of gfp activity at days post-infiltration in nsp and days post-infiltration in nsp as seen under uv light ( fig. b and c) . in the infiltration experiments, fhvb plasmid was used as positive control and a mutated fhvb expressing plasmid as negative control. gfp reversal was further confirmed by analysing gfp transcript levels and the depletion of small rna population of gfp in the leaves using northern hybridisation. s rna and s rrna were used as loading controls for mrna and small rna northerns respectively. (fig. d) . while there was an increase in the gfp mrna levels in leaves infiltrated with nsp and nsp , gfp small rnas showed % and % decrease in their population in the leaves infiltrated by nsp and nsp respectively thereby providing evidence that chikv nsp and nsp are influencing small rna production post-transcription. even as the assays in different systems provided enough clarity as to presence of rnai suppressor activity in chikv nsp and nsp , the first strong proof for any rnai suppressors is its role in interfering with the rnai machinery. the most well known mechanism is the ability of vsrs to bind to dsrna/sirna population thereby preventing risc from acting upon them. this phenomenon has been evaluated by studying the binding of suppressor protein to double stranded rna population resulting in a shift in their mobility efficiency , . for this purpose, we expressed both nsp and nsp in the heterologous e.coli system. while we were able to produce nsp in its natural folded form, nsp aggregated into inclusion bodies and could not be purified, most probably owing to its large size. to overcome this problem, we expressed both chikv_nsp and chikv_nsp in sf cell line by transient transfection (supplementary fig. s ). using cell lysate of the transfected cell expressing nsp and nsp , we evaluated their ability to bind double stranded rna by using [γ p] labeled shrna of gfp and running the bound complex in a % polyacrylamide gel (fig. e) . binding of shrna to nsp and nsp proteins were evident by complex formation and shift that increased proportionally to protein concentration (fig. e -lane no. , , , ) . addition of cold shrna probe served as competitive inhibitor of binding reaction and the decrease in the intensity of the shift proved the binding specificity of the complex. untransfected sf lysate in the presence of non-specific inhibitor (salmon sperm dna) of varying concentrations and gfpshrna served as negative the y-axis shows % gfp reversion (mean ± sd). statistical significance was analysed using student's t-test and fisher's least significant difference (lsd) test using empty vector as control. the *symbol indicates a statistically significant difference in terms of the p value (p < . ). nsp , nsp and nsp were found statistically highly significant from lsd test (p value < . each) when compared with the vector (negative control) (alpha level of the test = . ). , , ) . these results clearly proved nsp and nsp bound to dsrna. taken all these findings together, we conclude that chikv nsp and chikv nsp are suppressors of rnai. suppressor proteins interfere with the host rnai components to inhibit the pathway. vsrs may block the dicing activity and thereby reduce formation of sirnas. alternately, vsrs may also impede loading of sirnas into the risc and consequently block functionalization of sirna loaded risc .a direct way to assess effect of vsrs on sirna formation is to estimate dicing enzyme activity in vitro in the presence of the vsrs. unfortunately, our attempts to purify chikv nsp in its active form were unsuccessful, hence we resorted to cell based facs assays to gain preliminary insight into possible suppression mechanism. using sf gfp cell line, we transfected dsrna or sirna-gfp and subsequently co-transfected with the vsrs to estimate the level of gfp reversal. we reasoned that gfp reversal was an indication of where the vsrs may act -if they acted during dicing, then the reversal would be evident upon co-transfection with dsrna+ vsrs and if they acted at the time of sirna loading into risc, it would be evident while using sirna+ vsrs. our results showed there was > % gfp reversion with both dsrna and sirna for both nsp and nsp ( fig. a and b) , thus highlighting that their mode of action may be downstream of risc loading in the rnai pathway. domain mapping of nsp and nsp reveal domain specific rnai suppressor activity. the non-structural proteins, nsp and nsp of chikv participate in several functions; nsp has been shown to have multiple functions including helicase, protease and ntpase activities [ ] [ ] [ ] [ ] . this protein is kda and consists of four domains, the aa long n-terminal domain, aa long domain that exhibits helicase activity, aa long protease domain and a aa long c terminal domain (fig. a) . similarly, nsp exhibits phosphatase activity and also consists of a macrodomain that has been well characterised . this is kda and consists of three domains (fig. c) . the macrodomain, present on the n-terminus, is a globular domain aa long and is evolutionary highly conserved. most of the activities exhibited by the protein are associated with this domain. the sequence of central part of nsp ( aa) is conserved only among alphaviruses and hence is termed alphavirus unique domain (aud). the c-terminal domain ( aa) is termed hypervariable region, since it has no sequence similarity even among alphaviruses. having confirmed that both nsp and nsp exhibit rnai suppressor activities, efforts were taken to characterise the proteins better and to identify those specific domains that were and ns b (dengue virus) were taken as positive controls and empty vector was used as negative control. statistical significance was analysed using student's t-test and fisher's least significant difference (lsd) test using empty vector as control. the *symbol indicates a statistically significant difference in terms of the p value (p < . ) and # symbol represents p-value > . . nsp (p-value = . ) and nsp (p-value = . ) were found statistically highly significant, while nsp was found not significant (p-value = . ) (alpha level of the test = . ). (c) fluorescent microscopic images of hek t cell line based assay. hek t cells were transfected with gfp plasmid and gfpshrna plasmid in separate wells. for gfp reversion assay gfpshrna and individual vsr plasmid were co-transfected and cells were checked for fluorescence under microscope at h post transfection. scientific reports | : | doi: . /srep responsible for rnai suppressor activity. for this purpose, each of the domains was cloned individually in pib/ v -his topo vector and transfected into the sf sensor line. for convenience, the domains were labelled as n d , n d , n d and n d in nsp and n d , n d and n d in nsp . in case of nsp , n d , the domain that is responsible for helicase activity showed maximum reversion of gfp expression and in nsp , n d , i.e., the macrodomain showed maximum reversion ( fig. b and d) . rna binding motifs are conserved across alphaviruses. it is well known that most of the vsrs possess certain sequence characteristics to function as an rnai suppressor. presence of rna binding motifs and presence of gw/wg motifs has been shown to be important features required for binding to dsrna/sirna and argonaute loading . to understand the sequence structure of the domains that showed maximum suppressor activity in nsp and nsp , we identified the rna binding motifs within the domains showing maximum reversions in both the vsrs (fig. a and c) . furthermore, we hypothesized that these features are important amongst all species of alphavirus. to test this hypothesis, we aligned the sequences of all known alphaviruses nsp and nsp . to date, complete sequence information is available for alphavirus species. as expected, all rna binding motifs were conserved in both nsp and nsp proteins across all species (fig. b and d) . additionally, we observed the presence of one gw motif in domain of nsp in all species. taking all these aspects, we hypothesize the rnai suppressor activity of chikv nsp and nsp may be conserved across all alphaviruses. rna binding motifs contribute to vsr activity of chikv nsp and nsp . having identified the conserved rna binding motifs in nsp and nsp , we proceeded to evaluate the impact these motifs have in rnai suppressor activity in the vsrs. substituting all or most of the amino acids in motifs with alanine through site directed mutagenesis, we generated mutants. three mutants were generated in nsp with different permutations and combinations of substitutions, while two mutants were generated in nsp . schematic representation of mutant generation is depicted in fig. a and b. gfp reversal in these mutants were evaluated using sf rnai sensor line and the results are shown in fig. c . further, we performed mobility shift assays to evaluate the binding capacities of the domains and the mutants generated using [γ p] labeled shrna of gfp and running the bound complex in a % polyacrylamide gel (fig. d) . complex formation due to dose dependent binding of shrna to the mutants of nsp and nsp are evident (lane , , and respectively). in the same manner, binding efficiencies of the domains to shrna are demonstrated in lane , , and respectively. the results clearly show the involvement of the rna binding motifs in the binding to dsrna and we conclude that these motifs play a direct role in rnai suppressor activity. nsp -sf lysate in the presence of non-specific inhibitor (salmon sperm dna) and gfpshrna served as negative controls (lane , , ). alphaviruses are maintained in nature by alteration between mosquitoes and human host. while their infections in mosquitoes are mainly asymptomatic and persistent, in vertebrate hosts, the infection is acute and self limiting . efficiency of virus replication in both vector and host depends not only on the expression of its own proteins but also on its ability to counteract upon host antiviral defence. mosquito innate immune system poses a serious challenge to virus infection and activates different defence pathways including toll pathway, imd (immune deficiency) pathway, jak stat pathway and rnai pathway . rnai pathway is a major antiviral defence mechanism employed by insects that results in degradation of viral rna thereby curbing infection [ ] [ ] [ ] [ ] . viruses employ numerous strategies to evade host rnai response, including mutations in or near the target to bring conformational changes rendering risc inaccessible and vsrs which inhibit or compete with dicer/other rnai components, thus decreasing the amounts of viably loaded risc complexes , , . earlier reports have suggested that arboviruses may possess weak suppressors that have been yet undetected due to inappropriate assay conditions , . the speculations of absence of vsrs in arboviruses were eliminated when denv proteins showed suppressor activity . however, since alphaviruses do not cause as severe illness as the flavivirus, it was assumed that vsr activity against rnai defence might be detrimental for the host, thus reducing virus transmission , . using an in-house developed gfp reversion assay , , we report that chikv nsp and nsp are rnai suppressors. certain vsrs such as geminivirus nuclear protein ac have been previously shown to transactivate host genes . we confirmed the specificity of the gfp reversion by checking nsp and nsp for non-specific promoter activity (supplementary fig. s ). the non-structural proteins, nsp and nsp of chikv participate in several functions; nsp has been shown to have multiple functions including helicase, protease and ntpase activities , [ ] [ ] [ ] . nsp has been shown to interact the most with host factors than any other chikv protein, mainly due to its dual role as key component of viral replication complex and also as an important virulence factor inhibiting the host antiviral response . in our study, nsp has shown stronger vsr activity in comparison to nsp . earlier reports have shown this protein exhibits anti-ifn activity , emphasising on the overlapping nature of interferon and rnai pathway. domain mapping of this protein revealed the domain that possessed helicase activity showed maximum activity for rnai suppression. interestingly, helicases such as heif of dictyostelium, b of wuhan nodavirus and ns of dengue virus has been proven to exhibit vsr activity , , . of interest is the presence of a gw motif, conserved across all alphavirus species studied, that is known to be important for binding to dsrna/sirna and argonaute loading . the crystal structure of chikv nsp macrodomain reveals that this domain possesses rna-binding activity . this protein is also known to modulate pathogenicity in mice . our study has provided evidence of an additional role of the macrodomain and clearly elucidates it role in pathogenicity as a viral suppressor of rnai. the existence of both nsp and nsp in both the nucleus as well in the cytoplasmic fraction , may help in their interactions with the various components of the risc complex. alphavirus species exhibit sequence conservation amongst several of their proteins. motifs that are involved in direct interactions with both the vector as well as the host have been shown to be conserved even in the otherwise non-conserved regions . through our study we have established all rna binding motifs of chikv nsp and nsp domains that possess vsr activity are conserved across alphavirus species. it is speculated that these proteins may participate in similar functions in the other alphavirus species as well. taken together, the current study has identified chikv proteins nsp and nsp to exhibit rnai suppressor activity in the in-vitro system that was further demonstrated in its natural hosts, namely, an aedes and mammalian cell line. the domains responsible for suppressor activity was further identified and their suppressor strength evaluated based on mutations introduced in the rna binding motifs of these domains. finally, these rna binding motifs were found to be conserved across alphavirus species. it is to be noted however, the vsr activities of nsp and nsp exhibited by in-vitro assays could be strengthened further by a direct demonstration employing in-vivo infection system. supplementary table s . to find the putative rnai suppressors in chikv, a previously developed rnai sensor cell line constitutively expressing gfpshrna was used. briefly, . million sf gfpshrna cells were seeded in a well plate in complete media and transfected with μ g of chikv gene plasmids using cellfectinii reagent (invitrogen) as per manufacturer's protocol to transiently express the chikv proteins. empty pib/v -his topo vector was used as negative control. previously published dengue virus suppressor ns b and flock house virus protein b (fhvb ) were used as positive controls. facs analysis was done after h for gfp expression in transfected cells. sf cell line with and without gfp were used as facs gating control. gfp specific dsrna and sirna mediated reversion assay was carried out similarly in sf gfp stable cell line in well plate. briefly, μ g vsr plasmid was co-transfected with . μ g gfp dsrna plasmid and . pmole sirna in separate wells. scrambled sirna was used as gfp silencing negative control. facs analysis was done similarly after h. rnai suppression assay in natural host cell lines. aag (aedes aegypti) cells were transfected using attractene reagent (qiagen). briefly, . million aag cells were co-transfected with gfpshrna plasmid and chikv-pib/v -his topo plasmid. gfp levels were analyzed with facs after h. gfp reversion assay in human host was done using hek t cells. each chikv-pcdna . + plasmid was co-transfected with gfpshrna plasmid using jet prime reagent (polyplus transfection) in jet prime buffer, followed by facs analysis after h. activated cell sorting (facs) with facs calibur (bd biosciences). media from cells was removed and the cells were washed and resuspended in μ l of facs grade pbs. respective cell lines without gfp were used specifically for gating the region of negative control. cell quest software (becton-dickinson, franklin lakes, nj, usa) was used to analyse the percentage of gfp reversion in , cells against fl -h.percentage gfp reversion data was represented as mean ± sd. the experiments were repeated at least four times in triplicates. statistical analysis of experimental data was conducted using graphpad prism (version ). two tailed student's t test and fisher's least significant difference (lsd) test were performed to check significance of the data and p-values < . were considered significant. validation of rnai suppression in nicotiana leaves. nsp and nsp genes were separately cloned in pbi vector for transformation in agrobacterium. the secondary culture was incubated in fresh yem and acetosyringone for h, followed by infiltration in transgenic nicotiana leaves which has gfpshrna stably integrated. the culture was infiltrated on dorsal side of the leaves. the leaves were analysed in uv transilluminator , , and days post infiltration (dpi). a previously known suppressor fhvb was used as positive control and mutated fhvb was used as negative control. fected with all genes of chikv cloned in pib vector, nsp /m pib, nsp /m pib and vsr domains cloned in pib plasmids separately. the cells were harvested after h and lysed in lysis buffer ( mm tris hcl and mm nacl) containing protease inhibitor cocktail (roche). whole cell lysate concentration was determined using bradford protein assay (bio-rad laboratories). protein extracts ( μ g) were subjected to % reducing page and western blotting was done using anti-his (mice) primary antibody (sigma), followed by ap conjugated (mice) secondary antibody. western blotting to detect changes in gfp expression levels. sf sensor cell line was transfected with nsp pib and nsp pib plasmids separately and cells were harvested after h. μ g total cell lysate was run on % sds page and western blotting was done using anti-gfp primary antibody (santa cruz) and ap conjugated secondary antibody. levels of gapdh proteins were used as housekeeping control. electrophoretic mobility shift assay. gfpshrna binding ability of of different proteins was evaluated using electrophoretic mobility shift assay (emsa). sf cells transfected with nsp , nsp , mutants and domains plasmids were analysed along with untransfected sf cell line as negative control. gfpshrna probe was synthesised from sigma as a self complementary bp dna oligonucleotide with a hairpin loop. binding reaction was setup with different concentrations of cell lysate, x binding buffer ( mm hepes and mm nacl), [γ − p] atp labelled shrna probe ( , cpm per reaction) and μ g of salmon sperm dna (thermo scientific). unlabelled shrna was added as cold probe to check specificity of the binding. the complex was resolved on % polyacrylamide non-denaturing gel (pre-run for h at °c) in x tbe buffer at v at °c. gels were dried and exposed for auto-radiography. the scanning was performed using typhoon variable mode imager (amersham biosciences). northern blotting. nicotiana leaves infiltrated with vsr plasmids were harvested and total rna was isolated using gitc method. for mrna northern. μ g total rna was run on % agarose gel. rna was transferred onto hybond-n + membrane (ge healthcare) overnight by capillary transfer. rna was immobilised on the membrane by uv cross linking at , × μ j. northern blotting for mrna was performed using [γ − p] atp labelled shrna probe. for small rna northern. rna was isolated using gitc method followed by small rna enrichment. μ g rna was run on % page containing m urea. rna was transferred onto a hybond-n + membrane (ge healthcare) for min at v and immobilised by uv cross-linking at , × μ j. gfp probe was prepared by dig labelling kit (roche, usa). northern hybridisation and development was performed according to instruction manual. small rna marker (neb) lane was cut from the blot and radioactively labelled with [γ − p] atp separately. domain mapping of the chikv rnai suppressors in sensor cell line. sf sensor cell line was transfected with μ g of each domain plasmid using cellfectin ii reagent, followed by facs analysis after h. complete gene plasmid and fhvb were used as positive controls and empty vector was used as negative control. alignment of rna binding motifs of alphaviruses. nsp and nsp sequences of other alphaviruses were fetched from ncbi and aligned with chikv new delhi strain ind- -del using mega software. the rna binding motifs were predicted using bindn software and checked for conservation amongst alphaviruses. site directed mutagenesis in nsp and nsp pib/v -his topo plasmids. nsp and nsp -pib clones were mutated using quickchange multi site directed mutagenesis kit (agilent technologies) for mutating different sites in the same plasmid simultaneously. briefly, ng plasmid was mutated using different primers binding to the same dna strand with pcr, followed by dpni digestion and transformation. the mutations were confirmed by sequencing from macrogen. gfp reversion was assayed in sf sensor cell line. wild type vsr plasmids were used as positive controls. rna-based antiviral immunity suppression of gene silencing: a general strategy used by diverse dna and rna viruses of plants rnai function, diversity, and loss in the fungal kingdom mosquito rnai is the major innate immune pathway controlling arbovirus infection and transmission dicing of viral replication intermediates during silencing of latent drosophila viruses small silencing rnas: an expanding universe the rna silencing endonuclease argonaute mediates specific antiviral immunity in drosophila melanogaster the rest is silence dicing and slicing: the core machinery of the rna interference pathway the many faces of rnai the pirna pathway in flies: highlights and future directions. current opinion in genetics & development antiviral immunity directed by small rnas double-stranded rna binding may be a general plant rna viral strategy to suppress rna silencing mechanism of induction and suppression of antiviral immunity directed by virus-derived small rnas in drosophila rna binding by a novel helical fold of b protein from wuhan nodavirus mediates the suppression of rna interference and promotes b dimerization suppression of rna silencing by flock house virus b protein is mediated through its interaction with the paz domain of dicer novel drosophila viruses encode host-specific suppressors of rnai present and future arboviral threats advances in dissecting mosquito innate immune responses to arbovirus infection induction and suppression of tick cell antiviral rnai responses by tick-borne flaviviruses arbovirus evolution in vivo is constrained by host alternation mosquitoes put the brake on arbovirus evolution: experimental evolution reveals slower mutation accumulation in mosquito than vertebrate cells the role of rnai and micrornas in animal virus replication and antiviral immunity the structure of the flock house virus b protein, a viral suppressor of rna interference, shows a novel mode of double-stranded rna recognition cricket paralysis virus antagonizes argonaute to modulate antiviral defense in drosophila role of rna interference (rnai) in dengue virus replication and identification of ns b as an rnai suppressor noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells alphaviruses (togaviridae) and flaviviruses (flaviviridae the old world and new world alphaviruses use different virus-specific proteins for induction of transcriptional shutoff evolution of rna viruses the alphaviruses: gene expression, replication, and evolution functions of alphavirus nonstructural proteins in rna replication the crystal structures of chikungunya and venezuelan equine encephalitis virus nsp macro domains define a conserved adenosine binding pocket a structural and functional perspective of alphavirus replication and assembly mapping of rna-temperature-sensitive mutants of sindbis virus: assignment of complementation groups a, b, and g to nonstructural proteins suppression of rna interference increases alphavirus replication and virus-associated mortality in aedes aegypti mosquitoes the long and short of antiviral defense: small rna-based immunity in insects the a accessory protein of severe acute respiratory syndrome coronavirus acts as an rna silencing suppressor processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the c-terminal half of nsp and functions both in cis and in trans proteolytic processing of semliki forest virus-specific non-structural polyprotein by nsp protease identification of the active site residues in the nsp proteinase of sindbis virus site-specific protease activity of the carboxyl-terminal domain of semliki forest virus replicase protein nsp evasion of the innate immune response: the old world alphavirus nsp protein induces rapid degradation of rpb , a catalytic subunit of rna polymerase ii viral rna silencing suppressors (rss): novel strategy of viruses to ablate the host rna interference (rnai) defense system the host defense of drosophila melanogaster rna interference against viruses: strike and counterstrike virus counterdefense: diverse strategies for evading the rna-silencing immunity dengue ns , an rnai suppressor, modulates the human-mirna pathways through its interacting partner alphavirus-derived small rnas modulate pathogenesis in disease vector mosquitoes suppression of rna silencing by a geminivirus nuclear protein, ac , correlates with transactivation of host genes mapping of chikungunya virus interactions with host proteins identified nsp as a highly connected viral component chikungunya virus nonstructural protein inhibits type i/ii interferon-stimulated jak-stat signaling helf, a putative rna helicase acts as a nuclear suppressor of rnai but not antisense mediated gene silencing the nsp macro domain is important for sindbis virus replication in neurons and neurovirulence in mice different types of nsp -containing protein complexes in sindbis virusinfected cells nuclear localization of semliki forest virus-specific nonstructural protein nsp sh domain-mediated recruitment of host cell amphiphysins by alphavirus nsp promotes viral rna replication this work was supported by the financial grant from department of biotechnology (bt/pr / agr/ / / ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. km acknowledges council of scientific and industrial research (csir, india) for the fellowship. we thank dr. alain kohl for providing aag cell line. we are grateful to dr. sunil mukherjee for the discussions and beneficial scientific inputs. we also thank pavan kakumani for providing the ns b-pib construct and sudhanshu sekhar das for fhvb and fhvb m constructs. geetha sundaresan's involvement in agrobacterium infiltration assays is acknowledged. key: cord- -swlkjtyo authors: arzt, jonathan; branan, matthew a.; delgado, amy h.; yadav, shankar; moreno-torres, karla i.; tildesley, michael j.; stenfeldt, carolina title: quantitative impacts of incubation phase transmission of foot-and-mouth disease virus date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: swlkjtyo the current investigation applied a bayesian modeling approach to a unique experimental transmission study to estimate the occurrence of transmission of foot-and-mouth disease (fmd) during the incubation phase amongst group-housed pigs. the primary outcome was that transmission occurred approximately one day prior to development of visible signs of disease (posterior median hours, % ci: . – . ). updated disease state durations were incorporated into a simulation model to examine the importance of addressing preclinical transmission in the face of robust response measures. simulation of fmd outbreaks in the us pig production sector demonstrated that including a preclinical infectious period of one day would result in a % increase in the median number of farms affected ( additional farms and , pigs euthanized) compared to the scenario of no preclinical transmission, assuming suboptimal outbreak response. these findings emphasize the importance of considering transmission of fmd during the incubation phase in modeling and response planning. interventions . the proportion of transmission that occurs during the incubation phase is commonly denoted by theta (θ) , . a low value of θ indicates correspondingly low extent of transmission prior to observation of clinical disease. although a low θ is relatively uncommon, this has been associated with infectious diseases for which control or eradication programs have been highly successful such as smallpox and rinderpest , . at the other end of the spectrum are diseases for which θ is remarkably high, such as hiv/aids, which are notoriously difficult to control . closely related to the concept of θ is the temporal disparity between incubation and latency which represents the period of infectiousness included within the incubation period, defined herein as ω (omega; fig. ). while θ is intrinsically linked to the total length of the infectious period (often difficult to measure), ω can be estimated without having knowledge of the termination of infectiousness. additionally, the basic reproduction number (r ) of a pathogen is frequently cited to project and characterize an infectious disease epidemic. this quantity is defined as the average number of secondary cases generated by a typical primary case during its entire period of infectiousness, in a completely susceptible population and in the absence of control measures , . thus, in addition to aspects intrinsic to the specific host-pathogen combination, r will also be affected by numerous extrinsic factors at the population level. combining estimates for r , θ, and ω provides a robust background for evaluating the importance of preclinical transmission in successful modeling of disease spread and control interventions. this current study focused on investigating the concept of θ and ω for direct contact transmission of a virulent strain of foot-and-mouth disease virus (fmdv) amongst juvenile domestic pigs, and examining the impact of pre-clinical transmission on simulated outbreak size and severity within a us swine production system assuming either optimal or suboptimal response conditions. fmdv is a highly contagious viral pathogen of cloven-hoofed domestic and wild animals (genus: aphthovirus, family: picornaviridae) , . the virus, which is the causative agent of foot-and-mouth disease (fmd), has been associated with multiple major disease epidemics in previously free regions in recent years [ ] [ ] [ ] . given the highly contagious nature of the disease, the ability to predict potential fmd dissemination in a naïve population and assess the effectiveness of control interventions can result in critical improvements in preparedness and emergency response for food animal production systems . however, data-driven modeling is intrinsically and precariously dependent upon the appropriateness of the data used for parameterization. recent work examining incubation phase transmission of fmdv amongst cattle has been inconsistent between two studies suggesting either a high value of θ for dairy cattle or a low value for calves , . in contrast to this, a similar investigation demonstrated the occurrence of substantial transmission of fmdv during the incubation phase in group-housed pigs . this current report provides novel modeling approaches based upon the data obtained from this recent transmission study . the findings presented herein suggest a difference between the duration of the latent-and incubation periods (pre-clinical infectiousness; ω) in fmdv-infected pigs that is statistically significantly greater than , and a resultant θ that is larger than previously suggested in studies of fmdv transmission in cattle. additionally, simulating fmd outbreak scenarios using fixed and incrementally increasing values of ω demonstrated significant influence of incubation phase transmission on the size and duration of fmd outbreaks in us pig production systems, even in the presence of rigorous control strategies. fmdv transmission study. the experimental basis for the analyses presented herein consisted of a transmission study in which groups of naïve pigs were exposed to a group of fmdv-infected "donor pigs" through successive eight hour time periods (fig. ) . successful transmission from donors was determined by confirmed occurrence of fmd in contact-exposed pigs subsequent to exposure. the progression of infection was monitored and quantitated in both donors and contact-exposed pigs through measurements of viral genomic rna in serum and oropharyngeal fluids (opf). in brief, all pigs in the donor group were confirmed to have been infected by needle-free oropharyngeal inoculation with fmdv and were shedding fmdv rna in opf throughout the figure . definitions used to characterize distinct periods of infectious diseases. the latent period (e; green arrow) begins at the time of infection and ends at the onset of the infectious period. (i; red arrow). the incubation period (c; blue arrow) starts at the time of infection and ends at the appearance of clinical signs of disease (purple arrow). the difference between incubation and latency is denoted by ω (omega), and can have either a positive or negative value depending on whether transition to the infectious period occurs before or after appearance of clinical signs. the ratio of transmission occurring during incubation, and transmission occurring through the total infectious period is denoted by θ (theta; not shown). contact transmission trial. viremia in donor pigs was first detected at hours post inoculation (hpi), whereas fever (rectal temperatures > °c) and vesicular lesions were observed simultaneously at hpi ( supplementary fig. s ). there were no transmission events between the donors and contact groups or , which had been exposed to the donor pigs from to hpi, and to hpi, respectively. contrastingly, all pigs in contact groups through , corresponding to exposure from through hpi of the donors, developed clinical fmd (fig. ) . on this basis it was experimentally demonstrated that transmission from donor pigs had occurred at least hours prior to detection of clinical disease . a bayesian model was used to estimate the length of the latent, incubation, and infectious periods of the donor pigs based on data from the transmission trial . the model estimated the latent period to last slightly longer than one day (median hours, % ci - hours) ( table , fig. ). the incubation period was estimated to be approximately days (median hours, % ci: - ), and the total infectious period was estimated to be approximately days (median hours, % ci: - hours). the posterior median latent period was shorter than the prior median latent period (fig. a ). using the prior information, the latent period ended approximately hours after inoculation. by the posterior information, the length of the latent period was updated to last about hours (table ) . thus, the latent period is likely longer than a day and the effect of the observed data was large enough to influence the diffuse prior information (table ; figure . experimental design, fmdv transmission study. seven groups of five pigs each were exposed to five fmdv-infected donor pigs through successive eight hour exposure periods. contact groups were housed in separate isolation rooms before and after exposure to the donor pigs. the time points in the figure represent beginning (green) and end (red) of exposure for each contact group in relation to inoculation of the donor pigs. there was no transmission of infection to contact groups and (exposed from - , and - hours post infection of donors, respectively). all pigs in contact groups through developed clinical fmd after exposure to the donors. , incubation (c), and infectious (i) periods and their means (μ e, μ c, and μ i ) are expressed in terms of hours for a typical member of the donor animal group. parameter estimates for θ are in terms of a proportion, and β is expressed in terms of transmission rate. www.nature.com/scientificreports www.nature.com/scientificreports/ fig. a) . in contrast to this, the durations of the incubation and total infectious periods by posterior distributions were not significantly different from the prior distributions (fig. b,c) . a specific interest of this part of the analysis was to determine the duration of infectiousness during the incubation period (ω) as well as the proportion of transmission that occurred during this subclinical infectious period relative to total transmission (θ). the estimation of θ relies on the length of the total infectious period, which is comprised by both subclinical and clinical phases. assuming a median total infectious period of hours as estimated by the bayesian model, θ was estimated to be . . the duration of the subclinical phase of infectiousness corresponds to the difference between incubation and latent periods, both of which were modeled based upon directly measured experimental data. given the posterior medians of the latent ( hours) and incubation ( hours) periods, the difference between these values indicates a median duration of subclinical infectiousness (ω) of approximately hours ( % ci: . - . hours; table ). estimating the sensitivity of θ to variations in duration of infectious period. theta (θ) represents the fraction of transmission that occurs during the incubation phase in relation to transmission occurring through the total infectious period and is thereby intimately linked to the duration of the total infectious period. yet, no study has effectively measured the duration of infectiousness in fmdv-infected animals experimentally; rather, modeling studies have universally used proxies to generate estimates for this variable. in order to explore the sensitivity of θ to variations of the infectious period, θ was modeled using step-wise increasing durations of infectiousness ranging from to days, while keeping ω at the modeled estimate of . hours. the maximum resultant value of θ, which was based on modeling an infectious period of day was . ( % ci . - . ; supplementary table s ). contrastingly, if modeling a duration of infectiousness of days, the resulting estimate of θ was . ( % ci . - . ; supplementary table s ). as expected, θ varied inversely with duration of infectiousness throughout the sensitivity analysis. because transmission is rarely directly quantified in experimental or field studies, we tested the ability of our bayesian model to characterize transmission using various forms of more commonly available proxy measures for table ) . in order to test the reliability of these proxies to predict transmission, the bayesian model of donor pig infection dynamics was repeated to compare the model outcomes when the onset of infectiousness in donor pigs was defined based on the proxy measures rather than confirmed transmission events (cte-standard). the most noteworthy findings from this approach were the substantial changes in the modeled estimates of the duration of latency and subclinical infectiousness (ω) when defining the onset of infectiousness by either the occurrence of clinical signs of fmd in donor pigs or by any detection of fmdv rna in opf. defining the onset of infectiousness by the occurrence of clinical signs in donors led to a hour discrepancy of latency compared to the cte-standard, with a prolonged latent period of hours ( % ci - hours), and an ω of − . hours (fig. , supplementary table s ) . similarly, detection of (any) fmdv rna in opf as indicator of infectiousness resulted in a shortened latent period lasting only hours ( % ci - hours) and a resultant duration of subclinical infectiousness (ω) of hours ( % ci - hours; fig. , supplementary table s ). defining the onset of infectiousness by detection of fmdv rna in opf above a threshold of . log gcn/ml led to marginally decreased durations of latency and ω, whereas use of the proxy, detection of fmdv in serum led to the closest estimate to the cte-standard (fig. , supplementary table s ). our bayesian modeling of infection dynamics in fmdv-infected pigs estimated the occurrence of a subclinical infectious period (ω) of hours (table ). the practical ramification of this finding is that pigs that are infected with fmdv are capable of transmitting disease for approximately day prior to the development of any visible signs of infection, which could lead to further disease spread through animal movements and indirect contacts before a producer realizes through clinical observation that there is a health problem in the herd. in order to assess the impact of the duration of the subclinical infectious period in fmdv-infected pigs on outbreak size and duration, we performed a series of fmd outbreak simulations with the us national fmd model (interspread plus (isp) version . model software ), which can account for the complex movements and interactions seen in intensive pig production systems. simulations were run on a reduced farm population file composed of , swine operations with different production types and a total of , , pigs, in the eastern united states ( supplementary fig. s ). surveillance and control measures were modeled including passive and active surveillance in zones around infected premises, movement restrictions in the k zone around infected premises, and depopulation of infected premises. under optimal conditions, detection of the index case was based on the onset of clinical signs within the herd and depopulation occurred within to days (depending on herd size) at a rate of farms/day. for the suboptimal response, initial detection was delayed days, and depopulation was delayed days. for both the optimal and suboptimal responses, the duration of subclinical infectiousness was varied from to days, and the effect on outbreak size and duration examined. overall, incremental increases of subclinical infectious period (ω) led to substantial increases in the size and severity of simulated outbreaks under both categories of outbreak responses. when outbreak response conditions were assumed to be optimal, a -day subclinical infectious period (ω = day) as compared to absence of subclinical infectiousness (ω = day), resulted in a % increase of the median number of infected farms, necessitating euthanasia of , more pigs, and an increase of the median outbreak duration from to days ( fig. and supplementary table s ). when the outbreak response was assumed to be suboptimal, the effects of increasing ω were more profound, with a one-day subclinical infectious period resulting in median outbreak size increasing by farms ( %), outbreak duration increasing from days to days, and euthanasia of , additional pigs required ( fig. and supplementary table s ). further increases of ω resulted in corresponding increases in outbreak size and severity, under both optimal and suboptimal outbreak response conditions (fig. ) . specifically, each incremental increase in omega led to a longer duration of the outbreak, with some increments having significant effect. the greatest magnitude of effect that was estimated occurred with a -day subclinical infectious period (ω = day) combined with suboptimal outbreak response which resulted in . % of farms, and . % of all pigs in the population ( , , pigs) becoming infected. numeric values between and , with . increments corresponding to observations of lesions in contact-exposed pigs table . proxy measures used to determine the onset of infectiousness. www.nature.com/scientificreports www.nature.com/scientificreports/ effective control of infectious disease outbreaks is dependent upon rapid identification of infected individuals and mitigation of risks that could otherwise cause widespread dissemination of contagion. in the absence of an outbreak, data-driven mathematical models that can estimate disease spread and the impacts of control interventions can offer valuable insights to enable planning and preparedness . the ultimate goal of such modeling is to guide and assess the effectiveness of control measures to minimize disease impacts, quantitated as morbidity, mortality, economic loss, or other relevant metrics , . the overarching objective of the current investigation was to use detailed data from a carefully executed experimental study of fmdv transmission to model the durations of distinct stages of disease in fmdv-infected pigs, and to highlight the importance of addressing incubation phase (subclinical) transmission in fmd models. in order to explore the concept of disease spread before clinical detection, it was of particular interest to estimate the latent period (time from infection until onset of infectiousness) in relation to the incubation period (time from infection until appearance of clinical signs of disease). the estimated parameters were subsequently used to explore the impact of alterations in the duration of subclinical infectiousness on simulated fmd outbreaks in commercial swine production systems under both optimal and suboptimal response conditions. disease modeling is critically dependent upon input parameters that closely reflect the intrinsic properties of the infectious agent as well as externally variable features of the modeled scenario (e.g. population composition and density, contact networks and patterns, topographical characteristics, meteorological variations and availability of resources) , . the simulations in this current investigation utilized the national fmd model developed by the us department of agriculture to simulate fmd outbreaks within the us pig production system. this the proxy measures consisted of detection of fmdv rna in blood ("viremia"), detection of fmdv shedding in oropharyngeal fluid (opf) either above the assay lower limit detection ("any" shedding), or above a defined threshold of . log fmdv rna copies per ml ("threshold" shedding), or detection of clinical signs of fmd. the duration of latency (a) was underestimated compared to the cte-standard, when using fmdv shedding in opf ("any" or "threshold" shedding) to define the onset of infectiousness. detection of viremia as a proxy of infectiousness led to an estimated latent duration that was close to the cte-standard, whereas defining infectiousness by detection of clinical signs overestimated the duration of latency. the duration of subclinical infectiousness (b) and the proportion of transmission during the incubation phase (c) were underestimated when the onset of infectiousness was based on detection of clinical signs. the estimates based on the remaining four proxy-measures were less dispersed. for both parameters, detection of viremia provided the estimates closest to the cte-standard, whereas detection of fmdv shedding in opf provided slightly higher estimates. www.nature.com/scientificreports www.nature.com/scientificreports/ model is adapted to specific conditions within the us agricultural system, and parameterized to reflect the complex and overlapping outbreak response measures identified in the us national fmd outbreak response plan . additionally, the model is flexible and allows for adjustment of select parameters, as was performed in the current study to explore the effect of preclinical transmission on simulated outbreaks. similar to other disease spread models, this model is built upon assumptions concerning specific characteristics of the us production system for which it was designed. extrapolation of the modeled output should therefore be done with caution. however, under the given circumstances, the outcome of the outbreak simulations included herein serve to emphasize the critical impact that the occurrence of disease transmission during the incubation phase may have on the magnitude of an fmd outbreak. parameters for modeling of disease outbreaks are usually derived from meta-analyses figure . output of fmd outbreak simulations based on increasing durations ( - days) of subclinical infectiousness under optimal and suboptimal outbreak response conditions. ribbon plots for the cumulative number of infected pigs (a) and ridge plots for the epidemic curve (b) when modeled using incrementally increasing durations of subclinical infectiousness (ω) and assuming optimal (left panels) and suboptimal (right panels) outbreak responses. the lower edge, central line, and upper edge of the plots represent th percentile, median, and th percentile, respectively for the specific duration of the subclinical infectiousness (ω). www.nature.com/scientificreports www.nature.com/scientificreports/ of published experimental investigations that were not originally designed to assess disease transmission , . bayesian methods offer an alternative approach to inferring epidemiologic parameters from transmission experiments, which can improve our understanding of the latent and infectious periods , . regardless of the method used, in the absence of data explicitly describing actual disease transmission, different proxy measures must be used to define the transition between distinct stages of disease. the use of proxies often results in the assumption that the onset of infectiousness is defined either by the first evidence of infection in a given individual (pathogen detection), or by the appearance of visible clinical signs of disease. in the current investigation, the proxy measure of high-threshold shedding was interpreted to be the most relevant predictor of transmissibility of fmdv. onset of viremia was also correlated with infectiousness, which is likely due to the role of viremia in causing systemic dissemination and shedding of virus. as demonstrated in this investigation, inappropriate use of proxy measures to define the onset of infectiousness in replacement of actual transmission data can lead to drastic misinterpretations of the transmission potential of infected animals. these misinterpretations may in turn affect fmd outbreak simulations, resulting in underestimation of outbreak size and severity, leading to unrealistic estimates of resource needs and misguided guidance on the appropriate application of control interventions. specifically, underestimating the potential for disease transmission during the incubation phase may lead to wider dissemination of the outbreak than anticipated, and subsequent failure of reactive control interventions such as vaccination. contrastingly, overestimating disease transmission can promote excessively aggressive countermeasures and lead to destruction of large numbers of healthy animals and associated economic losses. this may result in substantial animal welfare issues and added economic losses as products and animals from unaffected farms cannot enter the production chain. appropriately balanced interventions are thus critical to effectively control disease spread while striving to maintain business continuity. the current investigation demonstrated that the modeled onset of infectiousness in fmdv-infected pigs occurred approximately one day prior to appearance of clinical signs of disease, as was consistent with the primary descriptive data from these experiments . this is consistent with earlier work which demonstrated fmd transmission during the incubation phase in pigs, lambs, and cattle . however, one published work contradicts these findings by suggesting that fmd is unlikely to be transmitted before the onset of clinical disease . although there is limited published information on this subject, the consensus of available data suggests that subclinical transmission of fmd does occur, indicating that incubation is longer than latency and the resultant ω is greater than zero. this result was concluded based on the significant disparity between the durations of incubation and latency which indicated a distinct period of subclinical infectiousness that was demonstrated by experimentation and verified by modeling. due to the limited group sizes and ubiquitous infection in groups to which transmission occurred, it was not possible to estimate r in the current investigation. however, previous investigations have estimated the basic reproduction ratio, r , for fmdv within groups of non-vaccinated pigs in experimental settings to be as high as . ( % ci: . - . ) or ( % ci: - ) . the combination of a generally high r for within-group transmission of fmdv in pigs, and a positive value for ω as determined in the current study suggests that the potential for fmdv transmission during the incubation phase should be recognized when modeling fmd outbreak scenarios. our estimate for the proportion of preclinical transmission of fmdv that occurs in pigs was %, falling between values reported for sars (θ < %) and smallpox ( < θ < %) . fraser et al. identified a relationship between r , θ, and the effectiveness of control interventions to bring an outbreak under control. as r and θ increase, control interventions must be highly effective, and multiple interventions are required in order bring the outbreak under control. estimated values of r and θ for fmdv suggest that control of epidemics is dependent on using multiple, highly effective control interventions. interestingly, these results may also suggest that isolation and contact tracing, if conducted nearly perfectly, could eventually be sufficient to prevent epidemic propagation. however, in the absence of significant technological advances, such interventions are unlikely to be implemented perfectly in livestock populations, and the timelines required for disease control could be much longer than when more interventions are enacted simultaneously. the potentially profound consequences of fmdv incursions into regions previously free of fmd can, to a great extent, be attributed to the highly contagious nature of the virus. this was demonstrated in the current study by the maximum impact simulated example of infection of over , , pigs when high ω was combined with a suboptimal outbreak response. although fmdv can be transmitted via a multitude of both direct and indirect routes , movement of infected animals has been identified as the most significant risk for dissemination of infection during the early phase of an outbreak . this current investigation demonstrated the relevance of fmdv transmission during the incubation period in group-housed pigs. furthermore, it was demonstrated that the duration of subclinical infectiousness had significant effects on spread and duration of simulated fmd outbreaks. the data used for modeling of disease stage durations in the current study were derived from experiments in which pigs were infected with one specific fmdv strain, under experimental conditions. the virus strain and host criteria were chosen based upon extensive experience in our laboratory with these conditions and the assumption that the virus and conditions were representative of most virulent fmdvs. it is possible that disease dynamics in the field may differ due to strain-specific variations in virulence, as well as differences in animal age, health status, and housing conditions. thus, it should be emphasized that the modeled output presented herein is based on experimental conditions, and represents our best estimate of what could be expected to occur under natural conditions in the field. the findings presented herein demonstrate the importance of considering and elucidating the intricacies of key epidemiologic parameters, including preclinical infectiousness, the importance of understanding the relationships between proxy measures of disease status and infectiousness, and the subsequent value of incorporating these detailed parameters into disease spread models. additionally, improved understanding of the influence of animal-level disease dynamics upon dissemination of fmd outbreaks may lead to improved approaches to www.nature.com/scientificreports www.nature.com/scientificreports/ surveillance and diagnostic testing to further refine control measures and maximize effectiveness while limiting undesired consequences for the agricultural industries. animal experiment. the data used in this current investigation were derived from an experimental trial designed to evaluate the onset of infectiousness in relation to the appearance of clinical disease in fmdv-infected pigs (fig. ) . a detailed description of the experimental study and clinical findings has been published previously . animal experiments were carried out within bsl -ag facilities at plum island animal disease center, new york. all procedures were carried out in accordance with guidelines specified within the associated experimental protocol (protocol - -r), and were approved by the plum island animal disease center institutional animal care and use committee. in brief, the study included groups of pigs of approximately - weeks of age (~ kg), of which one group was infected with fmdv a cruzeiro through simulated-natural inoculation . the remaining groups of pigs (contact groups - ) were sequentially exposed to the infected donor pigs through hours of successive co-habitation within a designated isolation room (fig. ) . after exposure to the donor pigs, contact-exposed pigs were moved into separate isolation rooms and were monitored for development of fmd. samples collected were oropharyngeal swabs to assess virus shedding and blood samples to measure viremia. clinical examinations and sample collection were done at - hour intervals after exposure. the choice to use fmdv a cruzeiro for these experiments was based upon numerous previous experiments in our laboratory which had demonstrated that this strain was consistently virulent and transmissible in pigs , - . data analysis. definitions. the end of latency for the donor pigs was defined as the beginning of the hour contact exposure period during which the first successful transmission event occurred. it was not possible to attribute transmission events to specific individuals as donors and contact pigs were allowed to move freely within the exposure room. thus, the earliest observed transmission event was used to define the transition from latent to infectious periods for all donor pigs. the end of the incubation period for the donor pigs was determined for each pig individually by the first detection of vesicular lesions, which for all donor pigs coincided with detection of fever (rectal temperature ≥ °c). fmdv shedding was defined by continuous detection of fmdv rna in opf. viremia was defined by detection of fmdv rna in serum. a bayesian model was fitted to the data for the purpose of estimating the length of three distinct periods the donor pigs were expected to traverse: the latent, incubation, and infectious periods. a modeling approach was adapted from that published by charleston et al. . this model describes the relationship among the observed transmission successes and the unobserved latent, incubation, and infectious periods as well as the hyperparameters describing the distribution of those periods in the following way: : start/end time of challenge i of donor j. p ij : probability of successful transmission in challenge i of donor j. β: transmission rate. t ij : time during challenge i for which donor j is infectious. e j : latent period for donor j. c j : incubation period for donor j. i j : infectious period for donor j. μ e , μ c , μ i , σ e , σ c , σ i , ρ ec : hyperparameters for latent, incubation, and infectious period prior distributions (in order; the means for the three periods, the standard deviations for the three periods, and the correlation between the latent and incubation periods). α e , η e , α c , η c , α i , η i : hyperparameters for the means and standard deviations for the latent, incubation, and infectious period prior distributions (in order, the mean and standard deviation for the mean of the prior latent distribution, the mean and standard deviation for the mean of the prior incubation distribution, the mean and standard deviation for the mean of the prior infectious distribution). the likelihood above describes the relationship between the observed transmission event data with the unobserved latent, incubation, and infectious periods, while the prior information (table ) was based on accumulated data from previous investigations carried out under similar conditions [ ] [ ] [ ] [ ] , or were left diffuse in the absence of such information. these sources of information were combined and the posterior distribution over the parameters (latent, incubation, and infectious periods along with the means of the three periods) given the observed data ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ (the outcomes of the transmission events) was estimated. this distribution is proportional to the product of the likelihood and the joint prior distribution for all of the parameters. as the posterior conditional distributions for at least the latent period, infectious period, and the transmission rate were intractable, a numerical rather than an analytic approach was pursued. the model was coded using just another gibbs sampler (jags) software designed to perform markov chain monte carlo (mcmc) simulations . this version of the model required two sacrifices: the latent and incubation periods could not be jointly lognormal, and the prior hyperparameter variance terms (σ e , σ c, and σ i ) were held fixed due to mcmc chain convergence issues as a result of unidentifiability issues. the result of the former being that the latent and incubation periods are assumed independent a priori. the proportion of infection that occurs prior to onset of clinical disease, denoted as θ, is a function of the quantities estimated by the model described above. using the jags version of the model adapted from charleston et al. and assuming that latent and incubation periods are independent a priori, θ is described in as follows: in addition to θ, we propose herein a distinct parameter (ω; omega) that allows more precise and direct inference about pre-clinical infectiousness to be made regardless of the total duration of the infectious period by representing the disparity between incubation and latency (fig. ) . estimating fluctuations of θ due to variations in duration of infectious period. as total duration of infectiousness was not experimentally evaluated in the current study, information about the length of the infectious period is drawn almost solely from the prior information. in order to evaluate the sensitivity of θ to variations in the total infectious period, θ was estimated as described above, but using incrementally increased integer values of infectious duration ranging from to days. modeling infection dynamics using different proxy measures to determine the onset of infectiousness. four different proxy measures (table ) were evaluated for their ability to predict disease transmission. the outcome of modeling transmission using the defined proxy measures was compared to the standard model, which was based on confirmed transmission of fmdv to contact-exposed pigs (confirmed transmission event (cte) -standard). the four evaluated proxies were defined as follows: (a) detection of fmdv rna in serum, (b) detection of any fmdv rna in opf, (c) detection of fmdv rna in opf above a threshold of . log gcn/ml, which had previously been associated with successful transmission of fmdv (d) clinical signs of fmd. the data were input into a bayesian model via five distinct indicator matrices, one for each measure of transmission, in which the number of rows equaled the number of contact animals-hours-post-inoculation combinations and the number of columns represented the five donor pigs. each entry in the matrices was either a , if the donor animal met the criteria for successful transmission for the given transmission metric (table ) during exposure to the contact animal represented by that row, or a if the donor animal did not meet the criteria for successful transmission given transmission metric at that time. for the cte-standard transmission metric, which was defined by confirmed transmission to contact-exposed pigs, the vector for all donors was identical as the effect of individual donors on contact animal could not be determined as both sets of animals were exposed to one another in groups. simulation modeling of an fmd outbreak using estimated transmission parameters. the objective of the fmd outbreak simulations described herein was to evaluate the effect of altering the duration of the subclinical infectious period (omega; ω) on spread and duration of an outbreak of fmd in a us pig production sector. estimates of disease stage durations were derived from modeling of animal-level infection dynamics ( table ). the originally modeled output consisted of estimates for the durations of latent (e), incubation (c) and infectious (i) periods. the durations of the subclinical infectious period (ω = c − e) and clinical infectious period (i c = i − c) were derived from these outputs. in addition to the baseline scenario (ω = day), five omega values ( day, days, days, days, and days) were subjectively selected to evaluate the effect on altering the duration of the subclinical infectious period on the spread and duration of simulated outbreaks. while choosing different omega values, the durations of latent and clinical infectious periods were kept constant (similar to baseline scenario) except for the scenario in which ω = day of omega where the latent duration was set to days (supplementary table s ). the within-herd (wh) software version . . available through the north american animal disease spread model was used to estimate herd-level parameters based on the modeled animal-level fmd disease stage durations. a spatial microsimulation model called the farm location and agricultural production simulator (flaps) was used to generate a synthetic population file of , farms with , , total pigs representative of pig production systems in the eastern united states (supplementary fig. s ). the us pig farms (with essential attributes for isp such as identification number, herd size, type of farms, and cartesian coordinates) located in the great lake, north east and south east regions were included in the model scenarios. about % (n = , ) of the total pig farms in the united states are located in these regions. amongst these farms, % were commercial farms with a median herd size of pigs (range: to , ) and % were small-scale enterprises with a median herd size of pigs (range: to ). farm-type specific movement parameters and contact rates were assigned to reflect differences in movements between commercial and small-scale enterprises. fmd outbreak simulations were performed using interspread plus (isp) version . model software . the isp www.nature.com/scientificreports www.nature.com/scientificreports/ is a state-transition, stochastic and spatial modeling tool for the simulation of fmd and other similar diseases . twelve fmd outbreak scenarios were developed in isp representing optimal and suboptimal outbreak response conditions for each of distinct values for the duration of subclinical infectiousness: day, day, days, days, days, and days of omega, respectively (supplementary table s ). the unit of interest in the model was the individual farms. fmd epidemics were initiated from single farms. after exposure to fmdv, the susceptible farms were modeled to transit into latent, subclinically infectious, clinically infectious, and depopulated states. the spread of fmdv from an infected to susceptible farms was modeled to occur through direct contact, indirect contact, and local spread (supplementary note). the daily probability of transmission of fmd virus from infected farms to susceptible farm was calculated as the hypergeometric probability of shipping at least one infected animal off of an infected farm given the average herd size, shipment size, and the number of infected animals in a herd on a given day ( supplementary fig. s , supplementary note). once an infected farm was detected, several control strategies were imposed simultaneously as is typically performed in response to outbreaks in fmd-free areas. control strategies included zoning of control areas, tracing of animal movements, animal movement restrictions, depopulation of the infected farms, and surveillances as delineated in the national response plan for fmd (supplementary note). for each of the omega scenarios, two overall control strategies (optimal and suboptimal) were separately simulated. in the optimal control strategy, the detection of infected farms through passive surveillance was modeled to occur just after onset of clinical signs, which was further delayed by days in suboptimal control category. additionally, the delay in depopulation of detected farms was (small farms) to days (big farms) in the optimal control category, which was delayed by more days to represent the suboptimal control. the major outputs parameters were outbreak size (number of infected farms and pigs), epidemic duration (days from onset of infection to end of epidemic), time between onset of infection to detection, and daily new infected farms due to each of the incorporated omega values. the median and interquartile range of outbreak size and epidemic duration were reported. the data analyses were performed using sas (sas institute inc., nc, usa, ) and microsoft excel (microsoft excel, redmond, washington, ). the kruskal-wallis test with bonferroni corrections was performed for multiple group comparisons for the outcomes from various omega values. a p-value of ≤ . % was considered for statistical significance. all data generated or analyzed during this study are included in this published article and its supplementary information files. table . prior distributions and estimates used to model animal level infection dynamics. posterior estimates for the latent (e), incubation (c), and infectious (i) periods, their means (μ e, μ c, and μ i ), and their standard deviations (σ e , σ c , and σ i ) are expressed in terms of hours for a typical member of the donor animal group. parameter estimates for θ are in terms of a proportion, and β is expressed in terms of transmission rate. mathematical prediction in infection models of foot-and-mouth disease transmission dynamics and control of ebola virus disease (evd): a review data-driven models of foot-and-mouth disease dynamics: a review decision-making for foot-and-mouth disease control: objectives matter bayesian inference of epidemiological parameters from transmission experiments simulation modelling of a hypothetical introduction of foot-andmouth disease into alberta managing complexity: simplifying assumptions of footand-mouth disease models for swine transmission of foot-and-mouth disease virus during the incubation period in pigs planning for smallpox outbreaks factors that make an infectious disease outbreak controllable relationship between clinical signs and transmission of an infectious disease and the implications for control global eradication of rinderpest. yea or nay? infectious diseases of humans: dynamics and control on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations the pathogenesis of foot-and-mouth disease ii: viral pathways in swine, small ruminants, and wildlife; myotropism, chronic syndromes, and molecular virus-host interactions foot-and-mouth disease description of recent foot and mouth disease outbreaks in nonendemic areas: exploring the relationship between early detection and epidemic size reemergence of foot-and-mouth disease the foot-and-mouth disease epidemic in japan. the journal of veterinary medical science/the foot and mouth disease virus transmission during the incubation period of the disease in piglets, lambs, calves, and dairy cows quantifying the value of perfect information in emergency vaccination campaigns parameter values for epidemiological models of footand-mouth disease in swine parameterization of the duration of infection stages of serotype o foot-and-mouth disease virus: an analytical review and meta-analysis with application to simulation models a meta-analysis quantifying transmission parameters of fmdv strain o taiwan among non-vaccinated and vaccinated pigs the pathogenesis and diagnosis of foot-and-mouth disease relative risks of the uncontrollable (airborne) spread of fmd by different species infection dynamics of foot-and-mouth disease virus in pigs using two novel simulated-natural inoculation methods early events in the pathogenesis of foot-and-mouth disease in pigs; identification of oropharyngeal tonsils as sites of primary and sustained viral replication detection of foot-and-mouth disease virus rna and capsid protein in lymphoid tissues of convalescent pigs does not indicate existence of a carrier state direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences american animal disease spread model (naadsm) development team simulating the distribution of individual livestock farms and their populations in the united states: an example using domestic swine (sus scrofa domesticus) farms interspread plus: a spatial and stochastic simulation model of disease in animal populations pauszek are thanked for supporting sample processing and laboratory analyses. c.s. and j.a. conceived and coordinated the study, executed the animal experiments and drafted the manuscript. m.b. performed the bayesian analysis and contributed to data interpretation. k.m. and s.y. performed simulation modeling and contributed to data interpretation. a.d. coordinated and oversaw data analyses and contributed scientific content. m.t. critically reviewed data analyses and contributed scientific content. all authors have reviewed and revised the manuscript and approved the final product. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -iiw w authors: ding, xibing; jin, shuqing; tong, yao; jiang, xi; chen, zhixia; mei, shuya; zhang, liming; billiar, timothy r.; li, quan title: tlr signaling induces tlr up-regulation in alveolar macrophages during acute lung injury date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: iiw w acute lung injury is a life-threatening inflammatory response caused by severe infection. toll-like receptors in alveolar macrophages (amΦ) recognize the molecular constituents of pathogens and activate the host’s innate immune responses. numerous studies have documented the importance of tlr-tlr cross talk, but few studies have specifically addressed the relationship between tlr and tlr . we explored a novel mechanism of tlr up-regulation that is induced by lps-tlr signaling in a dose- and time-dependent manner in amΦ from c bl/ mice, while the lps-induced tlr expression was significantly reduced in tlr (−/−) and myd (−/−) mice and following pretreatment with a nf-κb inhibitor. the enhanced tlr up-regulation in amΦ augmented the expression of cytokines and chemokines in response to sequential challenges with lps and poly i:c, a tlr ligand, which was physiologically associated with amplified amΦ-induced pmn migration into lung alveoli. our study demonstrates that the synergistic effect between tlr and tlr in macrophages is an important determinant in acute lung injury and, more importantly, that tlr up-regulation is dependent on tlr -myd -nf-κb signaling. these results raise the possibility that bacterial infections can induce sensitivity to viral infections, which may have important implications for the therapeutic manipulation of the innate immune system. mouse amΦ were isolated from the balf of wild-type (wt) mice and stimulated with lps ( , . , . , , or μ g/ml) in dmem containing % fbs for h. the graphed values represent the mean ± sem. five mice were analyzed per group. (c,d) mouse amΦ were isolated from the balf of wt mice and stimulated with lps ( μ g/ml) in dmem containing % fbs for - h. the graphed values represent the mean ± sem. five mice were analyzed per group. (a,c) a western blot of tlr protein expression in amΦ . actin expression was identified to normalize the densitometry of tlr expression. (b,d) rt-pcr analysis was used to evaluate tlr mrna expression in amΦ . β -actin mrna was detected for normalizing the value of tlr mrna. (e,f) qrt-pcr analysis was used to evaluate tlr mrna expression in amΦ . *p < . compared with the other groups. immunity against some viral infections. numerous studies have documented the importance of tlr cross talk, and multiple signaling pathways contribute to synergistic tlr ligand-dependent cytokine expression. in , alexopoulou l. et al. reported in nature that following an intraperitoneal injection of lps into mice, dramatic up-regulation of the expression of tlr mrna was observed in all tissues except the thymus, suggesting that the nuclei are stained with dapi (blue). the images were acquired using evosfl fluorescence microscopy. (d) qrt-pcr analysis was used to evaluate tlr mrna expression in amΦ . *p < . compared with the other groups; **p < . compared with the other groups. the expression of tlr is inducible. of note, a particularly high expression level of tlr mrna was observed in lung tissue. these results raised the possibility that bacterial infection can induce sensitivity to viral infection. this observation prompted us to further investigate the mechanism of tlr -tlr cross talk in amΦ . in the present study, using both an in vivo ali mouse model and the in vitro culture of amΦ from tlr −/− and myd −/− mice, we demonstrate that lps up-regulation of tlr in amΦ is dependent on tlr -myd -nf-κ b signaling. the functional relevance of the amplification in tlr -induced tlr expression in amΦ was demonstrated by a marked increase in the expression of the chemokines and cytokines, including macrophage inflammatory protein- (mip- ), macrophage chemoattractant protein- (mcp- ), tumor necrosis factor-alpha (tnf-α ), and interleukin- (il- ), in the ali mouse model. furthermore, we observed alveolar-capillary permeability and the induction of polymorphonuclear leukocyte (pmn) migration in response to sequential challenges with lps and poly i:c. thus, tlr up-regulation in amΦ is dependent on tlr -myd -nf-κ b signaling, which may represent an important mechanism responsible for amplifying pmn migration. to detect the expression of tlr in lps-induced amΦ , which were isolated from the bronchoalveolar lavage fluid (balf) of wild-type (wt) mice, lps was administered at different dosages ( , . , . , , and μ g/ml). tlr expression increased with . μ g/ml but was markedly increased following treatment with μ g/ml lps, and the increase in tlr expression trended back to the basal level at μ g/ml lps (fig. a,b ). as shown in fig. c , a μ g/ml lps challenge of amΦ from wt mice was associated with an increase in tlr protein expression at h and a more marked increase at h, while the increase in tlr expression trended back to the basal level at h. the tlr mrna expression changes shown in fig. d paralleled the protein expression changes. in addition, we obtained the same results using quantitative pcr (fig. e,f) . these results suggest that lps, a tlr ligand, can induce tlr expression in amΦ . lps up-regulates tlr expression in amΦ through tlr -myd -nf-κb signaling. as shown in fig. a , lps ( μ g/ml) challenge of amΦ , which were isolated from wt mice, resulted in an increase in tlr protein expression at h and a marked increase at h. however, in amΦ from tlr −/− mice, lps failed to induce tlr expression ( fig. a) , indicating that tlr signaling mediates the lps-induced up-regulation of tlr . all tlrs, with the exception of tlr , utilize myd , and tlr /myd signaling pathway is the primary signaling pathway used to induce the expression of pro-inflammatory cytokines . however, tlr can signal through both myd -dependent and -independent pathways, and the myd -independent signaling for tlr results in the production of type i ifns . to address the role of myd in mediating the lps-tlr -induced up-regulation of tlr , lps ( μ g/ml) was administered to amΦ from myd −/− mice, and tlr expression was assessed. as shown in fig. a , myd deficiency prevented lps-induced up-regulation of tlr . the changes in the mrna expression of tlr are shown in fig. b , and these results corresponded to the observed changes in protein expression. as shown in fig. c , the localization of tlr was further investigated in amΦ by immunofluorescence. in the absence of lps, tlr was found within small vesicles throughout the cytoplasm. tlr -containing vesicles increased in a time-dependent manner in amΦ from wt mice, but not in tlr −/− or myd −/− mice, and similar results were observed using quantitative pcr (fig. d) . can occur in the presence of myd . nf-κ b p was detected using nuclear protein extracts from amΦ to determine whether lps-tlr up-regulation of tlr was the result of activation of nf-κ b. as shown in fig. a , in response to lps in wt mice, the expression of nf-κ b p increased at . h and reached a significant level at . h. however, in tlr −/− mice, lps failed to induce nuclear nf-κ b p expression. to address the role of nf-κ b in mediating the lps-induced up-regulation of tlr , we determined the effects of ikk-nbd, an nf-κ b inhibitor , , on lps-induced tlr expression in amΦ . as shown in fig. b ,c, ikk-nbd ( μ m) prevented the lps-induced up-regulation of tlr mrna and protein expression in amΦ isolated from wt mice. we observed similar results by quantitative pcr (fig. d) . these results demonstrate that nf-κ b signaling plays a role in mediating tlr -tlr cross talk. to address the physiological relevance of lps/tlr activation and tlr expression in amΦ , we assessed mip- expression in the lungs using sequential challenges of lps and poly i:c. injection of lps at time followed by a saline injection (group ) at h resulted in a marked increase in mip- expression by h after lps challenge, which was further increased at h and then returned to the basal level by h after lps challenge (fig. a) . saline injection at time and poly i:c injection ( μ g/g body weight, intratracheally administered) (group ) at h caused a very small increase in mip- expression. however, the sequential injection of lps at time and poly the results show the effects of sequential challenges with lps and poly i:c on mip- expression in the lungs. in wt mice, lps ( μ g/g) or saline (sal) was injected intratracheally at time h, and hours later, poly i:c ( μ g/g) or sal was injected. in tlr −/− and tlr −/− mice, lps was injected intratracheally ( μ g/g) at time h, and poly i:c ( μ g/g) was injected intratracheally at h. lung tissue was harvested at the indicated times, and mip- protein expression was detected using western blotting (a,c), with actin being used to normalize the densitometry of mip- expression. additionally, mip- mrna expression was detected using rt-pcr (b), and qrt-pcr was used to detect mip- mrna expression in amΦ (d). β -actin mrna was used to normalize the value of mip- mrna expression. the graphed values represent the mean ± sem. five mice were analyzed per group. **p < . compared with the other groups; ***p < . compared with the other groups. because amΦ production of chemokines, such as mip- , is an important determinant of pmn migration, we also addressed the role of lps/tlr -mediated up-regulation of tlr in regulating pmn migration in the lungs. pmns were counted in the balf of wt, tlr −/− , and tlr −/− mice. in the lps (group ) and poly i:c (group ) only injection groups, mip- caused a slight increase in pmn migration in wt mice when compared with the saline control (group ). sequential injection of poly i:c at h after the initial lps injection (group ) markedly increased pmn migration in wt mice compared with the other groups. however, sequential challenge with lps and poly i:c did not significantly increase pmn migration in tlr −/− mice compared with wt mice (fig. ) . moreover, there was a similar increase in amΦ after sequential challenge with lps and poly i:c in tlr −/− mice (group ) when compared to lps alone (group ). additionally, the observed mip- expression level in the lung was consistent with pmn migration. together, these data show the important role of tlr signaling and amΦ mip- expression in mediating pmn migration in response to the up-regulation of tlr expression. to address the effect of lps/tlr -mediated activation of tlr in amΦ on inflammatory cytokines, we assessed tnf-α and il- in the serum and balf, as well as the chemokines mip- and mcp- in the balf, following sequential intratracheal challenges with lps and poly i:c. in the ali animal model, lps was injected intratracheally at time , and poly i:c was injected intratracheally at h (the time point when tlr was up-regulated in amΦ as described above). enzyme-linked immunosorbent assays (elisas) were used to assess mip- and mcp- in the balf as well as il- and tnf-α in both the balf and serum at h. lps (group ) or poly i:c (group ) alone induced a slight increase in mip- , mcp- , il- , and tnf-α when compared with the saline control (group ) (fig. ) , whereas sequential challenge with lps and poly i:c (group ) stimulated a marked increase in mip- , mcp- , il- , and tnf-α expression. however, the sequential challenge with lps and poly i:c did not significantly increase mip- , mcp- , il- , and tnf-α in tlr −/− mice (group ) when compared to wt mice (group ). the sequential challenge with lps and poly i:c also led to a similar increase in tlr −/− mice (group ) when compared to the lps alone group (group ) (fig. ) . because chemokine-dependent pmn migration and inflammatory cytokine secretion are important determinants of ali, we next addressed the role of lps/tlr -mediated up-regulation of tlr in alveolar-capillary permeability using evan's blue, which binds albumin, in wt, tlr −/− , and tlr −/− mice. we assessed permeability at , , , and h after lps/poly i:c administration. as shown in the results show the effects of sequential challenges with lps and poly i:c on pmn migration into the airspaces. in wt mice, lps ( μ g/g) or saline (sal) was injected intratracheally at time h, and h later, poly i:c ( μ g/g) or sal was injected. in tlr −/− and tlr −/− mice, lps ( μ g/g) was injected at time h, and poly i:c ( μ g/g) was injected intratracheally at h. pmn numbers in balf were counted at the times indicated. the graphed values represent the mean ± sem. five mice were analyzed per group. **p < . ; ***p < . compared between the groups as indicated. scientific reports | : | doi: . /srep weight, intratracheally administered) at h resulted in minimal changes (group ). however, the sequential injection of lps at time and poly i:c injection at h (at a time when tlr was up-regulated) in wt mice (group ) led to augmented changes in histopathology, including diffuse interstitial edema and inflammatory cell infiltration, compared with the single lps or poly i:c challenge. in contrast, the sequential injection of poly i:c at h after lps failed to induce augmented histopathology changes in tlr −/− mice (group ) compared with wt mice. in addition, we did not observe augmented histopathology changes in tlr mice (group ) and instead observed a change similar to that observed for lps alone (group ). taken together, these data show the important role of lps/tlr signaling in the up-regulation of tlr expression in ali. amΦ , which are located in the alveolar compartment of the lungs, provide a key initiation signal for ali [ ] [ ] [ ] . previous studies have shown that the later phase of ali is neutrophil-dependent , while amΦ contribute to the acute phase of lung injury. once activated by bacteria or viruses, amΦ generate and release a multitude of mediators, such as cytokines (il- , tnf-α ) and chemokines (mcp- , mip- ) , . these mediators act as chemoattractants for the migration of large numbers of activated inflammatory cells, such as monocytes and neutrophils, into the airspaces . synergy between viral and bacterial tlr signaling, which leads to amplification of the inflammatory response, has been reported previously . additionally, tian x. et al. found that poly i:c enhanced susceptibility to secondary pulmonary infections by bacteria . co-stimulation with lps and poly i:c markedly enhances the immune response, although the mechanism for this combined effect remains poorly understood. alexopoulou l. et al. found that when lps was injected intraperitoneally, tlr expression in lung tissue was dramatically up-regulated, which suggests that the expression of tlr is inducible. pan et al. provided direct evidence that lps induces tlr expression via a tlr -myd -irak-traf -nf-κ b-dependent signaling pathway in human the results show the effects of sequential challenges with lps and poly i:c on alveolar-capillary permeability and histological changes in the lung. in wt mice, lps ( μ g/g) or saline (sal) was injected intratracheally at time h, and h later, poly i:c ( μ g/g) or sal was injected. in tlr −/− and tlr −/− mice, lps ( μ g/g) was injected at h, and poly i:c ( μ g/g) was injected intratracheally at h. lung tissue and serum were harvested at the times indicated, and alveolar-capillary permeability was detected by using evan's blue staining (a). lung tissue was collected at the time indicated and subjected to hematoxylin and eosin (h&e) staining (x magnification) (b). **p < . ; ***p < . compared between the groups as indicated. scientific reports | : | doi: . /srep peripheral blood monocytes and monocytic cell lines, such as thp- cells. furthermore, we observed that lps could induce tlr expression in a dose-and time-dependent manner in amΦ . the stimulation of tlr by lps induces the release of critical proinflammatory cytokines that are necessary to activate a potent immune response. indeed, lps/tlr signaling has been intensively studied in recent years [ ] [ ] [ ] . in our study, the role of tlr signaling in regulating tlr expression was clearly shown using tlr −/− mice. lps challenge in wt mice induced the up-regulation of tlr , whereas this response was impaired in tlr −/− mice. the major adaptor molecules that bind to the intracellular domain of tlr are myd and trif , , and our results showed that myd mediated the tlr -tlr cross talk, as lps challenge of myd −/− mice failed to induce tlr expression. furthermore, the activation of nf-κ b was associated with an increase in tlr expression after lps challenge and a reduced expression of tlr in amΦ in which nf-κ b was inhibited by ikk-nbd. therefore, these results demonstrate an important role for tlr -myd -nf-κ b in mediating tlr expression. ali is caused by an uncontrolled systemic inflammatory response that results from direct (aspiration, pneumonia, ventilation-induced lung injury, etc.) or indirect injury (sepsis, hemorrhagic shock, etc.), leading to the activation of amΦ and the sequestration of neutrophils . excessive recruitment of leukocytes is critical to the pathogenesis of ali. neutrophils are the first leukocytes to be recruited to sites of inflammation in response to chemokines released by activated amΦ . specifically, stimulation of amΦ leads to the release of chemokines, which induce neutrophils to migrate from the intravascular space across the endothelium and epithelium into the airspaces . according to the relative position of the cysteine residues, chemokines have been classified into four subfamilies (cxc, cc, c, and cx c). among these, mip- , which is also known as cxcl , is thought to play a major role in mediating neutrophil recruitment . belperio j.a. et al. found that lung expression of mip- was correlated with lung injury and neutrophil sequestration during the pathogenesis of ventilation-induced lung injury, and cxcr −/− mice show a marked reduction in neutrophil sequestration and lung injury. additionally, mip- was shown to be up-regulated in the lungs and balf of animals, which was associated with neutrophil accumulation in the lungs after lps administration [ ] [ ] [ ] . furthermore, villar j. et al. showed that a cxcl polymorphism is associated with better outcomes in patients with severe sepsis . in the present study, we observed that lps/tlr signaling up-regulated tlr expression in amΦ . this cross talk between tlr and tlr in amΦ resulted in the amplification of cytokine (il- , tnf-α ) and chemokine (mip- , mcp- ) expression in response to lps and poly i:c, which activate tlr and tlr , respectively, and subsequently led to enhanced pmn sequestration into the lung, which was found to be correlated with ali based on the assessment of alveolar-capillary permeability and histological sections of lung tissue. thus, the present study demonstrates a novel mechanism by which lps can induce amΦ to up-regulate tlr expression, through a tlr -myd -nf-κ b-dependent pathway, thereby sensitizing amΦ to tlr ligands and promoting enhanced lung inflammation (fig. ) . in clinical scenarios, a variety of pathogens are involved in lung infections. in particular, viral infections are a significant risk factor for acquiring bacterial infections , . tlr is thought to be a major mediator of the cellular response to viral infection, because it responds to dsrna, a common by-product of viral replication . tlr has been implicated in infections by mouse cytomegalovirus (mcmv), reoviruses, lymphocytic choriomeningitis virus (lcmv), and influenza a virus (iav) . tlr −/− mice are more resistant to lethal west nile virus, a mosquito-borne ssrna flavivirus, which causes human disease of variable severity , and show reduced inflammation and lethality upon iav infection , . thus, up-regulation of tlr may contribute to an increased lung immune response to viral infection following a bacterial infection. despite the relatively well-known role of viral infections in promoting bacterial infections, it is still not clear whether bacterial infections also promote viral infections. therefore, our results are the first to offer new insight into this topic. there is also a growing body of evidence showing that most patients have a mixture of bacterial and viral infection , , and the cross talk between tlr and tlr induces a synergistic inflammatory response in cases of mixed infection. cameron r.j. et al. found that bacterial and viral pathogens interact to cause additional increases in inflammatory markers and an exacerbation of disease severity in patients with chronic obstructive pulmonary disease (copd). tlr has been shown to respond to dsrna, a replication intermediary for many viruses, but the heterologous rna released from necrotic cells or that is generated by in vitro transcription, such as mrna, also stimulates tlr signaling and induces immune activation. indeed, cavassani ka observed tlr activation during experimental polymicrobial sepsis and ischemia gut injury in the absence of an exogenous viral stimulus, and tlr −/− mice were protected from the lethal effects of sustained inflammation. moreover, treatment with a tlr antibody could attenuate the tissue injury associated with gut ischemia and significantly decrease sepsis-induced mortality. therefore, tlr serves as a regulator of the amplification of the immune response as well as an endogenous sensor of necrosis, independent of viral activation. our study showed that up-regulation of tlr significantly amplified il- , tnf-α , mcp- , and mip- expression, which then enhanced pmn migration and finally led to ali. tlr cross talk has obvious advantages for the host in protecting against infectious agents, because it enhances the initial immune reaction to pathogen infection and better primes the host for mounting a more robust adaptive immune response. the concept that multiple tlr-ligand interactions are required for effective host resistance to pathogens has important implications for the design of vaccinations and immunotherapies against infectious diseases. several studies have convincingly shown the improved efficacy of treatment with multiple tlr ligands compared with single tlr ligands in stimulating cellular immune responses in vivo. for example, co-administration of poly i:c and cpg odns increased serum cytokine production in a mouse tumor model when compared to the administration of either of the ligands alone . bone-marrow-derived dcs exposed to both poly i:c and a tlr ligand more effectively stimulated cytotoxic t lymphocyte responses compared to dcs exposed to either tlr ligand alone . these studies provide an important conceptual foundation to examine the protective efficacy of multiple tlr-ligand combinations in vaccines against infectious diseases. however, synergistic amplification of the inflammation response may be detrimental, because it can result in immune over-activation, which hampers immune homeostasis. as a consequence of an overactive response, the function of various organ systems may be compromised, resulting in multiple organ dysfunction syndrome (mods) and death. cytokines (tnf-α and il- ) are important components of the immune system because they act as messengers between cells, but they are also involved in many pathological aspects of the cascade leading to systemic inflammatory response syndrome (sirs) and ultimately mods. according to the two-hit hypothesis , patients who survive the initial inflammatory insult may die following a relatively minor second event that would not normally be life-threatening. in this study, viral (poly i:c) stimulation as a relatively minor secondary insult, led to an exaggerated secondary inflammatory response. thus, understanding the mechanism of this two-hit phenomenon may help to devise novel therapeutic strategies to prevent overwhelming and life-threatening inflammatory conditions such as septic shock and trauma-induced sirs. animals. male c bl/ wild type mice were purchased from the laboratory animal research center of shanghai. tlr knockout (tlr −/− ) mice, myd knockout (myd −/− ) mice and tlr knockout (tlr −/− ) were obtained from dr. billiar's lab at the university of pittsburgh. all mice used are on a c bl/ background. all experimental protocols involving animals were approved by institution animals care and use committee of tongji and pittsburgh university. the experiments were performed in accordance with the national institutes of health guidelines for the use of laboratory animals. mice were - weeks of age at the time of experiments and were maintained on standard rodent chow and water ad libitum. animals were anesthetized with mg/ kg ketamine and mg/kg xylazine administered intraperitoneally. animals were intratracheally administered lps ( μ g/g body wt; escherichia coli o :b ; sigma, st. louis, mo) in μ l of saline (sal) or sal at first time, then after h intratracheally administered poly i:c ( μ g/g body wt; - - ; invivogen) in μ l of saline (sal) or sal alone at the second time using a microspray syringe. the animals were randomly in one of four groups: sal/sal, sal/poly i:c, lps/sal, and lps/poly i:c. at various time points, a g sterile catheter was inserted into the trachea to collect bronchoalveolar lavage fluid (balf) as previously described . blood samples were immediately obtained by cardiac puncture and transferred to the laboratory for analysis of cytokines (il- , tnf-α ), chemokines (mip- , mcp- ) in serum and balf by elisa. pmn counts in balf were determined on wright-giemsa-stained slides. briefly, total cell counts were determined on a grid hemocytometer. then a total of cells were counted in cross-section per sample, and the number of pmns was calculated as the total cell count times the percentage in the balf sample. the lungs were rapidly removed from all mice and washed in ice-cold saline. half of the lung tissues were stored at − °c prior to biochemical analyses including mip- expression in lung lysates was measured by western blotting and rt-pcr, while the other half of the lung tissue was fixed in % formalin solution in preparation for histopathological analyses. for histological analysis, lung tissue samples were fixed in % paraformaldehyde in pbs overnight at °c. the samples were then dehydrated, embedded in paraffin, and cut into μ m sections. after deparaffinization, the tissues were stained with hematoxylin and eosin (h&e) for histological analysis. lung sections were scored for lung injury, including the following: ( ) alveolar and capillary edema, ( ) intravascular and peri-bronchial influx of inflammatory cells, ( ) thickness of the alveolar wall, and ( ) hemorrhage. the items were semiquantitatively scored as none, minimal, light, moderate, or severe (score , scientific reports | : | doi: . /srep , , or , respectively) by a pathologist blinded to the experimental group. the lung injury score was obtained by averaging the score from the animals within each group. amΦ isolation. bal was performed as previously described . normally, the bal fluid contains ~ % of amΦ and ~ % of other cells, including pmn and lymphocytes. the immunomagnetic separation system was used to isolate amΦ . magnetic nanoparticle-conjugated antibody (cd b microbeads, miltenyi biotec) was chosen to label and remove pmn and lymphocytes. the resulting cells consisted of > % macrophages, and cell viability was > %. amΦ from wild type, tlr −/− , tlr −/− and myd −/− mice were cultured in dmem containing % fbs and μ l/ml penicillin/streptomycin for days, then they were washed three times with pbs and the medium was changed to low-serum medium ( % fbs). cells were stimulated with lps (nc, . , . , , μ g/ml) for - h in dmem containing % fbs at a concentration of × cells/ml of medium. tlr expression in the amΦ lysates was measured by western blotting and rt-pcr. evans blue albumin (eba) as previous described , . evans blue ( . % eb, sigma-aldrich, st louis, mo, usa) was dissolved in ca + /mg + -free phosphate-buffered saline (pbs; sigma-aldrich), and conjugated to albumin ( % eba) that was prepared by adding bovine serum albumin (sigma-aldrich). after thoroughly dissolving by gently stirring with a magnetic bar, the eba solution was filtered through a . μ m syringe filter and aliquots were stored at − °c until use. each aliquot was used only once for each experiment. to evaluate alveolar-capillary barrier function, eba ( mg/kg body weight) was injected into the internal jugular vein h before euthanasia and lung harvesting. blood samples were obtained from the right heart, and the pulmonary vasculature was subsequently infused with ml pbs. the right lung was ligated at the level of the right mainstem bronchus, excised, blotted dry, weighed and stored in liquid nitrogen until these samples were used for eba analysis. after freeze/thaw, the lung tissue was homogenized in ml pbs and incubated with an additional ml of formamide (sigma-aldrich) ( h; °c). formamide extracts were centrifuged ( , g × min; °c), and the centrifuged supernatants were collected to quantify lung eba content using a dual-wavelength ( nm and nm) spectrophotometric method. pulmonary eba absorbance at nm was corrected by a correction factor with eba absorbance at nm. the eba permeability index was calculated by dividing pulmonary eba absorbance at nm/g of lung tissue by plasma eba absorbance at nm. nuclear protein extraction. nuclear protein extracts were prepared from amΦ following the kit instructions of thermo scientific (ne-per nuclear and cytoplasmic extraction reagents, , thermo fisher). amΦ were harvested with trypsin-edta and then centrifuge at × g for minutes. after washing cells twice with pbs, pellet by centrifugation at × g for minutes and discarded the supernatant. adding cer i (protease inhibitors and pmsf) to the cell pellet, vortex the tube vigorously for s, then incubated the tube on ice for minutes. added cer ii to the tube and vortex for s on the highest setting, incubated the tube on ice for minute. the tube was centrifuged for minutes at , × g, then transferred the supernatant (cytoplasmic extract) to a clean pre-chilled tube. nuclei pellet was suspended in ner and vortex for s. placed the sample on ice and continued vortexing for s every minutes, for a total of minutes. after centrifuged at , × g for minutes, supernatants containing nuclear proteins were frozen in liquid nitrogen in small aliquots and store at − °c. protein quantification was performed using bca protein assay. western blot analysis. amΦ and lung tissues were lysated in radio-immunoprecipitation lysis buffer (ripa), protease inhibitors (roche, mannheim, germany) and phenylmethylsulfonyl fluoride (pmsf). protein concentrations were subsequently determined by standard bca assay. after addition of × sodium dodecyl sulfate (sds) loading buffer, equivalent amounts of protein were heated ( °c; min) and separated by gel electrophoresis using a % sds-polyacrylamide electrophoresis gel. resolved proteins were then transferred to a nitrocellulose membrane and blocked with tris-buffered saline containing tween- (tbst) and % nonfat milk ( h; °c). nitrocellulose membranes were incubated overnight at °c with primary antibody against tlr (ab ; abcam, hong kong, china), nf-κ b p (ab , abcam, hong kong, china), abcam, mip- (ab , hong kong, china), pcna (ab , abcam, hong kong, china) and β -actin (ab , abcam, hong kong, china). the membranes were washed in tbst three times, incubated with secondary antibody ( - irdye mouse-anti-rabbit secondary antibody; licor biosciences, lincoln, ne, usa) for h at °c and then washed in tbst three additional times. the membranes were determined by using an odyssey image analysis system (licor biosciences). western blots were quantitated using quantity one software (bio-rad, foster city, ca, usa) and normalized to β -actin and pcna signal. total rna was extracted from amΦ and lung tissues using the trizol reagent (sigma-aldrich) and following the manufacturer's instructions. total rna was then reverse transcribed using a primescript rt reagent kit (takara bio inc. shiga, japan). primers for tlr amplification ( bp) were: position forward ′ -gtgagatacaacgtagctgactg- ′ , position reverse ′ -tcctgcatccaagatagcaagt- ′ . primers for mip- amplification ( bp) were: position forward ′ -ccaaccaccaggctacagg- ′ , position reverse ′ -gcgtcacactcaagctctg- ′ . primers for β -actin amplification ( bp) were: position forward ′ -ggctgtattcccctccatcg- ′ , position reverse ′ -ccagttggtaacaatgccatgt- ′ . the product of reverse transcription was amplified by following the premix taq version . instructions (takara bio inc.). pcr products were separated using a % agarose gel and identified by sybr green staining. expression of mrna was quantitated using image lab software (bio-rad) and normalized to the β -actin signal. scientific reports | : | doi: . /srep quantitative real-time pcr. quantitative real-time pcr (qpcr) reactions were performed using fast sybr green master mix (thermo fisher) in applied biosystems ht fast real-time pcr system according to the manufacturer's instructions. the cycling conditions were °c for min followed by cycles of °c for s, °c for s. at the end of the last cycle, the temperature was increased from °c to °c ( . °c/s) to produce a melting curve. the specificity of amplification was assessed for each sample by melting curve analysis. each pcr product showed a single peak. the size of the amplicon was checked by electrophoresis. agarose gel electrophoresis revealed a single product of the expected size. the analysis of the expression of tlr relative to the β -actin was performed with software in relative quantification mode following the manufacturer's instruction. the following criteria of sequences of all primers used in this study were applied in the course of designing the primers: product size from to bp, primer size from to bp, and a mean melting temperature of °c. immunofluorescence staining of cells and florescence microscopy. amΦ were cultured for a defined time period, fixed in % paraformaldehye/ × pbs for min. cells were washed three timers with × pbs and permeabilized using . % triton x- in × pbs, and blocked with % bsa for min and sequentially administered primary antibody and secondary antibody (alexa- -conjugated donkey anti rabbit secondary antibody). nuclei were stained with dapi for immunofluoresence analysis. stained cells were examined and recorded using evosfl fluorescence microscopy. statistics. the data are presented as the means ± sem of the indicated number of experiments and analyzed using anova; post hoc testing was performed using the bonferroni modification of the t-test. the individual studies performed throughout this work represent at least five independent studies. power analyses were performed by using a type i error probability of . , with a power of . , to determine the sample size necessary to reject the null hypothesis. all statistical analyses were carried out using the graphpad prism program. has mortality from acute respiratory distress syndrome decreased over time? a systematic review the acute respiratory distress syndrome: pathogenesis and treatment role of chemokines in the pathogenesis of acute lung injury hemorrhagic shock-activated neutrophils augment tlr signalinginduced tlr upregulation in alveolar macrophages: role in hemorrhage-primed lung inflammation tlr-signaling networks: an integration of adaptor molecules, kinases, and cross-talk toll-like receptor signaling pathways the macrophage response towards lps and its control through the p (mapk)-stat axis toll-like receptors and innate immunity tlr : interferon induction by double-stranded rna including poly(i:c) the toll-like receptor :dsrna signaling complex beyond dsrna: toll-like receptor signalling in rna-induced immune responses establishment of monoclonal antibody against human toll-like receptor that blocks double-stranded rna-mediated signaling subcellular localization of toll-like receptor in human dendritic cells mrna is an endogenous ligand for toll-like receptor tlr is an endogenous sensor of tissue necrosis during acute inflammatory events recognition of double-stranded rna and activation of nf-kappab by toll-like receptor cytomegalovirus enhances macrophage tlr expression and myd -mediated signal transduction to potentiate inducible inflammatory responses type i interferon signaling contributes to the bias that tolllike receptor exhibits for signaling mediated by the adaptor protein trif selective inhibition of nf-kappab activation by a peptide that blocks the interaction of nemo with the ikappab kinase complex tlr signaling induces tlr expression in endothelial cells via neutrophil nadph oxidase alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury diverse macrophage populations mediate acute lung inflammation and resolution role of resident alveolar macrophages in leukocyte traffic 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mercurial nature of neutrophils: still an enigma in ards? tnfalpha and mip- : role in particle-induced inflammation and regulation by oxidative stress critical role for cxcr and cxcr ligands during the pathogenesis of ventilator-induced lung injury syndecan- shedding facilitates the resolution of neutrophilic inflammation by removing sequestered cxc chemokines darc on rbc limits lung injury by balancing compartmental distribution of cxc chemokines role for macrophage inflammatory protein- in lipopolysaccharideinduced lung injury in rats a cxcl polymorphism is associated with better outcomes in patients with severe sepsis respiratory syncytial virus and staphylococcus aureus coinfection in children hospitalized with pneumonia incidence and characteristics of viral community-acquired pneumonia in adults transcriptional signaling by double-stranded rna: role of tlr toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis involvement of toll-like receptor in the immune response of lung epithelial cells to double-stranded rna and influenza a virus detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia virus infection in exacerbations of chronic obstructive pulmonary disease requiring ventilation mixed bacterial-viral infections in septic children with leukemia synergistic activation of innate immunity by double-stranded rna and cpg dna promotes enhanced antitumor activity synergistic activation of dendritic cells by combined toll-like receptor ligation induces superior ctl responses in vivo modeling the two-hit hypothesis for evaluating strategies to prevent organ injury after shock/resuscitation rgd peptides protects against acute lung injury in septic mice through wisp -integrin β pathway inhibition wnt -inducible signaling pathway protein contributes to ventilator-induced lung injury competing financial interests: the authors declare no competing financial interests. key: cord- - rxv fy authors: welch, david; buonanno, manuela; grilj, veljko; shuryak, igor; crickmore, connor; bigelow, alan w.; randers-pehrson, gerhard; johnson, gary w.; brenner, david j. title: far-uvc light: a new tool to control the spread of airborne-mediated microbial diseases date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: rxv fy airborne-mediated microbial diseases such as influenza and tuberculosis represent major public health challenges. a direct approach to prevent airborne transmission is inactivation of airborne pathogens, and the airborne antimicrobial potential of uvc ultraviolet light has long been established; however, its widespread use in public settings is limited because conventional uvc light sources are both carcinogenic and cataractogenic. by contrast, we have previously shown that far-uvc light ( – nm) efficiently inactivates bacteria without harm to exposed mammalian skin. this is because, due to its strong absorbance in biological materials, far-uvc light cannot penetrate even the outer (non living) layers of human skin or eye; however, because bacteria and viruses are of micrometer or smaller dimensions, far-uvc can penetrate and inactivate them. we show for the first time that far-uvc efficiently inactivates airborne aerosolized viruses, with a very low dose of mj/cm( ) of -nm light inactivating > % of aerosolized h n influenza virus. continuous very low dose-rate far-uvc light in indoor public locations is a promising, safe and inexpensive tool to reduce the spread of airborne-mediated microbial diseases. cells in a particular field of view, while green fluorescence indicated the integration of live influenza a (h n ) viruses into the cells. results from the zero-dose control studies (fig. , top left) confirmed that the aerosol irradiation chamber efficiently transmitted the aerosolized viruses through the system, after which the live virus efficiently infected the test mammalian epithelial cells. figure shows the surviving fraction, as a function of the incident -nm far-uvc dose, of exposed h n aerosolized viruses, as measured by the number of focus forming units in incubated epithelial cells relative to unexposed controls. linear regressions (see below) showed that the survival results were consistent with a classical exponential uv disinfection model with rate constant k = . cm /mj ( % confidence intervals . - . cm / mj). the overall model fit was good, with a coefficient of determination, r = . , which suggests that most of the variability in virus survival was explained by the exponential model. the rate constant of . cm /mj corresponds to an inactivation cross-section (dose required to inactivate % of the exposed viruses) of d = . mj/cm ( % confidence intervals . - . mj/cm ). we have developed an approach to uv-based sterilization using single-wavelength far-uvc light generated by filtered excilamps, which selectively inactivate microorganisms, but does not produce biological damage to exposed mammalian cells and tissues [ ] [ ] [ ] . the approach is based on biophysical principles in that far-uvc light can traverse and therefore inactivate bacteria and viruses which are typically micrometer dimensions or smaller, whereas due to its strong absorbance in biological materials, far-uvc light cannot penetrate even the outer dead-cell layers of human skin, nor the outer tear layer on the surface of the eye. here we applied this approach to test the efficacy of the -nm far-uvc light to inactivate influenza a virus (h n ) carried by aerosols in a benchtop aerosol uv irradiation chamber, which generated aerosol droplets of sizes similar to those generated by human coughing and breathing. aerosolized viruses flowing through the irradiation chamber were exposed to uvc emitting lamps placed in front of the chamber window. as shown in fig. , inactivation of influenza a virus (h n ) by -nm far-uvc light follows a typical exponential disinfection model, with an inactivation cross-section of d = . mj/cm ( % ci: . - . ). for comparison, using a similar experimental arrangement, but using a conventional nm germicidal uvc lamp, mcdevitt et al. found a d value of . mj/cm ( % ci: . - . ) for h n virus. thus as we , and others [ ] [ ] [ ] reported in earlier studies for bacterial inactivation, -nm far-uvc light and -nm broad-spectrum germicidal light are also comparable in their efficiencies for aerosolized viral inactivation. other recent work comparing viral inactivation across the uvc spectrum has shown variations in efficiency are expected, but in general both regions of the spectrum are effective in inactivation, though the precise cause of inactivation may differ , . however as discussed above, based on biophysical considerations and in contrast to the known human health safety issues associated with conventional germicidal -nm broad-spectrum uvc light, far-uvc light does not appear to be cytotoxic to exposed human cells and tissues in vitro or in vivo [ ] [ ] [ ] . if these results are confirmed in other scenarios, it follows that the use of overhead low-level far-uvc light in public locations may represent a safe and efficient methodology for limiting the transmission and spread of airborne-mediated microbial diseases such as influenza and tuberculosis. in fact the potential use of ultraviolet light for airborne disinfection is by no means new, and was first demonstrated more than years ago , . as applied more recently, airborne ultraviolet germicidal irradiation (uvgi) utilizes conventional germicidal uvc light in the upper part of the room, with louvers to prevent direct exposure of potentially occupied room areas . this results in blocking more than % of the uv radiation exiting the uvgi fixture, with substantial decrease in effectiveness . by contrast, use of low-level far-uvc fixtures, which are potentially safe for human exposure, could provide the desired antimicrobial benefits without the accompanying human health concerns of conventional germicidal lamp uvgi. a key advantage of the uvc based approach, which is in clear contrast to vaccination approaches, is that uvc light is likely to be effective against all airborne microbes. for example, while there will almost certainly be variations in uvc inactivation efficiency as different influenza strains appear, they are unlikely to be large , . likewise, as multi-drug-resistant variants of bacteria emerge, their uvc inactivation efficiencies are also unlikely to change greatly . in conclusion, we have shown for the first time that very low doses of far-uvc light efficiently inactivate airborne viruses carried by aerosols. for example, a very low dose of mj/cm of -nm light inactivates > % of airborne h n virus. our results indicate that far-uvc light is a powerful and inexpensive approach for prevention and reduction of airborne viral infections without the human health hazards inherent with conventional germicidal uvc lamps. if these results are confirmed in other scenarios, it follows that the use of overhead very low level far-uvc light in public locations may represent a safe and efficient methodology for limiting the transmission and spread of airborne-mediated microbial diseases. public locations such as hospitals, doctors' offices, schools, airports and airplanes might be considered here. this approach may help limit seasonal influenza epidemics, transmission of tuberculosis, as well as major pandemics. far-uvc lamps. we used a bank of three excimer lamps containing a kr-cl gas mixture that predominantly emits at nm , . the exit window of each lamp was covered with a custom bandpass filter designed to remove all but the dominant emission wavelength as previously described . each bandpass filter (omega optical, brattleboro, vt) had a center wavelength of nm and a full width at half maximum (fwhm) of nm and enables > % transmission at nm. a uv spectrometer (spm- -bt , photon control, bc, canada) with a sensitivity range between nm and nm was utilized to verify the nm emission spectrum. a deuterium lamp standard with a nist-traceable spectral irradiance (newport model , irvine, ca) was used to radiometrically calibrate the uv spectrometer. an sm- ozone monitor (aeroqual, avondale, auckland, new zealand) measured the ozone generation from the lamps to be < . ppm, which is not a significant level to provide an antimicrobial effect to aerosolized viruses . far-uvc dosimetry. optical power measurements were performed using an -uv/db low-power uv enhanced silicon photodetector with an -r optical power meter (newport, irvine, ca). additional dosimetry to determine the uniformity of the uv exposure was performed using far-uvc sensitive film as described in our previous work , . this film has a high spatial resolution with the ability to resolve features to at least µm, and exhibits a nearly ideal cosine response , . measurements were taken between experiments therefore allowing placement of sensors inside the chamber. scientific reports | ( ) : | doi: . /s - - -w a range of far-uvc exposures, from . µj/cm up to . mj/cm , were used to define a response calibration curve. films were scanned as bit rgb tiff images at dpi using an epson perfection v photo flatbed scanner (epson, japan) and analyzed with radiochromic film analysis software to calculate the total exposure based on measured changes in optical density. measurements using both a silicon detector and uv sensitive films were combined to compute the total dose received by a particle traversing the exposure window. the three vertically stacked lamps produced a nearly uniform dose distribution along the vertical axis thus every particle passing horizontally through the irradiation chamber received an identical dose. the lamp width ( mm) was smaller than the width of the irradiation chamber window ( mm) so the lamp power was higher near the center of the irradiation chamber window compared to the edge. the uv sensitive film indicated a power of approximately µw/cm in the center third of the window and µw/cm for the outer thirds. the silicon detector was used to quantify the reflectivity of the aluminum sheet at approximately % of the incident power. combining this data allowed the calculation of the average total dose of . mj/cm to a particle traversing the window in seconds. additionally, the silicon detector was used to confirm the attenuation of -nm light through a single sheet of plastic film was %. the addition of one or two sheets of plastic film between the lamps and the irradiation chamber window yielded average doses of . mj/cm and . mj/cm , respectively. benchtop aerosol irradiation chamber. a one-pass, dynamic aerosol / virus irradiation chamber was constructed in a similar configuration to that used by ko et al. , lai et al. , and mcdevitt et al. , . a schematic overview of the system is shown in fig. and is pictured in fig. . aerosolized viruses were generated by adding a virus solution into a high-output extended aerosol respiratory therapy (heart) nebulizer (westmed, tucson, az) and operated using a dual-head pump (thermo fisher - - fk, waltham, ma) with an input flow rate of l/min. the aerosolized virus flowed into the irradiation chamber where it was mixed with independently controlled inputs of humidified and dried air. humidified air was produced by bubbling air through water, while dry air was provided by passing air through a desiccant air dryer (x - - , wilkerson corp, richland, mi). adjusting the ratio of humid and dry air enabled control of the relative humidity (rh) within the irradiation chamber which, along with the nebulizer settings, determined the aerosol particle size distribution. an optimal rh value of % resulted in a distribution of aerosol particle sizes similar to the natural distribution from human coughing and breathing, which has been shown to be distributed around approximately µm, with a significant tail of particles less than µm - . after combining the humidity control inputs with the aerosolized virus, input flow was directed through a series of baffles that promoted droplet drying and mixing to produce an even particle distribution and stable humidity . the rh and temperature inside the irradiation chamber were monitored using an omega rh meter (omega engineering inc., stamford, ct) immediately following the baffles. a hal technologies hal-hpc particle sizer (fontana, ca) was adjoined to the irradiation chamber to allow for sampling of particle sizes throughout operation. during uv exposure, the -nm lamps were placed cm from the irradiation chamber window. the lamps were directed at the cm × . cm chamber window which was constructed of -µm thick uv transparent plastic film (topas x , topas advanced polymers, florence, ky), and which had a transmission of ~ % at nm. the wall of the irradiation chamber opposite the transparent window was constructed with polished aluminum in order to reflect a portion of the uvc light back through the exposure region, therefore increasing the overall exposure dose by having photons pass in both directions. the depth of the irradiation chamber between the window and the aluminum panel was . cm, creating a total exposure volume of . l. flow of the aerosols continues out of the irradiation chamber to a set of three way valves that could be configured to either pass through a bypass channel (used when no sampling was required), or a biosampler (skc inc, eighty four, pa) used to collect the virus. the biosampler uses sonic flow impingement upon a liquid surface to collect aerosols when operated at an air flow of . l/min. finally, flow continued out of the system through a final hepa filter and to a vacuum pump (wp , emd millipore, billerica, ma). the vacuum pump at the end of the system powered flow through the irradiation chamber. the flow rate through the system was governed by the biosampler. given the flow rate and the total exposure volume of the irradiation chamber, . l, a single aerosol droplet passed through the exposure volume in approximately seconds. the entire irradiation chamber was set up inside a certified class ii type a biosafety cabinet (labconco, kansas city, mo). all air inputs and outputs were equipped with hepa filters (ge healthcare bio-sciences, pittsburgh, pa) to prevent unwanted contamination from entering the chamber as well as to block any of the virus from releasing into the environment. irradiation chamber performance. the custom irradiation chamber simulated the transmission of aerosolized viruses produced via human coughing and breathing. the chamber operated at a relative humidity of % which resulted in a particle size distribution of % between . µm and . µm, % between . µm and . µm, and % > . µm. a comparison to published ranges of particle size distributions is shown in table . aerosolized viruses were efficiently transmitted through the system as evidenced from the control (zero exposure) showing clear virus integration (fig. , top left) . , ( ) where k is the uv inactivation rate constant or susceptibility factor (cm /mj). the regression was performed with the intercept term set to zero, which represents the definition of % relative survival at zero uv dose. bootstrap % confidence intervals for the parameter k were calculated using r . . software . the virus inactivation cross section, d , which is the uv dose that inactivates % of the exposed virus, was calculated as d = −ln[ − . ]/k. global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study aerosol transmission is an important mode of influenza a virus spread evidence of airborne transmission of the severe acute respiratory syndrome virus control of air-borne microorganisms by ultraviolet floor irradiation ultraviolet germicidal irradiation handbook: uvgi for air and surface disinfection viability of b. coli exposed to ultra-violet radiation in 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spectrum the history of ultraviolet germicidal irradiation for air disinfection upper-room ultraviolet germicidal irradiation (uvgi) for air disinfection: a symposium in print spatial distribution of fluence rate from upper-room ultraviolet germicidal irradiation: experimental validation of a computer-aided design tool dielectric-barrier-discharge excilamp in mixtures of krypton and molecular chlorine krcl barrier-discharge excilamps: energy characteristics and applications a new ozone-based method for virus inactivation: preliminary study unlaminated gafchromic ebt film for ultraviolet radiation monitoring measurement of uv emission from a diffusing optical fiber using radiochromic film radiation technology for polymers angular dependence of the efficiency of the uv sensor polysulphone film on multichannel film dosimetry with channel-independent perturbations influence of relative humidity on particle size and uv sensitivity of serratia marcescens and mycobacterium bovis bcg aerosols size and uv germicidal irradiation susceptibility of serratia marcescens when aerosolized from different suspending media characterization of uvc light sensitivity of vaccinia virus characterization of expiration air jets and droplet size distributions immediately at the mouth opening the size distribution of droplets in the exhaled breath of healthy human subjects size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities the log transformation is special r: a language for data analysis and graphics this work was supported by the shostack foundation and also nih grant r ai - . we thank dr. rea dabelic from the department of environmental health sciences, mailman school of public health at columbia university for her expertise and training with viral cell culture. d.w., m.b. and v.g. designed and performed experiments, analyzed the data, and wrote the manuscript; i.s. analyzed the data; c.c. and a.w.b. designed the irradiation chamber; g.w.j. constructed the irradiation chamber; d.j.b and g.r.-p. supervised, contributed conceptual advice, and wrote the manuscript. all authors discussed the results and commented on the manuscript. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -wtnf p authors: chen, xiaojuan; tu, chongzhi; qin, tao; zhu, liqi; yin, yinyan; yang, qian title: retinoic acid facilitates inactivated transmissible gastroenteritis virus induction of cd (+) t-cell migration to the porcine gut date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: wtnf p the digestive tract is the entry site for transmissible gastroenteritis virus (tgev). tgev transmission can be prevented if local immunity is established with increased lymphocytes. the current parenteral mode of vaccination stimulates systemic immunity well, but it does not induce sufficient mucosal immunity. retinoic acid (ra) plays an important role in the induction of cells that imprint gut-homing molecules. we examined whether ra assist parenteral vaccination of pigs could improve mucosal immunity. we demonstrated that elevated numbers of gut-homing cd (+) t cells (which express α β and ccr molecules) were presented in porcine inguinal lymph nodes and were recruited to the small intestine by ra. intestinal mucosal immunity (iga titre) and systemic immunity (serum igg titre) were enhanced by ra. therefore, we hypothesized that ra could induce dcs to form an immature mucosal phenotype and could recruit them to the small intestinal submucosa. porcine t-cells expressed β integrin and ccr receptors and migrated to ccl by a mechanism that was dependent of activation by ra-pretreated dcs, rather than direct activation by ra. together, our results provide powerful evidence that ra can assist whole inactivated tgev (wi-tgev) via subcutaneous (s.c.) immunization to generate intestinal immunity, and offer new vaccination strategies against tgev. transmissible gastroenteritis (tge), which is caused by transmissible gastroenteritis virus (tgev), is a highly contagious disease in newborn piglets . after entering the digestive tract, tgev can replicate in intestinal enterocytes and then induce enteritis and watery diarrhoea . both live and killed tgev vaccines (intramuscular route or subcutaneous injection) are currently available to control tge; however, they are not always successful . these vaccination strategies can stimulate systemic immunity well; however, they do not induce sufficient mucosal immunity, especially the induction of local, virus-specific siga antibodies . determining how to induce a mucosal immune response and improve local immunity in the intestine is important in preventing enteropathogen infection. excellent induction of mucosal immunity depends on the inductive and effector sites . the mucosal immune mechanism includes naive lymphocyte activation in classical inductive sites (such as intestinal peyer's patches), after which the sensitized lymphocytes migrate to the blood circulation before homing to effector sites (such as the intestinal epithelium or lamina propria) and differentiating into effector lymphocytes that contribute to immunity . effective viral clearance requires the rapid migration of effector t cells to the site of intestinal infection. intestinal lymphocyte homing includes lymphocytes selectively passing through the postcapillary venule and migrating directly to the intestinal epithelium or lamina propria. t cells migrating to the intestine require the expression of specific receptors, including homing receptors, such as α β -integrin and ccr , and their corresponding ligands (i.e., addressin-cell adhesion molecule , madcam ) on endothelial cells from intestinal postcapillary venules as well as ligands (such as ccl ) on the intestinal epithelium , . ccr /ccl interactions can induce the homing of effector t and b cells to the gut , . additionally, these interactions can guide plasmacytoid dendritic cells (dcs) to the intestine , . retinoic acid (ra), a vitamin a metabolite, has emerged as a critical factor in mucosal immune responses . ra induces intestinal cytokines generation , and iga responses , , , and ra supplementation reduces morbidity and mortality due to enteric infectious diseases . furthermore, ra was shown to stimulate t cell reagents. retinoic acid (ra), -(and )-carboxyfluorescein diacetate succinimidyl ester(cfda-se), bovine serum albumin (bsa), lps (from escherichia coli :b ), were purchased from sigma-aldrich, saint louis, usa. fitc-conjugated mouse anti-pig cd α ( - - ) monoclonal antibody (mabs), rat anti-mouse integrin β (na/le) mabs, were purchased from bd biosciences, usa. fitc-conjugated mouse anti-human cd (kd ) mabs, fitc or pe-conjugated mouse anti-pig swine workshop cluster a (swc a) ( - mabs, pe-conjugated mouse anti-pig cd ( - - ) mabs, rabbit anti-human ccr (e ) mabs, rabbit anti-human ccr mabs (extracellular domain), pe/cy -conjugated rat anti-mouse cd b (m / ) mabs, rabbit anti-human cd (sp ) mabs, ro - were purchased from abcam, hongkong. fitc-conjugated mouse anti-pig sla-dr ( e / ) mabs, pe-conjugated mouse anti-human hla-dp (hl- ) mabs were obtained from lifespan biosciences, usa. rabbit anti pig igg, goat anti pig iga antibody were purchased from bethy laboratories, usa. pe-conjugated goat anti-rat igg antibody was bought from santa cruz biotechnology, texas, usa. purified tgev s-ad protein . purified porcine ccl protein was generated in our lab. dylight -conjugated goat anti-rabbit igg antibody, dylight -conjugated goat anti-rabbit igg antibody, dylight -conjugated goat anti-rabbit igg antibody were purchased from multiscience, hangzhou, china. abc-based system (biotinylated goat anti rabbit igg antibody) was used as the secondary antibody with dab as a chromogen was (boster, wuhan, china). porcine intestinal epithelial cell line (ipec-j ) and swine testicle (st) cell lines were purchased from jennio biotech, guangzhou, china. vaccine. the tgev strain (shxb, wild-virulent strain) was supplied by the jiangsu academy of agricultural sciences (nanjing, china) . the viral % tissue culture infectious dose (tcid ) of the shxb tgev strain was × . tcid / ul. the viruses were inactivated by ultraviolet radiation (uv) for h and tested for complete loss of infectivity by inoculation into st cells for three passages . ra was dissolved with dimethyl sulfoxide (dmso) according to the manufacturer's instructions. an ra working solution was diluted with corn oil (changshouhua, shandong, china). immunization. piglets were randomly divided into groups, with pigs per group. the first group was immunized via the subcutanoues route with ml of corn oil into the right groin as a control, the second group was immunized via the s.c. route with ml of ra ( mg/ml in corn oil), the third group was immunized via the s.c. route with ml of wi-tgev ( × . tcid / ul), the fourth group was immunized via the s.c. route with ml of wi-tgev ( × . tcid / ul) combined with ml of ra ( mg/ml in corn oil), and the last group was orally immunized with ml of wi-tgev ( × . tcid / ul) ( table ). all of the piglets were immunized at the age of days and given a booster immunization at the age of days on the same side. all of the animals were anaesthetized and killed days after the booster immunization. sample collection. on days , , , , after the first vaccination, four pigs were sampled randomly from each group for determination of serum and faecal tgev-specific antibody. the serum was separated by centrifugation and stored at − °c for tgev-specific igg antibody detection. for tgev-specific iga antibody detection, . g faecal sample was resuspended with ml of pbs, and the supernatant was collected by centrifugation and stored at − °c for tgev-specific iga antibody detection. on days after first vaccination, pigs were exsanguinated, the ileum and the right side of the inguinal lymph nodes (ilns) were stored in pbs. and then cells from the ileum and ilns were isolated and analyzed by flow cytometry . a portion of the ileum was triturated with a mortar and liquid nitrogen, and the homogenate was weighed and dissolved in pbs at a dilution of mg per μl. the supernatants of these homogenates were collected after centrifugation for the detection of specific iga antibodies. after dilution, the homogenate supernatant protein concentrations were measured using a bca protein assay kit (thermo scientific pierce). the ileal tissue was either frozen in liquid nitrogen and stored at − °c for immunofluorescence detection or fixed in bouin's liquid for immunohistochemical detection. flow cytometry analyses. cells from the ileum and ilns were isolated as previously described . the cells were washed twice with cold pbs and stained with specific fluorescent antibodies at °c for . h per the manufacturer's guidelines. to determining the levels of homing receptor on cd + cells, cells were stained with antibodies against integrin β (bd, , . μg for cells), ccr (abcam, ab , . μg for cells) and cd α (bd, , μg for cells). to examination of tissue dc phenotypes, cells were stained with antibodies against cd b (abcam, ab , . μg for cells) and swc a (abcam, ab , . μg for cells), or cd (abcam, ab , μl for cells) and hla-dp (lsbio, ls-c / , μl for cells). to analysing bm-dc phenotypes, cells were stained with antibodies against cd a and swc a (abcam, ab , μg for cells), hla-dp and swc a, or integrin β , ccr and swc a. the respective isotype controls were used as negative controls. after three pbs washes, in each sample were acquired × cells by flow cytometry with a bd facs calibur (bd biosciences, us) , . data were analyzed using flowjo . (tree star). siga and cd + t lymphocyte detection. paraffin-embedded ileal sections were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol . after being blocked with % bovine serum for min, the sections were incubated with the primary antibodies overnight at °c, followed by incubation with secondary antibodies at room temperature for h. the iga + cells were labelled with goat anti-pig iga followed by alexa fluor donkey anti-goat igg. the cd + cells were labelled with rabbit anti-human cd igg followed by biotinylated goat anti-rabbit igg, and then the sections were sealed with a coverslip for examination. the respective isotype controls were used as negative controls. the sections were visualized with an axioplan microscope (zeiss, oberkochen, germany), × magnification. specific igg and iga detection. the tgev-specific igg and tgev-specific secretory iga (siga) levels were measured with enzyme-linked immunosorbent assays (elisas) as previously described . briefly, elisa plates were coated with . μg of purified recombinant tgev s-ad protein (the major antigenic sites a and d in tgev)/well at °c overnight. following protein removal, the plates were blocked with . % (wt/vol) bovine serum albumin (bsa) in pbs for h at °c and then incubated with μl of samples for h at °c. after washing with pbst, μl of hrp-conjugated rabbit anti-pig igg or goat anti-pig iga antibody was added at a : , dilution and incubated for h at °c. the plates were washed times and incubated with , ′ , , ′ -tetramethylbenzidine (tmb). after minutes, the reaction was arrested with sulphuric acid ( m), and the absorbance was read at nm with a microplate reader. swine bm-dcs were isolated as per our advanced methods . briefly, bone marrow was extracted from the femurs of piglets and treated with red blood cell lysing buffer. the bone marrow cells were differentiated into dcs by resuspending the cells in complete medium (rpmi- (invitrogen) supplemented with % foetal bovine serum (fbs) (wisent, ca), % penicillin/streptomycin, ng ml − porcine granulocyte-macrophage colony-stimulating factor (gm-csf), and ng ml − porcine il- (prospec, ness ziona, israel) and plated at × cells per ml in -well plates. non-adherent granulocytes were removed by discarding the culture medium after h of culture. on day of culture, the clusters were harvested and subcultured overnight so that the adherent cells could be removed. non-adherent cells were collected after days of culture, washed, and used as immature dcs for subsequent studies. in vivo homing assay. three piglets were used in vivo homing assay. porcine peripheral blood mononuclear cells (pbmcs) were isolated from the blood of piglets by density centrifugation using histopaque ( . g l − ) (sigma). to isolate t cells, pbmcs were labelled with a mouse anti-cd antibody (abcam, hong kong) followed by incubation with rat anti-mouse igg microbeads (macs; miltenyi biotec, germany) . cd + t cells were cultured with or without ra for h. thereafter, the cells were divided into two parts, one part was detected with flow cytometry to determining the homing receptor levels on cd + cells, cells were stained with antibodies against integrin β , ccr and cd α , and the other part was harvested for the in vivo homing assay. for the in vivo homing assay, the cells treated with ra were labelled with cell tracker cm-dii (life technologies, eugene), and the cells treated without ra were labelled with cfda-se (invitrogen), respectively, according to the manufacturer's instructions. thereafter, the cells were centrifuged and extensively washed. then, million cells from each preparation were mixed and intravenously injected into recipient piglets (n = ). the recipients were construction of porcine ccl . the dna fragment of porcine ccl (nm_ . , life technologies, eugene) was amplified from porcine genome used the primers f-ggactcagatctcgagg ccaccatgaggccgtggctcctggcc and r-tctggaacatcgtatgggtatggtcct ggaatagctgttg, and inserted into the lentiviral vector plvx-acgfp . lentiviral production was achieved through calcium phosphate transfection of plp , plp , plp/vsvg (invitrogen) plasmids. lentivirus packaging referenced literature . for infection × ipec-j cells were plated per cm dish a day prior to infection. at the day of infection the viral supernatant was supplemented with polybrene ( mg/ml, life technologies, eugene) and added to the cells for h. the multiplicity of infection was set to to obtain single copy integration of the synthetic gene circuit. porcine ccl high-expressed clone was cultured to collect supernatant and activity was assayed by nanodrop uv-vis spectrophotometer (thermo scientific, usa). transwell migration assay. migration assays were performed as previously described using -well falcon cell culture inserts with μm pores (corning, new york, usa). cd + t cells were isolated from pmbcs with the same treatment as above. ra, ro - or ra plus ro - were added to the bm-dcs at different concentrations for h before use. following repeated washes, pre-treated bm-dcs were mixed together with t cells at × /well (dc/t-cell ratios of : ) for days. thereafter, the mixed cells were collected for transwell migration assays. to induce cell migration rpmi- medium ( μl) containing ccl ( ng/ml) was placed in the lower chamber, and rpmi- medium ( μl) containing × mixed cells (dcs mixed with t cells) was placed in the upper chamber. after h of incubation at °c, to determine the amount of cd + cells in the lower chamber, the migrated cells were collected, we counted the number of cd + cells by flow cytometry. statistical analysis. all of the data were expressed as the means ± s.d. and analyzed with spss . . the data were analyzed with non-parametric tests, the unpaired mann-whitney for the in vivo data and the paired wilcoxon for the comparisons between conditions with cells from the same piglet. a p value < . was considered statistically significant. in the intestinal mucosal immune system, cellular immune responses play an important central role in the outcome of several viral infections . virus-specific cd + t cells play a central role in controlling and eliminating most pathogen infections. to the best of our knowledge, there are limited reports of studies focused on the design of cd + t cells-based vaccines in mucosal immunity , . when given together with ra, exogenous antigens can effectively stimulate lymphocytes, which have an increased expression of gut-homing receptors and decrease the expression of skin-homing receptors on lymphocyte surfaces. these research studies indicated that the skin could replace peyer's patches as an outstanding mucosal immune inductive site . as we known, the area of abdominal skin was large and the temperature was constant, which was conducive to the vaccine absorption. furthermore, there was less nerves distribution in abdominal skin, which was beneficial to reduce the injection pain and stress. inguinal lymph node was located in the groin, so in our study the skin on the right groin was selected as an immune inductive site. we subcutaneously immunized piglets with corn oil, ra, or wi-tgev alone or in combination with ra, or we orally immunized piglets with wi-tgev alone. to analyze whether ra has the capability of generating gut-homing cells; therefore, cells were isolated from the right side of the inguinal lymph nodes (ilns) and ileum after immunization. intriguingly, the flow cytometry data from the ilns showed that the number of cd + cells that expressed β -integrin and ccr was significantly increased after ra administration compared with the control (fig. a ,b, p < . ). furthermore, the s.c. administration of ra plus tgev increased the number of gut-homing cd + cells expressing β -integrin and ccr compared with the s.c. administration of tgev alone in the ilns (fig. a ,b, p < . ). lymphocyte homing to the small intestine is believed to play a crucial role at mucosal immunity effector sites, and cd + effector t cells in intestinal epithelium can effectively prevent or directly limit infection at the entrance site of enteric viruses . furthermore, effector t cells maintain the epithelial barrier and tissue homeostasis . in our study, the ileum was selected as the mucosal immune effector site. additionally, we assessed the level of cd + cell homing to the ileum. notably, the flow cytometry data from the ileum showed that following the ra plus tgev s.c. and oral tgev treatments, the gut tropism receptor frequency on cd + cells was remarkably enhanced compared with the controls (fig. c ,d, p < . ). next, we further evaluated the number of cd + cells in the porcine ileum with immunohistochemistry. in the cross-sectional view, cd + t cells were gathered in the epithelial layer and villous lamina propria (fig. e, × magnification) . the ra plus tgev s.c. administration substantially expanded the areas with cd + t cells compared with the controls (fig. f , p < . ), although these levels were lower than those observed with the oral tgev treatment. next, to verify whether ra could assist wi-tgev in enhancing the mucosal and systemic immune responses, we determined the local secretion iga and systemic igg, immunization methods as above. our immunofluorescence results showed that the iga-secreting cells were mainly gathered in the villous lamina propria or around the ileal glands (fig. b-f) . in the ileum, the areas of iga-secreting cells of the ra plus tgev s.c. and oral tgev treatment were significantly increased than control (p < . ), and the ra plus tgev s.c. treatment significantly increased the areas containing iga-secreting cells compared with the tgev s.c. alone treatment (fig. g, p < . ) , however, this level was slightly less than the level observed following the oral tgev delivery. furthermore, we noted that the iga-secreting cells displayed iga and ccr co-expression in the villous lamina propria after the ra plus tgev challenge (fig. h-j) . a similar finding was observed in the faeces, in which the subcutaneous ra plus tgev immunization obviously increased the tgev-specific iga levels compared with the s.c. tgev alone treatment at days , and (elisa method) (fig. k, p < . ) , and this effect lasted longer than that of the oral tgev immunization. for the systemic immune response, in the serum, we found that the tgev-specific igg titres in the ra plus tgev s.c. treatment group remained at a high level during the entire immunity period ( - days) and were substantially greater than those in the other groups at day (fig. l, p < . ) . these observations demonstrated that ra-assisted tgev immunization could recruit iga-secreting cells to the ileum and induce a robust antigen-specific iga response in the faeces. dcs are the main cellular element that controls t lymphocyte activation and regulation , which is critical for subsequent immunity responses of the intestinal mucosa. porcine small intestine dcs are express mhcii (sla-dp), cd a (swc a), cd , cd r , or cd b , . to determine whether ra in the vaccine could recruit dcs to the small intestine, we isolated intestinal cells from piglets and determined the dc numbers in the lamina propria by flow cytometry. very few hla-dp + cd + dcs (fig. a,c) or swc a + cd b + dcs (fig. b,d) migrated to the small intestinal lamina propria following tgev s.c. treatment. nevertheless, the s.c. administration of ra plus tgev and oral tgev (positive control) significantly enhanced the frequency of these migrated dcs compared with tgev s.c. treatment (fig. c,d, p < . ) . these findings suggested that s.c. ra treatment could effectively facilitate tgev not only by increasing the cd + cell numbers (fig. c,d) but also by increasing the dc numbers in the lamina propria of the ileum. observed that ra-assisted tgev treatment increased the dc numbers in the porcine intestine; however, the mechanism impacts dcs migration is unclear. a previous study showed that ra induced gut-homing receptors on mice bm-dcs in a narrow time window and had a stringent dose response . thus, we examined whether ra could induce porcine bm-dcs to express gut-homing receptors. after we directly treated porcine bm-dcs with ra for or h in vitro, the expression levels of the gut-homing receptors integrin β and ccr were decreased in a dose-independent manner (fig. a,b) , nm ra treatment significantly reduced the expression of the gut-homing receptors on dcs compared with controls (p < . ). next, to test whether ra could affect porcine bm-dc maturation, we analyzed the expression of the phenotype markers cd a and hla-dr. after bm-dcs were incubated with ra for or h, the percentages of swc a + cd a + (fig. c,d) and swc a + hla-dr + (fig. e ,f) dcs were decreased in a dose-independent manner, but there was no significant difference. to better understand whether ra could directly imprint gut-homing-specific receptors on porcine t cells, unactivated t cells were directly treated with or without ra in vitro, and then the homing receptors β integrin and ccr were detected with flow cytometry. however, there was no significant difference between their levels of homing receptor levels on cd + cells with (up to nm) or without ra treatment (fig. a) . next, to evaluate whether ra-treated t cells could preferentially home to the small intestine, we performed competitive homing assay in vivo. t cells were isolated from a piglet and treated with or without ra, labelled with cm-dii (red, molecular probes) or cfse (green) for long-term cellular labeling. equal numbers of cells from the two populations were mixed and adoptively transferred into piglets via intravenous injection. twenty-four hours later, the mixed cells that homed to the ileal villi were detected by flow cytometry. however, the two populations homed equally into the ileum. there was no significant difference between the levels of homing cells in the piglets with or without ra-treated t cells (fig. b-f) . observations of the intestinal villi and peyer's patches (pps) in cryosections further confirmed the flow cytometry results of homing cells (fig. g,h) . these data of t-cell gut-homing further confirmed the above results that ra did not directly increase expression of gut-homing receptors on t cells. for a deficiency of gut-homing receptors on t cells, t cells could not recruited into ileum. thus, ra did not directly induce t-cell homing to the gut in the piglets. in an mlr assay, ra-treated dcs cocultured with cd t cells, flow cytometry analyses were performed by collecting the mixed cells after days incubation to determine t-cell proliferation and the homing receptor expression levels on t cells (fig. a) . the results showed that the bm-dcs treated by ra stimulated t-cell proliferation were increased in a dose-independent manner at bm-dc/t-cell ratios of : (fig. b) and : (fig. c) , but there was no significant difference compared with the untreated bm-dc group. additionally, the β integrin and ccr expression levels on t cells were detected with flow cytometry. the data showed that , nm ra-treated bm-dcs induced cd + cells to highly express β integrin and ccr compared with the untreated bm-dc group (fig. d , p < . , bm-dc/t-cell ratios of : ). at bm-dc/t-cell ratios of : , , nm ra-treated bm-dcs still induced cd + cells to highly express β integrin and ccr , but there was no significant difference compared with the untreated bm-dc group (fig. e) . to verify whether the porcine cells that expressed ccr could respond to ccl (ccr ligand), we evaluated the migration capacity of t cells towards ccl (fig. f) , which is constitutively expressed by intestinal epithelial cells . all of the t cells when activated by ra-activated dcs showed strong responsiveness to ccl , the number of migrated cells with an ra dose-independent increase (fig. g) . notably, the maximum responsiveness to ccl relied on exogenous ra ( , nm) conditioning. on the contrary, the ability of the t cells to migrate from the apical side to the basolateral side of the transwell filters was significantly reduced after ra receptor agonists (rars) (ro- - , , to , nm) were added with ra ( , nm) to the culture (fig. h) , the number of migrated cells among , or , nm ro- - plus ra or ra treatments was no significant difference, which was consistent with a previous observation . taken together, the cell migration assay results revealed a strong chemotactic response in t cells when activated by ra-activated dcs. these data demonstrated that ra-pretreated bmdcs could activate t cells to express high functional levels of the gut-homing receptor ccr , as well as to migrate towards the porcine chemokine ccl . current tgev vaccines are effective in protecting neonatal piglets from tgev infection. two types of tge vaccine are currently licensed for commercial use, including live attenuated vaccines, which are delivered orally, and inactivated vaccines, which are administered via parenteral inoculation to induce immunity . although live attenuated vaccines induce efficient protective immunity, live viruses are unsafe and can revert to virulence. in the development of a vaccine, safety and effectiveness are important considerations. in recent years, inactivated vaccines have been known for their safety and have been given higher priority in the pig industry, but inactivated vaccines require multiple booster immunizations and/or supplementation with adjuvants, because they are less immunogenic than live vaccines. the initial tgev infection occurs in the digestive tract. the oral route of vaccine delivery should be an effective method to prevent viral adhesion and colonization, thus decreasing viral shedding in the digestive tract . however, the oral route for vaccine delivery is the most challenging and difficult to achieve, particularly for inactivated vaccines. the efficacies of oral inactivated vaccines are currently poor, mainly because inactivated antigens must overcome a series of mucosal barriers, including mucus, digestive fluid, and compact epithelium, before they are captured by submucosal antigen-presenting cells (apcs) . thus, oral administration generally requires a large concentration of antigens to achieve effective immune protection levels . in this study, the antigen dose for the oral immunization (positive control) was times greater than that of the subcutaneous immunization, which significantly increased the production cost of the vaccines. an ideal tgev vaccine should induce both local mucosal and systemic immune responses. one effective strategy to improve local immunity involves inducing lymphocyte trafficking to the intestinal mucosa. ra is critical for inducing gut-homing receptors on t cells, which enhances their migration capacity to the intestinal mucosa . our data confirmed that ra plus tgev s.c. could generate a great number of gut-homing cd + t cells in the inguinal lymph nodes below the subcutaneous immunization site. this was consistent with a previous report that subcutaneous ra-assisted antigen treated mice had increased lymphocytes at the injection site lymph nodes and these cells had up-regulated gut-homing receptor levels and down-regulated skin-homing receptor levels . these results suggested that ra played an important role in immune induction. cd + effector t cells in the intestinal epithelium can effectively prevent or directly limit enteric virus infection , as well as maintain the epithelial barrier and tissue homeostasis . the in vivo assay in our study demonstrated that using ra plus tgev s.c. could greatly increase the number of cd + cells expressing β integrin and ccr in the ileum. however, the cd + cells in the s.c. tgev alone treated mice did not show significant changes, indicating that the ra was necessary to see cd + cell increase in β integrin and ccr . furthermore, it was recently reported that ccr expression on t cells may also regulate the localization and ability of mature cd α α intestinal intraepithelial lymphocytes in the small intestinal epithelium to influence mucosal immune responses. cd α α t cells of intraepithelial lymphocytes (iel) that likely function as regulatory t cells , these cd α α t cell subsets in gut maintain gastrointestinal homeostasis . in agreement with the migration of gut-homing cd + cells, our observations showed that after ra plus tgev s.c. treatment, many cd + t cells were recruited to the intestinal villi and lamina propria, and these data also showed that ra-assisted antigen s.c. can establish a stronger and faster cellular immune response to defend against foreign pathogens . siga plays an important role in reducing viral adhesion and capturing invasive viruses . thus, a large amount of iga-secreting cell homing to the gut is important to produce iga antibody against intestinal pathogens . ra is required to induce gut-tropic iga-secreting cells in mice and humans , , . in our study, iga-secreting cells were, remarkably, recruited to the intestinal lamina propria after the s.c. ra-tgev treatment, which was in line with the subsequent strongly initiated iga antibody response in the local porcine intestinal mucosa. furthermore, the ccr expression on the iga-secreting cells helped their cellular recruitment, and this was consistent with a previous report that ra was critical for the generation of gut-homing receptors on iga-secreting cells . interestingly, ra plus tgev immunization generated a long-lasting specific-siga response in the faeces, even though it was lower than the levels following the oral immunization, implying that an enhancement in siga secretion may be conducive to tgev eliminate and infection resistance in the gut. however, it is worth mentioning that the antigen dose in the oral immunization was times higher than that in the subcutaneous immunization in our study. thus, at equal amount of antigen, it is possible that ra addition increases iga secretion even above the secretion seen in response to the oral vaccine. therefore, the ra administration in the subcutaneous immunization not only improved tgev mucosal humoral immune responses but also was less expensive. moreover, serum igg plays a vital role in systemic immune responses, particularly for protecting against invasive pathogens that enter the bloodstream . ra combined with the s.c. tgev injection indeed induced a pronounced igg response, which could compensate for the disadvantages of the oral immunization. dcs have unique capacities to induce primary t-cell responses , , . ra has an important influence on dc differentiation, maturation and functions, such as the capacity for dcs to induce iga in peyer's patches . in our study, we found that ra could induce wi-tgev to increase the dc number in the intestinal lamina propria of piglets in vivo. the immature dcs (idcs) that are located beneath the intestinal epithelium play an important role in monitoring and capturing antigen . this implied that the dcs that were recruited following the ra-assisted tgev s.c. treatment had the characteristics of immature dcs, as they patrolled the intestinal tract, captured antigens in the intestinal lumina, quickly migrated to mlns for antigen presentation and improved the porcine mucosal immunity. dcs are derived from proliferating precursors in the bone marrow that migrate via the blood to lymphoid and non-lymphoid tissues . to investigate the mechanisms by which ra acted on dcs, we directly treated porcine bm-dcs with ra (in vitro), however, the ra treatment was unable to up-regulate the gut-homing-specific molecules as in mice , even with a wide range of doses. the possible reason for this may have been due to the species differences between porcine and mice. our results also showed that ra couldn't up-regulated the expression of maturation phenotype molecules on porcine bm-dcs in vitro, such as swc a, cd a, and hla-dr molecule expression; these findings were supported by a previous study showing that ra treatment endowed bm-dcs with immature phenotypes via the down-regulation of cd / , cd , and mhcii expression in mice . these observations suggest that ra inhibits porcine bm-dc maturation, and they may help explain our previous observation that the dcs recruited to the intestinal lamina propria following ra-tgev treatment were immature dcs that were conductive to capturing antigen and initiating a specific antibody response. given that ra failed to directly induce gut-homing receptors on dcs, we wondered whether ra could induce porcine lymphocytes to express the integrin β and ccr receptors. however, in the porcine in vitro and competitive homing experiments, ra was unable to directly induce unactivated t cells to express gut-homing-specific receptors, and it also failed in inducing t-cell migration to the small bowel. we wondered about the mechanisms that induce cd + t cells to express the integrin β and ccr receptors after ra-assisted tgev s.c. challenge scientific reports | : | doi: . /srep in vivo. some studies have suggested that dcs are crucial in regulating downstream activation and differentiation of t cells , . moreover, some evidence has demonstrated that cd + t cells and iga-secreting b cells express gut-homing receptors via intestinal dc interactions , . the above analysis tempts us to speculate that ra may regulate dcs in the porcine inguinal lymph nodes to imprint gut tropism receptors on lymphocytes. intriguingly, our data confirmed that ra cooperated with bm-dcs in promoting t cell proliferation, suggesting that ra-pretreated bm-dcs may have a powerful capacity to transfer antigens to t cells. our data further indicated that t cells expressed high gut-homing-specific receptor levels following incubation with ra-pretreated bm-dcs, implying that porcine ra-pretreated bm-dcs may have the ability to imprint integrin β and ccr receptor expression on lymphocytes. moreover, a cell migration assay was performed using transwell inserts, and the t cells that were treated with ra and dcs displayed a strong chemotactic response to porcine ccl , which was consistent with a recent study in mice that showed a powerful homing capacity for these cells. additionally, the capacity of ra-pretreated dcs to induce gut-homing receptors on responding t cells was blocked by an rar antagonist, and those t cells lost the ability to migrate, supporting the hypothesis that ra is indispensable for generating gut tropic t cells in piglets. in view of the in vitro study, the ra-pretreated bm-dcs showed a significant ability to activate t cells to express gut-homing receptors for migration. one possible mechanism for this, with regard to the ra-pretreated dcs, is that ra could act as a "mucosal adjuvant" to induce mucosal dc and lymphocyte activation , . furthermore, exogenous ra promotes intestinal lamina propria dcs to produce endogenous ra , which guarantees gut-homing α β and ccr expression by effector t cells . thus, another possibility is that the ra-pretreated dcs may also generate or combine with other cytokines to jointly promote gut-homing molecule expression, such as ra inducing mucosal dcs to secrete tgf β and il- , which enhance gut-homing receptors on lymphocytes . however, the mechanistic knowledge by which porcine intestinal dc subsets develop is still limited; therefore, further studies will be needed to obtain insights into these potential mechanisms. viral diarrhoea is the second major cause of death and a main reason for malnutrition in children under five years of age. compared to murine models, porcine models have substantial advantages, including physiological, anatomical and genetic similarities to the human intestine as well as defence mechanisms against disease similar to those of humans , , and these models have recently been widely applied to studies of intestinal immunity , gut microbiota , nosogenesis of rotavirus infection in newborns , and even organ transplantation . our research regarding s.c. ra-assisted antigen treatment in the piglet model may helpful to guide the prevention and control of viral diarrhoea in children. taken together, our results demonstrate that ra provides a great opportunity for novel vaccine designs. ra interacts with dcs to guide lymphocyte migration to the intestinal tract, which enhances both mucosal and systemic immunity and is thus beneficial in preventing tgev transmission between piglets and reducing the risk of diarrhoea. the molecular biology of coronaviruses effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants mucosal immunization and adjuvants the mucosal immune system: features of inductive and effector sites to consider in mucosal immunization and vaccine development cellular and molecular mechanisms for induction of mucosal immunity dendritic cells in intestinal immune regulation generation of 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in neonatal piglets initial in vivo experience of pig artery patch transplantation in baboons using mutant mhc (ciita-dn) pigs this work was supported by national natural science foundation of china ( ) and priority academic program development of jiangsu higher education institutions (papd). x.c. design and performed all the experiments, analyzed the data and drafted the manuscript, c.t. and l.z. performed the immunofluorescence histochemical and immunohistochemical, t.q. and y.y. did dendritic cell isolation and culture, performed flow cytometry analyses, participated in the statistical analyses and writing the manuscript, q.y. supervised the experiment and participated in the experiment designs, q.y. conceived and coordinated the study, participated in writing the manuscript. all the authors read and approved the final manuscript. the authors declare no competing financial interests. key: cord- - uoozj authors: fujimoto, yousuke; hasegawa, shunji; matsushige, takeshi; wakiguchi, hiroyuki; nakamura, tamaki; hasegawa, hideki; nakajima, noriko; ainai, akira; oga, atsunori; itoh, hiroshi; shirabe, komei; toda, shoichi; atsuta, ryo; morishima, tsuneo; ohga, shouichi title: pulmonary inflammation and cytokine dynamics of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during a(h n )pdm influenza infection date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: uoozj asthmatic patients present more rapid progression of respiratory distress after a(h n )pdm influenza infection than after seasonal infection. here, we sought to clarify the pathophysiology of early deterioration in asthmatic patients after a(h n )pdm infection. cytokine levels and virus titres in bronchoalveolar lavage fluid from mice with and without asthma after a(h n )pdm or seasonal h n infection were examined. in asthma/a(h n )pdm mice, il- and tnf-α levels peaked at days post-infection and were higher than those in all other groups. ifn-γ levels in asthma/a(h n )pdm mice at days post-infection were higher than in all other mice at any time point, whereas at days post-infection, the levels were lowest in asthma/a(h n )pdm mice. virus titres in asthma/a(h n )pdm mice were highest at days post-infection, and decreased by days post-infection, although the levels at this time point were still higher than that in any other group. histopathological examination showed more inflammatory cell infiltration and lung tissue destruction in the asthma/a(h n )pdm group than in any other group. the distinct cytokine profiles in a(h n )pdm -infected asthmatic mice indicated excessive inflammation and virus replication within a few days after infection. thus, bronchial asthma could be a more exacerbating factor for pandemic influenza infection than for seasonal influenza infection. the incidence of a(h n )pdm viral infection was significantly higher in children with asthma than in children without asthma . paediatric patients with a(h n )pdm infection showed milder symptoms than those with seasonal h n infection. however, severe respiratory issues, including pneumonia and acute respiratory distress syndrome (ards), have been reported in children and young adults with a(h n )pdm infection , [ ] [ ] [ ] . bronchial asthma increases the risk of hospital and intensive care admission in infants and children [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we previously reported that a(h n )pdm infection, but not seasonal h n infection, induces severe pulmonary inflammation in a mouse model of asthma days after infection , . however, we observed that the duration of the latent period for a(h n )pdm infection is shorter than days, and patients present earlier progression of pulmonary disease and systemic conditions after infection. since exacerbation was frequently observed in asthmatic patients after viral infection, we suspect that a cytokine storm, including inflammatory cytokines (interleukin [il]- and tumour necrosis factor [tnf]-α), anti-inflammatory cytokines (il- ), anti-viral cytokines (interferon [ifn]-γ), and helper t (th) cytokines (il- and il- ), may occur in the lung following a(h n )pdm infection. however, little is known about the early host response against a(h n )pdm infection in patients with underlying bronchial asthma. in the present study, we investigated the sequential changes in intra-tracheal cytokine production, viral loads, and pulmonary inflammation in a mouse model of bronchial asthma during the first days after a(h n )pdm or seasonal h n influenza infection. inflammatory cytokine concentrations in bronchoalveolar lavage (bal) fluid. il- , tnf-α, and il- β concentrations in bal fluid obtained , , and days post-infection are shown in fig. . although the mean il- levels in asthmatic mice challenged with a(h n )pdm were low ( . pg/ml) at days post-infection, the levels were markedly increased (to . pg/ml) at days post-infection, and the levels in all groups remained high at days post-infection. in contrast, il- levels in a(h n )pdm -challenged control mice were . pg/ml at days post-infection, peaked at . pg/ml by days post-infection, and remained at similar levels at days post-infection. after challenge with influenza a/puerto rico, il- levels in asthmatic mice slowly increased to . pg/ml by days post-infection, and then reached . pg/ml at days post-infection. in contrast, il- levels in control mice increased to . pg/ml at days post-infection, similar to the levels after a(h n )pdm infection. il- levels in control mice at days post-infection exceeded those of asthmatic mice at this time point (p = . ), and peaked to . pg/ml at days post-infection. the tnf-α levels in asthmatic/a(h n )pdm mice increased to . pg/ml at days post-infection, which was the highest for all groups, and levels remained high at days post-infection (p = . ). in contrast, tnf-α levels in the control/a(h n )pdm group increased to . pg/ml at days post-infection, and peaked to . pg/ml by days post-infection (p = . ). after seasonal virus infection, tnf-α levels in asthmatic mice increased to only . pg/ml at days post-infection, which was similar to the levels in control/a(h n )pdm mice (p = . ), and these levels were maintained until days post-infection. in contrast, the levels in control mice increased to . pg/ml by days post-seasonal virus infection, which were similar to those in asthma/a(h n )pdm mice (p = . ), and these levels were maintained until days post-infection. elevations in il- and tnf-α levels in bal fluid were not observed in the two mock-infected groups. the bal il- β levels in control ( . pg/ml) and asthmatic mice ( . pg/ml) after seasonal infection were significantly higher than the a(h n )pdm -infected groups (control/a(h n )pdm : . pg/ml, asthmatic/a(h n )pdm : . pg/ml), respectively. the early increasing pattern of il- and tnf-α levels (but not il- β) in asthmatic mice after a(h n )pdm infection (but not control mice), was in contrast to the dynamics observed in a/puerto rico-infected mice. other cytokines in bal fluid. ifn-γ levels in asthmatic/a(h n )pdm mice significantly increased to . pg/ml by days post-infection, which was the highest among all mice at this time point (vs. control/a(h n )pdm , p = . ; vs. asthma/seasonal, p = . ; vs. control/seasonal, p = . ), and then levels increased to . pg/ml at days post-infection (p = . ). ifn-γ levels in control mice at days after a(h n )pdm infection increased to . pg/ml, which was significantly lower than the levels in asthmatic/a(h n )pdm mice at days post-infection (p = . ). however, ifn-γ levels in control/a(h n )pdm mice peaked at . pg/ml at days post-infection, which were the highest of all the groups. il- levels were undetectable in all mice until days post-infection. at days post-infection, the levels in asthmatic/a(h n )pdm mice increased to . pg/ml, which was the highest of all the groups (vs. control/a(h n )pdm , p = . ; vs. asthma/seasonal, p = . ; vs. control/seasonal, p = . ). il- levels in control/a(h n )pdm mice increased to . pg/ml at days post-infection. these levels were similar to those in control/seasonal mice ( . pg/ml, p = . ) but higher than those in asthma/seasonal mice ( . pg/ml, p = . ). after seasonal virus infection, the ifn-γ levels in asthmatic mice increased to . pg/ml at days post-infection, which were similar to the levels in a/puerto rico-challenged control and asthmatic mice at days post-infection, but were lower than those in asthmatic/a(h n )pdm mice at days post-infection (p = . ). the ifn-γ levels in asthmatic/seasonal mice then increased to . pg/ml by days post-infection, which were lower than that in any other group at this time point (vs. asthmatic/a(h n )pdm , p = . ; vs. control/a(h n )pdm , p = . ; and vs. control/seasonal, p = . ). ifn-γ levels in control/seasonal mice only increased to . pg/ml at days post-infection, but then increased to . pg/ml by days post-infection (p = . ). neither il- levels nor ifn-γ levels were elevated in non-infected groups. bal il- levels in all asthmatic groups were higher than those in all non-asthmatic control groups, but the differences were not statistically significant. additionally, the levels in the control/a(h n )pdm and asthma/a(h n )pdm groups increased from to days post-infection. no significant differences in the levels of il- , il- , or il- a, were observed among the groups, and the levels were all below the detection limits. p < . , † † p < . ; control/seasonal vs. asthma/seasonal: || p < . , || || p < . ; control/a(h n )pdm vs. control/seasonal: p < . ; asthma/a(h n )pdm vs. asthma/seasonal: § p < . , § § p < . ; asthma/a(h n ) pdm vs. control/seasonal: $$ p < . ; and control/a(h n )pdm vs. asthma/seasonal: ‡ p < . , ‡ ‡ p < . ; mock/control or mock/asthma vs. each group: *p < . , **p < . ; day vs. day , ¶ ¶ p < . ; day vs. day : fig. . the numbers of total cells, lymphocytes, cd + cells, and cd + cells in all infected groups were increased at days post-infection, and then decreased at days post-infection. the numbers of cd + cells in the asthma/a(h n )pdm and control/seasonal groups were maintained until days post-infection. the number of lymphocytes in control/seasonal group were higher than that in control/a(h n ) pdm at days post-infection, however, there were no significant differences between control/seasonal and asthma/a(h n )pdm , control/a(h n )pdm and asthma/a(h n )pdm groups, respectively. additionally, the numbers of cd + cells, neutrophils and eosinophils on the days and cd + cells on the days post-infection were higher in the asthmatic/a(h n )pdm group than other groups respectively, but not statistically. in contrast, the numbers in the control/mock and asthma/mock groups were lower than those in the infected groups. virus titres in bal fluid. the virus titres in bal fluid at , , and days post-infection are shown in fig. . the mean titres at days post-infection in the a(h n )pdm -infected groups (asthma/a(h n )pdm : . × pfu/ml and control/a(h n )pdm : . × pfu/ml) were higher than those in the seasonal-infected groups (asthma/seasonal: . × pfu/ml, control/seasonal: not detected); however, these differences were not statistically significant. the mean titre in the asthmatic/a(h n )pdm group at days post-infection ( . × pfu/ml) was the highest of all groups, and the differences were significant (vs. control/a(h n )pdm , p = . ; vs. asthma/seasonal, p = . ), with the exception of the control/seasonal group. the virus titre in the asthmatic/a(h n )pdm group at days post-infection ( . × pfu/ml) was higher than those in any other groups at this time point (vs. control/a(h n )pdm , p = . ; vs. asthma/seasonal, p = . ; vs. control/seasonal, p = . ). after a(h n )pdm infection, the titre in asthmatic mice at days post-infection was higher than the titre at days post-infection (p = . ). the virus titres in control mice were also higher at days post-infection than at days post-infection (p = . ) after challenge with seasonal h n . histopathological findings in the lungs. figure shows the h&e staining of lung tissues from mice at (a), (b) and (c) days post-infection. the degrees of inflammatory cell infiltration and abscess formation in the asthma/a(h n )pdm group were more remarkable than in the control/a(h n )pdm , asthma/seasonal, and control/seasonal groups on , and days post-infection. in addition, on days post-infection, they were most severe in asthma/a(h n )pdm mice, compared with other mice. nucleoprotein antigen (infa-np) in the lungs of mice after a(h n )pdm or seasonal infection were observed by immunohistochemistry (fig. ) . infa-np was detected in the epithelial cells and suspected macrophages in the lungs of asthmatic/a(h n )pdm (a, day ) and control/a(h n )pdm group mice (a, day ) since the early phase to day (b) after infection, but not in mice from seasonal infected groups. infiltration of various inflammatory cells was noted, mainly in the alveolar walls in all four infected groups and more around the bronchioles in the a(h n )pdm -infected groups, than in the seasonal h n -infected groups. only the asthmatic/a(h n ) pdm group showed abscess formation with severe inflammation. the notable findings in the present study were the early peak in both il- and tnf-α levels, the high inflammatory cell infiltration in bal fluids, and the severe pulmonary inflammation at days post-infection in asthmatic/a(h n )pdm mice. the pulmonary cytokine storm at days post-infection in asthma/a(h n )pdm mice may mirror the rapid exacerbation observed in asthmatic patients . in contrast, the delayed peak in il- levels and insufficient surge of ifn-γ levels in a(h n )pdm mice at days post-infection could lead to ineffective exclusion of the viruses. the early potent inflammation associated with high viral loads in the lungs of asthmatic/a(h n )pdm mice may corroborate the rapid progression of asthmatic patients during outbreaks of pandemic virus infection. because the dynamics of il- β was different from those of il- and tnf-α levels, il- β may play other roles after influenza virus infection. in addition, the il- levels were increased in only the asthma/a(h n )pdm group after the infection. il- may be involved in pathophysiology of a(h n )pdm infection in asthmatic children. although il- , il- , and il- a may be involved in the pathogenesis of bronchial asthma and influenza infection, they were undetectable in the bal fluid from mice in this study. this may be explained by the cytokines' short half-lives and/or limited roles in this microenvironment. in asthma/a(h n )pdm group, the number of cd + cells at days and cd + cells at days post-infection were higher than those of other groups, respectively. these results show that cd + cells may act anti-viral function during influenza infection. we could not recognized whether these were th or th lymphocytes, however both cd + and cd + cells may play important roles in pathophysiology of a(h n )pdm -infected asthmatic patients. the histopathological findings in the early phase of infection in asthmatic/a(h n )pdm mice were severe pneumonia with abscess formation, and were not observed in any other groups (fig. ) . these results demonstrated that pulmonary inflammation in asthmatic mice is induced beginning in the early phase of a(h n )pdm infection, which mirrors the finding that a(h n )pdm infection in asthmatic children induces severe pulmonary complications, including pneumonia, atelectasis, etc., after a shorter incubation period than with seasonal virus infection. after a(h n )pdm infection, the viral loads in bal fluid from asthmatic mice were higher than those from control mice, which was not typically observed after seasonal infection (fig. ) . immunostaining of the virus showed many infa-np-positive cells in the lungs of asthmatic/a(h n ) pdm and control/a(h n )pdm mice since early phase after viral infection, but not in the lungs of mice from seasonal h n groups, as shown in fig. . previous reports demonstrated that a(h n )pdm viral proteins were detected in damaged type ii pneumocytes, epithelial cells, and infiltrated macrophages in the lung by immunohistochemistry [ ] [ ] [ ] . in addition, at autopsy after a(h n )pdm infection, acute diffuse alveolar damage was observed . avian influenza viruses preferentially bind to saα - gal, which is expressed on distal bronchioles and type ii pneumocytes in the lower respiratory tract . in contrast, seasonal h n influenza viruses bind to saα - gal, which is expressed on epithelial cells in the upper respiratory tract. a(h n )pdm virus binds to both saα - gal and saα - gal , . predominant replication of a(h n )pdm virus in the lower respiratory tract, compared with that of seasonal influenza virus, could explain the distinct viral loads shown in fig. . these findings suggested that a(h n )pdm virus may induce severe pneumonia in asthmatic patients, which is much less likely in seasonal influenza-infected asthmatic patients or non-asthmatic patients. we control/seasonal, : asthma/seasonal, : control/mock, : asthma/mock. control/a(h n )pdm vs. asthma/ a(h n )pdm : † p < . , † † p < . ; control/seasonal vs. asthma/seasonal: || p < . , || || p < . ; control/ a(h n )pdm vs. control/seasonal: *p < . ; asthma/a(h n )pdm vs. asthma/seasonal: § p < . , § § p < . ; asthma/a(h n )pdm vs. control/seasonal: $$ p < . ; and control/a(h n )pdm vs. asthma/seasonal: ‡ p < . , ‡ ‡ p < . ; mock group vs. each group: *p < . , **p < . ; day vs. day , ¶ ¶ p < . ; day vs. day : ## p < . . concluded that severe pulmonary complications are caused not only by the characteristics of the infecting viruses but also by factors in the host defence of asthmatic children during a(h n )pdm infection. in the present study, cytokine levels in bal fluid ( fig. ) appeared not to be associated with the lung histopathology. the histopathological analyses showed that airway inflammation was augmented in asthmatic mice when compared to control mice infected with either a(h n )pdm or seasonal influenza (fig. ) . however, bal fluid cytokine levels showed no paralleled alterations. in fact, inflammatory cytokine levels in the non-infected groups were equivocally low in mice with or without bronchial asthma, which suggested that these histopathological changes without detectable cytokine elevations, were independent of asthma. at days post-infection, ifn-γ levels in bal fluid were significantly higher in the asthmatic/a(h n ) pdm group than in the control/a(h n )pdm group, in contrast to the pattern at days post-infection. a previous report also showed that ifn-γ levels in asthmatic mice were lower than those in control mice at days after a(h n )pdm infection . virus titres in the asthmatic/a(h n )pdm group at both and days post-infection were significantly higher than those in control mice, and the titres of asthmatic/a(h n )pdm mice at days post-infection were the highest among all groups at both and days post-infection. in contrast, the virus titres of the control/seasonal group were significantly lower than those of the asthmatic/seasonal group at both and days post-infection. ifn-γ levels were reportedly reduced in bronchial asthmatic patients, indicating an alteration in the cytokine milieu, with excess production of th cytokines and decreased production of th cytokines , , and it has been reported that bronchial asthma patients show suppressed innate immunity [ ] [ ] [ ] . ifn-γ is produced by th cells, cd + t (cytotoxic t) cells, natural killer (nk) cells, and nkt cells , , and in our study, ifn-γ levels were elevated against the high virus load in the asthmatic/a(h n )pdm group at days post-infection, but were not sufficiently augmented at days post-infection. pulmonary inflammation, through not only the ifn-γ pathway but also other inflammatory molecules, might be involved in the exacerbation observed in a(h n )pdm -infected asthma patients. we have some limited reasons for the unexplainable reciprocal pattern of virus titres in a(h n )pdm -and seasonal influenza-infected asthmatic mice. the viral titre may depend on both the specificity of these viruses and the distinctive host defences in asthmatic individuals. however, further investigations are needed to characterize the immune responses against a(h n )pdm infection in asthmatic patients. in this study, the lung tissues of asthmatic/a(h n )pdm and control/a(h n )pdm mice were positive for infa-np antigens, whereas the lung tissues of mice in seasonal groups were not, even though virus titres were detected in the bal fluid of all infected groups. the reasons for this discrepancy may be the lower affinity for the virus receptors present in the lower respiratory tract or some yet unknown properties of the polyclonal antibodies and/or viral strains used, although the reason remains unclear. further research should be directed toward immunohistochemical studies of the upper respiratory tract along with lung function and airway hyperresponsiveness. in conclusion, a(h n )pdm infection can induce more severe pulmonary inflammation in patients with bronchial asthma than seasonal h n infection, based on the dynamics of early excessive production of inflammatory cytokines and the reciprocal depression of anti-viral cytokines, along with high viral loads in a mouse model of bronchial asthma. sensitization of mice and allergen challenge. balb/c mice (age: - weeks) were obtained from chiyoda kaihatsu co., ltd. (tokyo, japan) and were sensitized and challenged with grade ii ovalbumin (ova; sigma-aldrich., st. louis, mo, usa), as previously described , . all animal procedures were approved by the institutional animal care and use committee of yamaguchi university (no. -s ), and all methods were control/seasonal, ▲: asthma/seasonal. data are the mean ± sd of three independent experiments. control/ a(h n )pdm vs. asthma/a(h n )pdm : † p < . , † † p < . ; control/seasonal vs. asthma/seasonal: ||p < . , || ||p < . ; asthma/a(h n )pdm vs. asthma/seasonal: § p < . , § § p < . ; day vs. day , ¶ ¶ p < . ; day vs. day : ## p < . . conducted in accordance with the approved guidelines. this study was performed independently of our previous reports , . viruses, infection of mice, and preparation of bal fluid. mouse-adapted a(h n )pdm (strain: a/narita/ / ) or seasonal h n (strain: a/puerto rico) viruses were provided by the national institute of infectious diseases (tokyo, japan). on day , influenza virus (concentration: × pfu/ μl) or vehicle (mock-infection) was administered intranasally to mice. then, mice were euthanized at , , or days post-infection, and samples were collected. bal fluids were collected on day , , and ( , , and days post-infection) with three consecutive -ml instillations of phosphate-buffered saline (pbs) at room temperature. the collected bal fluid was centrifuged at , rpm for min at °c, and the supernatants were stored at − °c for estimation of cytokine levels and virus titres. pg/ml, respectively. measurement of cd + cells, cd + cells, eosinophils, and neutrophils in bal fluid. cell pellets were resuspended in pbs and stained with fluorescein isothiocyanate (fitc)-conjugated anti-cd (bd biosciences) and allophycocyanin (apc)-conjugated anti-cd (bd biosciences) antibodies; erythrocytes were lysed by the addition of facs lysing solution epidemiology of pandemic influenza a (h n ) deaths in the united states factors associated with death or hospitalization due to pandemic influenza a (h n ) infection in california fatal cases associated with pandemic influenza a (h n ) reported in greece does glycosylation as a modifier of original antigenic sin explain the case age distribution and unusual toxicity in pandemic novel h n influenza? hospitalized patients with h n influenza in the united states risk factors for severe outcomes following influenza a (h n ) infection: a global pooled analysis increased h n infection rate in children with asthma pneumonia and respiratory failure from swine-origin influenza a (h n ) in mexico acute respiratory distress syndrome induced by a swine h n variant in mice three children with plastic bronchitis associated with h n influenza virus infection plastic bronchitis in three children associated with influenza a(h n ) virus infection a retrospective cross-sectional study of risk factors and clinical spectrum of children admitted to hospital with pandemic h n influenza as compared to influenza a characteristics of atopic children with pandemic h n influenza viral infection: pandemic h n influenza reveals 'occult' asthma of childhood analysis of bronchoalveolar lavage fluid in a mouse model of bronchial asthma and h n infection cytokine profile of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during seasonal h n infection sudden death of a patient with pandemic influenza (a/h n pdm) virus infection by acute respiratory distress syndrome influenza a viruses target type ii pneumocytes in the human lung increased severity of pandemic influenza a virus subtype h n infection in alveolar type ii cells from patients with pulmonary fibrosis histopathological and immunohistochemical findings of autopsy cases with h n virus infection receptor-binding specificity of pandemic influenza a (h n ) virus determined by carbohydrate microarray the immune profile associated with acute allergic asthma accelerates clearance of influenza virus interferon-gamma: a historical perspective rhinovirus-induced interferon-gamma and airway responsiveness in asthma il- is more potent than il- in provoking il- -producing nuocytes (type innate lymphoid cells) and airway contraction influenza enhances caspase- in bronchial epithelial cells from asthmatic volunteers and is associated with pathogenesis innate immunity to influenza in chronic airways diseases inhibition of nk cell activity by il- allows vaccinia virus to induce severe skin lesions in a mouse model of eczema vaccinatum nk cells and nkt cells in innate defense against viral infections a mutant h n influenza virus uses an alternative activation mechanism in tmprss knockout mice by loss of an oligosaccharide in the hemagglutinin stalk region we thank dr. takashi plaque assay. plaque assays were performed as described previously , . briefly, madin-darby canine kidney (mdck) cells (lonza, walkersville, md, usa) were maintained at °c in a humidified % co chamber under stationary conditions. each well of a -well plate was seeded with × cells and cultured in α-minimum essential medium (mem; gibco/invitrogen, carlsbad, ca, usa) containing % foetal bovine serum (fbs), units/ml penicillin (gibco), and μg/ml streptomycin (gibco). after two washes with serum-free dulbecco's modified eagle's medium (dmem; gibco/invitrogen), the cells were maintained in serum-free dmem at °c for h. then, each well was overlaid with μl of diluted bal ( − , − , and − dilutions) and incubated at °c for h. after one wash in serum-free dmem, the cells were overlaid with serum-free dmem containing . % agarose (becton, dickinson and company, sparks, md, usa), . % diethylaminoethyl-dextran (sigma-aldrich), and μg/ml trypsin (sigma-aldrich). the cells were cultured at °c for h, fixed in % formaldehyde (wako pure chemical industries, ltd., osaka, japan), and then stained with . % methylene blue (wako pure chemical industries, ltd.). each experiment was performed in duplicate.histological and immunohistochemical examination of the lungs. lung tissues were fixed in % buffered formalin for h at room temperature and then embedded in paraffin. serial sections ( -µm thick) were cut and stained with hematoxylin and eosin (h&e; muto pure chemicals co., ltd., tokyo, japan). the distribution of viral antigens was examined by immunological staining with a rabbit polyclonal antibody against infa-np, which recognize not only a(h n )pdm and seasonal h n , but also h n influenza . specific antigen-antibody reactions were visualized by , ′-diaminobenzidine tetrahydrochloride staining with the envision rabbit/hrp system (dako cytomation). the stained sections were observed by light microscopy to evaluate the degree of pulmonary inflammation and localization of a(h n )pdm -infected cells.statistical analysis. the differences between groups were analysed by the mann-whitney u test. p values less than . were considered statistically significant. all analyses and calculations were performed using spss version . (spss inc., chicago, il, usa). yousuke fujimoto, shunji hasegawa, and shouichi ohga were the principal investigators who take primary responsibility for the study. yousuke fujimoto, shunji hasegawa, takeshi matsushige, hiroyuki wakiguchi, and tamaki nakamura performed the mouse experiments. hideki hasegawa, noriko nakajima, akira ainai, atsunori oga, and hiroshi itoh performed the histopathological examinations. komei shirabe, shoichi toda, ryo atsuta, and tsuneo morishima supported this study with helpful discussions. yousuke fujimoto, shunji hasegwa, and shouichi ohga wrote the first draft of the manuscript. competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -o y wygc authors: shirvani, edris; paldurai, anandan; manoharan, vinoth k.; varghese, berin p.; samal, siba k. title: a recombinant newcastle disease virus (ndv) expressing s protein of infectious bronchitis virus (ibv) protects chickens against ibv and ndv date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: o y wygc infectious bronchitis virus (ibv) causes a highly contagious respiratory, reproductive and urogenital tract disease in chickens worldwide, resulting in substantial economic losses for the poultry industry. currently, live-attenuated ibv vaccines are used to control the disease. however, safety, attenuation and immunization outcomes of current vaccines are not guaranteed. several studies indicate that attenuated ibv vaccine strains contribute to the emergence of variant viruses in the field due to mutations and recombination. therefore, there is a need to develop a stable and safe ibv vaccine that will not create variant viruses. in this study, we generated recombinant newcastle disease viruses (rndvs) expressing the s , s and s proteins of ibv using reverse genetics technology. our results showed that the rndv expressing the s protein of ibv provided better protection than the rndv expressing s or s protein of ibv, indicating that the s protein is the best protective antigen of ibv. immunization of -week-old spf chickens with the rndv expressing s protein elicited ibv-specific neutralizing antibodies and provided complete protection against virulent ibv and virulent ndv challenges. these results suggest that the rndv expressing the s protein of ibv is a safe and effective bivalent vaccine candidate for both ibv and ndv. generation of rndvs expressing s , s or s protein of ibv. the expression cassettes containing the codon optimized s , s , s and non-codon optimized s genes of ibv were cloned into the cdna encoding the complete antigenome of ndv strain lasota, using the pmei site, between p and m genes (fig. ) . the correct sequences of genes cloned into full length cdna of ndv were confirmed by nucleotide sequence analysis. infectious recombinant ndvs containing s , s and s genes of ibv were recovered from all cdnas. the sequences of s , s and s genes present in the rndvs were confirmed by rt-pcr. to evaluate genetic stability of rndv expressing codon optimized s protein, the viruses were passaged five times in -day-old embryonated specific pathogen free (spf) chicken eggs. the nucleotide sequence analysis of the s gene showed that the inserted orf were maintained without any adventitious mutations. evaluation of the expression of the s , s and s proteins of ibv. the expression of codon optimized s , and s proteins and non-codon optimized s protein of ibv strain mass- by rndv constructs was detected by western blot analysis in df- cells using a chicken polyclonal anti ibv serum ( fig. a -upper panel and b). as the expression of non-codon optimized s was not detected clearly in the first attempt ( fig. a) , we detected it in another attempt (fig. b) . the expression level of codon optimized s protein of ibv was significantly higher than that of the non-codon optimized s protein of ibv. for the codon optimized s protein of ibv expressed from rndv ( fig probably represent uncleaved s protein (s ) or polymeric forms of s protein. the ~ kd band represents s or s subunit of cleaved s protein of ibv. in the case of rndv/ibv-s strain ( fig. a-lane ) , there are two bands (~ - kda) on top, representing polymeric folded forms of s protein, a ~ kda band and a ~ kda band representing s subunit. the expression of s protein from a transcription cassette in which the signal peptide sequences of s protein was not fused with s gene was not detected (data not shown). lane of fig. a and lane of fig. b represent rndv as control. lane of panel a represents non-infected df- cells. these results showed that codon optimized s and s proteins of ibv were expressed efficiently. the non-codon optimized s protein was also expressed from rndv, but not efficiently and not consistently. a monoclonal anti-ndv/hn antibody was used to detect a ~ kda of hn protein of ndv in lysates, confirming similar level of ndv protein in each lane ( fig. a-lower panel) . we further evaluated incorporation of ibv s and s proteins into ndv virions. the rndvs expressing codon optimized s and s proteins and rndv expressing non-codon optimized s protein were inoculated into eggs, days after inoculation, viral particles in infected allantoic fluid were partially purified and analyzed by western blot (fig. c-upper panel) . two bands (~ - kda) on top, representing s protein, a ~ kda band and a ~ kda band representing s or s subunit of cleaved s protein, were detected in purified particles of rndv expressing codon optimized s protein by western blot analysis (fig. c-lane ) . the lane of fig. c shows two bands (~ - kda) on top, representing polymeric folded of s protein, a ~ kda band and a ~ kda band representing s subunit. the lane of fig. c represents purified rndv control and lane of fig. c shows purified rndv expressing non-codon optimized s protein. these results suggested that the codon optimized s and s proteins of ibv expressed by rndvs were incorporated into rndv particles. a monoclonal anti-ndv/hn antibody was used to detect a ~ kda of hn protein of ndv in partially purified virions, confirming similar level of ndv protein in each lane (fig. c-lower panel) . the expression of codon optimized s protein expressed from four individual rndv constructs were detected by western blot analysis in lysates (fig. a) and supernatant (fig. b ) of infected df- cells, using a chicken polyclonal anti ibv serum. the lanes - represent infected df- cell lysates of rndv, rndv/s , rndv/ s + ibv-s-tm&ct, rndv/s (cs−) + ndv-f-tm&ct and rndv/s (cs+) + ndv-f-tm&ct, respectively. a ~ kda band representing expression of s by rndv/s + ibv-s-tm&ct, rndv/s (cs−) + ndv-f-tm&ct, and rndv/s (cs+) + ndv-f-tm&ct in lysate of df- cells (fig. a -lanes - ) and rndv/s in infected df- cell supernatant (fig. b-lane ) was observed. our attempts to detect the incorporation of the s protein into ndv envelope were not successful, due to the difficulties in the detection of very low level of s protein by western blot analysis (data not shown). our results showed that the s protein was expressed at very low level by all the rndvs based on western blot analysis. only the unmodified s protein was detected in the cell culture supernatant. growth characteristics of rndv constructs. the recovered rndvs were passaged in -day-old embryonated spf chicken eggs. all the viruses were able to replicate well in eggs (≥ hau/ml). rndv/s , rndv/ s (cs+) + ndv-f-tm&ct, rndv/s , rndv/codon optimized-s and rndv were evaluated in the presence of exogenous protease in df- cells (fig. ) . compared to the parental virus, rndv expressing codon optimized s protein of ibv grew slightly less efficiently. the maximum titer of parental virus reached . tcid /ml at hours post infection, whereas the maximum titer of rndv expressing codon optimized s gene of ibv reached . tcid /ml at hours post infection. these results indicated that presence of s, s and s genes did not significantly affect the growth characteristics of rndv. the protective efficacy of rndvs expressing s , s or s protein of ibv in chickens against a virulent ibv challenge. ibv protection experiment . to evaluate the protective efficacy of rndvs expressing s , s or s protein of ibv, spf chicks were immunized at -day-old age with each virus via oculanasal (on) route. at three weeks post-immunization, chickens were challenged with virulent ibv strain mass- . the severity scores of ibv clinical signs were recorded twice a day for days post-challenge (fig. a ). compared to chickens immunized with parental rndv and chickens inoculated with pbs, chickens immunized with rndvs expressing figure . schematic diagram of recombinant ndv constructs containing ibv genes. seven transcription cassettes including; - ) four versions of codon optimized s subunit of s gene of ibv strain mass- ; namely, (a) s subunit of s gene ( nt), (b) s subunit of s gene ( nt) fused with n-terminus of transmembrane and cytoplasmic tail of s gene ( nt), (c) s subunit of s gene ( nt) containing five putative cleavage site residues of s gene fused with n-terminus of transmembrane and cytoplasmic tail of f gene of ndv ( nt). in this construct, five c-terminus putative cleavage site residues of s gene (rrfrr) plus the first serine (s) residue of n-terminus of transmembrane and cytoplasmic tail of f gene of ndv provides six putative cleavage site residues of s protein of ibv strain mass- (rrfrr/s). (d) s gene ( nt) without cleavage site residues of s gene fused with n-terminus of transmembrane and cytoplasmic tail of f gene of ndv ( nt), ) the n-terminus of codon optimized s gene of ibv strain mass- ( nt) fused with c-terminus of signal peptide sequence of s gene ( nt), ) the codon-optimized s gene ( nt) and ) the non-codon optimized s gene of ibv strain mass- ( nt) were flanked into individual plasmids containing cdna of lasota between p and m genes using pmei site. each transcription cassette contains the orf of foreign gene with the addition of pmei restriction enzyme site sequence, nt of ndv utr, ge signal of ndv, one t nucleotide as intergenic sequence, gs signal of ndv, nucleotides for maintaining the rule of six and kozak sequence. scientific reports | ( ) : | doi: . /s - - - codon optimized s, s or s protein of ibv showed significantly less severe of clinical signs (p < . ). among groups of chickens immunized with rndvs expressing codon optimized s , s or s protein, the group immunized with rndv expressing codon optimized s protein showed the least severity of clinical signs (p < . ). in order to evaluate the efficacy of rndvs expressing s , s or s protein of ibv in preventing shedding of virulent ibv challenge virus in immunized chickens, on day five post-challenge, tracheal swab samples were collected from chickens of each group and were evaluated for the viral load by rt-qpcr. our results did not show significant difference in virus shedding among groups of immunized chickens at day five post challenge (fig. b) . however, the results of the inoculation of the tracheal swab samples into -day-old embrynated chicken eggs showed that out of ( . %) chickens vaccinated with rndv expressing codon optimized s protein of ibv and out of ( %) of non-infected chickens were shedding virus in trachea, respectively, whereas out of ( %) of chickens of all other groups were shedding virus in the trachea (data not shown). ibv protection experiment . to evaluate the protective efficacy of rndv expressing codon optimized s protein of ibv in adult chickens, spf chickens were immunized at -week-old age. the protective efficacy of rndv two bands (~ - kda) on top represent uncleaved s protein (s ) or polymeric forms of s or s protein (c-lane ). the ~ kda and the ~ kda band represent s or s subunit of cleaved s protein (c-lane ). the two bands (~ - kda) on top represent polymeric forms of s protein, the ~ kda band and the ~ kda expressing codon optimized s gene of ibv was determined by challenging the immunized chickens with the world organization for animal health (oie) recommended dose ( . eid ) of virulent ibv strain mass- at week post-immunization . the severity scores of ibv clinical signs were recorded twice a day for days post-challenge (fig. a ). compared to chickens inoculated with pbs, chickens immunized with rndv expressing codon optimized s protein of ibv and chickens immunized with a commercial live attenuated ibv vaccine showed significantly less severe clinical signs (p < . ). in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at day following challenge with a figure . western blot analysis of rndv expressing s protein of ibv. the expression of codon optimized s protein of ibv expressed from four individual rndvs expressing four different expression cassettes of s protein were detected using western blot in cell lysates (a) and cell supernatant (b) of infected df- cells infected with rndvs, using a chicken polyclonal anti ibv serum. the lanes - represent cell lysates of rndv, rndv/s , rndv/s + ibv-s-tm&ct, rndv/s (cs−)+ndv-f-tm&ct, rndv/s (cs+) + ndv-f-tm&ct, respectively. a ~ kd band represent expression of s protein by rndv/s + ibv-s-tm&ct, rndv/ s (cs−) + ndv-f-tm&ct and rndv/s (cs+) + ndv-f-tm&ct in infected df- cell lysate (a lanes [ ] [ ] [ ] and rndv/s in infected df- cell supernatant (b-lane ). the full-length gel is presented in supplementary figure s . the severity scores of ibv clinical signs include; ocular discharge, nasal discharge and difficulty in breathing ( = normal, = presence of mild ocular discharge, mild nasal discharge and or sneezing = presence of heavy ocular discharge and or heavy nasal discharge with mild tracheal rales and mouth breathing and or coughing = heavy ocular discharge and heavy nasal discharge with sever tracheal rales and mouth breathing, gasping, dyspnea and or severe respiratory distress) were recorded twice a day for each chicken for days after challenge. the severity scores represent as average scores of clinical signs measured for each chicken over days. (b) relative viral load determined by rt-qpcr in tracheal swab samples at day five following virulent ibv challenge. the relative viral load expressed as mean reciprocal ± sem log . whereas chickens inoculated with pbs showed high levels of viral load in the trachea (p < . ). however, compared to chickens immunized with a commercial ibv vaccine, chickens immunized with rndv expressing codon optimized s showed slightly less viral load in the trachea (fig. b ). ibv protection experiment . to evaluate the protective efficacy of rndv expressing codon optimized s protein of ibv in adult chickens against a higher dose of virulent ibv challenge, spf chickens were immunized at -week-old age. the protective efficacy of rndv expressing codon optimized s gene of ibv was determined by challenging the immunized chickens with . eid virulent ibv strain mass- at week post-immunization. the severity scores of ibv clinical signs were recorded twice a day for days post-challenge (fig. a ). compared to chickens immunized with rndv and chickens inoculated with pbs, chickens immunized with rndv expressing codon optimized s protein of ibv and chickens immunized with a commercial live attenuated ibv vaccine showed significantly less severe clinical signs (p < . ). in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at days following challenge with virulent ibv, the tracheal swab samples collected from five chickens of each group were analyzed for the ibv specific lesions in chicken embryo. our results showed that out of ( %) chickens vaccinated with rndv expressing codon optimized s protein of ibv and out of ( %) chickens vaccinated with a commercial ibv vaccine were shedding virus in trachea, respectively, whereas out of ( %) of chickens immunized with parental rndv and out of ( %) of chickens inoculated with pbs were shedding virus in the trachea (fig. c) . the tracheal swab samples collected from five chickens of each group were also analyzed for the viral load by rt-qpcr. our results showed that chickens vaccinated with rndv expressing codon optimized s protein of ibv showed low levels of viral load in the trachea and chickens vaccinated with a commercial ibv vaccine showed very low levels of viral load in the trachea, whereas chickens inoculated with pbs and rndv showed high levels of viral load in the trachea. compared to chickens immunized with rndv expressing codon optimized s protein, chickens immunized with a commercial ibv vaccine showed less viral load in the trachea (p < . ) (fig. b ). ibv protection experiment . to evaluate the effect of the route of inoculation of virulent ib challenge virus on the outcomes of the protective efficacy of rndv expressing codon optimized s protein of ibv, spf chicks were immunized at -day-old age. the protective efficacy of rndv expressing codon optimized s gene of ibv was determined by challenging the immunized chickens with eid virulent ibv strain mass- by the intraocular route at week post-immunization. this route of challenge has been specified in usda-cfr- for ibv . the severity scores of ibv clinical signs were recorded twice a day for days post-challenge. compared to chickens immunized with rndv and unvaccinated chickens, chickens immunized with rndv expressing codon optimized s protein of ibv and chickens immunized with a commercial live attenuated ibv vaccine showed significantly less severe clinical signs. however, compared to chickens immunized with commercial ibv vaccine, chickens immunized with rndv expressing s protein showed less severe clinical signs (p < . ) (fig. a) . in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at days following challenge with virulent ibv, the tracheal swab samples collected from all chickens of each group were analyzed for the ibv specific lesions in chicken embryos. our results showed that out of ( %) chickens vaccinated with rndv expressing codon optimized s protein of ibv and out of ( %) chickens vaccinated with a commercial ibv vaccine were shedding virus in trachea, respectively, whereas out of ( %) of chickens immunized with parental rndv and out of ( %) of unvaccinated chickens, infected with ibv, were shedding virus in the trachea (fig. b) . the protective efficacy of rndvs against a highly virulent ndv challenge. to evaluate the protective efficacy of rndv expressing s gene of ibv against a virulent ndv strain, groups of five -day-old chicks were immunized with rndv, rndv expressing codon optimized s protein and pbs. three weeks after immunization, chickens were challenged with virulent ndv strain texas gb in our bsl- plus facility. our results showed that all chickens immunized with the rndv and rndv expressing codon optimized s gene of ibv survived after highly virulent ndv challenge, while all chickens in pbs group died at day and post-challenge (fig. a) . antibodies produced against ibv and ndv. hemagglutination inhibition (hi) assay using a standard protocol of oie was used to assess the level of antibodies mounted against ndv in serum samples of chickens days after immunization. the results showed that hi titers of ndv was detected in serum samples of all chickens immunized with rndv and rndv expressing codon optimized s protein. there was no significant differences observed among hi titers against ndv in serum samples of chickens from groups immunized with rndv and rndv expressing s protein (fig. b ). virus neutralization assay was performed according to a standard protocol of oie to assess the level of neutralizing antibodies mounted against ibv strain mass- in serum samples of chickens at days after immunization. the results showed that the neutralizing antibodies against ibv were detected in serum samples of chickens immunized with rndv expressing codon optimized s protein of ibv and with commercial live attenuated ibv vaccine (fig. d) . neutralizing antibodies against ibv were not detected in : dilution of a serum sample from a chicken immunized with empty rndv vector. this result showed that the rndv expressing codon optimized s protein of ibv induces neutralizing antibodies against ibv. this study was conducted to compare the protective efficacies of s , s , and s proteins of ibv using rndv as a vaccine vector. the s , s , and s genes of ibv strain mass- were individually inserted between the p and m genes of rndv strain lasota. this site was chosen because it has been identified as the optimal site for insertion of foreign genes into ndv genome , - . four different versions of ibv s gene were used to identify the version that is expressed at the highest level and incorporated into ndv particles. we were able to recover all the recombinant viruses and their growth characteristics were similar to rlasota. however, the recombinant viruses containing ibv s gene grew slightly slowly than the parental virus. the viruses were stable after passages in spf chicken embryos. western blot analysis showed that chicken codon optimized s and s proteins were expressed at much higher levels and were incorporated into ndv particles. whereas, all the four versions of s protein were detected at very low levels by western blot analysis. it is noteworthy that the unmodified s protein was detected in the infected cell culture supernatant, indicating that the modification of s protein probably caused retention of the protein in the cell. these results suggest that the s protein acts as a chaperone to assist in the folding of the s protein. the s protein is folded incorrectly in the absence of s protein and the new structure probably causes loss of some conformational epitopes for ibv antibodies. in the first ibv protection experiment, we found that -day-old chicks immunized with rndv expressing the s protein of ibv conferred better protection from disease compared to -day-old chicks immunized with rndvs expressing either s or s protein of ibv. our results showed that the s protein, which contains both s and s proteins, is the best protective antigen of ibv. the s protein lacks major neutralizing epitopes which are present in the s protein, hence it is not an effective antigen. the s protein contains major neutralizing epitopes, but it losses some conformational epitopes when expressed separately. although in the first study we showed that rndv expressing s protein provided enhanced protection, it could not reduce virus shedding, indicating that we needed to determine whether the elimination of virus shedding would probably require a much higher level of immune response than that is induced by rndv expressing s protein or would require the optimization of the ibv protection study. in this study, our results support previous reports that rndv vectored ibv vaccines prevent disease but do not stop virus shedding , . these results also support the recent report that a spike ectodomain subunit vaccine protects chickens against ibv . in the second ibv protection experiment, we investigated whether age at immunization influences the outcome of ibv challenge. our results showed that a single immunization of -week-old chickens with rndv expressing s protein completely protected chickens against ibv challenge based on disease and viral load in tracheas. indeed, the level of protection conferred by rndv expressing s protein was similar to that of a commercial ibv vaccine. however chickens immunized with either commercial live attenuated ibv vaccine or rndv expressing ibv s protein showed very low levels of tracheal viral load. this showed that protection was greater when the chickens were immunized at an age when their immune system is relatively well developed. in the third ibv protection experiment, we showed that rndv expressing ibv s protein protects adult chickens against a higher dose of virulent ibv challenge. however, compared to standard challenge dose of virulent ibv, a higher challenge dose of virulent ibv caused higher levels of tracheal viral load in adult chickens immunized with rndv expressing ibv s protein and low levels of tracheal viral load in chickens immunized with commercial live attenuated ibv vaccine. our result showed that although both the age of immunization and dose of challenge virus affect the results of ibv challenge, the influence of the age of immunization is greater than the effect of the dose of challenge virus. our results also showed that when we challenged adult immunized chickens with standard challenge dose of virulent ibv, rndv expressing s protein showed slightly better protection than a commercial live ibv vaccine, based on disease and viral shedding in trachea, but when the adult immunized chickens were challenged with a higher dose of virulent ibv, commercial live ibv vaccine showed slightly better protection than rndv expressing s protein. hence to compare the efficacy of rndv expressing s protein of ibv with the efficacy of live attenuated ibv vaccine, a large ibv protection study using commercial chickens is needed. in the fourth ibv protection experiment, we showed that rndv expressing ibv s protein protected young chickens against virulent ibv challenge by the intraocular route. the route of challenge has been recommended by usda-cfr- . our results showed that compared to infection of chickens with virulent ibv by the oculanasal route, infection of chickens with virulent ibv by the intraocular route, caused much lower levels of tracheal viral load in young chickens immunized with rndv expressing ibv s protein and low levels of tracheal viral load in chickens immunized with commercial live attenuated ibv vaccine. our results showed that the route of the challenge virus inoculation affected the results of the tracheal virus shedding in young chickens immunized by rndv expressing s protein following ibv challenge; however, it did not affect the outcomes of the severity of clinical signs. although our studies showed that the rndv expressing the s protein and commercial live ibv vaccine provided comparable protection, rndv expressing the s protein has several advantages over live ibv vaccines in controlling ib in the field. (i) ndv vectored ibv vaccine is highly safe in -day-old chicks, (ii) it will not create new vaccine derived variant viruses, which is a major concern in using live modified ibv vaccines, (iii) a single vaccine can be used to control both ndv and ibv, (iv) we believe that the level of immunity induced by the ndv vectored vaccine against ibv is probably sufficient to completely stop ibv infection in field condition, and (v) the immune response of ndv vectored vaccine can be enhanced by prime-boost vaccination strategy. in summary, we have shown that although the s and s proteins of ibv are known to contain virus neutralizing epitopes, the presence of the whole s protein is necessary for eliciting a strong protective immune response. the s protein is the antigen of choice for any vectored ibv vaccine. ndv is an attractive vaccine vector for ibv, because it can be used as a bivalent vaccine. our results suggest that a recombinant ndv vectored ibv vaccine is the vaccine of choice for controlling ibv infection in the field. cells were obtained from the american type culture collection (atcc, manassas, va). they were grown in dulbecco's minimal essential medium (dmem) containing % fetal bovine serum (fbs). the recombinant avirulent ndv strain lasota was generated previously in our laboratory using reverse genetics . the rndv and rndvs expressing chicken codon optimized s , s and s genes and non-codon optimized s gene of ibv strain mass- were grown in -day-old embryonated spf chicken eggs at °c. the virulent ibv strain mass- was propagated in -day-old spf embryonated chicken eggs and harvested five days after infection. the titer of virus in harvested allantoic fluid was determined by % embryo infectious dose (eid ) method. briefly, ten-fold serial dilutions of ibv strain mass- was inoculated into -day-old embryonated spf chicken eggs. seven days after inoculation, infected embryos were examined for ibv specific lesions such as stunting or curling. the titer of virus was calculated using reed and muench method .the modified vaccinia virus strain ankara expressing t rna polymerase (mva-t ) was propagatd in monolayer primary chicken embryo fibroblast cells. generation of rndvs containing s , s or s gene of ibv. a plasmid containing full-length antigenomic cdna of ndv strain lasota has been constructed previously . in order to develop an effective ibv vaccine the maximum neutralizing epitopes with correct conformation are needed to be displayed. most neutralizing epitopes are located in the s protein. in this study seven transcription cassettes containing s, s or s genes of ibv were constructed to identify the best protective antigen for the development of ndv vectored ibv vaccines. the s, s and s genes were chicken codon optimized for higher level of expression in chickens. the following transcription cassettes were designed: (i) a transcription cassette containing the s gene of ibv strain mass- ( nt) was designed to determine whether the expression of the whole s gene from ndv will lead to display the maximum neutralizing epitopes in correct conformation, (ii) a transcription cassette containing the s subunit of s gene ( nt) of ibv fused with c-terminus of signal peptide sequence of s gene ( nt) was constructed for transport of the protein from the cell (iii) a transcription cassette containing the s subunit of s gene ( nt) was designed to determine the protective efficacy of s protein, (iv) a transcription cassette containing the s gene ( nt) fused with n-terminus of transmembrane and cytoplasmic tail of s gene ( nt) was designed for incorporation into ndv envelop, (v) a transcription cassette containing the s subunit of s gene without s protein cleavage site residues ( nt) fused with n-terminus of transmembrane and cytoplasmic tail of ndv f gene ( nt) was designed for incorporation of the s protein into envelope of ndv. (vi) a transcription cassette containing the s subunit of s gene containing s protein cleavage site residues ( nt) fused with n-terminus of transmembrane and cytoplasmic tail of ndv f gene ( nt) was designed to incorporate the s protein into ndv envelope and also to know whether adding the cleavage site residues has any effect on the fusion of two scientific reports | ( ) : | doi: . /s - - - proteins, and (vii) a transcription cassette containing the non-codon optimized s gene ( nt) was constructed to compare the level of protein expression between the codon optimized and non-codon optimized s genes. ndv genome contains six genes: nucleocapsid(n), phosphoprotein(p), matrix(m), fusion(f), hemagglutinin-neuraminidase(hn) and large(l). the genes are ordered ′-n-p-m-f-hn-l- ′. the beginning and the end of each gene contain conserved transcriptional sequences known as the gene-start (gs) and gene-end (ge), respectively. between the genes, there are gene junctions . any of the gene junctions is a potential insertion site for the transcription cassette of a foreign gene. however, we and others have found that the intergenic region between the p and m genes is a good site for expression of most foreign genes , [ ] [ ] [ ] [ ] . the transcription cassettes containing ibv genes contained pmei restriction enzyme sequence, nt of untranslated region (utr) of ndv, ndv ge signal, one t nucleotide as intergenic sequence, ndv gs signal, extra nucleotides to maintain the rule of six , , kozak sequence at the upstream of foreign gene orfs and pmei restriction enzyme sequence at downstream of foreign gene orf. the transcription cassettes of codon optimized and non-codon optimized s gene were digested from two commercially synthesized (genscript; puc -ibv-mass- -s syn) plasmids containing codon optimized (genscript; optimization on gallus gallus codons using optimumgene tm pso algorithm) and non-codon optimized s gene of ibv strain mass- (genbank accession no. ay . ), respectively. the transcription cassettes of codon optimized s and s genes were amplified from the commercially synthesized plasmid containing codon optimized s gene of ibv strain mass- and cloned into individual shuttle vectors (pgem ® -t easy vector, promega corporation). then the flanking dna of transcription cassettes were digested from shuttle vectors. the transcription cassettes derived from shuttle vectors were cloned into complete individual plasmids containing cdna of rlasota at p and m gene junction using pmei site (fig. ) . the correct sequences of the foreign genes were confirmed by nucleotide sequence analysis. rndvs containing the ibv genes were recovered by reverse genetics as described previously .briefly, each full length cdna was co-transfected with three expression plasmids containing n, p or l gene of ndv strain lasota into mva-t infected hep- cells. three days post-transfection, µl of supernatant of transfected cells were inoculated in - day-old spf embryonated chicken eggs. after three days, haemaglutination test was used to detect infected allantoic fluids collected from eggs. rndvs containing s gene, s gene fused with transmembrane and cytoplasmic tail of ibv s gene, s gene containing cleavage site residues of s gene of ibv fused with transmembrane and cytoplasmic tail of ndv f gene, s gene without cleavage site residues of s gene fused with transmembrane and cytoplasmic tail of ndv f gene, s gene, codon optimized s gene and non-codon optimized s gene were named rndv/s , rndv/s + ibv-s-tm&ct, rndv/s (cs+) + ndv-f-tm&ct, rndv/s (cs−) + ndv-f-tm&ct, rndv/s , rndv/codon optimized-s and rndv/non-codon optimized-s, respectively. the ibv genes were amplified from the rndv constructs by rt-pcr. expression of s , s and s proteins of ibv. confluent monolayers of df- cells were infected at a multiplicity of infection (moi) of . - . with rndv strain lasota, rndv/s , rndv/s + ibv-s-tm&ct, rndv/ s (cs+) + ndv-f-tm&ct, rndv/s (cs−) + ndv-f-tm&ct, rndv/s , rndv/codon optimized-s or rndv/ non-codon optimized-s. df- cells were harvested hours post-infection, lysed and analyzed by western blot. a polyclonal chicken anti-ibv strain mass- was used to detect the expression of s , s and s proteins of ibv. to determine the incorporation of ibv proteins into ndv envelope, rndv, rndv/s , rndv/codon optimized-s and rndv/non-codon optimized-s were inoculated into -day-old embryonated spf chicken eggs. three days after incubation, recombinant viral particles from infected allantoic fluids were partially purified by sucrose density gradient centrifugation and analyzed by western blot analysis. a monoclonal anti-ndv/hn antibody also was used to detect hn protein of ndv in lysates and purified virions by one more western blot analysis. growth characteristics of rndv constructs. in order to determine the growth kinetics of rndvs expressing s , s or s protein of ibv, confluent monolayers of df- cells in -well tissue culture plates were infected at a moi of . with rndv, rndv/s (cs+) + ndv-f-tm&ct, rndv/s and rndv/codon optimized-s and adsorbed for minutes at °c. after adsorption, cells were washed with pbs, then incubated with dmem containing % fbs and % fresh spf chicken egg allantoic fluid at °c in presence of % co . aliquots of µl of supernatant from infected cells were collected and replaced with fresh dmem including fbs at intervals of hours until hours post-infection. the titer of virus in the harvested samples was determined by tcid method in df- cells in -well tissue culture plates. the protective efficacy of rndvs expressing s , s and s protein of ibv against virulent ibv challenge. based on the level of expression of s , s and s proteins of ibv from rndvs, rndv/ s (cs+) + ndv-f-tm&ct, rndv/s , and rndv/codon optimized-s viruses were selected for animal study to evaluate their protective efficacy against virulent ibv challenge. ibv protection experiment . in this study, the protective efficacy of rndvs expressing s , s or s protein of ibv strain mass- were evaluated in -day-old spf chicks. briefly, a total of eighty -day-old chicks were divided into five groups of fifteen each and one group of five. chicks of the first four groups were inoculated with eid of rndv, rndv/s (cs+) + ndv-f-tm&ct, rndv/s and rndv/codon optimized-s strains via oculonasal route. the fifteen chicks of group five and five chicks of group six were inoculated with pbs. three weeks after immunization, all immunized chickens, were challenged with . eid of virulent ibv strain mass- . this challenge virus dose was determined by an experimental chicken infection study. the severity scores of clinical signs of ibv including, nasal discharge, ocular discharge and difficulty in breathing ( = normal, = presence of mild ocular discharge, mild nasal discharge and or sneezing = presence of heavy ocular discharge and or heavy nasal discharge with mild tracheal rales and mouth breathing and or coughing = heavy ocular discharge and heavy nasal discharge with sever tracheal rales and mouth breathing, gasping, dyspnea and or severe scientific reports | ( ) : | doi: . /s - - - respiratory distress) were recorded twice a day for days post-challenge. in order to evaluate protective efficacy of rndvs expressing s , s and s genes of ibv in preventing shedding of virulent ibv in immunized chickens, at day five post-challenge, tracheal swab samples were collected from fifteen birds of each group and placed in . ml serum free dmem with x antibiotics. the swab samples were analyzed for quantification of viral rna using an ibv-n gene-specific rt-qpcr. ibv protection experiment . in this study, the protective efficacy of rndv expressing codon optimized s protein of ibv was evaluated in -week-old spf chickens against the oie recommended dose of virulent ibv challenge . a total of twenty -week-old spf chickens were divided into four groups of five each. five chickens of groups one and two were inoculated with eid of rndv and rndv/codon optimized-s, respectively, via oculanasal route. five chickens of group three were inoculated with recommended doses of a commercial live attenuated mass-type ibv vaccine via oculanasal route and chickens of group four were inoculated with pbs. three weeks after immunization, chickens of all groups were challenged with . eid of virulent ibv strain mass- by the oculonasal route. the severity scores of clinical signs of ibv, described in ibv protection experiment , were recorded for days post-challenge. in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at day post-challenge, tracheal swab samples were collected from twenty chickens and placed in . ml serum free dmem with x antibiotic. the swab samples were analyzed for quantification of viral rna using an ibv-n gene-specific rt-qpcr. ibv protection experiment . in this study, the protective efficacy of rndv expressing codon optimized s protein of ibv was evaluated in -week-old spf chickens against a higher dose of virulent ibv challenge. a total of thirty two -week-old spf chickens were divided into four groups of eight each. eight chickens of group one and two were inoculated with eid of rndv and rndv/codon optimized-s, respectively, via oculanasal route. eight chickens of group three were inoculated with recommended doses of a commercial live attenuated mass-type ibv vaccine via oculanasal route and chickens of group four were inoculated with pbs. three weeks after immunization, chickens of all groups were challenged with . eid of virulent ibv strain mass- by the oculonasal route. the severity scores of clinical signs of ibv, described in ibv protection experiment , were recorded for days post-challenge. at day post-challenge, three chickens from each group were euthanized for tracheal ciliostasis analysis (data not shown). in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, tracheal swab samples were collected from five chickens from each group and placed in . ml serum free dmem with x antibiotic. each fluid was tested for ibv specific lesions on chicken embryo by inoculation ( . ml) of one -day-old embryonated spf chicken egg. the swab samples were also analyzed for quantification of viral rna using an ibv-n gene-specific rt-qpcr. the swab samples collected from two non-vaccinated spf chickens involved in another ibv protection study also were used as control. ibv protection experiment . in this study, the protective efficacy of rndv expressing codon optimized s protein of ibv was evaluated in -day-old spf chicks against virulent ib challenge virus infected by the intraocular route. intraocular route was used, because this route of ibv challenge has been specified by the usda-cfr- . a total of forty five -day-old spf chickens were divided into three groups of ten each and three groups of five each. ten chickens of group one and two were inoculated with eid of rndv, rndv/ codon optimized-s, respectively, via oculanasal route. ten chickens of group three were inoculated with one recommended dose of a commercial live attenuated mass-type ibv vaccine via oculanasal route and chickens of groups four to six were left non-vaccinated. three weeks after immunization, chickens of all groups one to four were challenged with eid of virulent ibv strain mass- by the intraocular route, chickens of group five were challenged with eid of virulent ibv strain mass- by the oculanasal route, and chickens of group six were left non-infected. the severity scores of clinical signs of ibv, described in ibv protection experiment , were recorded for days post-challenge. in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at day post-challenge, tracheal swab samples were collected from all chickens of each group and placed in ml serum free dmem with x antibiotic. each fluid was tested for ibv specific lesions on chicken embryo by inoculation with . ml to each of five -day-old embryonated spf chicken egg. the sample was considered positive for virus shedding, if any of the five embryos showed ibv lesions. we performed all experiments involving virulent ibv in our usda approved biosafety level- and biosafety level- plus facilities following the guidelines and approval of the animal care and use committee (iacuc), university of maryland. the protective efficacy of rndv expressing s protein of ibv against virulent ndv challenge. the protective efficacy of rndv expressing s protein of ibv strain mass- was evaluated against a virulent ndv strain gb texas challenge in our biosafety level (bsl- ) plus facility. briefly, a total of fifteen -day-old chicks were divided into three groups of five each. chicks of two groups were inoculated with eid of rndv and rndv/ibv-codon optimized-s via oculonasal route. the five chickens of group three were inoculated with pbs. three weeks after immunization, blood samples of all birds were collected for ndv antibody response analysis and challenged with one hundred % chicken lethal dose (cld ) of the highly virulent ndv strain gb texas via oculonasal route. the chickens were observed daily for days after challenge for mortality with clinical signs of disease (neurological signs included torticollis, paralysis, and prostration). we performed the experiment involving virulent ndv in our usda approved biosafety level- plus facility following the guidelines and approval of the animal care and use committee (iacuc), university of maryland. scientific reports | ( ) : | doi: . /s - - - serological analysis. the level of antibodies induced against ndv and ibv were evaluated. the serum samples were collected three weeks post-immunization. hemagglutination inhibition (hi) assay using a standard protocol oie was used to assess the level of antibody titer mounted against ndv in chickens immunized by rndvs . the virus neutralization assay according to oie was used to measure the level of neutralizing antibodies mounted against ibv . briefly, serum samples of three birds from the group immunized with rndv expressing codon optimized s protein of ibv and serum samples of three birds from the group immunized with commercial ibv vaccine group were incubated at °c for minutes. one hundred eid of ibv strain mass- was mixed with fold dilutions of antiserum and incubated for hour at °c. one hundred µl of each serum and virus mixture was inoculated into three -day-old embryonated spf chicken eggs. to confirm that at least eid of virus was inoculated into each egg, three eggs were inoculated with µl of pbs containing eid of ibv. three eggs were inoculated with µl of pbs as negative control. three eggs were inoculated with a mixture of eid of ibv and a dilution of : of a randomly selected serum sample collected from a bird immunized with rndv strain lasota as vector control. the eggs were incubated at °c and were observed daily for dead chicken embryos for days post inoculation. the serum titers were calculated according to the method of reed and muench , based on mortality and ibv specific lesions on chicken embryos. quantitative reverse transcription-polymerase chain reaction (rt-qpcr). rna was extracted using trizol reagent (invitrogen) from tracheal swab samples collected from chickens. the first strand cdna was synthesized using thermo scientific revertaid reverse transcriptase (rt). sybr green rt-qpcr was performed using a specific primer pair set: (a) n gene - forward primer: ′ gaccagccgctaacctgaat ′ and (b) n gene - reverse primer: ′ gtcctccgtctgaaaaccgt ′ amplifying nt of n gene of ibv strain mass- . pcrs were performed using a bio-rad cfx cycler. each µl reaction was carried out using µl of cdna, µl of itaq universal sybr green supermix (bio-rad), µl of forward and reverse primers and µl of nuclease free water. forty cycles of pcr at °c for s (denaturation), °c for s (annealing), and °c for s (elongation) followed by melting curve analysis that consisted of °c for s and °c for s. a serial fold dilution of cdna synthesized from extracted rna of allantoic fluid stock of a virulent ibv strain mass- with . eid /ml was used to establish the standard curve. the cdna synthesized from extracted rna of allantoic fluid stock of a virulent ibv strain mass- and the cdna synthesized from extracted rna of swab sample solution were served as positive and negative controls, respectively. melting point analysis was used to confirm the specificity of the test. statistical analysis. data were analyzed among groups by one-way-anova test. the student t-test was used to compare two groups. to avoid bias, all animal experiments were designed as blinded studies. avian infectious bronchitis. version adopted by the world assembly of delegates of the oie in may the long view: years of infectious bronchitis research infectious bronchitis coronavirus avian infectious 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against heterologous avian influenza h n virus challenge a recombinant newcastle disease virus (ndv) expressing infectious laryngotracheitis virus (iltv) surface glycoprotein d protects against highly virulent iltv and ndv challenges in chickens code of federal regulations, standard requirements for ibv vaccines p and m gene junction is the optimal insertion site in newcastle disease virus vaccine vector for foreign gene expression avian paramyxovirus type- as a vaccine vector: identification of a genome location for high level expression of a foreign gene optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses recombinant newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication protection against infectious bronchitis virus by spike ectodomain subunit vaccine high-level expression of a foreign gene from the most -proximal locus of a recombinant newcastle disease virus a simple method of estimating fifty percent endpoints the rule of six, a basic feature for efficient replication of sendai virus defective interfering rna we would like to thank dr. laura crespi sanglas and all our laboratory members for their excellent technical assistance. we also thank girmay gebreluul and johanna lavigne for their animal study assistance. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -n rgqyv authors: schuh, amy j.; amman, brian r.; sealy, tara s.; flietstra, timothy d.; guito, jonathan c.; nichol, stuart t.; towner, jonathan s. title: comparative analysis of serologic cross-reactivity using convalescent sera from filovirus-experimentally infected fruit bats date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: n rgqyv with the exception of reston and bombali viruses, the marburgviruses and ebolaviruses (family filoviridae) cause outbreaks of viral hemorrhagic fever in sub-saharan africa. the egyptian rousette bat (erb) is a natural reservoir host for the marburgviruses and evidence suggests that bats are also natural reservoirs for the ebolaviruses. although the search for the natural reservoirs of the ebolaviruses has largely involved serosurveillance of the bat population, there are no validated serological assays to screen bat sera for ebolavirus-specific igg antibodies. here, we generate filovirus-specific antisera by prime-boost immunization of groups of captive erbs with all seven known culturable filoviruses. after validating a system of filovirus-specific indirect elisas utilizing infectious-based virus antigens for detection of virus-specific igg antibodies from bat sera, we assess the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigens. this data is then used to generate a filovirus antibody fingerprint that can predict which of the filovirus species in the system is most antigenically similar to the species responsible for past infection. our filovirus igg indirect elisa system will be a critical tool for identifying bat species with high ebolavirus seroprevalence rates to target for longitudinal studies aimed at establishing natural reservoir host-ebolavirus relationships. unlike the spillover events leading to marburgvirus disease, those for ebolavirus disease have been difficult to associate with a specific environment, such as caves. furthermore, the evidence linking ebolavirus spillover to a particular species of bat is largely circumstantial. for example, the and index cases of sudan virus disease both worked in a cotton factory in sudan, where a retrospective investigation eight to nine months after the initial outbreak identified a large roof colony of trevor's free-tailed bats (mops trevori) directly over the working area of the index case . the putative index case of the ebola virus disease outbreak in the kasaï-occidental province of the drc was said to have purchased bats for consumption following a reported annual migration of hammer-headed bats (hypsignathus monstrosus) and franquet's epauletted fruit bats (epomops franquetti) . lastly, the presumed index case of the ebola virus disease outbreak that started in guinea was reported to have played in a tree hollow, where dna traces of mops condylurus were later identified . the majority of research directed towards the identification of the natural reservoir hosts of the ebolaviruses has consisted of cross-sectional surveillance of the sub-saharan african and asian bat population for evidence of active and past ebolavirus infection. the predominance of cross-sectional studies can be attributed to the large number of bat species residing within the geographic range of ebolavirus circulation that require investigation, as well as the challenges associated with obtaining diagnostic specimens from a mammal that is difficult to capture due to its elusive nocturnal nature and ability to fly. collectively, ebola virus rna has been detected in three bat species captured in the republic of the congo and gabon (hypsignathus monstrosus, epomops franquetti and myonycteris torquata) and reston virus rna has been reportedly detected in four bat species captured in the philippines (chaerephon plicatus, cynopterus brachyotis, miniopterus australis and miniopterus schreibersii) . yet, infectious virus was not isolated from any of these species , , indicating that they are either dead-end virus hosts, had cleared virus infection prior to sampling, or that current filovirus isolation techniques lack the sensitivity to recover infectious virus from specimens with low viral loads. serological reactivity of bat sera with ebolavirus antigens using indirect elisas, indirect fluorescent tests, bead-based multiplex assays and/or western blots has been reported in bats representing at least species throughout sub-saharan africa and asia [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, interpretation of this data has been exceedingly difficult due to multiple reasons that include: ( ) the absence of a panel of positive and negative bat sera for initial serological assay validation and continuing quality control, ( ) the use of various uncharacterized recombinant filovirus antigens, and ( ) the application of different statistical methods for establishing cutoff values for seropositivity. nonetheless, ebolavirus serosurveillance offers several distinct advantages compared to surveillance for active virus infection including a longer diagnostic window (i.e., length of time a diagnostic test can detect evidence of infection), no knowledge of virus-tissue tropism and no requirement for destructive sampling. although erbs are natural reservoir hosts of the marburgviruses only, they develop a robust virus-specific igg antibody response following experimental inoculation with the ebolaviruses , . in this study, we generate high quality, virus-specific antisera by prime-boost immunization of captive erbs with marburg virus, ravn virus, ebola virus, bundibugyo virus, taï forest virus, sudan virus or reston virus. these virus-specific antisera, along with a set of filovirus-naïve bat sera, are then used to validate a series of indirect elisas that utilize non-recombinant, infectious-based filovirus antigens for the detection of virus-specific igg antibodies from bats. we then assess the level of serological cross-reactivity between each virus-specific antiserum and viral antigen and use this information to generate a filovirus antibody fingerprint that is able to predict which of the six filovirus species in our system is most antigenically similar to the species responsible for past infection. filovirus-specific antisera generated from erbs. the bats used to generate the filovirus-specific antisera in this study originated from an erb breeding colony . a total of erbs were divided into seven groups (table ) (table ) , prepared in . ml of sterile dulbecco's modified eagle's medium (thermo fisher scientific inc., waltham, ma, usa). after eight weeks, the bat groups were subcutaneously challenged with log tcid of homologous virus. the bats were euthanized two to four weeks thereafter by cardiac exsanguination under isoflurane anesthesia, followed by an overdose of isoflurane. whole blood collected into serum separator tubes (fisher scientific, grand island, ny, usa) at euthanasia was allowed to clot and then centrifuged at , g for min. the sera were transferred into polypropylene cryoelite wheaton vials (dwk life sciences, millville, nj, usa), and then temporarily stored under liquid nitrogen vapors in the biosafety level four laboratory (bsl- ) until they were double bagged, passed through a dunk tank containing % micro-chem plus (national chemical www.nature.com/scientificreports www.nature.com/scientificreports/ laboratories incorporated, inc., philadelphia, pa, usa) and transported to the gamma-cell irradiator. the doublebagged serum aliquots were sandwiched between layers of dry ice and then exposed to × rads of gamma-cell irradiation using a cobalt- source. filovirus-naïve sera collected from erbs. sera obtained from filovirus-naïve bats from the breeding colony and negative control bats from previous experimental studies , [ ] [ ] [ ] were used as filovirus-naïve sera. these sera were collected and processed in the same manner as outlined above. husbandry. all bats were group-housed according to filovirus inoculum in a climate controlled bsl- animal area, with a h day/ h night cycle. bats received a quantity of fresh fruit, supplemented with protein/vitamin powder, equal to their body mass daily and water ad libitum. (table ) and uninfected control antigen lysate for the filovirus igg indirect elisas were prepared as previously described [ ] [ ] [ ] . briefly, roller bottles of vero e cells were inoculated with virus and the cultures were harvested when ≥ % of cells exhibited evidence of infection by immunofluorescence assay. filovirus antigen lysates were prepared from the cultures by detergent basic buffer extraction of infected cells. uninfected control antigen lysate was generated in the absence of virus, but otherwise prepared in the exact same manner. filovirus igg indirect elisas. each filovirus-specific antiserum (n = ) and filovirus-naïve serum (n = ) was tested for reactivity against each filovirus antigen lysate (n = ) and uninfected control antigen lysate (n = ; supplementary fig. s ). wells of -well elisa plates were coated ( µl) with the dilution of filovirus antigen lysate (diluent: pbs containing % thimerosal) that was found to result in optimal reactivity with sera pooled from each of the filovirus-infected bat groups ( : for the ebola, bundibugyo, taï forest and reston virus lysates, and : for the marburg, ravn and sudan virus lysates) and corresponding wells were coated with an equivalent dilution of uninfected control lysate ( : or : ). after incubation overnight at °c, the plates were washed with pbs containing . % tween- (pbs-t) and µl of serum diluent (pbs containing % skim milk and . % tween- ) was added to each well of the plate. after min, µl of a : dilution of gamma-irradiated bat serum pre-diluted in masterplate diluent (pbs containing % skim milk powder, . % tween- and % thimerosal) was added to the first well of the plate and four-fold serial dilutions were www.nature.com/scientificreports www.nature.com/scientificreports/ performed. final bat serum concentrations were : , : , : and : . following a hr incubation at °c, the plates were washed with pbs-t and µl of a : , dilution of goat anti-bat igg conjugated to horseradish peroxidase (bethyl laboratories, montgomery, tx, usa, cat#: a - p, lot#: a - p- ) in serum diluent was added to the plates. the manufacturer notes that this antibody reacts specifically with bat igg and with light chains common to other immunoglobulins. after incubation for hr at °c, the plates were washed with pbs-t, µl of the two-component abts peroxidase system (kpl, gaithersburg, md, usa) was added, and the plates were allowed to incubate for min at °c. the plates were then read on a microplate spectrophotometer set at nm. the optical density (od) values of each four-fold serial dilution were visually inspected to ensure linearity. to negate non-specific background reactivity, adjusted od values were calculated by subtracting the ods at each four-fold dilution of wells coated with uninfected control antigen lysate from ods at corresponding wells coated with filovirus antigen lysate. the adjusted sum od value was determined by summing the adjusted od values at each four-fold serial dilution. the average adjusted sum od of duplicate runs was reported. notably, performing each indirect elisa (seven filovirus antigen lysates and two dilutions of uninfected control antigen lysate) in duplicate required only µl of serum. influence of sex and age on the reactivity of antisera with homologous antigen. the breeding colony was founded from ugandan wild-caught, filovirus-naïve adult (≥ yr) erbs . for founder bats, age in years was determined by subtracting the date of capture from the date of euthanasia and then adding one year. for bats born in captivity, age in years was determined by subtracting the date of birth from the date of euthanasia. the bats were then classified into three age categories: < yr, ≥ yr-≤ yrs and > yrs. a two-way anova was performed to determine if serological reactivity of bat sera with homologous antigen was significantly influenced (p < . ) by sex, age category, or the interaction between sex and age category (spss statistics , ibm software, armonk, ny, usa). for each filovirus-specific igg indirect elisa, quadratic discriminant analysis was used to determine a cutoff value for seropositivity by considering: ( ) the reactivity of the filovirus-specific antisera group with homologous antigen, ( ) the reactivity of the filovirus-naïve sera group with that same antigen, and ( ) the prior probability of the filovirus-specific antisera group (vba for microsoft access , microsoft office professional plus , redmond, wa, usa). the prior probability of each filovirus-specific antisera group was set at . . this value represents the overall population seroprevalence of marburgvirus in its natural reservoir host, the erb . we assume that the seroprevalence of the ebolaviruses in their respective natural reservoir bat hosts approximates this value. however, the prior probability has relatively little influence on determining the magnitude of the cutoff value for seropositivity. the cutoff value for each filovirus-specific indirect elisa represents the value where the posterior probabilities for the reactivity of the filovirus-specific antisera with homologous antigen and the reactivity of filovirus-naïve sera with this same antigen are equal. sensitivity and specificity of the indirect elisas. for each indirect elisa at its specified seropositivity cutoff value, the filovirus-specific antiserum and filovirus-naïve serum samples were classified as true positive, false positive, false negative or true negative. the sensitivity of each assay was then calculated by dividing the number of individuals with a positive test result by the number of individuals with a history of past infection (experimentally inoculated with that particular virus), and the specificity of each assay was calculated by dividing the number of individuals without a history of past infection (filovirus-naïve group) by the number of individuals with a negative test result. level of serological cross-reactivity. the level of serological cross-reactivity between each group of filovirus-specific antisera and each heterologous virus antigen was calculated by dividing the number of antisera positive for reactivity with a virus antigen by the total number of antisera tested against that antigen (e.g., number of marburg virus antisera positive for reactivity with ravn virus antigen/number of marburg virus antisera tested for reactivity against ravn antigen), and was expressed as a percentage. after the nucleotide sequences of the filovirus isolates used to inoculate the bats in this study were retrieved from genbank, the coding regions were translated into amino acids and aligned using the muscle algorithm (geneious . . , biomatters limited, auckland, new zealand). percent identity values were then calculated from pairwise amino acid comparisons generated from the virus protein alignment. a pearson's product-moment correlation was performed to assess the relationship between percent serological cross-reactivity and percent amino acid identity (spss statistics , ibm software, armonk, ny, usa). antibody fingerprint analysis. quadratic discriminant analysis was used to predict which one of the seven filoviruses in the system was most antigenically similar to the filovirus responsible for past infection by considering the relative reactivity of each filovirus-specific antiserum and filovirus-naïve serum with each filovirus antigen, as well as their covariance (visual basic for microsoft access). first, prior probabilities were calculated for the eight classes used in the analysis (seven filovirus antigen classes and one negative class). second, classification was performed by calculating posterior probabilities for the inclusion of an antisera/sera sample in each class and then assigning the sample to the class with the highest probability. evaluating the performance and discriminatory ability of the filovirus igg indirect elisa system. to evaluate the ability of our system comprising seven filovirus-specific indirect elisas to predict the filovirus species most antigenically similar to the species responsible for past infection, we tested seven marburg virus convalescent serum or whole blood samples collected from experimentally inoculated erbs. five of these samples www.nature.com/scientificreports www.nature.com/scientificreports/ were collected four weeks post primary marburg virus inoculation , , while two of the samples were collected at and weeks post primary inoculation following a "natural" boost (i.e., marburg virus-specific antibody levels waned in these bats and then increased following contact with infectious cagemates) . serological reactivity with homologous filovirus antigen is not influenced by age or sex. a total of erbs were used in this study that was performed over a two-year time span. the total number of bats dedicated to this study, as well as the size and composition of each group was dependent on the reproductive capacity of the erb breeding colony, as well as the characteristics and number of bats needed for other experimental studies. the bats were divided into seven groups, with group sizes ranging from - individuals (table ) . two groups of bats had approximately equal sex ratios (ravn and taï forest), while two groups were comprised of mostly female individuals (bundibugyo and sudan), and the remaining five groups were comprised of all male (reston) or all female (marburg and ebola) individuals. two groups of bats were comprised solely of individuals < yr of age (bundibugyo and taï forest), while the majority of individuals in three groups were ≥ -≤ yrs of age (marburg, ebola and reston) and the majority of individuals in two groups were > yrs of age (ravn and sudan). despite the observed variability in group composition, a two-way anova revealed that serological reactivity with homologous filovirus antigen was not significantly (p > . ) influenced by sex (f( , ) = . ), age (f( , ) = . ), or the interaction between sex and age (f( , ) = . ). filovirus-specific indirect elisas are highly sensitive and specific. with the exception of the igg antibody indirect elisa using marburg virus antigen ( % sensitivity and % specificity; fig. a and table ), all of the assays exhibited % sensitivity and specificity ( fig. and table ). the mean group adjusted sum od values ranged from . (± . sd) for the igg antibody indirect elisa using marburg virus antigen to . (± . sd) for the igg antibody indirect elisa using ebola virus antigen. strong positive correlation between filovirus serological cross-reactivity and filovirus protein amino acid identity. figure and table show the level of serological cross-reactivity between each group of bat filovirus-specific antisera and the six heterologous filovirus antigens. the consistent magnitudes of serological reactivity of the individual bat antisera across all six heterologous filovirus antigens highlights the robust performance of our filovirus igg indirect elisa system. strong serological cross-reactivity was observed between marburg virus antisera and ravn virus antigen ( %), and ravn virus antisera and marburg virus antigen ( %) (fig. a,b) . (fig. e-g) . very limited cross-reactivity was observed between marburgvirus antisera and ebolavirus antigen ( %: majority of antisera-antigen combinations; %: marburg virus antisera versus taï forest virus antigen, ravn virus antisera versus bundibugyo and taï forest virus antigens; %: ravn virus antisera versus reston virus antigen), and ebolavirus antisera tested against marburgvirus antigen ( . %: sudan virus antisera versus marburg virus antigen; . % sudan virus antisera versus ravn virus antigen) (fig. ) . a pearson's product-moment correlation revealed that there was a statistically significant, strong positive correlation between percent filovirus serological cross-reactivity and percent filovirus amino acid identity (r = . , n = , p < . ). filovirus serological cross-reactivity dataset used to develop an antibody fingerprint classification system. we used the adjusted sum od data (each serum tested against each antigen) generated from bats intentionally infected with each of the seven known culturable filoviruses to develop a classification system (fingerprint) to predict which filovirus elicited the antibody response. supplementary table s shows posterior probability support values for classification of each filovirus-specific antiserum or filovirus-naïve serum sample into eight pre-defined classes (seven filovirus antigen classes and one negative class), with probabilities of each row in the table summing to . . each sample was assigned to the class with the highest posterior probability (i.e., highest degree of certainty). overall, all of the samples ( / ) were correctly classified, with posterior probabilities in support of the true class (i.e., filovirus used to prime-boost each bat) ranging from . to . . this indicates that this system, using seven independent filovirus igg indirect elisas, has the ability to establish an antibody fingerprint from filovirus convalescent bat sera that can be used to predict which of the filovirus species in the system is the most antigenically similar to the species responsible for past infection (see supplementary note for an example of how the antibody fingerprinting was performed in this study). to further assess the performance and predictive ability of our filovirus igg indirect elisa system, we tested five marburg virus convalescent sera or whole blood samples collected four weeks post primary marburg virus experimental inoculation and two whole blood samples collected at and weeks post primary experimental inoculation following a "natural" boost (i.e., marburg virus-specific antibody levels waned and then increased following contact with infectious cagemates). as expected, marburgvirus-specific igg antibody levels were markedly lower in these seven convalescent serum and whole blood samples compared to marburgvirus-specific igg antibody levels in the serum samples from the prime-boosted bats in this study (fig. a) . however, all samples collected after primary marburg virus inoculation or following a "natural" boost were classified as marburgvirus igg antibody positive. the antibody fingerprint analysis predicted that three of these bats were previously infected with marburg virus (sample ids , and with marburg posterior probability values of . , . , and . , respectively) and four were previously infected with ravn virus (sample ids , , and with ravn posterior probability values of . , . , . and . , respectively; fig. b ). www.nature.com/scientificreports www.nature.com/scientificreports/ in this study, we generated filovirus-specific antisera from prime-boosted erbs and collected filovirus-naïve sera to validate and characterize an indirect elisa system that utilizes non-recombinant, infectious-based filovirus antigens for the detection of virus-specific igg antibodies from bats. initial validation of the system revealed that the individual filovirus-specific igg indirect elisas exhibited high sensitivity ( - %) and specificity ( %). similar to previous studies using human sera, we observed limited to no serological igg cross-reactivity between the filovirus genera ( - %) [ ] [ ] [ ] , varying levels of serological igg cross-reactivity between the ebolavirus species table for statistics on the reactivity of each filovirus antigen lysate with homologous antisera and filovirus-naïve sera, as well as for the sensitivity and specificity of each of the filovirus igg indirect elisa. www.nature.com/scientificreports www.nature.com/scientificreports/ ( - %) , and strong serological igg cross-reactivity within the species marburg marburgvirus ( %). the low level of serological igg cross-reactivity between some of the ebolavirus species underscores the necessity of performing all assays within the system to avoid false negative results. although significant levels of serological igg cross-reactivity were observed between the prime-boost filovirus-specific antisera and some of the filovirus antigens, when the overall covariance of the seven-individual indirect elisas in the system were considered, we were able to predict the filovirus species responsible for past infection % of the time using as little as μl of sera (each serum was tested against each antigen in duplicate). further evaluation of the performance and predictive ability of our system using seven marburg virus convalescent serum or whole blood samples collected after primary experimental infection with marburg virus (or primary experimental infection with marburg virus plus a "natural" boost) confirmed that the system was able to predict the filovirus species most likely responsible for past infection. as expected, the filovirus igg indirect elisa system was not able to correctly predict whether past infection was due to marburg virus or ravn virus % of the time. this finding supports the current classification of marburg and ravn viruses into a single virus species, marburg marburgvirus . the robustness and discriminatory power of our system results from its underlying components and various control points. we employed infectious-based filovirus antigens, rather than recombinant virus protein antigens, as a strategy to increase both the sensitivity (i.e., ability of the filovirus igg indirect elisa system to correctly identify those with past exposure to a filovirus as filovirus igg antibody positive) and specificity (i.e., ability of the filovirus igg indirect elisa system to correctly identify those with no past exposure to a filovirus as filovirus igg antibody negative) of the system. although recombinant filovirus antigens can be generated in large quantities and do not require a bsl- laboratory for production, the use of single recombinant virus proteins for antibody detection can lead to false negative results if an individual's antibody repertoire is not directed against the particular recombinant virus protein that was employed , [ ] [ ] [ ] [ ] [ ] [ ] . alternatively, false positive serological results can occur when recombinant virus antigens unknowingly share similar epitopes with other virus antigens for which the study population possesses antibodies against , . false positive results can also arise from failing to account for non-specific serological reactivity resulting from the presence of non-virus contaminants in an antigen preparation. here, we negated non-specific serological reactivity to vero e cell components by subtracting the reactivity of wells coated with uninfected antigen lysate from the reactivity of corresponding wells coated with filovirus antigen lysate. while the majority of previously published ebolavirus serosurveys of bats used recombinant filovirus antigens generated in bacterial expression systems, not all of the studies implemented procedures to negate non-specific reactivity between residual bacterial epitopes in the antigen preparation and antibacterial antibodies in bat sera , . like the majority of published ebolavirus serosurveys of bats, we performed serial serum dilutions to assess for linearity and specificity , , , [ ] [ ] [ ] [ ] . most importantly, we included pooled filovirus-specific bat antisera as a positive control and pooled filovirus-naïve bat sera as a negative control in every run to ensure that our filovirus igg indirect elisa system continually performed as expected. using filovirus-specific antisera collected from bats that were prime-boosted at the same time not only allowed us to thoroughly examine the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigen, but also provided the opportunity to use this knowledge to evaluate previously published ebolavirus serosurveys of bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . four out of the previously published ebolavirus serosurveys of bats used ebola virus antigen only to detect ebolavirus igg antibodies , [ ] [ ] [ ] and another four of these serosurveys used ebola and reston antigens only to detect ebolavirus igg antibodies , [ ] [ ] [ ] . based on the low serological cross-reactivity between ebola virus antigen and sudan virus antisera ( . %) and reston virus antigen and sudan virus antisera ( . %) observed in our study, we predict that the serosurveys using ebola virus antigen or ebola and reston virus antigens only would have missed the opportunity to detect bats previously infected with sudan virus. likewise, due to the high level of serological cross reactivity between ebolavirus-specific antisera and heterologous ebolavirus antigen that we observed in our study, it is possible that the serological reactivity of bat sera with ebola or reston virus antigens in the previous serosurveys was due to past infection with one of the other known ebolaviruses or an undescribed ebolavirus. although we successfully used quadratic discriminant analysis to predict which of the filovirus species in the system was most antigenically similar to the species responsible for past infection, only nucleotide sequence data can be used to ascertain infection with a particular filovirus. furthermore, incrimination of an ebolavirus-natural reservoir host relationship requires consistent detection of both active (rna and virus isolation from tissues/bodily fluids suggestive of a transmission mechanism) and past infection with a particular ebolavirus through space and time. www.nature.com/scientificreports www.nature.com/scientificreports/ while our filovirus igg indirect elisa system enables the robust detection of virus-specific igg antibodies from bats and was able to predict the filovirus species most likely responsible for past infection, it does have some limitations. first, this system was validated using virus-specific antisera from prime-boosted bats and sera from table for statistics on the cross-reactivity between each filovirus-specific bat antisera and heterologous filovirus antigen. www.nature.com/scientificreports www.nature.com/scientificreports/ filovirus-naïve bats. although all seven of the marburg virus convalescent whole blood and serum samples collected from erbs after primary experimental marburg virus inoculation or following a "natural" boost were marburgvirus igg antibody positive, these samples had markedly lower marburgvirus-specific igg antibody levels compared to samples collected from the bats in this study that were prime-boosted with one of seven filoviruses. this suggests that the sensitivity of the individual filovirus igg indirect elisas may be lower than what was reported here ( - %) using filovirus-specific antisera from prime-boosted bats and filovirus naïve bat sera. however, statistical determination of seropositivity cutoff values for the indirect elisas were largely driven by the variance surrounding the mean reactivity of the filovirus-naïve sera with each of the virus antigens. it is likely that circulation and long-term maintenance of the ebolaviruses in their natural reservoir hosts leads to multiple virus exposures throughout the lifetime of an individual, resulting in boosting of virus-specific igg antibody levels. "natural" boosting of virus-specific igg antibody levels in erbs that had been previously infected with marburg virus was observed during a recent experimental transmission study shortly after documenting virus infection and seroconversion in naïve contact bats . others have reported periodic boosting of rabies virus neutralizing antibody titers in a wild colony of big brown bats (eptesicus fuscus) . second, we used a goat anti-bat igg conjugated to horseradish peroxidase as the secondary antibody in our system. this antibody was generated using www.nature.com/scientificreports www.nature.com/scientificreports/ sera from erbs, as well as sera from nine other bat species comprising four chiropteran families (manufacturer product datasheet). while this antibody has been confirmed to react with sera from these chiropteran species and has been used with success to detect igg antibodies in sera from > bat species comprising seven chiropteran families [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it is possible that its reactivity with igg antibodies from divergent bat species might diminish. if this is the case, the concentration of secondary antibody may need to be optimized prior to testing a new species of bat or an alternative secondary antibody may need to be used. third, our filovirus igg indirect elisa system includes seven virus antigens that represent all of the reported culturable filoviruses. given the recent discoveries of filovirus rna from divergent bat families in geographically distant locations ( ), we expect that there are a number of undiscovered ebolaviruses that continue to circulate in nature. however, we believe that the genetic diversity of ebolavirus antigens (up to . %) included in our system will allow for the detection of past infection with a novel ebolavirus, and prediction of the ebolavirus species that is most antigenically similar to the species responsible for past infection. we intend to use our filovirus igg indirect elisa system to screen archived and incoming specimens from cross-sectional serosurveys of the bat population of sub-saharan africa and asia for evidence of past filovirus infection. if other specimen types exist for bat species identified as filovirus seropositive, they will be tested for evidence of active infection using virus-specific qrt-pcr, pan-filovirus rt-pcr and/or virus isolation techniques. bat species positive for evidence of active filovirus infection or species exhibiting a seroprevalence equivalent to or larger than that of marburgvirus in erbs (~ % total population seroprevalence) will be targeted for longitudinal studies aimed at collecting a wide range of specimens for evidence of active and past ebolavirus infection. while most of the virus-specific antisera generated in this study will be pooled to use as positive controls in our indirect filovirus elisa system, some will be reserved for investigations aimed at determining the mechanisms by which bats clear and control filovirus infections through virus neutralization and cross-neutralization assays, as well as epitope mapping studies. any remaining filovirus-specific bat antisera may be used to develop a pan-ebolavirus indirect elisa that utilizes a mixture of non-recombinant, infectious based antigens from culturable ebolaviruses, as well as validate the performance of indirect elisas that use recombinant filovirus proteins to detect filovirus antibodies. ecology of filoviruses taxonomy of the order mononegavirales: second update epidemiology of ebola (subtype reston) virus in the philippines the discovery of bombali virus adds further support for bats as hosts of ebolaviruses field aspects of the marburg virus outbreak: . primate supply marburg-virus disease in kenya report of a who/international study team. ebola haemorrhagic fever in sudan report of an international commission. ebola haemorrhagic fever in zaire human infection due to ebola virus, subtype cote d'ivoire: clinical and biologic presentation 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corresponding author upon request. we thank the diagnostics team from the viral special pathogens branch at the centers for disease control and prevention (cdc) for preparing the filovirus antigen lysates used in this study. we would also like to thank peter eworonsky, lester slough and eddie jackson from the comparative medicine branch at the cdc for providing care to the bats during this study. this study was partially funded by dtra grant hdtra- - - , subaward s- - . the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. supplementary information accompanies this paper at https://doi.org/ . /s - - -z. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -dvfmpm authors: ai, hannan; ai, yuncan; meng, fanmei title: genomelandscaper: landscape analysis of genome-fingerprints maps assessing chromosome architecture date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dvfmpm assessing correctness of an assembled chromosome architecture is a central challenge. we create a geometric analysis method (called genomelandscaper) to conduct landscape analysis of genome-fingerprints maps (gfm), trace large-scale repetitive regions, and assess their impacts on the global architectures of assembled chromosomes. we develop an alignment-free method for phylogenetics analysis. the human y chromosomes (grch.chry, huref.chry and yh.chry) are analysed as a proof-of-concept study. we construct a galaxy of genome-fingerprints maps (ggfm) for them, and a landscape compatibility among relatives is observed. but a long sharp straight line on the ggfm breaks such a landscape compatibility, distinguishing grch p .chry (and throughout grch p .chry) from grch p .chry, huref.chry and yh.chry. we delete a . -mbp target segment to rescue the landscape compatibility, matching the antecedent grch p .chry. we re-locate it into the modelled centromeric and pericentromeric region of grch p .chry, matching a gap placeholder of grch p .chry. we decompose it into sub-constituents (such as bacs, interspersed repeats, and tandem repeats) and trace their homologues by phylogenetics analysis. we elucidate that most examined tandem repeats are of reasonable quality, but the bac-sized repeats, u c ( . kbp) and u c ( . kbp), are likely over-repeated. these results offer unique insights into the centromeric and pericentromeric regions of the human y chromosomes. centromeres in the human reference genome becomes an urgent interest, which intrigues the field to create new methods. this study addresses the above critical issues and aim to ( ) display discrepancies among multiple assemblies of a chromosome, ( ) detect misassembled segments of its chromosome architecture, ( ) delete the misassembled segments, and ( ) decompose the segment into sub-constituents and trace their homologues that contributed to the misassembling. as such, one can detect misassembling and determine to replace a misassembled artefact or maintain a true intrinsic segment. our hypotheses are: ( ) there should be a landscape compatibility among relatives under comparison, and ( ) the more the relatives were compared at a large scale, the easier the misassembled architectures could be detected in a big-picture view, thus overcoming the aforementioned central challenge. based on our genomefingerprinter algorithm , here we establish a method (called genomelandscaper) to construct a galaxy of genome-fingerprints maps (ggfm), which comprises a set of genome-fingerprints maps (gfm) that are simultaneously constructed for a set of chromosomes under comparison. hence we can compare a set of chromosomes in a big-picture view at a large scale. to compare a number of large and divergent genomes, we also develop an alignment-free method for phylogenetics analysis. as a proof-of-concept study, we create a ggfm for the human y chromosomes (grch.chry , , huref.chry [ ] [ ] [ ] and yh.chry ) and conduct assessments on their global architectures. this study establishes a method to retrospectively assess the correctness of assembled chromosome architectures by means of evaluating the quality of their multiple assemblies, which is crucial to assess, re-construct and use complex genomes. principles of the genomelandscaper method. to conduct landscape analysis of genome-fingerprints maps (gfm) and retrospectively assess the global architectures of assembled chromosomes, we establish the genomelandscaper method based on our genomefingerprinter algorithm . the steps of working flow with key features are illustrated by using the primates mitochondria genomes (< . kbp, kilos of base pairs) including homo sapiens, pan troglodytes, gorilla gorilla, macaca fascicularis and macaca mulatta (fig. ) . our genomefingerprinter algorithm circularises a linear sequence (fig. a) to avoid interference from arbitrary cut-off sites of the sequence . it defines a set of functions for the three-dimensional coordinates (xn,yn, zn) (fig. b) , where n is the order number of a base (a, t or u, g and c) of a sequence (in length of n) : each component of the coordinates is a function of distribution difference between the assigned two base-types, reflecting the distribution bias between them. we speculate that a certain continuous distribution bias along with a given sequence should yield a line along an axis. a line along the x axis illustrates a continuous distribution bias of purine (a plus g) over pyridine (c plus t); a line along the y axis indicates a continuous distribution bias of amino-nucleotides (a plus c) over keto-nucleotides(g plus t); and a line along the z axis demonstrates a continuous distribution bias of weak hydrogen bonds (a plus t) over strong hydrogen bonds (c plus g). using the three-dimensional coordinates (fig. b) , we can transform a genome into a genome-fingerprints map (gfm) by trajectory-plotting (fig. d) , contour-plotting (fig. e ) and scatter-plotting (fig. f ), respectively. . d ) and the set of d-maps (fig. d, e) can represent the given genome, respectively. we operate map-to-map comparison, instead of alignment-based base-to-base comparison. this alignment-free method relieves computation burdens and allows us to compare a number of large genomes and divergent genomes in a big-picture view at a large scale. feature . calculation of a weighted distance matrix. the geometric centre (x , y , z ) of a gfm solely represents the given genome and is regarded as a point in three-dimensional space, thus a weighted euclidean distance between two points, the i th (x i, yi, z i) and the j th (x j, yj, z j), is calculated by the formula: where σ x , σ y and σ z is the standard deviation for each axis; and m is the number of genomes to be compared, which determines a m × m symmetric matrix whose elements are d (i,j) . we weight the geometrical mean of radiuses of the circularised d-maps (fig. d) to discriminate a pair of overlapped d-maps (sharing a geometric centre with different shapes). to consider the effect of angles on the eigenvectors (c k i , c k j ) between two genomes, we integrate other factors into the weighted euclidean distance matrix, referring to : hence a final m × m symmetric matrix, whose elements are d (i,j) , is created (fig. g) . feature . construction of a phylogenetic tree and a phylogentic network. the weighted distance matrix (fig. g) can be transformed into a phylogenetic tree (fig. h ) and a phylogenetic network (fig. i ) by conventional software [ ] [ ] [ ] [ ] [ ] . we construct a phylogenetic tree (fig. h , left) using fastme software based on our weighted distance matrix. we construct a bootstrap consensus tree (fig. h , right) using the mega package under the minimum-evolution (me) model. two un-rooted trees are approximate to one another, indicating that our alignment-free and bootstrap-free method has an adequate approximation (fig. h) . such an approximation has an advantage in analysing a number of large genomes (e.g., mbp, millions of base pairs) and divergent sequences (e.g., variations in size, gap, and divergence). if necessary, our method can be applied to analyse each set of disturbed sequences that are created by traditional bootstrap approaches [ ] [ ] [ ] [ ] [ ] ; but the traditional bootstrap approaches may not work when disturbing large genomes and divergent sequences due to algorithmic and computational constraints. frameworks of using the genomelandscaper method. the frameworks of using the genomelandscaper method are exemplified by a proof-of-concept study on the human y chromosomes (grch.chry , , huref. chry - and yh.chry ). we develop an itinerary throughout the next sections: ( ) constructing a galaxy of genome-fingerprints maps, ( ) detecting discrepancies among multiple assemblies, ( ) deleting the misassembled chromosome architecture, ( ) re-locating the deleted target segment, ( ) tracing bacs of the deleted target segment, ( ) tracing interspersed repeats of the deleted target segment, and ( ) tracing tandem repeats of the deleted target segment. notably, instead of intending to conduct the straightforward de novo assembling, our goal is to provide a novel method to retrospectively assess the correctness of assembled chromosome architectures by means of evaluating the quality of their multiple assemblies, so that one can detect misassembling and determine to replace a misassembled segment or maintain a true intrinsic segment. constructing a galaxy of genome-fingerprints maps. a compact ggfm contains one d-trajectory map (x-y-z) and three d-trajectory maps (x-y, x-z, y-z) for at least one genome (fig. a) . alternatively, we create a d-contour map (x-y) ( fig. b-f ) for each genome to demonstrate its high-resolution genome fingerprints. huref.chry ( . mbp) produces . gb (gigabytes) of coordinates. we took hours (fig. a ) and hours (fig. b) to construct two figures for one genome huref.chry (fig. a,b) , respectively, on a high performance workstation (dell precision t ) with gb (gigabytes) physical memory installed with origin pro . ( -bit) software. such two forms (fig. a,b) are expensive to construct, which hampers their practical applications to a set of genomes under comparison. to create another alternative form (fig. ) , we separately construct the d-trajectory map (x-y-z) and d-trajectory maps (x-y, x-z, y-z, x-length, y-length, z-length) and combine them (fig. ) . we calculate six genomes (in total . mbp) of the human y chromosomes (huref.chry, yh.chry, grch p . chry, grch p .chry, grch p .chry and grch p .chry) to create the three-dimensional coordinates (in detecting discrepancies among the six assemblies. we compare the human y chromosomes on the ggfm (figs and ). huref.chry is distinct due to its incomplete sequence at q-arm terminus. yh.chry and grch p .chry share an architecture, as confirmed by their similar d-contour maps (fig. c,d) . grch . comparison of global architecture among the human y chromosomes. an alternative form of ggfm is presented. huref.chry (red) is distinct due to its incompleteness of q-arm terminus. yh.chry (green) is almost overlapped by grch p .chry (blue). grch p .chry (purple) and grch p .chry (cyan) are completely overlapped by grch p .chry (yellow), demonstrating they are identical. compared to grch p .chry (blue), the descendents grch p .chry (purple), grch p .chry (cyan) and grch p . chry (yellow) have an extra turning-changed long sharp straight line (marked by a black arrow), respectively. chry has multiple releases (e.g., grch p .chry, grch p .chry and grch p .chry), but they are factually identical (fig. e,f) . the d-contour maps indicate that grch p .chry is distinct from grch p .chry and grch p .chry ( fig. d-f ). there is an extra turning-changed long sharp straight line on the ggfm (fig. ) , which can distinguish grch p .chry from grch p .chry (and throughout grch p .chry). hence we intuitively display and detect the incredible discrepancies of chromosome architecture among huref.chry, yh.chry, grch p .chry, grch p .chry, grch p .chry and grch p .chry (fig. ) . these findings validate that grch.chry and yh.chry have a better quality of chromosome architecture over huref.chry, and suggest that grch p .chry or grch p .chry is likely misassembled (fig. ). deleting the misassembled chromosome architecture. to evaluate which one is likely misassembled, we need to understand the mechanism of how the turning-changed long sharp straight line occurs between grch p .chry and grch p .chry (fig. ) . the logic is simple with no biases: if the antecedent grch p .chry were (and should have been over time) correct, then the descendent grch p .chry with a newly-introduced extra segment should be incorrect; and vice versa. to simplify the logic of validations, here we deliberately hypothesise that the turning-changed long sharp straight line on the ggfm (fig. ) is resulted from likely misassembling of grch p .chry. to test this, we delete the identified segment corresponding to the long sharp straight line on the ggfm (fig. ) . specifically, we delete a . -mbp (from , , bp to , , bp) target segment (supplementary dataset ) from the prior-cleaned grch p .chry, as guided by the turning-changed long sharp straight line on the d-trajectory map (x-length) of the ggfm (fig. a ). as expected, the resulting re-assembled form (reass.grch p .chry) of grch p .chry does roughly match its antecedent grch p .chry on the ggfm (fig. ) . hence, we name a proofreading errors-deletion (pred) for this operation of target deletion guided by the ggfm (fig. a) . such a pred-deletion rescues the harmonious state of chromosome architecture among the relatives on the ggfm (fig. ) . accordingly, we name a landscape compatibility for such an observed harmonious state. to justify such a rescue ( fig. ) , we decompose and analyse sub-constituents of the . -mbp target segment (supplementary dataset ). before that, we must exclude interferences by the telomeric and centromeric regions that were masked by ns in grch p .chry. we prior-deleted ns before conducting the pred-deletion, which ensures the deleted . -mbp target segment (supplementary dataset ) is devoid of ns. as anticipated, we scan it but find no tandem repeats (ttaggg)n, regardless of dispersed copies of single ttaggg element. these data conclude the . -mbp segment (supplementary dataset ) is not from a telomeric region featured by the known tandem repeats (ttaggg)n , thus leaving the centromeric region to be a suspect. re-locating the deleted target segment. we re-locate the . -mbp target segment (supplementary dataset ) against the newest assembly grch p (gcf_ . ). the ucsc human blat analysis indicates a match in centromeric region (fig. a) . the ncbi genome data viewer (fig. b) illustrates that it fits in a broad region of grch p .chry with three blocks ( to justify our findings (fig. ) , we survey the literatures but find no documents describing how such a megabase-sized target segment was assembled step by step. we track out that block i ( . kbp) was documented to be the dyz alpha satellite array in a centromeric database that was created from the huref wgs reads library , whereas block ii ( . kbp) and block iii ( . kbp) are unclear (fig. ) about their assembling processes that caused dramatic changes from grch p .chry to grch p .chry (and throughout grch p ) (figs and ) . we conclude that the . -mbp target segment (supplementary dataset ) does locate in the centromeric and pericentromeric region of grch p .chry (and throughout grch p .chry) (figs , and ), which encourages us to trace its sub-constituents that contributed to the observed turning-changed long sharp straight line on the ggfm (figs and ). tracing bacs of the deleted target segment. blast search against ncbi nr/nt database with the . -mbp segment (supplementary dataset ) shows no hits over the entire megabase-sized sequence, but traces homologous bacs (> . kbp) (fig. a) . with cover > % and identity > %, we choose bacs to compose a dataset for phylogenetics analysis (fig. b-g) . the results demonstrate that the traced bacs are divergent homologues stemmed from the human (h. sapiens) autosomal chr , chr , chr and chr as well as from the chimpanzee (p. troglodytes) autosomal chr and sex chry (fig. b, c) , and imply that these bacs might be shared or contaminated. given that the . -mbp target segment (supplementary dataset ) presents debuting in grch p .chry (rather than in grch p .chry) (figs and ), but absents from huref.chry and yh.chry that did not use bacs for sequencing and assembling, they are unlikely shared. note that our method is , times faster to create a distance matrix for such chosen bacs. our method took only minute to calculate genome fingerprints and create a weighted distance matrix, but the mega package took hours to complete base-to-base alignments and calculate a pair-wise distance matrix (i.e., the mega distance matrix). we use the mega package to construct traditional bootstrap consensus trees, both the me (minimum-evolution) tree (fig. b ) and the nj (neighbour-joining) tree (fig. c) have a low confidence at arguable sub-branches (e.g., containing fp . , ac_ . , ac_ . , ac_ . and ac_ . ). in contrast, we use our weighted distance matrix to construct an nj tree (fig. d ) by neighor. exe (from the phylip package) and a fastme tree (fig. e ) by fastme software (an update version of me). our trees (fig. d,e) demonstrate a better resolution at the questionable sub-branches observed on the opposite mega trees (fig. b,c) . further, we construct two phylogenetic networks (fig. f,g) using splitstree software , scientific reports | ( ) : | doi: . /s - - - based on our weighted distance matrix and the mega distance matrix, respectively. they are approximate to one another, but ours (fig. f) has a better resolution for discriminating the major discrepancies (e.g., fp . , ac . and fo . ) observed on the phylogentic trees (fig. b-e) . accordingly, we track out that fp . ( . kbp, h. sapiens chr clone ch - o ) has a . -kbp segment of , copies of a -bp (tggaa) unit (i.e., a cluster of (tggaa) ); ac . ( . kbp, p. troglodytes chr clone ch - l ) has a . -kbp segment of copies of a -bp unit; and fo . ( . kbp, h. sapiens chr clone rp - f ) has a . -kbp segment of , copies of a -bp (tcatt) unit. these findings demonstrate that our method has a better resolution for taxa containing high copy numbers of repeats. we thus use our method to construct phylogenetic networks throughout the next sections when dealing with a number of large and divergent sequences, on which traditional approaches may not work. table s ), preventing us from exhaustively analysing them one by one, we intend to evaluate the chosen examples of predicted long repeats (e.g., ltr retrotransposons and satellite dna). hence we conduct de novo predictions of long repeats from the . -mbp segment (supplementary dataset ), trace homologues, and analyse evolutionary relationships. using ltr-finder software , we predict ltr retrotransposons (fig. a,b) that are dispersed on a . -kbp cluster (from , bp to , bp) of the . -mbp segment (supplementary dataset ). we use each of them to do the blast search against the ncbi nr/nt database and select top hits (if applicable) to compose a dataset for phylogenetics analysis. such ltr retrotransposons are mono-centred on the phylogenetic network (fig. c) , coinciding with their close locations (fig. a,b) . these findings weaken the impacts of interspersed repeats, thus strengthen the impacts of tandem repeats to be elucidated. tracing tandem repeats of the deleted target segment. using trf software , we predict , tandem repeats (supplementary dataset ) , more than the records ( , tandem repeats) in the repeatmask/ repbase database (supplementary table s ). the trf program can list "period size" and "consensus size", which are usually the same. we use the former for simple notation throughout this paper. we name core-units (cus) (e.g., u for a -bp monomer) and core-unit repeats (curs) (e.g., u c for , copies of u). a cur is composed of multiple copies of a cu. the core-unit repeats (curs) here are equivalent to the higher-order repeats (hor) elsewhere . for instance, u c containing , copies of u (a -bp monomer) yields a . -kbp segment (from to , bp) of the . -mbp segment, which corresponds and phylogenetic networks (f,g). the bootstrap consensus me (b) and nj (c) trees constructed by the mega package have a low confidence at arguable sub-branches. but the nj (d) and fastme (e) trees constructed by our method have a better resolution at the questionable sub-branches. the splitstree networks (f,g) constructed by using our weighted distance matrix (f) and the mega distance matrix (g), respectively, illustrate that our method has a better resolution for the taxa containing high copy numbers of repeats (e.g., we track out that fp . has a . -kbp segment of (tggaa) in the main text). to block i ( . kbp) in the assigned centromeric region of grch p .chry (fig. ) . likewise, u c containing , copies of u (a -bp monomer, gaatg) yields a . -kbp segment (from , to , , bp) of the . -mbp segment, which corresponds to a part of block iii ( . kbp) in the assigned pericentromeric region of grch p .chry (fig. ) . each is close to a bac's size (> . kbp). we choose long tandem repeats to do the blast search against the ncbi nr/nt database, and select top hits (if applicable) from each search to compose a dataset for pursuing our phylogenetics analysis. we construct phylogenetic networks both at the cus level (fig. a) and at the curs level (fig. b-e) . under the circumstances tested, all cus (fig. a ) and most curs (fig. c-e) with their traceable homologues demonstrate random distributions, respectively, in a harmonious state (i.e., a landscape compatibility), regardless of certain outliers (fig. b) . such random distributions on the phylogenetic networks, both at the cus level (fig. a) and at the curs level (fig. c-e) , demonstrate individual landscape compatibility among relatives. but the orphan bac-sized curs (e.g., u c and u c) indicate distribution biases to be exceptional outliers on the phylogenetic network (fig. b) , betraying such an observed landscape compatibility. hence we group the curs into two categories. category contains most curs that randomly distribute on the phylogenetic networks (fig. c-e) , obeying the observed landscape compatibility. category contains the orphan curs that distribute as exceptional outliers on the phylogenetic network (fig. b) , , bp) . these data conclude that all cus (fig. a ) and most curs (fig. c-e) are of reasonable quality, having traceable homologues; but the orphan bac-sized curs (fig. b) this paper presents a novel geometric analysis method (called genomelandscaper) (fig. ) to conduct landscape analysis of genome-fingerprints maps (gfm) in order to trace large-scale repetitive regions and retrospectively assess their impacts on global architectures of assembled chromosomes (figs , , , , , and ) . we develop an alignment-free and bootstrap-free method for phylogenetics analysis. this study also sets up an itinerary of using the genomelandscaper method (figs , , , , and ) . as a proof-of-concept study, we created a galaxy of genome-fingerprints maps (ggfm) (figs , and ) for the human y chromosomes (grch.chry , , huref. chry [ ] [ ] [ ] and yh.chry ) and conducted multifaceted assessments on their global architectures (figs , , , , , and ). through data-mining approach without prior knowledge or biases (fig. ) , our data-driven computational analyses (figs , , , , and ) uncovered and characterised the questionable . -mbp target segment (supplementary dataset ) that distinguished grch p .chry (and throughout grch p .chry) from grch p .chry, huref.chry and yh.chry (figs , , and ) . we elucidated that ( ) it was the . -mbp target segment (supplementary dataset ) , identified in the modelled centromeric and pericentromeric region debuting in grch p .chry throughout grch p .chry (fig. ) , that contributed to the observed long sharp straight line on the ggfm (figs and ) ; and ( ) the orphan bac-sized curs (fig. b) dataset and dataset ) . this proof-of-concept study validates the efficacy of our genomelandscaper method (fig. ). hence we have established an effective method to display, detect, delete and analyse the large-scale repetitive regions, thus retrospectively assessing their impacts on the global architectures of assembled chromosomes. we expect that the genomelandscaper method could be broadly applicable to understanding and assessing the yet-untouchable centromeric and pericentromeric regions of variants from the human , genomes project , and other mammalian genomes. such endeavours should benefit improvements of the reference genome grch and the , genomes , , which is valuable for precise medicine since the roles of centromeres in chromosomal behaviours and clinical diseases are increasingly appreciated , . we would emphasise the technical features of our genomelandscaper method (fig. ) . first, our genomefingerprinter algorithm circularises a linear sequence (fig. a) to avoid interference from arbitrary cut-off sites of a sequence , thus ensures the circularised d-map (figs d and a) solely representing the given sequence. this feature improves the accuracy of computing a distance matrix (fig. g) based on values of geometric centres of the circularised d-maps (figs d and a) . second, we operate map-to-map comparison (figs , and ) , instead of base-to-base comparison (see fig. ), to relieve computation burdens. this alignment-free method allows us comparing large genomes in a big-picture view at a large scale (figs , and ) . third, we weight the geometrical mean of radiuses (equation ( )) of the circularised d-maps to create a weighted distance matrix (fig. g) , thus discriminate a pair of overlapped d-maps that share a geometric centre but have different shapes. this feature improves the accuracy of clustering both distant and close relatives (figs , and ) . fourth, we create a weighted distance matrix (fig. g) using the values of geometric centres of the circularised d-maps (fig. d) , and conduct calculations in the mathematical real number system (equations ( ) to ( )). whenever calculating the same set of sequences should result in the same weighted distance matrix (fig. g) , thus leading to the sole phylogenetic tree (fig. h) and phylogenetic network (fig. i) . as exemplified by the small-sized mitochondria genomes (< . kbp) of the primates, our tree was approximate to the traditional bootstrap consensus tree (fig. ) . so did the moderate-sized bac clones (> . kbp) (fig. ) . our nj and fastme trees (fig. d,e) and splitstree network (fig. f) have a better resolution for the taxa containing high copy numbers of repeats. our alignment-free and bootstrap-free method is faster and has worked effectively for cluster approximations in our cases (figs , and ) . altogether, these features allow us analysing a number of large genomes (mbp) (figs , and ) and divergent sequences (variations in size, gap, and divergence) (figs , and ) , on which the traditional approaches - may not work. we have demonstrated main findings with significance throughout the proof-of-concept study. we have found that there is a landscape compatibility of chromosome architecture among relatives under comparison (figs and ) . a misassembled segment can be detected if and only if it breaks such a landscape compatibility (fig. ) , which can guide the pred-deletion of the misassembled segment (fig. ) . as such, we have traced down the questionable . -mbp target segment (supplementary dataset ) from grch p .chry throughout grch p . chry (figs , and ) . furthermore, we have found multiple lines of evidence on its likely misassembling of the . -mbp target segment (supplementary dataset ). first, we located it in the modelled centromeric and pericentromeric region of grch p .chry (fig. ) . second, we decomposed it into sub-constituents such as bacs (fig. ) , interspersed repeats (fig. ) and tandem repeats (fig. ) . and we traced back their homologues from heterogeneous chromosomes beyond the human y chromosome (figs , and ) . third, we elucidated that among the examined tandem repeats, all cus (fig. a ) and most curs (fig. c-e) were of reasonable quality, but the orphan bac-sized curs (up to . % of the . -mbp segment) were likely over-repeated (fig. b) . such data-driven analyses without prior knowledge or biases (fig. ) should offer an informative starting-point for the community to retrospectively verify the modelled centromeric and pericentromeric regions that caused dramatic changes from grch p .chry to grch p .chy (and throughout grch p .chry) (figs , , and ) . this proof-of-concept study demonstrates the power of our genomelandscaper method (fig. ) , as discussed below. the mode of map-to-map (instead of base-to-base, see fig. ) comparison (figs , and ) not only overcomes computational constraints at a large scale, but also performs a holistic comparison in a big-picture view, thus bypassing the lack of a "true" sequence being referred to. further, our genomefingerprinter algorithm only calculates non-n (a, t, g and c) bases, bypassing gaps and linking two non-n bases adjacent to the distal ends of a homopolymer of ns, thus presents the effective genome. these advantages allow us to assess incomplete genomes regardless of gaps, sizes and divergences. a batch of incomplete genomes can be displayed as a ggfm (figs , and ) and compared at the chromosome architecture level. as a result, we have detected the incredible discrepancies among the human y chromosomes (grch.chry , , huref.chry [ ] [ ] [ ] and yh.chry ), as well as the four releases (grch p .chry, grch p .chry, grch p .chry and grch p .chry) of the grch.chry assembly per se (figs , and ) . there is an observed individual landscape compatibility among relatives on the ggfm (fig. ) and the phylogenetic network (fig. a,c-e) , respectively. this feature enables us to display and detect a misassembled chromosome architecture in a big-picture view at a large scale (fig. ) (figs and ) exhibits its own sensitivity, an extent of disturbing the landscape compatibility among relatives. the d-trajectory map (x-y-z) (fig. a) and three d-trajectory maps (x-y, x-z, y-z) (figs d-f and d-f) are the most sensitive, another two d-trajectory maps (x-length, y-length) (figs a,b and a,b) are less sensitive, and the d-trajectory map (z-length) (figs c and c) is the least sensitive. hence, the components xn and yn of the three-dimensional coordinates carry more information about the distribution biases of bases. the d-trajectory map (x-y-z) (figs. a) and d-trajectory map (x-y) (figs d and d ) amplify such effects of both xn and yn on disturbing the landscape compatibility, thus yielding more complex and sensitive profiles. two d-trajectory maps (x-length, y-length) (figs a,b and a ,b) are easier (due to simplicity) to guide the pred-deletion of an identified misassembled segment (figs and ) . in addition, the d-contour map (x-y) (fig. b-f ) with high-resolution genome fingerprints is the most sensitive in discriminating close relatives (huref.chry, yh.chry, grch p .chry and grch p .chry). the proofreading errors-deletion (i.e., the pred-deletion) guided by a ggfm is an efficient means for deleting a misassembled segment (fig. ) . we deliberately hypothesised that the extra turning-changed long sharp straight line on the ggfm (fig. ) resulted from likely misassembling of grch p .chry, when compared to its antecedent grch p .chry. this hypothesis simplified the logic of validations: if the antecedent were (and should have been over time) correct, then the descendent with a newly-introduced extra segment should be incorrect; and vice versa. accordingly, we conducted the pred-deletion of the . -mbp segment (supplementary dataset ) , guided by the turning-changed long sharp straight line on the ggfm (fig. a) . to justify its likely misassembling, we found multiple lines of evidence. first, the pred-deletion of the . -mbp target segment (supplementary dataset ) from grch p .chry (fig. ) did rescue the landscape compatibility, matching its antecedent grch p .chry. second, its sub-constituents such as bacs (fig. ) , ltr retrotransposons (fig. ) , and tandem repeats (fig. ) (figs and ) . furthermore, to justify our findings from data-driven computational analyses, we conducted retrospective researches in literatures. we tracked out the centromere model representations debuting in grch p .chry (dyz . mbp) and grch p .chrx (dxz . mbp), where the assigned gap placeholders were replaced by the models (dyz and dxz ) that were simulated based on a centromere database derived from the huref wgs reads library . grch p .chry bears a modelled centromere , , rather than a real one that was assembled from original reads. these facts are consistent with our findings. first, the . -mbp target segment (supplementary dataset ) of grch p .chry (from , , bp to , , bp) roughly equals the assigned gap placeholder of grch p .chry (from , , bp to , , bp). second, the . -mbp segment covers three blocks that are scattered on grch p .chry (fig. ) . block i ( . kbp) (fig. ) was documented to be the dyz ( . mbp) alpha satellite array that was simulated from the huref wgs reads library . block ii ( . kbp) and block iii ( . kbp) (fig. ) remained unclear about their assembling processes that have caused dramatic changes from grch p .chry to grch p .chry throughout grch p .chry (figs , and ). third, the orphan bac-sized curs (fig. b) are the individual parts of block i ( . kbp) and block iii ( . kbp), which locate in the centromeric and pericentromeric regions of grch p .chry (fig. (figs and b) . fourth, these orphan bac-sized curs are exceptional outliers on the phylogenetic network (fig. b) . they are scattered on and up to . % of the . -mbp segment (supplementary dataset and dataset ), which mainly contributed to the turning-changed long sharp straight line on the ggfm (figs and ) . such findings coincide with the fact that the monomer ordering of the chromosome-specific alpha-satellite repeats was proportional to that observed in the initial read database, but the long-range ordering of repeats was inferred by a graph-based simulation in the modelled centromere of grch p .chry , [ ] [ ] [ ] . we conclude that the orphan bac-sized curs (fig. b) are likely over-repeated, lacking traceable homologues at this moment. meanwhile, the human satellites hsat and hsat were documented to be composed of -bp (e.g., cattc, gaatg) repeats with diverged arrangements and constituted . % of the human genome, occupying heterochromatic blocks adjacent to centromeric regions (see reference no. and others therein). by using the same graph-based simulation method , the subfamily-specific -mers for hsat and hsat were recently modelled (each cluster is small, < kbp) in the pericentromeric regions adjacent to centromeres of the human chromosomes and y . the sizes of the predominant hsat -rich arrays on the y chromosomes were estimated to distribute differently within the distinct y haplogroups . the sizes of hsat a (dyz ) arrays from individuals varied over an order of magnitude ( to mbp) . thus we would suggest that both the core k-mers (i.e. cus) and their copy numbers (i.e., curs) should be equally concerned on the case-by-case basis. the modelled centromeric and pericentromeric regions debuting in grch p .chry are not yet true, linear assemblies , , ; rather, they are modelled reference regions to be used for mimicking and mapping target short-reads at this stage. hence the true cus and curs discussed in our cases remain to be elucidated in the future. with and without the modelled centromeric and pericentromeric regions, the two counterparts (grch p . chry and grch p .chry) have provided excellent cases for a proof-of-concept study via data-mining without prior knowledge or biases, thus have validated the efficacy and demonstrated the power of our genomelandscaper method. the itinerary has worked effectively and should be generally applicable. however, we remind that it deserves to investigate whether the . -mbp segment (supplementary dataset ) is a true intrinsic segment on the human y chromosome. theoretically, pcr reactions could be determinate, but could be undoable in this case owing to two-faceted difficulties: amplification of the . -mbp segment (supplementary dataset ) as a whole is technically unachievable, while amplifying sizable parts of its elements is challenging in designing proper primers. the desired primers must cover the sub-constituents, such as bacs (fig. ) , ltr retrotranspons (fig. ) , and tandem repeats (fig. ) , but also avoid similar sequences that might be scattered on the human y chromosome. these tasks are hardly achievable due to the natures of eukaryotes repeats , . historical experimental data (including physical mapping, see reference no. and others therein) would be inadequate at the level of single-base resolution to discriminate arguable sequences of repeats in genomics. we would recommend sequencing and assembling the real centromeric and pericentromeric regions of the human y chromosomes by future technology (once it is applicable) , [ ] [ ] [ ] . with true, linear reference regions for pursuing our ggfm comparisons, one could ultimately justify whether the . -mbp segment (supplementary dataset ) containing the orphan bac-sized curs (fig. b) might result from an artefact or should be a true intrinsic segment. therefore, the graph-based simulation method modelled the centromeric and pericentromeric regions in the human reference genome grch , which opened a door to model the yet-untouchable centromeric and pericentromeric regions of mammalian genomes. our genomelandscaper method offers a geometric analysis means to retrospectively assess such modelled regions, which opens a window to assess their impacts on the global architectures of assembled chromosomes. we calculated the three-dimensional coordinates for a genome using our genomefingerprinter algorithm . we transformed a genome into a genome-fingerprints map (gfm); and a set of gfms were simultaneously constructed to create a galaxy of genome-fingerprints maps (ggfm). we calculated a weighted euclidean distance matrix (as described in results section). we used the weighted distance matrix to create a phylogenetic tree using fastme . software under the minimum-evolution (me) model, and using neighbor.exe (from the phylip package . ) under the neighbour-joining (nj) model. we used the weighted distance matrix to create a phylogenetic network using splitstree . software , under the neighbour-net model. default parameters were applied. we used the mega package to conduct pair-wise and multiple alignments by clustal w (with iub dna weight matrix, transition weight . , gap opening penalty , gap extension penalty . ) , and create a minimum-evolution (me) tree and a neighbour-joining (nj) tree (both with bootstrap replicates , maximum composite likelihood model, nucleotide substitutions, transition and transversion) . the pair-wise deletion model was used for the treatment of gaps and missing subset data . given sequence, we searched it against repeatmasker/repbase database (at http://www.girinst.org/censor/index. php) to retrieve the registered repeats . we used ltr-finder software and tfr software to conduct de novo predictions from the given sequence in order to predict ltr retrotransposons and tandem repeats, respectively. the parameters for ltr-finder were default, while for tfr were ( / / / / / / ). evaluation of grch and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly the genomes project consortium. a global reference for human genetic variation genetic variation and the de novo assembly of human genomes the sequence of the human genome whole-genome shotgun assembly and comparison of human genome assemblies the diploid genome sequence of an individual human de novo assembly of a haplotype-resolved human genome direct determination of diploid genome sequences canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation hinge: long-read assembly achieves optimal repeat resolution fast and accurate de novo genome assembly from long uncorrected reads international human genome sequencing consortium. initial sequencing and analysis of the human genome international human genome sequencing consortium. finishing the euchromatic sequence of the human genome an assessment of the sequence gaps: unfinished business in a finished human genome analysis of the centromeric regions of the human genome assembly centromere reference models for human chromosomes x and y satellite arrays modernizing reference genome assemblies extending reference assembly models the genome fingerprint and the universal genome fingerprint analysis for systematic comparative genomics coronavirus phylogeny based on a geometric approach fastme . : a comprehensive, accurate and fast distance-based phylogeny inference program molecular evolutionary genetics analysis version . phylip: phylogeny inference package (version . ) analysing and visualizing evolutionary data application of phylogenetic networks in evolutionary studies the ucsc genome browser database: update repbase update, a database of repetitive elements in eukaryotic genomes ltr_finder: an efficient tool for the prediction of full-length ltr retrotransposons tandem repeats finder: a program to analyze dna sequences sequences associated with centromere competency in the human genome genomic variation within alpha satellite dna influences centromere location on human chromosomes with metastable epialleles genomic characterization of large heterochromatic gaps in the human genome assembly the impact of retrotransposons on human genome evolution single-molecule sequencing resolves the detailed structure of complex satellite dna loci in drosophila melanogaster hapcut : robust and accurate haplotype assembly for diverse sequencing technologies improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - h monh authors: lee, seung hoon; park, jin-sil; byun, jae-kyung; jhun, jooyeon; jung, kyungah; seo, hyeon-beom; moon, young-mee; kim, ho-youn; park, sung-hwan; cho, mi-la title: pten ameliorates autoimmune arthritis through down-regulating stat activation with reciprocal balance of th and tregs date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: h monh pten is a tyrosine phosphatase with significant function in inhibiting stat activation. recently, inactivation of stat has been demonstrated as a therapeutic candidate for autoimmune arthritis. the expression of pten controlled by p regulates autoimmune arthritis through modulating the balance between th and treg. we hypothesized that pten regulated by p might reduce cia severity and inflammatory response via inhibiting stat activation. our results revealed that pten could ameliorate experimental autoimmune arthritis by reducing stat activity and th differentiation. systemic infusion of pten overexpression downregulated cia severity. in addition, pten overexpression decreased the activation of t cells and modulated reciprocal differentiation of th and treg cells. we observed that pten expression downregulated by p deficiency induced the activation of stat . loss of p exacerbated autoimmune arthritis and dysregulated the population of th and treg. these data suggest that induction of stat -modulatory activity of pten may be a therapeutic target for rheumatoid arthritis therapy. spontaneous inflammatory disorder . pten production is associated with tumor protein p involving in the reduction of autoimmune inflammatory response . it has been demonstrated that transcription of pten is controlled by p . we hypothesized that pten could attenuate the development of autoimmune arthritis by reducing stat activation and th cells differentiation. previously, we have reported that p could control autoimmune arthritis through stat mediated balance between th and treg . the present study was conducted to identify whether pten had therapeutic potential related to p in autoimmune arthritis. thus, we evaluated the therapeutic efficacy of pten in experimental autoimmune arthritis. overexpression of pten ameliorates cia development. to determine whether pten had anti-arthritic effect, cia induced mice were injected with either pten overexpression or mock vector once weekly. pten overexpression significantly downregulated the severity of arthritis in cia induced mice (fig. a) . the concentrations of total igg, igg , and igg a in the serum were significantly decreased in mice injected with pten overexpression compared to mock group (fig. b) . pten overexpression significantly reduced the degree of inflammation, bone damage, and cartilage damage (fig. c) . immunohistochemical analysis revealed that injection with pten overexpression vector significantly suppressed the expression of proinflammatory cytokines and osteoclastogenesis related factor such as rankl and trap in joints compared to cia mice treated with mock vector (fig. d,e) . our results suggested that pten overexpression could suppress cia severity, thus reducing inflammatory response and osteoclastogenesis in joint. to determine whether pten overexpression could attenuate dysregulated balance between th and treg, we examined the differentiation of th and treg in cia mice. the overexpression of pten reduced th differentiation in the spleen tissues of cia induced mice. however, the differentiation of treg cell was promoted in cia induced mice injected with pten overexpression vector ( fig. a) . it is well documented that t cell activation is involved in the pathogenesis of ra . in mixed lymphocyte reaction (mlr), the alloreactive t cell response was decreased in lbrm transfected with pten overexpression compared to that in the control (fig. b) . additionally, pten overexpression decreased the number of il- producing cd + p-stat + or p-stat + t cells. however, the number of cd + p-foxp + t cells in the spleen tissues of cia induced mice was significantly upregulated compared to that in the spleen tissues of mice treated with mock vector based on immunofluorescence confocal microscopy ( fig. c,d) . these data suggested that pten overexpression ameliorated the imbalance between th and treg in cia. loss of p induces stat activation. previously, p has been known as a modulator of stat activation through significant reducing stat phosphorylation and stat dna binding activity . the phosphorylation levels of stat tyr and ser in p deficient mice splenocytes were increased compared to those of wt mice splenocytes (fig. a,b) . cells isolated from wt or p deficient mice were cultured under the condition of th or th . il- production in p −/− mice was significantly increased compared to that in wt mice (fig. c,d) . gene expression of il- and ccl which causes recruiting of il- expressing cells in wt mice splenocytes was increased after stimulation with tgf-β and il- . but, mrna expression of il- and ccl was reduced significantly by p activator, nutlin- a. in contrast, bpv(hopic), the inhibitor of pten, promoted gene expression of il- and ccl (fig. e ). gene expression of pten was enhanced significantly (c) spleens of cia mice were subjected to immunostaining for cd + il- or cd + cd + foxp + cells. (original magnification, × ) (d) spleens of cia mice were subjected to confocal staining for cd + pstat y + or cd + pstat s + cells (original magnification, × ). the number of cells was counted in four independent quadrants. data are presented as mean ± sd of three independent experiments (*p < . , ***p < . , n = ). by nutlin- a. however, pifithrin-α , the inhibitor of p , significantly decreased the mrna level of pten in mice splenocytes (fig. f ). these results suggested that the inhibition of p under inflammatory milieu of ra might have enhanced inflammation. additionally, pten expression could be regulated by p dependent manner. recently, p has been demonstrated as a mediator of balance between th and treg in ra . cells of wt or p deficient mice in normal state were cultured under th or th condition. the expression of il- was significantly increased (fig. a ). gene expression of il- in splenocytes isolated from p deficient mice was also significantly increased compare to that in wt mice (fig. b ). t cell activation was profoundly upregulated in cells isolated from p deficient mice compared to that in wt mice (fig. c ). th differentiation in p deficient mice was significantly increased whereas treg differentiation was significantly reduced compared to that in wt mice (fig. d ). these data suggested that p deficiency could lead to t cell activation and imbalance between th and treg. p deficiency exacerbates cia severity by upregulating inflammation. based on arthritis scores, it was found that p deficiency exacerbated cia progression in vivo (fig. a ). serum levels of total igg, igg , and igg a were significantly increased in p deficiency mice compared to those in wt mice (fig. b) . moreover, histological analysis showed that paws and ankles of p deficiency arthritis mice had higher degree of inflammation with bone damage and cartilage damage (fig. c ). the expression of proinflammatory cytokines in joints were significantly upregulated in p deficiency arthritis mice compared to that in wt mice (fig. d ). our results suggested that p deficiency might have failed to regulate inflammatory response, thus worsening local inflammatory milieu. treg. since pten expression is regulated by p , we investigated pten expression in p deficiency mice with cia. gene expression levels of pten in splenic cd + t cells, splenocytes, and draining lymph nodes isolated from p deficiency mice with cia were decreased significantly compared to those in wt mice with cia (fig. a) . since ra can result in dysregulated balance of th /treg , we also measured the balance between th and treg. the mrna level of th related molecules including il- was increased, whereas the mrna expression of treg cell-related molecules such as foxp was enhanced in p deficiency mice with cia (fig. b ). in addition, the number of cd + p-stat + t cells in the spleen tissues of p deficiency mice with cia was significantly downregulated compared to that in the spleen tissues of wt mice with cia based on immunofluorescence confocal microscopy (fig. c) . however, loss of p promoted the number of il- producing cd + p-stat + or p-stat + t cells in the spleen tissues based on immunofluorescence confocal microscopy (fig. d) . moreover, pten expression was significantly downregulated in p deficient mice compared to that in wt mice (fig. e) . these results demonstrated that p deficiency could accelerate the imbalance between th and treg in cia. until now, pten has been investigated extensively as a tumor suppressor with a role in cell metabolism, motility, and tumor microenvironment . recently, pten has been observed to be associated with cell differentiation as a phosphatase . in addition, pten is revealed to have therapeutic effect in rat with cia . however, little is known about the process of pten function associated with p in ra. here, we studied the therapeutic activity of pten in ra and identified a new mechanism of ra regulation. the most notable observation of this investigation is that pten can attenuate ra via reciprocal differentiation of th /treg. to our knowledge, this is the first research to provide evidence that pten could be used for ra therapy through regulating th /treg balance. previously, a number of documents have demonstrated that imbalance between th and treg can contribute to ra , . modulation of th and treg cells has an important role in ra therapy , . the activation of stat prolongs foxp production in treg, while stat activation increases the differentiation of th cells . moreover, binding of p-stat and p-stat can competitively regulate il- transcription . our results demonstrated that foxp + t cells were induced by pten overexpression while p deficiency significantly induced p-stat + t cells. this could be due to the diminishment of t cell transcriptional regulators such as foxp and socs and the enhancement of rorγ t, runx , and batf. in addition, pten decreased the number of treg cells while th differentiation was promoted in ra mice model. hence, pten might be another efficient mechanism regulated by which p through controlling th /treg balance. tumor protein (p ), a tumor suppressor factor, is essential for cellular response to dna damage. it plays a key role in several gene expression as a resourceful transcription factor under stressful conditions. it has been demonstrated that p is involved in a variety of cellular signal pathways, cell proliferation, and apoptosis [ ] [ ] [ ] . although p function has been recognized mainly in the cell cycle, emerging evidence has suggested that p has important role not only in apoptosis, cell differentiation, and dna repair, but also in the modulation of stat-mediated th cells . loss of p can promote the progression of antigen-induced arthritis and increase activated t cell differentiation . in this study, pten expression was found to be regulated in p dependent manner. additionally, p deficiency aggravated cia severity and reduced pten expression. these results suggested that pten could have therapeutic effect in autoimmune arthritis through p . il- is a typical proinflammatory cytokine inducing the expression of il- , - and tnf-α - . it has been documented that il- can upregulate proinflammatory cytokines including il- and il- and aggravate joint inflammation of ra through activating cd + t cells , . th secreting il- performs a key role in the pathogenesis of ra. th frequency and il- level are strikingly correlated with ra development. it has been documented that th can result in excessive inflammation in patients with ra . our data revealed that pten overexpression reduced the activation of t cells and that the loss of p enhanced the proliferation of th cells. since il- expression is well known to induce ra development, suggesting a novel therapeutic strategy to modulate ra via pten expression. (a,b) relative mrna levels of pten and factors such as il- , rorγ t, and foxp involved in the differentiation of th and treg in splenic cd + t cells, splenocytes, and draining lymph nodes from cia induced wt or p −/− mice were assessed by real-time pcr. data are presented as mean ± sd of three independent experiments (*p < . , **p < . ). (c) spleens of cia mice were subjected to immunostaining for cd + il- or cd + cd + foxp + cells. (d) spleens of cia mice were subjected to confocal staining for cd + pstat y + or cd + pstat s + cells. the number of cells was counted in four independent quadrants. data are presented as mean ± sd of three independent experiments (*p < . , ***p < . , n = ). scientific reports | : | doi: . /srep granulocyte-macrophage colony-stimulating factor (gm-csf), an immune modulatory cytokine, performs a significant role in immune tolerance and attenuates autoimmune disorder . it has been suggested that gm-csf suppressed progression of autoimmune disease via induction of tregs . previously, immune tolerance can be a good strategy for cia therapy. indeed, immune tolerance induction using cii showed therapeutic effect in vivo and in vitro . cd + tregs can reveal therapeutic implications in cii involved-disease inducing immune tolerance . the therapeutic effect of pten in cia may be involved in upregulation of gm-csf and immune tolerance. thus, further study will be needed to confirm therapeutic activity of pten related with gm-csf expression and immune tolerance. we analyzed the gene expression of p and stat in cd + t cells of healthy individuals and ra patients from the national center for biotechnology information gene expression omnibus database (gse ). the gse database contains healthy subjects and ra patients with clinic and pathological information. we observed that the relative mrna level of stat of ra patients was promoted significantly compared to that of healthy individuals in this database. the mrna expression of p in ra patients was downregulated significantly compared to that in healthy individuals. the lack of p might have aggravated ra progression and induced stat activation. in this study, we demonstrated that pten overexpression could reduce cia progression and that the loss of p enhanced the expression of stat . the function of pten and p in stat activation has already been studied in previous investigations , . recently, p deficiency has revealed correlation with ra severity inducing th differentiation . however, our study demonstrated significant mechanism of pten associated with p in the development of cia via reciprocally regulating th and treg. this preliminary evidence suggested that upregulating pten could be a strong therapeutic strategy for the treatment of ra. ethics statement. the animal care committee of the catholic university of korea approved the experimental protocol, and all the experimental procedures were carried out in accordance with the protocols approved by the animal research ethics committee at the catholic university of korea. all procedures performed followed the ethical guidelines for animal studies. animals. male dba /j mice and c bl/ mice at - weeks old (orient, korea) were maintained in groups of five in polycarbonate cages in a specific pathogen-free environment. they were provided free access to standard mouse chow (ralston purina, gray summit, mo) and water ad libitum. mice harboring the p -null allele with a c bl/ mice background (b . s -trp tm tyj/j) were obtained from the jackson laboratory. collagen-induced arthritis (cia) was induced in dba /j mice (each group: n = ). mice were immunized with μ g of chicken cii (chondrex inc., redmond, wa, usa) dissolved overnight in . n acetic acid ( mg/ml) in complete freund's adjuvant or incomplete freund's adjuvant (chondrex inc). the immunization was performed intradermally into the base of the tail. cia was induced in the p −/− strain mice as described previously . eight days after immunization, mice were injected intravenously with μ g of pten or mock vector in ml of saline over a -second period. after days, the same mice received intramuscular injection of μ g of pten or mock vector in the left leg with electrical stimulation (electroporation) using a -gauge needle insulin syringe for hydrodynamic-based procedures. two days later, mice received an intramuscular injection of μ g of pten or mock in the right leg through electroporation. clinical scoring and histological assessment of arthritis. arthritis score was measured visually twice per week based on the appearance of arthritis in the joints and graded according to williams et al. . the joints of each mouse were fixed in % formalin, decalcified in % edta, and embedded in paraffin wax for histological analysis. hematoxylin-eosin (h&e) stained sections were scored for inflammation, destruction of cartilage, and bone damage according to published criteria , . real-time polymerase chain reaction (pcr). total flow cytometry. flow cytometry was conducted as described previously , . cells were immunostained with various combinations of fluorescent antibodies against cd , cd , foxp , ifn-γ , il- , and il- (ebioscience, san diego, ca, usa). prior to intracellular staining, cells were restimulated with phorbol myristate acetate (pma; ng/ml) and ionomycin ( ng/ml) for hours in the presence of golgistop (bd biosciences). for analysis of treg cells, cells were surface labeled with cd and cd , followed by fixation, permeabilization and intracellular staining with foxp was perfirmed per the manufaturer's protocol. flow cytometry was performed on a facscalibur flow cytometer (bd biosciences). the data was analyzed using the flowjo software (tree star, ashland, or,usa). elisa. enzyme-linked immunosorbent assay (elisa) was conducted as described previously , . briefly, blood was obtained from the orbital sinus of mice. serum levels of igg antibodies were measured using a commercially available elisa kit (bethyl laboratories, montgomery, tx, usa). horseradish peroxidase (hrp) activity was measured using tetramethyl benzidine as substrate (ebioscience, san diego, ca, usa). scientific reports | : | doi: . /srep staining for confocal microscopy analysis. tissue cryosections ( μ m thick) were fixed with acetone and stained with fitc-, pe-, percp-cy . -, or apc-conjugated monoclonal antibodies against mouse cd , pstat (tyr , ser ), pstat , il- , and foxp (ebioscience). after incubation at °c overnight, stained sections were visualized through confocal microscopy (lsm meta; zeiss, göttingen, germany). immunohistochemistry. immunohistochemistry was performed using the vectastain abc kit. tissues were first incubated with primary anti-c-jun and anti-c-fos antibodies overnight at °c. the primary antibody was detected with a biotinylated secondary antibody followed by incubation with a streptavidin-peroxidase complex for h. dab chromogen was added to obtain colored product. transfection. pten vector purchased from addgene (plasmid# ) was used to generate the overexpression of pten. mock and pten vector constructs were transfected into lbrm (mice t lymphoma cell line) cells using amaxa d-nucleofector x unit according to the manufacturer's recommendations with program dn- (lonza). splenocytes were harvested in ack lysis buffer, washed, and resuspended in complete culture medium (rpmi supplemented with % [v/v] heat-inactivated fetal calf serum). to purify splenic cd + t cells, splenocytes were incubated with anti-cd -coated magnetic beads, and cd + t cells were isolated using magnetic-activated cell sorting (macs) separation columns (miltenyi biotec). the cells were pretreated with pifithrin-α , nutlin- a (cayman chemical) or bpv(hopic) (santa cruz biotechnology) and then stimulated under the required polarizing conditions. aliquots of × cd + t cells (responders) were cultured with × irradiated ( , cgy) apcs in -well plates containing μ l/well of complete medium, at °c in a humidified % (v/v) co /air atmosphere. cells were pulsed with μ ci of tritiated thymidine ( western blot. western blot was performed as described previously , . proteins were loaded onto % polyacrylamide gels and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transferring to nitrocellulose membranes (invitrogen life technologies, carlsbad, ca, usa). membranes were blocked with % (w/v) non-fat milk in tris-buffered saline containing . % tween- for h followed by incubation with antibodies against p-stat y , p-stat s , t-stat (cell signaling), and β -actin (santa cruz biotechnology) overnight at °c. immunoreactivity was determined using enhanced chemiluminescence reagents (amersham biosciences, piscataway, nj, usa). statistical analysis. data were presented as means ± standard deviations (sd). statistical analysis was performed with nonparametric mann-whitney u test using graphpad prism v. . . one-way analysis of variance (anova) and bonferroni's post hoc test were used for multiple comparisons. statistical significance was considered when p value was less than . . disturbed th /treg balance in patients with rheumatoid arthritis pro-inflammatory cytokines in rheumatoid arthritis: pathogenetic and therapeutic aspects th cells, but not th cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin- a production high levels of il- in rheumatoid arthritis patients: il- triggers in vitro il- production via cyclosporin a-sensitive mechanism stat and nf-kappab signal pathway is required for il- -mediated il- production in spontaneous arthritis animal model il- receptor antagonist-deficient mice stat and stat direct development of il- -secreting th cells stat regulates cytokine-mediated generation of inflammatory helper t cells mechanisms underlying lineage commitment and plasticity of helper cd + t cells metformin attenuates experimental autoimmune arthritis through reciprocal regulation of th /treg balance and osteoclastogenesis sta- , a promising stat- inhibitor that reciprocally regulates th and treg cells, inhibits osteoclastogenesis in mice and humans and alleviates autoimmune inflammation in an experimental model of rheumatoid arthritis perturbations of the akt signaling pathway in human cancer activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor pten at sites of invasive growth and destruction pten is a negative regulator of stat activation in human papillomavirus-infected cells treg cells require the phosphatase pten to restrain th and tfh cell responses tumor suppressor p inhibits autoimmune inflammation and macrophage function regulation of pten transcription by p p controls autoimmune arthritis via stat-mediated regulation of the th cell/treg cell balance in mice the central role of t cells in rheumatoid arthritis p regulates stat phosphorylation and dna binding activity in human prostate cancer cells expressing constitutively active stat th cytokines stimulate ccl expression in keratinocytes in vitro and in vivo: implications for psoriasis pathogenesis the functions and regulation of the pten tumour suppressor the protein phosphatase activity of pten is essential for regulating neural stem cell differentiation amelioration of collagen-induced arthritis in rats by adenovirus-mediated pten gene transfer the t(reg)/th cell balance: a new paradigm for autoimmunity rebamipide suppresses collagen-induced arthritis through reciprocal regulation of th /treg cell differentiation and heme oxygenase induction human immunodeficiency: connecting stat , th and human mucosal immunity opposing regulation of the locus encoding il- through direct, reciprocal actions of stat and stat the complexity of p modulation: emerging patterns from divergent signals wild-type p induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin- p regulation by post-translational modification and nuclear retention in response to diverse stresses the tumour suppressor gene p modulates the severity of antigen-induced arthritis and the systemic immune response il- is produced by th cells and drives il- production in a stat -dependent manner potential new targets in arthritis therapy: interleukin (il)- and its relation to tumour necrosis factor and il- in experimental arthritis il- can promote tumor growth through an il- -stat signaling pathway il- induces production of il- and il- in rheumatoid arthritis synovial fibroblasts via nf-kappab-and pi -kinase/akt-dependent pathways il- induces the production of il- in rheumatoid arthritis gm-csf: an immune modulatory cytokine that can suppress autoimmunity granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: effects on regulatory t cells eye-mediated immune tolerance to type ii collagen in arthritis-prone strains of mice type ii collagen induces peripheral tolerance in balb/c mice via the generation of cd + t regulatory cells protocol for the induction of arthritis in c bl/ mice anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis trance/rankl knockout mice are protected from bone erosion in a serum transfer model of arthritis blockade of pi kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis a novel pancreatic beta-cell targeting bispecific-antibody (bsab) can prevent the development of type diabetes in nod mice the novel role of il- ligation to il- receptor in myeloid cells of rheumatoid arthritis and collagen-induced arthritis human monoclonal antibodies against highly conserved hr and hr domains of the sars-cov spike protein are more broadly neutralizing identification of a novel toll-like receptor endogenous ligand in rheumatoid arthritis synovial fluid that can provoke arthritic joint inflammation identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay conceived and designed the study, interpreted the data, and made critical revisions of the manuscript for important intellectual content supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord- -rb xdzro authors: zheng, xuexing; wong, gary; zhao, yongkun; wang, hualei; he, shihua; bi, yuhai; chen, weijin; jin, hongli; gai, weiwei; chu, di; cao, zengguo; wang, chong; fan, quanshui; chi, hang; gao, yuwei; wang, tiecheng; feng, na; yan, feihu; huang, geng; zheng, ying; li, nan; li, yuetao; qian, jun; zou, yong; kobinger, gary; gao, george fu; qiu, xiangguo; yang, songtao; xia, xianzhu title: treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from ebola virus infection date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: rb xdzro recent successes with monoclonal antibody cocktails zmapp(tm) and mil against ebola virus (ebov) infections have reignited interest in antibody-based therapeutics. since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. we produced purified equine antisera from horses hyperimmunized with ebov virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. balb/c mice were given up to mg of purified equine antisera per animal, at minutes, or days post-infection (dpi), in which all animals survived. to decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate f(ab′)( ) fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. full protection was achieved with when treatment was initiated at dpi, but the suboptimal protection observed with the minute and dpi groups demonstrate that in addition to virus neutralization, other fc-dependent antibody mechanisms may also contribute to survival. guinea pigs given mg of antisera or f(ab′)( ) at or starting at or dpi were also fully protected from ebov infection. these results justify future efficacy studies for purified equine products in nhps. of these reasons means that there are high personal risks involved with working on ebov in a laboratory setting, and as such it is classified as a biosafety level (bsl- ) agent. in the spring of , a new ebov variant emerged in the west african nation of guinea , an area in which the virus had not been previously reported. the outbreak soon spread to neighbouring sierra leone and liberia. occasionally, cases have been exported into other countries through travel; with countries located in africa, europe and north america all having recorded ebov cases imported by travel, or repatriation of infected citizens. as of mid-december , there are over , fatalities and , infections , the largest evd outbreak in history. although the outbreak is now largely under control with no reported cases since the week of november th , , the virus had shown itself at the peak of the outbreak to be extremely resistant to traditional containment methods designed to curb ebov transmission. passive immunotherapy with sera of animal origin has been used for over years to treat bacterial and viral infections, envenomations and drug intoxications. in , a report demonstrated that the passive transfer of igg from nonhuman primate (nhp) survivors of ebov disease to naive nhps was sufficient to confer post-exposure protection against ebov challenge in all animals . building on these findings, cocktails of monoclonal antibodies (mabs) raised against the ebov glycoprotein (gp) were soon shown afterwards to be effective in the treatment of ebov disease , . this culminated in the development of zmapp tm , a combination of three mabs produced in genetically modified tobacco plants, which were shown to reverse advanced ebov disease in experimentally-infected nhps , and may have provided a survival benefit when administered to ebov-infected patients . a second antibody cocktail (mil- ), which was based on zmapp tm and produced in modified cho cells, were later shown to have similar efficacy to zmapp tm in nhps . therefore, passive immunotherapy is an extremely promising approach to control ebov disease. due to the ease of management, high antibody yield and low risk of human contamination by virus or adventitious agents, horses are the most commonly used animal species in the production of hyperimmune sera. immunization itself is standardized and performed under optimal conditions for both personnel and animals. passive immunotherapy is still commonly used in countries that are still resource-poor medically, and treatment with immune globulin against rabies virus and clostridium tetani infections remains frequent in africa, asia and latin america. the lower manufacturing costs of hyperimmune equine antisera therefore represents an attractive alternate avenue of treatment, especially to developing and third-world countries, compared to the more costly production process of ebov gp-specific mabs. to investigate further, we first prepared purified anti-ebov antisera via the immunization of horses with ebov enveloped virus-like particles (evlp), which consists of ebov viral protein (vp ) and gp. the equine antisera was then tested in vitro using a neutralization assay with recombinant ebov expressing egfp (ebov-egfp), as well as in vivo against a lethal challenge with mouse-adapted ebov (ma-ebov) in immunocompetent balb/c mice. to investigate whether ebov infections can be controlled by virus neutralization alone, and to prevent the possible induction of serum sickness in humans that would be administered antisera, the post-exposure efficacy of f(ab′ ) (immunoglobulin treated with pepsin to remove the fc regions of the antibody) were also investigated side-by-side with equine antisera in all experiments as a potential alternate treatment. guinea pigs then were used to confirm the efficacy results from mouse studies, due to their status as a higher phylogenic species which more closely models hallmarks of evd in humans. additionally, prior to efficacy studies both the equine antisera and f(ab′ ) had been evaluated for safety in the peking union medical college center for new drug safety evaluation, chinese academy of medical sciences, which is certified by the food and drug agency of the people's republic of china. both equine-derived products were found to meet safety standards for clinical use in china. immunization of horses and production of equine antibody products. the horses were immunized with evlp produced from the infection of sf cells with rbv-vp -gp. the filamentous evlp were observed under an electron microscope (supplementary figure ) and confirmed to be morphologically similar to ebov. immunoblotting of rbv-vp -gp infected sf cell lysates demonstrated that the evlp contained ebov gp and vp (supplementary figure ) . three horses were immunized intramuscularly (im) with injections of evlp over weeks and the hyperimmune sera were collected from each animal at specified timepoints ( fig. a) to determine the serum titers by neutralization assay against a recombinant hiv- virus pseudotyped with ebov gp. a pseudotyped-virus was used for these studies to confirm the in vitro efficacy of the antisera preparations under biosafety level (bsl- ) conditions, before subsequent studies in the bsl- laboratory. neutralizing serum titers were detectable after the th immunization at week , and increased until the th immunization at week (fig. b) . the serum neutralizing antibody titers of two horses were : , after seven immunizations, and was : , for the third animal. the equine antisera (purified by ammonium sulphate-based precipitation) was then digested with pepsin at a concentration of - u/ml for to min and purified to generate f(ab′ ) fragments. the purity of the antisera and f(ab′ ) preparations, as determined by sds-page followed by thin layer chromatography, was determined to be % and %, respectively (fig. ) . approximately ml of purified antisera could be obtained from each horse during each collection, with the stock concentration between - mg/ml. up to - collections can be performed on each horse, therefore each hyperimmunized horse yields between to g of purified antisera. in vitro characterization of equine antisera and f(ab') . the equine antisera and f(ab′ ) products were first characterized against a laboratory generated ebov-egfp for their potential to neutralize ebov. equine antisera and f(ab′ ) were found to possess levels of similar neutralizing activity, with the half effective maximal concentration (ec ) of the products to be . μg/ml and . μg/ml, respectively (fig. ) . moreover, the neutralization activities of both the equine antisera and f(ab′ ) were complete at a concentration of . μg/ml, scientific reports | : | doi: . /srep indicating that the two products were indistinguishable from each other in terms of neutralization at high concentrations. ebov-specific igg in the equine antisera preparations were also determined by elisa. serial -fold the neutralizing activities of equine antisera and f(ab′ ) against ebov-egfp were compared over different concentrations (x-axis). total fluorescence from infected veroe cells at dpi were shown as a percentage of the fluorescence observed with the pbs control, which is set at % (y-axis). samples were processed in triplicate, and error bars indicate standard error. data shown in this figure are representative of three independent neutralization studies with f(ab′ ) or antisera. dilutions of stock antisera with a concentration of - mg/ml were assayed in triplicate over three independent experiments, and the titer was determined to be between , and , endpoint dilutions. were first assessed in guinea pigs, and found to be - hours and - days, respectively (supplementary table ). the protective efficacy of equine antisera and f(ab′ ) were assessed over three experiments in balb/c mice. the first experiment was to compare the efficacy of antisera and f(ab′ ) treatments side-by-side. groups of mice were given intraperitoneal (ip) injections of f(ab′ ) at μg per dose, twice daily for days (b.i.d. × d), starting at either or days post-infection (dpi) with a uniformly lethal dose of ma-ebov. for comparison, groups of mice were administered an ip injection of equine igg at μg per dose (q.d. × d), at either or dpi. control mice were given an equal volume of pbs in place of the treatment (q.d. × d) at dpi. pbs was used instead of non-specific immunoglobulin or f(ab′ ) , because previous studies involving passive transfer of non-specific antisera in mice and nhps did not result in protection , , and thus survival due to the non-specific effects of serum proteins is considered unlikely. control mice lost approximately % of its body weight over the course of the experiment and none survived, with a mean time to death (mtd) of . ± . dpi. five of eight mice given f(ab′ ) starting at dpi survived (p-value < . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi; however, none of the mice given f(ab′ ) starting at dpi survived the challenge (p-value = . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi (fig. a,b) . five of eight mice given antisera at dpi survived (p-value < . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi (fig. c ). three of eight mice given antisera at dpi survived (p-value = . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi (fig. d ). comparing groups with equal treatment times, there was no statistical difference between f(ab′ ) at or dpi (p-value . and . , respectively). however, given that multiple administrations of f(ab′ ) were required to achieve similar protection levels demonstrated by a single injection of antisera, the results suggest that equine antisera is a superior product to f(ab′ ) in terms of efficacy, possibly due to a longer in vivo half-life of equine antiseras. since f(ab′ ) appear to be promising early in ma-ebov infection, the dosage of this treatment was increased for a second experiment. groups of - mice were given ip injections of f(ab′ ) at or mg per dose, twice daily for days (b.i.d. × d), starting at either minutes or dpi with ma-ebov. control mice were treated with pbs (q.d. × d) at dpi. control mice lost approximately . % of its body weight over the course of the experiment and none survived, with a mtd of . ± . dpi. partial survival was observed when treatment began minutes after challenge. four of nine mice in the mg group survived (p-value < . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi, whereas four of ten mice in the mg group survived (p-value < . , compared with pbs group), with an average weight loss of . % and an mtd of . ± . dpi (fig. a,b) . in contrast, all mice survived if treatment was initiated at dpi, with negligible weight loss (less than %) observed in both the and mg groups (p-value < . , compared with pbs group), indicating that protection was complete. these results indicate that f(ab′ ) can contribute to protection from ma-ebov, but only within a certain timeframe after challenge. the efficacy of equine antisera at higher doses was then investigated in a third experiment. groups of mice were given ip injections of antisera at mg per dose (q.d. × d), at minutes, or dpi with ma-ebov. control mice were treated with pbs (q.d. × d) at dpi. control mice lost approximately . % of its body weight over the course of the experiment and none survived, with a mtd of . ± . dpi. all mice treated with antisera at minutes and at dpi survived with negligible weight loss (p-value < . , compared with pbs group). nine of ten mice treated with antisera at dpi also survived the challenge (p-value < . , compared with pbs group), with the lone non-survivor dying at dpi and the average peak weight loss determined to be . % within this treatment group (fig. a,b) . these results again indicate that equine antisera, compared to f(ab′ ) is able to offer a greater contribution to protection from ma-ebov in the mouse model due to the need for fewer administrations to achieve a similar level of protection. guinea pigs were then used to confirm the protective effects from antisera and f(ab′ ) in a higher phylogenic species for ebov challenge, since these animals better mimic hallmarks of human ebov infections, such as coagulation abnormalities . groups of animals were given a single ip injection of antisera (q.d. × d) starting at or dpi with ga-ebov, or twice-daily ip injections of f(ab′ ) for three days (b.i.d. × d), starting at or dpi. each injection dose contained mg antisera or f(ab′ ) . a group of five control animals were given a single injection of pbs at dpi. control guinea pigs lost approximately . % of its body weight over the course of the experiment and none survived, with a mtd of . ± . dpi. all guinea pigs that were given antisera at or dpi survived (p-values = . for both groups, compared with pbs group), with no clinical signs of disease or significant weight loss (fig. a,b) . in addition, animals that were given f(ab′ ) starting at or dpi survived ga-ebov challenge (p-values = . for both groups, compared with pbs group) without signs of disease or significant weight loss (fig. a,b) . the disparity between the efficacy of f(ab′ ) between mice and guinea pigs may be attributed to a much higher dosage of f(ab′ ) administered per weight: each f(ab′ ) dose for guinea pigs was × higher than those given to mice, but the guinea pigs used in this experiment were only - times bigger than the mice by weight. the lack of bsl- laboratories globally, trained personnel as well as the rigors of working under high biocontainment conditions have severely hampered basic research with ebov, leading to a dearth of vaccines and therapeutics for use in humans. these weaknesses were exposed with the - ebov crises, and highlight the value and need for basic and translational science that occurs prior to an impending threat. in an effort to save lives, several experimental candidate treatments that had been tested for efficacy in mice or nhps, in addition to approved or nearly-approved drugs developed against unrelated pathogens, were expedited for use in humans under compassionate circumstances, with varying degrees of success . of these, antibody-based treatments appear to hold the most promise in the clinic and thus should be further investigated for use in humans. passive immunotherapy against ebov had been tried in the past. towards the end of the ebov outbreak in kikwit, zaïre (presently democratic republic of the congo), the passive transfer of whole blood from ebov survivors to patients resulted in the survival of seven out of eight recipients . however, safety concerns with blood transfusions, such as the spreading of blood-borne diseases, allergic reactions and concerns of inefficacy mean that this approach is controversial and will likely not be used unless a better alternative was unavailable. in the s, russian investigators had prepared hyperimmunized antisera from horses vaccinated with inactivated ebov, which were shown to be effective when administered at a dose of mg/kg to baboons, with % survival ( survivors out of ) when given hours before challenge, and % ( survivors out of ) when given immediately after challenge , although baboons are known to be more resistant to ebov infection compared to other nhp species . the survival benefits of the equine antisera were limited to a delay in the onset of viremia clinical symptoms when tested in cynomolgus macaques immediately after challenge, at a dose of mg for an animal weighing between - kg . comparing to successful passive immunotherapy studies with purified igg from nhps ( mg/kg), as well as zmapp and mil- ( mg/kg), the dosage for the cynomolgus macaque experiment was likely too low at the time. use of equine antisera as an emergency post-exposure treatment against ebov has been approved in russia, and several investigators that had been accidentally exposed to the virus have been administered this treatment, although it is not clear if they had actually been infected . the results of this study show that both equine antisera and f(ab′ ) preparations, which can be rapidly produced in large quantities at a lower cost compared to mabs, are effective in the post-exposure treatment of ma-ebov challenged mice and ga-ebov infected guinea pigs. f(ab′ ) was developed in this study due to past documented issues with antisera administrations, in which equine botulinum toxin and anti-snake venom antisera was shown to produce serum sickness at high doses . however, mice that were given a single dose of antisera demonstrated more complete protection over a greater period of time compared to multiple injections of f(ab′ ) . this suggests that f(ab′ ) has a shorter half-life compared to igg (which has been confirmed in this study), and/or that virus neutralization plays a partial role in survival from ebov. the observation that administering f(ab′ ) at dpi is more efficacious than when the same treatment was given at minutes post-exposure (fig. a ) was also observed in past studies with therapeutic ebov gp-specific monoclonal antibodies , , and suggests that virus neutralization may play a bigger role in protection at a later timepoint after ebov challenge. with regards to neutralization, past reports have shown that while elevated antisera levels in nhps vaccinated against ebov correlated with survival, levels of specific neutralizing antibodies did not . it is a possible explanation as to why mab kz , which was originally selected for its ability to neutralize ebov , failed to protect nhps when given at a dose of mg/kg starting day before challenge , and suggests that in the future, the screening of potentially efficacious antibodies against ebov should not be based solely on the ability of the antibody to neutralize virus. fc-dependent antibody functions, which include antibody dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neonatal fragment crystallizable receptor (fcr)-mediated cross-presentation, likely play a role in protection. for instance, studies in mice against yellow fever virus and west nile virus infections have shown that the protective mechanisms of monoclonal antibodies are dependent on fcr , . furthermore, complement component c q has been previously shown in influenza virus and west nile virus infections to directly enhance the neutralizing activities of antibodies , . these mechanisms are not mutually exclusive, but determining their relative importance of each proposed mechanism to efficacy and survival will yield further insight into the specific mechanisms in which antibodies help protect against ebov disease. furthermore, the findings of this study justify the testing of equine igg in a higher phylogenic species for ebov, such as nhps, and may result in the development of a safe and economical method for the production of an effective ebov therapeutic. ethics statement. mice animals. balb/c mice and hartley guinea pigs were purchased from a commercial supplier (charles river). the animals were kept in sterile, autoclaved cages and provided sterilized food and water ad libitum. animals were also provided red, plastic shelters inside the cages as an added source of stimulation. horses were purchased from a commercial supplier (red hill military horse farm) and provided food and water ad libitum. the sequences of ebov gp and vp , with lengths of , and bp, respectively, were derived from genbank (zaire ebolavirus strain zaire , complete genome; genbank accession no. ay ). the gp and vp genes were cloned into the puc- vector, named puc-gp and puc-vp , and then inserted into pfastbac dual vector (life technologies) under the polyhedrin promoter and p promoter respectively, resulting in plasmid pfastbac dual-vp -gp. the purified plasmid was transformed into dh ™ bac e. coli (life technologies) for transposition into a bacmid. a cellfection ® ii reagent (life technologies) was used according to manufacturer instructions to generate recombinant baculoviruses co-expressing ebola vp and gp (rbv-vp -gp). evlp production and inspection. to produce evlp, sf cells were infected with rbv-vp -gp at a multiplicity of infection (moi) of , and incubated at °c for days. culture supernatants were harvested and centrifuged at × g for min to remove cells, and then pelleted by ultracentrifugation at , × g for min at °c. the pellets were re-suspended in pbs and purified through a - - % discontinuous sucrose gradient at , × g for min at °c. the evlp band obtained between % and % density range was collected, washed, and re-suspended in pbs . for immunoblotting, evlp and control sample (cell culture supernatant) were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions, transferred onto a polyvinylidene fluoride (pvdf) membrane (whatman, kent, uk) and then probed with mouse anti-ebov gp polyclonal antibody (prokaryotic expression gp protein immunized mice) or mouse anti-ebov vp monoclonal antibody (ab , abcam) at a dilution of : overnight at °c. the sample was then incubated with horseradish peroxidase (hrp)-conjugated goat anti-mouse secondary antibody at a dilution of : (millipore, boston, ma, usa) for min at °c. the pvdf membrane was colored with a chemiluminescence solution (pierce biotechnology, rockford, il, usa). for electron microscopy, evlp were applied onto a carbon-coated formvar grid, which was immediately stained with % phosphotungstic acid and then observed by a transmission electron microscope. horse immunizations. horses (n = ) were vaccinated intramuscularly (subcutaneous multi-point injection) with either or mg of evlps emulsified in freund's complete adjuvant/freund's incomplete adjuvant at weeks , , , , , and ( times). blood samples were collected from the jugular vein at week prior to the first immunization and week after each immunization, and the sera were stored at − °c until further analysis. fragmentation and purification of equine antibody products. the equine antisera and f(ab′ ) were produced at changchun institute of biological products co., ltd., using the large-scale, current good manufacturing practice compatible equine antiserum manufacturing platform. equine antisera and f(ab′ ) preparations were characterized according to guidelines set forth by chinese pharmacopoeia ( edition), including appearance, color, visible foreign matter, ph value, f(ab′ ) and immunoglobulin content, and sterility. for equine antisera, the blood was taken from the jugular vein of immunized horses, and the sera were separated h later. the horse sera were diluted -fold with pbs, centrifuged at , rpm for min, and supernatants were subjected to filtration through a . μm filter. the antisera were purified by ammonium sulphate precipitation , followed by salt column, and then stored in . % nacl solution. for f(ab′ ) : horse sera were diluted -fold and the ph was adjusted to . using m hcl, and then the antisera were subjected to shock digestion at °c for min with pepsin, and . m naoh was added to terminate digestion. the digestion products were subjected to salt column purification, followed by protein a column. the flow-through fluid was harvested and subjected to ultrafiltration to remove the pepsin and small molecular proteins, and the f(ab′ ) was stored in . % nacl solution. sds-page and thin layer chromatography. the purified equine antisera and f(ab′ ) samples were mixed with non-reducing (without β -mercaptoethanol) protein sample buffer, heated at °c for min, and then subjected to sds-page ( % gel, staining for h and destaining for h); and then the target fractions in the gel were examined by thin layer chromatography scanner (cs- , shimadzu), (transmission, zigzag scan, dual wavelength, swing width: mm, delta y: . mm) to determine the purity of equine antisera and f(ab′ ) . elisa. ebov gp expressed by e. coli bl- was diluted with . m bicarbonate buffer ph . to a final concentration of μg/ml, and coated on -well elisa plates overnight at o c ( μl/well). after blocking with angiotensin converting enzyme (ace) buffer (bio-rad, california, usa) at °c for h, the plates were incubated with μl of serial -fold dilutions of stock equine antisera in triplicate at °c for . h. after washing, μl of hrp-conjugated goat anti-horse igg (comwin biotech co., ltd. beijing, china) diluted : in pbs- . % tween was added to the wells and incubated at °c for min. after washing, μl of substrate ′, , ′ -tetramethylbenzidine (tmb, sigma) was added and incubated at room temperature for min. the reaction was stopped by adding μl of . m h so , and the absorbance was measured at nm. a dilution was considered positive if the absorbance reading was at least twice that of the negative control (pbs) at the same dilution. the igg endpoint titer was calculated as the highest dilution still showing a positive result. the assay was performed independently three times. half-life studies in guinea pigs. group of guinea pigs were administered an ip injection of ml purified equine antisera (neutralization titer : ) or f(ab′ ) (neutralization titer : ). blood samples were collected at , , , , , , , , , , h post-injection, and sera were used for determination of neutralization titer against a recombinant hiv- virus pseudotyped with ebov gp. the time range post-infection in which the titer decreases by % is considered the half-life. each sample was performed in triplicate. determining the protective efficacy of equine antisera. balb/c mice, - week old, female and weighing between - g, were randomly assigned into groups of - mice. all animals were challenged intraperitoneally (ip) with a dose of × ld mouse-adapted ebov (ma-ebov, usamriid/balb/c-lab/ cod/ /mayinga-ma-p ) in μl dmem. the treatment was given ip (q.d. × d) at or days post-infection (dpi) with . mg equine anti-ebov antisera per mouse, or in a subsequent experiment, given ip at minutes, or dpi with mg equine anti-ebov antisera per mouse. the control group was given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. female strain hartley guinea pigs, - week old and weighing between and g, were randomly assigned into groups of animals. all animals were challenged ip with × ld guinea pig-adapted ebov (ga-ebov, vector/c.porcellus-lab/cod/ / mayinga-gpa-p ) in ml dmem. the treatment was given ip (q.d. × d) at or dpi with mg equine anti-ebov igg per animal. a control group of guinea pigs were given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. determining the protective efficacy of f(ab′) . balb/c mice, - week old, female and weighing between - g, were randomly assigned into groups of - mice. all mice were challenged ip with a dose of × ld ma-ebov in μl dmem. the treatment was given ip at or dpi with μg f(ab′ ) per mouse, or in a subsequent experiment, given ip at minutes, or dpi with either mg or mg of f(ab′ ) per mouse. the treatment was given via ip twice a day for days (b.i.d. × d). the control group was given the same volume of pbs as a mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. female strain hartley guinea pigs, - week old, were randomly assigned into groups of animals. all animals were challenged ip with × ld guinea pig-adapted ebov in ml dmem. the treatment was given ip at or dpi with mg f(ab′ ) per animal. the treatment was given via ip twice a day for days (b.i.d. × d). a control group of guinea pigs were given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. pseudotyped virus neutralization assay. titers of equine antisera and f(ab′ ) from horses was tested in an neutralization assay in huh- cells against recombinant hiv- virus pseudotyped with ebov gp. the method for generating pseudotyped viruses was described in a previous publication . briefly, pseudotyped virus containing supernatants were incubated either with serially diluted horse sera at °c for h, before addition to pre-plated target cells in -well culture plates (density of cells/well). cells were re-fed fresh medium h after addition, and followed by lysing cells at h using cell lysis buffer (promega) and transferring the lysates into -well luminometer plates. luciferase substrate (promega) was added to the plates, and the relative luciferase activity was determined. the inhibition of pseudotyped was presented as % inhibition. the highest serum dilution giving over % reduction of luciferase activity was regarded as the neutralizing antibody titer. ebov-egfp neutralization assay. serial two-fold dilutions of f(ab′ ) or antisera (between . to . μg/ml) were incubated with plaque forming units (pfu) of ebov expressing egfp (ebov-egfp, nml/h.sapiens-lab/cod/ /mayinga-egfp-p ) at °c for h, transferred to vero e cells and incubated at °c for h, and then replaced with dmem supplemented with % fetal bovine serum. control wells contained pbs instead of f(ab′ ) or antisera, and all samples were repeated in triplicate. the plates were fixed with % phosphate buffered formalin at h after infection, and scored for the intensity of egfp using the biotek synergy ht microplate reader. the results were expressed as a percentage of the fluorescence reading with the control (which is set at %), and fitted to a -parameter logistic curve (graphpad). statistical analysis. the p-values for rodent studies were determined using the log-rank (mantel-cox) test. calculated values of less than . were considered statistically significant. all in vivo studies were performed once. outbreaks chronology: ebola virus disease clinical management of filovirus-infected patients organization of patient care during the ebola hemorrhagic fever epidemic in kikwit, democratic republic of the congo emergence of zaire ebola virus disease in guinea ebola situation report - postexposure antibody prophylaxis protects nonhuman primates from filovirus disease delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies reversion of advanced ebola virus disease in nonhuman primates with zmapp clinical care of two patients with ebola virus disease in the united states two-mab cocktail protects macaques against the makona variant of ebola virus use of a reduced ( -dose) vaccine schedule for postexposure prophylaxis to prevent human rabies passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal ebola virus infection without complete inhibition of viral replication postexposure antibody prophylaxis protects nonhuman primates from filovirus disease animal models for ebola and marburg virus infections backs against the wall: novel and existing strategies used during the - ebola virus outbreak treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee from whole blood to component therapy: the economic, supply/demand need for implementation of component therapy in sub-saharan africa disease modeling for ebola and marburg viruses passive immunization of ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses defense against filoviruses used as biological weapons hypersensitivity reactions associated with botulinal antitoxin epitopes involved in antibody-mediated protection from ebola virus ebola gp-specific monoclonal antibodies protect mice and guinea pigs from lethal ebola virus infection. plos neglected tropical diseases , e immune parameters correlate with protection against ebola virus infection in rodents and nonhuman primates ebola virus can be effectively neutralized by antibody produced in natural human infection neutralizing antibody fails to impact the course of ebola virus infection in monkeys the fc portion of antibody to yellow fever virus ns is a determinant of protection against yf encephalitis in mice antibodies against west nile virus nonstructural protein ns prevent lethal infection through fc gamma receptor-dependent and -independent mechanisms natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity complement protein c q reduces the stoichiometric threshold for antibody-mediated neutralization of west nile virus ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies a quantitative immunochemical measure of the primary interaction between i bsa and antibody structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus x.z., g.w., y.z., h.w., s.h., y.b., x.q., s.y. and x.x. performed the experiments and analyzed the results. x.z., g.w., g.k., g.f.g., x.q., s.y. and x.x. wrote the manuscript. w.c., h.j., w.g., d.c., z.c., c.w., q.f., h.c., y.g., t.w., n.f., f.y., g.h., y.z., n.l., y.l. and j.q. prepared the horse antisera. g.f.g., x.q., s.y. and x.x. jointly supervised the work. all authors reviewed the manuscript. key: cord- -k jxvzfi authors: noh, ji yeong; jeong, dae gwin; yoon, sun-woo; kim, ji hyung; choi, yong gun; kang, shien-young; kim, hye kwon title: isolation and characterization of novel bat paramyxovirus b - potentially belonging to the proposed genus shaanvirus date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: k jxvzfi the bat paramyxovirus b - was first isolated in korea in this study. using the isolated virus, we could obtain not only genomic information, but also several biological characteristics of the virus. in the phylogenetic analysis, the virus was found to belong to the recently proposed genus shaanvirus. through sequence analyses and in vitro testing, the isolated virus was also found to have haemagglutinin-neuraminidase (hn) protein as one of the structural proteins. when mouse antiserum was generated against the isolated virus and tested, it was cross-reactive to human parainfluenza virus in an indirect immunofluorescence assay but could not cross-neutralize human parainfluenza virus . in addition, the bat paramyxovirus b - was not infectious in the mouse model. collectively, this study provided basic information on further classification of the bat paramyxovirus b - and related viruses in the proposed genus shaanvirus. bats are considered a reservoir of severe emerging infectious diseases. severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), nipah virus, hendra virus, and ebola virus are all thought to be bat-borne viruses , . notably, bats also host major mammalian paramyxoviruses from the family paramyxoviridae, order monone-gavirales , . while henipaviruses (nipah and hendra viruses) in south east asia and australia are associated with fruit bats , other paramyxoviruses have been detected not only in fruit bats but in insectivorous bats worldwide [ ] [ ] [ ] [ ] . a potential pathway for nipah virus transmission from bats to humans was found to be associated with a human-bat interface, specifically date palm sap shared by bats and humans . in addition, serological evidence of possible human infection with a bat-originated paramyxovirus, tioman virus , reinforces the epidemiological role of bats in the emergence of pathogens such as paramyxoviruses in humans. in addition to these bat paramyxoviruses with zoonotic potential, other new paramyxoviruses have been reported. these include several new mammalian paramyxoviruses such as beilong virus and j virus, which remain unassigned under the family paramyxoviridae . recent bat-associated paramyxoviruses were proposed to be grouped in a separate phylogenetic clade within a potentially separate genus such as shaanvirus which was distantly related to jeilongvirus . in addition, novel strains of bat paramyxoviruses in diverse genera have been reported continuously [ ] [ ] [ ] . based on the recent papers, bat paramyxoviruses found worldwide to date have belonged to the genera rubulavirus, morbillivirus, henipavirus and the unclassified proposed genera shaanvirus. expanded classifications for grouping newly identified viruses in bats can be accomplished by further studying the biological characteristics of novel paramyxoviruses as well as genome characterization . in this study, active surveillance was performed to reveal paramyxoviruses circulating in korean bats. a total of bat samples were collected at sites in natural bat habitats and tested for the possible existence of paramyxoviruses. identification of novel bat paramyxovirus b - . in this study, bat samples were collected at sites from natural bat habitats and tested for the possible existence of paramyxoviruses (supplementary table s ). based on the rt-semi-nested pcr using the consensus paramyxovirus primers targeting the rdrp region , five samples were positive and confirmed to be bat paramyxoviruses by sequencing. b - was from a urine sample of miniopterus schreibersii at bt cave in hapcheon in march , and b - was from a feces sample of miniopterus schreibersii at g cave in danyang in april . b - and b - were from feces samples of murina ussuriensis at oj cave in youngwol and at s cave in pyungchang in july , respectively. b - was from a urine sample of miniopterus schreibersii at blr cave in seogwipo in december . among these, one paramyxovirus (b - ) was successfully isolated. the isolated virus, bat paramyxovirus b - , was isolated from the korean bat miniopterus schreibersii. the virus was successfully cultivated after three blind passages in the marc- cell line (fig. ) , which is a clone of the ma- cell line derived from kidney epithelial cells of an african green monkey . viral particles were observed in a pellet of infected marc- cells under a transmission electron microscope (fig. b) . in terms of viral growth, infectious viruses were increased in the culture supernatant and infected cells from hours post inoculation (hpi) and were consistently released for hpi, as measured using tissue culture infectious dose (tcid ) method (fig. c ). infectivity in c bl/ mice. when -week-old female c bl/ mice were inoculated via the intranasal and intragastric routes with tissue culture infectious dose (tcid) /ml or . tcid /ml of bat paramyxovirus b - , we found no evidence of infection, i.e. no viral shedding, histopathological findings, or seroconversion. characterization of the bat paramyxovirus b - . using high throughput sequencing (hiseq. sequencing system based on the transcriptome de novo sequencing platform), , contigs totaling , , base pairs (bps) with an average length of bps were obtained. in the taxonomy annotation with mg-rast, , contigs were annotated. while most of the annotated sequences associated with eukaryotes, sequences were associated with family paramyxoviridae. the , bases of viral genomic sequences were obtained by rt-pcr using primers designed based on the paramyxoviridae-associated contigs and race sequencing. the genomic sequence of the virus encodes open reading frames (orfs) ( fig. a) , which is consistent with bat paramyxovirus strain ms-parav/anhui (kc ) . in addition, maximum-likelihood trees showed that the bat paramyxovirus b - strain was closely related to the bat paramyxovirus ms-parav/anhui (kc ) and btmi-parav/qh (kj ) strains rather than beilong virus, j-virus, and tailam virus (figs b and s ). even though the hn amino acids sequences were similar to those from viruses in the proposed genera shaanvirus, it was also related to that of sendai virus and human parainfluenza virus , which belong to a different genus, respirovirus showing (table ) . based on the partial rna-dependent rna polymerase (rdrp) gene, the bat paramyxovirus isolates and other korean bat paramyxoviruses belong to the genus shaanvirus, with . - . % nucleotide similarities (fig. c ). this suggests a single, closely related group of paramyxoviruses circulating in korea. consistent with the genetic findings, the bat paramyxovirus b - was found to have haemagglutinin and neuraminidase activities in the haemadsorption assay ( . % chicken red blood cells in pbs (ph . )) and neuraminidase assay, respectively (fig. ). in the preliminary studies of mouse antisera (balb/ ca mice) against human respiratory syncytial virus a (kbpv-vr- ), human mumps virus (kbpv-vr- ) and human parainfluenza virus (kbpv-vr- ), the antisera against human parainfluenza virus (kbpv-vr- ) was only cross-reactive with the bat paramyxovirus b - as determined using the immunofluorescence assay. therefore, further cross-reactivity and cross-neutralizing tests between human parainfluenza virus (kbpv-vr- ) and the bat paramyxovirus b - were performed. as shown in table , partial cross-reactivities were observed between the two viruses in the indirect immunofluorescence assay, even though they belong to different genera. among the six mouse sera against the bat paramyxovirus b - , two sera were cross-reactive to human parainfluenza virus (kbpv-vr- ) with and as the end-point titers for the fluorescent signals. in addition, among three pooled sera (two mouse sera each) against human parainfluenza virus (kbpv-vr- ), one pooled serum was cross-reactive to the bat paramyxovirus b - with as the end-point titer for the fluorescent signal ( table , fig. ). while the mouse antisera neutralized the homologous virus with - serum neutralizing titer, they did not neutralize the heterologous virus (table ). the bat paramyxovirus b - in this study was similar to viruses in the recently proposed genera shaanvirus and jeilongvirus. the genus jeilongvirus includes beilong virus, which was first isolated from human kidney mesangial cells , and j virus, first isolated from mice . these two viruses were only recently proposed to form jeilongvirus as a distinct genus . in addition, the newly discovered bat paramyxoviruses, ms-parav/ anhui and btml-parav/qh , were recently proposed to form the additional genus shaanvirus due to their genomic length and sequence identity differences . however, their taxonomy has not yet been confirmed by the international committee on taxonomy of viruses (ictv, https://talk.ictvonline.org). the proposed genera shaanvirus and jeilongvirus have two novel genes putatively encoding sh (small hydrophobic) and tm (transmembrane) proteins , . two viral genes were also located between the fusion and haemagglutinin-neuraminidase genes of bat paramyxovirus b - . further, their nucleotide and amino acids reference viruses belonging to paramyxoviridae were included and aligned by mafft. the phylogenetic tree was generated by the maximum-likelihood method with , replicates of bootstrap sampling and the kimura -parameter model using mega . bat paramyxovirus b - isolated in this study is indicated with the blue box. (c) phylogenetic analysis based on partial rna-dependent rna polymerase (rdrp) nucleotide sequences of bat paramyxoviruses detected in the korean bats and other reference viruses belonging to paramyxoviridae. the phylogenetic tree was generated by the maximum-likelihood method with , replicates of bootstrap sampling and the kimura -parameter model using mega . korean bat paramyxoviruses are indicated by blue boxes. sequences were similar to the sh and tm genes identified from bat paramyxovirus strain ms-parav/anhui . therefore, the isolated bat paramyxovirus b - might belong to the proposed genus shaanvirus rather than jeilongvirus. the bat paramyxovirus b - in this study was predicted to have a haemagglutinin-neuraminidase (hn) protein as an attachment glycoprotein in the sequence analysis. consistent with the genetic findings, the bat paramyxovirus b - was found to have haemagglutinin and neuraminidase activities in the haemadsorption assay ( . % chicken red blood cells in pbs (ph . )) and neuraminidase assay, respectively ( fig. a,b) . notably, even though the hn amino acids sequences were similar to those from viruses in the proposed genera shaanvirus, it was also related to that of sendai virus and human parainfluenza virus , which belong to a different genus, respirovirus (table ) . maximum-likelihood tree analysis based on the full hn amino acid sequences also showed that the bat paramyxovirus b - was related to viruses in the genus respirovirus ( supplementary fig. s ). therefore, the b - hn amino acid sequence was compared to that of human parainfluenza virus (kbpv-vr- ), and six regions that have at least six conserved amino acids were found. these regions were located on the sialidase superfamily domain and were structurally gathered together ( supplementary fig. s ). among the seven neuraminidase active sites (r, d, e, r, r, y, and e ), two of these (e at the third site and y) were located within the conserved amino acid sequences. these bioinformatics findings might indicate that the hn proteins of bat paramyxovirus b - and human parainfluenza virus (kbpv-vr- ) are genetically related, even though they are in different genera. further study on their antigenic and evolutionary relationship should be followed up. notably, in a previous study, bat serum (diluted / ) from african wild bat, micropteropus pusillus was reactive with human parainfluenza virus in an indirect immunofluorescence assay . there may be two possibilities to explain this. first, the african bat might have been infected with a human parainfluenza virus -related virus in the genus respirovirus that has not yet been found. however, as there has been no evidence of bat paramyxoviruses related to the genus respirovirus in recent studies , , , the first possibility seems low. second, the bat might have been infected by other bat paramyxoviruses which were cross-reactive with human parainfluenza virus . in fact, in this study, when mouse antisera were made and tested against bat paramyxovirus b - and human parainfluenza virus (kbpv-vr- ), the two viruses were partially cross-reactive to each other in an indirect immunofluorescence assay. this result provided an evidence that there could be cross-reactivity between viruses belonging to different genera. therefore, the bat paramyxovirus b - -related viruses circulating in bats might be one of the reasons why the wild bat sera was reactive with human parainfluenza virus in the previous study. however, we could not find the cross-neutralization between bat paramyxovirus b - and human parainfluenza virus . based on the recent studies, cross-neutralization was found between the viruses in the same genus of paramyxoviruses: sendai virus and human parainfluenza virus , which both belong to the genus respirovirus , and human mumps virus and african bat mumps virus, which both belong to the genus rubulavirus . further, the engaged proteins in the cross-reactivity between bat paramyxovirus b - and human parainfluenza virus had not been revealed. therefore, although bat paramyxovirus b - in this study was cross-reactive with human parainfluenza virus in indirect immunofluorescence assay, it is difficult to say that the bat paramyxovirus b - was antigenically related with human parainfluenza virus . since a number of paramyxoviruses have been newly found around world, their classification has been difficult due to the limited criteria, such as sequence alone . additional input from the biological context may be helpful. in this study, a bat paramyxovirus b - belonging to the proposed genus shaanvirus was first isolated. through virus isolation, we could obtain not only nucleotide sequence information, but also several biological characteristics of the virus. its putative hn protein was demonstrated to have haemagglutinin and neuraminidase activities. mouse antisera against the bat paramyxovirus b - was also cross-reactive to human parainfluenza virus in the indirect immunofluorescence assay, but could not cross-neutralize human parainfluenza virus . additionally, the bat paramyxovirus b - was not infectious in -week-old female c bl/ mice. therefore, this study provided basic information on further classification of the bat paramyxovirus b - and related viruses in the proposed genus shaanvirus. samples. in , a total of samples were collected at sites in natural bat habitats. most samples were collected as fresh guano under the colony of bats. in some cases, bats were captured by a fine mesh net to collect fresh feces as well as oropharyngeal and urine swabs, and the animals were then immediately released. the collected samples were placed into transport medium (noble bio. co. ltd) in a % suspension and were transported to the lab (bsl ) for further analysis. the major bat species at the collection sites were determined based on morphology and on previous data from bats' roosting sites table s ). rna extracted from bat samples was tested by rt-semi-nested pcr using consensus paramyxovirus primers targeting the rdrp region . the nucleotide sequences of the positive samples were determined by cosmogenetech co. ltd. five of the bat samples were confirmed positive to the bat paramyxoviruses by sequencing. virus isolation was performed from the rt-semi-nested pcr-positive samples including feces, urine, and oropharyngeal swabs from bats. each bat sample was diluted -fold with fresh dulbecco modified eagle medium (dmem) and filtered with a . -μm filter before being inoculated into the marc cell line, a clone from the ma- cell line from kidney epithelial cells of an african green monkey ; blind passaging was performed up to three times. the cytopathic effect (cpe) was confirmed in cells infected with one sample (b - ) of five pcr-positive samples. next, a monolayer of marc was infected with bat paramyxovirus (b - ) at a multiplicity of infection (moi) of . . two days post-infection, the infected cells were harvested and pelleted. the infected cell pellet was fixed with . % glutaraldehyde in pbs (ph . ) for hours and sent for transmission electron microscopy (tem) to the advanced analysis center in the korean institute of science and technology in korea. briefly, after washing with pbs the cells were post-fixed in % osmium tetroxide and dehydrated in an ethanol series ( %, %, %, %, %, and % (two times)). then, the sample was transited with % propylene oxide and embedded in epoxy resin. the embedded sample was sectioned into - nm sections with an ultramicrotome (ultra cut c; leica), stained with % uranyl acetate and lead citrate on a copper grid, and examined using tem mode on a cryotecnai f (fei ltd., usa). culture supernatant and freeze-thawed cell lysate were centrifuged at , × g for minutes to remove cell pellets and then used for viral titration. the infectious viruses were quantified from the prepared samples using the tissue culture infectious dose (tcid ) method. briefly, the prepared samples were -fold serially diluted and inoculated to monolayers of marc- cells in wells. the highest dilution showing % of viral infection was obtained by observing cytopathic effects with a light inverted microscope (ix s f- , olympus corporation). the tcid was calculated using the spearman-karber method. genomic sequencing and analyses. a virus isolate showing cytopathic effects (cpes) was filtered through a . μm filter and ultra-centrifuged at , × g for hour. each pellet was suspended in µl of x digestion buffer (turbo dna free kit; ambion) and treated with turbo dnase; the suspension was incubated at °c for min. the rna from the suspension was isolated using the trizol ls reagent according to the manufacturer's instructions (ambion). the extracted rna was submitted to macrogen (seoul, korea) for high throughput sequencing in a hiseq. sequencing system based on the transcriptome de novo sequencing platform. the obtained viral contigs were analysed and annotated by the mg-rast server . the cut-off for the annotation was − maximum e-value, % minimum percentage identity, and for minimum alignment length. the viral genomic sequences were obtained by rt-pcr using primers based on the paramyxoviridae-associated contigs obtained by high throughput sequencing. additionally, ′ and ′ end sequences were obtained by race sequencing based on pcr using the adapter-oligo dtvn primer from bionics co. ltd. for human parainfluenza virus (kbpv-vr- ), the , bases of the hn gene were obtained by rt-nested pcr following the previously published method . the obtained genomic rna sequences and rt-pcr-positive amplicons were further analysed with related sequences in genbank using mafft and mega version . genomic sequence data generated in this study have been deposited in genbank under accession number mg . the full hn sequence of human parainfluenza virus (kbpv-vr- ) has also been deposited in genbank under accession number mg . the hn amino acid sequences of human parainfluenza virus (kbpv-vr- ) and bat paramyxovirus were further compared with the bioedit program . putative hn protein structures were predicted using the phyre website and visualized with the ucsf chimera . . program . haemadsorption and neuraminidase assays. to investigate the activity of the hn protein, the haemadsorption assay was used. monolayer cultures of marc cells in -well plates were inoculated with the bat paramyxovirus b - at an moi of . . after hours, the cells were gently washed with pbs and incubated for hour with . % chicken red blood cells (rbcs) in pbs (ph . ) at room temperature. then, the cells were washed with pbs twice to remove unbound rbcs and observed under a microscope. in addition, the neuraminidase assay was performed with the viral isolate using the na-fluor influenza neuraminidase assay kit (thermo) according to the manufacturer's protocol. the virus was diluted -fold with the na-fluor × assay buffer in a black -well plate and incubated for hour at °c with the na-fluor substrate. after adding the stop solution, the plate was read at the excitation and emission wavelengths of nm and nm, respectively, using a perkinelmer victor fluorometer. a total of -week-old female c bl/ mice maintained under specific-pathogen-free (spf) conditions were divided into two groups of negative controls (n = each) and four inoculation groups (n = each). the inoculation groups were inoculated with µl of tcid or . tcid of the bat paramyxovirus b - via intra-gastric administration routes, and µl of tcid or . tcid of the bat paramyxovirus b - via the intranasal administration routes. each inoculation was conducted twice with week in between. weight loss was monitored daily for up to weeks. the lungs, liver, brain, and intestine were harvested at or days post-challenge, fixed with % paraformaldehyde in pbs, ph = . , and submitted to biolead inc., korea, for histopathological examination. additionally, rna was extracted from each tissue and from oral swabs and fecal samples collected daily for days post-challenge. the extracted rna was tested using rt-semi-nested pcr with the consensus paramyxovirus primers . in addition, real time pcr was performed with newly designed primers and probes targeting regions of the membrane and fusion protein. briefly, the designed primers were as follows: forward primer: pvm-f ′-cccaggagtatggttatcaagtgagg- ′; reverse primer: pvm-r ′-tcca ttgggctctctttgtttgc- ′; taqman probe: pvm-p ′-fam-cccatcccagaccagccacca gaccc-tamra- ′ real time rt-pcr was performed as follows: reverse transcription at °c for min, followed by pcr − °c for min, cycling times at °c for sec, and °c for sec. using the -fold diluted virus (bat paramyxovirus b - ), standard curves were performed for every reaction. generation of mouse antisera against bat paramyxovirus b - and human paramyxoviruses. we next observed preliminary cross-reactivity in indirect immunofluorescence assay between several human viruses belonging to the family paramyxoviridae and the bat paramyxovirus b - . -week-old female balb/ca mice maintained under spf conditions were intramuscularly inoculated twice, with weeks in between, with a : ratio mixture of addavax adjuvant and live virus stock (bat paramyxovirus b - , human respiratory syncytial virus a (kbpv-vr- ), human mumps virus (kbpv-vr- ), and human parainfluenza virus (kbpv-vr- ), respectively, from the korea bank for pathogenic viruses. two weeks after the last immunization, antisera against the virus were obtained by cardiac puncture for further studies related to cross-reactivity in indirect immunofluorescence assay. mouse antisera against the bat paramyxovirus b - ( . tcid /ml) and human parainfluenza virus (kbpv-vr- , . tcid /ml) were further compared through serum titration using the indirect immunofluorescence assay and serum neutralization test. the washed cells were fixed with % ethanol for hour at − °c and rehydrated with pbs (ph . ) for min at room temperature. primary antibodies used in the experiments were the mouse antisera against bat paramyxovirus b - and human parainfluenza virus (kbpv-vr- ). the sera were diluted with % culture supernatants from the cell lines in fresh dmem; the corresponding mock-infected cells were treated in the same way as the target virus culture. then, the sera were serially diluted -fold from an initial dilution of : and added to the fixed infected or mock-infected cells and incubated for hour at room temperature. the plates were washed three times with pbs containing with the same serum diluent and was added to the plates and incubated for hour at room temperature. after three washes with pbs-t, the plates were viewed with a fluorescence microscope they were then diluted -fold from an initial dilution of : in -well plates and mixed with tcid of each virus. the plates were then incubated at °c for hour. the virus-serum mixtures for the bat paramyxovirus b - and human parainfluenza virus (kbpv-vr- ) were added to monolayer cultures of marc and llc-mk cells, 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analysis ucsf chimera-a visualization system for exploratory research and analysis data availability. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -iif lnez authors: linster, martin; do, lien anh ha; minh, ngo ngoc quang; chen, yihui; zhe, zhu; tuan, tran anh; tuan, ha manh; su, yvonne c. f.; van doorn, h. rogier; moorthy, mahesh; smith, gavin j. d. title: clinical and molecular epidemiology of human parainfluenza viruses – in children from viet nam date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: iif lnez hpivs are serologically and genetically grouped into four species that account for up to % of all hospitalizations due to acute respiratory infection in children under the age of five. genetic and epidemiological data for the four hpivs derived from two pediatric cohorts in viet nam are presented. respiratory samples were screened for hpiv – by real-time pcr. demographic and clinical data of patients infected with different hpiv were compared. we used a hemi-nested pcr approach to generate viral genome sequences from hpiv-positive samples and conducted a comprehensive phylogenetic analysis. in total, samples tested positive for hpiv. hpiv was most commonly detected in our cohort and co-detections of hpiv with other respiratory viruses were found. phylogenetic analyses suggest local endemic circulation as well as punctuated introductions of new hpiv lineages. viral gene flow analysis revealed that viet nam is a net importer of viral genetic diversity. epidemiological analyses imply similar disease severity for all hpiv species. hpiv sequences from viet nam formed local clusters and were interspersed with sequences from diverse geographic regions. combined, this new knowledge will help to investigate global hpiv circulation patterns in more detail and ultimately define more suitable vaccine strains. viruses can infect individuals from all age groups. consequently, they likely form one gene pool with endemic circulation across all ages. factors contributing to successful transmission include shedding of the virus at sufficiently high titers, from the upper respiratory tract, environmental stability of the virion and ability to initiate infection upon contact with a new host. hpivs replicate abundantly in the tracheal epithelium facilitating transmission from the upper respiratory tract and are likely highly infectious, as was demonstrated for a closely related murine virus , . transmission of hpivs is by close contact and short-range transmission by respiratory droplets with minimal aerosol transmission . hpiv also retains its infectivity on inanimate objects for up to hours and its incubation period as found in volunteer studies was estimated at - days . no specific antivirals or vaccines to treat and prevent hpiv infection are currently available. periodicity and magnitude of hpiv epidemics is likely determined by susceptibility to infection, partial cross-protective immunity and differences in virus transmissibility [ ] [ ] [ ] [ ] . disease presentation and severity varies between hpiv species, geographic locations and over time . however, our understanding of the molecular epidemiology and global transmission of hpiv is limited since sequence information to date is mainly restricted to the hemagglutinin-neuraminidase (hn) gene, while full genome data is sparse and biased towards the americas. the present study describes species-specific clinical presentation, the genetic variability and hpiv circulation in viet nam. the outcome of rsv infection in hospitalized children under years of age presenting with acute lower respiratory infection (alri) in ho chi minh city was described previously . a second study was conducted enrolling children under years of age presenting with acute respiratory infection (ari) to outpatient departments. respiratory samples from both cohorts that were positive for hpiv were sourced into the current study for sequencing purposes. using bayesian phylogenetic methods, we also infer the importation of hpiv into viet nam from viral sequence data and report high viral diversity within the country. patients and samples. samples were sourced from two previous acute respiratory infection (ari) cohorts among in-and outpatients, that were conducted at children's hospital and in ho chi minh city, viet nam during years and . detailed information on patient enrolment, sample collection and clinical parameters for the inpatient study are provided in do et al. . briefly, children under years of age were eligible if admitted for a lower respiratory tract infection with an onset of symptoms less than days prior to recruitment. patients with uncomplicated non-respiratory or non-infectious respiratory causes for hospitalisation were excluded. in the outpatient study children under years of age, living in ho chi minh city, and with a clinical diagnosis of acute respiratory infection (defined as presenting with cough, sore throat, runny nose or nasal congestion for less than days as the chief complaint) with no underlying illness except asthma were enrolled at the outpatient clinic of children's hospital . three types of respiratory specimens (nasal swabs, throat swabs, and nasopharyngeal aspirates) were collected on admission by trained personnel using standard operating procedures, and were placed in viral transport medium . specimens were kept at °c for a maximum of h and then aliquoted and stored at − °c until further processing. rna was extracted and viral nucleic acid was detected as described previously [ ] [ ] [ ] . the studies were approved by the scientific and ethical committee of the hospital for tropical diseases, the institutional review boards of children's hospital and and the oxford university tropical research ethical committee under the approval numbers . for the inpatient and . for the outpatient study. written informed consent was obtained from parents or legal guardians of all children enrolled in the study. pcr amplification and sequencing. complementary dna (cdna) was prepared from hpiv-positive samples as previously described and shipped to duke-nus for pcr amplification and sanger sequencing. for sequencing, hpiv-positive samples were further amplified in a hemi-nested pcr approach with newly designed species-specific primers targeting overlapping regions of the viral genome (table s ) . briefly, the first-round pcr was conducted in or separate reactions and amplified an 'outer' pcr product of about ~ kb collectively covering the entire genome of hpiv (~ kb for hpiv - , ~ kb for hpiv ). these initial pcr products were further amplified in a second-round pcr yielding separate overlapping 'inner' products of . - . kb. amplicons were gel-purified and sequenced with pcr primers. if required, pcrs and sequencing reactions using additional primers were performed to achieve full genome coverage. low quality reads were manually curated, primer sequences were removed and contigs were assembled in geneious version . . nucleotide alignments of the hn gene of all hpiv species and whole genome sequences of hpiv were created using mafft . phylogenetic analysis. all available hn gene sequences of the hpiv species were downloaded from the niaid virus pathogen database and analysis resource (vipr) and annotated with sample collection date and country. temporal phylogenies of individual hpiv species were inferred in beast v . . using a strict clock, the hky substitution model, and a constant coalescent tree prior. mcmc chains were run for million steps with sampling every , generations. run convergence was confirmed in tracer v . after % burn-in removal . maximum clade credibility (mcc) trees were generated using tree annotator v . . the phylogeny of hpiv was further inferred from available whole-genome sequences. all phylogenetic trees were visualized using of migration in and out of viet nam were inferred using an asymmetric substitution model with the bayesian stochastic search variable selection (bssvs) option. two independent runs of million generations were combined using logcombiner v . . after removal of appropriate burn-in and the mean migration rates were extracted from the combined log file. epidemiological analysis. we analysed the clinical characteristics of hpiv species from the two studies from which our samples were sourced. demographics (age, sex) and clinical metadata (duration of illness at presentation, clinical symptoms pulse, respiratory rate, total episode duration, icu admission, clinical diagnosis and disease outcome) recorded during the original studies were used. for each of the studies, we assessed the variability of parameters (age, duration of illness, pulse, and respiratory rate) across hpiv species with a kruskal-wallis test with a dunn post-test. proportions of demographic and clinical variables were compared between hpiv species with a chi-squared test with yates' correction. the clinical diagnosis at presentation and follow-up were classified as upper respiratory infection (rhinitis, rhinopharyngitis or laryngitis) or bronchiolitis/ pneumonia or any lower respiratory infection (either bronchitis, croup, bronchiolitis or pneumonia). a p-value of < . was considered significant. all statistical analyses were performed on graphpad prism . d for mac software (graphpad software, la jolla, california usa). data availability. all data generated or analysed during this study are included in this published article (and its supplementary information files). sequence data generated on this study has been deposited in the ncbi repository (https://www.ncbi.nlm.nih.gov/nuccore). any additional information is available from the authors on request. a total of cdna samples tested positive for hpiv (n = , , and for hpiv - , respectively). of these, patient samples tested positive for only a single hpiv species while were co-infections of two hpiv species; hpiv and hpiv (n = ), hpiv and hpiv (n = ), hpiv and hpiv (n = ) ( table ). the remaining ( %) hpiv-positive samples also tested positive for other respiratory viruses. in general, hpiv and hpiv were more commonly associated with viral co-infections, with rhinovirus/enterovirus most frequently co-detected, being present in of ( %) of hpiv infections. notably, bocavirus was found as a co-infection with each hpiv species, and was found in of ( %) hpiv-positive samples. we first examined the clinical presentation of hpiv in the outpatient study that recruited children under years with ari. in this study, the median age and gender distribution of infected children and frequency of clinical presentation (fever, cough, sore throat, runny nose and nasal congestion) as well as vital signs (pulse and respiratory rate) and duration of illness at presentation were similar between hpiv species (p > . ). for all the hpiv species, presentation as an illness with wheeze was more common than with crepitations. next, we compared the clinical diagnosis at presentation and follow-up. the proportion of pneumonia or bronchiolitis at presentation was / ( %), / ( %), / ( %), / ( %) for hpiv - , respectively. further, the proportion presenting as lower respiratory infection was high both at presentation [ / ( %), / ( %), / ( %) and / ( %)] and follow-up [ / ( %), / ( %), / ( %), / ( %)] for hpiv - , respectively, indicating a high morbidity and poor resolution of symptoms. the inpatient study consisted of children under years of age with a diagnosis of alri . the age, gender distribution and clinical presentation were similar between viral species. for all hpiv species, at least one clinical sign of alri (rapid respiratory rate or intercostal in-drawing) was present in the majority of children [hpiv / ( %), hpiv / ( %), hpiv / ( %), hpiv / ( %)]. bronchiolitis was more commonly seen with hpiv , and ( / , %; / , %; / , %; respectively) compared to hpiv ( / , %). complete clinical recovery at days post-admission was infrequent for hpiv and hpiv ( / , / , % each, respectively) compared to hpiv and hpiv ( / , % and / , %, respectively) (p > . ). the duration of the alri episode as inferred by a telephonic interview at - months, was similar for all hpiv species (p > . ). oxygen therapy at admission was only required for a subset of patients infected with hpiv ( / , %) and hpiv ( / , %), while only the two hpiv infected patients were admitted to intensive care. we were able to generate sequences from ( %) of the available cdna samples from both studies. in total, we obtained viral gene sequences, wherein were above % total gene coverage. for co-infections, sequences were obtained from the hpiv species with the lower c q value. the individual virus names, sampling dates, sequenced viral genes and corresponding accession numbers are listed in supplementary table . we reconstructed the temporal phylogeny using the newly generated full-length hn gene of hpiv - (n = , , and , respectively) and publicly available data (n = , , and , respectively). results indicated that hpiv has been circulating in humans since the 's, with time to the most recent common ancestor (tmrca) estimated as ( % highest posterior density (hpd): - ) ( table ). since the mid- s, hpiv has diverged into two distinct monophyletic lineages (clades and ) that co-circulate worldwide. all vietnamese viruses sequenced within this study clustered in clade along with viruses from argentina, japan and usa (fig. a) . limited genetic data is available for hpiv and hpiv and this may influence the estimation of dates and these results must therefore be interpreted with caution. the mean tmrca for hpiv was estimated as ( % hpd: - ). hpiv also diverged into two clades with clade viruses predominant in the s until they were superseded by clade viruses during the early s (fig. b) . all vietnamese hpiv viruses sequenced from this study, as well as all sequences obtained after the year from asia, belong to clade . the mean tmrca of the hpiv viruses was estimated at ( % hpd: - ), while the tmrcas for hpiv a and hpiv b were estimated as ( % hpd: - ) and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , respectively. the single vietnamese hpiv sequence grouped with hpiv a subspecies viruses but was relatively distant and in a basal position to other known hpiv a virus sequences. the mean tmrca of hpiv was estimated at ( % hpd: - ), which is similar to the age of hpiv (table ). hpiv has also diverged into two major monophyletic clades (clades and in fig. ) . in marked contrast to other hpivs, the hpiv phylogeny exhibits more extensive lineage diversification in the s, with some indication of continuous genetic drift, indicated by a ladder-like phylogeny , that is more evident within the clade than clade ( fig. a) . viral lineage turnover is also present, whereby older virus strains become extinct and are replaced by newer strains. clade viruses also demonstrated greater diversification of multiple independent lineages and inter-continental mixing of the viruses than clade viruses. our results also show that the hpiv infections in viet nam during - resulted from at least five independent virus introductions that are highlighted as clades v -v in fig. a . the viruses detected in viet nam were generally closely related to viruses from east asia, although clades comprised viruses from diverse geographic areas (for examples see fig. b and c). in clades v and v there is one cluster each of hpiv viruses from viet nam that were detected over a period of - months, suggesting sustained endemic circulation. the mean tmrca of clade v was estimated as ( % hpd: [ ] [ ] [ ] suggesting that these viruses may have been circulating in viet nam for at least years. however, we cannot exclude the possibility that this may be due to inadequate global sampling and sequencing of hpiv viruses. we also reconstructed the hpiv phylogeny using newly generated whole genomes and publicly available genomes. the whole-genome hpiv phylogeny was largely congruent with the hn phylogeny, showing multiple clades of viruses that circulate globally (supplementary fig. ) . we further performed phylogeographic analysis to investigate the extent of hpiv migration into and out of viet nam during - . our results show that the total diffusion rates of hpiv into viet nam from all other areas are significantly greater than from viet nam into all other regions . this indicates that viet nam was not acting as a source population for epidemics elsewhere but was a net importer of hpiv diversity in - . this study reaffirms the described clinical presentation of hpiv species . in both cohorts, hpiv-infected patients presented within the clinical spectrum of both auri and alri. characteristic clinical features could not be attributed to a specific hpiv species. among outpatients, all four hpiv species predominately manifested with alri (bronchitis, bronchiolitis, pneumonia). this is remarkable in the case of hpiv , which was previously known to cause mild clinical illness infection with all hpiv species at initial consultation, with only incomplete resolution of symptoms at follow-up, is an indication of high disease severity in this cohort. in the inpatient cohort, which in contrast to the outpatient cohort used clinically manifesting alri as an inclusion criterion, two cases of hpiv infection resulted in admission to the intensive care unit, possibly indicating enhanced pathogenicity of hpiv over the other species as suggested previously , , , although the episode duration was similar for all hpiv species. all hpiv species have been detected in diverse geographical regions and several circulation patterns have been suggested , . in our study, viral co-detections were common among samples positive for all hpiv species. our rate of viral co-infections of % is within previous reports and highlights the importance for screening for several viral pathogens simultaneously . rhino-/enteroviruses were most commonly found in combination with hpiv, possibly due to longer duration of shedding with the former and/or frequent orofecal transmission in paediatric patients . notably, co-detection of bocavirus was found among all hpiv species and in both cohorts. furthermore, the complete absence of influenza virus co-infection in both cohorts is remarkable. human adenovirus, human respiratory syncytial virus, human metapneumovirus, seasonal coronaviruses, and human parechovirus were only incidentally detected in combination with hpiv. the concurrent detection of hpiv and hpiv occurred in nine instances in the outpatient cohort, suggesting this combination to be rather common. hpivs are usually detected throughout the year, but enhanced epidemic transmission probably occurs during the colder months of the year. fry et al. described an interaction of hpiv and hpiv detections in the us . in our study, we found co-detection of hpivs year-round, however, no distinct peak was observed. this is likely due to the study design as the original studies among in-and out-patients were conducted over a limited time span. we here report a high number of co-detections, which is in line with previous acute respiratory infection studies , . however, we were unable to identify specific clinical symptoms associated with individual hpiv species, and future studies employing an inclusive case definition are needed to identify risk factors for severe disease in cases of hpiv infection and co-infection. several factors might have contributed to the fact that not all detected hpivs could be amplified and sequenced. first, suboptimal primer design is likely due to the limited number of available sequences per hpiv species available in public repositories and unknown sequence diversity; second, we did not retest positive qpcr results allowing for false-positive test results; and third, the rna might have degraded after their initial collection and during long-term storage and shipping. phylogenetic analysis for hpiv and hpiv was restricted due to low sequence numbers from viet nam and globally. two groups of hpiv co-circulate globally but only restricted diversity was detected in this study. viet nam viruses belong to clade and are most closely related to virus sequences from argentina, japan and usa. in contrast, hpiv sequences from viet nam belonged to multiple clades and grouped with viruses from diverse geographical regions. multiple introductions of hpiv into viet nam were observed in and , and in two cases these may have resulted in sustained endemic transmission, while clades comprised of viruses from different geographical areas suggests frequent transmission between those areas. in addition, phylogeographic analysis indicated that viet nam was a net importer of hpiv diversity. while other viruses such as influenza a/h n viruses and rsv circulate globally [ ] [ ] [ ] [ ] , this has not 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thank carmen butt, ainie zahir, divaashini ravichandran, and lai xing yi for excellent technical assistance. this study was supported by the duke-nus signature research programme funded by the ministry of health, singapore and by research grants from the national medical research council (nmrc/ cirg/ / ) and the ministry of health (moh/cdphrg/ / ), singapore. the original studies in which the samples were collected were funded by the wellcome trust of great britain. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -b dk tk authors: liang, xun; sun, leqiang; yu, teng; pan, yongfei; wang, dongdong; hu, xueying; fu, zhenfang; he, qigai; cao, gang title: a crispr/cas and cre/lox system-based express vaccine development strategy against re-emerging pseudorabies virus date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: b dk tk virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. here we present an express vaccine development strategy based on crispr/cas and cre/lox system against re-emerging pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in china. by crispr/cas system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using cre/lox system for vaccine safety concern. notably, single cell facs technology was applied to further promote virus purification efficiency. the combination of these state-of-art technologies greatly accelerated vaccine development. finally, vaccination and challenge experiments proved this vaccine candidate’s protective efficacy in pigs and the promise to control current pseudorabies outbreak. this is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. it may pave the way for future express antiviral vaccine development. infectious diseases caused by novel emerging and re-emerging viruses pose a continuous threat to our health and economy [ ] [ ] [ ] [ ] . vaccination is the most effective way to prevent viral infectious diseases, which saves millions of lives every year, yet the current long and laborious journey to develop antiviral vaccines is very inefficient . more challengingly, virus evolves rapidly to escape the old vaccine-induced immunity by changing its genome architect . therefore, new biotechnologies to facilitate and accelerate vaccine development against novel emerging and re-emerging viruses are desperately needed, for the endless arms race with the dynamically evolving virus. although the genome of dna viruses is relatively more stable than rna viruses . there are many studies reported outbreaks of infectious diseases caused by re-emerging dna virus, such as adenovirus, herpes simplex virus (hsv), chicken pox virus (varicella), hepatitis b virus (hbv), cytomegalovirus (cmv), etc. , , . recently, the pseudorabies virus (prv), a model herpes virus, was widely prevalent in vaccinated pig farms in china, and caused tremendous economic loss in the swine industry [ ] [ ] [ ] [ ] [ ] . prv is a member of the alpha herpesvirinae subfamily and constitutes approximately k double strand dna genome . prv infection caused pseudorabies is one of the most devastating swine infectious diseases in the swine industry worldwide . it has been well controlled for decades by using attenuated and gene deletion vaccines. however, in spite of the great efforts on prv vaccination, pseudorabies re-emerged as one of the top swine epidemic diseases in recent times, most likely due to prv mutation caused antigenic drift , . it is conceivable that this phenomenon is occurring in the entire virus community. the laborious and time-consuming traditional vaccine development strategies, including attenuated vaccines and gene deletion vaccines require many rounds of plaque purification or passages and cannot meet the urgent demand for new vaccines. development of inactivated vaccine is much faster, but requires high dose administration and is generally less effective. thus, there is an imperative need for novel technologies that could speed up and simplify vaccine development. recently, a revolutionary gene-editing technology termed clustered regularly interspaced palindromic repeats (crispr)/associated (cas ) system provided a versatile tool for gene editing [ ] [ ] [ ] . with guide rna (grna), cas and its mutant cas n protein can specifically break or nick the targeting dna with high efficiency [ ] [ ] [ ] . subsequently, this will cause indels in the target region by non-homologous end joining (nhej) dna damage repair or foreign genes knocked-in through homologous recombination (hr) in the presence of homologous dna donor [ ] [ ] [ ] . these two pathways are deliberately manipulated for gene editing purposes in different organisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the technology of gene editing in dna viruses is at its infancy. there is evidence that this system can be applied for gene editing in dna viruses, such as hsv, prv, adenovirus and hbv [ ] [ ] [ ] . yet, this technology has neither been demonstrated for simultaneous multi-gene deletion in viral genome nor for successful vaccine development. cre/lox system is another high efficient technology extensively used for gene manipulation in many species . lox sites derived from bacteriophage contains several variants with specific self-compatibility, such as lox p, lox n and lox . all together, application of these modern powerful technologies may potentially accelerate vaccine development. the aim of the present study is, therefore, to isolate the re-emerging prv and use it as a model virus to establish a fast and cost-effective technology for express vaccine development. isolation, characterization and sequence analysis of re-emerging prv virulent strain. since , prv re-emerged and escaped the old vaccine-induced immunity, causing widespread pseudorabies outbreaks in vaccinated pig farms in china . in order to isolate new prv virulent variants resistant to the current vaccines, prv-pcr-positive brain samples of aborted fetus from vaccinated pigs were homogenated and inoculated onto pk- cells after bacteria filter. a new prv virulent strain (hnx) was isolated by plaque purification. the major antigens, glycoprotein b (gb), gc and gd of prv were then amplified by pcr and subjected to sequencing. as shown in fig. a -c, the gb, gc and gd amino acid sequences of prv hnx displayed some variations and deletion, when compared with the bartha strain and previous pandemic ea strain. to further characterize the virulence of prv hnx strain, prv-free piglets were intramascularly vaccinated with bartha-k , a classical attenuated live vaccine, and then challenged with prv hnx or ea strain ( tcid ). the group challenged with prv hnx strain developed typical pseudorabies symptoms, whereas no symptoms was observed by the ea and the uninfected control groups (data not shown). this was further confirmed by the fever (rectal temperature > . °c) in prv hnx infected group, but not in prv ea infected and uninfected control groups (fig. d) . moreover, the virus neutralization titer of antisera was measured days after vaccination. compared to the neutralization titer of antisera against prv ea, the titer against prv hnx was significantly lower, suggesting the insufficient prophylactic effect of this vaccine against the re-emerging prv hnx strain (fig. e ). collectively, these data demonstrate that the re-emerging prv strain can escape the immunity induced by the current commercial vaccines, thus posing an urgent demand for new vaccines. prv multi-virulent genes recombination via crispr/cas n system. to establish an express vaccine development strategy, two highly efficient gene edit systems, crispr/cas and cre/lox system were employed. meanwhile single cell facs technique was also applied to simplify and accelerate the plaque purification as shown in fig. a . to avoid false positive signals caused by fluorescent gene expression in homologous recombination donor template, the expressions of gfp and mcherry were driven by endogenous viral promoter, which occurs only after precise dna homologous recombination. gfp and mcherry genes were flanked with lox p and lox n pairs respectively to facilitate fluorescent marker genes excision (fig. b) . thymidine kinase (tk) and ge are the major virulent genes of prv and also the primary targets for prv gene deletion vaccines . the simultaneous deletion of tk and ge genes was achieved by crispr/cas system mediated homologous recombination. firstly, sgrnas target to tk and ge genes (designed by web based tool http://crispr.mit.edu/) and dna donors containing fluorescent selection genes and homologous arms were co-transfected to hek t cells (fig. a) . eight hours post transfection, cells were then infected with prv hnx with different moi (multiplicity of infection) and incubated until recombinant virus expressing red and/or green fluorescence were observed (fig. b) . notably, no green/red overlapping cells were observed in the control group (data not shown). next, the recombinant viruses were collected and inoculated to pk cells (p ). pk cells with fluorescence were then subjected to facs and plated one cell per well to -well plate pre-cultured with pk cells. the wells showing maximum green and red overlapping signals were then subjected one round of plaque purification to obtain the pure recombinant viruses (p ) (fig. c,d) . as the viruses with tk gene replicate much faster and may thus outgrow the tk gene deleted viruses, we applied bvdu (bromovinyl deoxyuridine), an inhibitor of tk, to suppress wt prv or ge single deletion prv. finally, the purity of recombinant virus was validated by pcr amplification. as shown in fig. e , the positive control pcr bands were observed in all the the samples except h o excision of double fluorescent marker genes via cre-lox based system. due to vaccine safety concerns and regulation, all the selection markers in vaccine candidate need to be completely removed. to this end, the incompatible loxp and loxn sites were inserted into the homologous recombination dna donor, flanking gfp and mchrrey genes respectively in the same orientation (fig. b) . hek t cells were used for cre recombinase gene transfection and it mediated gene excision, because of the high transfection and expression efficiency of this cell line. as shown in fig. a , the cre recombinase gene transfected hek t cells were infected with prv-hnx-tk − /ge − gfp + mcherry + recombinant prv with various moi ( . , , and ). after hours of incubation, the cre recombinase transfected cells exhibit significant less fluorescence compared to the control with mock transfection (fig. b,c) . the first generation of cre-treated recombinant viruses were harvested after infection at an moi of , which showed the highest efficiency for gene excision (p ). double fluorescent gene excision was achieved through three rounds of plaque purification as shown in fig. d . thus, by virtue of these genome-editing tools, the re-emerging prv hnx was successfully engineered into a prv-hnx-tk − /ge − recombinant virus (p ). prophylactic effect of prv-hnx-tk − /ge − in mice and piglets. the safety of this novel vaccine candidate was firstly assessed in mice. all mice injected with prv hnx group died, three to five days later, whereas all the prv-hnx-tk − /ge − injected mice remained healthy (fig. a) . next, the prv-hnx-tk − /ge − vaccinated mice were challenged with the re-emerging prv hnx, prv ea or a standard wt virus strain prv-becker with gfp in ge position. fig. b -d demonstrated that vaccination with prv-hnx-tk − /ge − provides complete protection against the previous and re-emerging prv strains. this was further supported by the presence of extensive gfp signals in brain sections of unvaccinated mice but not in that of the vaccinated group (fig. e) . next, sixteen of prv-free piglets were randomly divided into two groups (one group immunized with prv-hnx-tk − /ge − and the other with dmem) and subsequently challenged with current pandemic prv ( tcid ) intranasally at days post vaccination. death, anti-gb antibodies, and clinical signs including body temperature, respiratory distress, depression, anorexia, cough, convulsions, ataxia, itching, and weight were measured or recorded. no typical pseudorabies clinical signs were observed in prv-hnx-tk − /ge − vaccinated group, whereas severe pseudorabies symptoms were developed in the control group (data not shown). as shown in fig. a , this vaccine candidate can completely protect piglets against the current pandemic prv strain, while only two piglets survived in the control group. in this line, anti-gb antibody was significantly increased post vaccination in vaccinated piglets (fig. b) . the fever (rectal temperature > . °c) and daily weight gain of vaccinated group were significantly more than that of the control group (fig. c,d) . next, the dead and surviving piglets were subjected to histopathological analysis. piglets from the control group exhibited severe pathological damages in brain, such as obvious meningeal inflammation, microglia nodule and perivascular cuff, whereas just a mild meningeal inflammation were observed in the vaccinated piglets even after challenge with high dose of virus ( tcid ) (fig. e,f) . together, these data demonstrated the potential of this vaccine candidate for protection against pseudorabies. despite of the interdisciplinary efforts, it is still time-consuming and laborious to develop vaccines against the constantly changing viruses . in this study, we isolated a re-emerging prv and used it as a model to establish a fast and cost-effective technology for express vaccine development. the traditional way to develop multiple gene deletion vaccine involves several steps of single gene recombination and marker gene excision. to obtain a prv double gene deletion vaccine candidate, it requires about twenty rounds of time-consuming plaque purification . we combined two highly efficient gene edit systems, crispr/ cas system and cre/lox system to increase the multi-gene editing efficiency in viral genome. meanwhile, single cell facs technology was also employed to further promote virus purification efficiency. we can indeed obtain prv-hnx-tk − /ge − /gfp + /mcherry + recombinant prv by just a single round of plaque purification, whereas it requires about ten rounds of plaque purification by traditional strategy. moreover, owning to the high efficiency of cre recombinase and incompatible loxp and loxn sites, the multiple marker genes excision can be achieved simultaneously in one step followed by just three rounds of plaque purification. of note, in our study, the expression of selection fluorescent genes was driven by endogenous viral promoter, which occurs only after precise dna homologous recombination. in this scenario, we could avoid the false positive due to basal fluorescent gene expression and can thus utilize single cell sorting as an update for traditional time-consuming viral plaque purification. together, this strategy greatly facilitates and accelerates prv vaccine development to simply one step of simultaneous multiple-gene recombination mediated by crispr/cas system, and one step of simultaneous multiple marker genes excision mediated by cre/lox system. it requires just only four rounds of plaque purification in total to obtain a double gene deletion vaccine candidate. this strategy could also be applied to vaccine development against other dna virus such as hsv, cmv, adenovirus, vzv, duck hepatitis virus (dhv), bovine herpesvirus (bhv) and epstein barr virus (ebv). yet the crispr/cas system is mostly applied in dna editing, accumulating data suggest that this versatile technology could also work effectively in the rna world. o'connell et al. demonstrated that trans-complementation of protospacer adjacent motif presenting oligonucleotides (pammers) can indeed stimulate site-specific targeting and cleavage of ssrna by cas endonuclease . recently, francisella novicida cas has been shown to have the capability of targeting endogenous bacterial rna and was engineered to target and inhibit hepatitis c virus, a ssrna virus . it is conceivable that with the advances in this dynamic research field, crispr/cas system could be very possibly employed to develop anti-rna virus vaccines. moreover, as recombinant prv and hsv are extensively exploited for neural circuit tracing, our strategy could also significantly simplify the recombinant viral tracer engineering and could thus contribute to neuroscience research as well . while it has been shown that the crispr system can be used in viral genome editing , , the application of this technology in viruses is at its infancy. crispr-based simultaneous multiple viral genes editing and vaccine development has never been reported. here, we simultaneously replaced two main virulent prv genes, ge and tk, by gfp and mcherry respectively using crispr/cas system assisted homologous recombination. in contrary to a recent study which showed the crispr/cas system could only work with extracted prv viral genome dna , our data proved this system is also able to directly edit prv genome during viral infection. in this line, it has been reported that crispr system introduced into host cells during adenovirus and hsv replication can indeed robustly stimulate dna breakages in the targeted genomes with high frequency . this discrepancy could be possibly due to the application of tk gene inhibitors in our experiments to prevent wt viruses outgrow the gene deletion viruses. generally, the genome of dna viruses is relatively more stable than that of rna viruses , however, there are many studies reporting outbreaks of infectious diseases caused by newly or re-emerging dna viruses , , . here, we isolated a re-emerging, prv hnx resistant to the classical prv vaccines. the sequence analysis revealed several mutations/deletion in gb, gc and gd of prv. it would be of great interesting to further investigate whether these mutations/deletions could cause the antigenic drift, and if so, what is the molecular mechanism leading to the resistance to the vaccine induced immunity in the swine population , , . here, our animal experiments in mouse and piglets validated the protective effect of this gene editing technologies based new vaccine candidate, demonstrating its potential in controlling the current pseudorabies outbreak in the swine industry. together, we combined several state-of-art modern biotechnologies to establish a novel strategy for express vaccine development and obtained, to our knowledge, the first successful gene editing technologies based vaccine candidate. this new generation biotechnologies based strategy might open an express avenue for future anti-viral vaccine development. plasmid construction, viral genomic preparation and pcr amplification. the guide rnas were cloned into sgrna/cas n expression vector (addgene plasmid px ) as previously described . ge and tk homologous arms were amplified from prv hnx strain by pcr. gehm -loxp-gfp-loxp-gehm and tkhm -loxn-mcherry-loxn-tkhm donor templates were constructed using overlapping pcr. the gfp were inserted into ge position in prv-becker bac (a kind gift from dr. l. w. enquist) of which the virus were rescued in pk cell as previously described . the prv genomic dna was extracted using the tianamp virus dna kit (tiangen) following the manufacturer's protocol. all the sequence of primers, sgrnas, donor templates and antigenic genes are listed in supplementary data. dmem with % fbs, and streptomycin/penicillin. dna was transfected into hek t and pk cells using lipofectamine. prv infection was performed as previously described . the culture medium was changed to dmem with % fbs after transfection and viral infection. dna synthesis, sequencing and analysis. the pcr primer synthesis and dna sequencing were done by shanghai sangon biotch, china. gb, gc, and gd gene sequences of prv ea strain obtained from ncbi were analyzed by bioedit software. virus preparation, propagation and plaque purification. virus infected pk cells were harvested at hours post-infection. after three rounds of freeze-thaw, the cell lysate was centrifuged for minutes at , rpm/min. the supernatant were used for next propagation or stored at − °c. for virus purification, the infected pk cells were covered by mixture with × dmem and . % low melting-point agarose ( μ g ml − bvdu when necessary). after - hours, well-separated plaques were picked up by pipette tip and was pipetted into micro-tubes containing μ l serum free dmem for next propagation or plaque purification. fluorescence-activated cell sorting. the fluorescence-activated cell sorting was performed on moflo xdp sorter (beckman coulter) following the manufacturer's protocol. pk cells were infected with the first generation recombinant prv at an moi of . . the single cells expressing fluorescent proteins were sorted into -well plate pre-seeded with , pk cells. animal experiments. female balb/c mice were immunized with . tcid prv-hnx tk − /ge − and inoculated subcutaneously with prv hnx, prv ea or prv becker two weeks later. for piglet experiments, tcid prv-hnx tk − /ge − or prv bartha-k injected intramuscularly for immunization, booster vaccination was performed days later. the piglets were intranasally challenged with prv at days post second immunization. clinical signs were recorded daily for up to days. all of the animal experiments were approved by the research ethics committee, huazhong agricultural university, hubei, china (hzaumo - ). all the animal experiments were carried out in accordance with the recommendations in the guide for the care and use of laboratory animals from research ethics committee, huazhong agricultural university, hubei, china. elisa, neutralization assay and histopathology. the serum samples of prv-specific gb antibodies were evaluated using commercial elisa kits according to the manufacturer's directions (idexx, usa). the neutralizing antibody against prv was tested as described previously . the brain tissues were fixed with % paraformaldehyde solution at room temperature for days and then processed by routine histopathological procedures as previously described . clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study emergence of zaire ebola virus disease in guinea geometric constraints dominate the antigenic evolution of influenza h n hemagglutinin notes from the field: outbreak of skin lesions among high school wrestlers-arizona vaccine process technology emerging and re-emerging viruses: a global challenge 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system heritable gene targeting in the mouse and rat using a crispr-cas system efficient genome editing in zebrafish using a crispr-cas system genome engineering in saccharomyces cerevisiae using crispr-cas systems genome engineering of drosophila with the crispr rna-guided cas nuclease one-step generation of mice carrying mutations in multiple genes by crispr/cas-mediated genome engineering high-efficiency targeted editing of large viral genomes by rna-guided nucleases a simple and rapid approach to manipulate pseudorabies virus genome by crispr/cas system crispr/cas cleavage of viral dna efficiently suppresses hepatitis b virus programmable rna recognition and cleavage by crispr/cas cas -mediated targeting of viral rna in eukaryotic cells transneuronal circuit analysis with pseudorabies viruses a self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis oral immunization of wild boar and domestic pigs with attenuated live vaccine protects against pseudorabies virus infection handbook of histopathological and histochemical techniques: including museum techniques statistical analysis. the significant differences of the animal experiments were analyzed using two-way anova or/and t-test in the graphpad prism (version . ) software (san diego, ca). differences were considered statistically significant when p < . . g.c. and x.l. wrote the main manuscript. t.y. and x.l. prepared for the figure ; l.s. and x.l. for figures ; x.l. for the figure - individually; y.p., d.w., x.h. and x.l. for the figure . z.f., q.h. and g.c. contributed to study design and manuscript writing and proofreading. supplementary information accompanies this paper at http://www.nature.com/srep key: cord- -ybikajhh authors: nyarko, prince b.; tarr, sarah j.; aniweh, yaw; stewart, lindsay b.; conway, david j.; awandare, gordon a. title: investigating a plasmodium falciparum erythrocyte invasion phenotype switch at the whole transcriptome level date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: ybikajhh the central role that erythrocyte invasion plays in plasmodium falciparum survival and reproduction makes this process an attractive target for therapeutic or vaccine development. however, multiple invasion-related genes with complementary and overlapping functions afford the parasite the plasticity to vary ligands used for invasion, leading to phenotypic variation and immune evasion. overcoming the challenge posed by redundant ligands requires a deeper understanding of conditions that select for variant phenotypes and the molecular mediators. while host factors including receptor heterogeneity and acquired immune responses may drive parasite phenotypic variation, we have previously shown that host-independent changes in invasion phenotype can be achieved by continuous culturing of the w mef and dd p. falciparum strains in moving suspension as opposed to static conditions. here, we have used a highly biologically replicated whole transcriptome sequencing approach to identify the molecular signatures of variation associated with the phenotype switch. the data show increased expression of particular invasion-related genes in switched parasites, as well as a large number of genes encoding proteins that are either exported or form part of the export machinery. the genes with most markedly increased expression included members of the erythrocyte binding antigens (eba), reticulocyte binding homologues (rh), surface associated interspersed proteins (surfin), exported protein family (epf ) and plasmodium helical interspersed sub-telomeric (phist) gene families. the data indicate changes in expression of a repertoire of genes not previously associated with erythrocyte invasion phenotypes, suggesting the possibility that moving suspension culture may also select for other traits. some of the sialic acid-dependent strains (particularly w mef, dd and csl ) have the propensity to switch invasion phenotype when selected on sialic acid-deficient erythrocytes , , . recently, we discovered another remarkable feature of w mef and dd . when continuously cultivated in moving suspension cultures, these strains gradually gain the ability to invade neuraminidase-treated (sialic acid-deficient) erythrocytes, but remain sialic acid-dependent when kept in continuous static cultures . this observation was surprising given that no modifications were made to either the parasites or erythrocytes used for culturing. interestingly, the observed phenotype selected by moving suspension culture was similar to that achieved in other experiments by selection on sialic acid-deficient erythrocytes, and similarly associated with greatly increased expression of the rh and eba genes. we considered the possibility that the phenotypic switch to sialic acid-independent invasion may also involve genes that are not yet known to be associated with different invasion pathways. to this end, we conducted whole transcriptome analysis of schizonts from parasite cultures that had switched invasion pathways during suspension culture, compared with schizonts from unswitched static cultures, using highly biologically replicated samples . this has revealed the differential expression of a larger repertoire of genes likely to be involved in invasion phenotype switching, including known invasion ligands as well as gene families not previously implicated in erythrocyte invasion. parasite schizont samples were generated for baseline (baseline; bl), moving suspension (suspended; sp) and static control (static; st) cultures of p. falciparum w mef and dd strains, using eight replicate cultures of each condition. the switched invasion phenotypes of all sp culture replicates were confirmed, with invasion assays showing that they had acquired the ability to invade neuraminidase-treated erythrocytes ( fig. ) , while st cultures remained similar to bl. schizonts were harvested from each culture replicate and total rna extracted for sequencing. quality controls were performed at the rna extraction, library preparation and sequencing steps. after quality control filtering, whole transcriptome data for analysis were obtained for eight replicates for each of the w mef and dd baseline cultures (w mef bl and dd bl, respectively), eight replicates of the dd suspended cultures (dd sp), five replicates of the w mef suspended cultures (w mef sp), and two replicates of each of the w mef and dd static control cultures (w mef st and dd st, respectively), and analysed using the deseq r package . samples that could not be sequenced had insufficient rna (≥ ng) for library preparation. reads were uniquely mapped to a total of p. falciparum genes (supplementary tables s and s ) , with very wide variation in mapping depth among the genes as expected (transformed log fpkm ranging from . to . ; supplementary table s ). principal component analysis (pca) showed overlaps among members of sample groups, with no specific group clustering ( supplementary fig. s ). this was expected since we envisioned only subtle differences to exist between suspended and static cultured parasites. nonetheless, to test the robustness of our approach, we checked for outliers using default parameters of the damirseq package and removed samples with mean absolute correlation ≤ . . we then performed differential expression analysis with deseq using retained samples while correcting for any batch effects (designated here as corrected analysis). a correlation of the fold change values for corrected vs uncorrected analysis shows a near perfect linear relationship, with a correlation coefficient of . ( supplementary fig. s ). furthermore, we correlated the fpkm values of all samples to the fpkms of data from a previous time-course study and confirmed that all samples had peak expression at - hours post invasion ( supplementary fig. s ). this was considered as a validation of well-synchronized cultures and therefore, qualifying all samples for inclusion in the subsequent analyses. based on the results of our aforementioned quality checks we resolved to include all samples in our primary analysis. and their respective suspended cultures were monitored at regular intervals by invasion assays. baseline cultures were confirmed to be sa-dependent, while suspended parasites gradually acquired sa-independent invasion phenotype with increasing length of time in culture. schizonts were harvested at baseline, and weeks and , respectively for suspended w mef (a) and dd (b). broken lines indicate suspended replicates which were not sequenced. www.nature.com/scientificreports www.nature.com/scientificreports/ to limit statistical noise and false discovery, we first compared the st cultures to bl cultures to determine the extent of any background noise generated from culturing in general. we observed no significantly differentially expressed genes between st and bl cultures in either strain when an adjusted p value < . was applied as the level of significance (supplementary tables s and s ). this was as expected, since st cultures were phenotypically similar to bl cultures. this observation also allowed us to confidently use the highly replicated bl cultures as an alternative to st cultures as comparator against sp cultures in the differential expression analysis. a more stringent adjusted p value (< . ) was however applied for the transcriptome screen to identify significantly differentially expressed genes in the sp cultures relative to bl cultures. to investigate the molecular mechanisms associated with the switching of parasites when cultured in continuous suspension, we first examined differential gene expression in w mef sp relative to w mef bl cultures. focusing on genes with log fold change > (more than four-fold difference) and adjusted p value < . , we identified genes to be differentially expressed, of which and had higher and lower transcript levels, respectively, in w mef sp relative to w mef bl ( fig. and supplementary table s ). these differentially expressed genes are widely distributed in all chromosomes of the p. falciparum genome ( supplementary fig. s ). we further compared individual sp replicates to the pooled bl samples and observed a similar trend in differential gene expression among replicates as was observed in the bulk analysis ( supplementary fig. s ). despite replicate showing a generally reduced fold difference, the topmost genes had expression patterns similar to the other replicates ( supplementary fig. s ). removal of this replicate did not alter the results for the initial analysis. gene ontology analysis of the most significantly differentially expressed genes showed enrichment for a number of terms (p < . ; supplementary table s ) , with heparin binding, sulfur compound binding and glycosaminoglycan binding representing the most enriched terms (p < . for all). among the most significantly differentially expressed genes in our dataset were those involved in invasion. these included eba , eba , rh , rh a, rh b, rh , rhoptry neck protein (ron ) and gpi-anchored micronemal antigen (gama) (fig. and supplementary table s ). additionally, a large number of virulence associated genes, including members of the surface-associated interspersed protein (surfin) family, exported protein family (epf ), ring exported protein (rex ) and interspersed repeat antigen (fira); all of which are exported into host erythrocytes, were significantly expressed at higher levels in sp parasites relative to bl (fig. ) . other differentially expressed genes included those encoding erythrocyte membrane protein trafficking proteins (ptp and ptp ), ring-infected erythrocyte surface antigen (resa and resa ), bromodomain protein (bdp ), members of the apiap transcription factors including ap -g, ap -o, chromatin modifying proteins such as lysine-specific histone demethylase (lsd ), and proteins of unknown functions (supplementary table s ) . to further test these observations, we performed differential expression analyses between the whole transcriptome of static (w mef st) and suspended w mef (w mef sp) grown for the same length of time, and compared the expression profile to that observed for bl vs sp analysis. the results show concordance in differential expression between sp cultures and either the respective bl or st cultures, with overall spearman rank correlation coefficient of . (fig. ) . a closer look at the differentially expressed genes show similar patterns, with many invasion-related and exported protein genes being more highly expressed in sp cultures relative to their st counterparts. furthermore, we tested the robustness of this approach by pooling all static samples (st + bl) and compared them to sp samples in a differential expression analysis. a correlation plot of the sp vs either baseline only or pooled static samples gave a strong positive linear correlation, with a correlation coefficient of . ( supplementary fig. s ). focusing on gene families, with the exception of rh , all the rh genes previously implicated in invasion (rh , rh a, rh b and rh ) were significantly upregulated in sp parasites relative to bl ( fig. a and supplementary fig. s ). among the eba genes, eba and eba showed significantly higher expression in sp parasites (the pseudogene eba is co-regulated with rh ) , while the others showed a trend in the same direction although at www.nature.com/scientificreports www.nature.com/scientificreports/ insignificant levels (fig. a) . identification of large numbers of highly expressed exported protein genes prompted us to take a closer look at the transcriptome-wide pattern of some exported protein family members. we observed higher expression levels for all members of the surfin family in sp parasites relative to bl, with members having more than two-fold increased expression ( fig. b and supplementary fig. s ). the genes encoding surfin . and surfin . which have been localized to merozoites , , had the largest change in expression levels within this gene family. interestingly, all genes belonging to the maurer's cleft associated exported protein family (epf ) also had higher transcript levels in sp parasites compared to bl cultures (fig. c) . likewise, fifteen genes belonging to the plasmodium helical interspersed sub-telomeric (phist) family, had more than two-fold increased expression in sp parasites (fig. d) . the phist protein genes included the ring-infected erythrocyte surface antigen (resa ) and pf d _ ; both of which have previously been shown to be essential for parasite survival . also included in the differentially expressed genes were those coding for transcription factors, with notable ones being ap -g and other members of the apiap gene family, as well as bromodomain protein among others (supplementary fig. s and supplementary table s ). epigenetically regulated genes are differentially expressed between moving suspension and static cultures. previous data have shown that the switched suspended culture phenotype is reversible, and that expression of rh and eba is epigenetically controlled , , , . therefore, we sought to find out the extent of expression of epigenetically controlled genes among the larger number of differentially expressed genes here. heterochromatin protein (hp ) occupancy at the promoter region is a marker for epigenetic gene silencing , , and can be used as a proxy for the identification of epigenetically regulated genes. cross-referencing our data with a previously compiled list of hp -associated genes , we identified genes in our data to have hp occupancy. of these, ( . %) were significantly differentially expressed in sp parasites (supplementary table s ) , indicating a high representation of epigenetically controlled genes (odds ratio . , p = . × − ), with ( . %) of these genes expressed at higher levels in sp parasites. a recent study identified a large proportion of p. falciparum genes to be required for parasite survival . we thus cross-referenced our list of differentially expressed genes with those designated as essential or dispensable. a total of ( . %) of the essential genes were differentially expressed in w mef sp relative to w mef bl, whereas ( %) of the dispensable genes were differentially expressed. this shows that a significantly lower than random proportion of essential genes were differentially expressed (odds ratio . , p = . × − ). genes differentially expressed in suspended dd relative to baseline. having identified genes that are significantly differentially expressed between sp and bl cultures of w mef, we performed similar differential expression analyses for the dd clone. generally, the range of fold change values observed for differential expression between dd sp and dd bl were lower (− . to . ) compared to those in w mef (− . to . ) (supplementary fig. s ), potentially because dd was cultured for a shorter time ( rather than weeks) or a possible biological difference which might exist between the two apparently isogenic strains when grown in suspension conditions. using a cutoff of -fold difference between groups and adjusted p value < . , twelve genes were identified to be differentially expressed between dd sp and dd bl, all of which were more highly expressed in dd sp (supplementary fig. s ) . a less stringent cutoff of more than -fold difference identified genes to be significantly differentially expressed, with of these expressed at higher levels in sp parasites ( fig. a and supplementary table s ). the pattern of gene expression was similar to that of w mef, with rh and eba among the most highly differentially expressed genes. additionally, a number of exported protein genes as well as functionally unknown ones were more highly expressed in dd sp relative to dd bl. a comparison of differential gene expression in w mef with dd showed a significant positive correlation (fig. b) , with the directionality of expression of most of the highly significantly expressed genes in w mef remaining similar in dd (supplementary table s and supplementary fig. s ). similar to w mef, majority of the individual biological replicates showed concordance with the results from the pooled analyses ( supplementary fig. s ) . analysis of the genes differentially expressed between dd sp and dd bl for enriched gene ontology terms (p < . ) identified protein kinase activity and translocation of peptides or proteins into host cell cytoplasm to be among the most enriched terms (supplementary table s ). a comparison of the levels of differential expression of the eba and rh gene between st and sp parasites in the current rna sequencing study and our previous rt-qpcr analysis shows a strong positive correlation of results from the two studies ( supplementary fig. ). the specific roles of parasite ligands in erythrocyte invasion phenotype variation are not well-understood and appear to differ between parasite strains , , , . these previous studies have shown that invasion phenotype switching has mainly been through the differential expression of members of the rh or eba gene families. we recently demonstrated that the phenotypic switching in p. falciparum w mef and dd clones could be achieved through simply culturing them in moving suspension rather than static conditions . in this report, we have investigated this phenomenon further using whole transcriptome analyses. we show significantly increased expression of four rh and two eba genes in sp parasites relative to bl controls, all of which have been implicated in previous invasion phenotype switching studies, except eba , [ ] [ ] [ ] , , . the mechanistic relevance of the differential expression of multiple invasion-related genes in sp parasites is not yet known, but it is notable that the encoded proteins possess erythrocyte binding properties and have been shown to function during early interactions leading to invasion , , . in contrast, the invasion-related genes ama and rh which function during the later stages of invasion were not significantly differentially expressed in sp parasites, suggesting that moving suspension culture may possibly select for parasites with stronger interactions to fasten onto the erythrocyte surface prior to entry. the expression of rh a and rh b are of interest, as a recent forward genetic screen showed the rh locus to be associated with invasion phenotype switch, with higher expression of these ligands leading to increased sensitivity to chymotrypsin treatment . our previous phenotypic data showed that switched sp parasites were more sensitive to chymotrypsin treatment compared to st parasites , thus the increased expression of these ligand genes in the switched sp parasites here suggests a possible role of rh a and rh b in the phenotypic switch. increased expression of rh b and rh correlates with sialic acid-independent invasion by clinical isolates of p. falciparum , suggesting that the two ligands may function cooperatively. host erythrocyte remodeling by exported parasite proteins leading to increased adherence and resistance to stress is a prominent virulent property of p. falciparum , , [ ] [ ] [ ] [ ] [ ] . increased rigidity and adhesion of infected erythrocytes enhance sequestration through cytoadherence and rosetting, both of which are associated with disease severity . a substantial number of exported proteins were highly expressed in suspended parasites, prominent ones being the surfins and epf . both surfin . and surfin . have been localized to the parasitophorous vacuole and merozoite surface, with surfin . suggested to have a role in merozoite invasion , , . available data show that surfin . forms a complex with glutamate-rich protein (glurp) and ron in both schizonts and free merozoite, with anti-surfin . antibodies partially inhibiting erythrocyte invasion . additionally, surfin . is exported to the erythrocyte surface , with deletion of its gene resulting in reduced erythrocyte membrane rigidity . surfin . , though annotated as a pseudogene, has been shown to be expressed as a functional protein, with its knockdown resulting in impaired merozoite formation during schizogony . the epf family are maurer's cleft associated proteins whose reduced expression results in deficient merozoite release . efficient merozoite release increases the chances of successful invasion and thus higher parasite growth, which is a hallmark of parasites grown under flow conditions , - . the phist proteins are known to be exported to various locations including both the host cell periphery and cytosol, as well as the parasite's parasitophorous vacuole, maurer's cleft and merozoite surface [ ] [ ] [ ] [ ] . they function by interacting with host erythrocyte cytoskeletal components , , , as well as parasite-specific proteins such as pfemp , skeleton binding protein (sbp ), and knob associated histidine rich protein (kahrp) , , , ultimately leading to cytoadherension and resistance to stress , . phist proteins such as members of the pfemp trafficking protein (ptp) contribute to cytoadherence by mediating the successful trafficking of pfemp from the maurer's cleft to the host cell surface . additionally, ptp mediate cell-cell communication by trafficking exosome-like vesicles between infected erythrocytes, a phenomenon which modulates host immune response and increase gametocytogenesis [ ] [ ] [ ] . the contents of these vesicles range from parasite dna, rna and pfemp proteins , . elevated expression of exported protein genes in sp parasites may thus have physiological relevance, as these proteins could potentially affect the rigidity of the erythrocyte membrane to withstand the stress imposed by moving suspension culture conditions, mediate effective communication among parasites, modulate gametocyte production, etc. another plausible hypothesis for the increased expression of the exported protein genes is to increase the adherent properties of erythrocytes in preparation for stronger binding to possible endothelial molecules in an in vivo setting. another feature of the current data is the differential expression of genes involved in transcription regulation. we observed increased expression of some members of the apiap transcription factor family which are involved in the regulation of different processes and stages during parasite development [ ] [ ] [ ] . notably, ap -g, which regulates gametocyte conversion , - is among the most significantly differentially expressed genes in w mef sp . this gene is epigenetically controlled, and thus the observation could be a consequence of global upregulation of epigenetic-regulated genes. however, a recent study has shown that ap -g interacts with ap -i to drive the expression of invasion related genes, potentially to increase the invasion efficiency of sexually committed scientific reports | ( ) : | https://doi.org/ . /s - - -y www.nature.com/scientificreports www.nature.com/scientificreports/ merozoites . given the length of time of culture adaptation and the observation that sp parasites grow better than st parasites , we postulate that the expressed ap -g, if functional, is to enhance parasite outgrowth by increasing invasion efficiency. another ap with increased expression in suspended parasites is ap -exp which regulates the expression of clonally variant genes . this is unsurprising given the extent of increased expression of exported protein genes in the current dataset. it is presently unclear how the moving suspension culture condition selects for the phenotype and transcriptional change seen. if parasites have the cellular machinery to sense culture motion, they might alter their phenotype in response, reminiscent of nutrient sensing by the sucrose non-fermenting (snf )-related serine/ threonine protein kinase kin . a more general possibility is that a subset of parasites that grow better under such conditions are selected over time, given the length of time it takes for the majority of parasites under suspension culture to acquire the neuraminidase-resistant erythrocyte invasion phenotype. the significant enrichment in expression of epigenetically controlled genes in the sp cultures relative to st has offered a clue about the possible mechanisms for the changes in gene expression. elucidating these mechanisms that connect culture agitation with transcriptional changes that mediate phenotypic variation will be the focus of our future studies. parasite culturing and schizonts harvesting. ethical approval was obtained from the institutional review board of the noguchi memorial institute for medical research, university of ghana, and all methods used in the study were in accordance with the guidelines and regulations provided by the ethical committee. all human erythrocytes used in this study were obtained with written informed consent of the donors. plasmodium falciparum strains w mef and dd were cultured as previously described . briefly, parasites were cultured at °c in rpmi- (sigma) supplemented with . % albumax ii (gibco), mg hypoxanthine, g/l sodium bicarbonate (sigma) and µg/ml gentamicin sulfate (sigma) using human group o + erythrocytes at % hematocrit in a mixed gas environment of % nitrogen, % co , and % oxygen (air liquide, birmingham, united kingdom). cultures were initially grown under static culture conditions for the generation of baseline schizont material for analysis, and the sialic acid-dependent invasion phenotype of both strains was confirmed using a previously described procedure . the true positive discovery rate of differentially expressed genes should increase significantly by using a larger number of biological replicates , with six replicates having previously been recommended for differential expression analyses between different strains of plasmodium falciparum . this study aimed at identifying differentially expressed genes between parasites of the same strain grown under different conditions, thus we projected to study each condition with eight biological replicates. parasites were maintained as ml cultures at % hematocrit, and at about % parasitemia with more than % rings, the ring stages were selected by treatment with % sorbitol . these parasites were cultured to develop to schizont stages, the schizonts were purified by percoll-alanine discontinuous gradient centrifugation , diluted with five-fold more fresh erythrocytes, µm e added to prevent egress and cultured for a -hour period to allow schizont maturation . mature schizonts were purified by percoll-alanine, homogenized with µl of trizol reagent (ambion/life technologies, carlsbad, california) and stored at − °c until processing for rna extraction. after successful harvesting of schizonts from baseline cultures, each of the experimental replicates was divided into two flasks, one kept in a static incubator (static cultures, st) and the other kept on an orbital shaker rotating at rpm (moving suspended cultures, sp); making st and sp replicates for each strain. the invasion phenotypes of sp cultures were monitored weekly until invasion efficiency into neuraminidase treated erythrocytes exceeded % relative to invasion into untreated erythrocytes, indicating that most parasites had the switched phenotype. all replicates reached this threshold after - weeks of continuous culturing. upon switching, schizonts were harvested from all replicate cultures as described above. rna extraction and processing. frozen trizol-homogenized schizonts were thawed at room temperature and total rna was extracted by the trizol method (invitrogen corp., carlsbad, ca). rna pellets were dissolved in µl rnase-free water. the rna was purified and dna removed by dnase i digestion on rneasy mini columns (qiagen, heidelberg, germany), following which rna was eluted in µl rnase-free water, and quantified by qubit high sensitivity rna assay (thermo fisher scientific). for samples containing at least ng rna, the rna integrity was checked on an agilent bioanalyzer using rna nano reagents and chips (agilent genomics, waldbronn, germany). whole mrna transcriptome library preparation and sequencing was performed using methods as previously described . briefly, rna sequencing libraries were prepared with truseq stranded mrna library prep kit (illumina) using ng - µg rna. quality of libraries were validated on an agilent bioanalyzer using dna reagents and chips (agilent genomics, waldbronn, germany) to quantify library sizes and confirm the absence of primer dimers. libraries were quantified using a kapa universal library quantification kit (roche diagnostics limited) on a fast real-time pcr system (thermo fisher scientific) and library concentrations were adjusted for library size. pooled libraries of - pm concentrations were sequenced on a miseq system (illumina) using a miseq reagent kit v (illumina) with × cycles. parasite rnaseq data analyses. paired-end fastq files were aligned using hisat (default alignment parameters) , duplicate reads removed with picard and converted to indexed bam files using samtools . bam files were visualized with artemis to confirm that majority (> %) of the reads aligned to exons with minimal overlaps within introns. the bam files were then filtered to exclude reads with mapq scores below . reads were counted using the summarizeoverlaps feature of the genomicalignments package in r, against the p. falciparum d version . reference genome sequence using an annotation file that had masked out extremely ( ) : | https://doi.org/ . /s - - -y www.nature.com/scientificreports www.nature.com/scientificreports/ polymorphic gene regions, duplicated genes, and the var, rifin and stevor large subtelomeric gene families so that they were not included in the analysis . differential expression analysis was conducted with deseq in r. briefly, deseqdataset was constructed from the summarizedexperiment object and low expressed genes filtered out. to model for batch effect, "batch" was included in the design formula. fragments per kilobase of transcript per million mapped reads (fpkm) were made from the deseqdataset. results tables were generated from the deseqdataset using wald test and differential expression analyses conducted with the deseq results function. a combination of differential expression (log fold change values) and level of significance (benjamini-hochberg adjusted p values) were used to identify significantly differentially expressed genes between sp parasites and their corresponding bl or st cultures. karyotype was constructed with the karyoploter package in r. principal component analyses were performed with deseq using default parameters and plotted with ggplot. all rna sequencing data are available for access at gene expression omnibus (https://www.ncbi.nlm.nih.gov/geo/), accession number: gse . global malaria mortality between and : a systematic analysis malaria: global progress - and future challenges 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karyoploter: an r/bioconductor package to plot customizable genomes displaying arbitrary data we are grateful to saikou bah of waccbip and suzanne hocking of lshtm for various contributions. this work was supported by funds from a world bank african centres of excellence grant (ace -waccbip: awandare) and a deltas africa grant (del- - : awandare). prince nyarko was supported by waccbip-world bank ace masters fellowship and waccbip-deltas student-visitor fellowship, while yaw aniweh was supported by a waccbip-deltas postdoctoral fellowship. the deltas africa initiative is an independent funding scheme of the african academy of sciences (aas)'s alliance for accelerating excellence in science in africa (aesa) and supported by the new partnership for africa's development planning and coordinating agency (nepad agency) with funding from the wellcome trust [ /z/ /z: awandare) and the uk government. the views expressed in this publication are those of the author(s) and not necessarily those of aas, nepad agency, wellcome trust or the uk government. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -y.correspondence and requests for materials should be addressed to g.a.a.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -k hzbbx authors: fan, hanlu; du, xiaohong; zhang, jingyuan; zheng, han; lu, xiaohui; wu, qihui; li, haifeng; wang, han; shi, yi; gao, george; zhou, zhuan; tan, dun-xian; li, xiangdong title: selective inhibition of ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: k hzbbx the ebola crisis occurred in west-africa highlights the urgency for its clinical treatments. currently, no food and drug administration (fda)-approved therapeutics are available. several fda-approved drugs, including selective estrogen receptor modulators (serms), possess selective anti-ebola activities. however, the inhibitory mechanisms of these drugs remain elusive. by analyzing the structures of serms and their incidental biological activity (cholesterol accumulation), we hypothesized that this incidental biological activity induced by serms could be a plausible mechanism as to their inhibitory effects on ebola infection. herein, we demonstrated that the same dosages of serms which induced cholesterol accumulation also inhibited ebola infection. serms reduced the cellular sphingosine and subsequently caused endolysosomal calcium accumulation, which in turn led to blocking the ebola entry. our study clarified the specific anti-ebola mechanism of serms, even the cationic amphiphilic drugs (cads), this mechanism led to the endolysosomal calcium as a critical target for development of anti-ebola drugs. serms selectively inhibit the entry of the ebola pseudovirion, but not through the estrogen receptor pathway. as all stages of ebola entry (binding to and internalization from the cell surface, as well as trafficking to and fusion with the limiting membrane of endolysosome) are mediated by trimeric gp spikes arrayed around the ebola particles , , so the pseudovirion model with ebola gp can simulate the stages of wild-type ebola entry, and several groups have studied the ebola entry mechanism and screened the anti-ebola entry drugs with ebola pseudovirion model , , , . we choose the pnl - -luc-r − e − based ebola pseudovirion to study the ebola entry, and the ebola vlp-mcherry (composed by gp, vp and vp -mcherry) to study the ebola internalization. then, we treated the hepatocellular carcinoma cell line hepg with two serms (tamoxifen and clomiphene), and infected them with the ebola pseudovirion. both serms selectively inhibited the ebola entry more than %; however, they did not inhibit the entry of the vsv or influenza a (a/wsn/ (h n ), wsn) pseudovirions (fig. a) . treatment with estrogen and estrogen receptor (er) antagonists did not inhibit the ebola pseudovirion entry (fig. a) , excluding the possibility that serms inhibit the ebola entry through the er pathway. to determine the effect of serms on ebola internalization, we transfected the hepg cells with the tpc -egfp and lamp -bfp vectors, and infected the cells with ebola vlp-mcherry. co-localization of tpc -egfp, lamp -bfp and vlp-mcherry demonstrated that serms did not inhibit ebola internalization to the tpc + lysosome; rather, they blocked ebola vlp release from the lysosome (fig. b) . it was reported that u a, also a cad, interacts with and inhibits npc , and also inhibits the ebola entry , . we also observed that u a selectively inhibited the entry of ebola pseudovirion (no entry inhibition was observed for the vsv or wsn pseudovirions) and did not inhibited ebola vlp internalization to the tpc + lysosome ( supplementary fig. s a, s b) . considering the critical role of npc domain c in ebola entry in our previous study and the similar structural properties of the cads displaying selective anti-ebola activity , we speculated that the cads with selective anti-ebola activity may target npc to inhibit ebola entry. to test this possibility, we expressed and purified the domains a and c of npc , . then, we carried out microscale thermophoresis (mst) and isothermal titration calorimetry (itc) to detect the interactions between tamoxifen, clomiphene, or u a and the npc domains a and c. in all cases, we did not detect interactions by mst or itc ( supplementary fig. s a , s b, respectively). considering the similar structural properties and the reported incidental biological activities of the cads, it is possible that the incidental biological activities of the serms are related to the mechanism of ebola entry inhibition by the serms. to verify this hypothesis, we compared the effective serms dosages for both cholesterol accumulation and ebola entry inhibition. the ebola pseudovirion assay demonstrated that serms inhibit ebola entry starting from μ m (fig. a) . filipin staining also indicated that serms induce cholesterol accumulation at approximately μ m (fig. b,d, supplementary fig. s ). therefore, the dosages of serms required to induce cholesterol accumulation matches the dosages of serms required for ebola entry inhibition, which implies that this incidental biological activity of serms are related to the ebola entry inhibition mechanism. intriguingly, the cholesterol accumulation appeared after h of serms incubation (fig. c,e) , whereas the ebola virus has already entered the cytoplasm at approximately h . thus, we speculated that intercellular changes within h after serm treatment should mediate the cholesterol accumulation and the inhibition of ebola entry. (a) effects of e , ici, tamoxifen, and clomiphene on ebola/wsn/vsv pseudovirion entry. hepg cells were pretreated with nm e , nm ici, μ m tamoxifen, and μ m clomiphene for h and infected by ebola/wsn/vsv pseudovirion with compounds for h. the cells were then lysed, and the luciferase assay was carried out. nh cl serves as a negative control. (b) representative images of colocalization of vlps (red, marked by mcheery-vp ) with tpc (green, marked with egfp) and lamp (blue, marked with bfp) from μ m tamoxifen or μ m clomiphene treated hepg . white arrows indicate examples of colocalization. data are expressed as the means ± sem (n = ). significant differences versus control group are presented by asterisks (*), **p < . , ***p < . , ns means no significance. serms reduce the cellular sphingosine. as an ebola entry inhibitor, u a induces sphingosine accumulation within min; moreover, sphingosine treatment also induces cholesterol accumulation . we speculated that serms may act in the same way as u a by inducing accumulation of sphingosine and cholesterol. to detect the effect of serms on sphingosine storage, we carried out the liquid chromatography-tandem mass spectrometry (lc-ms/ms). surprisingly, both u a and serms significantly decreased the sphingosine of hepg and hela cell lines after h treatment (fig. , supplementary fig. s ). serms upregulate the endolysosomal calcium levels. it has been reported that the sphingosine could regulate the levels of endolysosomal calcium , and we demonstrated that serms could decrease sphingosine in this study. to establish a correlation between serms and endolysosomal calcium, we imaged gly-phe β -naphthylamide (gpn)-sensitive (endolysosomal) calcium release to detect the effect of serms on endolysosomal calcium levels. the results demonstrated that both serms and u a significantly up-regulated the calcium release from the gpn-sensitive endolysosomal calcium store after h treatment (fig. a-c) . to determine whether endolysosomal calcium is involved in the mechanism of ebola entry inhibition, we treated hepg cell line with a high-affinity rhod-dextran to chelate intraluminal calcium, and infected the cells with ebola, wsn, or vsv pseudovirions. interestingly, chelation of endolysosomal calcium significantly and specifically inhibited the entry of ebola, while no inhibitory effects were observed for the vsv or wsn pseudovirions (fig. a) . chelation did not inhibit the internalization of ebola vlp to the tpc + lysosome (fig. c) . moreover, chelation of endolysosomal calcium also induced the cholesterol accumulation (fig. b ). serms were identified as new and specific inhibitors for ebola infection from a screen of fda approved drugs , , , ; however, the definite mechanism by which the serms inhibit the infection of ebola is still uncertain. because the possibilities of the disturbed acidification, cathepsin activity, or internalization of ebola have been excluded previously , we attempted to ascertain the relationship between the inhibition of ebola infection and the adverse effect of serms. in this study, our results indicated that both tamoxifen and clomiphene specifically inhibit the release of ebola from tpc + lysosome, and this inhibition is independent of the estrogen receptor pathway. entry of ebola virus into the cells, which is mainly mediated by its sole glycoprotein (gp) , , is initiated by the binding of ebola gp to c-type lectins and the viral envelope phosphatidylserine to phosphatidylserine receptors . ebola virion is thought to be internalized into endolysosome primarily through micropinocytosis [ ] [ ] [ ] , meanwhile the heavily glycosylated mucin-like domain and glycan cap of gp is removed by the low ph-dependent cathepsins l and b, resulting in a -to -kda gp , . proteolytic processing of gp expose the gp receptor binding domain (rbd), which interact with the npc domain c within the endolysosome , . this interaction between gp and npc domain c and low ph conditions are essential for subsequent membrane fusion , , . however, whether there is other membrane fusion trigger factor remains unclear. recently, it has been proven that tpc plays a key role in ebola virus infection and the ebola virus enters cells through the endolysosome that contain both npc and tpc , . although the definite role of tpc is uncertain, as a calcium channel, it may involve the movement of endosomes containing ebola virus or the membrane fusion. previous results had demonstrated that npc mediates the entry of ebola, and some small molecular inhibitors block the infection of ebola by disturbing the interactions between npc domain c and ebola primed gp . thus, we first speculated that the cads with selective anti-ebola activity may interact with npc to disturb the interaction between npc domain c and ebola primed gp. however, we did not detect any interactions of tamoxifen/ clomiphene/u a with npc domains a or c. in accordance with our results, another group also showed that clomiphene and u a do not disturb the interaction of npc domain c and ebola primed gp . recently, a study demonstrated that u a interacts with the npc sterol-sensing domain (ssd) to inhibit npc function and cholesterol transport; however, the authors emphasized that the inhibition of ebola requires -fold higher concentrations of u a. they therefore excluded the possibility that u a interacts with the ssd of npc to inhibit the ebola entry . thus, these data demonstrate that npc is not the target of cads during ebola entry inhibition. until now, approximately fda approved drugs have been shown to have selective anti-ebola activities, and a structural analysis revealed that of the drugs are cads (supplementary table s ). the majority of these cads having selective anti-ebola activity have the similar structural properties (pka > , logp > ) (supplementary fig. s a ). based on their structural properties, most cads are protonated, trapped, and finally concentrated in the acid endolysosome compartment . due to the liposolubility of cads, they insert into the lipid bilayer of endolysosome and disturb the metabolism of lipids and the transport of several components, leading to the dip, cholesterol accumulation, and/or functional inhibition of asm (supplementary fig. s b) . moreover, most cads induce their incidental biological activities at micromolar concentrations, which match the concentrations of inhibition ebola infection by cads. thus, it is possible that cads inhibit ebola entry through their incidental biological activities. a filipin staining and ebola pseudovirion entry assay demonstrated that serms and u a induce the cholesterol accumulation and inhibit the ebola entry at the same concentration (approximately μ m); however, the cholesterol accumulation ( h) appeared after ebola entry into the cytoplasm ( h), implying that something changed within h after the serms treatment to mediate the cholesterol accumulation and the inhibition of ebola entry. in contrast with this previous report, our data demonstrated that serms and u a decrease the sphingosine within h. this discrepancy might be due to the different cell types and/or the different treatment dosages of u a used. in agreement with our results, much research has demonstrated that cads can induce the detachment of asm protein from the inner endolysosome membranes leading to inactivation. several cads have been identified as functional inhibitor of asm , and elojeimy et al. have demonstrated that some cads also inhibit the activity of ac . due to the metabolic pathways of sphingomyelin, the inhibition of asm and ac both decrease the cellular sphingosine. it has been reported that ebola infection requires asm activity , so cads could inhibit ebola entry through the inhibition of asm activity. however, how asm is involved in ebola infection is still unknown; future studies are needed to better understand the molecular mechanism of asm and ebola entry. it is well known that intracellular calcium signals involve in vesicular fusion, but whether the fusion of the ebola and endolysosomal membranes also requires a calcium signal is still unclear. in vitro, the conformational change of membrane-bound ebola fusion peptide, which is required for the vesicle fusion, is dependent on calcium concentrations . moreover, the proteins in endolysosomal calcium channels, tpc and tpc , recently have been identified as key players in ebola entry. the inhibitors for tpcs can block nicotinic acid adenine dinucleotide phosphate (naadp)-stimulated calcium outflow and ebola infection . the sphingosine treatment could induce the deregulation of endolysosomal calcium , and a subsequent study demonstrated that tpc and tpc (mainly tpc ) mediate the endolysosomal calcium response to sphingosine . consistent with these results, we proved that serms and u a decrease the sphingosine and increase the endolysosomal calcium. in addition, chelating luminal endocytic calcium with high-affinity rhod-dextran also blocks ebola infection, but not the internalization of ebola vlp to tpc + lysosome. taken together, these results demonstrated that the endolysosomal calcium is involved in ebola infection and is also possible involved in the ebola-endolysosome membrane fusion event. recently, zhao et al. reported that toremifene can bind to and destabilize the ebola gp trimer, triggering premature release of gp , thereby preventing fusion between the viral and endolysosomal membranes . this study revealed the mechanism of ebola inhibition by toremifene, however, binding and destabilize gp may not play the major role in other situation. firstly, the author informed that residues lining the binding site are highly conserved among filoviruses except marburg virus (marv), suggesting that marv may not bind these drugs, but it has been proven that toremifene and clomiphene (even other cads) could effectively inhibit the infection of marv , . secondly, compared to the ethyl chlorine of toremifene, the corresponding ethyl group in tamoxifen and chlorine in clomiphene make weaker interations with the binding sites in ebola gp , and the obvious structural differences of raloxifene (and other cads with anti-ebola activities) with toremifene make the interactions more weaker (supplementary fig. s ). however, all these drugs can effectively inhibit the ebola infection , , , . thirdly, many of cads with anti-ebola activities inhibit ebola and/or marv infection at similar kinetics (inhibition > % at μ m) , , , , which is hard explained by interaction with gp. therefore, the binding with gp plays a major role in ebola inhibition by toremifene, but may not in the ebola or marv inhibition by toremifene, tamoxifen, clomiphene, raloxifene or u a (even other cads with anti-ebola activities). in this study, we demonstrated that serms inhibit ebola pseudovion entry and induce cholesterol accumulation at equal dosages, and they can reduce the cellular sphingosine and upregulate the endolysosomal calcium levels which is regulated by sphingosine through tpc and tpc , moreover, chelation of endolysosomal calcium significantly and specifically inhibited the entry of ebola. based on previous studies and our results, we proposed a hypothesis of the ebola infection (fig. ) . under normal conditions, ebola is internalized by the npc + /tpc + endolysosome , in which the primed gp interacts with npc domain c . the asm and ac in the endolysosome then hydrolyze sphingomyelin to sphingosine . the elevated sphingosine induces endolysosomal calcium outflow through tpc and tpc . both the local calcium flux and the interaction of primed gp with npc domain c induce conformational changes in the ebola fusion peptide, triggering the membrane fusion during the ebola infection . when treated with cads that have selective anti-ebola activities, including serms and u a, the cads are protonated and trapped inside the acidic endolysosome . due to their structural properties, these concentrated cads induce the detachment of the asm protein from membranes, which inactivates the asm and in turn leads to a decrease in sphingosine reagents. β -estradiol (e ), ici , (ici; an er antagonist), tamoxifen, clomiphene citrate, u a, and filipin were purchased from sigma-aldrich (sigma, us). d-erythro-sphingosine (sph) and c -d-erythrosphingosine (c -sph) were purchased from avanti polar lipids (alabastar, us). high-affinity (with the strong calcium buffering) rhod-dextran was purchased from thermo fisher (life technology, us). dmso (sigma, us) was used as solvent for tamoxifen and clomiphene citrate, pbs was used for u a and high-affinity rhoddextran, and methanol (sigma, us) was used for sph and c -sph. cell and plasmids. the human hepatoma cell line hepg , hela cervical carcinoma cell line and the human embryonic kidney (hek) t cell line were obtained from the cell resource center, peking union medical college (licensed from atcc). hepg was maintained in eagle's minimum essential medium (gibco invitrogen, us), hela and t was maintained in dulbecco's modified eagle's medium high glucose (dmem-hg; gibco invitrogen, us) supplemented with % fbs (gibco invitrogen, us) and u/ml penicillin (sigma, us) and μ g/ml streptomycin (sigma, us). the cells were cultured at °c in a humidified atmosphere of % co . the ebola (gueckedou strain) gp, ebola vp , vsv gp, wsn ha/na and pnl - -luc-r − e − vector have been generated as described previously , . we deleted the mucin ( - aa) of ebola gp by overlap-pcr to reduce the cytotoxicity of ebola gp. the tpc -egfp and lamp -bfp expression vector were constructed by inserting tpc and lamp to pegfp-n and pbfp-n vectors. expression and purifications of the a and c domains of npc . the npc domains a and c were constructed, expressed and purified as previously described , . briefly, sf cells infected with npc domain a ( - , n q/n q/n q) baculovirus were used to infect hi- cells. after incubation for h at °c, cells were pelleted by centrifugation, and the medium was concentrated by tangential flow filtration. the concentrated medium was applied to a ni-nta column. fractions containing npc -a were pooled, concentrated. the concentrated npc -a was then applied to a superdex gel filtration column. the cdna encoding npc domain c ( - ) was cloned into pet a vector. the npc domain c was expressed in escherichia coli strain bl (de ) as an inclusion body and then refolded in vitro using the method previously described . the refolded npc -c was then concentrated and purified by gel filtration on a hiload / superdex pg column (ge healthcare). all itc experiments were carried out at °c on an itc instrument (microcal, us). the sample cell contained npc domain a or c ( μ m), and tamoxifen/clomiphene/ u a ( μ m of each compound) was added in injections with μ l of each. data were processed using origin software (microcal, us). each experiment was duplicated three times. microscale thermophoresis. affinity measurements using mst were carried out with a monolith nt. instrument (nanotemper technologies, germany). npc domain a was labeled using the nanotemper nhs nt- labeling kit (nanotemper technologies, germany). the labeling reaction was performed according to the manufacturer's instructions and applying a final concentration of μ m protein with a x molar excess of dye at rt for min in the dark. free dye was eliminated using the supplied dye-removal columns equilibrated with the npc domain a storage buffer. tamoxifen, clomiphene or u a was diluted in the npc domain a storage buffer creating a dilution series of : dilutions ( μ m to . nm for tamoxifen and clomiphene; and μ m to . nm for u a). for the thermophoresis experiment, each ligand dilution was mixed : with labeled npc domain a, incubated for min at rt, before applying samples to monolith nt standard treated capillaries (nanotemper technologies, germany). thermophoresis was measured at rt with % led power and %/ % mst ir-laser power. data from at least three independently performed experiments were analyzed (nt analysis software version . . , nanotemper technologies, germany) using the signal from thermophoresis+ t-jump. each experiment was duplicated three times. for ebola entry and trafficking assays, ebola pseudovirion and vlps were prepared by transfecting hek t cells using polyethyle-nimine with four plasmids: codon-optimized Δ mucin ( - aa) ebola (gueckedou strain) gp and hiv based pnl - -luc-r − e − for ebola pseudovirion. ebola gp, ebola vp and ebola vp with mcherry fused to its n terminus for ebola vlp. the plasmids were transfected at a ratio of : and : : , respectively. vsv and wsn pseudovirions were generated in an identical fashion, but with the substitution of a plasmid encoding vsv gp or wsn ha/na. culture supernatant at hour posttransfection (hpt) was collected and replaced with the fresh media. a second harvest was conducted at hpt. all harvests were pooled and cleared of cell debris by two sequential centrifugations at g for min at °c. vlps in the cleared supernatant were then pelleted through % sucrose [in mm hepes, mm nacl, ph . (hnb)] at , g ( , rpm) in an sw rotor for hours at °c. pelleted vlps were then resuspended overnight in % sucrose (in hnb; / of original volume of culture medium collected), aliquoted, and stored at − °c. approximately h before the experiment, × hepg cells were plated in each well of a -well dish. cells were then pretreated for min with the indicated concentration of inhibitor or with dmso vehicle in medium without fbs. these drug concentrations were maintained in all subsequent steps of the experiment until lysis. after the preincubation period, the cells were infected with μ l of ebola pseudovirion for h and then lysed for the luciferase assay according to the manufacturer's instructions (galen, china). ebola trafficking assay. hepg cells were transfected with tpc -egfp and lamp -bfp plasmids using lipofectamine (invitrogen, us). at hpt, hepg cells were pretreated with dmso, μ m tamoxifen, clomiphene, or u a for h. hepg cells were then incubated with ebola vlp for h, and then fixed. the cells were mounted and then imaged by the nikon a confocal microscope system (nikon, japan). filipin staining. cholesterol accumulation was monitored by staining hepg cells with filipin. the day before an experiment, , cells were plated on glass coverslips in a -well plate. the next day, cells were treated with inhibitors at the indicated concentrations for h. after fixation with % paraformaldehyde, cells were washed twice with pbs, incubated in mg/ml filipin (sigma, us) in pbs for h, and washed times with pbs, after which the coverslips were mounted and imaged on a zeiss axio observer fluorescence microscope (carl zeiss, germany). representative images were inverted for clarity and are shown with uniform adjustments to brightness and contrast across all images. sphingosine measurement. we performed sphingosine measurements using lc-ms/ms (thermo, us) as previously described . briefly, cells were washed twice with ice-cold pbs, then cell pellets containing approximately ∼ × cells were harvested by scraping cells with ice-cold pbs and transferred to pre-chilled . ml new eppendorf tube on ice. the suspended cells were centrifuged at rpm for min at °c to remove pbs, then cell pellets were resuspended in μ l distilled water for further sample preparation. cell pellets were stored at − °c until use. μ l of hepg cell suspension, μ l of internal standards ( ng/ml for c -sph) and μ l of methanol were added into a . ml eppendorf tube. the mixture was vortex-mixed for s followed by centrifuged at , rpm for min, then supernatant was transferred to clean glass vials, and μ l of the supernatant was injected into the lc-ms/ms system for analysis. the autosampler was set at °c. endolysosomal calcium assay. the endolysosome calcium assay was carried out as previously described with some modifications . hepg cells were pre-incubated with μ m tamoxifen, clomiphene or u a for h, and loaded with μ m membrane-permeable calcium indicators fluo -am (abcam, us) for mins at room scientific reports | : | doi: . /srep temperature, then placed into calcium-free buffer for confocal live imaging, repeating two different rois for each treatment. the μ m thapsigargin (tg, sigma, us) was loaded at s for s to release the endoplasmic reticulum calcium, and μ m gly-phe β -naphthylamide (gpn, sigma, us) was loaded at s for s to release the acidic compartment (endolysosomal) calcium. the gpn induced calcium release was quantified with image j software version . (image processing and analysis in java, nih, bethesda, md; http://rsb.info.nih.gov/ij/download.html). statistical analysis. the data were analyzed for statistical significance with the spss . . package (spss inc., us). data for all groups were first tested for normality with the shapiro-wilk test. if the group's data were normally distributed, they were compared using a one-way analysis of variance. p values < . were regarded as statistically significant. all values are presented as the means ± sem (standard error of mean). all of the graphs were generated with graphpad prism . (graphpad software inc., us). ebola haemorrhagic fever ebola virus: unravelling pathogenesis to combat a deadly disease multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection fda-approved selective estrogen receptor modulators inhibit ebola virus infection reversion of advanced ebola virus disease in nonhuman primates with zmapp a screen of approved drugs and molecular probes identifies therapeutics with anti-ebola virus activity identification of compounds that block ebola virus-like particle entry via a repurposing screen of approved drugs. emerging microbes & infections , e ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection chinese hamster ovary cell lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for npc expression ebola virus entry requires the host-programmed recognition of an intracellular receptor ebolavirus glycoprotein directs fusion through npc + endolysosomes ebola outbreak in west africa -case counts correlates of protective immunity for ebola vaccines: implications for regulatory approval by the animal rule cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change toremifene interacts with and destabilizes the ebola virus glycoprotein cationic amphiphilic drug-induced phospholipidosis in vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid based methodologies lipidosis induced by amphiphilic cationic drugs drug-induced phospholipidosis a toxicogenomic approach to drug-induced phospholipidosis: analysis of its induction mechanism and establishment of a novel in vitro screening system inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism mechanistic review of drug-induced steatohepatitis drug-induced hepatic steatosis incidence and risk factors for non-alcoholic steatohepatitis in females treated with tamoxifen for breast cancer the association of nonalcoholic steatohepatitis and tamoxifen in patients with breast cancer functional inhibitors of acid sphingomyelinase (fiasmas): a novel pharmacological group of drugs with broad clinical applications. cellular physiology and biochemistry: international journal of experimental cellular physiology identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model ebolavirus glycoprotein structure and mechanism of entry structure of the ebola virus glycoprotein bound to an antibody from a human survivor discovery, synthesis, and biological evaluation of a novel group of selective inhibitors of filoviral entry identification of a small-molecule entry inhibitor for filoviruses the transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in npc cells ebola viral glycoprotein bound to its endosomal receptor niemann-pick c structure of n-terminal domain of npc reveals distinct subdomains for binding and transfer of cholesterol ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step niemann-pick disease type c is a sphingosine storage disease that causes deregulation of lysosomal calcium ebola virus entry: a curious and complex series of events the ebola virus glycoprotein mediates entry via a nonclassical dynamin-dependent macropinocytic pathway ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein ebola virus entry requires the host-programmed recognition of an intracellular receptor ebola viral glycoprotein bound to its endosomal receptor niemann-pick c structure and function of the complete internal fusion loop from ebolavirus glycoprotein ebola virus. two-pore channels control ebola virus host cell entry and are drug targets for disease treatment ebolavirus glycoprotein directs fusion through npc (+ ) endolysosomes identification of npc as the target of u a, an inhibitor of lysosomal cholesterol export and ebola infection new insights on the use of desipramine as an inhibitor for acid ceramidase ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection calcium-dependent conformational changes of membrane-bound ebola fusion peptide drive vesicle fusion niemann-pick disease type c is a sphingosine storage disease that causes deregulation of lysosomal calcium intracellular sphingosine releases calcium from lysosomes ebolavirus glycoprotein directs fusion through npc + endolysosomes proteolytic activation of the influenza virus hemagglutinin studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha plasticity of human cd alpha alpha binding to peptide-hla-a* simultaneous determination of sphingosine and sphingosine -phosphate in biological samples by liquid chromatography-tandem mass spectrometry this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- -qr ukfti authors: tabraue-chávez, mavys; luque-gonzález, maría angélica; marín-romero, antonio; sánchez-martín, rosario maría; escobedo-araque, pablo; pernagallo, salvatore; díaz-mochón, juan josé title: a colorimetric strategy based on dynamic chemistry for direct detection of trypanosomatid species date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: qr ukfti leishmaniasis and chagas disease are endemic in many countries, and re-emerging in the developed countries. a rapid and accurate diagnosis is important for early treatment for reducing the duration of infection as well as for preventing further potential health complications. in this work, we have developed a novel colorimetric molecular assay that integrates nucleic acid analysis by dynamic chemistry (chemnat) with reverse dot-blot hybridization in an array format for a rapid and easy discrimination of leishmania major and trypanosoma cruzi. the assay consists of a singleplex pcr step that amplifies a highly homologous dna sequence which encodes for the rna component of the large ribosome subunit. the amplicons of the two different parasites differ between them by single nucleotide variations, known as “single nucleotide fingerprint” (snf) markers. the snf markers can be easily identified by naked eye using a novel micro spin-tube device "spin-tube", as each of them creates a specific spot pattern. moreover, the direct use of ribosomal rna without requiring the pcr pre-amplification step is also feasible, further increasing the simplicity of the assay. the molecular assay delivers sensitivity capable of identifying up to . copies per µl with single mismatch specificity. the spin-tube thus represents an innovative solution providing benefits in terms of time, cost, and simplicity, all of which are crucial for the diagnosis of infectious disease in developing countries. conserved region amplification and abasic probes design. pcr amplification was carried out to amplify the conserved region encoding for the s ribosomal rna delta genes. as shown from the multiple sequence alignment in fig. a , the pcr amplification of the target sequence was performed using a single pair of primers (singleplex pcr) which amplifies any of the parasite species present in the sample. the target region is highly conserved among the trypanosomatids t. cruzi and l. major, except for five snfs. amplified fragment analysis was performed using the previously described spin-tube. briefly, abasic pna probes, pna and pna were designed in order to hybridize efficiently with the amplified single strand sense dna, with the important particularity that the abasic positions in the pna probes are opposing the nucleobases under interrogation. once the abasic pna probes hybridize with target sequences, the smart-c-biotin dynamic incorporation takes place, enabling the unequivocal identification of the parasite present in the amplicon sample, because of the unique colorimetric pattern that each trypanosomatid amplicon generates. as shown in fig. b , the abasic pna probe was designed so that its abasic site lies opposite a guanidine nucleotide (at position + of the amplicon), www.nature.com/scientificreports www.nature.com/scientificreports/ irrespective of the species, so as to give a positive result for both parasites, thus confirming the presence of one or both trypanosomatids in the sample (see red arrow in fig. a ). at the same time, the abasic pna is the probe that enables discrimination of the trypanosomatid species. as the target parasite t. cruzi has a guanidine at the position that lines up with the "abasic" position in the abasic pna probe (snf in fig. b ), the smart-c-biotin incorporation is templated, thereby covalently incorporating the biotin tag into the probe attached to the membrane, resulting in a blue spot after applying the labelling and color development protocol. dna coming from l. major has the same sequence but with an adenosine at the snf position (instead of a guanine, see fig. b ). this dna hybridizes with pna but smart-c-biotin is not incorporated and so no signal is detected on abasic pna . in summary, this approach requires two specific molecular events to create a signal: (i) perfect hybridization between nucleic acid strands and complementary abasic pna probes; and, (ii) specific molecular recognition, through guanidine-cytosine base-pairing to allow smart-c-biotin incorporation onto the abasic site of the pna probes (fig. c ). spin-tube fabrication. the chemnat technology with its colorimetric reverse dot blot assay (colorimetric chemnat assay) was integrated into a novel micro device, known as "spin-tube" (fig. a ). as shown in fig. b , the spin-tube consists of: (i) a centrifuge collection tube; (ii) an internal column for the assay; (iii) a nylon membrane (pre-spotted with abasic pna probes) immobilized onto the bottom of the column via a plastic pressure ring (iv). the abasic probes were amino-pegylated at their n-terminal end and printed onto nylon membranes containing pre-activated carboxyl groups (fig. c ). an important stage in the construction of the spin-tube, apart from the immobilization of abasic pnas, was the definition of the array probe layout (fig. d ). probes with fixed concentrations were printed with an automatic nano-plotter onto the nylon membrane, and optimization of signal strength and best signal to background ratios was undertaken. taking into account the mm membrane diameter, it was decided to create a × array. abasic pna and pna were printed onto two parallel rows of three spots each ( features in total, in red and blue in fig. d respectively) . two control biotin-labelled dna oligomers were printed on the top row of the array, to identify array orientation and provide a labelling internal control (in yellow in fig. d ). following printing, performance of probes was initially checked by using synthetic mimic dna oligomers and smart-c-biotin incorporation (data not shown). abasic pna probes design and synthesis. abasic pna and pna were synthesized with amino-pegylated groups in order to be covalently bonded and immobilized onto the solid substrate (nylon membranes). as previously described by our group, abasic neutral pna probes immobilized onto solid surfaces have been found to lack stability and can exhibit a degree of undesirable deformation, flexing and/or bending . this affects the performance (e.g., specificity and/or sensitivity) of the abasic pna probes in this assay -preventing maximum target binding and assay performance. abasic pna and pna were designed and synthesized with these solid surface challenges in mind, adding pna monomers containing propanoic acid residues at their gamma positions across the probe backbone, improving the probe performance and reducing self-aggregation ( fig. b and fig. s in si). moreover, these gamma modifications give rise to a sterocenter, hence creating chiral figure . spin-tube assay: merging of dynamic chemistry with a simple colorimetric end-point assay in a novel micro spin-tube device for analysing "single nucleotide fingerprint" (snf) markers. step : modification of abasic pnas, which are immobilized onto nylon membranes following specific spot patterns, with the biotinylated aldehyde-modified cytosine (smart-c-biotin). this process requires three steps: (i) perfect hybridization between abasic pnas and pcr amplicons; (ii) generation of a reversible iminium specie between the secondary amine of the "abasic" unit and the aldehyde group of the smart-c-biotin nucleobase driven by the templating nucleobase. in this case, smart-c-biotin incorporation can be just templated by a guanidine as otherwise the iminium specie is not stable enough to be reduced; and (iii) reduction of the iminium specie by sodium cyanoborohydride to yield a non-reversible tertiary amine within the pna printed onto the nylon membrane. step : (iv) biotin labelling with streptavidin alkaline phosphatase (streptavidin-alp); (v) incubation with the chromogenic substrate (nbt/bcip, nitro blue tetrazolium chloride/ -bromo- -chloro- -indolyl phosphate) which generates a blue precipitate. step : data analyses by naked-eye reading. time for the assay: min pcr amplification; min dynamic chemistry reaction; min color development procedure, ± h total). ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ pnas molecules, being just the l-pna monomers the ones producing pna oligomers capable of hybridizing complementary natural nucleic acids [ ] [ ] [ ] [ ] . the result was that the l-propanoic abasic pna probes bound to nylon membrane were more readily available to hybridize to complementary nucleic acid strands. still unknown was if such abasic pna probes would prove to be less prone to flexing, bending or otherwise deforming from their "normal" linear configuration . the modifications introduced led to the two probes demonstrating improved specificity and improved sensitivity towards a base complementary to the nucleobase of the dna amplicon to be characterized. the abasic position monomer was also modified with this propanoic acid chain at gamma position and with the same stereochemistry allowing a better dynamic incorporation of the smart-c-biotin because of the spatial orientation of the free secondary amine of the "blank" position ( fig. s in si). labelled smart nucleobase for the colorimetric detection. while our previous mass spectrometry method allowed parasite species detection and characterization by measurement of molecular weight differences between abasic pna probes and aldehyde-modified nucleobases (smart-bases), in this spin-tube platform it was required that the smart-base were biotin-labelled (figs s -s in si) , . here the color development reaction depends on the biotin recognition by a streptavidin-alkaline phosphatase complex, that transforms a colorless chromogenic substrate (nbt/bcip, nitro blue tetrazolium chloride/ -bromo- -chloro- -indolyl phosphate) into a blue precipitate each time there is a biotin tag attached to the membrane. the blue spots emerging correspond to those abasic pna probes which have successfully hybridized with the complementary pcr products and in whose abasic positions smart-c-biotin has been incorporated through a guanosine template (fig. ) . the smart-c-biotin bears a peg spacer to increase water solubility while at the same time distancing the nitrogenous base involved in the hydrogen bonding recognition from the biotin tag responsible for the color development . www.nature.com/scientificreports www.nature.com/scientificreports/ sequence analysis of genomic dna. the spin-tube platform was validated using pcr products from genomic dna (gdna) of both parasites. singleplex pcr was carried out to amplify the highly conserved segment of dna containing the two single nucleotides under interrogation (fig. ) . dna amplicon products were denatured and then together with the dynamic chemistry reaction reagents added directly into the internal column of the spin-tube that supports the nylon membrane for the color-development assay (fig. ) . subsequently, the reaction was carried out at a constant temperature of °c avoiding the need to make use of sophisticated apparatus. single stranded dna hybridizes with the abasic pnas acting as template molecules, driving the error free incorporation of smart-c-biotin into the specific chemical pocket. finally, the incorporated smart-c-biotin is labelled with a streptavidin-alp, so that a colorimetric signal pattern was generated when the chromogenic substrate is added. the signal pattern generated by the protocol allows the visual or photographic imaging of which species cause the patient infection. l. major gdna generated a signal only at the abasic pna probe, as its abasic site lies opposite a guanidine at + position from the ′-terminus of amplicon (see red arrow in fig. ) . while, t. cruzi gdna generated signals for both abasic pna probes. the abasic sites of both pna probes lie opposite a guanidine, allowing the incorporation of smart-c-biotin with its biotin tag, thus resulting in a blue spot after its incorporation. results in fig. assay specificity. bioinformatic along with experimental analysis were carried out to discard the possibility that the presence of human gdna on the samples could affect the singleplex pcr. initially, a primer blast study was carried out using the ncbi primer blast tool and no human hits came out. primers specificity against human gdna was experimentally checked. ng of human gdna were used as pcr template for its amplification with the set of primers described in our study and neither colorimetric signals nor bands were detected using the spin-tube and capillary electrophoresis analysis respectively (fig. , column : a and b), hence probing the specificity when human gdna is present. we also mixed ng of human gdna with ng of dna from both trypanosomatid species in order to perform pcr and amplicon analysis by capillary electrophoresis and spin-tube approaches (fig. , column : a and b and column : a and b). it was confirmed that amplicon formation of the trypanosomatid species and their detection were not affected by the presence of the human gdna. in order to clarify the specificity of the pcr towards other microorganisms, an in silico study was carried out (data not shown). s ribosomal rna delta gene sequences from leishamania spp., trypanosoma brucei and trypanosoma cruzi were aligned using embl-emi tool for multiple sequence alignment clustal omega to check that not only the primers but also the designed abasic pna probes were suitable for the spin-tube and maldi-tof approaches. this tool was also used to study which other leishmania species were suitable to be www.nature.com/scientificreports www.nature.com/scientificreports/ submitted to this singleplex pcr. forward and reverse primers were blasted towards other organisms using ncbi primer blast tool. it was found that mainly genome from trypanosomatids species matched the designed primers. although, a few other genome sequences could also be amplified by our designed primers (schistosoma mansoni, leptomonas pyrrhocoris, aspergillus sclerotioniger and aspergillus eucalypticola), all these infectious agents cause diseases with clinical manifestations very different from those manifested in leishmaniasis and chagas disease and therefore other diagnostic paths would be proposed. another key factor of the assay spin-tube is the signal emerges after the dynamic incorporation of smart-c-biotin into the abasic site of the abasic pna probes. even if off-target sequences are amplified, no colored spots will be observed. so, dna strands coming from any off-target species will not create a signal, as that dna strends will not have precise homology with both forward and reverse primers, as well as the region complementary to the abasic pna within a narrow area of its genome. assay sensitivity. clinically relevant sensitivity was achieved using the amplification and detection platform. the assay was challenged by interrogating different pcr amplification reactions, using decreasing amounts of t. cruzi gdna as starting material. important to note in this study, biotin markers were printed on the top row of the array, rather than (fig. a) . instead of simple circular plastic-ring (see red arrow in fig. s -d in si), here for the construction of spin-tube, pressure plastic-ring with three plastic teeth (see white arrow in fig. s -d in si) were used. six different amounts of gdna ( ng, ng, . ng, . ng, . ng, . ng) were studied (table s in si). pcr amplifications were validated and quantified by capillary electrophoresis. all but the two lowest amounts of gdna ( . ng and . ng) produced amplicons which were able to be detected and quantified by capillary electrophoresis (fig. b) . the amplification products were then used as template for the dynamic incorporation of smart-c-biotin within the spin-tube. positive signals were obtained for both abasic pnas as anticipated, confirming the presence of t. cruzi (fig. c) . a relative quantification of the signal intensity was carried out using imagej software. average background signal intensity was taken and subtracted from all the measurements. biotin signal intensity (marker) was used as reference representing % intensity. the signal intensity for each abasic pna probe was extracted and expressed as a percentage of the biotin signal. almost no difference was observed by eye for the membranes in which and ng of t. cruzi gdna was used as template for the pcr, whereas the relative signal intensity quantification www.nature.com/scientificreports www.nature.com/scientificreports/ by imagej software showed a slight lowering of signal as the genomic amounts decreased (fig. c) . the lowest point which could be detected was . ng or . copies/µl of template (see si, table s for the correlation of ng of gdna and copy number). no results were observed for the two lowest genomic quantities, respectively . and . ng. these results confirm what had been already observed using capillary electrophoresis, further confirming that the limit of detection of the dynamic incorporation assay depends on the pcr yield to provide sufficient template. reactions in which not enough copies of amplicons were created were mis-called as parasite free, being false negatives. negative control (made with h o as template for the pcr) was also called as parasite free, being a true negative result. analysis of spiked parasite gdna in blood. verma decreasing amounts of pcr products were used as templates for the dynamic chemistry reaction to incorporate the smart-c-biotin. positive signals were obtained on both abasic pna probes with pcr starting concentration from ng up to . ng ( . copies/µl) of template what coincides with the last pcr product that was able to be detected by capillary electrophoresis. a percentage of the relative intensity was calculated using as % signal the average of the three biotin marker spots. www.nature.com/scientificreports www.nature.com/scientificreports/ experiment was performed. two human blood samples were spiked-in with and . ng of l. major gdna and total dna was extracted. those extracted dnas were used as template for pcr amplifications and the generated amplicons were analyzed by capillary electrophoresis (fig. a ) and by the spin-tube (fig. b) . the results confirmed that the spin-tube is able to detect a minimum of . ng of l. major gdna spiked-in μl of whole blood that equals to , parasite per ml of blood according the previous publication . direct sequence analysis of ribosomal rna. since the target nucleic acid of the assay was the sense strand dna of the gene coding for the s rrna delta unit, rna could also be used as a target nucleic acid using the dynamic chemistry approach (fig. a) . recently, the dynamic chemistry approach was used to quantify circulating mirnas , demonstrating its feasibility to detect both dna and rna molecules. direct detection of ribosomal rna would avoid performing dna pre-amplification steps by pcr and denaturation and could significantly simplify and shorten the protocol. this would allow its implementation in developing and resources-limited countries. a proof-of-concept study was carried out using rna as the templating nucleic acid. total rna from both parasites l. major and t. cruzi was used. rna quality and quantitation were determined using an agilent bioanalyzer and rna nano kit (fig. s in si) . total rna was fragmented enzymatically, breaking down rna strands into small segments to facilitate the subsequent hybridization with complementary abasic pna probes. rna fragmentation was checked using rna pico kit on an agilent bioanalyzer as shown in si, fig. s . dynamic incorporation of smart-c-biotin was performed using µm of smart-c-biotin and mm of sodium cyanoborohydride for hour. the results obtained coincided with those obtained when using pcr products. l. major gave positive results for pna and t. cruzi provided a positive signal for both pna probes (fig. b) . this was a breakthrough achievement, demonstrating for the first time that direct, pcr free parasite identification from rna samples was achieved. in addition, a much shorter protocol was developed while at the same time reducing the possibility of cross-contamination. this demostrated that rna can also be considered as a biomarker source for the chemnat approach and a future application of the spin-tube. general. all chemicals were obtained from sigma aldrich and used as received. scd buffer was prepared from x saline sodium citrate (ssc) and . % sodium dodecyl sulphate (sds) with the ph adjusted to . using hcl. all synthetic dna oligomers (desalted) were purchased from microsynth ag (balgach, switzerland). the two abasic pna probes (abasic pna and pna ) were synthesized by destina genomica sl (spain) using standard solid-phase synthesis techniques on an intavis bioanalytical instruments multiprep cf synthesizer (intavis ag gmh, germany). aqueous solutions of abasic pna probes were prepared and concentrations were determined using a nanodrop nd- spectrophotometer version . . . (thermo fisher scientific) using as extinction coefficient values (Ɛ ) either . , . , . and . (mm − cm − ) for c, t, a, g, respectively. smart-c-biotin was prepared by destina genomica sl (spain) as reported elsewhere . buffers and dilution reagents were provided by master diagnostica sl (spain). the composition of reagents i to vi is property of vitro sa (spain). genomic dna samples. parasitic protozoa genomic dnas (gdnas) were purchased from the american type culture collection (atcc). respectively, gdna from t. cruzi strain tulahuen with atcc id d and gdna from l. major with atcc id d. human gdna was extracted from mum- b cell line provided by j.c. rodríguez-manzaneque's laboratory. target nucleic acid selection. multiple sequence alignment of the s rrna delta genes of l. major vs. t. cruzi was carried out using the clustal omega, a free on-line available multiple sequence alignment tool offered by embl-ebi. blast studies were carried out with the ncbi primer blast tool (https://www.ncbi.nlm.nih.gov/tools/ primer-blast/primertool.cgi?ctg_time= &job_key=nt_qawtpcueufxn hho sgqbjnpjej nsa). pcr amplification. alignment results were used to design a single pair of primers able to amplify the target region of both parasites. primers ( '- ') sequences were: forward: gattgtgaagggatctcgcag and reverse: tctggcttagaggcgttca. pcr amplification was performed on a veriti -well thermal cycler (thermo fisher scientific). cycling conditions for pcr were as follows: ( ) initial denaturation at °c for min; rna fragmentation. rna was extracted from parasites as described elsewhere . rna quality assessment and quantitation were determined using the agilent bioanalyzer and rna pico kit (fig. s in si) . nebnext magnesium rna fragmentation module protocol was used to fragment rna. the following reagents were mixed in a sterile pcr tube: - µl of purified rna containing - µg of total rna, µl of rna fragmentation buffer and complete with nuclease-free water up to µl. the mixture was incubated at °c for minutes (to get fragments of around -mer length). then the tube was transferred to ice and µl of rna fragmentation stop solution were added. the fragmented rna was cleaned up using ethanol precipitation: µl of fragmented rna, µl of m sodium acetate ph . and µl of % ethanol. the mixture was incubated at - °c for minutes and then centrifuged at , r.p.m. for minutes at °c and ethanol was removed carefully. the pellet was washed with µl of % ethanol, centrifuged and removed the ethanol. finally, the pellet was air-dry for up to minutes at room temperature and re-suspended in . µl of nuclease-free water. to assess the yield and size distribution of the fragmented rna, µl of a -fold dilution was analyzed using the agilent bioanalyzer and rna pico kit analysis (fig. s in si) . www.nature.com/scientificreports www.nature.com/scientificreports/ abasic pna probes spotting. an automatic immobilization of the probes on the membranes was done using personal arrayer (capitalbio corporation, china). immunodyne abc membrane was purchased from pall corporation (us). abasic pna probe spotting solutions were prepared to have the following final reagents concentrations . mg/ml amaranth dye, . m sodium bicarbonate, % of dmso and µm abasic pna probe. spin-tube: smart-c-biotin dynamic incorporation reaction on membranes and colorimetric readout. reaction mixtures with a final volume of µl were prepared by mixing: µl of pcr products, µl of smart-c-biotin ( µm), µl of sodium cyanoborohydride (nabh cn) - mm in water, and µl of scd buffer. protocol: all steps were performed using µl and spin-tube devices were centrifuged to discard the solutions between each step. membranes were incubated for minutes at °c with µl of scd buffer. the reaction mixture was added and incubated at °c for minutes. upon completion of the incubation, three post-reaction washing steps were carried out using reagent i (pre-heated at °c). this was followed by a blocking step in which membranes were incubated with reagent ii for minutes at rt. the enzymatic reaction was carried out by incubating the membranes with reagent iii at rt for minutes. four post-enzymatic reaction washing steps with reagent iv were carried out. the chromogen solution (reagent v) was added to the membranes and left at °c for minutes. finally, three post-chromogen washing steps with reagent vi were carried out. membranes were able to be analyzed. sensitivity and specificity of the assay. to assess the specificity of the assay, four tests were carried out: ( ) one in which the pcr contained water instead of gdnas and ( ) another one in which ng of human gdna was used as template; and two more tests in which ng of human gdna were mixed with other ng of each parasite (fig. ) . on the other hand, for the sensitivity study, a range of pcr amplification reactions were performed using decreasing amounts of starting t. cruzi gdna. six different concentration points ( -fold dilutions) plus a negative control (water) were used in triplicate. all pcr amplification products were analyzed by capillary electrophoresis to determine the amount, if any, of amplicon had been generated (fig. for specificity and fig. and table s for sensitivity). expected size of amplicons were detected when the pcr was performed using the four highest amount of parasitic gdnas while no bands were detected neither when using just human gdna nor when using . and . pg/µl (fig. b ). negative control (water) has not shown any signal, being truly a negative and so being effectively called as parasite free. l. major gdna spike-in experiments on blood. dna was extracted from µl of human whole blood samples using qiaamp dna blood mini kit (qiagen, germany) following manufacturer's guidelines. after proteinase k treatment, two samples were spiked with and . ng of l. major gdna. then, the next steps of the dna extraction protocol were followed as recommended. samples were eluted in µl of elution buffer ae. dna was quantified with nanodrop nd- spectrophotometer version . . . (thermo fisher scientific). pcr reactions were performed with a fix amount of dna, ng. after that, pcr were analyzed by capillary electrophoresis (using the agilent bioanalyzer and dna kit) and by the spin-tube. our previous snf sequence analysis by mass-based assay (maldi-tof) for trypanosomiasis identification has been further developed onto a new ultra-low-cost, easy and fast to use (~ hours/test) spin-tube device. this novel device was designed to combine the dynamic chemical approach for nucleic acid reading (chemnat technology) with a colorimetric method in a plastic column and nylon membrane device (spin-tube). the spin-tube accurately distinguish and identify chagas disease vs. leishmaniasis. the test consists of a singleplex pcr to amplify a highly conserved sequence of dna, that encodes the rna component of the large ribosome subunit containing snfs from the two different parasite species under interrogation. amplicon identification to single base resolution was achieved. dynamic chemistry enables preformans in a simple spin-tube. the assay allows for a naked-eye read-out of the unique colorimetric patterns coming for sample analysis. clinical treatment decisions can be made without any ancillary equipment. the proposed spin-tube assay not only allows the detection of the presence of trypanosomatid pathogens, but also differential diagnosis of leishmaniasis vs. chagas disease. multiplexing is achieved by coupling various target-specific abasic probes onto nylon membranes using different array layouts and patterns. incorporation of smart-biotin only into target sequences ensure high specificity. clinically relevant sensitivity was obtained using our amplification and detection platform, down to a level of detection of . ng/µl ( . copies per µl) of gdna from pathogen. moreover, . ng of l. major gdna was successfully detected when spiked-in with human blood samples. the assay has demonstrated a clinically relevant sensitivity and specificity . a remarkable achievement was for the first time to succeed in the direct detection of ribosomal rna. it opens up the possibility for direct detection of trypanosomatids from biological fluids without any pre-amplification or pre-labelling of target nucleic acids. this breakthrough provides a prototype assay for an innovative pcr free product with many inherent benefits such as lower cross-contamination risk, simplification of protocol, reduction of time-to-results, significantly lower cost, to insure far lower risk of assay result errors. concluding, we believe that the spin-tube developed by our group provides an accurate tool conbianing high sensitivity and specificity, permitting rapid identification and differential diagnosis of chagas disease and leishmaniasis. its clinical value will be an improved patient monitoring and therapeutic decision making. the spin-tube opens-up the promise of repertoire of assays for other infectious diseases, such as malaria and tuberculosis. all data and 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transcriptomes of trypanosomes in mixtures with mammalian cells: the effects of parasite purification and selective cdna amplification this research work has received funding from junta de andalucía, consejería de economía e innovación (project number -bio ), the spanish ministerio de economía y competitividad (grants ctq - , bio - -r, fpi grant bes- - ). this research was partially supported by the th european community framework program (fp -people- -cig-project number ). the unidad de excelencia de química aplicada a biomedicina y medioambiente of the university of granada. the authors thank destina genomica sl for the support throughout the entire period work. we also thank you mr. hugh ilyine for proofreading and j.c. rodríguez-manzaneque's laboratory for providing human gdna samples. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - enr authors: nam, gyu-hwi; mishra, anshuman; gim, jeong-an; lee, hee-eun; jo, ara; yoon, dahye; kim, ahran; kim, woo-jin; ahn, kung; kim, do-hyung; kim, suhkmann; cha, hee-jae; choi, yung hyun; park, chan-il; kim, heui-soo title: gene expression profiles alteration after infection of virus, bacteria, and parasite in the olive flounder (paralichthys olivaceus) date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: enr olive flounder (paralichthys olivaceus) is one of economically valuable fish species in the east asia. in comparison with its economic importance, available genomic information of the olive flounder is very limited. the mass mortality caused by variety of pathogens (virus, bacteria and parasites) is main problem in aquaculture industry, including in olive flounder culture. in this study, we carried out transcriptome analysis using the olive flounder gill tissues after infection of three types of pathogens (virus; viral hemorrhagic septicemia virus, bacteria; streptococcus parauberis, and parasite; miamiensis avidus), respectively. as a result, we identified total , differentially expressed genes (deg) from viral infection, , from bacterial infection, and from parasite infection, respectively. to investigate the effects of pathogenic infection on immune response, we analyzed gene ontology (go) enrichment analysis with degs and sorted immune-related go terms per three pathogen groups. especially, we verified various go terms, and genes in these terms showed down-regulated expression pattern. in addition, we identified common genes ( up-regulated and down-regulated) present in three pathogen infection groups. our goals are to provide plenty of genomic knowledge about olive flounder transcripts for further research and report genes, which were changed in their expression after specific pathogen infection. viruses, viral hemorrhagic septicemia virus (vhsv) is affiliated to novirhabdovirus genus, which is a member of the rhabdoviridae family . the six gene were contained in the vhsv genome of about k bases and each of them coded nucleoprotein (n), phosphoprotein (p), matrix protein (m), glycoprotein (g), nonstructural viral protein (nv), and rna polymerase (l) in the following order '-n-p-m-g-nv-l- ' . infection of vhsv results in contagious viral hemorrhagic septicemia (vhs) in diverse fish species regardless of their inhabitation; seawater or freshwater . in east asia, a lot of infection cases into olive flounder have been reported steadily, since vhsv was detected in middle of s [ ] [ ] [ ] [ ] . a variety of scuticociliates have been reported as cause of scuticociliatosis in marine species including turbot, guppy, and southern bluefin tuna [ ] [ ] [ ] . in olive flounder, disease has been reported to be causing from various scuticociliates; uronema marinum, pseudocohnilembus persalinus, philasterides dicentrarchi, miamiensis avidus [ ] [ ] [ ] [ ] . interestingly, judging from infection experiments using various scuticociliates plus identification outcome of isolates acquired from olive flounders with symptom of ulcers and haemorrhages, miamiensis avidus was suggested as the major aetiologic agent of scuticociliatosis because of high pathogenicity and mortality rate compared with other scuticociliates , . infection of bacteria could sustain serious damage to fish. streptococcosis is known to be caused by a variety of streptococcic species; streptococcus parauberis, streptococcus iniae, streptococcus difficilis, lactococcus garvieae, lactococcus piscium, vagococcus salmoninarum, and carnobacterium piscicola, and has become major nuisance in olive flounder farms [ ] [ ] [ ] [ ] . in particular, streptococcus iniae, lactococcus garvieae, and streptococcus parauberis have been introduced to be related with streptococcosis in olive flounder [ ] [ ] [ ] [ ] [ ] . the main issue of aquaculture industry is to reduce economic loss by preventing mortality of fish from various pathogens. a large number of immunologic studies have been proceeded about various immune-related gens against pathogen infection , [ ] [ ] [ ] [ ] . a huge quantity of genomic information from next generation sequencing (ngs) technique has been gradually increasing for the last few years, indicating that researchers could approach more comprehensive understanding view about genome of organisms than when they research a single gene level. with development of wide-sized analysis methods, it is not difficult to figure out change of gene expression level after any chemical treatment or environmental change. recently, studies to identify large-scale genes were conducted in the olive flounder genome for researches about vaccine, gonadal development, and sex determination [ ] [ ] [ ] . in particular, characterizing of immune-related genes was reported in olive flounder spleen tissue . a lot of studies reported earlier were focused on gene expression analysis of single pathogen and specifically defined the expression pattern of limited genes [ ] [ ] [ ] [ ] [ ] . further, infection by two or more pathogens were reported in the olive flounder genome , . in order to solve these problem, we need plentiful genomic information to respond rapidly to multiple infection of pathogens. however, researches, which were comprehensively analysed about change of gene expression pattern by different type of pathogens, have not been reported in the olive flounder genome, so far. in this research, we identified differentially expressed genes (degs) by transcriptome analysis and conducted gene ontology (go) analysis with genes identified. then, we tried to find important genes which showed consistently meaningful expression change in the results of three infection experiments. as a result, we determined up-regulated genes and down-regulated genes in common after infection of three pathogens. we aimed to provide essential genome information which is related with pathogen infection and explore the various consequences related to differential infections and find out the common strategies against specific candidates involved in disease progression in natural habitat of aquaculture. statistical summary of transcriptome analysis. to profile gene expression after infection of three pathogens (vhsv, streptococcus parauberis, and miamiensis avidus), transcriptome analysis was conducted using gill tissues of olive flounders, respectively. we prepared twelve olive flounders (three un-infected individuals as control, three virus-infected, three bacteria-infected, and three parasite-infected individuals) to raise confidence. to gain the sufficient number of transcripts, twelve independent rna samples acquired from normal and pathogen-infected olive flounder gill tissues were employed for construction of cdna library. then, these cdna libraries were sequenced using illumina hiseq , generating the numbers of approximately . million, . million, and . million raw reads from three control samples, . million, . million, and . million raw reads from bacteria-infected samples, . million, . million, and . million raw reads from virus-infected samples, . million, . million, and . million raw reads from parasite-infected samples, respectively (table ) . after trimming of low-quality reads and adaptor sequences, the number of clean reads acquired from control samples were average . million reads from control samples, average . million reads from bacteria-infected samples, average . million reads from virus-infected samples, and average . million reads from parasite-infected samples, respectively. then, we checked gene coverage whether the reads that we acquired are sufficient for quantitative gene expression analysis ( supplementary fig. ). the clean reads were assembled into , transcript sequences acquired from transcriptome analysis, we identified total , genes involving novel , genes from transcript sequences using interproscan database and non-redundant protein database in the ncbi ( table ). to figure out the effects of external pathogen for gene expression, we sorted out genes which showed expressional change after pathogens infection having p-value of < . when compared with control sample. as shown in table and fig. , the largest numbers of gene expression change were shown in vhsv infection group; total , degs were identified from transcriptome analysis. we showed information of degs derived from viral infection in supplementary to explore the functional enrichment of these degs, we performed go enrichment analysis using david tool table ). prediction following viral infection, we identified the degs with p-value of < . after infection with bacteria (streptococcus parauberis) and parasite (miamiensis avidus) ( table and table ). miamiensis avidus affected gene expression pattern in olive flounder genome and selected genes which showed expression change (supplementary table ). we identified degs caused by infection of miamiensis avidus; description samples u-i u-i u-i b-i b-i b-i v-i v-i v-i p-i p-i p-i number distributional pattern of total degs acquired from transcriptome analysis. after comparison of gene expression level among twelve transcriptome analysis, we identified degs after pathogen infection. then, we focused on selection of genes showing expression change pattern after three types of pathogen infection in common. as a result, we summarized up-regulated genes and down-regulated genes, respectively (fig. ) . we analyzed these degs to identify their gene symbol correctly using their sequences in non-redundant protein database of the ncbi database, and degs were annotated (table ) . we showed the rest of unannotated degs in supplementary table . with development of sequencing technique, numerous genomic researches have been reported to understand infection results by virus , , , bactria , , , and parasite in the olive flounder genome. a fundamental way to overcome disease outbreak from external pathogens is to approach from their genome level. it is essential to expand quantity of genomic information in pursuance of biological research about any target. investigation of overall gene expression change after pathogen infection would provide clues of cause of biological damage. gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed. phylogenetic diversity of pathogens (virus, bacteria and parasite) is also responsible for differential expression of genes in diseases. each individual pathogen causes disease in a different way, which makes it challenging to understand the basic biology of infection. in this study, we understood the relation between three types of pathogen infection and differential gene expression in the olive flounder genome through transcriptome analysis, respectively. the diverse pathogens used in this study, carry specific antigenic variations, which refers to the specific mechanism by which an infectious agent infect the fish and progress the disease. transcriptome analysis help us to understand the progression of disease in fish through pathogen infection based on diversity of pathogen (virus, bacteria and parasite). this study shows differentially expressed genes were up-and down-regulated at different extend in fish tissue. interestingly, virus and bacteria have more down-regulated genes while parasite have more up-regulated genes. this data signifies the fact that fish immune system interacts with bacteria and virus with the same strategy, while with parasite different due to difference in mode of infections between them. for efficient prevention against pathogen, it is important to understand which genes were activated/repressed after pathogen infection because their expression change means variation of metabolic system in body. in this study, we identified total number of , in vhsv infection group, , in streptococcus parauberis infection group, and degs from miamiensis avidus infection group, respectively (table ). given the difference in the number of degs among three pathogen groups, these results seemed that virus had the most impact on gene expression mechanism in the olive flounder genome among three pathogens. interestingly, , degs ( % of all degs) showed down-regulated pattern after viral infection. this phenomenon that global gene expression was decreased by viral infection must cause pathogenic disease by affecting immune-related gene expression level, finally leads to death. this view was supported by our findings (supplementary table ), which showed expression decrease pattern of all genes in the immune-related go terms. especially, go terms in viral infection group showed that all genes tend to be down-regulated after pathogen infection, indicating loss of resistance against pathogens by down-regulating the expression of immune-related genes. like this situation, functional information of genes acquired from go enrichment could help researcher to figure out critical biological pathway against any external factor. the immune mechanism in fishes is composed of a set of cellular and humoral system and divided into innate (inherit), and adaptive (acquired) substances. the understanding of fish immune system structure and function is essential for the development of new technologies and products to improve productivity. the transcriptome analysis bring exposure to basic difference in expression profile of all pathogens in the host. these differences were due to nature of parasite, their mode of infection, antigenic variations and many other factors. additionally, along with all above differences, disease progressed in host due to external surface variation of pathogens (viral, parasite and bacterial) and their appropriate recognition by host immune systems for making the basis to initiate microbial clearance , . disease research requires the knowledge of important key factors like method of avoiding host immune surveillance, antigenic variations, subversion of immune responses through phagocyte and inhibition of cytokines and chemokines in common with pathogen infections (viral, bacterial and parasite) , . on the other hand, the disease progression was different in accordance to type of pathogens. in case of viral infection, understanding of complement inhibition and blockade of cellular immunity is the most important, while parasite and bacterial infections required knowledge and research of innate pathway and acquired immunity , . our analysis indicates the complexity and difference of expression profile could be due to all the above reasons. in addition, important basis of fish vaccine is depending on innate and adaptive immunity . there were many vaccine types which depend upon antigens, live microorganisms or specific dna segment of pathogens or polyvalent vaccines. all above vaccines required complete knowledge of pathogenicity and deep research of efficacy . our study clearly indicates about various immune and antigenic genes which can be chosen for pathways analysis and use for therapeutic agents or as some vaccine candidates (supplementary table ) . despite of their different infection pathway, we wondered common degs which were affected from infection. as shown in fig. , total degs ( up-regulation and down-regulation) were identified in common in three pathogens, and of up-regulated and of degs were annotated by genomic database (table ; , genes (bacteria), and genes (parasites). these genes were specific for each pathogen so can be used as candidate genes for vaccination or therapeutic agents. in case of the down-regulation of gene (virus), gene (bacteria) and gene (parasites) in infection of fish, these genes were specific for specific pathogens so can be used as a diagnosis marker for specific pathogens. c-x -c motif containing , col a ; collagen type i alpha chain, col a ; collagen type i alpha chain, slc a ; solute carrier family member ) which showed highest expression change (down regulation) with fold change (log ) of ' <− . ' on at least two infection groups. in this study, we investigated all the above candidate genes and found their role in disease progression. we have listed these genes and their role in below headings. anpep, called as gene aminopeptidase n (apn), is metallopeptidase that exerts strong influence on various immune response mechanisms. for example, apn has been known to cause decomposition of cytokines and peptides used by neurons [ ] [ ] [ ] , and acts as receptor for viruses , . in addition, relation between expression level of apn and stimulated t-cell was reported . recently, it was suggested that apn controlled the balance of innate immune and adaptive immune by regulating tlr signal transduction pathway in myeloid cells . bglap, also known as osteocalcin, is a noncollagenous protein, mainly found in bone, which needs vitamin k for its synthesis. this protein was thought to play a role in calcium ion homeostasis and used as biological marker for bone formation . in addition, it concerns in endocrine regulation, especially in digestive system, by stimulating release of insulin hormone from β-cell of the pancreas and adiponectin hormone from fat cells, respectively . as well as these function, it has been reported to take a role in promotion of energy availability and sexual maturation of male by stimulation of testosterone biosynthesis , . cmc , called as mtcp , has been mainly reported to be related in various diseases. it was reported that mtcp gene affected t-cell homeostasis prior to process of leukemogenesis in transgenic mice . although the function of this gene has been entirely discovered, regulation error of mtcp gene affected on cell survival and cell growth . besides, this gene was known to be related in the pathogenesis of a subset of t-cell lymphoproliferative diseases , . col a and col a encodes the pro-α chain and the pro-α chain protein, respectively. the type i collagen, which is comprised by two pro-α chains and one pro-α chain, plays a role in reinforcement and support in most of all connective tissues such as bone, cartilage, skin, and tendon, and offers those tissues rigidity and elasticity , . this protein was reported to stimulate expression of pro-inflammatory cytokines and professional phagocytes in teleost fish gilthead seabream , . in addition, it has been reported that receptor-mediated interaction which is formed in between cells and collagen molecule might affect in wound healing, inflammatory, and immune response by activating various factors such as cytokines, growth factor, and matrix metalloprotease , [ ] [ ] [ ] [ ] [ ] . slc a , also is known urea transporter (hut ), is important gene involved in urea transport and play role in physiology. in mammals, two types of urea transporter (slc a and slc a ) has been reported and were regulated by vasopressin hormone . the kidney uses urea to maintain the appropriate concentration and volume of blood. without control of these proteins, organism would result in extreme damage in urinary system. besides, a previous study has reported that genetic variation including nucleotide change is known to significantly influence blood pressure (bp) and metabolism syndrome , . as shown in supplementary table , immune-related degs were revealed as results of three pathogens infection. infection of pathogens caused activation of immune system to respond to invasion of harmful external elements, indicated that change of expression level of immune-related genes. the down-regulation of gene could sequentially influence on expression of various molecules positioned in down-streams in metabolic pathway. in this view, cd , which is one of down-regulated genes by infection of three pathogens on common, was reported to inactivate interleukin . representative function of this cytokine is to induce migration of neutrophils and granulocytes toward infection site. in addition, absence of cd considerably improves cross-presentation of soluble antigen via regulation of receptor-mediated uptake . thus, decrease of cd expression consequentially might activate immune response in the olive flounder. mtcp gene induced malignant t-cell transformation and was related in the leukemogenic process of mature t-cell proliferation . this gene was thought to maintain balance of immune response by t-cell. the innate immune system mediates the initial inflammatory response by pathogen infection or injury. for rapid response against external pathogens, infected cells secrete various cytokines to induce effector cells and complements. the type i collagen, which is comprised by proteins coded from col a and col a , was involved in the expression of pro-inflammatory cytokines in the innate immune system. however, two genes (col a and col a ) showed decreasing expression pattern after infection in our study. given sampling period ( days from infection) of olive flounders for this study, it might be explained that the adaptive immune response was activated in the olive flounder genome. it is hard to understand comprehensively about immune system of fish genome. however, our results were expected to contribute for further study by extend of genomic knowledge in the olive flounder. in conclusion, this study is helpful in understanding infection of the diversified pathogens (antigenic variation) and their role in disease progression in the olive flounder. the differentially expressed genes identified from transcriptome analysis using three types of pathogens could be useful to study the basic diagnosis and therapeutic mechanisms, and offer opportunities for designing the appropriate vaccines or drug targets for pathogen specific candidate genes. because of lack of genomic information or using one external infection factor, previous studies have been limited to understand global expression pattern of whole genes in the olive flounder genome. we hope that this research would contribute to achieve great outcome in various biological field. ethical statement. all experiments with the olive flounders in this study were carried out in accordance with the guidelines and regulation approved by ethical committee of pukyong national university. preparation of olive flounder gill tissues. gill tissues from twelve olive flounder (bw = ~ g, n = / group) including healthy and infected fish with each pathogen were used for this study. briefly, healthy fish (non-challenged), sampled fish at days post challenge (dpc) with s. parauberis at . × cfu/fish in / seawater of °c, sampled fish at dpc with vhsv at pfu/fish in / seawater of construction of cdna libraries for transcriptome analysis. building of transcriptome libraries were conducted by illumina's truseq rna protocol, and - μg of total rna were used in each samples. ampure xp beads (beckman coulter) and ambion fragmentation reagents kit (ambion, austin, tx) were used for extraction of poly(a)+ rna and their fragment, respectively. as the following steps, cdna synthesis, end-repair, a-base addition, and ligation of the illumina indexed adapters were carried out according to illumina's protocol. the size-selected - bp cdna fragments were loaded on a % nusieve : (lonza) agarose gel for libraries. the cdna fragments were recovered using qiaex ii gel extraction reagents (qiagen), and amplified using phusion dna polymerase (new england biolabs) for pcr cycles. the amplified libraries were purified by ampure xp beads, their concentration and product sizes were assessed on an agilent bioanalyzer. sequencing of paired-end libraries were conducted with the illumina hiseq , ( × nucleotide read length). transcriptome analysis and differential gene expression. transcriptome analysis were carried out with the rnaseq tuxedo protocol. mapping of sequences were conducted against the olive flounder draft genome (submitted at present) using tophat v . . with default options for paired-end sequences. transcripts expression were estimated using the cufflinks program v . . . total sequencing reads were subjected to preprocessing as follows: adapter trimming was performed using cutadapt with default parameters, and quality trimming (q ) was performed using fastqc with default parameters. processed reads were mapped to the olive flounder draft genome (submitted at present) using tophat and cufflink with default parameters . the differential analysis was performed using cuffdiff using default parameters. further, the fpkm values from cuffdiff were normalized and quantitated using r package tag count comparison (tcc) to determine statistical significance (e.g., p values) and differential expression (e.g., fold changes.). through these statistics analysis, we sorted degs having p < . and showed them as results. gene ontology analysis. deg set for go analysis was acquired from transcriptome analysis. degs were annotated from interproscan database and non-redundant protein database in the ncbi. david and uniprot tool were used for exploring the functional enrichment of these degs 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cross-presentation and t cell responses by inhibiting receptor-mediated antigen uptake differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks tcc: an r package for comparing tag count data with robust normalization strategies this research was a part of the project titled "omics based on fishery disease control technology development and industrialization ( ), " funded by the ministry of oceans and fisheries, korea. g.n. wrote the main manuscript text, a.m. and j.g. supported arrangement of contents in manuscript, h.l. and a.j. supported to complete fig. , d.y. and a.k. prepared fish tissue samples, w.k. provided olive flounder genome reference sequence, k.a. provided bioinformatic advice for this study, d.k., s.k., h.c., y.c. and c.p. reviewed the manuscript, h.k. supervised entire flow of the manuscript and reviewed contents. supplementary information accompanies this paper at https://doi.org/ . /s - - -y.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -e ihpe i authors: chang, ya-chun; huang, kuo-tung; chen, yu-mu; wang, chin-chou; wang, yi-hsi; tseng, chia-cheng; lin, meng-chih; fang, wen-feng title: ventilator dependence risk score for the prediction of prolonged mechanical ventilation in patients who survive sepsis/septic shock with respiratory failure date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: e ihpe i we intended to develop a scoring system to predict mechanical ventilator dependence in patients who survive sepsis/septic shock with respiratory failure. this study evaluated adult patients in medical intensive care units (icus) between august to october , who had survived for over days and received aggressive treatment. the risk factors for ventilator dependence were determined. we then constructed a ventilator dependence (vd) risk score using the identified risk factors. the ventilator dependence risk score was calculated as the sum of the following four variables after being adjusted by proportion to the beta coefficient. we assigned a history of previous stroke, a score of one point, platelet count less than , /μl a score of one point, ph value less than . a score of two points, and the fraction of inspired oxygen on admission day over % as two points. the area under the curve in the derivation group was . (p < . ). we then applied the vd risk score for validation on patients. the area under the curve in the validation group was . (p = . ). vd risk score could be applied to predict prolonged mechanical ventilation in patients who survive sepsis/septic shock. imposes a financial burden on patients, families, and the healthcare facility. furthermore, it decreases the patient's quality of life. we sought to determine the risk factors for ventilator dependence in patients who survive sepsis and septic shock. patient characteristics and findings. a total of patients with sepsis or septic shock and acute respiratory failure requiring mechanical ventilation were admitted to the medical intensive care unit in the kaohsiung chang gung memorial hospital from august to october . of the patients, patients were enrolled in the study (fig. ) . for validation, we collected data from sepsis/septic shock patients with mechanical ventilation admitted to the medical icu from november to november and patients were included in the study (fig. ) . among the patients studied, . % were admitted from the emergency room. the site of suspected infection was of pulmonary origin in . % (table ) . patients ( %) were ventilator-dependent (on the ventilator for at least days), and ( %) patients were ventilator-independent (needing ventilator support for less than days). the mean age (standard deviation (sd)) of ventilator-dependent patients was older ( . ( . ) vs. . ( . ) , p < . ) ( table ) . no statistical differences were observed in the apache ii score (p = . ) or the initial sofa score (p = . ) between the ventilator-dependent and ventilator-independent groups. however, there was a significant difference between the ventilator-dependent and ventilator-independent groups regarding previous stroke (p = . in the univariate analysis) ( table ) . hematology, biochemistry, fio , and pao /fio were collected for further analysis ( table ). the platelet count was lower on admission day in the ventilator-dependent group compared with the ventilator-independent ventilator dependence risk score. we constructed a ventilator dependence risk score using individual risk factors, which were first identified from the univariate analysis. risk factors on admission day , day and day in univariate analysis included white blood cells, hemoglobin, platelet, prothrombin time, inr, ast, alt, bun, creatinine, na, k, c-reactive protein, albumin, lactate, procalcitonin, ph, paco , bicarbonate, fio , pao / fio , etc. variables on admission day , admission day and admission day , which were possibly associated with ventilator dependence in the univariate analysis (p < . ), were included in a multivariate analysis model. a total of variables were included in the multivariate analysis model, such as age > years, fio > %, history of stroke, ph < . , platelet < , /μl, history of coronary artery disease, hemoglobin, bun, c-reactive protein, pao /fio and paco on admission day . after stepwise method, the independent factors associated with ventilator dependence were identified to build a score. a clinical score (vd risk score) was calculated based on four variables independently associated with ventilator dependence in the multivariate analysis, including previous stroke, thrombocytopenia, acidosis, and higher fio ( table ). the ventilator dependence risk score was calculated as the sum of these four variables after adjusting by proportion to beta coefficient. we assigned a history of stroke one point, platelet count on admission day of less than , /μl one point, ph value on admission day of less than . two points, and the fraction of inspired oxygen on admission day over % two points (table ) . receiver operating characteristic curves were plotted in fig. . the area under the curve (auc) of the ventilator dependence risk score was . with p value < . . a ventilator dependence risk score equal to or more than one point yielded . % sensitivity and . % specificity. sofa score for ventilator dependence prediction. we tested sequential organ failure assessment (sofa) score on admission day and day to predict ventilator dependence on sepsis and septic shock patients with respiratory failure. the area under the curve (auc) of the sofa score on admission day was . and the auc of the sofa score on admission day was . . we also tested and found that sofa pao /fio subscore and gcs subscore on admission day could help predict ventilator dependence on sepsis and septic shock patients with significant difference in univariate analysis ( table ). the area under the curve of the pao /fio subscore on admission day was . and the auc of the gcs subscore on admission day was . (fig. ). validation ventilator dependence risk score. for validation, we collected data from sepsis/septic shock patients with mechanical ventilation admitted to the medical icu from november to november . we used the ventilator dependence risk score to predict ventilator dependence in these patients. patient characteristics and underlying disease are collected in revealed as table . the auc of the ventilator dependence risk score was . and the p-value was . (fig. ) . a ventilator dependence risk score equal to or more than one point yielded . % sensitivity and . % specificity. we also found the auc of ventilator dependence risk score was . in the cancer group and p-value was . . auc of ventilator dependence risk score was . in the chronic kidney disease group (p = . ) (fig. ). in this study, we identified risk factors for prolonged mechanical ventilation in patients who survived sepsis and septic shock. these included a history of stroke, and data collected on day (thrombocytopenia, acidosis, and a higher fraction of inspired oxygen). the ventilator dependence risk score can help easily predict prolonged mechanical ventilation. we chose biochemical and physiological variables from day to incorporate into our score, as opposed to day or day , which would each have advantages and disadvantages. for example, it is too late to predict ventilator dependency using day data. on the other hand, with multiple factors and different treatment response, it is difficult to predict ventilator dependency from day data. with aggressive treatment in the first week, day data can help identify which patients face a substantial risk of becoming long-term ventilator-dependent. patients who have suffered a stroke in the past often have respiratory dysfunction due to respiratory drive impairment. according to a previous study, respiratory function depends on numerous neurologic structures, which extend from the cerebral cortex to the medulla; complications after an injury to the respiratory center could lead to prolonged mechanical ventilation , . therefore, a previous stroke is an independent risk factor for predicting prolonged ventilator use. sepsis is a life-threatening organ dysfunction caused by a disproportionate host response to infection and it involves complex mechanisms . during sepsis, platelet numbers decrease due to increased platelet destruction. sepsis may result in hypercoagulation due to fibrin deposition and platelet activation. this leads to the formation of micro-thrombi as a host defense mechanism against pathogens, in which platelets play a crucial role. in extreme situations, this may progress to disseminated intravascular coagulation (dic), with severe thrombocytopenia and coagulation system impairment [ ] [ ] [ ] . platelet dysfunction during sepsis correlates with a poorer prognosis. thus, the morphology, number, and function of platelets may be used as biomarkers for the risk stratification of patients with sepsis . although we excluded very ill patients with decreased platelet counts who expired within days (in our series, average * /μl), the platelet count on day could differentiate the ventilator-dependent and independent groups on day . a relatively low platelet count on admission day suggests that a septic patient has not completely recovered and may have greater risk of ventilator dependence. although there was a significantly lower hemoglobin level in the ventilator-dependent group, it is hard to suggest that bleeding caused by thrombocytopenia is causing weaning failure. the hemoglobin level in both groups was greater than g/dl. acidosis is increased acidity (hydrogen ion concentration) in the blood and other body tissues. it occurs when the arterial ph falls below . . sepsis can cause tissue hypoperfusion and the accumulation of lactate, which causes metabolic acidosis . acidosis resolution in survivors was attributable to a decrease in strong ion gap and lactate levels . additionally, respiratory acidosis can be due to the accumulation of carbon dioxide in the lungs, which indicates poor lung functioning . our data revealed that arterial blood gas acidosis on day was one of the independent risk factors predicting ventilator dependence. we did not find statistical differences between groups on higher lactate levels or vasopressor use trends in ventilator dependent patients. acidosis could be non gap metabolic acidosis from hyperchloremia and fluid overload. in addition, either sepsis progression or poor lung functioning may have caused the resulting acidosis. fraction of inspired oxygen (fio ) is the fraction or percentage of oxygen in the volume being measured. it is used to represent the percentage of oxygen participating in gas exchange. according to a study by diniz et al., fio levels sufficient to ensure a spo ≥ % do not alter breathing patterns or trigger clinical changes in weaning patients . the fio level was enough to represent the oxygen status of the ventilated patient. our data revealed that a higher fraction of inspired oxygen demand was associated with greater risk of ventilator dependence in patients with sepsis or septic shock. applying the ventilator dependence risk score to predict prolonged ventilator dependence can help us communicate with the family, enable quick adjustment of the treatment strategy, and ensure more efficient allocation of medical resources. in addition, it is clinically applicable. the score includes two components. one component is uncorrectable, such as previous stroke history; the other component is correctable if treatment is successful, such as thrombocytopenia, acidosis, and fraction of inspired oxygen. we do not suggest correction of thrombocytopenia and acidosis by blood transfusion and bicarbonate use, as there are inherent risks with platelet transfusion and bicarbonate infusion. however, the clinical physician should make the best efforts in correcting underlying progressive sepsis to avoid prolonged ventilator use. we do not routinely use subcutaneous heparin for prophylaxis of deep vein thrombosis or pulmonary embolism in taiwan. therefore, we seldom have heparin induced thrombocytopenia patients. in our study group, we had no patients with sepsis and pulmonary embolism concurrently. however, we should keep the possibility in mind. as some components of our ventilator dependency risk score are similar to sofa values, we tested sofa score for ventilator dependence prediction. we found the area under the curve (auc) of the ventilator dependence risk score ( . ) was better than the sofa score on admission day and day . however, components of sofa score (pulmonary subscore: pao /fio and gcs subscore) on admission day were significant for predicting ventilator dependence in univariate analysis (p < . ). despite these findings, the pao /fio and gcs auc were not better than the ventilator dependence risk score auc (fig. ) . in fact, we have previously described an immune dysfunction scoring system for predicting -day mortality in septic patients, with better discrimination than sofa score; this system was valid and reproducible. the above cases were from part of the current sepsis cohort, who agreed for immune function assessment . however, in the present study, we are focused on ventilator dependency amongst patients who survive sepsis more than days. combining those tools, we can predict long term ventilator dependence and predict survival. the area under the curve (auc) of the ventilator dependence risk score was . in our study group and the auc of the ventilator dependence risk score was . in the validation group. after further analysis of the validation group, we found the auc of ventilator dependence risk score was . for sepsis with cancer group and the auc was . for sepsis with chronic kidney disease group. we are actively studying the effect of co-morbidity on the outcomes of patients with sepsis, although it is out of the scope of this study. our previous study revealed that among patients admitted to the icu with sepsis, those with underlying active cancer had higher baseline levels of plasma il- , higher trend of g-csf and higher mortality rate than those without active cancer . our ventilator dependence risk score could help predict who will need prolonged mechanical ventilation. we did not exclude patients with tuberculosis or severe immunosuppression (human immunodeficiency virus (hiv), oncologic, solid-organ or bone marrow transplantation). our score can also be used for these patients. septic patients admitted to the hospital or the intensive care unit are usually screened for contamination with multi-resistant bacteria and subjected to collection of blood cultures and respiratory secretions. as in our previous study , multi-resistant bacteria or specific pathogens influence survival in patients with ventilator associated pneumonia. the phenomenon was not shown for ventilator dependency . most of our patients came from er ( . %) and most of our blood culture showed no growth. we suggest that multi-resistant bacteria may not influence prediction of prolonged mechanical ventilation. however, further study may be needed to determine the effect. renal replacement therapy could be a risk factor. however, there was no statistical significance in univariate analysis. in addition, the sofa renal subscores did not differ between ventilator dependent and independent patients. therefore, renal replacement therapy was not used in the scoring system. in , sellares j et al. described that copd, increased heart rate and paco during the spontaneous breathing trial independently predicted prolonged weaning. however, our studied group had small proportion of copd ( . % in ventilator-dependent group and . % in ventilator-independent group) ( table ). in addition, we did not routinely record heart rate and paco during the spontaneous breathing trial. therefore, paco and heart rate during the spontaneous breathing trial were not included in our scoring model. extubation failure before day may be an additional prognostic parameter for ventilator dependence. however, in our study population, no extubation failure before day was noted. the limitations of the study include the retrospective study design and possible selection bias. however, first, we used prospectively collected data and screened consecutive patients. second, we excluded patients who died within days, which may have masked some predictors associated with both mortality and ventilator dependence. however, mortality prediction was beyond the scope of this study. the application of the score focused on patients who survived sepsis/septic shock with acute respiratory failure on admission day . this patient group was not completely recovered and needed further treatment and strategic decision making. from our results, the data from day is enough to calculate the score, which makes it feasible to use for predicting ventilator dependence. patients require mechanical ventilation due to either pulmonary function problems or neurological function problems. in patients with sepsis, both components may co-exist. it is difficult to delineate what proportion of patients requiring prolonged mechanical ventilation is attributed to pulmonary or neurological problems. we did not incorporate any data on the patients' pulmonary system mechanics or respiratory muscle strength (respiratory system compliance or resistance, maximal exhaled tidal volume, negative inspiratory force, rapid shallow breathing index) that are typically studied during weaning from mechanical ventilation . it is partially because of some missing data owing to the retrospective characteristic of the study, which makes it difficult to analyze. most importantly, obtaining parameters like static compliance requires an additional procedure such as paralysis and muscle relaxant, which may add risk to those patients with unstable severe sepsis. for easy application to patients with sepsis and septic shock, we chose to incorporate data easily checked in clinical practice. it is now well known that sepsis and multi organ failure can cause neurological dysfunction by way of critical illness neuropathy and myopathy (i.e., icu acquired weakness), which can cause difficulty weaning from mechanical ventilation due to diaphragmatic weakness. sepsis and multiple organ dysfunction are the most common and well accepted risk factors for icu acquired weakness. some other risk factors like ards, neuromuscular blockade, glucose control, and steroid use are missing from the analysis due to the retrospective study design. those particular entities deserve attention. the diagnosis of icu acquired weakness is often clinical with emg support, which is not often conducted in routine clinical practice. with respect to neurological function, we note a significant difference in the groups with the history of prior stroke. we did not have complete data differentiating hemorrhagic or ischemic strokes. in addition, the functional status or delirium data were also lacking. we attempted to use gcs (the required data are already present within the apache and sofa scores) but the results showed poor discrimination. those issues need to be explored further in the future. a valuable tool to predict which septic patients will need prolonged mechanical ventilation may have not only therapeutic ramifications, but also significant financial and social implications. as shown in table , patients requiring long term mechanical ventilation have significantly longer icu stay and in hospital mortality. it is primarily due to the medical acuity. however, in part, it is also due to a paucity of ventilator weaning facilities. patients who require long term mechanical ventilation are often difficult to place, leading to longer hospital stays than expected for their given illness. we did not discuss diagnosis of ards in this study. the pao /fio were comparable between the two groups. in the same period, our colleagues participated in a multiple center study showing the effects of ards and fluid balance on outcomes. over resuscitation leads to fluid overload and pulmonary edema, and hypoxia, which may influence ventilator dependence. we found a negative day - cumulative fluid balance was associated with a lower mortality rate in critically ill patients with influenza . we are now exploring whether cumulative fluid balance predicts ventilator dependency. we need to further evaluate an association between over resuscitation and ventilator dependence in the future. ventilator dependence risk score, including a history of stroke and data from day (thrombocytopenia, acidosis, and the higher fraction of inspired oxygen), can be applied to predict prolonged mechanical ventilation in patients who survive sepsis and septic shock. setting and study design. this retrospective study was conducted in three medical icus, including beds at kaohsiung chang gung memorial hospital, a , -bed tertiary teaching hospital in southern taiwan. consecutive adult patients (aged ≥ years) with acute respiratory failure on admission to the medical icu with sepsis/septic shock were surveyed from august to october through chart review. we excluded patients who passed away within days, those whose families requested palliative treatment before day , and those who were already long-term mechanical ventilator dependent. the enrolled patients were divided into two groups, i.e., ventilator-independent and ventilator-dependent, according to their ventilator status at the time of ventilator use on day from the chart review (fig. ) . we also collected data from sepsis/septic shock patients with respiratory failure who were admitted to the medical icu from november to november as the validation group (fig. ) . the study was approved by the institutional review board (irb) of chang gung memorial hospital and the requirements to obtain informed consent from patients were waived by irb ( - c). we confirmed that all methods were performed in accordance with the relevant guidelines and regulations. definitions. long-term ventilator dependence in patients was defined as the need for mechanical ventilation for more than six hours per day for more than days . sepsis was defined as a life-threatening organ dysfunction due to a disproportionate host response to infection . patients with septic shock were identified by a vasopressor requirement to maintain a mean arterial pressure of > mmhg in the clinical condition , . all enrolled patients met the new criteria for sepsis and required mechanical ventilation at the time of admission to the icu. moreover, they survived for at least days after admission to the icu. iv titratable sedation was applied if the patient's condition required and the titration protocol was standardized by medical intensive care unit. the standard clinical practices for weaning the patient from mechanical ventilation were performed during the study period (i.e., pressure support and spontaneous breathing trials). clinical data were retrieved from medical records and included age, gender, sequential organ failure assessment (sofa) score , acute physiological assessment and chronic health evaluation ii (apache ii) score , charlson index and underlying comorbidities , , and other clinical factors possibly related to prolonged ventilator use. we also collected hematology, biochemistry, fraction of inspired oxygen (fio ) and pao /fio on admission day to follow up on the patient's condition. all variables were evaluated as possible risk factors of prolonged ventilator use. score construction and calculation. categorical variables were analyzed using the chi-squared test, and continuous variables were compared using the student's t-test. a two-tailed p value of < . was considered to indicate a significant result. univariate analysis was used to identify significant risk factors associated with ventilator dependence. variables associated with ventilator dependence in the univariate analysis (p < . ) were included in a multivariate analysis model. using the stepwise method, the independent factors associated with ventilator dependence were identified to build a score using the hosmer-lemeshow goodness-of-fit test. a clinical score (vd risk score) was calculated based on four variables independently associated with ventilator dependence in the multivariate analysis. the number of points assigned to each variable in the vd score was adjusted according to proportion to beta coefficient in the regression model. the vd risk score is the sum of the points for these four variables. the receiver operating characteristic (roc) curve was used to evaluate the performance of the vd risk score. all statistical analysis was performed using the spss . software package (spss inc., chicago, il, usa). sepsis: pathophysiology and clinical management healthcare cost and utilization project (hcup) statistical briefs epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care management of severe sepsis in patients admitted to asian intensive care units: prospective cohort study assessment of the worldwide burden of critical illness: the intensive care over nations (icon) audit assessment of global incidence and mortality of hospital-treated sepsis. current estimates and limitations population burden of long-term survivorship after severe sepsis in older americans benchmarking the incidence and mortality of severe sepsis in the united states surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock initial resuscitation guided by the surviving sepsis campaign recommendations and early echocardiographic assessment of hemodynamics in intensive care unit septic patients: a pilot study management of patients requiring prolonged mechanical ventilation: report of a namdrc consensus conference weaning from prolonged mechanical ventilation prolonged mechanical ventilation in critically ill patients: epidemiology, outcomes and modelling the potential cost consequences of establishing a regional weaning unit factors predicting ventilator dependence in patients with ventilator-associated pneumonia physiologic determinants of ventilator dependence in long-term mechanically ventilated patients chronic ventilator dependence in elderly patients prediction of prolonged ventilator dependence in children by respiratory function measurements early prediction of prolonged ventilator dependence in thermally injured patients application of the sequential organ failure assessment (sofa) score to bacteremic icu patients weaning failure in mechanical ventilation. ondine's curse: a clinical case and review respiratory dysfunction in stroke histone deacetylase (hdac ) attenuates lipopolysaccharide (lps)-induced inflammation by regulating pai- expression beyond thrombosis: the versatile platelet in critical illness the role of platelets in sepsis understanding platelet dysfunction in sepsis metabolic acidosis in patients with severe sepsis and septic shock: a longitudinal quantitative study breathing pattern in weaning patients: comparison of two inspired oxygen fractions development and validation of immune dysfunction score to predict -day mortality of sepsis patients immune profiles and clinical outcomes between sepsis patients with or without active cancer requiring admission to intensive care units impact of clinical severity index, infective pathogens, and initial empiric antibiotic use on hospital mortality in patients with ventilator-associated pneumonia predictors of prolonged weaning and survival during ventilator weaning in a respiratory icu a prospective study of indexes predicting the outcome of trials of weaning from mechanical ventilation association of day cumulative fluid balance with mortality in critically ill patients with influenza: a multicenter retrospective cohort study in taiwan assessment of clinical criteria for sepsis: for the third international consensus definitions for sepsis and septic shock (sepsis- ) developing a new definition and assessing new clinical criteria for septic shock: for the third international consensus definitions for sepsis and septic shock (sepsis- ) the third international consensus definitions for sepsis and septic shock (sepsis- ) comparison of the sequential organ failure assessment, acute physiology and chronic health evaluation ii scoring system, and trauma and injury severity score method for predicting the outcomes of intensive care unit trauma patients importance of pre-existing co-morbidities for prognosis of septicemia in critically ill patients impact of different measures of comorbid disease on predicted mortality of intensive care unit patients the work was supported in part by a grant from the chang gung medical foundation (chang gung memorial hospital) grant (cmrpg b , cmrpg b , cmrpg b , cmrpg f , and cmrpg f to w.-f. fang). we would like to thank all the staff and clinicians in the intensive care unit who participated in this study, for their support. we also thank the biostatistics center, kaohsiung chang gung memorial hospital, for statistics consultation. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -utsvsja authors: uematsu, takayuki; iizasa, ei’ichi; kobayashi, noritada; yoshida, hiroki; hara, hiromitsu title: loss of card -mediated innate activation attenuates severe influenza pneumonia without compromising host viral immunity date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: utsvsja influenza virus (ifv) infection is a common cause of severe viral pneumonia associated with acute respiratory distress syndrome (ards), which is difficult to control with general immunosuppressive therapy including corticosteroids due to the unfavorable effect on viral replication. studies have suggested that the excessive activation of the innate immunity by ifv is responsible for severe pathologies. in this study, we focused on card , a signaling adaptor known to regulate innate immune activation through multiple innate sensor proteins, and investigated its role in anti-ifv defense and lung pathogenesis in a mouse model recapitulating severe influenza pneumonia with ards. we found that influenza pneumonia was dramatically attenuated in card -deficient mice, which showed improved mortality with reduced inflammatory cytokines and chemokines in the infected lungs. however, viral clearance, type-i interferon production, and the development of anti-viral b and t cell immunity were not compromised by card deficiency. syk or card -deficient dcs but not macrophages showed impaired cytokine but not type-i interferon production in response to ifv in vitro, indicating a possible role for the syk-card pathway in dcs in excessive inflammation of ifv-infected lungs. therefore, inhibition of this pathway is an ideal therapeutic target for severe influenza pneumonia without affecting viral clearance. the host's innate immune system including macrophages and dendritic cells (dcs) to secrete inflammatory cytokines/chemokines, such as il- , tnf, cxcl and cxcl , , which play crucial roles in the pathogenesis of ards , [ ] [ ] [ ] . therefore, inhibition of cytokine/chemokine production via targeting the host's innate immune system may lead to the development of effective treatment options for ards. caspase recruitment domain family member (card ) is an adaptor protein that delivers nf-κ b activating signals through multiple innate sensor proteins, such as a wide array of c-type lectin (clr) and immunoglobulin (ig)-superfamily receptors that are coupled to immunoreceptor tyrosine-based activation motifs (itams) [ ] [ ] [ ] , as well as cytoplasmic rna sensors, such as rig-i and mda , and the dna sensor rad , . several lines of evidence have implied a role for card -mediated innate activation pathways in influenza pathogenesis and immunity. the clr dendritic cell-specific icam- grabbing non-integrin (dc-sign), which has a cytoplasmic itam-like sequence, was shown to bind influenza virus a , . additionally, card was found to be required for the production of inflammatory cytokines after infection with rna viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, by regulating nf-κ b signaling through rig-i and mda . however, the roles of card in influenza pneumonia, as well as in protection against ifv are yet to be elucidated. in this study, we demonstrate a role for card -mediated innate activation in influenza pathogenesis and immunity. our results showed that the card pathway was involved in fatal influenza pneumonia mediated by inflammatory cytokine/chemokine production, whereas it was dispensable for type-i interferon production as well as the development of anti-viral acquired immunity. therefore, inhibition of this pathway may represent an ideal target for the treatment of severe influenza pneumonia without affecting viral clearance. adapted ifv a/pr/ / strain (pr ) shows similar lung pathology to human ards - , we intratracheally infected wild-type (wt: c bl/ ) and card -/mice with a lethal dose ( pfu/mouse) of pr to determine whether card -mediated innate immune responses contributed to severe influenza pneumonia. we observed that pr -infected wt mice appeared visibly ill with ruffled fur and reduced oral intake during the to days after infection, whereas card -/mice appeared more active than wt mice. consistent with their activity, the final survival rate up to day after infection was dramatically improved in card -/mice (~ %) as compared with wt mice (~ %; fig. a ). histopathological analysis of ifv-infected lungs on days and revealed that lung inflammation, which was the most obvious on day in wt mice, was much less severe in card -/mice (fig. b) . given that inflammatory cytokines/chemokines were linked to lung damage in severe influenza pneumonia [ ] [ ] [ ] [ ] [ ] [ ] , we analyzed infiltrated cells and cytokine/chemokine levels in bronchoalveolar lavage fluid (balf) collected from the ifv-infected lungs of wt and card -/mice. consistent with the pathological data, the number of cd + balf cells were significantly fewer in the lungs of card −/− mice than in those of wt mice on day , with a marked decrease in the numbers of t cells and neutrophils ( fig. c ) that were the two main infiltrated subpopulations at this time point (supplementary fig. s ). however, the numbers of b cells, nk cells, macrophages, cdcs, and pdcs were not significantly affected by the card deficiency. the balf levels of inflammatory cytokines and chemokines, i.e., il- , tnf-α , ccl /mip- α , cxcl /kc, and cxcl /ip- , which have been reported to contribute to lung pathology , , , peaked on day and were considerably lower in the card −/− mice than in the wt mice (fig. d ). however, no reduction in cytokine/chemokine levels was evident on day , indicating that a card -mediated innate response controls cytokine/chemokine production at an early time point after ifv infection and influences subsequent inflammatory cell recruitment and lung pathology at a later time point. collectively, the loss of card attenuated severe influenza pneumonia and improved host mortality. card deficiency does not compromise anti-viral protective immunity. activation of innate immunity in response to ifv regulates anti-viral protective immunity . to evaluate the impact of card deficiency on anti-ifv protection, we first analyzed the viral burden in the ifv-infected lungs of wt and card -/mice. notably, we found that card deficiency did not increase virus titers and the amount of viral rna in the lungs, but instead significantly accelerated viral clearance in the later stage (day ) of infection ( fig. a) . type-i interferons (ifn-α /β ) are the primary factors that induce host resistance to ifv , . in contrast to cytokine/chemokine production, card deficiency did not significantly affect ifn-α /β production in ifv-infected lungs (fig. b) . the type ii interferon ifn-γ is not essential for viral clearance , but its presence during ifv infection ameliorates the severity of inflammation and lung injury . ifn-γ production was significantly more in the lungs of card −/− mice than in those of wt mice on day but not on day (fig. b) ; thus, this increase might contribute to the improvement of lung pathology in card −/− mice. moreover, the expression of ip- encoded by an ifn-responsive gene was significantly impaired in card −/− mice on day (fig. d) , albeit no significant reduction was seen in either type i or type ii interferons, suggesting that synergistic signals of interferons and various cytokines/chemokines are required for the higher expression of ip- at an early phase of infection. next, we examined the induction of virus-specific cd + t cells using the h- d b tetramer coupled with a viral nuclear protein (np)-derived peptide (np - ) , . the percentages of np-specific cd + t cells in the draining lymph nodes of the lungs on day were comparable between wt and card −/− mice (fig. c) . to evaluate the impact of card deficiency on acquired immunity to ifv, wt and card −/− mice were immunized with a sub-lethal dose ( / of ld ) of the pr strain and rechallenged with a high lethal dose ( ld ) of pr after weeks. we found that card deficiency did not alter viral clearance after the challenge (fig. d ). next, we assessed the induction of humoral and cellular acquired immunity to ifv in card -/mice. we re-infected wt and card -/mice on day after their first infection (representing the second infection) and analyzed the production of virus-specific antibodies as well as the development of virus-specific cd + t cells on day after the second infection. we found that the card deficiency affected neither the production of virus-specific igg in the serum nor virus-specific iga in the lung mucosa (fig. e) . additionally, card deficiency did not affect the ifn-γ response by splenic cd + t cells specific to a np antigen epitope np - (fig. f) . collectively, the card pathway is dispensable for the induction of protective immunity against both the primary and secondary ifv infections, and its deficiency even improves primary viral clearance upon lethal-dose infections probably because of improvement in lung damage and elevated ifn-γ production. card deficiency impairs inflammatory cytokine, but not type-i interferon, production by dcs. because macrophages and dcs are known to be an early source of inflammatory cytokines and type-i interferons in pulmonary ifv infection , , we examined whether card was required for their production by myeloid cells in response to ifv. thioglycolate-induced peritoneal macrophages (tg-mfs), bone marrow-derived macrophages (bmmfs), alveolar macrophages (amfs), conventional dcs (cdcs), or flt ligand-induced plasmacytoid dcs (flt l-dcs) prepared from wt or card -/mice were brought into contact with pr in vitro and the production of il- , tnf-α , and ifn-α /β was measured. il- and tnf-α produced by tg-mfs (fig. a) or bmmfs (fig. b ) and those mrnas produced by amfs (fig. c ) in response to pr were not affected by card deficiency. as previously reported , , card was required for a cytokine response to ox-zymosan through dectin- -syk but dispensable for the response to lps through tlr -myd in cdcs ( supplementary fig. s a ). we found that card -/-cdcs (fig. d ) or flt l-dcs (fig. e ) produced significantly lower levels of il- and tnf-α than wt cells in response to pr . in contrast, ifn-α /β production was not compromised in card -/-cdcs or flt l-dcs. these findings suggest that card deficiency primarily affected dcs but not mfs regarding the production of inflammatory cytokines but not ifn-α /β in response to ifv. it has been reported that the card -ips- pathway mediates the cytokine response to rna viruses through rlhs , while card has been found to transmit signals from myeloid itam-coupled receptors via syk [ ] [ ] [ ] . thus, we evaluated the contributions of these pathways. ips- -deficient (ips- -/-) cdcs and myd -deficient (myd -/-) flt l-dcs produced significantly lower levels ifn-α /β than wt cells in response to pr (fig. d ,e), consistent with previous findings showing that the ifn-α /β response to rna viruses is mediated mainly through rig-i/ips- in cdcs, and through tlr /myd in pdcs , . in addition, il- and tnf-α production was impaired in ips- -/-cdcs and myd -/-flt l-dcs. lower levels of cytokine production were also observed in myd -/-cdcs, likely due to impaired activation through several tlrs (i.e. tlr , , and ) that sense virus nucleic acids , . we found that inducible syk-deficient (syk del/del ) cdcs and flt l-dcs produced significantly lower levels of inflammatory cytokines than wt cells similar to the responses of card -/-cdcs and flt l-dcs, whereas the syk deficiency did not affect cytokine production in response to lps (supplementary fig. s a ). consistent with these findings, treatment of wt cdcs (fig. a ) and flt l-dcs (fig. b) with the syk inhibitor bay - reduced the production of inflammatory cytokines but not type-i interferons in response to pr in a dose-dependent manner. the treatment with bay - reduced the cytokine response to ox-zymosan but not to lps, indicating that the effect of the inhibitor on cytokine expression depends on the stimulus but cannot be attributed to its general effect on cytokine gene expression ( supplementary fig. s b ). overall, these results suggest that the attenuation of influenza pneumonia by card deficiency was likely attributable to the reduced cytokine/chemokine production by pulmonary dcs through the syk-card pathway in response to ifv. we have shown that card -mediated activation of the innate immune system exacerbates influenza pneumonia in mice. card deficiency resulted in the reduction of inflammatory cytokine/chemokines and the infiltration of inflammatory cells in ifv-infected lungs, resulting in improved mouse mortality rates. given the pathological similarity between the mouse intratracheal infection model and influenza-associated ards in human - , we postulate that the card pathway contributes to the exacerbation of human influenza pneumonia. although the syk-card -mediated innate immune balf were analyzed on day and after the second infection. (f) ifv-specific adaptive t cell response. splenocytes from wt and card -/mice (n = per group) days after infection as in (e) were stimulated in vitro with ifv nucleoprotein peptide (np - ) for days. the culture supernatants were analyzed for ifn-γ production. data are presented as mean ± sd of triplicates. data are representative of three independent experiments. *p < . by student's t-test. scientific reports | : | doi: . /srep response is crucial for anti-fungal acquired immunity [ ] [ ] [ ] [ ] , our data showed that card was dispensable for anti-ifv immunity, as demonstrated by the findings that card deficiency did not alter viral burden, the elevation of ifn-α /β , or the induction of anti-viral adaptive t and b cell responses in the ifv-infected mice. thus, other innate mechanisms independent of the card pathway may play a more dominant role in protective immunity against ifv. innate immune sensors for ifv and its downstream signaling pathway have been well illustrated , , . in our study, card deficiency resulted in a selective reduction in inflammatory cytokines, but not ifn-α /β production, by both cdcs and flt l-dcs in response to ifv. this pattern of impairment was observed in ips- -/-cdcs, and was consistent with results from previous reports indicating that card selectively regulates the cytokine response through rig-i-ips-i in cdcs . additionally, we found that syk del/del and syk inhibitor-treated cdcs showed the same pattern of impairment as card -/or ips- -/-cdcs. on the other hand, in flt l-dcs known to release large amounts of inflammatory cytokines and type-i interferons upon recognition of ifv , , ips- deficiency affected neither cytokine or type-i interferon responses. however, syk deficiency in flt l-dcs, similar to that in cdcs, resulted in a selective reduction in inflammatory cytokines but not ifn-α /β . thus, it is likely that the syk-card pathway controls detrimental cytokine production by pulmonary dcs upon acute influenza infection. alternatively, because card deficiency impairs cytokine production by dcs following stimulation with several tlr ligands , it may be important to examine whether the syk-card pathway is involved in the tlr -meditated response to ifv. the involvement of the syk-card pathway implies the presence of itam-coupled receptors that sense ifv and stimulate cytokine production by dcs. it has been reported that an interaction between dc-sign, which possesses an itam-like "hemitam" motif, and the carbohydrate of ifv hemagglutinin induces maturation of dcs and promotes endocytosis of the virus , indicating the signal-activating capacity of dc-sign upon virus binding. however, it is unclear whether the dc-sign hemitam is capable of transmitting sufficient signals to produce inflammatory cytokines. with regard to other viruses, recognition of the dengue virus by the dap -associated clr clec a results in increased vascular permeability due to the overproduction of tnf-α , resulting in fatal outcomes . however, its ability to recognize the ifv remains unknown. thus, it is worth examining whether these clrs are involved in the cytokine response to ifv. it is also conceivable that damage-associated molecular patterns (damps), generated due to lung damage by ifv infection, may be ligands for some itam-coupled receptors. indeed, the fcrγ -associated clr mincle was shown to recognize sap , a component of the ribonucloprotein released from dead cells . in conclusion, card -mediated innate immune activation in pulmonary dcs acts to exacerbate severe influenza pneumonia, but is dispensable for host protection against ifv. thus, inhibiting the card pathway may represent a promising therapeutic target for the control of pivp without affecting viral clearance. mice. card -/-, syk flox/flox , ips- -/and myd -/mice have been previously described , [ ] [ ] [ ] . these mice were backcrossed at least times onto c bl/ mice. c bl/ mice were purchased from clea japan, inc. (tokyo, japan). the animals were housed in specific pathogen-free conditions. all experiments were approved by the institutional animal care and use committee for kitasato university medical center and animals were treated in accordance with the regulations for animal experiments in kitasato university. all surgeries were performed under ketamine hydrochloride/xylazine anesthesia, and all efforts were made to minimize suffering. antibodies and reagents. fluorescein isothiocyanate (fitc)-conjugated anti-f / (clone bm ), fitc-conjugated anti-cd r/b (clone ra - b ), fitc-conjugated cd (clone d ), phycoerythrin (pe)-conjugated anti-ly- g (clone a ), pe-conjugated anti-siglech (clone ), biotin-conjugated anti-nk . (clone pk ), biotin-conjugated anti-bst /pdca- (clone ), peridinin chlorophyll protein/cy . (percp/cy . )-conjugated anti-cd (clone -f ), phycoerythrin-cy (pc )-conjugated anti-cd ε (clone - c ) and pc -conjugated anti-cd c (clone n ) monoclonal antibodies were purchased from biolegend (san diego, ca). fitc-conjugated anti-cd (clone kt ) monoclonal antibodies, pe-conjugated h- d b tetramer coupled with a viral np-derived asnenmetm peptide (np - : asnenmetm), h- d b -restricted influenza np - peptide were purchased from mbl (nagoya, japan). syk inhibitor iv (bay - ) was purchased from merck millipore (billerica, ma). lps from escherichia coli :b was purchased from sigma-aldrich (st. louis, mo). naclo-oxidized zymosan was prepared as described . ifv infection in mice. c bl/ and card -/mice were anesthetized and infected with plaque forming units (pfu) (unless otherwise indicated) of a mouse-adapted ifv (a/pr/ / strain: h n isotype, kindly provided by the kitasato institute, tokyo, japan) by intratracheal administration as described - . histology. whole lungs were collected at , , and days after ifv infection. paraffin embedding and hematoxylin and eosin staining of tissues were performed using standard methodologies. . bal was carried out as described previously , . in brief, tracheas of mice were cannulated with . -mm diameter polyethylene catheters. lungs were instilled with ml of pre-warmed pbs containing mm edta, followed by the retrieval of lavage fluid aliquots. cells in the bal fluid (balf) were counted after red blood cell lysis and subjected to flow cytometric analysis. the supernatants of the balf were subjected to multi cytokine/chemokine expression analysis and cytokine elisa. cytomix cytokine bead assay (bender medsystems, vienna, austria), with the exception of ifn-β , which was measured by a verikine tm mouse interferon-β elisa kit (pbl interferonsource, piscataway, nj). cell culture supernatants from splenocytes were assayed using a specific elisa kit for ifn-γ (biolegend). cell culture supernatants from tg-mfs, bmmfs, cdcs, and flt l-dcs were assayed using specific elisa kits for il- , tnf-α (biolegend), ifn-α and ifn-β (pbl interferonsource). all measurements were performed in triplicate. for quantitative pcr (qpcr) analysis for cytokine mrnas, total rna was extracted from cells with reliaprep rna cell miniprep system (promega, madison, wi) and cdna was synthesized with primescript rt reagent kit (takara bio, shiga, japan) according to the manufacturer's instructions. qpcr was performed with the applied biosystems ht fast real time pcr system (life technologies). primer sequences were as follows: mouse il , forword, ′-cca ctt cac aag tcg gag gct ta - ′, and reverse, ′-gca agt gca tca tcg ttg ttc ata c - ′; and tnf forword, ′-gtt cta tgg ccc aga ccc tca c - ′, and reverse, ′-ggc acc act agt tgg ttg tct ttg - ′. viral copy numbers. mice were euthanized by intraperitoneal administration of sodium pentobarbital at , , or days after ifv infection. lung tissues were homogenized using a gentlemacs dissociator (miltenyi biotec) and rna was extracted with an isogen ii rna extraction kit (nippon gene, tokyo, japan). reverse transcription was conducted with the uni- primer ( ′-agc aaa agc agg - ′) and qpcr was preformed with primers specific for np (forward: ′-gat tgg tgg aat tgg acg at - ′; reverse: ′-aga gca cca ttc tct cta tt - ′) using the applied biosystems ht fast real time pcr system (life technologies, carlsbad, ca). the standard calibration curve for qpcr was obtained by stepwise dilution of the cloned np gene fragment with a known copy number. ifv rechallenge. wt and card -/mice were left uninfected or infected intratracheally with sublethal dose ( pfu/mouse: / ld ) of pr . twenty-eight days after the first infection, mice were challenged with a high lethal dose ( pfu/mouse: ld ) of pr . two days postchallenge, virus titers in the lungs were measured in a plaque assay in mdck cells. antigen-specific b cell responses. b cell-mediated humoral responses were measured as virion-specific immunoglobulin production by elisa, as previously described . briefly, -well elisa plates (corning) were coated with ultrasonicated influenza virion (a/pr/ / strain) at × pfu/ ml in a carbonate buffer (ph . ), and incubated overnight at °c. plates were then washed with pbs containing . % tween (wako pure chemical industries). serum and balf collected from mice at day after the secondary infection were serially diluted with pbs/tween containing % skim milk, applied onto the virion-coated plates, and incubated for h at room temperature. after washing, goat anti-mouse total igg or iga conjugated to horseradish peroxidase (jackson immunoresearch, baltimore pike, pa) was applied and incubated for h at room temperature. after washing, the plates were stained with a tmb substrate set (biolegend). the reaction was terminated with m h so (wako pure chemical industries) and the absorbance was measured. antigen-specific t cell responses. ifv-specific t cell responses were measured as viral np-specific ifn-γ secretion by splenocytes, as described previously , . briefly, × splenocytes extracted from mice -days after the secondary infection were seeded on -well cell culture plates (corning) and then stimulated with μ g/ml of np - peptides. after days of culture, supernatants were collected and analyzed for ifn-γ production by elisa (biolegend). tg-mfs were prepared as described . amfs were isolated from balf of wt or card -/mice. bmmfs, cdcs, or flt l-dcs were prepared by culturing bone marrow cells for - days with rpmi medium (wako pure chemical industries) supplemented with % fetal bovine serum (life technologies) and antibiotics ( iu/ml penicillin and μ g/ml streptomycin; sigma-aldrich) containing m-csf ( ng/ml, peprotech, rocky hill, nj), gm-csf ( ng/ml, peprotech) or human flt -ligand ( ng/ml, peprotech), respectively. the flt l-dcs derived from the culture contain constantly - % of pdca- + siglech + plasmacytoid dcs (pdcs) irrespective of deficiency in syk, card , ips- or myd . for syk deletion in vitro, cultured cells derived from syk flox/flox mice were incubated with . μ m of the active metabolite of tamoxifen, -hydroxytamoxifen (sigma-aldrich) for d. for stimulation of mfs/dcs of, × cells were seeded on -well culture plates (corning) and incubated overnight, followed by replacement of μ l of serum-free medium containing pfu (multiplicity of infection [m.o.i] = ) of pr . after h of incubation, unabsorbed viruses were removed and the cells were incubated for a further h in serum-containing medium. dcs were also stimulated with lps ( ng/ml) or naclo-oxidized zymosan ( μ g/ml) for h in serum-containing medium. the culture supernatants were assayed for il- , tnf-α , ifn-α , and ifn-β by elisa. for stimulation of amfs, × cells were seeded on -well culture plate (corning), stimulated as above with pfu (m.o.i = ) of pr for h, and total rnas were extracted for qpcr analysis for cytokine expression. for treatment of cells with a chemical syk inhibitor, wt dcs were incubated with . or μ m bay - for h prior to virus stimulation. cell viability. cell viability was determined by the mts assay using the celltiter aqueousone solution cell proliferation assay kit (promega). in brief, μ l of mts solution was added to cells in -well plates after in vitro ifv stimulation and the plates were incubated for h at °c, then the absorbance at nm was measured using a benchmark microplate reader (bio-rad, hercules, ca). statistical analysis. survival curves were generated by the kaplan-meier method, and statistical analyses were performed using the log-rank test. the statistical significance was assessed by student's t-tests. a p value < . was considered significant. avian influenza a (h n ) infection in humans the genesis of a pandemic influenza virus characterization of the reconstructed spanish influenza pandemic virus aberrant innate immune response in lethal infection of macaques with the influenza virus h n and pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice the acute respiratory distress syndrome acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome inhibition of the cytokine response does not protect against lethal h n influenza infection use of early corticosteroid therapy on icu admission in patients affected by severe pandemic (h n ) v influenza a infection identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury cxcl -cxcr enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin the lipid mediator protectin d inhibits influenza virus replication and improves severe influenza influenza-induced innate immunity: regulators of viral replication, respiratory tract pathology & adaptive immunity role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? systemic cytokine responses in patients with influenza-associated encephalopathy card controls a non-tlr signalling pathway for innate anti-fungal immunity the adaptor protein card is essential for the activation of myeloid cells through itam-associated and toll-like receptors card versus carma in innate and adaptive immunity recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production rad -card interactions link cytosolic dna sensing to il- β production dc-sign and immunoregulation dc-sign mediates avian h n influenza virus infection in cis and in trans critical role of il- receptor-associated kinase-m in regulating chemokine-dependent deleterious inflammation in murine influenza pneumonia innate and adaptive immune responses to viral infection and vaccination interferons, interferon-like cytokines, and their receptors the role of alpha/beta and gamma interferons in development of immunity to influenza a virus in mice production of interferon-gamma by influenza hemagglutinin-specific cd effector t cells influences the development of pulmonary immunopathology the epitopes of influenza nucleoprotein recognized by cytotoxic t lymphocytes can be defined with short synthetic peptides a previously unrecognized h- d(b)-restricted peptide prominent in the primary influenza a virus-specific cd (+ ) t-cell response is much less apparent following secondary challenge differential role of tlr-and rlr-signaling in the immune responses to influenza a virus infection and vaccination plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells dectin- is required for host defense against pneumocystis carinii but not against candida albicans dectin- is required for beta-glucan recognition and control of fungal infection c-type lectin mincle is an activating receptor for pathogenic fungus dectin- recognition of alpha-mannans and induction of th cell differentiation is essential for host defense against candida albicans pathogen recognition and innate immunity pattern recognition receptors and inflammation binding of dc-sign to the hemagglutinin of influenza a viruses supports virus replication in dc-sign expressing cells clec a is critical for dengue-virus-induced lethal disease mincle is an itam-coupled activating receptor that senses damaged cells syk-dependent signaling pathways in neutrophils and macrophages are indispensable in the pathogenesis of anti-collagen antibody-induced arthritis ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction targeted disruption of the myd gene results in loss of il- -and il- -mediated function beta-glucan derived from zymosan acts as an adjuvant for collagen-induced arthritis immunokinetics in severe pneumonia due to influenza virus and bacteria coinfection in mice universal primer set for the full-length amplification of all influenza a viruses interleukin- is critical in the pathogenesis of influenza a virus-induced acute lung injury we thank yoshiyuki miyazaki, mako nakaya, fumika mi-ichi, masanori yamazaki, naoko ozaki, sayoi sato, shinsuke yasukawa, mio kubota, honglian tong, takashi fukuyama, taiga yamazaki, tomoko fujita and rui yamamura for helpful discussions and technical support. we also thank dr. yumiko imai and dr. keiji kuba (akita university) for helpful discussions, and dr. shinobu suzuki (nippon boehringer ingelheim co., ltd) and dr. shizuo akira (osaka university) for kindly providing the . the funders had no role in the study design, data collection or analysis, the decision to publish, or the preparation of the manuscript. key: cord- -yxx xxio authors: lee, ing-kit; yang, zih-syuan; ng, hwee-yeong; li, lung-chih; huang, wen-chi; chen, yi-chun; tsai, ching-yen; lee, chien-te title: impaired production of immune mediators in dengue virus type -infected mononuclear cells of adults with end stage renal disease date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: yxx xxio chronic kidney disease is an epidemiologically identified risk factor for development of severe dengue in dengue-affected patients. however, available data on the immune pathogenesis in end stage renal disease (esrd) patients affected by dengue is insufficient. we performed an in vitro study to evaluate the sequential immunological reactions and viral load in dengue virus type -infected mononuclear cells of patients with esrd (n = ) and in healthy controls (n = ). the concentrations of interleukins (il)- receptor antagonist (ra), il- , il- , il- , il- , il- p , granulocyte-macrophage colony-stimulating factor (gm-csf), monocyte chemotactic protein- (mcp- ), macrophage inflammatory protein- b (mip- b), vascular endothelial growth factor (vegf), tumor necrosis factor (tnf)-α and viral load cycle threshold (ct) were measured in the dengue virus type -infected mononuclear cells at h, h, h, and h post-infection. we found in the esrd group significantly higher gm-csf and il- levels at h post-infection. however, il- , il- , il- p , tnf-α, mcp- , and mip- b levels were found significantly lower than in the control group. at h, h, and h post-infection, significantly lower levels of il- ra, il- , il- , il- , il- p , tnf-α, mcp- , and mip- b were detected in esrd group. concentration of vegf at h and h, and of gm-csf at h and h were also found to be lower in esrd group than in control group. compared with controls, the viral load ct values were significantly lower in esrd group at h and h post-infection no significant difference in viral load ct values between two groups was found at h and h post-infection. our study discloses that the expression of immune mediators of dengue-infected mononuclear cells is impaired in esrd patients. during the 's and 's, dengue was generally considered to be a pediatric disease , . however, in recent decades, advancing age has been reported to be a risk factor for patients with severe dengue, as the comorbidities associated with ageing pose a substantial risk for mortality in elderly patients [ ] [ ] [ ] . chronic kidney disease has been implicated in the development of severe dengue [ ] [ ] [ ] [ ] . thein et al., in a study of patients with dengue, reported that renal disorder increased odds of death in adults . kuo et al. reported that patients with chronic kidney disease had a higher mortality rate than those without chronic kidney disease . in a study of adults with dengue, chronic renal disease was found to be a risk factor that contributes to the development of acute kidney injury, which is associated with high morbidity and mortality of the affected patients . wong et al. found that end stage renal disease (esrd) is a risk factor for acute respiratory failure in dengue affected adults . esrd is also an important clinical predictor of gastrointestinal bleeding in adult patients with dengue . in fact, patients with esrd are more susceptible to infections (such as bacterial sepsis) with poor clinical outcomes compared with the general population . in spite of esrd being an increasingly common disease, there is lack of enough data on the immune pathogenesis in esrd patients affected by dengue. thus, thorough investigation is urgently required for the better understanding of immune responses in dengue-affected esrd patients. in the present study, we aim to explore the sequential immunological reactions and viral load in vitro by investigating dengue virus-infected mononuclear cells of adults with esrd, which would eventually make it possible to target interventions for an improved treatment plan for dengue infection. ethics statement. this study was approved by the institution review board of kaohsiung chang gung memorial hospital medical center, taiwan (document no. - b). written informed consent from participants was obtained. this research adhered to the principles of the declaration of helsinki. study period and participants. the study was conducted at kaohsiung chang gung memorial hospital from january , to december , . volunteers were recruited from those suffering from esrd, and from healthy controls. age is controlled in the study. esrd affected volunteers refer to individuals with chronic kidney disease undergoing hemodialysis therapy thrice a week. a ml sample of blood was obtained from each participant (esrd and healthy individuals). to avoid the influence of dialyzer on the expression of immune mediators, blood samples from esrd volunteers were collected before starting the dialysis session. the blood samples of all participants were tested for dengue virus-specific immunoglobulin igm and igg antibodies using a dengue blot detection kit (gene labs diagnostics, singapore), to determine any previous dengue infection . blood samples were anonymized after completion of demographic data collection. preparation of mononuclear cells. the whole blood from all participants (esrd and healthy individuals) was separated into plasma and blood cells (i.e., leukocytes and erythrocytes) by centrifugation at , rpm ( × g) for minutes. erythrocytes were removed from blood cells by . % dextran sedimentation. after removal of erythrocytes, leukocytes were further separated into mononuclear cells and neutrophils using a density gradient centrifugation ( g/ min in ficoll-paque plus, amersham biosciences corp.) in accordance with the procedures described elsewhere . after this, mononuclear cells from esrd and healthy individuals were suspended in rpmi medium (gibco) and seeded into a -well culture plate at a density of . × cells per well for day at °c. and in southern taiwan in which the denv has been the predominant serotype in these outbreaks . in our series, an in vitro model with mononuclear cells infected with denv was designed. denv (new guinea c strain, atcc) was obtained from the institute of preventive medicine, national defense medical center, taipei. the virus was propagated in aedes albopictus c / cells in eagle's minimal essential medium (gibco brl, grand island, n.y., usa) at °c for days. virus titers were determined based on a standard plaque forming unit (pfu) assay on baby hamster kidney- cells as described previously . the virus titers were adjusted to . × pfu/ml in rpmi (gibco brl) medium. to achieve sufficient virus-infected mononuclear cells and avoid excessive cellular apoptosis, we used the multiplicity of infection (moi) of , which has been proven appropriate previously . cells from the -well culture plates (at a density of . × cells per well) were inoculated with denv having moi of , and incubated at °c for hours. the infected mononuclear cells were washed to remove extracellular viruses and cultured in rpmi medium at °c in a % co incubator. the supernatants and cells were www.nature.com/scientificreports www.nature.com/scientificreports/ determination of denv cycle threshold (ct) values by real-time reverse transcription-polymerase chain reaction (rt-pcr). viral rna was extracted from cultured mononuclear cells (esrd and healthy individuals) to measure the ct value by real-time rt-pcr, as previously described . the forward primer, reverse primer, and taqman probe sequences for detecting denv were ′-ggcttagcgctcacatcca- ′, ′-gctggccaccctctcttctt- ′, and ′-fam-cgcccaccactatagctgccgga-tamra- ′, respectively. data and statistical analyses. measurements of immune mediators were expressed as mean value and standard deviation (pg/ml). viral load was determined by ct value of rt-pcr. mann-whitney u test was used to analyze differences in immune mediators and viral load at various time points ( h, h, h, and h post-infection) between the esrd and control groups. a p value of < . was considered statistically significant. all analyses were performed using spss . (chicago, il, usa). demographic characteristics of the participants. the cells are obtained from participants, including esrd patients ( men and women; median age . years) and healthy controls ( men and women; median age, . years). among the esrd affected participants, hypertension ( . %) was the most common comorbidity, followed by type diabetes mellitus ( . %). none of the participants in control group have underlying medical condition. all participants tested negative for dengue virus-specific immunoglobulins igm and igg antibodies, as determined by dengue blot detection kit . immune profiles in overall esrd affected participants and controls. figure immune profiles in esrd affected participants without diabetes. beyond viral factor, the current evidence shown diabetes is a potentially important co-factor that affects the outcome of dengue infection . to eliminate the impact of diabetes in production of immune mediators in esrd patients, subgroup analysis was performed between esrd participants without diabetes (n = ) and control group (n = ) (fig. ) . at h post-infection, il- and gm-csf were detected at higher concentrations, but there was a significant reduction of stimulated mononuclear cell production of il- , il- , il- p , tnf-α, mcp- , and mip- b in esrd without diabetes group than in control group. at h, h and h post-infection, the levels of il- ra, il- , il- , il- , il- p , tnf-α, mcp- , and mip- b were still low in esrd without diabetes group than in the control group. in addition, significantly lower concentration of vegf was observed at h post-infection in esrd without diabetes group in comparison to controls. chronic kidney disease has been found to be an epidemiologically identified risk factor for development of dengue hemorrhagic fever among dengue patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, most of the previously published research on dengue immune pathogenesis involved a general population rather than a population with a specific underlying disease such as esrd . in the present study, eleven immune mediators and viral load ct values were measured in denv -infected mononuclear cells of adults with esrd, and in healthy participants. uremic itself presents a complex multidimensional inflammatory condition . previous studies have shown that t lymphocytes from esrd patients are dysregulated and the plasma il- , il- , tnf-α and c-reactive protein concentrations were significantly higher in esrd patients than those pre-dialysis controls . in our series, apart from high levels of gm-csf and il- that were observed at h post-infection, impaired production of immune mediators was observed at a later stage post-infection in the esrd group. this finding is potentially valuable for understanding of the basis of immunopathology reactions and provides new insight in the management of esrd patients with dengue virus infection. patients with dengue virus infection may progress through three clinical phases, namely, the febrile phase, the critical phase and the recovery phase . our report describes an in vitro experiment for dengue virus infected mononuclear cells and analysis of immune mediators at different times after infection, in a setting that mimicked infection in human and the sequential immunological reactions. www.nature.com/scientificreports www.nature.com/scientificreports/ the levels of gm-csf and il- were significantly raised in overall esrd population as well as those non-diabetic esrd participants at h post-infection. gm-csf plays an important role in mediating the innate immune response that stimulates stem cells to produce granulocytes and facilitates development of the immune system to fight against infections . gm-csf has been reported to increase significantly in patients with severe dengue as compared mild dengue . il- is an important pro-inflammatory cytokine in determining the activation of t-helper response to stimulate cell-mediated immunity . kurane et al. in a study of children showed that the level of il- was highest one day before defervescence in patients with dengue hemorrhagic fever . although gm-csf and il- were expressed in higher concentrations at h post-infection in esrd group, the production of a large number of pro-and anti-inflammatory cytokines as well as chemokines, including il- , il- p , tnf-α, mcp- , mip- b, and il- was markedly decreased. these results suggest derangements of www.nature.com/scientificreports www.nature.com/scientificreports/ immune reaction between pro-and anti-inflammatory cytokines/chemokines in esrd patients during the early phase of dengue illness. the hallmark of severe dengue is a transient perturbation in blood vessel integrity, leading to an increase in vascular permeability that results in plasma leakage , . this change is most likely due to effects of biological mediators as a consequence of complex interactions between dengue virus and host innate and adaptive immune responses , , . for example, increased levels of tnf-α and soluble tnf-α receptors as well as vegf have been www.nature.com/scientificreports www.nature.com/scientificreports/ reported in dengue hemorrhagic fever cases , . il- , having anti-inflammatory properties counter-regulates the cascade of pro-inflammatory cytokines and it has been reported to correlate with disease severity remarkable, the results of the present study showed a significant impairment of il- ra, il- , il- , il- , il- p , tnf-α, mcp- , and mip- b expression at h, h, and h post-infection. vegf levels at h and h, and gm-csf levels at h and h were also reported to be low in esrd group. impaired cytokines/chemokines expression also has been found in esrd without diabetes group as well. our results have been somewhat inconsistent with the reported overwhelming immune activation that is associated with dengue disease severity , ; however, our findings clearly reflect the impairment of immune responses in vitro study of denv -infected mononuclear cells in esrd patients. uremia per se can also alter the immune system in esrd patients . the complete blood count is not performed in our series. previous studies, however, have shown that monocytes and monocyte-derived dendritic cells of esrd patients have impaired endocytosis and maturation . lisowska et al. reported that esrd patients have lower percentages of cd + and cd + t cells, as well as b cells in peripheral blood . in fact, several factors influence immunity in esrd patients, such as uremic toxin, malnutrition, chronic inflammation, and therapeutic dialysis [ ] [ ] [ ] [ ] . in this regard, our findings and previous reports indicate that esrd patients have immune dysregulation with impaired expression of immune mediators in case of dengue virus infection. although the role of pro-and anti-inflammatory cytokines/chemokines in the pathogenesis of vascular permeability in dengue-affected esrd patients is uncertain in our series, the impairment of immune responses indicate reduction of the activation of the immune system in dengue-affected esrd patients that can lead to increased risk of morbidity and mortality in case of secondary bacterial infection. notably, in a study of severe dengue patients with concurrent bacteremia, . % of them had esrd . of adult dengue-affected patients with bacteremia, % were found to have esrd . another finding recognized in this study is that significantly lower viral load ct values at h and h post-infection in esrd group than controls. however, no significant difference in viral load ct values between both groups was found at h and h post-infection. the relationship between the dengue viral load and the severity of disease is still controversial , . murgue et al. had reported that high levels of viral load might contribute to the pathogenesis of dengue hemorrhagic fever . in another study of confirmed adult dengue cases, chen et al. found that viral load was not significantly different between dengue fever and dengue hemorrhagic fever patients . further studies are needed to determine the role of viral load in the pathogenesis of severe dengue in esrd patients. a limitation of the present study is that it is unclear whether similar immunologic responses occur in secondary dengue infection in dengue-affected esrd patients. despite the limitation, this is the first study that provides a basis for understanding the role of immune mediators in esrd population with dengue infection. in conclusion, the results of our study disclose that impaired immunologic reactions of mononuclear cells to a denv infection in esrd individuals. this implies that caution needs to be exercised in the immune dysregulation of esrd population and it might be a key determinant for clinical outcome when treating dengue-affected esrd patients. our study highlights that further clinical investigation beyond in vitro study is needed to confirm this important finding. all data generated or analyzed during this study are included in this published 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homeostasis and activation of the immune system activation of t lymphocytes in dengue virus infections. high levels of soluble interleukin receptor, soluble cd , soluble cd , interleukin , and interferon-gamma in sera of children with dengue dengue-specific t cell responses in peripheral blood mononuclear cells obtained prior to secondary dengue virus infections in thai schoolchildren virus-induced decline in soluble vascular endothelial growth receptor is associated with plasma leakage in dengue hemorrhagic fever cell type specificity and host genetic polymorphisms influence antibody-dependent enhancement of dengue virus infection impaired cellular immune function in patients with end-stage renal failure uremia impairs monocyte and monocyte-derived dendritic cell function in hemodialysis patients hemodialysis affects phenotype and proliferation of cd -positive t lymphocytes the immunoregulatory function of vitamin d: implications in chronic kidney disease the exploration of risk factors of concurrent bacteraemia in patients critically ill with severe dengue diagnostic performance of procalcitonin for bacteremia in patients with severe dengue infection in the intensive care unit high levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: implications for pathogenesis altered t helper reaction but not increase of virus load in patients with dengue hemorrhagic fever i.k.l is supported by the grants from the national science council, executive yuan, taiwan ( - -b- a- ), and a grant from the kaohsiung chang gung memorial hospital, taiwan (cmrpg h ). the funders had no role in the design or interpretation of this work, or in the decision to submit for publication. the authors declare no competing interests. correspondence and requests for materials should be addressed to i.-k.l. or c.-t.l. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -iaktm a authors: soto-quintero, albanelly; guarrotxena, nekane; garcía, olga; quijada-garrido, isabel title: curcumin to promote the synthesis of silver nps and their self-assembly with a thermoresponsive polymer in core-shell nanohybrids date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: iaktm a this work presents a simple one-pot protocol to achieve core-doped shell nanohybrids comprising silver nanoparticles, curcumin and thermoresponsive polymeric shell taking advantage of the reducing properties of phenolic curcumin substance and its ability to decorate metallic surfaces. silver nanoparticles were synthesized, via sodium citrate and silver nitrate addition into a boiling aqueous solution of curcumin, monomers and surfactant. curcumin and sodium citrate promoted silver nucleation, acting as reducing and stabilizing agents. these curcumin-capped agnps enabled, after adding the radical polymerization initiator, the assembling of the growing polymer chains around the hydrophobic agnp surface. the resultant core-doped shell nanohybrids exhibit plasmonic, luminescent and volume thermoresponsive properties, with improved possibilities to be used as successful therapeutic platforms. in fact, the possibility to nanoconfine the synergistic antioxidant, antiviral, antibacterial features of silver and curcumin in one bioavailable hybrid paves the way to promising applications in the biomedical field. synthesis of ag@cur-p(meo ma) core-doped shell hybrid nps. ag@cur-p(meo ma) hybrid nanoparticles, from now on also called as (ag@cur-g), were synthesized through one-pot two-steps method (fig. ) . letter g denotes to thermoresponsive p(meo ma) polymer participation. a typical procedure for the synthesis is described below for sample ag@cur-g b (table ). in the first step (fig. b) , ag@cur nps (silver nps decorated with curcumin (table ) (fig. b) . the mixture was vigorously stirred for an additional min at reflux and then allowed to cool down slowly to room temperature. in the second step (fig. c) , p(meo ma) shell formation around ag@cur core was endowed by free radical precipitation polymerization (frpp) of meo ma and tegdma crosslinker. the solution was degassed by n gas for min at room temperature and polymerization was initiated by adding μl of aps solution ( . m) after temperature was raised to °c. the reaction was allowed to proceed for h at °c under stirring and n gas inlet (fig. c ). after that, the mixture was cooled down in an ice-cold water bath while it was exposed to the air. the resultant core-doped shell nps (ag@cur-g b) were purified by three centrifugation cycles by using successive decreasing centrifugal rates ( , and rpm) at °c for min. subsequent centrifugation rendered three supernatant fractions (f , f and f ) and a fourth fraction (f ) corresponding to the final precipitate from the third cycle. this procedure ensured the complete removal of empty polymer particles and excess of reactants. finally, nps pellet was redispersed in milli-q water and kept at °c in a refrigerator immediately prior to use. during the synthesis, different reaction parameters such as reaction temperature (first step, fig. b ) and curcumin and sodium citrate concentration were considered in order to study their influence on size, shape and concentration of agnps. ag@citrate nps without curcumin, as control samples, were also synthetized. synthesis of cur-p(meo ma) nps or (cur-g). the p(meo ma) crosslinked nanogels in the presence (cur-g) and absence (g , control sample) of curcumin were synthesized through precipitation polymerization strategy in water. a typical procedure for the synthesis is described below for sample cur-g (table ). in a pyrex tube equipped with a magnetic stirrer, μl of a solution of curcumin in ethanol ( . mmol), meo ma monomer ( . mmol), μl of tegdma crosslinker solution in ethanol ( . mmol), μl of sds aqueous solution ( . - mmol) and ml of milli-q water were added. after min n gas purge, polymerization was initiated by heated up to °c, followed by addition of μl of aps solution ( . m). after around min, the solution turned cloudy, indicating that polymerization started, and the solution was left to react for h. to stop the reaction, the solution was cooling down in an ice-cold water bath while the tube was opened to air. afterward, the sample was purified by centrifugation ( rpm, min, °c) and then the pellet redispersed in water. the final product was stored at °c until further use. methods for characterization. ftir spectra were recorded by perkin elmer spectrum two spectrometer using attenuated total reflectance (atr) accessory. uv-vis absorption spectra, at controlled temperature, table . summary of curcumin concentration, ζ-potential, z average diameter and polydispersity (pdi) by dls, volume temperature induced phase transition (vptt), swelling ratio (q), and maximum of curcumin absorbance (λ abs ) of cur-p(meo ma) nanogels. (a) curcumin wt% is the feed curcumin/polymer ratio, (b) ζ values were determined at °c were carried out in a cary bio-varian uv-vis spectrophotometer equipped with a peltier temperature control device. the particle size and zeta potential (ζ) of core-shell nps and agnps were determined by a zetasizer nano zs instrument (malvern instruments ltd, uk) equipped with a mw he-ne laser operating at a light source wavelength of nm and a fixed scattering angle of ° for detection. malvern dispersion software was used for data acquisition and analysis, applying the general purpose algorithm for calculating the size distribution. transmission electron microscopy (tem) images were recorded with a field emission scanning electron microscope (fesem) hitachi su- operated at kv in transmitted electron imaging mode (s-tem). energy-dispersive x-ray spectroscopy (edx) analysis to determine the elemental composition of the hybrid samples was performed using a bruker nano with x-flash detector coupled to the sem. data were recorded at an accelerating voltage of kv. the progress of sample purification by centrifugation was verified by uv-vis spectroscopy (nanodrop one thermo-scientific spectrometer). the centrifugation was performed by refrigerating micro centrifuge r (eppendorf ™ ) to obtain ag@cur-g and cur-g nanoparticle pellets. to determine the luminescence features, uv-vis absorption and fluorescence spectra were recorded on a perkin elmer lambda- and perkin elmer ls ob spectrophotometer, respectively. emission spectra of dilute solutions of ag@cur-g core-doped shell nanohybrids and cur-g nanogels in water and dilute solutions of fluorescein in naoh . m were recorded keeping absorbance at the excitation wavelength (λ exc = nm) less than . . quantum yields were obtained by comparing the studied samples with fluorescein standard by the following equation applicable for dilute solutions. where Φ f and Φ s are the photoluminescence qy of the sample and that of the standard fluorescein (Φ s = . ) , respectively; i e and i e(s) are the integrated intensity of the emission curves corresponding to the sample and standard; η and η (s) are the refractive indices of the sample and reference solution; and i a and i a(s) are the fraction of light absorbed by the sample and standard respectively estimated as: where a is the absorbance at λ exc . synthesis and characterization of ag@cur-p(meo ma) core-doped shell nanohybrids. the ag@cur-p(meo ma) core-doped shell hybrid nps were prepared by free radical precipitation polymerization (frpp) of the stimuli-responsive meo ma monomer in the presence of tegdma (crosslinking agent), using curcumin-decorated ag@cur nps as seeds (fig. ) . the presence of sds guaranteed the control of np size. among all the reagents involved in the process, silver nitrate and sodium citrate followed standard concentration procedures , . however, the dual key-role of curcumin, as reducing agent and growth-polymerization promoter in this specific synthesis, required additional investigation to understand and optimize the chemical variables (solubility, concentration and reaction temperature) in order to achieve homogeneous, monodisperse and mononuclear ag@cur-p(meo ma) core-shell nanohybrids. indeed, frpp is a useful and common strategy to synthesize particles of thermoresponsive polymers, as long as monomers are soluble at the reaction temperature and polymer precipitates when their chains reach a critical size. then, since water solubility of curcumin is rather poor; pure curcumin compounds needed to be first dissolved into an organic phase of ethanol, meo ma monomer and crosslinker (fig. a) . a subsequent injection of aqueous solution of sds surfactant followed by dropwise addition of water resulted in a good dispersion of monomers and curcumin. a color change from cloudy (at room temperature) to transparent yellowish dispersion (at the reaction temperature), indicated that both curcumin and monomers had been dissolved. note that curcumin solubility increases with temperature . when the desired temperature was reached, sodium citrate solution was added to the reaction mixture already containing curcumin; and finally, in the presence of the two reducing agents, silver nitrate was added to the reaction (fig. b) . agnps synthesis (outlined in fig. b ) was completed at two different temperatures, °c and °c (series -a and -b in table , respectively). after min of reaction, the flask was removed from the silicon bath and allowed to cool to room temperature. the as-synthesized silver nanocores had an average size around nm ( °c, series -a) and nm ( °c, series b) in diameter, respectively. to elucidate the role of curcumin as stabilizing agent, the sample obtained in this first step was purified by centrifugation to eliminate the excess of reagents that are not protecting the silver core. the presence of curcumin close to the agnp surface, attained in this first step, was confirmed by atr-ftir spectroscopy (fig. s ). as it can be observed in this figure, the spectrum corresponding to ag@cur (green line) is very similar to that of curcumin (blue line). the most prominent vibration band at ∼ cm − assigned to highly mixed vibration including c=o stretching appears in both spectra as well as other characteristic vibration bands of curcumin at cm − (mixed c=c and c=o stretching), cm − (deformation of ch ), cm − (deformation of phenyl rings), cm − (c-h out-of-plane aromatic motions). in this fig. s , spectra corresponding to sodium citrate and ag@citrate nps are also displayed. as it can be seen, two strong bands corresponding to the asymmetric and symmetric stretching of co − groups at cm − and at cm − , respectively appear with nearly similar intensity for the free sodium citrate spectrum (violet line); whereas for ag@citrate spectrum (orange line), the most prominent band corresponds to the symmetric stretch at cm − . in the spectrum corresponding to ag@cur nps (green line), this vibration band at cm − is www.nature.com/scientificreports www.nature.com/scientificreports/ absent and only the asymmetric stretching (∼ cm − ) can be hardly intuited overlapped with vibration bands from curcumin (blue line); these facts indicate that the affinity of sodium citrate for the silver surface is lower than that of curcumin. in addition, the bands attributed to c-h ( - cm − ) and c=o stretching vibrations of meo ma appear very weak, also suggesting a low interaction of the monomer with the silver surface. figure c outlines the polymer shell preparation. this second step was done without previous ag@cur sample purification. for the purpose of thermoresponsive-shell polymerization, ag@cur was first flushed with n for min at the lowest possible temperature (room temperature), with the aim of reducing water evaporation during nitrogen flow. subsequently, aps initiator was added and incubated for h in a closed flask under stirring at °c. the presence of hydrophobic curcumin nearby the metallic surface led to precipitation-polymerization of p(meo ma) around the agnps previously formed (fig. b,c) ; and the resulting ag@cur-p(meo ma) nanoparticles were born negatively charged due to the persulfate groups from the aps initiator, which promotes their colloidal stability. the successful encapsulation of ag@cur nps within p(meo ma) nanogels can be anticipated by the atr-ftir spectrum shown in fig. s corresponding to f of ag@cur-g b sample (table ) , which displays typical vibration bands attributed to both curcumin and p(meo ma). it should be noted that an attempt to encapsulate agnps synthesized solely using sodium citrate caused irreversible aggregation in the second polymerization step (fig. s ). this fact evidences the role of hydrophobic curcumin to provide both a protection to the agnps and a good interface between the polymer and the metal surface. well-defined ag@cur-p(meo ma) nanohybrids (table ) were obtained by selective removal of the silver-free nanogels by centrifugation (see experimental section). it should be taken into account that the number of empty polymer particles created during the polymerization process exceeded the number of agnps in the solution by about two orders of magnitude; and they should be removed from. as illustration, uv-vis spectra at different purification steps by centrifugation of the ag@cur-g b crude sample are displayed in fig. . initially, the spectrum of the crude shows an intense band at nm resulting from a combined contribution of curcumin absorbance and surface plasmon resonance (spr) due to the collective oscillations of conductions electrons of silver nps. note that spr maximum for spherical agnps can be typically tuned from to nm depending on their np size and shape as well as their surrounding dielectric medium. then, after the first and second centrifugation cycles, the supernatant fractions (f and f respectively) revealed absorption maxima around nm, indicating that the most predominant absorbance is due to curcumin embedded in p(meo ma) nps. the third centrifugation cycle, however, on the basis of the supernatant fraction (f ) and its corresponding precipitate (f ) evidenced only core-doped shell ag@cur-p(meo ma) nanoparticles content (absorption www.nature.com/scientificreports www.nature.com/scientificreports/ maxima at nm). therefore, the core-doped shell nanohybrids used for further characterization and analysis correspond to f fraction from each crude sample (table ) . moreover, curcumin trapped-polymeric nanogels (cur-p(meo ma)) were synthesized through a similar precipitation-polymerization strategy reported for polymer encapsulating of ag@cur (fig. c , ag@ cur-p(meo ma)) and using similar curcumin/polymer ratios (see experimental section and table ). the chemical structure of both, polymeric nps (cur-g) and hybrid nps (ag@cur-g) can be elucidated from the atr-ftir spectra shown in fig. . typical vibration bands (c=o stretching at cm − and c-o stretching at cm − ) corresponding to p(meo ma) are present in both types of nps, in addition to vibration bands at , , and cm − attributed to curcumin molecule. surprisingly, when comparing both cur-g and ag@cur-g nps, for similar curcumin/polymer ratios (tables and ), qualitative differences were observed in their respective spectra evolution (fig. ) . consistently with curcumin data (tables and ) , curcumin related vibration bands seemed to be differently affected by its content. cur-g series exhibited an increase tendency of intensity (fig. a,b) ; whereas ag@cur-g series (fig. c,d) barely varied. this observation could be explained through the key-implication of curcumin in our nuclei-growth approach (fig. ) which determines, at the end, the morphology of ag@cur-g nanohybrids. in fact, the specific silver-nucleation strategy (fig. b) makes feasible that certain amount of curcumin be preferably confined at the metal surface, being subsequently enclosed at higher concentration during p(meo ma) polymer growing shell onto silver core nps (ag@cur-g) than during polymer assembling along silver-free p(meo ma) nanogels formation (cur-g). s-tem micrographs in fig. evidence a core-shell morphology of the ag@cur-g hybrid nps with silver core formed at °c (series ag@cur-g#a, fig. a ) and at °c (series ag@cur-g#b, fig. b) . a mere glance to s-tem pictures revealed almost concentric nanostructuration of polymer shell coverage around metal core for both series of nanohybrids. both series were synthesized with similar curcumin/polymer ratios ( . , . , . and . wt%, table ). the resultant mean values of ag-core diameter ( nm at °c and nm at °c) for both series are collected in table . these differences can be attributed to the faster nucleation rate with increasing temperature that leads to smaller nps. edx spectra in fig. s confirm the presence of ag in two representative hybrid samples with nm (ag@cur-g a) and nm (ag@cur-g b) core diameter, the determined elemental composition is in agreement with ther core size of both nanohybrids. tables and . moreover, it seems that samples synthesized with the two highest curcumin ratios ( . wt%, fig. aiv -biv and . wt%, fig. av -bv) exhibit a more homogeneous spherical shape and a quasi monodistribution, especially, when synthesized at °c (fig. biv-v) . this is in contrast to what happens with the lowest curcumin concentration ( fig. aii and bii) where two silver np populations appeared during the synthesis. these morphological changes were found to affect the spr of the resulting core-doped shell au@cur-g hybrid nps. spr bands of ag@cur-g#a samples synthesized at °c (uv-vis, fig. ai ) are broader and red-shifted compared to those of ag@cur-g#b hybrid nps obtained at °c (uv-vis, fig. bi ). in fact, these wavelengths variations resulted from changes of refractive index of the medium surrounding the metal surface, due to polymer/curcumin reorganization around the two different ag-core sizes. correlative changes in color were also observed when different sizes of metal core were used by keeping all the other reaction parameters constant (table and uv-vis insets of fig. ). larger silver nps exhibited a visual reddish color and a wavelength red-shift. in fact, the λ max for ag@citrate of and nm are and nm respectively , so that red shifts of about - nm collected in table are in accordance to the polymer coating with shell thickness values shown in table . aware of the importance of chemical variables, as curcumin and citrate reducing agents, on the silver-nuclei and their impact on the polymer-growth, an additional experiment with a higher amount of sodium citrate was programmed. at this time, μl of a citrate solution . m was added to the reaction mixture instead of . m, in the presence of . wt% of curcumin, at °c. as expected, it resulted in two highly differentiated ag-core populations ( nm and nm) shown in fig. s . interestingly, agnps (∼ nm) were mostly formed at the limit around the polymeric vesicle boundary but also closely packed inside polymer networking-shell of nm of size (fig. s ). this behavior is attributed to the high nucleation of the nanoparticles due to the high concentration of citrate that causes a greater ion exchange and increases the total ionic strength in the solution, promoting the formation of particle agglomerates in some cases. then, in the regime of low curcumin concentration, phase separation can take place, being the smallest spherical cores produced by sodium citrate and the almost rod-like agnps promoted by curcumin. so, we hypothesize that an appropriate balance among both reducing agents (sodium citrate and curcumin) is needed to obtain more uniform agnp population. thermoresponsive properties. micro/nanogels of crosslinked p(meo ma) in aqueous solution exhibit a reversible volume decrease when raising temperature above the lower critical solution temperature (lcst) of linear p(meo ma). this temperature is known as temperature-induced volume phase transition (vptt) which can be followed by measuring the particle hydrodynamic size by dls. in fig. representative z-average vs temperature curves are shown for the series of ag@cur-g#b nps synthesized at °c ( fig. ai and table ) and of the cur-g# nanogels (fig. aii and table ). as can be observed the vptt values, calculated from the inflection www.nature.com/scientificreports www.nature.com/scientificreports/ point of the curve (fig. ai) , are around °c for the two hybrid nps series (ag@cur-g, table ), showing no dependence on the curcumin content. these results are in good agreement with the atr-ftir spectra (fig. ) which indicated rather similar curcumin content for ag@cur-g hybrids nps. on the contrary, cur-g nanogels (fig. aii ) exhibit slightly decreasing vptt values with increasing the curcumin content (table ). this implication is clearly evidenced at low temperature, where the decrease of the nanogel size is affected by the hydrophobicity feature of curcumin (fig. aii) . interestingly, hybrid nanoparticles exhibit a higher nps size than polymeric nanogels. a plausible explanation of this behavior can lie on the impact of metal core which acts as seed during the precipitation-polymerization; since hybrid nps may need to grow more in order to decrease the polymer-metal interfacial energy and to reach then the stabilization. swelling ratio (q) is an important parameter affecting nanogel properties that can be determined by dls. it can be directly calculated from the ratio between the nanogel volume (v) at swollen state and at the collapsed state : while for polymeric nanogels the volume is directly calculated from the particle radius (r) as v = / π r , for spherical hybrid nps, shell volumes at both temperatures are calculated as v(shell) = / π [r p -r c ]. r p is the hydrodynamic radius of the hybrid particle and r c the hydrodynamic radius of the ag core. table shows the swelling ratios for the silver-polymer (ag@cur-g) hybrid nps and table for the polymeric (cur-g) nanogels investigated. the observed temperature-responsive swelling ratios (q) were close to for the most of the hybrid nps and slightly higher for polymeric nanogels. indeed, for polymeric nanogels ( table ) a decrease of q results from the increase of curcumin in the polymeric nanogels, which could be attributed to a lower swelling ability since the hydrophobic balance increases. on the other hand, the fact that for ag@cur-g hybrid nps, q values show no dependence on the feed composition, could indicate that the final composition of the polymer p(meo ma) shell is rather similar, corroborating again the atr-ftir results (fig. d) . some cyclic heating-cooling experiments were done (data not shown), after observing the reversible collapse-swelling behavior of the samples (fig. ) . at low temperature, the steric protection by the hydrophilic (table ) and (aii) cur-p(meo ma) nanogels (table ) with different curcumin content. (b) ζ-potential determined by dls at °c and °c for colloidal samples of (bi) p(meo ma) nanogels (g ), (bii) agnps functionalized with . wt% curcumin content (ag@cur) before and (biii) after (meo ma) polymerization, ag@cur-p(meo ma) hybrid nps (ag@cur-g b). (c) uv-vis spectra (below and above vptt) of ag@cur-p(meo ma) hybrid nps with ag core synthesized at °c (ci) and °c (cii), and cur-p(meo ma) nanogel (ciii). all samples shown (c) were synthesized with . wt% curcumin/polymer ratio (table and table www.nature.com/scientificreports www.nature.com/scientificreports/ p(meo ma) shell provides good stability in the aqueous medium; whereas at higher temperature, the driving force of colloidal stability is the electrostatic repulsion among the particles revealed by ζ-potential measurements. tables and show ζ-potential values corresponding to ag@cur, ag@cur-g hybrid nps and cur-g nanogels at °c. all types of nps synthesized with curcumin exhibit a fairly high negative ζ-potential that would confer stability against aggregation, especially at temperature above the vptt, where the increase of hydrophobic balance in the polymer shell suddenly produces water expelling from the nanogel. the negative ζ-potential of the synthesized nps can be attributed to several origins. it is well well-known that during polymerization process, incorporation of initiator groups (anionic persulfate) into polymer chains introduces negative charges into micro/nanogel particles synthesized by frpp. additionally, in the specific synthesis of the nanohybrids with silver core explored in this work, the contribution of the remanent sodium citrate cannot be neglected. even more, the negative ζ-potential may arise from curcumin, as it has been reported by some authors , . certainly, in previous works, we found an increase of the ζ-potential at temperature above polymer collapse for both, thermoresponsive polymeric nanogels and hybrid nps with metallic au-core . this effect was attributed to the decrease of the particle size, which increases the charge density at the np surface. nevertheless, in the present investigation, we only observed this temperature-dependent effect on nanoparticles without curcumin (g in fig. bi) . indeed, hybrid nps exhibited a tunable ζ-potential temperature-dependent behavior before (ag@ cur) and after polymerization (ag@cur-g). so, initial ζ-potential diminution values with increasing temperature (fig. bii ) rapidly transited to high and almost invariant ζ-potential values with temperature (fig. biii) as polymerization progressed. these observations (table and fig. b ) so far can be explained through the antagonistic tendencies of the polymer shell and curcumin. the ability of the polymer shell to swell or collapse as function of temperature can tune the optical and luminescent properties of the synthesized nanoparticles. figure c displays representative uv-vis spectra (below and above vptt) for two nanohybrid samples with different core size (ag nm@cur-g a, fig. ci and ag nm@ cur-g b, fig. cii ) and nanogels (cur-g , fig. ciii ) synthesized with . wt% of curcumin content (table ). in uv-vis absorbance spectra, spr band of the nanohybrid with a higher silver diameter (fig. ci) red-shifted while broadening considerably with respect to spr band of the nanohybrid with a smaller silver diameter (fig. cii) . obviously, the spr band position and width was affected by the core-size and the temperature-induced variation of the electromagnetic field in its neighboring environment. the cur-g sample containing silver-free nanogel (fig. ciii) only showed the curcumin effect on nanogel swelling behavior which determines the refractive index of the polymer-gel with the surrounding aqueous medium. in addition, a reversible red-blue shift was observed, when the temperature increase-decrease the vptt of the polymer shell (fig. ) . when the external temperature rises above the vptt, the nanogel shell expels water dictated by the strengthening of hydrophobic interactions giving rise to (i) a decrease of the polymer shell thickness and (ii) an increase of the refractive index. at that point, the effect of refractive index augmentation exceeds to that of the shell thickness diminution; therefore a red-shift should be observed above the vptt for plasmonic-core@thermoresponsive-shell nanohybrids. fig. ci and cii illustrate this behavior for our ag-based thermoresponsive-polymer (core@shell) hybrid nps which correlates well with similar results reported for au-core based thermoresponsive core@shell nanogels . furthermore, the change of the spr maximum with temperature is more pronounced for larger silver core (fig. ci) as previously observed for core-shell nanohybrids with gold core . indeed for the nanohybrids with silver core of nm (series ag@cur-g#a), the Δλlspr max is about - nm whereas for the nm silver core ones (series ag@cur-g#b) the Δλlspr max is about - nm ( table ). the cur-g nanogel sample, without metallic core intervention (fig. ciii) , exhibits no so remarkable λ max absorption change with temperature, at similar curcumin/polymer ratio. www.nature.com/scientificreports www.nature.com/scientificreports/ luminescent properties. curcumin is a tautomeric compound that can occur in diketo and keto-enol forms which ratio strongly varies with the solvent. according to manolova et al. , in solvents as ethanol, only the enol-keto tautomer is present; whereas the addition of water leads to appearance of a new spectral band (about nm), which was attributed to the diketo tautomeric form. the keto-enol form, due to a strong intramolecular hydrogen bond, undergoes transformation into a totally delocalized π-system. due to the hydrophobic character of curcumin, it presents luminescence properties in organic solvents but it is almost non-fluorescent in aqueous solution. however, by complexation through hydrophobic interactions with proteins or with amphiphilic polymers as pluronic , or encapsulating curcumin in hydrophobic polymer nanoparticles , fluorescence enhancement in aqueous medium has been reported. previous to the luminescence investigation of the ag@cur-g hybrid nps, we investigated the absorption and emission of curcumin encapsulated in the thermoresponsive nanogels, cur-g (table and fig. s ). hydrophobic curcumin was engulfed by thermoresponsive nps during the polymerization process, based on the precipitation of the growing polymer at °c due to its unfavorable interaction with the water solvent above lcst (fig. ) . absorbance spectra for curcumin embedded in p(meo ma) nanogels (cur-g) at the polymer concentration of mg ml − show an increase of intensity proportional to the curcumin concentration (fig. s ). in agreement with previous reports of curcumin entrapped in hydrophobic or amphiphilic block copolymers , a main absorption band at - nm in addition to a shoulder about nm appear in fig. s . some authors attributed the signal at nm to the keto-enol tautomer , , whereas the band at nm, associated to the keto form and that typically appears in water, is absent in the present case. fig. s inset shows the linear dependence of the curcumin absorption maxima at nm vs molar concentration of curcumin according to the beer-lambert law for cur-g nanogels. table ). samples were excited at nm. ( fig. a emission spectra in water for the synthesized cur-g nanogels excited at nm evidence remarkable luminescence due to curcumin. in table , the emission maxima (λ em ) and qy, calculated with fluorescein standard, are collected. the emission maxima are about - nm in good agreement with the values reported by banerjee et al. for curcumin encapsulated in polymeric nanoparticles. the position of the emission maxima of curcumin is strongly dependent on the solvent; so by increasing the polarity from chloroform to water, the emission maximum is red-shifted and broadened from nm to nm . both the qy values and the emission maxima position for the present cur-g nanogels seem to indicate that curcumin is well protected by the hydrophobic environment provided by the polymer. with respect to the qy values, two facts have been observed: first, the qy value increases with the decrease of curcumin concentration in the nanogels (fig. a and table ) ; and, second, an amazing fluorescence enhancement is noticed by the temperature increase (fig. b) . the effect of curcumin concentration could be related to the protection that hydrophobic domains of the polymer offer to curcumin, which is enhanced at low curcumin/polymer ratio. the increase of luminescence with increasing temperature should be associated to the water expelled out of the nanogels when temperature rises above vptt. indeed, at lower curcumin ratio (fig. b) , the remarkable increase in absorbance at °c can be attributed to the higher polymer contribution. this reversible change in luminescence with temperature has table ). samples were excited at nm. ( www.nature.com/scientificreports www.nature.com/scientificreports/ been previously reported for fluorescent dyes as bodipy in thermoresponsive p(meo ma) linear polymers and hydrogels . in fig. and fig. s a different behavior can be found for hybrid nps. in first place, analyzing the qy values collected in table , it can be seen that nanohybrids´ fluorescence values are lower than those measured for cur-g nanogels. on the other hand, for both series of ag@cur-g hybrid nps, an increase of luminescence with increasing of curcumin concentration is detected, in opposite to what happened in cur-g nanogels (table and fig. a ). however we know that it can be an ¨apparent increase¨ since the absorption band consideration for estimating the qy values was not properly drawn up. note that this absorption band reflects the additive contribution from the overlapped silver and curcumin spr bands. then, on the basis of these considerations, at lower curcumin concentration, the real curcumin contribution to the final absorbance will be lower. the same miscalculation could arise when comparing qys of hybrid nps with those of polymeric nanogels, because for hybrids an important part of the absorbance arises from the spr of agnps. in last place, the temperature effect also departs from that effect observed for cur-g nanogels, where an increase of fluorescence occurred after polymer collapse on account of hydrophobicity augmentation. in the case of hybrid nps, a slight decrease of fluorescence is detected by moving away from room temperature. this experimental evidence suggests a change on the curcumin-polymer interaction due to the silver presence, or in this specific case, that the interaction of curcumin with silver played a major role, since the specific silver-nucleation strategy causes that certain amount of curcumin be confined at the metal surface. the fluorescence diminution that accompanies the temperature decrease may find its origin in the hydrophilicity-gain experienced by the thermoresponsive polymer shell and the subsequent quenching of curcumin emission induced by water. whereas, when increasing temperature, the polymer increases its hydrophobic balance, which should contribute to protect curcumin against water; nevertheless this positive effect could be overcome by the fact that after polymer collapse, curcumin can get too close to the metal surface and this effect should lead to a further emission quenching. similar observation was reported on the strong curcumin emission-quenching for other silver np-based hybrid systems by other authors , . conclusion in this work, we focused on the knowledge and application of one-pot protocol to obtain core-shell hybrid nanoparticles with the ability to encapsulate hydrophobic, bioactive, antioxidant, and low bioavailability molecules. the aim is to draw on the reductive capacity of some bioactive molecules as curcumin for the in situ formation of metallic nps, meanwhile their hydrophobic character leads the polymer nanostructuration in aqueous medium in the presence of the inorganic metal-core. therefore, new core-doped shell nanohybrids based on silver plasmonic core, thermoresponsive polymer shell and embedded luminescence and bioactive curcumin are easily obtained with potential bioapplications such as antimicrobial systems. furthermore, polymeric nanogels encapsulating curcumin were also achieved, which luminescent properties are strongly enhanced by increasing temperature. this last property could be tuned even with the minimum ratio of bioactive compound. functional microgels and microgel systems temperature-and ph-tunable plasmonic properties and sers efficiency of the silver nanoparticles within the dual stimuli-responsive microgels stimuli-responsive nanogel composites and their application in nanomedicine facile synthesis of gold/polymer nanocomposite particles using polymeric amine-based particles as dual reductants and templates a simple approach to obtain 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esipt process? fluorescence quantum yields and their relation to lifetimes of rhodamine g and fluorescein in nine solvents: improved absolute standards for quantum yields ¶ adsorption and surface-enhanced raman of dyes on silver and gold sols raman response of dithiolated nanoparticle linkers temperature-dependent spectroscopic evidences of curcumin in aqueous medium: a mechanistic study of its solubility and stability dft and experimental studies of the structure and vibrational spectra of curcumin a rapid method to estimate the concentration of citrate capped silver nanoparticles from uv-visible light spectra interfacial tension of turmeric nanoparticles optical properties of responsive hybrid au@polymer nanoparticles the effect of the water on the curcumin tautomerism: a quantitative approach fluorescence study of the curcumin-casein micelle complexation and its application as a drug nanocarrier to cancer cells organic additive, -methylsalicylic acid induces spontaneous structural transformation of aqueous pluronic triblock copolymer solution: a spectroscopic investigation of interaction of curcumin with pluronic micellar and vesicular aggregates synthesis of a self organizable curcumin derivative and investigation of its interaction with metals in % aqueous media bodipy-conjugated thermo-sensitive fluorescent polymers based on -( -methoxyethoxy)ethyl methacrylate the authors gratefully acknowledge the financial support provided by the spanish ministerio de ciencia, innovación y universidades (grant mat - -r and pgc - -b- ). a.s.-q. acknowledges conacyt for the grant epe - . the authors would like to thank david gómez vargas assistance with the sem measurements. key: cord- -wjab y g authors: copland, alastair; sparrow, adam; hart, peter; diogo, gil reynolds; paul, mathew; azuma, miyuki; reljic, rajko title: bacillus calmette-guérin induces pd-l expression on antigen-presenting cells via autocrine and paracrine interleukin-stat circuits date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: wjab y g bacillus calmette-guérin (bcg) is the only licensed vaccine for tuberculosis (tb), and is also used as an immunotherapy for bladder cancer and other malignancies due to its immunostimulatory properties. mycobacteria spp., however, are well known for their numerous immune evasion mechanisms that limit the true potential of their therapeutic use. one such major mechanism is the induction of programmed death ligand- (pd-l ), which mitigates adaptive immune responses. here, we sought to unravel the molecular pathways behind pd-l up-regulation on antigen-presenting cells (apcs) by bcg. we found that infection of apcs with bcg induced pd-l up-regulation, but that this did not depend on direct infection, suggesting a soluble mediator for this effect. bcg induced potent quantities of il- and il- , and the downstream transcription factor stat was hyper-phosphorylated. intracellular analyses revealed that levels of pd-l molecules were associated with the stat phosphorylation state, suggesting a causal link. neutralisation of the il- or il- cytokine receptors dampened stat phosphorylation and bcg-mediated up-regulation of pd-l on apcs. pharmacological inhibition of stat achieved the same effect, confirming an autocrine-paracrine cytokine loop as a mechanism for bcg-mediated up-regulation of pd-l . finally, an in vivo immunisation model showed that bcg vaccination under pd-l blockade could enhance antigen-specific memory cd t-cell responses. these novel findings could lead to refinement of bcg as both a vaccine for infectious disease and as a cancer immunotherapy. the correct balance of immune effector and regulatory responses depends on a number of molecular interactions between the antigen-presenting cell (apc) and t-cell. a key interaction for immunological tolerance is between the receptors programmed death-ligand (pd-l ) and programmed death- (pd- ). apc expression of pd-l leads to binding of this molecule to pd- on t-cells, resulting in activation of the tyrosine phosphatase shp- and dephosphorylation of critical kinases involved in t-cell receptor (tcr) signalling. blockade of this interaction diminishes treg frequencies , enhances th and th effector cell frequencies and increases cytokine production both in vitro and in vivo . the pd-l :pd- interaction has thus been targeted in immunological situations that feature restricted antigen presentation, t-cell anergy and immune tolerance as detrimental characteristicsnamely chronic infectious diseases and malignancies. in the latter scenario, clinical trials have demonstrated the remarkable efficacy of drugs developed to target these receptors, with up to % clinical response rates in some refractory cancers . worldwide, tuberculosis (tb) is the leading cause of death due to infectious disease. the only vaccine available is bacillus calmette-guérin (bcg), which shows only modest protection in adults and alarmingly low efficacy in developing countries, where tb mortality is highest. bcg (like its pathogenic relative, m. tuberculosis) can impede antigen presentation in vivo , , which is believed to contribute to the relatively low efficacy of the vaccine in humans. while mycobacteria-induced pd-l expression has been postulated as a major mechanism driving impaired antigen presentation , , it is currently not fully understood (i) the molecular mechanisms underpinning bcg-mediated pd-l up-regulation, and (ii) the immunological consequences of blocking this pathway during bcg immunisation. for over years, bcg has been employed as a front-line immunotherapy for bladder cancer , and has been used since the late s for malignant melanoma . although the mechanism of action remains to be completely elucidated, it is believed that the bacteria trigger an inflammatory response that leads to immune cell infiltration of the tumour site, thus facilitating immune-mediated clearance . this is likely to be mediated by innate (i.e. toll-like receptor) signalling, providing scope for improvement by concomitant engagement of the adaptive immune responses, which are known to be suppressed by pd-l . here, we show for the first time that bcg can induce the up-regulation of pd-l on both macrophages and dendritic cells (dcs) via autocrine/paracrine secretion of stat -activating cytokines, chiefly il- and il- . blockade of the pd-l receptor in vivo during bcg immunisation led to superior cd t-cell responses to recall antigen, thus highlighting the potential utility of this pathway in clinical settings. these findings provide new targets for improving bcg as both a tb vaccine and cancer immunotherapy. ethics. all bacteria. bcg strain pasteur was a kind gift from professor juraj ivanyi (king's college, london) and was grown according to previous reports , using standard microbiological techniques. bcg expressing green fluorescent protein (gfp; also from the ivanyi laboratory) was grown in identical conditions, but under selective media and agar containing μg/ml hygromycin b (sigma-aldrich). mice and immunisations. female c bl/ mice ( to weeks old) were obtained from charles river laboratories, uk. mice were administered mg of pd-l -blocking antibody mih or the rat igg a isotype control mac (kind gifts from professor anne cooke, university of cambridge) via the intraperitoneal (i.p.) route (day - ). twenty-four hours later (day ), mice received × cfu bcg subcutaneously (s.c.). mice then received booster immunisations of mih or mac ( mg per dose) on days , and . to confirm receptor blockade, mice were administered mg mih or mac via the i.p. route, followed by euthanasia at h, and immediate ex vivo staining of the splenocytes. cells were stained with a reported competing fluorochrome-conjugated α-pd-l clone ( f. g ) , which binds to the same epitope as mih , to test for successful receptor blockade (fig. ) . as an additional control for specificity, pd-l was also stained after mih or mac treatments. bone marrow-derived dcs were obtained as previously described . cells were > % cd c + by flow cytometry. dcs were stimulated in complete rpmi (rpmi- containing % fcs, mm l-glutamine, and μm β-mercaptoethanol ± u/ml penicillin/streptomycin -all from sigma-aldrich). for experiments involving macrophages, the cell line j . was used, and cells were grown and stimulated in complete dmem (same recipe as rpmi -sigma-aldrich). for bcg infections, bacteria were washed in complete media without antibiotics, and then apcs were inoculated at the designated moi. cells were cultured in a % co humidified incubator at °c. in some experiments, e. coli-derived lipopolysaccharide (lps; sigma-aldrich) was used at ng/ml. cytokines (peprotech, uk) were www.nature.com/scientificreports www.nature.com/scientificreports/ diluted in complete rpmi before administration. interleukin blocking antibodies (purchased from biolegend, uk) were pre-cultured with the cells for h before stimulation. stattic (tocris bioscience, uk) was diluted in dmso (sigma-aldrich) and cells were treated for h before infection. ex vivo immunogenicity assays. spleens were aseptically removed from euthanised mice, mechanically homogenised and treated with ack lysis buffer. cells were then counted and seeded at . × per well in complete rpmi, followed by treatment with μg/ml brefeldin a (sigma-aldrich). cells were stimulated with μg/ml ag b/acr (lionex, germany) or ppd (nibsc, uk) with μg/ml α-cd (biolegend) for hours before staining for flow cytometry. pma/ionomycin treatment ( ng/ml and μg/ml, respectively -sigma-aldrich) was used as a positive control and for staining boundaries (data not shown). for lymph node analysis, inguinal lymph nodes were excised from euthanised mice on the indicated day, followed by mechanical disruption, counting and immediate flow cytometric analysis. flow cytometry. in most experiments, cells were first washed in pbs and then incubated with : viability dye (efluor fixable viability dye; ebioscience) under fc receptor blockade ( : trustain; biolegend) for - minutes. cells were then washed in flow cytometry buffer (pbs (invitrogen) containing . % bsa and . % sodium azide -both from sigma-aldrich) and stained with the appropriate pre-titrated flow cytometry antibodies for m at °c. cells were sometimes fixed using biolegend fixative buffer before being acquired on a bd facscanto ii instrument and analysed using flowjo software. for assessing phosphorylated residues, cells were instead mildly fixed with fixative buffer for m at °c, and then permeabilised with a commercial methanol buffer (true-phos buffer -biolegend) for hour before staining, as described elsewhere . for intracellular cytokine staining, cells were fixed with fixative buffer, followed by permeabilisation with flow cytometry buffer containing . % saponin (sigma-aldrich). compensation was performed using ebioscience ultracomp enzyme-linked immunosorbent assay. cytokine levels in supernatants were measured using ebioscience ready-set-go elisa kits, according to the manufacturer's instructions. plates were read at nm on a tecan plate reader. prism software, using averaged technical replicates where possible. each statistical test and post-test is detailed in the relevant figure legends. a p value less than . was considered significant. dcs and macrophages are critical for the initiation of adaptive immunity. bcg can induce the up-regulation of pd-l expression on pulmonary dcs in mice , however it is unclear whether the same holds true for macrophages. dcs and macrophages were therefore infected with bcg over a range of multiplicities of infection (moi) and across two time-points ( h and h); surface expression of pd-l was assessed by flow cytometry. as shown in fig. a , the positive control lipopolysaccharide (lps) was able to significantly increase pd-l expression on both cell types (p < . ), with a striking > fold increase in macrophages at h. upon infection with bcg, both apc types expressed high levels of pd-l compared to the unstimulated control at h and h (p < . ), and a dose trend was observed for increasing moi in macrophages at h. next, a transgenic strain of bcg that expresses green fluorescent protein (gfp) was used to determine whether the up-regulation of pd-l depended on direct interaction between apcs and the bacteria. cells were gated by gfp fluorescence into gfp neg (i.e. "bystander") and gfp pos (i.e. infected) populations. these were then tested for pd-l expression. as anticipated, gfp pos infected cells displayed increased expression of pd-l that was proportionate to the infectious dose (fig. b) . surprisingly, however, gfp neg bystander cells also exhibited similar dose-dependent increases in pd-l expression to gfp pos cells, which was significantly up-regulated compared to the uninfected control at the highest moi (p < . ), and approximately % that of directly infected cells. in support of these observations, . µm filtration of bcg infection supernatants, when applied to new cells, was able to up-regulate pd-l ( supplementary fig. ). as expected, control supernatants from uninfected cells did not affect pd-l expression. these data strongly suggested that a soluble, secreted factor in culture was driving pd-l up-regulation by bcg. interestingly, bcg was able to up-regulate other members of the b family (cd , cd , pd-l ) to a certain degree, but with a pattern of skewed up-regulation of pd-l compared to cd (supplementary fig. ). bcg induces il- and il- production in tandem with stat phosphorylation. the murine pd-l gene (cd ) is under the control of complex regulatory networks and can be induced by a number of inflammatory cytokines or tlr ligands . many of these control mechanisms are cell type-specific. since mycobacteria are adept at driving stat activation , and since stat is capable of binding to the pd-l promoter in tolerogenic dcs , we hypothesised that this signalling pathway was mediating the observed effects of infection. dcs and macrophages were infected for h and intracellular flow cytometry was used to determine the levels of stat tyrosine residue phosphorylation. as can be observed in fig. a , bcg was able to hyper-phosphorylate the stat transcription factor compared to unstimulated cells (p < . ). notably, there was a trend for increased phosphorylation when comparing bcg to the positive control lps. we next hypothesised that apcs were producing stat -activating cytokines. stat can be activated weakly by il- and il- , www.nature.com/scientificreports www.nature.com/scientificreports/ but strongly by prototypical myeloid-type cytokines such as il- and il- . apcs were therefore infected with bcg (or stimulated with lps) for - h and levels of il- and il- were measured by elisa. bcg was found to induce potent quantities of il- in both dcs and macrophages at a range of moi ( fig. b ; p < . bcg vs unstimulated cells). bcg elicited il- in both cell types. strikingly, with regards to il- production, bcg greatly surpassed the ability of lps at moi = , with over -fold concentrations of this cytokine compared to the positive control. with the hypothesis that bcg was inducing stat -activating cytokines to up-regulate pd-l , we treated dcs and macrophages with different concentrations of il- and il- and measured pd-l up-regulation by flow cytometry. in dcs (fig. a) , il- played a dominant role in the up-regulation of pd-l expression compared to il- . when both cytokines were used in combination, there was only a marginal increase above the levels of pd-l expression conferred by il- . in macrophages, by contrast, il- and il- behaved similarly in terms of receptor up-regulation, and there was a combinatorial effect that was clearly evident at pg/ml. for both cell types, there was a dose-dependent effect, with pg/ml of any cytokine (or combination) being superior to pg/ml in up-regulating pd-l expression. next, macrophages were infected with bcg for h and cells were then permeabilised as before and co-stained for p-stat and pd-l . cells were then divided into pd-l lo and pd-l hi populations and then www.nature.com/scientificreports www.nature.com/scientificreports/ levels of p-stat were quantified. as shown in fig. b , pd-l lo cells exhibited significantly lower levels of stat phosphorylation compared to their pd-l hi counterparts (p < . ), with a doubling of fluorescence intensity in some experiments. taken together, these data suggested that bcg was utilising interleukin signalling pathways in order to up-regulate expression of pd-l . we next questioned whether direct intervention in the interleukin-stat axis could revert the up-regulation of pd-l by bcg. to this end, we employed a combination of monoclonal antibodies (mabs) that are known to block the il- and il- cytokine receptors, alongside the pharmacological inhibitor 'stattic': a well-characterised and highly specific small-molecule inhibitor of stat transcriptional activity . to test whether neutralising the biological activities of endogenous cytokines could reduce the increase in pd-l expression caused by bcg, cells were first pre-incubated under il- r (mab d a ) or il- r (mab b . a) blockade, or a combination of both, for h. isotype controls served as controls for non-specific activity. cells were then infected with bcg and after h, pd-l expression was determined (fig. a) . both il- and il- blockade significantly reduced pd-l expression (p < . ) compared to the isotype control baseline. as expected, the combination of blocking both receptors led to the greatest reduction in pd-l fluorescence (~ - %; p < . vs isotype control). in keeping with the hypothesised role of stat in bcg-induced pd-l expression, blockade of both receptors also led to a large reduction in stat phosphorylation, as shown in fig. b (bcg + isotype mfi: ; bcg + dual blockade mfi: ). to confirm that stat was mediating the up-regulation of pd-l expression by bcg, cells were then pre-treated with stattic or a vehicle control for hours before infection with a low dose of bacteria (fig. c) . lps served as a positive control, since it can induce pd-l expression via this pathway . after hours, the cells treated with μm stattic and infected with bcg showed a dramatically reduced expression level of pd-l compared to those treated with a vehicle control (p < . ), with an near- % reduction in pd-l expression. similar results were observed for lps. strikingly, resting cells treated with stattic actually increased pd-l expression compared to the vehicle control, although this was a non-significant trend with high variation. collectively, these data proved that bcg could modulate pd-l expression by interleukin-stat signalling circuits. proof-of-principle in vivo immunogenicity assay was next performed in order to establish that targeting the pd-l receptor during bcg immunisation could lead to increased t-cell function. a cytokine panel spanning www.nature.com/scientificreports www.nature.com/scientificreports/ several hallmark th /th cytokines (ifn-γ, il- , il- a, tnf-α) was used to determine t-cell reactogenicity in a vaccination model with recall mycobacterial antigens (fig. a) . first, the up-regulation of pd-l in vivo was determined by sub-cutaneous immunisation with bcg followed by characterisation of pd-l expression in the draining lymph nodes (supplementary fig. ) . it was found that bcg was able to up-regulate pd-l in mhc class ii high cd c + dcs, but unable to do the same in t-cells, confirming a specific effect in apcs. next, mice were treated with mg of the pd-l blocking antibody mih via the intraperitoneal route, followed by immunisation with subcutaneous bcg. mice were then given follow-up booster immunisations to maintain the pd-l blockade. an isotype control was used at the same concentration to control for non-specific effects. on day , splenocytes from immunised mice were then pulsed with either immunodominant and latency-associated antigens (ag b and acr, respectively) or a mixture of protein antigens (ppd). as shown in fig. b , there was a general trend for an increase in antigen-specific cytokine production in cd t-cells. under pd-l blockade, bcg induced significantly more ifn-γ in response to ag b/acr and ppd (ifn-γ: p < . ), compared to the isotype control. tnf-α production was also increased in response to ag b/acr (p < . ). regarding il- and il- a, there were non-significant increases caused by pd-l blockade under both antigen recall conditions. turning our attention to the cd t-cell compartment (fig. c) , we found that bcg was much poorer at inducing antigen-specific cytokine responses, as has been observed previously by others . with the exception of il- after ag b/acr pulsing (p < . ), splenocytes from mice that received bcg + isotype control were unable to produce more cytokines than splenocytes from mock-immunised mice. only two cytokines were found to be increased by pd-l blockade beyond the pbs control group in response to ag b/acr but not ppd: ifn-γ (p < . ) and tnf-α (p < . ), however these effects were marginal. thus we concluded that pd-l blockade can effectively boost cd -dependent t-cell immunity, with only marginal effects on boosting cd t-cell responses. it is long-recognised that bacteria (and indeed other pathogens) belonging to genetically distinct phyla are capable of modulating the repertoire of co-stimulatory and co-inhibitory molecules expressed on the apc surface , thus affirming the critical importance of said receptors in the directing of adaptive immune responses. mycobacteria represent an example of immune evasion par excellence due to their ancient history of co-evolution with mammalian immune systems ; indeed, virtually all pathogenic mycobacteria are obligate parasites, requiring intracellular www.nature.com/scientificreports www.nature.com/scientificreports/ resources in order to thrive and propagate. it is therefore not surprising that they are biologically equipped to effectively counter adaptive immune responses that would lead to their own clearance. a centrally important molecule in the control of t-cell immunity is pd-l . mice infected with mycobacteria harbour pulmonary dcs that express high levels of pd-l and restrict antigen presentation to cd t-cells , and pd-l blockade in blood and lung lavage from tb patients can enhance responses to mtb antigens, as seen by greater cytokine production and t-cell proliferation , . furthermore, pd-l blockade is able to rescue these cells from functional exhaustion, as demonstrated by the reversal of t-cell apoptosis . for bladder cancer, the tumour surface is reportedly decorated with high levels of pd-l molecules, and the tumour-infiltrating b-and t-cells express high levels of both pd-l and pd- , . the utilisation of bcg as a prophylactic (tb) or therapeutic (malignancy) treatment for these diseases, combined with a strategy to mitigate the effects of pd-l , could provide a strong advantage for the efficacy of bcg. in this study, for the first time, it was shown that bcg drives up-regulation of pd-l expression on apcs by autocrine/paracrine cytokine circuits that lead to stat phosphorylation and up-regulation of this co-inhibitory receptor (illustrated in fig. ) . it has been known for some time that bcg is a strong inducer of these cytokines via rudimentary tlr signalling, however it was not known that they were driving pd-l expression in a biphasic response. the observation that inhibition of stat did not lead to a decrease in pd-l expression in resting cells-indeed, there was instead a non-significant increase in expression-is consistent with the notion that the pd-l gene promoter requires distinct transcriptional apparatus during the steady-state and during active infection. this is in accord with the fact that dcs and macrophages displayed moderate levels of constitutive pd-l expression in the absence of any appreciable cytokine levels . physiologically, this is undoubtedly to prevent spontaneous activation of the adaptive immune system. using the mab mih , we were able to neutralise most of the pd-l receptor in vivo and augment the quantities of cytokine produced by cd t-cells in response to recall antigen in a proof-of-principle experiment. it is interesting to note that pd-l blockade in vivo did not greatly boost the antigen-specific cd t-cell responses beyond that of the mock-immunised control group. bcg is widely known to only minimally induce cytotoxic t-cell responses, with a strong bias towards cd responses . this may be due to so-called cd 'decoy antigens' , such as tb . , that serve to divert responses away from immunogenic epitopes for cytotoxic t-cells . whether the enhancement of cd t-cell immunogenicity in the absence of strong cd t-cell immunogenicity leads to better vaccine (i.e. in the case of tb) or immunotherapeutic (i.e. in the case of a carcinoma) outcomes warrants testing in disease-specific animal models. www.nature.com/scientificreports www.nature.com/scientificreports/ stat -activating cytokines, (ii) il- /il- cytokine receptors, (iii) the stat transcription factor and (iv) the pd-l receptor itself. stat presents a highly appealing therapeutic target other than its role in pd-l expression due to the fact that is a pleiotropic master controller of general tolerance in apcs. stat can suppress autophagy , nitric oxide production , il- production , and co-stimulatory molecule expression . given our new findings, targeting this pathway during vaccination with bcg (for tb or malignancy) could reap multiple therapeutic benefits. in conclusion, we have revealed novel molecular insights into how bcg up-regulates pd-l on apcs, allowing for improved immunogenicity to specific antigens, but also more intricate understanding of fundamental host-pathogen interactions. future work will focus on exploring this pathway in specific disease models that rely on bcg as a treatment, with the aim of bolstering immunological parameters, and ultimately, treatment efficacy. pd-l regulates the development, maintenance, and function of induced regulatory t cells interactions between pd- and pd-l promote tolerance by blocking the tcr-induced stop signal interference with pd-l /pd- co-stimulation during antigen presentation enhances the multifunctionality of antigenspecific t cells pd- blockade with nivolumab in relapsed or refractory hodgkin's lymphoma mycobacterium bovis bcg decreases mhc-ii expression in vivo on murine lung macrophages and dendritic cells during aerosol infection bcg vaccine mediated reduction in the mhc-ii expression of macrophages and dendritic cells is reversed by activation of toll-like receptors and enhanced expression of pd-l and ifn-gamma on dendritic cells is associated with bcg-induced th inhibition mycobacteria-responsive sonic hedgehog signaling mediates programmed death-ligand -and prostaglandin e -induced regulatory t cell expansion bcg immunotherapy of bladder cancer: years on bcg immunotherapy of malignant melanoma: summary of a seven-year experience bacillus calmette-guerin immunotherapy for urothelial carcinoma of the bladder mucosal delivery of fusion proteins with bacillus subtilis spores enhances protection against tuberculosis by bacillus calmette-guerin preferential contribution of b -h to programmed death- -mediated regulation of hapten-specific allergic inflammatory responses type diabetes development requires both cd + and cd + t cells and can be reversed by non-depleting antibodies targeting both t cell populations the programmed death- ligand :b - pathway restrains diabetogenic effector t cells in vivo synovial regulatory t cells occupy a discrete tcr niche in human arthritis and require local signals to stabilize foxp protein expression intrinsic and extrinsic control of expression of the immunoregulatory molecule pd-l in epithelial cells and squamous cell carcinoma arginine usage in mycobacteria-infected macrophages depends on autocrine-paracrine cytokine signaling pd-l expression on tolerogenic apcs is controlled by stat- stattic: a small-molecule inhibitor of stat activation and dimerization expression of pd-l and pd-l on human macrophages is up-regulated by hiv- and differentially modulated by il- systemic bcg immunization induces persistent lung mucosal multifunctional cd t(em) cells which expand following virulent mycobacterial challenge manipulation of costimulatory molecules by intracellular pathogens: veni, vidi, vici!! plos pathog , e host-pathogen coevolution in human tuberculosis dendritic cells in chronic mycobacterial granulomas restrict local anti-bacterial t cell response in a murine model pd- /pd-l pathway inhibits m.tb-specific cd (+) t-cell functions and phagocytosis of macrophages in active tuberculosis programmed death- (+) t cells inhibit effector t cells at the pathological site of miliary tuberculosis inhibiting the programmed death pathway rescues mycobacterium tuberculosisspecific interferon gamma-producing t cells from apoptosis in patients with pulmonary tuberculosis a review on the evolution of pd- /pd-l immunotherapy for bladder cancer: the future is now a review of the pd- /pd-l checkpoint in bladder cancer: from mediator of immune escape to target for treatment pd-l and pd-l are differentially regulated by th and th cells mycobacterium tuberculosis-specific cd + and cd + t cells differ in their capacity to recognize infected macrophages quantitative comparison of the efficiency of antibodies against s and s subunit of sars coronavirus spike protein in virus neutralization and blocking of receptor binding: implications for the functional roles of s subunit constitutive activation of stat by the src and jak tyrosine kinases participates in growth regulation of human breast carcinoma cells tlr-mediated stat and erk activation controls il- secretion by human b cells cytoplasmic stat represses autophagy by inhibiting pkr activity stat represses nitric oxide synthesis in human macrophages upon mycobacterium tuberculosis infection il- -independent stat activation by toxoplasma gondii mediates suppression of il- and tnf-alpha in host macrophages latency-associated protein acr impairs dendritic cell maturation and functionality: a possible mechanism of immune evasion by mycobacterium tuberculosis we would like to thank professor anne cooke (university of cambridge) for kindly providing the mih antibody and for helpful suggestions regarding the in vivo experiments. we would also like to thank dr david bending (university of birmingham) for advice regarding the manuscript. this work was funded by an e.u. horizon grant as part of the eliciting mucosal immunity in tuberculosis (emi-tb) consortium project. a.c. conceived the work, performed majority of experiments and co-wrote the manuscript; p.h., a.s. and g.r.d. helped with flow cytometry assays and in vivo pd-l blockade experiment; m.j.p. helped with statistical analysis; m.a. provided the anti-pd-l antibody and critically reviewed the manuscript; r.r. conceived and supported the work and co-wrote the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - . publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -nxw k y authors: zhang, yewu; wang, xiaofeng; li, yanfei; ma, jiaqi title: spatiotemporal analysis of influenza in china, – date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: nxw k y influenza is a major cause of morbidity and mortality worldwide, as well as in china. knowledge of the spatial and temporal characteristics of influenza is important in evaluating and developing disease control programs. this study aims to describe an accurate spatiotemporal pattern of influenza at the prefecture level and explore the risk factors associated with influenza incidence risk in mainland china from to . the incidence data of influenza were obtained from the chinese notifiable infectious disease reporting system (cnidrs). the besag york mollié (bym) model was extended to include temporal and space-time interaction terms. the parameters for this extended bayesian spatiotemporal model were estimated through integrated nested laplace approximations (inla) using the package r-inla in r. a total of , influenza cases were reported in mainland china in cnidrs from – . the yearly reported incidence rate of influenza increased . times over the study period, from . in to . in per , populations. the temporal term in the spatiotemporal model showed that much of the increase occurred during the last years of the study period. the risk factor analysis showed that the decreased number of influenza vaccines for sale, the new update of the influenza surveillance protocol, the increase in the rate of influenza a (h n )pdm among all processed specimens from influenza-like illness (ili) patients, and the increase in the latitude and longitude of geographic location were associated with an increase in the influenza incidence risk. after the adjusting for fixed covariate effects and time random effects, the map of the spatial structured term shows that high-risk areas clustered in the central part of china and the lowest-risk areas in the east and west. large space-time variations in influenza have been found since . in conclusion, an increasing trend of influenza was observed from to . the insufficient flu vaccine supplements, the newly emerging influenza a (h n )pdm and expansion of influenza surveillance efforts might be the major causes of the dramatic changes in outbreak and spatio-temporal epidemic patterns. clusters of prefectures with high relative risks of influenza were identified in the central part of china. future research with more risk factors at both national and local levels is necessary to explain the changing spatiotemporal patterns of influenza in china. influenza is associated with notable mortality and morbidity worldwide, as well as in china - . the behaviours of major epidemics and pandemics of influenza were complicated due to dramatic genetic changes, subtype circulation, wave patterning and virus replacement . influenza vaccination is the most effective means to prevent infection, severe disease and mortality . the world health assembly recommends vaccinating % of key risk groups against influenza . although seasonal influenza vaccination was introduced in , influenza vaccination is not yet included on the national immunization program (nip) in china . the average national vaccination coverage was reported to be just . - . % between and , . the overall number of flu vaccines approved for sale by china's national institute for food and drug control (nifdc) has decreased in recent years , . the low coverage rate and reduction in flu vaccine supplementation have raised much concern about the increased risk of influenza incidence in china. although new emerging influenza virus types and subtypes, such as avian influenza a h n [ ] [ ] [ ] [ ] , influenza a (h n )pdm [ ] [ ] [ ] , and influenza a h n , , have been reported continuously in china, the disease burden of influenza has been dominated by a(h n ), a(h n )pdm influenza viruses, pre-pandemic a(h n ) or influenza b in recent years, which account for the majority of cases . the influenza a(h n )pdm virus was first introduced to mainland china on may , , and has been one of the dominant viruses in the seasonal influenza epidemics since then . the effect of newly emerging influenza a(h n )pdm viruses on the geographic patterns and temporal trends of influenza across the whole country is still unknown. covariates associated with the reported incidence cases of influenza. the table . the crude odds ratios (ors) and adjusted ors in both the univariate poisson models and multivariate adjusted poisson model are statistically significant. after adjusting for other covariates, a spatially unstructured random effect term (v i ), a spatially structured conditional autoregression term (υ i ), a first-order random walk-correlated time variable (γ j ), and an interaction term for time and place (δ ij ) in the multivariate adjusted spatiotemporal model, the flu vaccines (per million doses), flu surveillance protocols, rate of influenza a (h n )pdm , latitude and longitude still remain statistically significant. holding all other covariates to zero and adjusting for spatiotemporal variation, every one million increase in the number of influenza vaccines for sale approved by the china food and drug administration was associated with a . % decrease in the influenza incidence risk ( % ci = . - . ). similarly, the new update of the influenza surveillance protocol in was related to a . % increase in the influenza incidence risk ( % ci = . - . ) compared to the protocol used in to . for every % increase in the rate of influenza a (h n )pdm among all processed specimens from ili patients, there was a . % increase in the influenza incidence risk ( % ci = . - . ). every one degree increase in the latitude and longitude was associated with a . % ( % ci = . ~ . ) and . % ( % ci = . ~ . ) increase in the influenza incidence risk, respectively. the spatial and temporal effects in spatiotemporal models with covariates. the spatial effects. the map of the spatially structured conditional autoregression term demonstrated areas of spatial patterning and similarity among prefectures. the spatially structured relative risk and posterior probabilities of spatially structured relative risk greater than . are presented in figs. and , respectively. table . deviance information criterion (dic) for five spatiotemporal models. abbreviations: d, posterior mean of the deviance; pd, the number of effective parameters; dic, the deviance information criterion, as a measure of the trade-off between model fit and complexity. note: model terms used in four models include an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); uncorrelated time (γ j ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ j ). θ ij represents the relative risk of area i at time j. * model , convolution + uncorrelated time (time iid), e.g., θ α ν table . risk analysis of covariates associated with reported cases of influenza. abbreviations: or, odds ratio; ci, confidence interval. * univariate poisson analysis models. ** multivariate adjusted poisson analysis model, which included all variables in the univariate analysis models. † multivariate adjusted spatiotemporal models, which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ ij ). ‡ total number of flu vaccines approved for sale by china's national institute for food and drug control (nifdc), which were rescaled to one million doses as one unit. data were collected from nifdc. # the convolutional spatial risk term, which includes both the spatially structured conditional autoregression term (υ i ) and the spatially unstructured random effect term (ν i ) at the prefecture level, identified areas at increased risk of influenza throughout the -year study period (fig. ) . posterior probabilities for an area's spatial risk estimate exceeding . are presented in fig. . the proportion of the total spatial heterogeneity explained by the spatially structured conditional autoregression term was . %. after adjusting for fixed covariate effects and time random effects, both the map of the spatial structured term and the convolutional spatial term show that high-risk areas clustered in the central part of china and the lowest-risk areas in the east, northwest and southwest. the higher-risk prefectures were mostly distributed in guangdong, guangxi, guizhou, hunan, jiangxi, zhejiang, hubei, anhui, henan, hebei, beijing, tianjin, gansu, ningxia, and inner mongolia. the lower-risk areas in the east included some prefectures in the shandong peninsula and the prefectures of heilongjiang, liaoning, and jilin provinces in the northeast. the northwest areas are composed of prefectures in tibet, qinghai and xinjiang, while the southwest areas include chongqing and prefectures in sichuan and yunnan provinces. the temporal trend. the relative risks of the -year study period, holding the covariates and spatial risk constant, were calculated by exponentiating the marginal first-order random walk-correlated time term (γ j ) in the spatiotemporal models of influenza risk with and without covariates. for the spatiotemporal model without . ** adjusted by convolutional spatial term, space-time interaction term, and covariates, e.g., . figure . map of the spatially structured relative risk ( υ e i ), spatiotemporal model of influenza incidence risk with covariates, china prefectures, - . note: the linear terms in the model of spatiotemporal model of influenza incidence risk with covariates were , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ ij ). www.nature.com/scientificreports www.nature.com/scientificreports/ covariates, an overall increasing trend was found in the temporal trend term in the -year study period. the risk of influenza remained low between and . a steep increase was observed in . it dropped slightly back to a low level and remained stable in and . a rapid increase was obvious in the last years (table ) (fig. ) . for the temporal trend term in the spatiotemporal model with covariates, the relative risks in the years from to were not significantly different from that in the spatiotemporal model with covariates. the relative risks in the model with covariates in and were significantly lower than those in the model without covariates. the lower boundary of the % confidence intervals in the model with covariates showed some levelling off in recent years. the differences between the spatiotemporal model with and without covariates indicated that the recent increases in influenza incidence risks could be partially explained by the fixed covariate effects. space-time interactions. the probability exceedances for the yearly space-time interactions are presented for the study period (fig. ) . these identify areas with residual spatial risk greater than . compared to the prefecture-wide risk after the fixed effects, unstructured, spatially structured, and time random effects are held constant. changing patterns and large variations among the yearly specific spatial distributions are shown in fig. . it is interesting that most of the higher-risk areas were western areas of china before , and most of the higher-risk areas are eastern or northern areas of china after . , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ ij ). , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ ij ). ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ based on the incidence data of influenza gained from the chinese notifiable infectious disease reporting system, we used the bayesian spatiotemporal model in this study to assess the space-time patterns of the influenza epidemic at the prefecture level in mainland china from to and explored several factors that may be associated with the changing spatial and temporal patterns in the influenza incidence risk. several potential factors may be associated with the rapid increasing trend of influenza in china. first, insufficient flu vaccine supplements and a low uptake rate might be associated with an increase in influenza incidence. the results of the final spatiotemporal model showed that every million increase in the number of influenza vaccines approved for sale by the china food and drug administration was associated with a . % decrease in the influenza incidence risk ( % ci = . - . ). the rapidly increased crude rates of influenza from to coincided with a large reduction in the numbers of vaccines approved for sale at the same time. the reductions in the numbers of vaccine supplements were mostly due to the outcomes of vaccine scandals related to improper vaccine storage and production in and , respectively , , . previous studies reported that uptake figures of the influenza vaccine averaged . % nationally and . % among urban elderly aged years and above in cities of china during the - and - influenza seasons, respectively , , . it is expected that the uptake may be even lower, as people lost their faith in the safety of domestically produced vaccines after the vaccine scandals in china . our results are consistent with the study in italy, which reported an association between vaccination coverage decline and influenza incidence among italian elderly . , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ ij ). www.nature.com/scientificreports www.nature.com/scientificreports/ second, currently circulating influenza strains in humans include influenza a (h n )pdm , influenza a (h n ) and influenza b viruses, (b/victoria and b/yamagata) , , . influenza a (h n )pdm has been reported to be the predominant subtype in recent years according to ili surveillance and is more likely to be the major cause of regional and widespread outbreaks . our study showed that for every % increase in the rate of influenza a (h n )pdm among all processed specimens from ili patients, there was a . % increase in the influenza incidence risk ( % ci = . - . ). shu et al. reported that the predominant subtype of seasonal influenza a (h n ) and b/yamagata could circulate from the south to the north of china from to . our study also found that every one degree increase in latitude and longitude was associated with a . % ( % ci = . ~ . ) and . % ( % ci = . ~ . ) increase in the influenza incidence risk, respectively. this result was consistent with the role of climatic factors in influenza transmission dynamics , . third, the greater effort in influenza surveillance and the use of new technologies may account for the rise in influenza incidence. in recent years, especially after the pandemic season, influenza surveillance has been expanded worldwide, as recommended by the world health organization (who) [ ] [ ] [ ] , , . as cnidrs includes all sentinel hospitals, sentinel hospitals are likely to report more cases of influenza to cnidrs. in addition, more hospitals have used electronic health information systems, which may improve both the quantity and quality of data collection and exchange from hospitals to cnidrs [ ] [ ] [ ] [ ] . fourth, the reporting on influenza a (h n )pdm , avian influenza a (h n ), highly pathogenic avian influenza (hpai) h n and avian h influenza has increased in recent years , which included all variables in the univariate analysis models; which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ ij ). www.nature.com/scientificreports www.nature.com/scientificreports/ media and public health campaigns against the new emerging virus have caused both the government and the public to be more concerned about influenza. the improved public perception of influenza may change people's health-seeking behaviours, especially in the epidemic seasons , . furthermore, enlarged coverage of health care insurance in both urban and rural areas in recent years in china may also induce people to use more health services , . a rapid increase in the numbers of airlines and high-speed railway transports in china has been reported in recent years . these factors would make it easy to transmit the influenza virus at a larger scale and in a shorter time across the country - . the spatial pattern. the bym model includes both a spatial conditional autoregression component and a heterogeneous random effect component. this structure allows us to know how much of the residual disease risk is due to spatially structured variation and how much is unstructured overdispersion . the spatially structured conditional autoregression term demonstrated areas of spatial patterning and similarity among prefectures. the results of spatially structured variation show a distinguished spatial pattern of risk of influenza across prefectures in china. the highest-risk areas clustered in the middle part of china, while the lowest-risk areas were distributed in the east, northwest and southwest. different patterns of influenza between the north and south in china were well reported , , , , , . in china, the line following the qinling mountain range in the west and the huaihe river in the east is often used to split the mainland into the north and the south . in this study, we observed clustering in both the north and the south in the middle part of china. the unique structured spatial patterns may be attributed to the shared risk factors among the neighbouring areas. this may be associated with similarities in the climatic zone, the predominant subtype of the virus at the time of epidemics, socioeconomic background or lifestyles. the last important factor should not be ignored. some studies reported that clustering of diseases may be a consequence of spatial heterogeneity in surveillance efforts , . the space-time interaction. the space-time interaction is a random effect term, which is interpreted as the residual effect after the unstructured, spatially structured and time effects are modelled and represent sporadic short-term outbreaks or clusters. the changes and circulations of virus subtypes may determine the characteristics of the space-time interaction terms. the year was the critical point according to the results of the spatiotemporal analysis. there are four types of ili activities: sporadic, local outbreak, regional outbreak and widespread outbreak in flunet (www. who.int/flunet), global influenza surveillance and response system (gisrs) , . since the first case of influenza a (h n )pdm was reported on may , , in mainland china, the type a (h n )pdm virus has been detected in all ili activities according to the data from flunet. the yearly ili activities may be partially associated with the changes and similarities in the patterns of the space-time interactions from to . from the flunet data mentioned above, we found that sporadic ili activities were dominant in , , and . correspondently, we found more areas with high relative risk in these years in the space-time term. this implies that the more sporadic the activities are, the larger the variations in the spatiotemporal distribution of the risk of influenza. in contrast, the large outbreaks account for most ili activities in the years , , and . few prefectures were observed to have a relative risk greater than or during that period. large outbreaks, especially large regional and widespread outbreaks, may reduce the differences in the incidence risk of influenza among the areas and times on a large scale. strengths. this work adds to the existing research on influenza epidemiology in the following ways. first, the study initially presents the spatiotemporal distributions with higher-resolution spatial data than has been reported in china for the last years, which allows more opportunity for focused investigations and interventions. next, we used the exceedance probabilities instead of the observed risk estimates to identify those areas for which the increased risk was highly unlikely to be due to chance. then, this study also provided a baseline model that can be extended to include social, economic, ecological, and environmental factors, as well as intervention measures to explore their associations with influenza. finally, the methods in this study offer practical tools for spatial analysis of other notifiable infectious diseases in cnidrs. there are some limitations to this study. cnidrs is a passive surveillance system, and accessibility to health facilities and patient visit behaviour may affect the number of cases reported. we collected both clinically diagnosed and laboratory-confirmed cases in cnidrs, so misdiagnosis and misreporting are unavoidable because it is difficult to distinguish influenza from other respiratory viruses without laboratory testing, especially in the non-epidemic seasons. this paper outlined the application of the bayesian spatiotemporal model to assess the relative disease risk of influenza at the prefecture level in mainland china. we observed an increased incidence trend of influenza from to that was fairly steady in the first years and increased rapidly in the last years. clusters of prefectures with high relative risk values concerning influenza incidence were identified in the central part of china. the identification of high-risk areas is especially a priority in china because the limited resources available for disease control need to be focused on the places most in need. we hypothesize that the insufficient flu vaccine supplements, low vaccine uptake, the newly emerging influenza a (h n )pdm and expansion of influenza surveillance efforts might be the major causes of the dramatic changes in outbreak and spatiotemporal epidemic patterns. future research with more risk factors at the national and local levels is necessary to explain the changing spatiotemporal patterns of influenza in china. model specifications for spatiotemporal analysis. the besag york mollié (bym) convolution model was used as a baseline model . using the notation of banerjee et al. , the bym model is as follows: • n is the number of areas. the y i counts of influenza cases in area i are independently identically poisson distributed. θ i is the risk for area i. e i is the number of expected cases of influenza in area i, which acts as an offset. • α quantifies the average incidence risk of influenza in all the prefectures. • ν i is a spatially unstructured random effects component that is i.i.d normally distributed with mean zero. • υ i is a spatially structured component using an intrinsic conditional autoregressive structure (icar). the random effect for each area ζ i is thus the sum of a spatially structured component υ i and an unstructured component ν i . it is termed a convolution prior , . the bym model was extended to include a linear term for space-time interaction and a nonparametric spatiotemporal time trend. possible random effects specifications for the temporal term include a linear time trend (β j ), a random time effect (γ j ), a first-order random walk (γ j ), a second-order autoregression (γ j ), etc. . four types of interactions are proposed in knorr-held ( ) , see knorr-held ( ) for a detailed description. in this study, we assume no spatial and temporal structure on the interaction, and therefore, δ ij ∼ normal( ; τ δ ). four candidate models were tested and compared: in model , the space-time interaction is a random effect term and is interpreted as the residual effect after the unstructured, spatially structured and time effects are modelled and represent sporadic short-term outbreaks or clusters. model selection was based on deviance information criteria (dic), which take into consideration the posterior mean deviance, a bayesian measure of model fit, and the complexity of the model. a smaller dic indicates a better fit of the model . the final linear model consisted of an intercept (α); a vector of national-level explanatory variables ∑ β = ( x ) k n k k for the yearly total number of lot release of influenza vaccines by the china food and drug administration, the positive rate of influenza a (h n )pdm among the number of ili specimens processed, the percentage of influenza a (h n )pdm among all the positive influenza specimens, and protocol changes; a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ j ); and an interaction term for time and place (δ ij ). the prefecture-specific structured and unstructured spatial risks of influenza compared to the whole spatial risk of all prefectures are obtained by applying an exponential transformation to the components of ν i and υ i , respectively. the relative risk of space-time interaction is computed by the exponentiation of the term δ ij . the exceedance probabilities of spatial risk and risk of space-time interaction were also calculated. the exceedance probability represents the posterior probabilities for an area's spatial risk estimate exceeding some pre-set value and has been proposed as a bayesian approach to hotspot identification , . all spatial models were computed using integrated nested laplace approximations (inla), which have been developed as a computationally efficient alternative to mcmc . all spatial analyses were conducted within microsoft r open version . using the r-inla package (version . . ). ethics approval. the authors assert that all of the procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and the helsinki declaration of as revised in . this article does not contain any studies of human or animal subjects performed by any of the authors. since this analysis was based on anonymous aggregated statistical data, patient informed consent and ethical committee approval were not required in china. disclaimer. the views expressed are those of the authors and do not 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surveillance data between epidemiological features of and changes in incidence of infectious diseases in china in the first decade after the sars outbreak: an observational trend study. the lancet infectious diseases hierarchical modeling and analysis for spatial data bayesian mapping of disease. markov chain monte carlo in practice bayesian measures of model complexity and fit cluster detection diagnostics for small area health data: with reference to evaluation of local likelihood models space-time bayesian small area disease risk models: development and evaluation with a focus on cluster detection approximate bayesian inference for latent gaussian models by using integrated nested laplace approximations this study was supported by grants from the key joint project for data center of the national natural science j.q. ma. conceived, designed, and supervised the study. y.w. zhang., x.f. wang. and y.f. li. collected and cleaned the data. y.w. zhang. analysed the data and wrote the drafts of the manuscript. j.q. ma. and y.w. zhang. interpreted the findings. all authors read and approved the final manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -g rxygef authors: leinisch, fabian; mariotti, michele; andersen, sofie hagel; lindemose, søren; hägglund, per; møllegaard, niels erik; davies, michael j. title: uv oxidation of cyclic amp receptor protein, a global bacterial gene regulator, decreases dna binding and cleaves dna at specific sites date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: g rxygef uv light is a widely-employed, and environmentally-sensitive bactericide but its mechanism of action is not fully defined. proteins are major chromophores and targets for damage due to their abundance, but the role of proteins in inducing damage to bound dna, and the effects on dna-protein interactions is less well characterized. in e. coli (and other gram-negative bacteria) the cyclic amp receptor protein (crp/cap) regulates more than genes. in this study we show that exposure of isolated dimeric crp-camp to uv modifies specific met, trp, tyr, and pro side-chains, induces inter-protein tyr -tyr cross-links, and decreases dna binding via oxidation of met /pro residues in close proximity at the crp dimer interface. uv exposure also modifies dna-bound camp-crp, with this resulting in dna cleavage at specific g/c residues within the sequence bound to crp, but not at other g/c sites. oxidation also increases crp dissociation from dna. the modifications at the crp dimer interface, and the site-specific dna strand cleavage are proposed to occur via oxidation of two species met residues (met and met , respectively) to reactive persulfoxides that damage neighbouring amino acids and dna bases. these data suggest that modification to crp, and bound dna, contributes to uv sensitivity. . effects of uv irradiation on crp-dna complexes and isolated crp assessed by gel mobility shift assays (a,b) and sds-page (c,d). panel a: gel mobility shift assay of crp-camp complexes ( µm camp) incubated in the dark, or exposed to uv ( min exposure, λ max nm, nm band width, w m − , normoxia), before addition to p-labeled dna in crp binding buffer (see materials and methods). dtt ( mm), nan ( mm) and mannitol ( mm) were present as indicated. lane : no crp; lanes - : crp at . , and nm respectively with no uv exposure; lane - : as lanes - , respectively, but with uv-exposed crp; lanes - : as lanes - , respectively, but with dtt; lanes - : as lanes - , respectively, but with nan ; lanes - : as lanes - , respectively, but with mannitol. panel b. pre-formed crp-camp ( µm)-dna complexes were incubated in the dark, or exposed to uv, in the absence or presence of additives (as panel a). lane : no crp; lanes - : crp at . , . , and nm, respectively, with no uv exposure; lane - : uvexposed crp complex at . , and nm, respectively; lanes - : as lanes - , but with dtt; lanes - : as lanes - , but with nan ; lanes - : as lanes - , but with mannitol. panel c. sds-page of isolated crp ( μm)-camp ( µm) complex incubated in the dark (lane ), or exposed to uv (as panel a) for (lane ) or min (lane ). samples were run under reducing conditions, before visualization with instantblue staining. reactions were carried out in buffer containing: mm tris-hcl (ph . ), mm kcl, . mm mgcl , mm edta, µg ml − bovine serum albumin, . % np- and µg ml − calf thymus dna. panel d. sds-page analysis of crp ( μm)-camp ( µm) complex incubated in the dark, or exposed to uv (as panel a), in the absence or presence of plasmid dna. lane : crp alone, no uv; lane : crp with μg plasmid dna, no uv; lane : crp alone with uv; lanes - : uv-exposed crp with . , and μg plasmid dna, respectively. reactions were carried out in buffer containing: mm tris-hcl (ph . ), mm kcl, . mm mgcl , mm edta, µg ml − bovine serum albumin and . % np- . d o enhanced the loss of the crp band and increased oligomer formation ( supplementary fig. b) , consistent with a role for o . nan decreased oligomer formation particularly when present at high concentration. dtt also decreased oligomer formation but to a lesser extent ( supplementary fig. c ). the presence of the poly-histidine tag on the recombinant protein did not impact on oligomer formation ( supplementary fig. d ). inclusion of plasmid dna in the camp-crp solutions had no effect on crp in the absence of uv (fig. d , lanes versus ), but with uv exposure, the presence of dna enhanced protein oligomerization, and decreased the intensity of the parent crp band (fig. d) . these data indicate that camp-crp is highly susceptible to uv-mediated damage, and that dna enhances this damage. crp-bound to dna gives rise to site specific dna damage when exposed to uv light. experiments were carried out to test whether uv exposure affects dna integrity (as assessed by denaturing gel electrophoresis) when camp-crp is bound at the strong crp binding site on the bp dna fragment. uv exposure of the dna/crp/camp complex resulted in specific strand cleavage ( fig. a , lane and fig. b) , which was not observed when dna alone was exposed to uv light ( fig. a, lanes and fig. b ). incubation of dna/crp/camp complexes in the dark did not induce dna damage (fig. c, lane ) . this crp-and uv-light dependent cleavage occurs at specific g/c base pairs (fig. d) in one of the half-sites, in the position where a strong kink is generated in the dna structure on binding of crp/camp . omission of camp, or denaturing of either the crp or dna before irradiation prevented this dna damage (data not shown). inclusion of mannitol and nan had no effect on the dna cleavage (fig. , lanes and ) , consistent with a localized and specific oxidation. in the absence of dtt, (usually present in the crp binding buffer), both cleavage at the specific g/c pair and limited cleavage at other sites was detected (fig. , lane ) . these data indicate that dna cleavage occurs via two mechanisms: a highly-specific process in the presence of dtt that involves a camp-crp-derived intermediate that reacts at specific nucleotides, and a less-selective and more limited process in the absence of dtt, that . uv exposure alters the chemical composition of crp in the absence and presence of dna. isolated crp-camp complex ( . and μm, respectively) was incubated in the dark, or exposed to uv light (λ max nm, nm band width, w m − , normoxia) for min, before analysis using uplc with pre-column derivatization and fluorescence detection (panels a,b) or lc-ms/ms (panels c-f). panel a: changes in amino acid composition determined by acid-hydrolysis and total amino acid analysis. data are expressed as % modification of the indicated amino acids (positive values indicating loss, negative values indicating formation) relative to the uv exposure control. mean ± sd from independent experiments. panel b: material balance for trp and tyr residues determined by uplc analysis with direct fluorescence detection on acid-hydrolysed uv-exposed crp-camp complex. levels of unmodified parent amino acid, formation of dopa, the total of nfk and kyn (as nfk is converted to kyn during acid hydrolysis) and unknown products (difference to control values) are indicated. mean ± sd from independent experiments. panel c: extent of modification at different amino acids (met, m; pro, p; ser, s; tyr, y; his, h; trp, w) detected by lc-ms/ms for control crp-camp, uv-exposed crp-camp, and preformed crp-camp-dna complex exposed to uv. modifications detected at individual sites (panels d-f) were summed and are expressed as a percentage of the total (native and modified) concentration of the amino acid detected. panels d-f: percentage extents of modification at individual amino acids in the crp sequence (indicated on horizontal axis as position number in sequence). panel d: control crp-www.nature.com/scientificreports www.nature.com/scientificreports/ involves diffusible oxidants that can react at multiple sites. thus, the presence of thiols, as would occur in vivo, limits dna damage to a highly-specific, uv-driven, process involving camp-crp-derived species. characterization of uv-induced modification on crp. oxidative damage to crp was examined using two complementary techniques: total amino acid analysis (uplc with fluorescence detection), and peptide mass mapping using lc-ms/ms, with the former providing data on the overall extents of modification, and the latter data on the sites of oxidation and the identity of the species formed. uplc analysis of native and oxidized crp showed that uv exposure of crp resulted in significant modification (relative to controls) to trp, tyr, ser, met and arg residues (fig. a) . lower extents of modification (< %) were detected at other residues. lys, pro or cys could not be quantified using this method. data for his are not reported, as the -his tag on the expressed crp confounds analysis. a significant increase in methionine sulfoxide, a major met oxidation product, was also detected. trp-and tyr-derived products were identified and quantified using uplc with direct fluorescence detection, with n-formylkynurenine (nfk)/kynurenine (kyn) detected from trp (~ %; quantified as the sum due to acid catalyzed conversion of nfk to kyn), and , -dihydroxyphenylalanine (dopa; ~ . %) detected from tyr (fig. b ). the discrepancy between the parent trp and tyr lost, and products detected ( fig. b ) indicates the formation of additional materials. lc-ms sequence coverage of control and uv-exposed protein was high (~ %; supplementary fig. ), with most of the missing sequence being a single peptide near the n-terminus. q-tof-ms analyses of the uv-exposed samples, showed that met was the most modified ( . %), followed by pro ( . %), ser ( . %) and tyr ( . %) (fig. c ). analysis using an orbitrap-fusion-ms, showed a similar order but slightly higher levels of modification (met, . %; pro, . %; tyr, . %; his, . %). controls showed only low levels of modification (fig. c ). no trp modifications were detected as these residues occur in non-detected peptides ( supplementary fig. ). the decreased extent of modification detected by ms relative to uplc likely reflects the incomplete sequence coverage, and search strategies that use predefined mass changes (m/z + , + , + da); uncharacterized modifications are therefore not detected. q-tof ms analysis of camp-crp-dna complexes exposed to uv light showed a similar pattern of damage, but higher extents of met modification ( . %; fig. c ). the modifications detected at met and pro were m/z + da species, consistent with met sulfoxide and hydroxylation of pro. sequence mapping indicates an uneven distribution of modifications (fig. ) . met was the most heavily modified, followed by met and met , then met and met . low levels of modification at met were also detected using the orbitrap-fusion machine. the data for crp exposed to uv in the presence of dna, showed higher levels of oxidation at met , , and ( fig. e versus f). in the presence of dna, met is the most extensively modified residue ( . %), with this situated very close to the dna strands. in contrast, met , which is also modified to a major extent is positioned close to the dimer interface, and near the camp binding site (fig. ) . the extent of modification at other sites (e.g. met , pro ) was not markedly affected by the presence of dna. the uv-induced crp cross-links ( fig. d ) were characterized using ms with h o labelling (reviewed ). this method provided strong evidence for a di-tyr cross-link between tyr and tyr in a cross-linked peptide (kemilsylnqgdfigelglfeegqer) (kaetlyyivk); this species was not detected in controls. this cross-linked peptide showed the expected − . da mass difference compared to the sum of the parent peptides, and a theoretical mass close to the experimental ( . vs . da; error . ppm). the ms spectrum showed the expected + da mass shift (at m/z . ) for a peptide with two carboxyl termini following h o digestion (fig. a) . ms/ms analysis (fig. b ) revealed multiple fragment ions that retain the cross-link site confirming the identification (b -b for the longer α peptide; y for the shorter β peptide). the distance distribution of the atoms forming this cross-link (fig. c,d) , are consistent with this being inter-protein (i.e. a cross-link between two chains on different dimer molecules), as the average calculated distances between these residues for a putative intra-chain link is . Å (shortest . Å), for an inter-chain link . Å (shortest . Å), values that are much larger than those over which such cross-links are likely to form (cf. data for zero-link chemical cross-linking reagents ). this conclusion is supported by the surface accessibility of these residues in crp structures (pdb structures o t, hif and wc ), and the oligomer formation detected by sds-page. uv light, and particularly uvc, is widely used as an environmentally-sensitive sterilization and bactericidal agent in the health sector, and in water and waste stream disinfection. uvb light, (λ - nm) can initiate direct dna damage and formation of oxidized nucleobases, cyclobutane pyrimidine dimers, and endonuclease-sensitive sites . proteins are also major uv absorbing species in biological systems due to both their abundance ( - mm in mammalian cells; - mm in plasma), and significant uvb absorption bands in the - nm region from trp, tyr and cystine residues [ ] [ ] [ ] [ ] . some proteins have longer wavelength absorptions arising from interaction of trp residues with metal cations or charged side-chains , or the presence of co-factors . the high abundance of proteins makes these major targets for oxidants , with trp, tyr, cys, cystine, his, and met side-chains being particularly prone to modification , , . camp complex. panel e: uv exposed crp-camp. panel f: crp-camp exposed to uv in presence of dna (as above). modifications at the indicated residues are given as the change in m/z (− , − , + , + , + , + , + ) detected by ms for the modifications included in the data searches. the majority of the modifications correspond to m/z + species assigned to the addition of one oxygen atom (formation of an alcohol at pro and tyr; generation of the sulfoxide from met). ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ as crp regulates a large number of transcription units in e. coli , and is a global regulator in other gram negative bacteria, uv-mediated damage to the crp-dna complex may have significant biological effects. the data presented here indicate that isolated camp-crp is sensitive to biologically-relevant uvb and uva wavelengths ( - nm) and doses, with this generating chemical modifications on specific side-chains, and structural expansions of the dimer-interface region from panels b and c, indicating the pro (yellow)-met (red) pairs on the individual monomers and the increased proximity of these species at the dimer interface in the dnabound structure (panel e) compared to the non-bound (panel d). modification at these residues, with conversion of met to the sulfoxide and pro to an alcohol are proposed to result in significant steric and electronic interactions that limit binding of uv-oxidised isolated camp-crp to dna (cf. fig. a) , and dissociation of camp-crp from dna when the bound complex is exposed to uv (cf. fig. b) . in panel e, the trp residues (orange) are also indicated as these residues move closer to the pro -met residues on dna binding. ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ changes (dimer/oligomer formation). significant uv-induced modification was detected at met, trp, tyr, pro, ser and arg. ms peptide mass mapping indicate that specific met, tyr, and pro residues are modified, with large differences in parent loss and % conversion to products between sites. the extent of modification at individual trp sites could not be determined, but the overall extent of trp oxidation was significant, with a proportion of this being nfk and kyn, together with other uncharacterized materials. oxidation at met yielded the sulfoxide, and tyr oxidation yielded both dopa and dityrosine. unusually, significant modification was also detected at ser and pro, with these not being typical uv targets. some of the ser loss may arise from their proximity to the bound camp . products from ser (probably carbonyls) were not detected, as these were not included in the ms modification database. the products from pro were detected as m/z + species, consistent with the formation of alcohols (hydroxypro , ). these chemical modifications are consistent with the observed structural changes. sds-page data indicates significant levels of non-reducible cross-links, supported by the ms detection of a covalent tyr -tyr (dityrosine) link, assigned as an inter-molecular linkage, as the calculated intra-chain and inter-chain distances are too large for such species, unless the protein adopts conformations markedly different to those of free crp, or dna-bound crp. whether dityrosine is the sole type of cross-link is however unclear. the sequence of these two peptides is indicated, together with the site of the di-tyr linkage between the two tyr (y) residues (vertical green line). a number of the detected fragment ions retain this cross-link confirming its location. panels c and d: rendering of the structure of the crp-camp complex (pdb structure: wc ) indicating the relative positions of the tyr and tyr residues both within the individual monomer chains, and between tyr when these are present on different monomer chains. the corresponding vectors for intra-chain (▪) and inter-chain (▪) cross-links are indicated. in panel d, these vectors are plotted as distance distributions for a putative intra-chain cross-link (blue bars: average . Å, . Å shortest) and a putative inter-chain cross-link (red bars: average . Å, shortest . Å). these high shortest and median distances (> Å) imply that the observed cross-links are inter-molecular in nature and occur between monomers in two different dimers. this conclusion is supported by the surface accessibility of the two tyr residues in both the dna-free and dna-bound forms (supplementary fig. ). ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ the binding of crp to dna is camp dependent, with no gel shift detected in the absence of camp. however, uv-induced modification of camp-crp had a marked effect on native camp-crp binding to dna. it is well established that camp-crp binding to dna is associated with structural changes at the dimer interface, with the monomer units rotating relative to each other (fig. b,c) . interestingly, two of the most heavily modified residues, met and pro , are in close proximity both to each other, and to the respective residues on the other monomer, at this interface (fig. b,c) . the distance between the imine nitrogen on pro, and the sulfur on met calculated as . Å. conversion of the met residue to the sulfoxide, or addition of an oh group to the pro ring, is predicted to provide significant adverse steric and electronic interactions between these sites, thereby preventing the rotation and structural changes required for binding of camp-crp to dna. these residues are close to trp , with the distance between c of trp and imine nitrogen of pro ~ . Å in the non-dna bound structure. this proximity rationalizes the high extent of modification at these residues. extensive modification was also detected at these residues when the camp-crp complex was bound to dna before uv exposure. oxidation at these same met and pro residues in the dna-bound complex, and their resulting unfavourable interactions, may promote dissociation of the dimer from the dna and rationalize the decreased binding seen in the gel shift assays (cf. fig. a, lanes versus ) . the modifications detected on both the protein, and also on the bound dna when the camp-crp-dna complex was exposed to uv light, occur in a selective and localized manner. thus, some of the met, tyr and pro residues present in crp were not modified, or only to a limited extent, whereas others are extensively altered (fig. e,f) . a marked selectivity was also detected for dna chain cleavage, with this only detected at g and c residues within the binding dna sequence despite the presence of alternative g/c sites. this dna strand cleavage required both uv light and the protein, pointing to highly-selective protein-mediated events. the requirement for o , and the lack of effect of mannitol, is consistent with a role for o generated via type photochemistry, probably arising from initial light absorption at either camp, or more likely the trp residues. camp absorbs light with λ max ~ nm with a rapid tailing to higher wavelengths and little significant absorption > nm. it should also be noted that crosslinking occurs in the absence of camp. in contrast, trp residues typically have λ max values at ~ nm with a significant tail up to ~ nm. however, λ max for trp residues varies with solvent polarity, ph, metal ion, cations and nearby charged groups; thus some trp residues show absorption bands in the visible region . these data indicate that the trp residues in crp are the likely uv-absorbing chromophores. freely-diffusible o would be expected to react rapidly with all accessible target residues -both on the protein and the dna chains, which is not observed. furthermore, the presence of other materials in the reaction buffers, including dtt, would be expected to remove a large proportion of free-diffusible oxidants. thus, we propose that the selective damage detected at the dimer interface, and also to the bound dna chains, arises from the reaction of some of the , and selective oxidants. reaction of these species with a target results in the formation of met sulfoxide from the persulfoxide, and oxidation of the target. such protein-bound, met-derived persulfoxides formed by o , would therefore rationalize the oxidation at met /pro at the dimer interface, and met and the neighbouring g/c bases in the bound dna. overall, these data indicate that exposure to low levels of uvb and uva light (λ - nm) can result in highly selective and specific damage to camp-crp complexes, which then affects the binding of the complex to its target dna sequence. furthermore uv-induced oxidation of the dna bound complex appears to both promote dissociation of the bound crp, and induce site-specific cleavage of the bound dna. these data may provide a mechanism for damage to gram negative bacteria by uvb light via interference with the critical action of crp in gene transcription in these species. protein purification. the e. coli crp clone (paskacrp jw ) was acquired from the aska (gfp-) collection. the version used contained a his-tag and no gfp. e. coli crp was purified from bl (de ) containing the paskacrp jw plasmid in which the crp coding sequence is cloned under lac promoter control in the his-tag vector pca n . cells were grown in lb medium supplemented with μg ml − chloramphenicol, and crp expression was induced at od . with mm isopropyl-β-d-thiogalactopyranoside. cells from l were then harvested by centrifugation and the pellet frozen overnight at − °c. native crp was purified by resuspending the pellet in lysis buffer ( mm sodium phosphate, mm sodium chloride and mm imidazole supplemented with a protease inhibitor cocktail) followed by sonication at °c. insoluble material was removed by centrifugation ( g, min), and the supernatant was then incubated with nickel-nitriloacetic acid agarose beads for h at °c, with gentle rocking. the agarose beads were then loaded in to a column and washed twice with four column volumes of wash buffer ( mm sodium phosphate, mm sodium chloride and mm imidazole). the crp was then eluted and collected using elution buffer ( mm sodium phosphate, mm nacl and mm imidazole). purified crp protein was then dialyzed twice against l dialysis buffer ( mm sodium phosphate, mm kcl, mm β-mercaptoethanol, ph . ) and stored at − °c. crp purity was assessed using sds-page with coomassie staining, and the protein concentration was determined by the bca method. crp protein lacking the poly-histidine tag was prepared as described previously . amino acid analysis by uplc with fluorescence detection. samples were prepared and subjected to acid hydrolysis using m methanesulfonic acid as previously , . the resulting amino acid mixtures were subjected to pre-column derivatization using o-phthaldialdehyde and separated by uplc with eluted materials detected by fluorescence (λ ex nm, λ em nm). identification and quantification were made versus standards, with the data were normalized to the ala content. detection and quantification of oxidation products by uplc. protein samples were hydrolysed and neutralized as described above, then analyzed as described previously , . samples were injected on to a reversed phase column (phenomenex kinetex evo) and separated by gradient elution. product elution was monitored using fluorescence detector channels parametrized according to the retention times of the products and their fluorescence maxima. data analysis was carried out with shimadzu lab solutions browser software. materials were identified and quantified by comparison with commercial standards. to compensate for any losses during processing, data are expressed relative to parent tyr. mass spectrometric analysis of oxidation products. the detection and quantification of oxidation products was determined on peptides generated by tryptic digestion as described previously [ ] [ ] [ ] . samples ( µg protein) were prepared as described above, then subjected to buffer exchange into mm ammonium bicarbonate buffer (abc buffer) using spin filters ( kda cut-off). reduction and alkylation was carried out using tris( -carboxyethyl)phosphine (tcep, mm) and chloroacetamide ( mm) solution in abc buffer. residual materials were removed by centrifugation, and the samples digested overnight at °c, using trypsin with . % deoxycholic acid. peptides were then collected by centrifugation and the removed by precipitation using formic acid. samples were then analyzed on either a bruker impact ii esi-qtof (bruker daltonics) or an orbitrap fusion mass spectrometer (thermo fisher). for the former separation was carried out using a dionex ultimate chromatography system (thermo fisher) with an aeris peptide xb c column ( cm, . μm particle size, . mm internal diameter). samples were eluted using a solvent gradient system over min, using acetonitrile with . % formic acid and . % dimethyl sulfoxide at a flow rate of μl min − . sampling rate was hz for ms - hz ms (idas, top ), and a scan range of to m/z. for the fusion system, samples were separated on an easy nlc chromatograph using a flow rate of nl min − and gradient of solvents a ( . % trifluoroacetic acid, tfa) and b ( % acetonitrile and . % tfa). data acquisition was carried out with a universal method consisting of a full ms orbitrap scan followed by data-dependent high-energy collisional dissociation ms/ms scans. data analysis was performed using maxquant (version . . . ) , with semi-specific tryptic constraints and a % peptide level false discovery rate. carbamidomethylation of cysteine was used as a fixed modification. data was filtered in order to extract peptide-spectrum matches corresponding to established oxidative modifications using a list of known oxidative modifications , with changes at met, his, tyr and pro as variable modifications , . the % modification at a particular site was estimated using label-free quantification ratios relative to the parent, as determined using maxquant. peaks with changes of > % were evaluated in respect to elution times, isotopic distribution and msms using skyline (version . ) . mass spectrometric analysis of cross-linked peptides. analysis of cross-linked peptides by ms was performed as described previously , . briefly, after tryptic peptides were digested in h o-or h o, they were subjected to solid-phase extraction on activated stagetip c reversed-phase discs, and peptides were then dried down (speedvac concentrator, mins), re-suspended in μl h o and h o water, respectively, and mixed at a : ratio immediately prior to analysis. mass spectrometric analysis was carried out on an orbitrap fusion mass spectrometer (thermo fisher) as described above. data acquisition was performed either with a universal ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ method characterized by a full ms orbitrap scan followed by data-dependent high-energy collisional dissociation (hcd) ms/ms scans, or a data-dependent method where a group of signals with mass shifts of , and da are selected for ms/ms. massai software (univ. of southern denmark, april ) was used to identify and verify cross-linked peptides. the following settings were used: fixed (carbamidomethylation of cys) and variable (met, his, and tyr oxidation) modifications; maximum missed tryptic cleavages; parent mass tolerance ppm; ms/ms peak tolerance . m/z. tyr-tyr, lys-tyr, lys-his, his-his, and arg-his were selected as potential cross-links. rendering of protein structures. protein structures o t and hif (crp with and without dna) were visualized and distance distributions obtained, using molmol using scripts generated in gnu octave , and the pdb nmr structure wc . for distance distributions, all mesomers/chemically equivalent atoms of the coordinate set capable of forming a cross-link were considered, resulting in a sample size of - . histograms were generated using a bin size of . Å. statistics. data are presented as means ± sd from three replicate independent experiments, with errors propagated when data are normalized to another parameter. statistical analysis was carried out using the packages available in excel with p < . taken as significant. the data that support the findings of this study are available from the corresponding author upon reasonable request. regulondb v . : tackling challenges to unify classic and high throughput knowledge of gene regulation in e. coli k- studies of the distribution of escherichia coli camp-receptor protein and rna polymerase along the e. coli chromosome a comprehensive library of dna-binding site matrices for proteins applied to the complete escherichia coli k- genome cyclic adenosine monophosphate receptor: loss of camp-dependent dna binding activity after proteolysis in the presence of cyclic adenosine monophosphate structural studies of protein nucleic-acid interaction -the sources of sequence-specific binding lac dna, rna polymerase and cyclic amp receptor protein, cyclic amp, lac repressor and inducer are essential elements for controlled lac transcription structure of catabolite gene activator protein at . a resolution suggests binding to left-handed b-dna catabolite activator protein: dna binding and transcription activation phylogeny of the bacterial superfamily of crp-fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs transcription activation by catabolite activator protein (cap) the chemical basis of radiation biology excited states and free radicals in biology and medicine singlet oxygen-mediated damage to proteins and its consequences photo-oxidation of proteins protein-bound kynurenine is a photosensitizer of oxidative damage protein oxidation and peroxidation structure of the cap-dna complex at . angstroms resolution: a complete picture of the protein-dna interface identification and characterization of protein cross-links induced by oxidative reactions chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes ultraviolet radiation-mediated damage to cellular dna the color of cation-pi interactions: subtleties of amine-tryptophan interaction energetics allow for radical-like visible absorbance and fluorescence cyclic-amp and bacterial cyclic-amp receptor proteins revisited: adaptation for different ecological niches nonenzymatic hydroxylations of proline and lysine by reduced oxygen derivatives recent advances in the analysis of oxidized proteins persulfoxide: key intermediate in reactions of singlet oxygen with sulfides complete set of orf clones of escherichia coli aska library (a complete set of e. coli k- orf archive): unique resources for biological research scanning calorimetric study of the thermal unfolding of catabolite activator protein from escherichia coli in the absence and presence of cyclic mononucleotides dissecting direct and indirect readout of camp receptor protein dna binding using an inosine and , -diaminopurine in vitro selection system peroxyl radical-and photo-oxidation of glucose -phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues quantification of protein modification by oxidants structural and functional changes in rnase a originating from tyrosine and histidine cross-linking and oxidation induced by singlet oxygen and peroxyl radicals mass-spectrometry-based identification of cross-links in proteins exposed to photo-oxidation and peroxyl radicals using o labeling and optimized tandem mass spectrometry fragmentation unrestricted mass spectrometric data analysis for identification, localization and quantification of oxidative protein modifications maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteomewide protein quantification andromeda: a peptide search engine integrated into the maxquant environment redox proteomics: chemical principles, methodological approaches and biological/ biomedical promises skyline: an open source document editor for creating and analyzing targeted proteomics experiments molmol: a program for display and analysis of macromolecular structures structural basis for camp-mediated allosteric control of the catabolite activator protein the authors are grateful to the novo nordisk foundation (grant: nnf oc to mjd) for financial support. we are grateful to neel louv-jansen for expert technical assistance. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -x.correspondence and requests for materials should be addressed to n.e.m. or m.j.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -fdb az v authors: casalino-matsuda, s. marina; wang, naizhen; ruhoff, peder t.; matsuda, hiroaki; nlend, marie c.; nair, aisha; szleifer, igal; beitel, greg j.; sznajder, jacob i.; sporn, peter h. s. title: hypercapnia alters expression of immune response, nucleosome assembly and lipid metabolism genes in differentiated human bronchial epithelial cells date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: fdb az v hypercapnia, the elevation of co( ) in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases, and is associated with increased risk of mortality. recent studies have shown that hypercapnia adversely affects innate immunity, host defense, lung edema clearance and cell proliferation. airway epithelial dysfunction is a feature of advanced lung disease, but the effect of hypercapnia on airway epithelium is unknown. thus, in the current study we examined the effect of normoxic hypercapnia ( % co( ) for h) vs normocapnia ( % co( )), on global gene expression in differentiated normal human airway epithelial cells. gene expression was assessed on affymetrix microarrays, and subjected to gene ontology analysis for biological process and cluster-network representation. we found that hypercapnia downregulated the expression of genes and upregulated . among these, major gene clusters linked to immune responses and nucleosome assembly were largely downregulated, while lipid metabolism genes were largely upregulated. the overwhelming majority of these genes were not previously known to be regulated by co( ). these changes in gene expression indicate the potential for hypercapnia to impact bronchial epithelial cell function in ways that may contribute to poor clinical outcomes in patients with severe acute or advanced chronic lung diseases. go biological process-associated gene clusters targeted by hypercapnia. major clusters from hypercapnia-downregulated genes are linked to immune response, nucleosome assembly, cell differentiation, oxidation reduction, and ion and lipid transport (fig. ) . clusters from upregulated genes induced by high co (fig. ) involve biological processes related to lipid metabolism, cholesterol biosynthesis, signal transduction, and transport. a number of these important clusters, labelled a-e in figs and , are analyzed in more detail in the following sections. their corresponding gene lists are depicted in figs , , and supplementary fig. . hypercapnia differentially regulates genes associated with innate immunity and nucleosome assembly. cluster a, represented in fig. a , includes hypercapnia-regulated genes involved in signal transduction, immune and inflammatory responses, and leukocyte chemotaxis. notably, tlr , multiple chemokines (ccl , cxcl , cxcl , cxcl , and cxcl ) and the il- receptor gene (il r) were all downregulated by elevated co . on the other hand, the il- receptor like gene (il rl ) was upregulated by hypercapnia. to validate the microarray results related to co -induced changes in key immunoregulatory genes, expression of cxcl , cxcl , ccl , ilr and tlr was also assessed by qpcr. we found that these genes were all downregulated at levels similar to those in the microarray analysis (fig. c) . indeed, the degree of co -induced downregulation of these transcripts assessed by qpcr and microarray was highly correlated (r = . ). in addition, to determine whether downregulation of a key immunoregulatory transcript by hypercapnia was accompanied by a similar change in protein expression, we assessed expression of tlr protein in differentiated nhbe cells. immunofluorescence microscopy ( fig. d ) and immunoblotting (fig. e ) both showed that exposure to % co for h decreased nhbe cell expression of tlr protein. full-length blots are included in supplementary fig. . taken together, these results suggest that hypercapnia would suppress airway epithelial innate immune response to microbial pathogens and other inflammatory stimuli. next, we analyzed cluster b, which includes hypercapnia-regulated genes that codify proteins involved in nucleosome assembly (fig. a) . the heat map in fig. b shows that hypercapnia downregulates genes encoding multiple family members of the core histones h a and h b , as well as the nucleosome assembly protein -like (nap l ), which regulates protein complex assembly, chromosome organization and dna metabolism. the only upregulated gene in cluster b is h f , encoding histone h , which is normally expressed in terminally differentiated and slowly dividing cells. to validate the microarray data from cluster b, we performed qpcr for selected transcripts whose expression was significantly altered in the microarray analysis. figure c shows that expression of the histone genes hist h ac, hist h bd, and hist h bk was downregulated by hypercapnia as assessed by qpcr, again similar to the microarray results. and others), cell surface receptor signaling (egfr, ifnar , il r and others) and apoptosis (bcl l , dapl , sema a and others). the impact of elevated co on expression of these genes would be expected to alter epithelial metabolism and barrier function, as well as innate immune and inflammatory responses. to our knowledge, the present study is the first to investigate the impact of hypercapnia on global gene expression in airway epithelial cells. of importance, we utilized primary nhbe cells cultured at ali to achieve a differentiated state closely resembling normal human bronchial epithelium. our principal finding was that hypercapnia altered expression of a small number of specific genes ( out of , transcripts assayed, or . %) in differentiated nhbe cells. of these, genes ( %) were downregulated, while ( %) were upregulated. thus, the effects of elevated co are highly selective, involving both differential repression and differential activation of specific gene subsets. the overwhelming majority of these genes were not previously known to be regulated by co . furthermore, gene ontogeny analysis showed enrichment of hypercapnia-regulated genes involved in a variety of fundamentally important cellular processes. altering expression of genes related to these processes would be expected to impart functional changes in bronchial epithelial cells, which could in turn influence the pathophysiology and outcomes of many respiratory diseases. our data show that hypercapnia alters expression of multiple components of the innate immune system, including downregulation of the il- receptor (il r); the neutrophil chemokines cxcl , cxcl and cxcl ; the mucosal-associated chemokines ccl and cxcl [ ] [ ] [ ] and importantly tlr . hypercapnia also upregulated cd and cd , which bind virus at the cell surface , . while hypercapnia downregulated tlr , it increased the expression of il rl , which has been shown to inhibit tlr activation defense against multiple respiratory pathogens [ ] [ ] [ ] [ ] [ ] . interestingly, airway epithelial tlr expression was reduced in patients with severe copd as compared to those with less severe copd , possibly due to hypercapnia in patients with advanced disease. reduced expression of immune response genes was also seen in the lungs of newborn mice exposed to moderate hypercapnia ( % co ) for the first two weeks of life . while the immune genes downregulated by hypercapnia in the newborn mice differed from those we found in nhbe cells, the mucosal immunity chemokine cxcl was commonly downregulated in both systems. taken together, these observations indicate that the airway epithelium is an important target for hypercapnic suppression of innate immune gene expression. this, along with the suppressive effects of elevated co on macrophage, neutrophil, alveolar epithelial cell functions [ ] [ ] [ ] [ ] [ ] [ ] likely contributes to the deleterious impact of elevated co on lung injury and host defense. another cluster impacted by hypercapnia includes genes related to nucleosome assembly, which also have antibacterial properties. the nucleosome consists of - base-pair-segments of dna wrapped around a histone octamer containing one (h -h ) tetramer, two h a-h b dimers, and histone chaperones or linkers that facilitate nucleosome assembly . regulation of nucleosome assembly following dna replication, dna repair and gene transcription is critical for the maintenance of genome stability and epigenetic information . within this gene cluster, hypercapnia downregulated transcripts for the core histones h a and h b , the histone chaperone nap l , and the linker histone h . downregulation of histone gene expression can be triggered by dna-damage or indirect inhibition of dna synthesis and might lead to alterations of chromatin structure that would influence transcriptional regulation of many genes and even genome stability . exchange of core histones with histone variants might also alter the chemical nature and physical properties of the nucleosome, thereby affecting distinct cellular processes . in addition, histones h a and h b also can inactivate endotoxin and function as antimicrobial proteins , . we also found that elevated co upregulated nhbe cell expression of cholesterol and fatty acid biosynthesis genes, while downregulating atp-binding cassette (abc) transporters, which promote the efflux of cholesterol and phospholipids from the cell . interestingly, enveloped viruses subvert preexisting lipids for viral entry and trafficking and also reprogram lipid synthesis and lipid distribution in lipid rafts to establish an optimal environment for their replication, assembly and egress . furthermore, host defense against viral infection involves interferon-mediated downregulation of sterol biosynthesis . thus, hypercapnia-induced cholesterol accumulation might contribute to the entry, replication, and shedding of respiratory viruses in the airways. as noted above, in a previous study, we showed that hypercapnia downregulates the tca cycle enzyme idh , resulting in mitochondrial dysfunction and impaired proliferation of fibroblasts and a lung epithelial cells . however, in the current study, hypercapnia did not alter idh expression in nhbe cells, indicating that co -mediated regulation of gene expression is cell-type-specific. on the other hand, a number of genes involved in mitochondrial function were regulated by hypercapnia in nhbe cells. among these, upregulated genes included acyl-coa dehydrogenase short/branched chain (acadsb) and acyl-coa synthetase short chain family member (acss ), which encode enzymes involved in fatty acid synthesis and oxidation . genes downregulated by elevated co included gamma-butyretaine hydroxlase (bbox ), which catalyzes synthesis of l-carnitine, an essential co-factor in beta-oxidation ; kynurenine -monooxygenase (kmo), an outer mitochondrial membrane protein that hydroxylates tryptophan to form kynurenine ; bcl interacting protein (bnip ), a bh domain protein with pro-apoptotic activity ; and mitochondrial assembly of ribosomal large subunit (malsu ), an inhibitor of translation at the mitochondrial ribosome . the diverse activities of these genes indicate the potential for hypercapnia to disrupt multiple mitochondrial functions in nhbe cells. while the current study does not reveal the molecular mechanism(s) underlying hypercapnia-induced changes in gene transcription, other recent work suggests a path to elucidating components of a putative co -induced signaling pathway leading to inhibition of innate immune gene expression and impaired host defense. we previously reported that elevated co inhibits expression of antimicrobial peptide genes and suppresses antibacterial host defense in drosophila , suggesting that the immunosuppressive effect of hypercapnia is evolutionarily conserved. using a genome-wide rnai screen, we identified a small number of genes whose expression is required for co -induced immunosuppression in drosophila cells, and which are conserved in mammalian systems . flies deficient in of one these genes, a zinc finger homeodomain transcription factor known as zfh , were protected from co -induced mortality associated with bacterial infection . this opens up the opportunity to test whether orthologues of zfh and other genes identified in the drosophila screen mediate hypercapnic immunosuppression in mice and ultimately in humans. alterations in expression of innate immune and other genes in airway epithelial cells may be of central importance in the co -induced increase in mortality of pseudomonas pneumonia we previously observed in mice . in addition, the suppressive effect of elevated co on immune gene expression in the airway epithelium, along with similar effects on immune cells, suggest a reason why severe copd and other lung disease associated with hypercapnia all carry a high risk of pulmonary infection. bacterial and viral infections are a principal cause of acute copd exacerbations [ ] [ ] [ ] [ ] , which are linked to the need for hospitalization and to mortality , . thus, co -induced alterations in airway epithelial gene expression may underlie the increase in mortality associated with hypercapnia in advanced copd, as well as community-acquired pneumonia , adenoviral lung infections and cystic fibrosis . it is notable in this regard that reducing hypercapnia with noninvasive ventilatory support has been shown to decrease hospital readmissions and mortality in patients with severe copd , . further investigation of the molecular mechanisms and mediators of co effects on gene expression may reveal targets for pharmacologic intervention to prevent hypercapnic immune suppression in patients with advanced respiratory disease. humans without known lung disease were obtained from a commercial source (lonza). the cells were plated on collagen-coated plastic dishes, grown to confluence in begm tm bronchial epithelial cell growth medium (lonza), and passaged after enzyme dissociation with trypsin . cells from passage- were seeded onto -mm, . μm pore size, polyester, transwell inserts (corning) at . × cells per insert ( . cm ) and cultured in a serum-free medium , comprised of : mixture of bebm (lonza): dmem (mediatech), supplemented with hydrocortisone ( . μg/ml), insulin ( μg/ml), transferrin ( μg/ml), epinephrine ( . μg/ml), triiodothyronine ( . ng/ml), epidermal growth factor ( . ng/ml), retinoic acid ( nm), bovine pituitary extract ( . %), gentamycin ( μg/ml), and amphotericin b ( ng/ml). after the cells reached confluence in submersion culture, the medium above the inserts was removed and the cells were maintained in ali culture for two more weeks, at which point differentiation to a pseudostratified mucociliary epithelium with characteristics of airway epithelium in vivo was established , . differentiation after ∼ wk on ali culture was confirmed by the presence of beating cilia and mucus production, as previously described . culture of nhbe cells up to the point of full differentiation was carried out in an atmosphere of humidified % co / % air at °c. hypercapnia exposure. after differentiation, nhbe cells were cultured in ali for an additional h in humidified % co / % o / % n (hypercapnia) or maintained in humidified % co / % air ( % co / % o / % n ; normocapnia), as control. the growth medium was pre-saturated with appropriate co concentration for h prior to the addition to the cells. the pco and ph of the pre-saturated media were measured using a phox plus blood gas analyzer (nova biomedical corp). for the normocapnia-and hypercapnia-equilibrated media, the pco s were and mmhg, and the corresponding ph values were . and . respectively. to determine whether hypercapnia induces cytotoxicity, lactate dehydrogenase (ldh) release to the apical and basolateral compartments was assessed using a colorimetric cytotoxicity detection kit (roche) according to the manufacturer's instructions. absorbance at nm was measured using a versamax tunable microplate reader (molecular devices). percent ldh release was calculated as the amount of ldh measured in the basolateral supernatant or apical wash divided by the total amount of ldh in the culture (ldh in cell lysates plus that measured in apical and basolateral compartments) times . mini kit (qiagen). quality and quantity of each rna sample were assessed using a bioanalyzer (agilent). rna was hybridized to genechip ® human genome u . plus array (affymetrix). a total of chips, each hybridized to a crna from different normocapnic (n = ) or hypercapnic (n = ) nhbe cell cultures were used in this study. the u . plus arrays contain probes for approximately , transcripts and variants, including over , well characterized human genes. fluorescent images were detected in a genechip ® scanner and expression data were extracted using the genechip operating system v . (affymetrix). assessed by a statistical linear model analysis using the bioconductor package limma , (https://www.bioconductor.org/help/faq/), in which an empirical bayes method is used to moderate the standard errors of the estimated log-fold changes of gene expression. the moderated t-statistic p-values derived from the limma analysis were further adjusted for multiple testing by benjamini and hochberg's method to control false discovery rate scientific reports | ( ) : | doi: . /s - - -x (fdr). many genes whose expression signals were below background were defined as "absent". transcripts absent in all samples were filtered out, leaving , probes corresponding to , genes in the downstream analysis. the lists of differentially expressed genes were obtained by the fdr criteria of < . and fold-change cutoff of > . . differential gene expression in hypercapnia versus normocapnia was depicted in a pie chart, volcano plot of statistical significance (−log p value) plotted against log fold change, and hierarchical clustering by pearson correlation represented as heat maps generated using heatmapper and gene-e (https://software.broadinstitute. org/gene-e/). over representation analysis (ora) of gene ontology (go) terms from biological processes of all genes downregulated or upregulated by hypercapnia were separately analyzed using the gene ontology analysis innatedb tool which utilizes a manually-curated knowledgebase of genes, proteins, interactions and signaling pathways involved in mammalian innate immune responses. results from the innate db analysis were confirmed using genego metacore (thomson reuter), a separately curated database and pathway analysis tool. microarray data have been deposited to the national center for biotechnology information (ncbi) gene expression omnibus (geo; http://www.ncbi.nlm.nih.gov/projects/geo) complied with miame standards (accession number gse ). network ontology analysis. subsequent analysis of global expression changes and ontology network assessment on the differentially selected genes was performed using mathematica ® v . (wolfram research, inc., mathematica, version . , champaign, il ( )). ontology groups were generated using inbuilt genomedata, matching the annotated genes with pre-defined processes and intracellular functions. two approaches were used for analysis of genome wide expression changes: unbiased measurements of intra-network gene expression and fold-change ranked segmentation. unbiased intra-network changes were assessed for cellular processes that contained at least five genes in the post-screen data. mean-fold change, the variance of the fold-change, and pearson correlation of expression were measured for each process. intra-network heterogeneity of relative expression was measured by calculating the standard deviation of the relative expression for genes within any given ontological process. for instance, if a gene was classified as belonging to both "nucleosome assembly" and "signal transduction", it was assigned to both groups and a connection between these processes was indicated. to further understand the impact of hypercapnia-induced differential gene expression, cluster domains of go biological processes containing or more genes and with at least connections were also generated using mathematica ® v . . these processes were broadly grouped based on gene function and by their connections. quantitative taqman real-time rt-pcr. total rna was isolated from nhbe cells and first-strand cdna was generated using multiscribe ™ mulv reverse transcriptase (applied biosystems). the first-strand cdna was used to quantitate the mrna levels by taqman real-time pcr system (applied biosystems). the level of expression of eukaryotic translation elongation factor alpha (eef a ) was used as reference, and fold change of target genes was calculated by the ∆∆ ct method . immunofluorescence staining for tlr . after exposure to normocapnia ( % co ) or hypercapnia ( % co ) for h, differentiated nhbe cells were fixed with ice-cold % acetone/ % methanol for min. cells were blocked in pbs containing % bsa and . % triton x- then double-stained with : polyclonal rabbit anti-human tlr antibody (h- , santa cruz biotechnology) followed by : alexa fluor -conjugated goat-anti-rabbit igg (red) (invitrogen), and : monoclonal mouse anti-human acetylated tubulin antibody (clone - b- , sigma) followed by : alexa fluor -conjugated goat anti-mouse igg (green) (invitrogen). nuclei were identified by staining with µg/ml hoescht (blue) (sigma). images were obtained using a nikon te inverted fluorescence microscope (nikon) equipped with a spot rt monochrome digital camera (diagnostic instruments). all images were captured with the same gain and exposure time using metamorph software. immunoblotting for tlr . after exposure to normocapnia ( % co ) or hypercapnia ( % co ) for h, differentiated nhbe cells were lysed in ripa buffer (santa cruz biotechnology) supplemented with pmsf, sodium orthovanadate and protease inhibitor cocktail. lysate proteins ( μg/well) were resolved by sds/page - % gradient gels and transferred to nitrocellulose (bio-rad laboratories). membranes were probed with polyclonal rabbit anti-human tlr (h- ) antibody followed by hrp-conjugated anti-rabbit secondary antibody (pierce). blots were stripped and re-probed with monoclonal mouse anti-human β-actin (abcam) followed by hrp-conjugated anti-mouse secondary antibody (pierce) to confirm the equal loading. the signals were detected using enhanced chemiluminescence supersignal west dura substrate kit (pierce). tlr /βactin ratios were assessed using imagej . statistical analysis. data are presented as means ± se. differences between two groups were assessed using student's t test. levene's test was used to analyze the homogeneity of variances. significance was accepted at p < . . deaths: preliminary data for severe hypercapnia in critically ill adult cystic fibrosis patients carbon dioxide and the critically ill-too little of a good thing acute respiratory failure in obstructive lung disease. 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suppresses specific drosophila innate immune responses and resistance to bacterial infection identification of drosophila zfh as a mediator of hypercapnic immune regulation by a genome-wide rna interference screen influenza infection and copd importance of viral and bacterial infections in chronic obstructive pulmonary disease exacerbations predicting chronic obstructive pulmonary disease hospitalizations based on concurrent influenza activity infection in the pathogenesis and course of chronic obstructive pulmonary disease standards for the diagnosis and treatment of patients with copd: a summary of the ats/ ers position paper global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease non-invasive positive pressure ventilation for the treatment of severe stable chronic obstructive pulmonary disease: a prospective, multicentre, randomised, controlled clinical trial. the lancet effect of home noninvasive ventilation with oxygen therapy vs oxygen therapy alone on hospital readmission or death after an acute copd exacerbation: a randomized clinical trial mucin gene expression during differentiation of human airway epithelia in vitro. muc and muc b are strongly induced mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells characterization of mucins from cultured normal human tracheobronchial epithelial cells epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium bioconductor: open software development for computational biology and bioinformatics limma powers differential expression analyses for rna-sequencing and microarray studies controlling the false discovery rate: a practical and powerful approach to multiple testing heatmapper: web-enabled heat mapping for all innatedb: systems biology of innate immunity and beyond-recent updates and continuing curation analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method nih image to imagej: years of image analysis contributed reagents or analytic tools this work was supported by national institutes of health grants r hl , r hl , r hl and r hl ; and by a merit review from the department of veterans affairs. supplementary information accompanies this paper at https://doi.org/ . /s - - -x.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -tfp idq authors: hale, alison c.; sánchez-vizcaíno, fernando; rowlingson, barry; radford, alan d.; giorgi, emanuele; o’brien, sarah j.; diggle, peter j. title: a real-time spatio-temporal syndromic surveillance system with application to small companion animals date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: tfp idq lack of disease surveillance in small companion animals worldwide has contributed to a deficit in our ability to detect and respond to outbreaks. in this paper we describe the first real-time syndromic surveillance system that conducts integrated spatio-temporal analysis of data from a national network of veterinary premises for the early detection of disease outbreaks in small animals. we illustrate the system’s performance using data relating to gastrointestinal disease in dogs and cats. the data consist of approximately one million electronic health records for dogs and cats, collected from uk veterinary premises between march and . for this illustration, the system predicts the relative reporting rate of gastrointestinal disease amongst all presentations, and updates its predictions as new data accrue. the system was able to detect simulated outbreaks of varying spatial geometry, extent and severity. the system is flexible: it generates outcomes that are easily interpretable; the user can set their own outbreak detection thresholds. the system provides the foundation for prompt detection and control of health threats in companion animals. in the uk through surveillance schemes such as the small animal veterinary surveillance network (savsnet) . savsnet harnesses the growing volume of patient electronic health records (ehrs) available from small animal practices and complementary data from diagnostic laboratories to improve animal and human health through rapid and actionable research and surveillance. here we propose a real-time syndromic surveillance system that uses a spatio-temporal model in conjunction with bayesian inference for the early detection of health-event outbreaks. specifically, we use a markov chain monte carlo (mcmc) algorithm to generate samples from the bayesian predictive distribution of the underlying spatio-temporal surface. these samples are then used to compute predictive probabilities at given thresholds; a high predictive probability at a particular location and time gives an early warning of a possible disease outbreak. the system provides end-users (i.e. practising veterinary surgeons) decision-support tools for immediate analysis and easy interpretation of their data. as an example, we apply our model to small companion animal ehrs collected over two years by savsnet from a large network of uk veterinary premises. we illustrate the feasibility of our proposed surveillance system using gastrointestinal (gi) disease in dogs and cats as an example. gastrointestinal (gi) disease is one of the four syndromes for which savsnet currently gathers information for every consultation it receives. gi disease affects animal welfare, can be expensive to manage and may be transmissible to other pets or, more rarely, to people . current approaches to preventing and controlling gi disease in companion animals have focussed on individuals or small groups of animals. this seems to have had little impact on gi disease, which remains one of the commonest reasons for presenting for veterinary care in the uk , , - , although precise data to confirm this has been lacking. a more coordinated population-scale approach to gi disease surveillance in companion animals is needed. this paper focuses on the early detection of a gi disease outbreak, which we define as an unexplained, spatially and temporally localised increase in the fraction of gi consultations amongst all consultations. we illustrate the performance of our proposed surveillance system on simulated gi disease outbreaks of varying spatial extent and severity. this is, to our knowledge, the first surveillance system that conducts integrated spatio-temporal analysis of data from a national network of veterinary practices so as to enable real-time detection of spatially and temporally localised changes in reporting patterns across the network. the paper is structured as follows. first, we give details of the savsnet and socioeconomic data used in this paper. we then give the rationale for our methodological approach, describe the spatio-temporal stochastic model that is the foundation of our surveillance system, and report the results of fitting our model to our savsnet-acquired data. we then simulate spatio-temporal gi outbreaks by perturbing the actual savsnet data in various ways to demonstrate the ability of the surveillance system to achieve timely outbreak-detection. finally, we discuss the similarities and differences between our proposed system and other approaches in the literature, and also extensions for joint human and veterinary surveillance. savsnet. data collection. data were collected electronically in near-real-time from volunteer veterinary premises or sites using a compatible version of the practice management system (pms) namely robovet (vetsolutions, edinburgh) and teleos systems ltd (birmingham). this study used data for dogs and cats collected over the period between st march and th february . in our analysis we included data from an increasing number of premises as they enrolled in the robovet and teleos systems. by th february we had data from practices (amounting to a total of distinct premises) located in england, wales and scotland. the data were extracted from consultations where a booked appointment was made to see a veterinary surgeon or nurse, including out-of-hours consultations. through the savsnet system a compulsory, single-question questionnaire is appended at the end of each consultation allowing the attending veterinary surgeon or nurse to categorise the main reason for the animal's presentation into syndromes (currently gi disease, respiratory disease, pruritus and renal disease) or other routine veterinary interventions (i.e., trauma, neoplasia, 'other sick' , vaccination, 'other healthy' or post-operative check-up). specifically, the definition provided to participating veterinary surgeons to categorise the animal presentation as gi disease is that the main reason for the animal's presentation are signs including but not limited to diarrhoea, vomiting, weight loss and poor appetite. a full description of the savsnet data collection protocol has been described by sánchez-vizcaíno et al. . the data for this study were gathered on a consultation-by-consultation basis, and include the date the animal was seen, unique identifiers for practice, premise and animal, the animal description (including species, breed, sex and date of birth), the syndromic level classification and the full postcode of each veterinary premise and pet owner. data were only gathered if the owner had not opted out of study participation. the collection and use of these data were approved by the university of liverpool's research ethics committee (reth ); as such all collection and use of these data were performed in accordance with the relevant guidelines and regulations. data management. text-based data for species and breed were cleaned to deal with misspellings or the use of non-standard terms by mapping to standard terms. a full description of this cleaning procedure has been described elsewhere . many breeds were present in the data set, some represented by only a few individuals, limiting the scope for analysis by breed. thus, for the purposes of this study, only the animal's classification as purebred or crossbred was used. to identify localised outbreaks we needed to geocode all postcodes. the text-based data for each owner's full postcode were automatically cleaned by applying mapping rules of typical misspellings (e.g. letter 'o' instead of zero). any remaining records containing erroneous postcodes were discarded from our outbreak prediction as they could not be geocoded. similarly, if the age of the animal was recorded outside the range to years then the record was excluded. savsnet records with missing data were removed before the analysis. if an animal attended a veterinary premise on more than one occasion during the study period we included all attendances outbreak detection modelling rationale. as noted earlier, we define an outbreak as an unexplained spatially and temporally localised increase in the fraction of gi consultations amongst all consultations. the term "unexplained" refers to the fact that, for reasons that are well understood, some areas or times of year will experience higher fractions of gi consultations than others because of spatial variation in the local population susceptibility or temporal variation in the region-wide susceptibility to gi. we adjust for these known effects using measured explanatory variables, as described below in the section on explanatory variable selection. we then equate "unexplained" to "stochastic" and include this in our model as a latent, spatially and temporally correlated process s i,t , where i denotes premise and t denotes time, in days. by definition, the expected value of each s i,t is zero, and our goal is to determine where and when its actual value is materially greater than zero. note that the natural pattern of gi consultations will always be subject to fluctuations in time and space that cannot be explained fully by measured variables. it follows that outbreak detection is not a statistical hypothesis-testing problem. our approach acknowledges this by the fact that the actual value of s i,t will never be exactly zero. our formal solution is therefore to calculate, for each premise i and day t, the predictive probability q (i.e. the probability conditional on all available data up to and including day t) that s i,t > l, where l is a user-specified threshold representing an effect large enough to be of practical concern. we then declare an outbreak affecting premise i if this probability exceeds q , the required positive predictive value per premise, say q = . or . . as with any prediction problem using observational data, it is not possible simultaneously to control both the positive and negative predictive probabilities. prediction model. to accommodate the spatial and temporal correlations that would characterise an outbreak of gi disease, we use a spatio-temporal mixed effects regression model, and fit the model using bayesian inference. we define our binary response variable y j,it to take the value if the j th consultation at the i th premise on day t is a gi disease presentation and otherwise. conditionally on an unobserved, spatio-temporally structured random effect s i,t , the y j,i,t are distributed as mutually independent bernoulli variables with probabilities p j,i,t defined by is the quantile function of the standard normal distribution. the vector d j,i,t denotes the set of explanatory variables and θ their associated regression parameters. we discuss selection of explanatory variables, d j,i,t , below. the spatio-temporally structured collection of random effects for all premises and days is written as , and we denote by τ and n, respectively, the total numbers of days and premises contained in the data-set. the complete vector s follows a multivariate normal distribution with mean zero and covariance matrix that incorporates the spatio-temporal context of the data. specifically, we assume that, conditionally on its past, s (t) follows a multivariate gaussian distribution with mean vector ϕ − s t ( ) and spatial covariance matrix Ω, which we construct as follows. firstly, we associate with premise i a polygon consisting of all points closer to premise i than to any other premise; the resulting polygons, v i are called voronoi polygons. secondly, we define the neighbours of i to be the set n(i) of premises whose voronoi polygons are contiguous with v i . finally, we define distance-decay weights where u ik is the distance between premises i and k, and δ is a scaling parameter with units of distance. we then specify the conditional distribution of each s i,t given all other s k,t to be normal with mean ρm it where . together, these modelling assumptions imply that the so-called full conditional distributions of the s i,t that together determine the joint distribution of s are of the form using these full conditional distributions, we can simulate from the bayesian predictive distribution of the random effects s i,t using an mcmc algorithm based on auxiliary variable techniques as described in section . of rue & held . our system is intended to be run in near-real-time, but the mcmc computations eventually become prohibitive as the time-span of the data, τ, grows. to counteract this, we run the mcmc algorithm on a moving nine-day window, which is long enough to capture the temporal correlation in our data; the magnitude of the within-premise autocorrelation of s i,t for a time lag of eight days is typically around . . over a time-window of this size, the effects of any systematic time-trend or seasonal effect on the fraction of gi consultations are negligible, which removes the need to include these as explicit terms in the model; see also section below on selection of explanatory variables. we adopt the following set of mutually independent priors for the model parameters: θ ~ mvn ( , i); log σ ~ n (− , ); ρ ~ uniform ( , ); ϕ ~ uniform ( , ); δ ~ uniform { , , …, } these were chosen to be vague, in the sense that they have little influence on the predictive inferences for the random effects s i,t that constitute the primary goal of the analysis. however, if inferences about the model parameters are required, samples from their bayesian joint posterior distribution are produced automatically as a by-product of the mcmc algorithm. outbreak detection. let e i,t denote the exceedence probability for premise i on day t, i.e. the probability that s i,t > l conditional on all available data up to and including day t, where l is the user-specified threshold value. to calculate the e i,t , we generate m posterior samples s s , , from the joint predictive distribution of the random effects s i,t using an mcmc algorithm, and calculate and otherwise. for this calculation to be accurate, we need the mcmc algorithm first to run for a sufficiently long time, called the burn-in period, to have reached convergence and then for a further m iterations to feed eq. ( ), where m is sufficiently large that the sampling error on the right-hand-side of ( ) is negligible. we used a burn-in period of , iterations, followed by m = , iterations. the spatio-temporal model was fitted using the r package 'caramellar' www.nature.com/scientificreports www.nature.com/scientificreports/ hence, they do not take account of spatial and/or temporal correlation. nevertheless, we can use a standard probit regression model to establish whether there is a prima-facie case for including each explanatory variable in our outbreak prediction model, eq. ( ), using the following rule. we retained an explanatory variable if its effect was nominally significant at the conventional % level. this inclusion rule is conservative in the sense that in the presence of spatial or temporal correlation the standard probit regression analysis is likely to over-state the significance of individual regression effects. for both species, this led us to discard the explanatory variables pet insurance, micro-chipping and neutering status and to retain the following: • the three-level factor 'country' for the pet owner's home address (i.e. england, scotland or wales); • the two-level factor 'weekday' with values and indicating if the consultation date is a weekend day (saturday, sunday or public holiday) or a working weekday (monday to friday), respectively -we considered using day of the week as a factor on levels, but this did not improve the fit significantly using a likelihood ratio (deviance difference) test; • the two-level factor 'gender' with values and corresponding to 'female' and 'male' , respectively; • the two-level factor 'purebred' with values and corresponding to crossbred or purebred, respectively; • the continuous variable ' age' denoting the animal's age, in years and age = age × age, both included because the quadratic term improves the model fit; • the continuous variable 'imd' , is the rescaled deprivation measure relating to the pet owner's home address (as described above in our section on data sources). as noted earlier, fitting the model to moving nine-day windows of data removes any long-term trend or seasonal effects. the resulting provisional glm is where p denotes the probability that a presentation of a dog or cat (depending on the species evaluated) to a savsnet veterinary premise is recorded as a gi disease consultation. the first two terms on the right-hand side of eq. ( ) capture the interaction between country and imd, so as to account for the fact that the three countries use different imd measures, whilst θ θ θ … , , , are regression parameters for the remaining explanatory variables in the model. the glm outputs for dogs and cats can be found as supplementary tables s and s , respectively. all computation was carried out using r version . . . our model's ability to identify an outbreak, i.e. its sensitivity, is influenced by factors including the outbreak's duration, spatial extent and the number of infected animals presenting at premises in the locality. in each of our simulations, we construct an outbreak by adding varying numbers of aberrant gi disease to the actual (baseline) savsnet-recorded cases in a specified set of premises over a specified number of consecutive days. simulation model. we use the actual savsnet total consultations for dogs during february , together with their associated explanatory variables, to simulate a step increase in the proportion of gi disease cases affecting one or more premises from a given day t , corresponding to february , by augmenting eq. ( ) with an extra term as follows ( ) where the indicator function i i for premise i has value for premise i and all days ≥ t t if premise i is affected by the outbreak, and has value otherwise. by varying the value of γ we can control the probability of a gi case at an affected premise. for each simulation, we proceed as follows: ( ) use the actual savsnet consultations during february to fit the no-outbreak model using eq. ( ) and to generate simulated realisations of s i,t ; ( ) for t t ≥ , use the actual explanatory variables and the simulated s i,t to compute p j,i,t using eq. ( ) with γ > ; ( ) use the computed values of p j,i,t to simulate case and control flags ( or respectively) and use these to reassign each actual savsnet data consultation as either a case or control. see supplementary material for detailed r-code. simulation scenarios. we applied our simulation model to three sets of premises, which we selected based on their numbers of neighbours, defined to be other premises within an km radius, with the additional constraint that none of the sets of premises were within each other's km radius. the selected sets of premises, which we designated as dense, medium and sparse, had , and neighbours, respectively. the savsnet data gave no indication that these selected premises are atypical or that they experienced a genuine outbreak during the top row of timeseries plots is the 'baseline' , that is the actual savsnet data without any simulated outbreak i.e. γ = . the subsequent rows from top to bottom depict increasing severities of simulated outbreak labelled according the probability of a case at premise i e.g. p = and so on. the columns, from left to right, relate to the density of the region; 'sparse' , 'medium' and 'dense' respectively. for each simulation we plot the timeseries of the predicted distribution of s i,t for premise i. in each time timeseries the solid black line is the predicted value of s i,t , shaded areas are pointwise %, % and % predictive intervals. as an aid to rapid interpretation, we use a traffic-light system: if the predictive probability, q, is above . (defined as 'very high') the light shows red, if above . ('high') orange, if above . (medium) yellow, otherwise ('low') green (no outbreak). the outbreak commences on th february. the more intense the outbreak is the more the traffic light system tends towards red. www.nature.com/scientificreports www.nature.com/scientificreports/ during february ( , and for dense, medium and sparse, respectively) and similar proportions of gi consultations ( . , . and . for dense, medium and sparse, respectively). using these three sets of premises, we simulated under different scenarios as follows. performance evaluation. we use each scenario to generate a simulated set of consultations for february , to which we fit our model using eq. ( ). to assess the capability of our model to detect outbreaks we then use the predictive distribution s i,t from which we compute summary statistics, including exceedence probabilities and times to detection. we set the positive predictive value of the system at q = . . we set values of the reporting threshold at l = , . and . . note that l = corresponds to an observed pattern exactly equal to expectation and is analogous to, although formally different from, using statistical rather than clinical significance in hypothesis testing. we do not recommend using l = in practice, but use it here only as a benchmark to compare the system's performance under different scenarios. in a genuine application, the threshold value l would be chosen to represent a clinically significant increase in reporting rate, and the positive predictive value q to balance sensitivity against specificity. note, in this context, that because s i,t is measured on the probit scale, the increase in the fraction of gi cases corresponding to a fixed increase in s i,t necessarily depends on the baseline fraction. for example, if the expected fraction is . , which corresponds to setting θ = d and s i,t = in eq. ( ), then a log( ) threshold for s i,t represents a fraction log( ) = . i.e. an increase of . . in contrast, for a baseline fraction . , a log( ) threshold now represents a fraction . , i.e. an increase of . . simulation results. for each of the three regions (sparse, medium, dense) we ran our model a hundred times on the baseline data, where each run had a different random seed; we did not detect any false-positives with l = . given the february baseline data, in table we report the credible intervals of the regression parameters estimated from the outbreak detection model's mcmc samples. our model detected a simulated outbreak in out of the outbreak scenarios when the reporting threshold was set at l = ( table ) . the model detected an outbreak on the first day of its actual onset in six scenarios, one day after onset in a further seven scenarios and two days after onset in a further one scenario (table ) . alerting timeliness was inversely related to outbreak severity (table ) . figures and give a more detailed illustration of the performance of our outbreak detection methodology in response to a step change in the proportion of cases, for schemes and respectively and with the threshold value l = . these figures also illustrate the use of a traffic-light system whereby, rather than fixing a single value for the positive predictive probability, q, we report a categorised value of the exceedence probabilities at each premise on each day to indicate the strength of the evidence for an outbreak. we focus on the sparse and dense sets of premises since the central premises of these two sets had almost identical numbers of consultations. recall that under scheme the outbreak affects only the central premise of each set. also, the prediction algorithm exploits the estimated spatial correlation amongst the fractions of gi cases at different premises. as a consequence, the system is better able to detect an outbreak at a single premise when this premise does not have close 'outbreak-free' neighbours whose fractions of gi cases are as expected. in effect, the model smooths its predictions over a range corresponding to its estimated correlation range; fig. shows an example of this phenomenon. this explains why, under scheme (fig. ) , the system delivers a stronger detection signal for the sparse than for the dense set. under scheme (fig. ) , the results for the sparse and dense sets are more similar. also, because the outbreak affects more premises in the medium, and dense sets, their results show generally stronger detection signals than in scheme , as indicated by the increased number of traffic-lights tending towards red in fig. compared with fig. . results of our model's performance using the reporting thresholds l = . and l = . are available in the supplementary files; see table s and figs s and s , and table s and figs s and s , respectively. for example, given scheme (density sparse and p = . ) then: with l = we detect an outbreak over the period to february (see fig. ); with l = . we also detect an outbreak, albeit less strongly, over the period to february (see fig. s in supplementary material); with l = . we do not detect the outbreak (see fig. s ). an increase in the reporting threshold value l necessarily reduces the probability that an outbreak will be declared and increases its time to detection (tables s and s , figs s -s ). this emphasises that the choice of l must be made in context and is unrelated to the inherent quality of the outbreak detection algorithm. setting the probability of a case to . and with l = , the model's performance was compared with similar models in the sparse, medium and dense regions: , . all the variation is accounted for by the latent term s i,t so in a real-world application this model would be more prone to false-positives; in the context of scheme our www.nature.com/scientificreports www.nature.com/scientificreports/ simulations showed this model to be more sensitive. comparing this model with the full model (eq. ) we find they are identical in terms of timeliness but the model without covariates shows more strength of the evidence for the outbreak in that the exceedence probabilities are higher overall. (b) model without spatial correlation -scheme . in the presence of the outbreak only occurring at the central premise we found this model to be more sensitive at detecting outbreaks since the surrounding premises will not influence, and hence reduce, the inferred effects of the outbreak at the single central premise. compared with the full model (with spatial correlation) we find this model to be identical in terms of timeliness for the sparse and dense regions, but the outbreak is now detected in the medium region with a one-day lag. overall, the exceedence probabilities are higher in all regions. (c) model without spatial correlation -scheme . with the outbreak spread over the neighbouring premises, this model was less sensitive as the neighbours did not influence, and therefore support, the detection of the outbreak. in particular we did not detect the outbreak in the medium and dense regions. syndromic surveillance systems offer the opportunity to enhance the public and animal health community's ability to detect, and respond quickly to, disease outbreaks . the last decade has seen a growth in the field of disease surveillance in companion animals, notably in the uk , and in the usa , . however, to the best of our knowledge, this is the first surveillance system that conducts integrated spatio-temporal analysis of data from a national network of veterinary practices so as to enable real-time detection of spatially and temporally localised changes in reporting rate patterns across the network. we have illustrated the applicability of our proposed surveillance system using gastrointestinal disease syndrome in dogs and cats as an example. the system is fed with electronic health records (ehrs) collected in real-time through savsnet from volunteer veterinary premises across the uk. we applied our system to simulated gi disease outbreaks of varying spatial extent and severity, amongst which the system was able to detect of the . had these been real outbreaks, the proposed surveillance system would have triggered timely investigations, which ultimately would have aided control strategies. the system requires the user to specify a reporting threshold corresponding to an increase in case incidence (reporting rate) that would be considered large enough to be of practical importance. given this reporting threshold, the system delivers the predictive probability, q, at each location (here, veterinary premise), that the threshold is currently exceeded. declaring an outbreak when this probability is greater than a specified value q is equivalent to fixing the positive predictive value of the system (per location, per day) at q . alternatively, reporting the actual value of q gives an indication of the strength of evidence for an outbreak. increasing the value of the reporting threshold, l, necessarily reduces the value of q and consequently increases the average time to detection of an outbreak at a fixed value of q . a critical component of a syndromic surveillance system is the application of optimal disease aberration detection methods. most of the methods used in veterinary and public health surveillance systems are concerned with detecting disease-outbreaks and health-related threats in time rather than in space [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, disease incidences vary naturally in both space and time. thus, for example, these techniques may be late at detecting outbreaks that start locally when the surveillance region is large . in contrast, our proposed method has the advantage of being able to directly incorporate data for each individual animal's consultation, including the date of the visit and the location of the pet's owner. in temporal aberration detection algorithms, explanatory variables such as seasonality and day-of-the-week effects would generally be incorporated, but most of these methods cannot easily include individual-level explanatory variables. www.nature.com/scientificreports www.nature.com/scientificreports/ earlier spatio-temporal aberration detection methods have been introduced by rogerson , . however, these approaches lack measures of uncertainty associated with the identified clusters and are unable to account for covariate information. also, they are based on an assessment of global pattern change throughout the fig. . and . the top and bottom rows relate to the density of the region, 'sparse' and 'dense' , respectively, and the left and right columns relate to simulation schemes and respectively. the simulated probability of a case at the premise in the centre of each region is p = . . to aid interpretation, we use the traffic-light system described in fig. caption, as such each coloured circle on the map is derived from the predicted distribution of s i,t at each corresponding premise. panels (a,c) show when the central premise has neighbours who are not experiencing an outbreak it is less able to detect the outbreak, panel (c), when compared to a premise without neighbours, panel (a). if the neighbours also experience an outbreak the system is then better able to detect this outbreak at central premise, panel (d), compared with when the neighbours did not experience an outbreak, panel (c). www.nature.com/scientificreports www.nature.com/scientificreports/ geographical area under study, as opposed to our method, which is used to detect the specific geographical location of an outbreak. prospective space-time scan statistics have also been used in syndromic surveillance systems for the early detection of disease outbreaks , . the space-time permutation scan statistic uses only case numbers, with no need for population-at-risk data and, in contrast to rogerson's methods, does operate locally in both space and time. this method may therefore be suitable for setting up surveillance systems in the small animal sector where only case numbers are available. however, it does not acknowledge the uncertainty associated with any identified clusters, cannot easily incorporate continuous covariates, and can only detect outbreaks characterised by excess cases within a specified, regular shaped affected area, for example a circle or ellipse. also, in our context the number of veterinary premises participating in savsnet can change over time due to the ongoing process of recruiting new premises and/or as a result of premises that could potentially stop being part of the project. this can lead to biased results if a space-time permutation model is used, as the method cannot distinguish an increase in cases due to a local population increase versus an increase in disease risk. our spatio-temporal model, in conjunction with a bayesian inferential framework, takes account of all sources of uncertainty in both parameter estimation and prediction, and is able to accommodate spatial, temporal and individual-level covariate information. other examples of bayesian approaches include markov models , bayesian information fusion networks and bayesian hierarchical models [ ] [ ] [ ] . an earlier near-real-time syndromic surveillance system in small animals has been developed in the usa utilising ehrs from a similar network of primary care veterinary hospitals . briefly, in this approach the daily proportion of patients with a given clinical or laboratory finding was contrasted with an equivalent average proportion from a historical comparison period allowing construction of the proportionate diagnostic outcome ratio (pdor) . our surveillance system builds upon a similar epidemiological metric by modelling the spatio-temporal reporting rate of gi disease in dogs and cats as a proportion of all presentations. the two approaches use different inferential methods: the us study uses confidence intervals for recognising aberrant health events, whilst our approach uses predictive probabilities of exceeding policy-relevant thresholds. a more important difference is that we use a bespoke model that incorporates spatio-temporal covariance structure, with the aim of detecting outbreaks that are spatially and temporally localised without imposing any artificial assumptions on the geometrical shape of an outbreak or the extent of spatial correlation in disease incidence. our inferential paradigm of predictive inference within a generalized linear mixed model could equally be applied in purely temporal surveillance settings where the aim is the timely detection of area-wide increases in reporting rate, but in that context we cannot claim the same level of novelty. another usa study explored the feasibility of using veterinary laboratory test orders as one of the data sources for syndromic surveillance in companion animals . the inherent biases associated with the use of laboratory data in veterinary medicine have been described elsewhere , [ ] [ ] [ ] . however, the results derived from shaffer et al. demonstrated the stability and timely availability of test order data for companion animals and the potential of using these data as a basis for outbreak detection. in addition to ehrs from veterinary practices, savsnet also receives routine downloads of diagnostic test results from commercial diagnostic laboratories throughout the uk . although laboratory test results are less timely than test orders, future research is warranted to explore whether the former data could be used to enhance the real-time syndromic surveillance system described here, which is based on real-time data from consultations in small animal premises. raising the reporting threshold, l, and/or the required positive predictive probability, q , increases the specificity of the system at the cost of reducing its sensitivity, and conversely. in our analysis of the simulated outbreaks, we chose different reporting thresholds to illustrate the performance of our system. however, in any substantive application, the specified reporting threshold can and should be adjusted so as best to reflect end-users' (i.e. veterinary surgeons in practice) preferred balance between sensitivity and specificity. a pragmatic choice would be to set the threshold to some proportion above the historic average at each premise. end-users (hereafter "analysts") of a real-time surveillance system will be responsible for receiving system outputs, interpreting them, and if necessary following up on alarms. therefore, in addition to flexibility, another important attribute of a surveillance system should be that it reports outcomes in an easily interpretable manner. our system generates outputs in the form of practice-specific time-series and maps that display the spatio-temporal evolution of gi disease risk over an area of interest in a user-friendly manner; see fig. . additionally, we have illustrated the use of a traffic-light device as a visual aid for analysts to quickly identify potential gi disease outbreaks on a given day at their own premises. the traffic-light device is based on predictive probabilities for exceedence of reporting thresholds that can be tailored to the analysts' needs. we intend to integrate our daily model-based predictions into the savsnet system so as to make them available to each participating premise through their savsnet web interface. this implementation will include the other two syndromes with outbreak potential that are currently recorded by savsnet (respiratory disease and pruritus). this syndromic surveillance system should be a step towards facilitating the prompt detection and control of health threats in companion animals throughout the uk. in addition, the identified temporal and geographical trends in specific syndromes can be a valuable contribution to the evidence-base when veterinarians are deciding how to treat individual animals in their practice. one of the challenges of conducting epidemiological studies in the small animal sector is that information about the population-at-risk (in our study defined as the overall population of small animals across the uk or target population) is generally lacking. this makes it impossible to measure parameters typically used in human health surveillance systems, such as the average incidence in a day or period of days. other methods must therefore be employed to approximate, for instance, an incidence rate ratio. evidence suggests that in countries with developed pet industries, a high proportion of owned pet animals (pets who may approximate the target population) attend a veterinary surgeon , . therefore, although no single data source can detect all outbreaks that may occur in companion animal populations, ehrs of the kind that are extensively collected from veterinary practices ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ in many developed countries may be the best available source to include in surveillance activities for increasing our capabilities to detect those outbreaks that result from both endemic and potential emerging pathogens. one limitation of this study is that the veterinary practices contributing data to our system were selected by convenience, based on their use of a compatible version of pms, and recruited on the basis of their willingness to take part in the savsnet project. hence, the data used in our system might not be representative of the source population (in our study defined as the overall veterinary-visiting population across the uk). for this reason, we aimed to develop a syndromic surveillance system to detect changes in the relative, rather than absolute, incidence of gi disease presentations in the small animal veterinary premises participating in savsnet. nevertheless, the practices included in the current study were widely distributed around the uk and represented . % of those practices that constituted the source population in . thus, the number and geographical extent of savsnet-participating practices is such that changes in the relative risk of gi disease in this large network of premises can act as a proxy for changes in the level of gi disease in the wider source population. a further limitation relates to missing data. over the spatial domain and time-period of the simulation we found that % of consultations do not record location and % do not record breed. as a result, in total about % of the data are discarded due to incomplete data, our methodology assumes that these data are missing completely at random so that there is no inherent bias in the spatial distribution of the available data. another limitation is that each animal was classified only by its breed-status (purebred or crossbred). as such, we were unable to adjust for breed-specific phenotypes that could have an impact on the incidence of gi disease presentations. however, overall the breed distribution in our study population is consistent with previous studies. labrador retriever was the most common dog breed in our population as it is in earlier studies , , . also, nineteen out of the top twenty-six dog breeds in our study population were also in the top twenty breeds listed by the kennel club . in future work we aim to identify additional means by which breeds can be effectively summarised according to both shared genotype and phenotype. we are aware that the detection of a high relative risk for gi disease could trigger a false alarm if it is due to a localised decrease in the incidence of diagnosing other syndrome/s and routine veterinary interventions, leading to a higher than expected fraction of gi disease consultations. conversely, a localised increase in the incidence of diagnosing other syndromes could conceal a genuine gi disease outbreak. if the goal is to detect anomalous patterns of absolute incidence rather than relative risk, then provided that data are available to calculate any changes in the population base of each premise our approach can be modified accordingly, for example by using a poisson log-linear version of our spatio-temporal mixed model rather than the current binomial probit-linear version. in order to understand and mitigate shared gi disease aetiologies between humans and animals it would be necessary to develop a 'one health' surveillance system that integrates human and veterinary healthcare databases. in future work, we intend to adapt the approach described in this paper to human gi disease surveillance by re-calibrating the model against data relating to human gi disease presentations at general practitioner surgeries. a further extension of the approach would then be to a bivariate model for the joint surveillance of veterinary and human gi disease risk. a suitable starting point for this would be to replace the single eq. ( ) by a pair of equations, j k t jk t t k t , , , , , where eqs. ( ) and ( ) describe the relative risk of gi at veterinary premise i and gp surgery k, respectively. a bivariate model would allow non-zero correlations between the s i,t and ′ s k t , corresponding to closely located pairs of veterinary premises and gp surgeries. we have demonstrated the feasibility of a real-time spatio-temporal syndromic surveillance system using as an example small animal veterinary premises in the uk. our detection algorithm uses bayesian predictive inference within a spatio-temporal model. the method demonstrated promising performance in detecting simulated outbreaks signals of varying spatial extent and severity at different reporting thresholds. the system is flexible: the reporting threshold of elevated risk and the positive predictive probability per premise and day may be set to whatever levels best meet the needs of a particular application; the system estimates the parameters of the model from historical data rather than imposing specific values for these, and can therefore be re-calibrated to detect outbreaks of any syndrome of interest. a traffic-light system based on exceedence probabilities offers a visual aid to rapid identification of potential outbreaks on a given day at each premise. we intend to implement the system on savsnet servers for the early detection of outbreaks in gi and in other syndromes that have outbreak potential and are routinely recorded in savsnet. the datasets generated and/or analysed during the current study are not publicly available due to issues of companion animal owner confidentiality, but are available on request from the savsnet data access and publication panel (savsnet@liverpool.ac.uk) for researchers who meet the criteria for access to confidential data. the r scripts used for pre-processing and analysing the data supporting this article can be found as supplementary material online. the r package 'precara' developed for pre-processing the data supporting this article is publicly available from the 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surveillance in canada epidemiology & animal health management, and industry branches of the nzva, proceedings of the epidemiology & animal health management branch of the nzva detecting emerging diseases in farm animals through clinical observations estimation of the number and demographics of companion dogs in the uk health status and population characteristics of dogs and cats examined at private veterinary practices in the united states breed registration statistics processes spatially referenced data (precara) we wish to thank data providers both in practice (vetsolutions, teleos, cvs and non-corporate practitioners) and in diagnostic laboratories, without whose support and participation, this research would not be possible. the study was conceived and designed by a. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to a.c.h. or f.s.-v. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - w apdk authors: course, christopher; chakraborty, mallinath title: management of respiratory distress syndrome in preterm infants in wales: a full audit cycle of a quality improvement project date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: w apdk respiratory distress syndrome (rds) is the commonest diagnosis after premature birth. we aimed to audit clinical practices before and after introduction of a national guideline in wales on rds management. anonymised, prospective data on all infants born at < weeks of gestation and cared for at one of the participating neonatal units in wales were collected in two six-month time periods in and . a national guideline was introduced in by the wales neonatal network. data collection included areas of antenatal management, delivery room stabilisation, invasive and non-invasive respiratory support, surfactant treatment and elements of supportive care. univariate and multivariate methods were used to compare data between the two epochs. comparing care before and after introduction of the national guideline, areas of significant improvement include use of targeted tidal volume ventilation, use of caffeine therapy, oxygen therapy post-surfactant and increasing early use of parenteral nutrition. areas of poorer management included levels of positive end expiratory pressures and timing of introduction of enteral feeds. little variation was seen between level two and three units, although gestational age was a significant independent variable for several practices, including delayed cord clamping, stabilisation with intubation, early enteral feeding and caffeine administration. a national guideline for management of rds in wales has significantly improved practice in several areas. however, despite a large volume of high-quality evidence and robust guidance, there remains a significant variation in some elements of best practice for rds management. further work should focus on education and training, especially for elements requiring cross-departmental work. prospective, anonymised audits of the management of rds in all preterm infants born at < weeks gestational age and cared for in a participating neonatal unit in wales were undertaken. the first round of data collection was undertaken over a six-month period between september and march . following the first round of data collection, a wales neonatal network guideline on the management of rds, based on the european consensus guideline, was introduced in july and disseminated throughout all welsh neonatal units . this represented the best-practice document for the management of rds in infants born at < weeks gestational age, concentrating on areas which were supported by grade a evidence (http://www.gradeworkinggroup.org/). a second round of data collection was undertaken between march and september , aiming to assess changes in practice by comparing the two cohorts. patient recruitment and data collection was undertaken through the welsh research and education network (wren; www.wrenpaediatrics.com) . all infants who were born at < weeks gestational age and were cared for in a welsh neonatal unit were eligible for the study and all units were approached to participate. the audit proforma was based upon the recommendations in the european consensus guideline on the management of rds in preterm infants , and concentrated on management areas which were supported by grade a evidence . an update on the consensus guideline was released in , which did not alter the main recommendations, therefore the same proforma was used for both data collection cycles. the proforma (supplementary table ) collected data on items in six domains including infant demographics, antenatal management, delivery room stabilisation, surfactant management, non-invasive ventilation management, mechanical ventilation strategies and other supportive care used (supplementary table descriptive statistics for all variables were produced. for variables with a known eligibility denominator, univariate statistical comparisons between cohorts were made using chi-squared and t-tests where appropriate. unadjusted and adjusted odds ratios (or and aor) with % confidence intervals (ci) were estimated between the cohorts by logistic regression analysis using data as baseline, adjusting for level of unit of delivery (either level two or three, categorical variable) and gestational age at delivery (continuous variable). two post-hoc subgroup analyses were performed between the two cohorts comparing infants born at below and above weeks' gestation, and for those born in level or level units. statistical significance for all analyses was set at p < . . the study was designed as a quality improvement project and implemented as local audits in all participating hospitals. local audit and governance departments of all hospitals approved the collection of routine clinical data. individual consent was not requested from parents for the collection of routine clinical data. anonymised data from each hospital were combined at the end of the study period for analysis; the authors had no access to any identifiable data. table ). demographic details for infants in both cohorts are presented in table , with no significant difference being found between cohorts. the spread of gestational ages for both cohorts can be seen in the supplementary fig. the results from the post-hoc subgroup analyses were broadly similar to the regression analyses. all results are presented in supplementary tables and . in the < -week gestation subgroup, a significant decrease was seen in the number of infants stabilised in fio - % ( . vs . % p =< . ). a significant decrease was also seen in dcc ( . % vs % p = . ). a significant increase in the number receiving ttv ( . vs . % p = . ) was observed. in the ≥ -weeks subgroup a significant decrease was seen in stabilisation in www.nature.com/scientificreports www.nature.com/scientificreports/ early management of rds can have a significant impact on later morbidity, especially on the development of cld . an extensive evidence base has grown over the past two decades on optimal management for these vulnerable infants , , . we present for the first time the assessment of rds management across several welsh neonatal units before and after the implementation of a national guideline. to our knowledge, this is the first time rds management has been reported in a relatively large cohort over time against the same recommendations in the uk. www.nature.com/scientificreports www.nature.com/scientificreports/ there were several notable improvements in management, the most significant of which are use of ttv-mode, fio management post-surfactant administration, increased caffeine prescribing, and increasing use of parenteral nutrition on day one of life. however, there are two areas which appear to have deteriorated: stabilising infants in the delivery room with an fio - % and use of cpap pressures of ≥ cmh o. both of these practices showed some variation depending on the unit level and ga, with the tertiary units being more likely to use higher cpap pressures, but less mature infants being more likely to be stabilised with a higher inspired oxygen fraction. owing to the data collection methodology, this could be secondary to documentation; however, it is important to emphasise the importance of not exposing premature infants to unnecessarily high fio during stabilisation. immediate management of an infant being born prematurely is a multi-disciplinary process in the delivery room. despite evidence supporting the efficacy of dcc in premature infants , rates remained low in both cohorts, with no significant improvement. however, there was significant evidence of a move towards dcc for more mature infants. additionally, minimal enteral feeding commencing on day one of life remains low and unimproved. this may be secondary to clinical concerns with the infant but may also be due to a lack of maternal expressed breast milk. this often requires support from the midwifery team in the first hours of life. this highlights the need for robust training and complementary guidance between departments to ensure optimal practice. the recently published sift trial has demonstrated the safety of establishing full enteral feeding early in preterm and very-low-birthweight infants, and this evidence will hopefully aid clinician's confidence in initiating early enteral feeding in practice. we observed variation in management in several areas based upon ga. more mature infants were more likely to be stabilised on non-invasive respiratory support. this may be due to the experience of those in attendance and general confidence within unit culture in managing more vulnerable infants with non-invasive ventilation. extremely preterm infants born in level two units may need intubation before transfer to a tertiary unit for further care. however, no variation in intubation was observed based on unit of delivery, although only a small proportion of these infants were delivered in level two units. further evidence on lisa in extreme preterm infants may improve the success of non-invasive respiratory support in this population. in addition, several local quality improvement projects are ongoing in many of the neonatal units in wales, and we hope to see improved practice in the next round of data collection. our findings are in keeping with the limited amount of published data on rds management against consensus guidance. a uk-based survey from found a significant number of units reluctant to use cpap as the primary ventilation mode for extremely premature infants, but ttv-mode use was increasingly popular for mechanically ventilated infants . single-centre retrospective audits have been published on adherence to aspects of previous consensus guidelines. retrospective audits have found variable use of prophylactic surfactant with less use in more mature infants , and good adherence on early management in a cohort of twenty infants < weeks' gestation . both audits examined much more limited aspects of older european consensus guidelines highlighting the need for robust training and education, but none published follow-up data to document changes. our study has several strengths. by capturing prospective data across multiple sites during the same time points, we have achieved a highly representative, contemporaneous impression of current practice across two epochs. this is the first report we are aware of describing changes of practice in rds management within a defined population over time in the uk, which demonstrates the impact of a unified national guideline on rds management. our data collection was restricted mainly to areas of practice supported by grade a evidence, and we used robust statistical methods to analyse reliable data. we believe this framework can be used in any neonatal network in the uk to document quality improvement in the management of rds in preterm infants. there are several limitations of our work. as with any multi-centre audit project there were missing data for variables, and this varied by cohort and data item ( supplementary figs. and ). the effect of incomplete participation of level-two units was partly mitigated as the majority of preterm infants were delivered in tertiary units. however, some welsh preterm infants born in non-participating units did not have their management audited, and there may be more variation in practice than appreciated. it was difficult to determine the number of eligible infants (denominator) for some interventions through the data collection process, although the majority of these were based on grade b and c evidence. we also did not collect data on other relevant clinical indicators like admission temperature, which may have an effect on the severity of rds; this will be added in future rounds of data collection. finally, since our study examined practice against an established guideline, data on the outcome of these infants were not collected; this remains an objective in future rounds of data-collection. our analysis demonstrates that certain desirable grade a evidence-based interventions are still yet to come into clinical practice. the edition of the european consensus guideline recommended a change in practice towards stabilising preterm infants on non-invasive respiratory support at delivery rather than elective intubation . additionally, it also recommended delayed cord clamping of at least sixty-seconds is practised where possible. our data demonstrates that intubation rates at delivery have remained unchanged despite introduction of the wales guideline, and the number of infants receiving delayed cord clamping remains low. there are quality improvement and training initiatives targeting these practices currently underway in welsh neonatal units, and this will form a major focus in the next cycle of data collection in this quality improvement project. in conclusion, this study highlights the successes and challenges with improving management and reducing the variation in practice of rds in wales. interestingly, our data suggests relatively limited variation between level two and three units. some important areas of practice have shown substantial improvement, but there remain areas of practice that are not in keeping with current best evidence, most importantly the low rates of extremely preterm infants being stabilised on non-invasive respiratory support. unified, national guidelines are a potentially powerful tool to effect change and reduce variation in practice. they are feasible to implement and can be established in other neonatal networks and nations of the uk. moving forward, an update to the european pathophysiology of respiratory distress syndrome chronic lung disease after premature birth lung function outcome at school age in very low birth weight children effect of preterm birth on later fev : a systematic review and meta-analysis respiratory health in pre-school and school age children following extremely preterm birth longitudinal evaluation of airway function years after preterm birth global, regional, and national estimates of levels of preterm birth in : a systematic review and modelling analysis respiratory support for preterm infants -the cochrane evidence and beyond european consensus guidelines on the management of neonatal respiratory distress syndrome in preterm infants- update respiratory distress syndrome uk trainee-led paediatric governance collaboratives: improving the lives of both trainees and children european consensus guidelines on the management of respiratory distress syndrome - update european consensus guidelines on the management of respiratory distress syndrome - update delayed vs early umbilical cord clamping for preterm infants: a systematic review and meta-analysis controlled trial of two incremental milk-feeding rates in preterm infants the optimist-a trial: evaluation of minimally-invasive surfactant therapy in preterm infants - weeks gestation current practice in early management of neonatal respiratory distress syndrome: is it evidence-based? an audit of management of respiratory distress syndrome in the context of the audit on adherence to european consensus guidelines on the management of neonatal respiratory distress syndrome in preterm infants we would like to thank the following for their assistance with data collection: c.c. and m.c. designed audit tools and authored and led implementation of the wales neonatal network rds guideline. c.c. led and coordinated data collection across wales, led data analysis of both audit cycles and authored the first draft of the manuscript. m.c. oversaw all data analysis and reviewed the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to m.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ri qsarz authors: yashima, nozomi; ito, takashi; kajiyama, kenji; maeda, hiroyuki; kakihana, yasuyuki; maruyama, ikuro title: leukocyte-derived extracellular dna contributes to abnormal pressure elevation in the extracorporeal circulation circuit date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ri qsarz an abnormal elevation in pressure is a serious complication involving the extracorporeal circulation circuit. clot formation might be associated with this complication, but the precise mechanism of an abnormal elevation in pressure has not been identified. we investigated sufficient conditions for in-circuit elevation in pressure using an ex vivo re-circulation circuit with porcine blood. specifically, we investigated the effect of blood conditions, the type of anticoagulation, and pro-inflammatory stimulation on in-circuit pressure. we also examined the cause of an abnormal elevation of in-circuit pressure by specifically degrading dna, rna, or protein components of an obstructed filter and by using immunofluorescent techniques. neither a change in temperature nor change in ph in the blood increased in-circuit pressure. in contrast, long-term storage of blood, pro-inflammatory stimulation by phorbol myristate acetate, and heparin administration significantly increased in-circuit pressure. abnormal in-circuit elevation in pressure was associated with deposition of extracellular dna on the outlet surface of the filter. administration of dnase resulted in a rapid decline of in-circuit pressure. in an ex vivo re-circulation circuit system, extracellular dna deposition on the filter is responsible for an abnormal in-circuit elevation in pressure. senescent leukocytes, stimulated leukocytes, and heparin exposure are associated with extracellular dna deposition. long-term storage of heparinized blood increases in-circuit pressure. using the ex vivo circuit system (fig. a) and porcine blood, we investigated sufficient conditions for an elevation of in-circuit pressure. in control conditions where porcine blood was freshly collected (day ) and unstimulated, in-circuit pressure was not increased. neither a change in temperature ranging from °c to °c nor a change in ph ranging from . to . in the circuit system increased in-circuit pressure (data not shown). in contrast, long-term storage of blood significantly increased in-circuit pressure (p < . ) if blood was anticoagulated with heparin ( fig. b) , while in-circuit pressure elevation was minimal if blood was anticoagulated with citrate (fig. c) . these findings suggest that the storage term of blood and the type of anticoagulation may be associated with an abnormal elevation in pressure. heparin administration in stored blood leads to extracellular dna deposition on the outlet of the filter. we then investigated whether heparin administration in citrated blood leads to elevation of in-circuit pressure. heparin administration did not increase in-circuit pressure (pre-vs post-heparin administration, p = . ) if blood was fresh (day ) (fig. a) . in contrast, heparin administration significantly increased in-circuit pressure (pre-vs post-heparin administration, p < . ) if blood was stored for days or longer. when day stored blood passed through the filter, a grossly visible sticky substance was adhered (video s ). immunofluorescent analysis showed that this sticky substance was composed of extracellular dna and fibrin(ogen) (fig. b ). in contrast, little extracellular dna was observed when day blood was passed through the filter, and some intact nuclei and fibrin(ogen) were observed (fig. b) . a three-dimensional reconstitution technique showed that the extracellular dna layer was located immediately downstream of the filter (video s and fig. s ). the fibrin(ogen) layer was located downstream of the dna layer (fig. s ) . these results suggest that dna is primarily responsible for obstruction of the circuit. long-term storage of heparinized blood causes elevation of in-circuit pressure. (a) a schema of the extracorporeal recirculation circuit is shown. the circuit consists of polyvinyl chloride (pvc) tubes, a pooling reservoir, a roller pump, a manometer, and a removable filter with a pore size of µm. (b) heparinized blood stored for , , , or days was administered to the circuit and in-circuit pressure was monitored for up to minutes. n = per group. (c) citrated blood stored for , , , or days was administered into the circuit and in-circuit pressure was monitored for up to minutes. n = per group. repeated measures analysis of variance models were used to analyze changes in the area under the curve (auc) over time. *p < . , **p < . . www.nature.com/scientificreports www.nature.com/scientificreports/ leukocytes play a role in elevation of in-circuit pressure. we examined whether leukocytes, the dominant nucleated cells in blood, contribute to elevation of in-circuit pressure. we prepared platelet-rich plasma (prp) in which erythrocytes and leukocytes were almost absent, but platelets were abundant. on day , in-circuit pressure was increased if heparin was administered in whole blood (fig. a,b) . in contrast, in-circuit pressure was not increased if heparin was administered in prp (fig. a,b) . decreases of platelets and leukocytes were evident before and after administration of heparin, respectively, in whole blood while they were less obvious in prp (fig. c ). hemolysis was accompanied by the elevation of in-circuit pressure (fig. s ) . in immunofluorescent analysis, extracellular dna was detected all over the filter through which whole blood passed, whereas extracellular dna deposition was not observed on the filter through which prp passed (fig. d) . these findings suggest that platelets and plasma, two important components in thrombus formation, are not sufficient for abnormal elevation in pressure, and leukocytes and/or erythrocytes play an essential role. extracellular dna contributes to elevation of in-circuit pressure. we next examined whether extracellular dna directly contributes to elevation of in-circuit pressure. we administered dnase into the partially obstructed circuit and monitored in-circuit pressure. dnase treatment immediately decreased in-circuit pressure to a baseline level at minutes ( . ± . mmhg with dnase treatment vs . ± . mmhg with no treatment, p < . ) (fig. a ). in contrast, rnase treatment did not decrease in-circuit pressure (fig. b) . proteinase treatment partially decreased in-circuit pressure. immunofluorescent analysis showed that extracellular dna figure . heparin administration in stored blood leads to extracellular dna deposition on the outlet of the filter. (a) citrated blood (cit-blood) stored for , , or days was administered to the circuit and then u/ml of unfractionated heparin was administered at minutes. in-circuit pressure was monitored for minutes. n = per group. the paired t-test was used to analyze the difference between in-circuit pressure at and minutes. *p < . , **p < . . (b) the filter was removed at minutes and analyzed by immunofluorescence. fibrin(ogen) was labeled in red and dna was labeled in blue. whole images of the filters (scale bars: mm) were analyzed by bz-x microscopy and magnified images (scale bars: μm) were analyzed by lsm confocal microscopy. representative images of n = are shown. on the surface of the filter was degraded by dnase treatment (fig. c ). to determine whether these events were specific to the removable filter used in this study or generally applicable to clinical conditions, we performed the same experiment using the clinically approved venous reservoir, oxygenator, and arterial line filter. in-circuit pressure was elevated at the oxygenator if heparin was administered in whole blood that was stored for days (fig. s b) . furthermore, dnase treatment immediately decreased in-circuit pressure. these findings indicate that extracellular dna is directly responsible for obstruction of the circuit. www.nature.com/scientificreports www.nature.com/scientificreports/ we also examined the possibility that endogenous dnase protects the filter from dna obstruction. we used edta, which can inhibit dnase activity by chelating divalent metal cations. inhibition of endogenous dnase by edta significantly increased in-circuit pressure if heparinized blood stored for days was used (edta positive vs negative: . ± . mmhg vs . ± . mmhg at minutes, p < . ) (fig. s ). in contrast, this effect was not observed if heparinized blood on day was used (edta positive vs negative: . ± . mmhg vs . ± . mmhg at minutes). these results suggest that edta by itself does not induce obstruction of the filter, but it can exacerbate extracellular dna-mediated obstruction of the filter. leukocyte stimulation leads to extracellular dna deposition and elevation of in-circuit pressure. finally, we examined whether in-circuit pressure could be elevated even if blood was fresh. to this end, we used fresh blood and monitored in-circuit pressure under two hypothetical circumstances: transfusion of old blood and inflammatory stimulation. heparin administration into the mixture of fresh blood, saline, and day stored blood did not increase in-circuit pressure significantly (fig. s a) . however, immunofluorescent analysis showed deposition of extracellular dna on the filter surface (fig. s b) . these findings suggest that transfusion of old blood is not sufficient for elevation of in-circuit pressure by itself but may become the genesis of pore-clogging. then, we examined whether leukocyte stimulation results in elevation of in-circuit pressure since previous studies have suggested that stimulated leukocytes are prone to releasing dna into the extracellular space . in-circuit pressure was increased if whole blood on day was stimulated with phorbol -miristate -acetate (pma) (fig. a) . heparin administration in pma-stimulated blood led to a significant further increase in in-circuit pressure (fig. a,b) . immunofluorescent analysis showed that pma stimulation induced deposition of extracellular dna all over the filter surface (fig. c) . these findings suggest that not only leukocyte senescence, but also leukocyte stimulation, promote elevation of in-circuit pressure. www.nature.com/scientificreports www.nature.com/scientificreports/ our study showed that leukocyte-derived extracellular dna induced an elevation of in-circuit pressure. furthermore, heparin injection caused leukocytes to release extracellular dna. previous studies have reported an abnormal elevation in pressure in cpb [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, no studies have suggested that extracellular dna can contribute to abnormal elevation in pressure. extracellular release of dna from neutrophils, called neutrophil extracellular traps (nets), was first described in infectious diseases , and the process was named netosis. this process is morphologically and functionally distinct from other forms of cell death , . recently, subsequent studies established that other immune cells also release extracellular dna (renamed etosis) in eosinophils , , basophils , , monocytes , macrophages , and lymphocytes . although etosis was induced by different stimuli for several minutes to hours in these studies, a unique study showed that high hemodynamic forces trigger rapid net release within minutes . our data are in good agreement with this previous study. we found that leukocytes, including neutrophils, were disrupted when they passed through the narrow site in the circuit, and extracellular dna was adhered downstream of the filter. the elevation of pressure in cpb has unique characteristics, including transient elevation of pressure in some cases , , . in some experiments in our study, the same phenomenon was observed, especially using heparinized blood, but not citrated blood. this finding can be explained by endogenous dnase activity. extracellular dna is degraded by endogenous dnase, leading to a gradual reduction in elevated in-circuit pressure. because this enzyme requires a divalent cation to activate, in-circuit pressure is not reduced in blood that is anticoagulated with citrate sodium. our results suggest that heparin is also associated with elevation of in-circuit pressure. heparin is the most common anticoagulant in cardiac surgery because its effects can be measured by monitoring the activated clotting time and reversed by protamine. with regard to the dose of heparin used in this study, u/ml corresponds approximately to u/kg and is thus reasonable in cpb settings . however, some reports have suggested that heparin itself affects functions of leukocytes, besides its role in anticoagulation. lazarowski et al. reported that heparin alone, ranging from . to u/ml, could induce in vitro aggregation of human polymorphonuclear neutrophils (pmns). cairo et al. reported that pmns stimulated with n-formylmethionyl-leucyl-phenylalanine were aggregated by heparin, although heparin itself did not promote aggregation at low doses. this finding indicates that even low heparin concentrations can promote aggregation of pmns if they are activated. they considered that this aggregation might be induced by heparin's highly negative charge and ability to locate the surface of the cell membrane. gebska et al. showed that apoptotic and necrotic leukocytes had a high affinity for heparin compared with healthy live leukocytes. in our study, aggregation of leukocytes and platelets was observed at day in heparinized blood, but not in citrated blood, in giemsa-stained thin blood smears (fig. s ) . some of the aggregates were more than µm. this aggregation of leukocytes led to difficulty for them passing through the filter, leading to physical stress and they were easily destroyed. accordingly, heparinized blood and blood after adding heparin are likely to raise in-circuit pressure. similarly, leukocytes stimulated with pma were aggregated with nuclear degeneration and cytoplasmic vacuolization (fig. s ) . a reasonable treatment of an abnormal elevation in pressure is to add dnase to the circuit. in our study, dnase treatment resulted in a rapid decrease of raised in-circuit pressure. a previous study created a mouse model of ventilator-induced lung injury and transfusion-related acute lung injury , which were associated with net formation in the lungs. this previous study showed that inhalational or intratracheal dnase treatment eliminated nets and improved arterial oxygen saturation in ventilator-induced lung injury. however, many dna fragments enter the patient's body if dnase is added to the oxygenator when an abnormal elevation in pressure occurs. dna fragments, which is one of the damage-associated molecular patterns, may induce an inflammatory response . therefore, overall effects of dnase treatment should be investigated in an in vivo animal model. a new oxygenator needs to be developed, which can prevent blood from excessive shear stress, as well as coagulation activity. our study suggested that extracellular dna from disrupted leukocytes contributed to elevation of in-circuit pressure. not only taking account of intra-oxygenator thrombosis but also taking a proper care of leukocyte disruption may be important. another strategy to suppress this phenomenon is optimization of anticoagulation therapy. as mentioned above, heparin affects leukocytes, as well as coagulation/anticoagulation factors. we consider that an excessive dose of heparin can be adverse and an optimal dose of heparin should be determined. our study has some limitations. first, we showed that leukocyte-derived extracellular dna contributed to an abnormal elevation in pressure in an ex vivo circuit, but it may be merely a partial explanation of abnormal elevation in pressure in clinical settings. other factors, such as thrombosis, may also be associated with abnormal elevation in pressure in some cases. these factors may induce this phenomenon simultaneously. further clinical investigations are required. second, we did not assess the contribution of platelets to the abnormal elevation in pressure in our ex vivo circuit. we measured the number of erythrocytes, leukocytes, and platelets in our experiments. when in-circuit pressure was elevated, the numbers of platelets and leukocytes were decreased (fig. c) . some clinical studies have also reported a decline in platelet count during cpb with or without an abnormal elevation in pressure , . platelets are presumed to initially attach to the filter, which leads to narrowing of the space for blood to pass through. in a microfluidic study that showed shear-induced netosis, platelets were accumulated and fluid shear stress was increased, which resulted in disruption of leukocytes . further investigations are required to understand the role of platelets in abnormal elevation in pressure in cpb. in conclusion, our study shows that leukocyte-derived extracellular dna contributes to abnormal in-circuit elevation of pressure in an ex vivo circuit. additionally, heparin can promote the release of extracellular dna from leukocytes. storage of porcine blood and platelet-rich plasma. all experiments were performed in accordance with the guidelines of kagoshima university, kagoshima, japan. porcine blood anticoagulated with u/ml of unfractionated heparin (mochida, tokyo, japan) or a : volume of . % sodium citrate was purchased from domestic animal resource development co., ltd., minami-kyushu, japan. using porcine blood anticoagulated with sodium citrate, platelet-rich plasma (prp) was prepared by centrifugation at × g for minutes. the numbers of erythrocytes, leukocytes, and platelets were analyzed with celltac α (nihon kohden corp., tokyo, japan). whole blood and prp were stored at °c for , , , or days, and was warmed slowly back to room temperature before use. preparation of the extracorporeal recirculation circuit. we developed an ex vivo recirculating circuit ( fig. a) using polyvinyl chloride tubes, a pooling reservoir (jms co., ltd., hiroshima, japan), a roller pump (stockert instrument gmbh, munich, germany), a manometer (migishita seiki mfg co., ltd., amagasaki, japan), and a removable filter with the pore size of µm (merck millipore kgaa, darmstadt, germany). in some experiments, a venous reservoir, an oxygenator, and an arterial line filter approved for clinical use were used instead of a removable filter (fig. s a ). for priming, the circuit was filled with ml of saline solution and the flow rate was set at l/min. treatment of porcine blood with acid, alkali, a low temperature, phorbol -miristate -acetate, or ethylenediaminetetraacetic acid. lactate (wako, osaka, japan) or sodium hydrate (wako) was added to the priming solution to adjust the ph of porcine blood to approximately . or . . the ph pathologic fibrin formation and cold-induced clotting of membrane oxygenators during cardiopulmonary bypass heparin-coated equipment reduces the risk of 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death program leads to neutrophil extracellular traps unconventional roles of the nadph oxidase: signaling, ion homeostasis, and cell death. science's stke eosinophil extracellular dna trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans eosinophil extracellular dna traps: molecular mechanisms and potential roles in disease basophils exhibit antibacterial activity through extracellular trap formation monosodium urate crystals induce extracellular dna traps in neutrophils, eosinophils, and basophils but not in mononuclear cells human blood monocytes are able to form extracellular traps extracellular traps and macrophages: new roles for the versatile phagocyte the lymphocytes stimulation induced dna release, a phenomenon similar to netosis hemodynamic force triggers rapid netosis within sterile thrombotic occlusions heparin sensitivity and resistance: management during cardiopulmonary bypass aggregation of human-neutrophils by heparin synergistic effect of heparin and chemotactic factor on polymorphonuclear leukocyte aggregation and degranulation high-affinity binding sites for heparin generated on leukocytes during apoptosis arise from nuclear structures segregated during cell death mechanical ventilation induces neutrophil extracellular trap formation extracellular dna traps are associated with the pathogenesis of trali in humans and mice neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance effect of surface coating on platelet count drop during cardiopulmonary bypass the authors thank for binita shrestha, phd, visiting research assistant scientist, department of medical chemistry, university of michigan, ann arbor, mi for technical support for immunofluorescent staining. we wish to thank joint research laboratory, kagoshima university graduate school of medical and dental sciences, for the use of their facilities. we thank ellen knapp, phd, from edanz group (www.edanzediting.com/ac) for editing a draft of this manuscript. this work was supported by grants from jms co., ltd. n.y. conducted the study, analyzed the data, and wrote the manuscript. t.i. designed the study, analyzed the data, wrote the manuscript, and revised the manuscript. k.k. designed the study, conducted the study, and approved the manuscript. h.m. designed the study and approved the manuscript. y.k. revised the manuscript. i.m. revised the manuscript. h.m. and k.k. work for jms co., ltd which develops devices in cardiopulmonary bypass. the other authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to t.i.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -nuep nim authors: dewald, lisa evans; johnson, joshua c.; gerhardt, dawn m.; torzewski, lisa m.; postnikova, elena; honko, anna n.; janosko, krisztina; huzella, louis; dowling, william e.; eakin, ann e.; osborn, blaire l.; gahagen, janet; tang, liang; green, carol e.; mirsalis, jon c.; holbrook, michael r.; jahrling, peter b.; dyall, julie; hensley, lisa e. title: in vivo activity of amodiaquine against ebola virus infection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: nuep nim during the ebola virus disease (evd) epidemic in western africa ( ‒ ), antimalarial treatment was administered to evd patients due to the high coexisting malaria burden in accordance with world health organization guidelines. in an ebola treatment center in liberia, evd patients receiving the combination antimalarial artesunate-amodiaquine had a lower risk of death compared to those treated with artemether-lumefantrine. as artemether and artesunate are derivatives of artemisinin, the beneficial anti-ebola virus (ebov) effect observed could possibly be attributed to the change from lumefantrine to amodiaquine. amodiaquine is a widely used antimalarial in the countries that experience outbreaks of evd and, therefore, holds promise as an approved drug that could be repurposed for treating ebov infections. we investigated the potential anti-ebov effect of amodiaquine in a well-characterized nonhuman primate model of evd. using a similar -day antimalarial dosing strategy as for human patients, plasma concentrations of amodiaquine in healthy animals were similar to those found in humans. however, the treatment regimen did not result in a survival benefit or decrease of disease signs in ebov-infected animals. while amodiaquine on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of amodiaquine when used in combination with artesunate or another antiviral. antiviral activity of amodiaquine and artesunate and their metabolites in cell culture. the antimalarial treatment asaq that was administered to evd patients in , is a coformulation of artesunate (as) and amodiaquine (aq). as and aq are rapidly metabolized in the liver to dihydroartemisinin (dha) and desethylamodiaquine (deaq), respectively. prior to in vivo evaluation, the drug asaq, its components, and the metabolites were characterized for their inhibitory effects on ebov (makona variant, ebov/mak) replication in cell culture ( table ). the data confirm previous reports that both aq and the metabolite deaq block ebov replication with similar activity (ic = . to . µm) in huh and vero e cell lines (ic = . to µm). in primary human macrophages, both aq and deaq exhibited elevated cytotoxicity, which precluded any interpretation of antiviral activity. in contrast, activity of as and its metabolite dha was weak or undetectable. combinatorial testing of aq and as or the metabolites, deaq and dha, did not reveal in vitro synergistic effects against ebov replication (data not shown). based on the in vitro data, the decision was made to evaluate aq in the nhp model of ebov infection. nhps with an aq dosing regimen similar to that used for evd patients in ebola treatment centers in . treatment consisted of a -day course of as-aq with the dose determined according to age of the patient. the dose range for aq in humans is . to mg/kg corresponding to a rhesus macaque equivalent dose range of . to . mg/kg based on body surface area . a pharmacokinetic (pk) study in rhesus macaques ( groups of males and females) was performed to monitor plasma concentrations of aq (fig. a ) and the active metabolite deaq (fig. b) . the study evaluated three daily oral doses of aq, using mg/kg or mg/kg. pharmacokinetics were determined by collecting blood samples at , . , , , , , , , , and hours (h) following dosing on day and day ( fig. ) . after days of dosing with mg/kg in healthy male and female macaques, aq was well-tolerated. the higher dose of mg/kg resulted in a variety of clinical observations including hypoactivity, shivering (muscle tremors) and/or diarrhea. extreme hypoactivity was observed in a single male of the mg/kg group that resolved after dosing ceased. animals in both the mg/kg and mg/kg groups had lower blood pressure readings than at predose on both dosing days, typically at and/or h post-dosing. clinical chemistry and hematology parameters for both doses were analyzed and fell within normal ranges with the exception of liver enzymes. alanine aminotransferase (alt) and aspartate aminotransferase (ast) were elevated . -to . -fold on day at h after the rd dose. the increases in alt and ast were seen in both male and female animals at both the and mg/kg dose regimens, though changes were not dose-related. these increases were considered toxicologically significant. aq and the metabolite deaq reached peak concentrations at . to h (t max ) after administration for both day and day dosing (supplementary tables and ). the elimination phase half-life (t / ) varied among the individual animals, ranging from to h for aq and to h for deaq. plasma concentrations of the metabolite deaq were higher than concentrations of the parent drug resulting in auc last , and auc inf values that were . -to -fold higher for deaq than for aq. the mg/kg aq dose resulted in c max for aq ( . - . ng/ml) and deaq ( - ng/ml) that were in a similar range as reported for the mg/kg dose in humans (aq: c max = . ± . ng/ml and deaq: c max = . ± . ng/ml) . based on the results of the pharmacokinetics study, the mg/kg aq dose was chosen for a study to evaluate the in vivo effect of aq in the nhp model of evd (study outline, supplementary fig. ). rhesus macaques were challenged intramuscularly (im) with a target dose of plaque-forming units (pfu) of ebov/mak (measured dose of pfu) on study day . the control group (n = , female, males) received vehicle treatment on days , , , , and postexposure. treatment group (n = , females, males) received a -day course of mg/kg of aq by oral administration on days , and after exposure to ebov. treatment group (n = , females, males) received a -day course of mg/kg of aq by oral administration on days , , and after exposure to ebov. all animals were euthanized or succumbed to disease by day postexposure (fig. a) . median times to disposition were days for placebo and treatment group , and days for treatment group with no significant difference between the placebo or treatment groups versus the treatment group (p = . and . , respectively). the animals in all groups became febrile by day or postexposure, followed by a marked decrease in body temperature preceding death (fig. b) . no significant difference in febrile illness or weight loss was observed between control and treatment groups (fig. b,c) . nhps had a mild reduction in activity and responsiveness by www.nature.com/scientificreports www.nature.com/scientificreports/ day postexposure with a severe decrease observed for most animals on day postexposure ( table ). loss of appetite and lymphadenopathy began on day to followed by dehydration and rash (petechial, maculopapular) on day postexposure. clinical signs did not differ significantly between the groups. viral loads in plasma were not reduced in treated animals. plasma viremia was quantified by rt-qpcr, and viral titer was determined by plaque assay. viral rna was first detected in the serum of some animals days after exposure, with a rapid and substantial increase by day for both placebo-and aq-treated animals. rna levels at necropsy were in the range of - viral rna copies/ml for all nhps (fig. a) . infectious viral titers followed a similar trend for all groups with a sharp increase in viral titer starting on day postexposure and reaching a peak plateau by day before animal death (fig. b ). viremia correlated with the onset of clinical signs of disease such as loss of appetite, lymphadenopathy, and fever. aq treatment did not reduce plasma viral rna copies or infectious virus titer regardless of when treatment was initiated. no significant difference in viral titers was observed between groups. amodiaquine-treated animals present with abnormal hematology and biochemical profiles and pathological disease that corresponds with ebola virus disease. the animals showed a similar hematological profile regardless of treatment. complete blood counts and serum chemistry analysis were performed at scheduled sampling points, before (days - , - , and ) and after (days , , , and at necropsy) exposure (figs. , ) . the chemistry values for aq-treated nhps followed the same trends as the control animal values. similar hematological and biochemical profiles were observed for all animals regardless of treatment both in aq-and placebo-treated animals. concurrent neutrophilia and thrombocytopenia were observed for aqand placebo-treated animals on day postexposure with ebov (fig. ) . as animals reached endpoint criteria, neutrophil levels decreased (fig. a) . the low-level neutrophil values on day postexposure were from necropsy samples only. nhps from all treatment groups exhibited a concomitant increase in serum creatinine and blood urea nitrogen levels, indicative of renal dysfunction often noted in nhps with concurrent evd (fig. a,d) . other biochemical abnormalities consistent with evd were also observed beginning on day postexposure, including substantial elevation of serum alt, ast, and gamma-glutamyl transpeptidase (ggt) levels that indicate hepatocellular damage (fig. b ,e,f). necropsies were performed on all animals to evaluate pathological disease associated with ebov infection following aq or placebo treatment. the pathologist was blinded to the group identification during necropsy and tissue evaluation. the pathological findings were similar in type and severity between treated and untreated animals, and the gross and histopathologic findings in all animals were consistent with typical evd in rhesus macaques. common gross findings included a cutaneous rash on the face, often extending to other regions of the body, dehydration, discoloration of the liver with increased friability, turgid spleen, and enlarged, often congested kidneys. at the microscopic level, all animals exhibited lymphoid depletion of the axillary lymph nodes, lymphoid depletion and necrosis of the spleen, and hepatocellular degeneration and necrosis. panniculitis and necrosis were observed at the virus challenge site of animals. samples from infected animals collected on days , , , and postexposure and on day of necropsy (days , or ) were analyzed for determination of plasma levels of aq and its metabolite deaq. data indicate negligible levels of aq in all but five plasma samples, each from a different animal and day (fig. a) . as the samples were diluted by a factor of . for decontamination protocol, the concentrations were likely below the limit of detection. plasma levels of the metabolite deaq were higher than aq and detectable in all treated animals from both nhp nhp nhp nhp nhp table . clinical scores for responsiveness of animals. responsiveness of unanesthetized animals was scored using the following criteria: (white) = alert, responsive, normal activity, free of disease signs or exhibits only resolved/resolving disease signs; = slightly diminished general activity, subdued but responds normally to external stimuli; = withdrawn, may have head down, fetal or hunched posture, or reduced response to external stimuli; = recumbent but able to rise if stimulated, or moderate to dramatically reduced response to external stimuli; = persistently recumbent, severely or completely unresponsive, or may have signs of respiratory distress. www.nature.com/scientificreports www.nature.com/scientificreports/ groups and (fig. b) . deaq was evident through day postexposure and/or necropsy in of animals in group and in all animals in group . plasma levels of deaq in ebov-infected nhps were compared at different time points (fig. ) . animals that were treated on days , and (group , fig. a ), had plasma deaq levels ranging from to ng/ml on days , , and postexposure. the highest deaq levels were detected in necropsy samples on day ( and ng/ ml). for animals treated on days , and postexposure (group , fig. b ), plasma concentrations ranged from - ng/ml on day . higher concentrations were detected on day ( - ng/ml) and day postexposure ( - ng/ml). plasma levels of deaq in infected nhps and healthy nhps were compared at select time points that were identical in the pk and the efficacy study. plasma samples from infected group animals on day correspond to pk-plasma samples taken at h after the rd dose. healthy animals had higher plasma levels of deaq ( - ng/ml) than infected animals ( - ng/ml) (fig. a) . similarly, plasma samples on day from infected group animals were in a lower range (n = , - ng/ml) than those of healthy animals at the corresponding time point ( h after rd dose, n = , - ng/ml) (fig. b) . on day , h after the rd dose, deaq levels in the plasma of two animals from group were in a similar range (n = , - ng/ml) as found in healthy animals (n = , - ng/ml) (fig. c,d) . repurposing drugs continues to be of interest to the healthcare professional community for the treatment of emerging and re-emerging hemorrhagic fever viruses such as ebola, marburg, and lassa viruses. extensive efforts to screen approved and established drugs for antiviral activity led to a panel of drugs with broad-spectrum antiviral activity profiles that are available, affordable, have well-characterized pk/safety profiles and could be used under the emergency use authorization (eua) mechanism , , , . while a number of fda-approved compounds have proven to be efficacious against ebov in vitro or in murine models of disease, clinical evaluation or evaluation in more relevant disease models, such as nhps, has been limited. aq is a well-known antimalarial drug with wide usage in the countries that experience outbreaks of evd. the drug has activity against several human pathogens of other virus families including corona-, flavi-, and bunyaviruses [ ] [ ] [ ] [ ] . a recent report showed that entry of lassa-gp pseudotyped virus was blocked by aq, indicating that the drug may also have value for the treatment of other viral hemorrhagic fevers . aq is known to have in vitro antiviral activity against ebov - . multiple mechanisms for antiviral activity have been proposed. aq may block ebov entry, which depends on the acidification of endosomes. as a cationic amphiphilic drug, aq can accumulate in the late endo/lysosome and, as a weak base, neutralize the acidic environment, thereby inhibiting cathepsin b activation required for fusion of the viral and endosomal membrane . aq has also been reported to bind and inhibit cathepsin b directly . in silico binding studies revealed that aq may also bind to the viral protein vp . we found that aq inhibits ebov replication in multiple cell types, including huh and vero e cells and primary human macrophages, with ic 's ranging from . - . um depending on experiment and the cell type (table ) . aq is rapidly metabolized to the active metabolite, deaq, following oral administration. in vitro studies confirmed that the active metabolite also has anti-ebov properties in huh and vero e cells, and primary human macrophages, with ic 's ranging from . - . µm µm depending on experiment and the cell type. in contrast, as and its metabolite dha demonstrated minimal to no in vitro antiviral activity against ebov when tested side-by-side to aq using our assay parameters. when used in combination, as and aq did not have a synergistic effect on ebov replication in vitro. whereas as could possibly act with aq to impact the whole body-system in response to ebov in a way that cannot be replicated in the in vitro assays, this study assumed that aq was the main contributing factor of the drug combination resulting in anti-ebov activity. www.nature.com/scientificreports www.nature.com/scientificreports/ the goal of the study was to treat animals with aq using a similar dosing strategy as for human patients, with a target blood concentration range of the parent compound aq of . ± . ng/ml . indeed, we found that a mg/kg aq dose using the -day treatment regimen resulted in correlating plasma levels of . - . ng/ ml in healthy nhps. however, this treatment regimen did not result in a survival benefit or decrease of disease symptoms in ebov-infected animals. these results are disappointing and highlight the importance of utilizing relevant animal models of evd to evaluate potential antivirals, including detailed characterization of pharmacokinetics and tolerability in the context of evd. whereas aq treatment did not lead to detectable beneficial effects on evd progression in nhps, there may be value in investigating novel aq derivatives that have been improved for anti-ebov potency and/or improved tolerability . a possible impact from aq side effects needs to be considered. rare instances of acute liver injury usually after prolonged treatment have been reported , . for this reason, aq and the combination drug asaq are recommended for use as a malaria treatment in endemic areas, but not for prophylaxis against malaria. the onset of hepatic injury is often associated with agranulocytosis. elevated liver enzymes, alt and ast, were observed in the healthy nhps during the pharmacokinetics study. given that the evd patients in were given a co-formulation of aq and as, it would have been of interest to test the combined regimen rather than just aq to evaluate potential implications of the two drugs on disease progression. whereas as has no measurable anti-ebov effect when tested in combination with aq in vitro, the drug could have an in vivo effect that is not captured in cell culture assays. as and artemether are derivatives of artemisinin and are both metabolized to dha, the active metabolite for the treatment of malaria. however, as www.nature.com/scientificreports www.nature.com/scientificreports/ is water soluble, and reaches peak concentration more rapidly with higher plasma c max than artemether, which could impact in vivo activity against ebov. another consideration is that as and aq may affect each other in terms of pk, drug metabolism, or disposition. for example, as co-administration with aq has been reported to reduce exposure to aq . questions remain on the possible contribution of as to the beneficial effect of the asaq treatment observed in evd patients . therefore, testing the co-formulation would have value, but without testing each drug singly as well, attribution of any observed effect to a specific component will be challenging. other confounding factors not measured in the gignoux study could have accounted for the beneficial effect of asaq . the potential impact of concurrent malaria and the type of antimalarial used in evd patients was not addressed in our study. in conclusion, treatment with aq did not have a beneficial effect on survival or the symptoms of evd in rhesus macaques under the conditions tested. whereas aq on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of aq when used in combination with another antiviral. www.nature.com/scientificreports www.nature.com/scientificreports/ cells and virus. vero e (atcc crl- , manassas, va), hela (atcc ccl- ), and huh (human hepatocellular carcinoma) cells were maintained following recommended protocols. human monocyte-derived macrophages (mdms) were generated as previously described . ebola virus/h.sapiens-tc/gin/ /makona-c (ebov/mak, genbank accession no. kx . ) was propagated in vero e cells (bei resources, niaid, nih: vero c (e ), african green monkey kidney, working bank #nr- ) as previously described . virus stock and challenge inoculum titers were determined by plaque assay on vero e cells as previously described . drugs and treatment preparation. in vitro studies. the antimalarial artesunate-amodiaquine (asaq winthrop) (sanofi-aventis, gentilly cedex, france) was solubilized by crushing the tablets and resuspending in dimethyl sulfoxide to prepare a solution with the aq component at a concentration of mm. the compounds, amodiaquine dihydrochloride dihydrate (#a ), artesunate (#a ), and dihydroartemisinin (#d ) were purchased from sigma-aldrich (saint louis, mo). n-desethylamodiaquine hydrochloride (#sc- ) was obtained from santa cruz biotechnology (dallas, tx). drugs were prepared as mm stocks in dimethyl sulfoxide. animal studies. source, formulation, and preparation of amodiaquine hydrochloride was the same for the pk and the efficacy studies. amodiaquine hydrochloride was obtained from us pharmacopeia (rockville, md; cat. ; lot j ) and a mg/ml aq (free base) solution was prepared in sterile water for injection (usp). cell-based testing of ebov antiviral agents. the cell-based ebov drug screen and cytotoxicity assays were performed as previously described . briefly, vero e and huh cells were seeded at × cells/well, and mdms at × cells/well in -well plates. after h, cells were treated with compounds at -fold dilutions starting from µm. the starting concentration of the asaq tablet suspension corresponded to µm of the aq base component. cells were infected with ebov/mak h after the addition of the drugs in biosafety level (bsl ) containment at multiplicity of infection (moi) of . - . . after h, plates were fixed with % neutral buffered formalin (richard-allan scientific), and ebov/mak was detected with a mouse antibody specific for ebov vp protein (#b-md -bd -ae , usamriid) followed by staining with alexa fluor ® goat anti-mouse igg (heavy + light chain) antibody (life technologies, grand island, ny) or with anti-mouse igg-peroxidase labeled antibody (kpl# - ). fluorescence or luminescence was quantified on a plate reader (infinite ® m pro, tecan us, morrisville, nc). the signal of treated infected wells was normalized to uninfected control wells and measured (in percent) relative to untreated infected wells. non-linear regression analysis was performed, and the % inhibitory concentrations (ic s) were calculated from fitted curves (log [agonist] versus response [variable slope] with constraint to remain above %) (graphpad software, la jolla, ca). the ebov drug screen assay was performed with three replicates for each drug concentration, and the assay was repeated at least twice for confirmation. to evaluate cytotoxicity, cells were treated with compounds as described above in absence of virus. at h after drug addition, cell viability was quantified using the celltiter glo luminescent cell viability assay kit (promega, madison, wi). www.nature.com/scientificreports www.nature.com/scientificreports/ pharmacokinetic analysis of amodiaquine in nonhuman primates. rhesus macaques (macaca mulatta) were obtained from covance research products (princeton, nj). two groups (n = , females and males per group) were dosed with or mg/kg amodiaquine hydrochloride (us pharmacopeia, rockville, md; cat. ; lot j ) orally via nasogastric intubation once daily for days. nhps were housed in stainless steel primary enclosures singly or in pairs if compatible. nhps were provided teklad certified global % protein primate diet (# c) and purified water ad libitum. clinical observations were conducted for days including the day of dosing. blood (maximal μl) was collected from cephalic vessels on day and day . on day , blood was collected pre-dose, . , , , , , , , and h post-dose (immediately prior to dosing on day ). on day , blood was collected h after dose (or h after dose ), immediately prior to dosing on day , and . , , , , , , , and h after dose . drug concentrations of aq and its metabolite, deaq, were determined in collected plasma samples using liquid chromatography with tandem mass spectrometry (lc-ms/ms). plasma samples (volume μl) were prepared by adding ml of methyl t-butyl ether. each tube was vortexed for approximately minutes (min) at maximal speed and centrifuged for min at , g to facilitate separation of the liquid phases. upper layers ( μl) were transferred to new tubes, and the solvent was removed under vacuum in a centrifugal evaporator. the dried residues were reconstituted with μl of internal standard solution ( ng/ml risperidone and ng/ml chloroquine in : [v:v] acetonitrile:water). the tubes were vortexed min and clarified by centrifugation ( , g) for min. the clarified extracts were transferred to high performance liquid chromatography vials containing glass inserts for subsequent lc-ms/ms analysis. lc-ms/ms was performed using a lc- ad pump system (shimadzu, columbia, md) and a qtrap mass spectrometer (sciex, concord, ontario, canada) in multiple reaction monitoring mode and a polaris c -a column ( × . mm, μm; agilent, ca), using gradient elution with . % formic acid in water and . % formic acid in acetonitrile as the mobile phase. the lower limit of quantitation for aq and deaq of the method was ng/ml. the following parameters and constants were determined: maximal plasma concentration (cmax), time to maximum plasma concentration (tmax), area under the plasma concentration-time curve to the last time point and extrapolated to infinity (auc last and auc inf ), terminal elimination half-life (t / ), apparent volume of distribution (v/f), and total clearance (cl/f) after oral administration. pk parameters were analyzed using phoenix ® winnonlin ® software (v . ; certara, princeton, nj) to perform noncompartmental data analysis for extravascular administration. for clinical pathology (serum chemistry, hematology), blood ( ml) was collected prior to dosing on day and approximately h after the final day dose. treatment and challenge of nonhuman primates. rhesus macaques of chinese origin (n = , adult, < years of age) were obtained from charles river laboratories (frederick, md). animals were singly housed in stainless steel primary enclosures and provided chow and purified (reverse osmosis) water ad libitum. the vehicle control group (group ; n = ) was treated with sterile water. two treatment groups were treated with aq orally once daily as a consecutive -day course of mg/kg free base ( mg/kg amodiaquine hydrochloride (us pharmacopeia, rockville, md; cat. ; lot j ). treatment began on the day of ebov exposure (group ; n = , females and males), or on day postexposure (group ; n = , females and males). all groups were challenged im with a target dose of pfu) of ebov/mak (actual dose = pfu). the plaque assay back-titration of the challenge material was initiated on the day of preparation (study day ) on vero e cells, and plaques were fixed with % formalin and stained with crystal violet days later. animals were observed and weighed daily. the vehicle control group (group , n = ) received an equivalent volume of sterile water (gibco, cat. a , lot ). weight was recorded on day - and day - , then daily starting one day before challenge until primates succumbed to disease. responsiveness of the animals was monitored following -point range, in which a score of met primary endpoint criteria and initiated secondary criteria evaluation (table ) . when nhps scored a for primary euthanasia criteria and their temperature was above °c, secondary euthanasia criteria were reviewed. an nhp had to meet at least two secondary criteria, blood urea nitrogen ≥ mg/dl, calcium ≤ . mg/dl, ggt ≥ u/dl, and/or creatinine ≥ . mg/dl, for required euthanasia. two rhesus macaques were euthanized based on their secondary criteria. six of the fifteen rhesus macaques were euthanized based on veterinary discretion, not based on their clinical score or secondary criteria. hematology and serum chemistry. complete blood count with leukocyte differential was performed from peripherally collected blood samples using vacuette k ethylenediaminetetraacetic acid (edta) tubes and a sysmex xt- iv hematology instrument using a preprogrammed monkey species profile (sysmex america, ny). plasma and serum were prepared by separation for min at ambient temperature with centrifuge set to rcf. serum chemistries were performed using the piccolo xpress analyzer and general chemistry discs (abaxis, abbott point of care, nj) from vacuette z serum clot activator tubes (greiner bio-one, monroe, nc). pathology and histology. all animals were humanely euthanatized in accordance with defined experimental endpoints, and gross necropsy was performed. tissue samples were inactivated by fixing for h in % neutral buffered formalin. following fixation and removal from the bsl- laboratory in accordance with standard operating procedures, tissue samples were routinely processed in a tissue-tek vip- automated vacuum infiltration processor (sakura finetek usa, torrance, california, usa) followed by paraffin embedding with a tissue-tek model tec unit (sakura finetek usa, torrance, ca, usa). using a standard semiautomated rotary microtome and lighted water flotation bath (leica biosystems, wetzlar, germany), tissue sections were cut at a thickness of µm and mounted on positively charged uncoated glass slides (thermofisher, waltham, ma, usa), air-dried at room temperature, stained with hematoxylin and eosin (h&e), and coverslipped for microscopic evaluation by the pathologist. plasma samples from rhesus macaques that had received a dose of aq at integrated research facility/ niaid (fort detrick, md) were analyzed for concentrations of aq and the metabolite deaq. prior to shipment to sri, the samples were diluted : . and irradiated at mrad using a cobalt- source to destroy any pathogens that may have been present in the plasma samples. a pilot study with spiked quality control standards confirmed that irradiation does not appreciably alter the level of aq in the plasma (data not shown). a liquid:liquid extraction method with methyl t-butyl ether was used to separate aq and deaq from rhesus macaque plasma. samples were analyzed by lc-ms/ms with a lower limit of quantitation (lloq) of ng/ml ( . ng/ml × . dilution factor) in plasma for both aq and deaq. aq and deaq concentrations were adjusted for the dilution factor of . for analysis in figs. and . the virus 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inhibit ebola virus infection amodiaquine induced agranulocytosis and liver damage tolerability and safety of artesunate-amodiaquine and artemether-lumefantrine fixed dose combinations for the treatment of uncomplicated plasmodium falciparum malaria: two open-label, randomized trials in nimba county evaluation of the activity of lamivudine and zidovudine against ebola virus high dose sertraline monotherapy fails to protect rhesus macaques from lethal challenge with ebola virus makona pathology of experimental ebola-zaire (mayinga) virus infection transmitted to guinea pigs by oral, conjunctival and tonsillar routes the findings and conclusions in this report do not necessarily reflect the views or policies of the us department of health and human services or of the institutions and companies affiliated with the authors. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - mmlk authors: zhang, yun; wei, ying; liu, kang; huang, mengjiao; li, ran; wang, yang; liu, qiliang; zheng, jing; xue, chunyi; cao, yongchang title: recombinant influenza h n virus with a substitution of h hemagglutinin transmembrane domain showed enhanced immunogenicity in mice and chicken date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: mmlk in recent years, avian influenza virus h n undergoing antigenic drift represents a threat to poultry farming as well as public health. current vaccines are restricted to inactivated vaccine strains and their related variants. in this study, a recombinant h n (h n -tm) strain with a replaced h hemagglutinin (ha) transmembrane (tm) domain was generated. virus assembly and viral protein composition were not affected by the transmembrane domain replacement. further, the recombinant tm-replaced h n -tm virus could provide better inter-clade protection in both mice and chickens against h n , suggesting that the h -tm-replacement could be considered as a strategy to develop efficient subtype-specific h n influenza vaccines. h n vaccine with the tm-replacement presented broadened protection with promoted hi titers in vaccinated animals using antiserum. furthermore, the level of ifnγ was also increased, when inactivated h n viruses were used as stimulant . therefore, the ha tm domain is considered to be a potential candidate site for vaccine development. in this study, we generated a recombinant h n wild type strain (h n -wt) and a recombinant h n strain with a h -tm domain replacement (h n -tm) utilizing reverse genetics system. the biological characteristics and immunogenicity between the two viruses were compared. our results showed that the replacement of transmembrane (tm) domain did not affect the virus assembly and viral protein composition in the recombinant h n viruses. however, the biological characteristics, such as virus growth, ratio of trimer, thermal stability, acidic resistance and fusion activity were altered, suggesting an important role of the tm domain in viral replication and pathogenicity. furthermore, the tm-replaced h n -tm strain exhibited better protection in both mice and chicken when challenged against different phylogenetic h n clades. replacement of h ha tm domain did not affect the assembly and viral protein compositions of recombinant h n viruses. to understand whether change of transmembrane (tm) domain can affect virus structure, we first observed the morphology of tm-replaced viruses rescued by reserve genetics. applying electron microscope, the recombined tm-replaced virus (h n -tm) showed typical surface spikes as the recombined wildtype (h n -wt) (fig. b) , suggesting the replacement of the transmembrane (tm) domain did not change the surface structure of the virus. sds-page showed that the expression levels of ha , ha , ha , np, and m proteins were comparable in the two viruses (fig. c) . full-length blot is presented in supplementary figure a . these results suggest that the replacement of h ha transmembrane (tm) domain does not affect the assembly and viral protein compositions of recombinant h n viruses. to further investigate whether the biological characteristics of the recombinant viruses were changed through the transmembrane (tm) domain replacement, mdck cells were infected with the two recombinant viruses. we found the tm-replaced virus (h n -tm) formed smaller plaques than the recombinant wildtype virus (h n -wt) ( fig. a) , suggesting a slow viral replication due to the transmembrane (tm) domain replacement in cells. this was further confirmed when growth curves was measured that the pfu titers of recombinant h n -tm were lower than that of h n -wt (fig. b) . the pfu titers of h n -tm were significantly decreased at h (p < . ), h (p < . ) and h (p < . ) post-infection, suggesting tm-replacement may have an effect on delaying virus replication. when inoculated in chicken embryos, the h n -wt showed . log eid / . ml for virus titer, while the recombinant h n -tm showed . log eid / . ml (fig. c) , indicating a low infectivity of h n -tm in chicken embryos. furthermore, our data suggest that the delay of the virus replication may affect virulence of the virus in chicken embryos. in influenza viruses, the stalk domain (ha ) is crucial for the stabilization of the ha trimer, in which its transmembrane (tm) domain plays an important role in virus anchor. to further investigate whether tm-replacement may affect the stability of the ha trimer, we analyzed content of covalent bond-linked trimers of the ha protein through non-reducing western blot. the result showed that the recombinant h n -tm had an increased trimeric composition of the ha under non-reducing condition, comparing to h n -wt (fig. a,b) . full-length blot is presented in supplementary figure b . the trimeric ha is important for receptor binding and membrane fusion activity. to analyze whether the fusion activity of recombinant viruses was altered, we utilized virus-induced erythrocyte hemolysis assay. at low ph ( - ), the trimeric ha undergoes conformational changes that trigger membrane fusion. there was no difference in fusion ability at ph . to ph . between the h n -wt and h n -tm recombinant viruses. however, h n -tm showed poorer fusion activity than that of h n -wt in the ph ranging from . to . (p < . ) (fig. c ). this data suggests that the tm domain plays an important role in restricting the virus-induced membrane fusion activity in acidic environment. to further investigate whether the alteration of fusion ability is due to a change in viral biological characteristics such as thermal or acidic resistances, we incubated the recombinant viruses at different temperature from °c to °c. the recombinant viruses h n -wt and h n -tm had similar thermal resistance under °c. and their ha titers decreased gradually while the temperatures increased. from °c, the ha titers of the recombinant h n -wt declined more than the h n -tm and the difference was significant (p < . ) (fig. d ). this data indicates that the recombinant h n -tm virus had an increased thermal resistance. we next examined the infectivity of the recombinant viruses in mdck cells in acid environment (ph . , . , . ). fluorescent labeled viruses could be detected in each sample at ph . (fig. e) , suggesting that the infectivity of recombinant viruses remained normal at a neutral environment. the fluorescence receded rapidly at ph . in both groups. at ph . , the fluorescence of the recombinant h n -wt could be barely detected, while the fluorescence of h n -tm could be still detected though the signal was weak. these data suggest that the recombinant h n -tm remains acid resistance at low ph ( . ), thus is still infectious under acid environment. taken together, the increased thermal and acidic resistances of the recombinant h n -tm virus suggest that the substitution in the transmembrane (tm) domain can affect the stability of the ha, therefore alters viral biological characteristics. to explore whether the increased ha trimers and increased thermal and acidic resistances could result in better immune responses thus providing better protection in mice, we first vaccinated six-week-old mice with inactivated recombinant h n -wt and h n -tm viruses twice to check antibody responses. a significant increase of the serum ha-specific igg antibody titers was observed in the h n -tm group (p < . ) (fig. a) . to determine the level of protection against challenges of homologous h n virus, mice were then challenged with x mld of mouse-adapted wild-type a/chicken/guangdong/yys / (h n ) virus in a different clade (fig. ). all mice in the control group succumbed to infection by day post infection, while all animals were completely protected in the h n -tm vaccinated group (fig. b ). comparing to the h n -tm vaccinated group, mice immunized with h n -wt showed significant weight loss as the pbs group from day post infection (fig. c) . these data indicate that tm-replacement could provide better cross-protection against inter-clade challenge in mice. since h n is an avian originated virus, we next explored whether a better immune responses could be induced by h n -tm in chickens. we performed elisa assay and hi assay to analyze the antibody responses in vaccinated chickens. in elisa, h ha protein was utilized as antigen. similar as the antibody response results in mice, we found that the igy titers of h n -tm vaccinated group were significantly higher, comparing to that of the h n -wt group (p < . ) (fig. a) (fig. ). when viruses from clades . . and . . were used as antigens, the hi titers of h n -tm vaccinated chicken sera were comparable with the h n -wt vaccinated group. however, when viruses from clades . . and . . were used as antigens, the hi titer of h n -tm group was significant higher than that of h n -wt (p < . ) (fig. b) . to further investigate the cross-protection reaction through tm replacement, chickens were challenged using different phylogenetic h n viruses from clade . . , . . and . . (table ) (fig. ). virus shedding determined by ha titers was utilized to elucidate protection provided by vaccines. chickens immunized with h n -wt and h n -tm showed complete protection challenge by clade . . . virus shedding was found in all groups immunized with pbs. no virus shedding was found in all groups immunized with h n -tm. at day post infection, the shedding rates of h n -wt groups was % (virus shedding was found in out of chickens) and . % (virus shedding was found in out of chickens), which are more than the h n -tm vaccinated groups, when challenged with viruses from branch . . or branch . . respectively. furthermore, the ha titers of virus shedding in h n -wt vaccinated group was significantly higher (p < . ) than that of (fig. c) . the ha titers of d.p.i were comparable in all h n -wt and h n -tm vaccinated groups (fig. d ). the differences in virus shedding suggest a better inter-clade cross-protection when the recombinant h n -tm virus was used as vaccine. in conclusion, our data suggest that the recombinant tm-replaced h n -tm recombinant virus can provide better inter-clade cross-protection than the wild type. one characteristic of influenza a virus is that it undergoes rapid antigenic variation, especially under immunological pressure. mutations on hemagglutinin (ha) and neuraminidase (na) usually cause antigenic drift and antigenic shift, thus resulting in virus immune escape. therefore, it is difficult for the existing avian influenza inactivated vaccines to deal with the new strains, due to lack of cross-immune protection. take h n viruses in china for instance, which was previously considered to share similar antigenicity profiles , , vaccine strains and their relevant variants are mainly used for h n virus protection in china. however, current existing vaccines could not fulfill their task to prevent the outbreaks of h n in - in poultry farming of china . moreover, recent studies found that some h n influenza viruses were originated from the vaccine strains through antigenic drift and these antigenic variants finally caused the catastrophe in poultry industry of china in - , even under the long-term vaccination programs . therefore, to prevent next outbreaks of further antigenic h n variants caused by immunological pressure, an effective, safe and subtype-specific h n vaccine which can provide cross-protection is required. the ha stem has recently drawn attention to be a new potential influenza candidate site for universal vaccine development. since the discovery of a series of broadly neutralizing monoclonal antibodies, the majority of which target to highly conserved region of ha stem, researchers began to discuss the potential to induce broad protective immunity [ ] [ ] [ ] [ ] . various attempts were conducted to stabilize the ha stem in order to induce broad protective immunity. the stability and immunogenicity of the stabilized ha proteins through fusion with trimerization sequences have shown increased broad protective immunity. for instance, researchers showed when the ha protein was fused to the foldon domain of fibritin from bacterial phage t or gcn p trimerization repeat, the fusion proteins showed increased stability and cross-reactive immunity , . further researchers showed that fused ectodomain of ha protein to ferritin could form nanoparticles and the nanoparticle vaccine could improve the potency and breadth of influenza virus immunity , suggesting the importance of ha protein stability in enhancing cross-protection. this plausible correlation of the major surface antigen stability and cross-immunity was also supported by other researches using s protein of sars virus that the trimerized forms could elicit higher levels of neutralizing antibodies . the biological significance of ha stability remains unclear and future work is required. our previous studies found that the transmembrane domain of h subtype ha protein had a unique microdomain, which was related to the stability and cross-immunity of influenza virus , [ ] [ ] [ ] . furthermore, we found that h -wt tm-dependent cross-protection could be transferred to other subtypes by replacing their tms with h -wt tm , suggesting a plausible correlation of ha stability and cross-immunity. recently, we developed a tm-replaced h n vaccine. mice vaccinated with h n -tm vaccine strain showed increased cross-reactive antibodies and were well protected against interclade h n viruses . consistent with the previous studies, our data indicated that tm replacement could actually alter the physical and chemical characteristics of h n virus. the delayed virus replication in tm-replaced viruses may contribute to low pathogenesis. the increased thermal and acidic resistances suggest that a modification on transmembrane (tm) domain might lead to structural change of the ha protein, thus leading to an alteration of the biological characteristics in the recombinant virus. when used as a vaccine, the recombined tm-replaced h n strain (h n -tm) could perform better subtype-specific protection and inter-clade cross-protection against different phylogenetic h n viruses compared to that of recombinant h n -wt in both mice and chickens, suggesting the tm-replaced h n vaccines are efficient in different species. the elicited ha-specific antibody level was comparable in both h n and h n viruses, when tm-replacement was introduced in the vaccine strains. therefore, our results have suggested a universal technique to improve the existing influenza h n inactivated virus vaccine. furthermore, according to our researches, this technique could be used to develop vaccines of various types of avian influenza viruses. this tm-replacement technique suggests a way to improve the existing vaccine immune broad-spectrum activity, prolong the shelf life of the vaccine. and therefore, it is a good candidate for prevention of avian influenza and the development of new generation of vaccines in the future. in conclusion, in this study, our results demonstrated that inter-clade cross-protection could be enhanced with tm replacement and therefore further suggested a plausible correlation of ha stability and cross immunity. our results demonstrate that the tm-replaced vaccine could be a more effective influenza vaccine candidate and thus suggest a strategy to develop effective subtype-specific h n vaccine or even heterosubtypic vaccines against influenza viruses. other questions like whether the effect of tm-replacement is h specific, which segment is responsible for transmembrane domain function, whether tm-replacement changes the virus structure and how tm-replacement influences virus's biological characteristics still remain unknown. and future work is required to answer these questions. virus rescue and reverse genetics. the plasmid phw was used for reserve genetics as described previously . to construct recombinant h n virus containing a tm from h ha, strain a/chicken/ guangdong/ / (h n ) (genbank accession no. kf . ) was selected and the tm domain was replaced with a tm from h ha using overlap pcr (fig. a) . for virus rescue, t cells were transfected with eight genome-sense plasmids using x-tremegene dna transfection reagent (roche) according to manufacturer's instruction. thirty six hours after transfection, tpck-treated trypsin (sigma) was added to the cells with a final concentration of . - μg/ml. the transfected cell culture supernatant was collected at - h post-transfection and used to passage onto -day-old spf embryonated chicken eggs for the propagation of the recombinant viruses. the rescued recombinant virus containing wt ha was designated as h n -wt, whereas the rescued virus containing h ha was designated as h n -tm. virus propagation and purification. both recombinant h n -wt and h n -tm viruses were propagated in -day-old spf embryonated chicken eggs. after h, the allantoic fluids were collected and inactivated with . % β-propiolactone (bpl) at °c for h. the inactivated viruses were tested by performing serial passages on spf embryonated chicken eggs. the purification was performed by centrifugation on - % sucrose density gradients as described previously . western blot and electron microscopy. ds-polyacrylamide gel electrophoresis (sds-page) was performed to validate the purified virions. non-reducing western blot was performed as previously described . anti-h n mouse serum ( : ) was used as a primary antibody. the negative staining of purified recombinant virions was done as described previously . briefly, virions were stained by the phosphotungstic acid buffer and the shape was photographed on jem- cx-ii electron microscope (jeol). thermal and acidic resistance assays. the resistance assay was performed as described before . briefly, the viruses with the same ha titer were incubated at a temperature ranging from to °c for min. the ha titres were measured subsequently when viruses were cooled down to room temperature. in the acidic resistance assay, the viruses with the same pfu were incubated in an acidic buffer ( mm hepes, mm mes in pbs) at ph . , . , and . at °c for min. the solutions were then adjusted to ph . . mdck cells were infected with the recombinant viruses in -well plates at an m.o.i. of for min at °c, then the media were replaced with serum-free media containing μg/ml tpck-trypsin. the infected plates were fixed with % paraformaldehyde, permeabilized with . % troton x- in pbs, and stained with the fitc-labeled mab against np (abcam). the cell images were taken under inverted fluorescence microscope (zeiss). virus-cell fusion assay. according to standard protocol described previously , viruses standardized to ha units were mixed with % chicken red blood cells (rbc). the ph was adjusted from . to . with addition of the sodium citrate buffer. after min incubation, supernatants were transferred to an elisa plate for determination of nadph content by optical density measurement ( nm) with a bio-tek elisa plate reader (bio-tek instruments). baseline nadph activity values were derived from samples without viruses that underwent identical treatment. scientific reports | ( ) : | doi: . /s - - -x animal challenge. -day-old spf chickens were housed in individual isolators under positive pressure and randomly divided into three groups. each group (n = ) was subcutaneously immunized together with mineral oil adjuvant once. blood samples were then collected two weeks after immunization. three weeks after immunization, vaccinated chickens were challenged intranasally with a dose ( × eid ) of the following h n viruses, a/chicken/guangdong/yys / (h n ) (genbank accession no. kj . ), a/chicken/ henan/ / (h n ) (genbank accession no. af . ) and a/ hebei/ / (h ) (genbank accession no. kc . ). swabs of the larynx and cloaca were collected at and days post infection to inoculate spf chicken embryos subsequently. allantoic fluid were collected after hours incubation at °c to determine ha titres. samples were regarded as expelling viruses when titers ≥ . six-week-old female balb/c mice were selected and intramuscularly immunized by inactivated h n -tm and h n -wt viruses, with freund's incomplete adjuvant respectively, twice on week and . blood samples were collected at week .three weeks after booster immunization, mice were challenged intranasally with mouse adapted a/chicken/guangdong/yys / (h n ) ( × mld ). survival rate and weight loss were monitored daily after the challenge. elisa for anti-ha igg antibodies. ha-specific anti-chicken immunoglobulin y (igg) isotype antibody titers in chicken sera and igg antibody titers in mice sera were determined using enzyme-linked immunosorbent assay (elisa). h ha protein with a concentration of μg/ml were coated, incubated with serial dilutions of each serum sample( °c for h) and detected by hrp-conjugated goat anti-chicken igg (proteintech) and hrp-conjugated goat anti-mouse igg antibodies (proteintech). optical densities were read at nm using a spectrophotometer (bio-tek). hemagglutination inhibition assay. μl of each influenza virus of four ha units was used in the hi assay. each sample was treated with receptor destroying enzyme (rde) and diluted in a -fold serial. the highest dilution of the serum able to inhibit hemagglutination was defined as the hi titer. statistical analysis. data were presented as mean ± sem from at least three independent experiments. unless otherwise noted, statistical analyses were performed using student's two-tailed t test. difference was considered statistically significant at p < . . data availability. the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. taken together, our data indicate that h tm domain is critical for biological characteristics and inter-clade cross-protection of influenza viruses, and subtype-specific h n cross-protection can be enhanced with h -tm replacement. our results suggest a 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work was supported by the guangdong science and technology plan ( a ); the guangdong natural science foundation ( a ); the guangzhou science and technology plan ( ) and "zhujiang talent program" overseas youth talent introduction program (post-doctoral program). y.c. and y.z. designed the experiments; y.z. and y.w. wrote the main manuscript text; y.z., k.l., y.w., m.h. j.z. and y.w. performed the experiments; y.z. prepared the figures and tables; r.l. and q.l. analyzed the data; c.x. revised the manuscript text. all authors reviewed the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - -x.competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -u xa nw authors: rodriguez, sergio e.; cross, robert w.; fenton, karla a.; bente, dennis a.; mire, chad e.; geisbert, thomas w. title: vesicular stomatitis virus-based vaccine protects mice against crimean-congo hemorrhagic fever date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: u xa nw crimean-congo hemorrhagic fever virus (cchfv), a tick-borne bunyavirus, can cause a life-threatening hemorrhagic syndrome in humans but not in its animal host. the virus is widely distributed throughout southeastern europe, the middle east, africa, and asia. disease management has proven difficult and there are no broadly licensed vaccines or therapeutics. recombinant vesicular stomatitis viruses (rvsv) expressing foreign glycoproteins (gp) have shown promise as experimental vaccines for several viral hemorrhagic fevers. here, we developed and assessed a replication competent rvsv vector expressing the cchfv glycoprotein precursor (gpc), which encodes cchfv structural glycoproteins. this construct drives strong expression of cchfv-gp, in vitro. using these vectors, we vaccinated stat- knock-out mice, an animal model for cchfv. the vector was tolerated and % efficacious against challenge from a clinical strain of cchfv. anti-cchfv-gp igg and neutralizing antibody titers were observed in surviving animals. this study demonstrates that a rvsv expressing only the cchfv-gp has the potential to serve as a replication competent vaccine platform against cchf infections. crimean-congo hemorrhagic fever virus (cchfv) is a highly pathogenic zoonotic agent in the orthonairovirus genus within nairoviridae . the principal reservoirs for cchfv are ixodid hard ticks primarily belonging to the genus hyalomma . these ticks maintain the virus in nature by feeding on small mammals, ungulates, and birds within thirty countries across the eastern hemisphere . human infection can occur from the bite of an infected tick, exposure to infected animal products, or through nosocomial transmission . cchfv case-fatality rates in most outbreaks range from - %, though higher rates have been documented in some instances , . cchfv is categorized as a category a priority pathogen by the us national institutes of health due to its associated morbidity and mortality, potential for public health/societal impact, as well as a lack of approved therapeutic options or us/eu licensed vaccines for treatment. the guanosine analogue ribavirin has been suggested as a therapeutic when given early in human infections; however, efficacy has not been clearly demonstrated in clinical trials for cchf . for these reasons, vaccines and therapeutic countermeasures against cchfv are currently under development. full or partial mature cchfv particles contain single stranded, tri-partite, negative sense rna genomes with small (s), medium (m), and large (l) segments, respectively encoding the structural nucleoprotein (np), two envelope proteins (g n and g c ) and the viral rna-dependent-rna-polymerase (rdrp) . the m-segment contains a . kilobase open-reading frame which codes for a glycoprotein precursor polypeptide (gpc) . host cell processing, cleavage events, and post-translational modifications of this gpc yield the two mature structural glycoproteins g n and g c , along with several non-structural glycoproteins which aid in structural g n and g c maturation [ ] [ ] [ ] [ ] [ ] [ ] . the two glycoproteins are likely responsible for pertinent events in the viral replication cycle such as viral attachment, cell entry, tissue tropism(s), and induction of protective immune response as seen similarly with other members of bunyavirales , . the latter has been demonstrated for cchfv using monoclonal antibodies (mab) directed against g n and g c , which have demonstrated in vitro neutralization in tissue culture and in vivo passive protection in suckling mice [ ] [ ] [ ] . these data suggest that the gpc would be an important antiviral target for therapeutic and vaccine efforts. our initial attempts using the dna clone recovery system designed by lawson et al., failed to produce infectious Δgrvsv with cchfv-gpc (fig. a) . to overcome this, we used a modification based on a system described by whitt relying on in trans vsv glycoprotein (g) complementation (vsv-g*) of Δgrvsv virions. this technique allowed for vsv-g, incorporation into recoveries to facilitate efficient assembly of the rvsvΔg-cchfv-gpc genome without the need for cchfv-gpc to participate in initial infection of recovered virions (fig. a) . we recovered a virion containing the cchfv-gpc in the genome with vsv-g complementation (designated vsv-g*-Δgrvsv-cchfv-gpc), which contributed to a single-cycle infection; unless vsv-g is provided in trans this virus will not replicate effectively in cell culture. after the initial recovery of vsv-g*-Δgrvsv-cchfv-gpc, this virus was passaged on vsv-g complemented bhk cells and passaged onto un-complemented ('normal') bhk cells. we were unable to isolate infectious virus from initial supernatants, however, seven total serial passages of supernatants on un-complemented bhk cells resulted in eventual cytopathic effect (cpe) in cell culture with foci/plaque formations appearing on the monolayers. these monolayers with cpe were harvested for rna and were stained for cchfv-gpc antigens via immunofluorescence assay and found to be positive (data not shown). this replication competent construct was designated Δgrvsv-cchfv-gpcΔ (fig. b) . sanger sequencing of both constructs, using primers for the vsv backbone and cchfv-gpc orf, was carried out which confirmed a rvsvΔg-cchfv-gpc genome and revealed several single nucleotide polymorphisms (snp) (data not shown). next generation sequencing (ngs) was then performed to confirm sanger results and further detail the snps within the entire genomes of both constructs (supplemental table ). ngs sequencing demonstrated fourteen identical single nucleotide polymorphisms (snp) preserved between the replication deficient and replication competent constructs; with the replication competent construct possessing four additional non-synonymous mutations within the cchfv-gpc (fig. b , supplemental table ); henceforth referred to as gpcΔ. two of these mutations within the cchfv-gpc of the replication competent construct, resulted in the truncation of fourteen amino acids off the c-terminal tail of the g c (fig. b) . to assess the growth kinetics compared to authentic cchfv, we performed single-cycle growth curve analysis on bhk cells infected with respective viruses at various time intervals up to hrs post infection (hpi) or full monolayer destruction. the rvsv-gfp (wild-type control) peaked in titer at approximately hpi, while cchfv prototype-strain: ibar peaked at hpi ( fig. a) . the replication competent pseudotype, Δgrvsv-cchfv-gpcΔ had peak titer, at approximately hpi ( fig. a) . to assess the expression of the cchfv-gpc in the vector, we performed an immunofluorescence assay, using mabs that bind to either the cchfv-g c or -g n . the assay revealed strong in vitro expression of both antigens (fig. b) . we additionally examined if cchfv-g c was incorporated onto the rvsv virion. we used coomassie staining and western blot analysis on gradient purified Δgrvsv-cchfv-gpcΔ and semi-purified rvsv-gfp and cchfv (as controls). through coomassie staining, differences in number and mobility of protein bands were detected among the wt rvsv-gfp and Δgrvsv-cchfv-gpcΔ (fig. c , lanes , ). the cchfv-gpc recombinant had additional protein bands below kda with analogous bands detected in cchfv virion pellets (fig. c , lanes , ). the cchfv-gpc recombinant also possessed more pronounced protein bands at kda, compared to our wt rvsv-gfp (fig. c , lanes , ). cchfv structural glycoproteins g n and g c present as bands of approximately at kda and kda, respectively (fig. c , lane ), on sds-page gels . there was no observable g n band found on the cchfv-gpc recombinant at kda; however, there were two distinct bands between - kda in the recombinant (fig. c, lanes ) . the Δgrvsv-cchfv-gpcΔ did not encode for a vsv-g (which was confirmed by our deep sequencing shown in supplemental table ), nor was it complemented with vsv-g after seven rounds of bhk passaging. a duplicate protein gel was run and further probed by western blotting using a mab previously identified to be specific for cchfv-g c by western blot (mab e , bei resources) as there is currently, no available antibody that probes solely mature g n by western blot. these western blot data demonstrated limited, non-specific binding to three vsv proteins as observed in lanes rvsv-gfp (fig. d , lane ), but showed strong signal to at least two antigens at approximately kda and kda for Δgrvsv-cchfv-gpcΔ (fig. d, lane ). this indicated that mature cchfv-g c and potentially a precursor molecule or an oligomeric form of cchfv-g c (due to the lack of β-mercaptoethanol in antigen preparations), are incorporated in/on Δgrvsv-cchfv-gpcΔ virions. the semi-purified cchfv also showed limited non-specific binding to antigens at kda (cchfv-np) and kda (cchfv-g n ), but distinct signal at kda (cchfv-g c ) (fig. d , lane ). to examine the ultrastructure of the Δgrvsv-cchfv-gpcΔ, transmission electron microscopy studies were conducted. particles of rvsv-gfp ('wild-type' vsv electron microscopy control) were observed between - nm (fig. e , top). consistent with other rvsv pseudotyped with bunyavirus gp , our Δgrvsv-cchfv-gpcΔ pseudotype maintained rhabdovirus morphology and classical bullet shape with coiled intra-virion structure (fig. e , bottom). particles were observed to have lengths ranging between - nm (fig. e , bottom). this apparent length increase of the Δgrvsv-cchfv-gpcΔ is likely due to the genome containing approximately , extra nucleotides in comparison to the rvsv-gfp genome (which is also slightly larger than native wild-type vsv indiana [genbank number nc_ . ]). additionally, immunolabeling of Δgrvsv-cchfv-gpcΔ was employed with mab to cchfv-g c and counterstained with nm gold conjugated secondary mab (fig. e, bottom) . immunolabeling demonstrated labeling of g c spikes on the virion surface of Δgrvsv-cchfv-gpcΔ (fig. e, bottom) . with the data supporting that the replication competent construct expressed cchfv-gp in vitro and additionally expressed cchfv-g c on the surface of the virion, an in vivo study was designed to test the ability of the construct to function as an experimental vaccine. the stat- −/− mouse model for cchfv was selected to test these constructs for protective efficacy . in pilot studies (supplemental fig. ) the replication deficient construct (vsv-g*-Δgrvsv-cchfv-gpc), failed to provide protection; however the replication competent (Δgrvsv-cchfv-gpcΔ) construct did demonstrate some protection depending on dose. based on the results of these initial pilot studies, we elected to adjust vaccine and challenge doses, and administered pfu/dose of the replication competent virus (Δgrvsv-cchfv-gpcΔ) to prime and boosted groups of five stat- −/− mice, respectively (fig. ) . as a control, we used pbs as a mock vaccination. a single mouse succumbed in the boosted group (leaving only four mice to be challenged in the boosted group) the day of boosting. at days post prime, all mice were challenged with pfu of cchfv strain turkey and were monitored for clinical signs, weights, and temperatures. we challenged with a human/clinical strain, turkey , designated throughout this work as turkey ; which has previously been published in genbank with the accession numbers ky (s-segment), ky (m-segment), and ky (l-segment) . mean time-to-death (mtd) was . days post infection (dpi), with a standard deviation (sd) +/− . dpi as demonstrated with the pbs control group (fig. a ). for both prime and boosted groups, % protection was observed out to dpi (fig. a) . the prime www.nature.com/scientificreports www.nature.com/scientificreports/ and pbs group displayed weight loss, while the boosted group did not show weight loss (fig. b ). all three groups displayed elevated temperatures for the first three days post challenge (fig. c) . these results indicate that the primed group showed a marked illness, while the boosted group did not display observable disease. to examine the humoral response in vaccinated mice, sera from these groups were analyzed for both igg to cchfv-gp and for the presence of cchfv neutralizing antibodies. a separate group of animals was vaccinated (n = , per group) at similar prime/boost doses and bled at days − and − to assess pre-challenged immune status. circulating iggs to cchfv-gp were detected in sera of both vaccinated groups, beginning at day − and were found to have increased substantially at the end of study (fig. a) . curiously, at the study end point, the prime group displayed higher titers compared to the prime plus boosted group (fig. a ). this indicates the prime only group had a higher concentration of igg at the study endpoint which recognized cchfv-gp, compared to the boosted group. the reciprocal titer of the pbs group had negligible igg responses to cchfv-gp (fig. a) . to determine the neutralizing activity of these sera, a plaque reduction neutralization test (prnt) was carried out using the turkey challenge isolate. as depicted in fig. b , the prime only group had neutralizing antibody titers with a prnt of < : , and the boosted group a prnt of < : at the study endpoint (fig. b ). our prnt positive control, hyper-immune mouse ascitic fluid (hmaf, kindly provided by t. ksiazek, galveston, texas) raised against cchfv exposed mice, showed a prnt of < : while the pbs control mice did not demonstrate a prnt value (fig. b ). particle preparations were stained with α-cchfv-g c mab e using an hrp-conjugated secondary. (e) transmission electron micrographs of rvsv-gfp and replication competent Δgrvsv-cchfv-gpcΔ particles from bhk cells, semi-purified using a % sucrose cushion or purified using iodixanol gradient centrifugation, negatively stained with % aqueous uranyl acetate, immunolabeled with α-cchfv-g c mab e and a secondary nm gold labeled secondary. samples were fixed with % glutaraldehyde. images were adjusted for brightness, contrast, and formatted for size for display purposes. coomassie gel and western blot images were cropped from the same image file at same imaging parameters for ease of viewing. gel and blots originals are available in supplemental information. other original raw data files available upon request. www.nature.com/scientificreports www.nature.com/scientificreports/ additionally, at study endpoints for each cohort, we examined tissues by immunohistochemistry (ihc) for the cchfv-np antigen. the control cohort had marked cchfv-np immunolabeling in hepatocytes within the liver sections (fig. a ) while liver sections from the prime only and boosted cohorts had no observable www.nature.com/scientificreports www.nature.com/scientificreports/ cchfv-np immunolabeling (fig. a,b) . we further examined the spleen tissue sections and observed that the control cohort had marked cchfv immunolabeling in mononuclear cells (fig. d) , and that the prime cohort had a cytoplasmic, mild, and diffuse immunolabeling of mononuclear cells primarily in the red pulp (fig. e ). the boosted cohort had no specific cchfv immunolabeling within the spleen sections (fig. f ). studies have had varied success expressing cchfv-gpc in trans on viral vectors , . rvsv with cchfv-gpc has likely been challenging due to the numerous post-translational modifications required to mature, and yield functional, cchfv-gpc , , , . additionally, the cchfv-gpc can form immature cchfv particles at the golgi and egress via vesicular transport , . unlike cchfv, vsv buds from the plasma membrane , which hampers efforts to recover a replicating Δgrvsv vector with cchfv-gpc expressed. we used a mammalian www.nature.com/scientificreports www.nature.com/scientificreports/ codon optimized cchfv-gpc which has demonstrated expression of cchfv-g c to the cell surface , while maintaining native cchfv-gpc maturation factors. we hypothesized that this expression due to codon optimization could facilitate shuttling of cchfv-gp to the plasma membrane where vsv could acquire and bud with functional components of the gpc in the viral envelope. although we were able to drive strong expression cchfv-gpc in vitro, we were unable to generate a replication competent pseudotype virus without initially using vsv-g* in trans. there have been multiple studies that have examined pseudotyping rvsv with a cchfv-gpc using a plasmid encoding a vsvΔg backbone. a luciferase reporter pseudotype with in trans expression of the cchfv-gpc was developed in t cells by shtanko et al. . suda et al., also performed similar pseudotyping with in trans expression in t cells with various full length gpc and modified gpc constructs containing truncated g c c-terminal/endodomain tails . these pseudotypes also had a luciferase or gfp orfs genomically encoded in the vsvΔg backbone . both published pseudotyped cchfv-gpc systems were used for neutralization or entry/infection studies; however, these constructs were not characterized by western blot or immune electron microscopy to understand what is on the pseudotype envelope; nor were either constructs self-replicating or capable of further expressing antigen elements of cchfv-gpc post infection , . with the tools currently available, we were able to detect by western blot and electron microscopy, a form of cchfv-g c that was present and functional on the surface of the Δgrvsv-cchfv-gpcΔ virion. the current situation with reagents to mature cchfv-g n limit assaying for this particular antigen, as commercially available antibodies only target the precursor molecule cchfv-preg n . immune electron microscopy demonstrated that cchfv-g c was incorporated on the surface of the virion. while we were able to immunolabel for cchfv-g c , page and coomassie staining analysis of our purified virion lysates did not reveal prominent protein bands at the kda position, the estimated size of mature g n . nonetheless, we did have three smaller protein bands in the Δgrvsv-cchfv-gpcΔ preparations. similar protein profiles have been shown in other vectored cchfv-gpc preparations, as shown by buttigieg et al., . it is possible that these protein bands may also correspond to vsv-m and -m by alternative initiation at downstream start codons present in the orf of vsv-m . while not the focus of this current study, these observations warrant further characterization should this vector be used as a tool to further study cchfv-gpc mechanisms. suda et al., have shown an increase in the amount of infectious pseudotype the more the cchfv-g c tail is truncated, up to a deletion of residues at the end of the c-terminal cchfv-g c tail . our data supports this region as a probable, or at the very least, contributory mechanism which enables the replication competent Δgrvsv-cchfv-gpcΔ pseudotype formation, as our pseudotype also was found to contain a c-terminal cchfv-g c tail truncation (fig. b) . there is likely a localization or retention signal within the transmembrane region or endodomain tail which is aberrated and fails to transport cchfv-g c solely to the intracellular compartments; this may in turn permit shuttling to the plasma membrane. once again this is not the focus of this study, although it will be examined in the future. it is interesting to note, that a similar motif has been demonstrated by other bunyaviruses , and has also been demonstrated with other vsv pseudotypes . structural cchfv-g c has been shown to be an important protein for cchfv and contains a putative receptor binding region for mammalian cell surface nucleolin , a class ii viral fusion domain , a neutralization epitope , and human linear b-cell epitope sites , . our replication competent rvsv vector can enable further exploration of these components at lower containment (bsl- ), without the need for transfections for in trans expression of cchfv-gpc onto vsvΔg systems. when exploring the use of rvsv expressing cchfv-gp as potential vaccines in immunocompromised mice, there was a concern of murine virulence, which has been observed for wild-type vsv , . studies with rvsv expressing hemorrhagic fever virus gp have demonstrated lethal outcomes in the stat- −/− model . this has hampered the stat- −/− animal platform from serving as a vaccine development tool, as 'vaccinated' mice succumb to a prime dosing . because of this information we conducted several pilot studies examining effective dosing and challenge strategies, for both the replication deficient (vsv-g*-Δgrvsv-cchfv-gpc) and replication competent (Δgrvsv-cchfv-gpcΔ) construct. demonstrating the safety profile of both constructs (supplemental fig. ) , neither group had attributed fatality due to the vaccination; however, only our replication competent construct demonstrated protection. in selecting the cchfv challenge isolate, we chose an isolate with low passage history and demonstrated clinical relevance (i.e., documented history of disease in humans). the turkey isolate, came from a clinical case . elisa and prnt experiments on study endpoint sera, demonstrate a humoral igg response to cchfv-gpc with observed neutralizing antibodies produced from the prime group that received a high ( pfu) dose of Δgrvsv-cchfv-gpcΔ (fig. a,b) . curiously, our boosted ( pfu) group had lower detectable antibodies to the cchfv-gpc with lower neutralization titers (fig. a,b) . clinical data for these groups following cchfv challenge show that the prime only group had elevated temperatures and weight loss, additionally two animals from this cohort displayed clinical signs including roughed fur indicating illness from the cchfv challenge (fig. b,c, data not shown) . while the boosted group did display elevated temperatures, there was neither substantial weight loss detected nor observable changes in clinical scoring for any of the animals (fig. b ,c, and data not shown). further analysis of tissues at study end point for these cohorts also revealed that while there was no immunolabeling in the liver of the vaccinated animals, the prime cohort had diffuse cytoplasmic immunolabeling of mononuclear cells in the spleen sections (fig. e ). this finding, along with the clinical scores, suggested that there was more replication of cchfv in the prime group compared to the boosted group. this could potentially lead to the higher anti-cchfv-gpc igg titers in the prime cohort at the study endpoint as the cchfv challenge may have served as a heterologous booster, increasing levels when compared to the Δgrvsv-cchfv-gpcΔ boosted cohort (fig. a,b) . regardless, protection was achieved by both regimens, although the boosted group data suggests that at study endpoint, the observed igg titers against cchfv-gpc along with lower neutralizing titers (prnt of < : ) are evidence of the ability to combat lethal cchfv infection in the stat- −/− mouse model after vaccination (fig. a,b) . www.nature.com/scientificreports www.nature.com/scientificreports/ correlates of protection against cchf have been difficult to define due to the multiple vaccine and delivery platforms examined to date . several cchfv experimental vaccines studies have identified cell-mediated and humoral involvement, with some instances of neutralizing antibody production , . in looking at what is known for human cchf, survivors mount a humoral response whereas those who succumb, typically lack an igg response . other studies have examined antibody and neutralizing responses from the various vaccine platforms. dna vaccines following a three round vaccination regimen have induced detectable antibodies with neutralizing capacity observed up to : in prnt dilutions and achieved % protection, depending on the antigens encoded on the dna plasmids , . a cell culture based inactivated virus vaccine achieved high igg titers ( : , ) and a high neutralizing response of : , ; however, this also required three vaccination rounds, an alum adjuvant, and conferred % protection against the clinical isolate turkey-kelkit in ifnar mice . antibody titers, neutralization capacity, and challenge virus presented here were similar, though through the vsv platform, we achieved greater protection with a single dose. these studies, along with our own, suggest other facets of the immune system are likely involved in conferring complete protection. as this study was a proof of concept study and not a correlate study, t cell responses were not evaluated, and could be contributory as other groups have shown , , . now that we have established that this new vector can provide protective benefit, our future studies will temporally examine the antibody and t cell repertoire after prime and boosting doses following Δgrvsv-cchfv-gpcΔ, but before cchfv challenge, as these would be informative for the stat- −/− mouse model. in the future, the use of the is murine model might provide additional insights using an intact murine immune systems for vaccinations with a lethal model for cchfv infections . ideally, an immunocompetent, larger animal model is greatly needed in the cchf field to further test the array of cchfv experimental vaccines which have shown promise in these mouse models. a promising step forward appears to be the nonhuman primate disease model that has been recently developed . however, cchf in this model appears strain and challenge route dependent. in conclusion, this study offers not only a tool to study the biology off cchfv as it relates to structural g c , but also serves to develop and characterize a replication competent pseudotype, in relation to cchf vaccine development. the replication competent construct provides up to % protection with an observed humoral response, with a single-injection from a human isolate challenge strain. this information is valuable in designing future studies in cchfv animal models, and establishes characterized tools to examine the biology of structural cchfv-g c in a pseudotyped rvsv system. ksiazek, utmb -world reference center for emerging viruses and arboviruses, galveston, tx), were propagated in vero e cells once, plus previous passages in suckling mice and vero cells since isolation. all in vitro and in vivo work with cchfv was performed in a biosafety level facility at the galveston national laboratory, university of texas medical branch, galveston, tx. all cell and viral stocks were tested and free of mycoplasma, by pcr kit (intronbio, gyungg-do, south korea). monoclonal antibodies (mab) mouse-α-cchfv-g c e and a , and mouse-α-cchfv-preg n antibody g were generated and characterized as described previously , . described antibodies are available at bei resources (atcc) except for a , which was kindly provided by united states armed forces research institute for infectious diseases, frederick, md. generation of Δgrvsv vectors. the rvsv vector was cloned and recovered from cdna as previously described . briefly, a bluescript backbone plasmid under t polymerase promoter control encoding a Δg, vsv indiana backbone expressing a chimeric zaire ebolavirus (chebov) glycoprotein (gp), was used as the vector (designated pvsvΔg-chebov-gp- ). this plasmid was modified by cutting out an existing chebov-gp coding sequence via mlui and nhei restriction sites, yielding a pvsvΔg vector. an insert, coding for the codon optimized open reading frame of the complete cchfv glycoprotein precursor (gpc), was digested and ligated into the pvsvΔg vector. the cchfv-gpc insert was created by overhang pcr mutagenesis; flanking a ′ mlui restriction site plus kozak sequence, and a ′ xbai restriction site, from a pcr amplified, codon optimized, pcagg-cchfv-gpc (kindly provided by j. kortekaas, central veterinary institute, lelystad, netherlands). this ligated and cloned plasmid, designated pvsvΔg-cchfv-gpc, was transfected into bhk cells that were also co-transfected with vsv protein n, p, g, and, l 'helper' plasmids under t promoter control and driven by infection (moi ) with recombinant vaccinia virus expressing t polymerase (rvv-tf - ; kindly provided by m. whitt). recovered virus, designated vsv-g*-Δgrvsv-cchfv-gpc, was collected - hrs post infection/ transfection, filtered twice through a . μm filter to remove vaccinia virus, plaque purified onto vsv-g* complemented bhk cells, and stored at − °c for further use. all plasmid maps and cloning primer sequences are available upon request. infections, enumeration, growth kinetics, and preparation of viral material. for infections of replication deficient vector, semi-confluent (~ - %) monolayers of bhk cells were transfected (lipofectamine www.nature.com/scientificreports www.nature.com/scientificreports/ ltx, invitrogen) with μg/ × cells using pcagg-vsv-g (indiana) . once monolayers displayed cell rounding and syncytia, they were infected at an moi of . with vsv-g*-Δgrvsv-cchfv-gpc. supernatants were harvested at hrs post infection (hpi) and clarified at , rpm for min at °c. confluent monolayers of bhk cells were infected with replication competent Δgrvsv-cchfv-gpcΔ at moi . for hr at °c with % co with rocking at min intervals and harvested/clarified at hpi. plaque assays for viral titrations were carried out in an analogous manner, with an overlay media final concentration of . % avicel rc- (fmc biopolymer, philadelphia, pa) in x eagles minimum essentials medium (mem) with % fbs and % p/s on bhk cells for - hpi. after incubations, overlays were aspirated and a % buffered formalin fix with a x crystal violet stain was incubated onto monolayers for one hr. plaques were enumerated and plaque forming units (pfu) were determined by averaging technical replicates per sample. single-cycle growth curves at moi . were performed by absorbing rvsv-gfp, Δgrvsv-cchfv-gpcΔ, or cchfv onto duplicate monolayers of bhk cells in six-well plates as described above. inoculum was aspirated, cells were washed three times with pbs, and d was added to monolayers and incubated at various time points indicated in fig. a . at designated time points, samples were collected, clarified, and stored at − °c until titrations were performed described above. cchfv-g c protein analysis. immunofluorescence analysis was carried out by infecting huh- monolayers at a moi of . for hpi. cells were fixed with % (w/v) paraformaldehyde, permeabilized with % triton x- and stained with : diluted mab a or g . cells were washed, blocked with % bsa, and incubated with a dilution of : , secondary goat-α-mouse mab conjugated to fluorescein isothiocyanate (fitc) (invitrogen). cells were mounted and counterstained with the nuclear stain of ′, -diamidino- -phenylindole (dapi) (invitrogen). stained cells were examined on a nikon eclipse ti-s fluorescence microscope. whole virions were analyzed by infecting bhk cell monolayers, semi-purifying clarified supernatants over a % sucrose cushion at , rpm for min at °c using a beckman sw ti rotor. viral pellets were lysed using np- with x protease inhibitor cocktail (invitrogen). recombinant vsv lysates were incubated at °c for min and protein subsequently quantified using bca protein assay per manufacturers' instructions (thermo fisher scientific, waltham, ma). per institutional inactivation protocols, cchfv lysates had a modified inactivation protocol using instead x laemmli sample buffer (lsb) (thermo fisher scientific) at °c for min boiling and transfer to a fresh tube. approximately ng of purified and semi-purified virion associated total protein was mixed : with x lsb (without β-mercaptoethanol) and run on - % gradient mini-protean tgx electrophoresis gels (bio-rad, hercules, california). coomasie staining was accomplished via incubating tgx gels in coomassie fluor orange protein gel stain (thermo fisher scientific) per manufacturers' instructions and imaged at nm on a gel doc xr+ gel documentation system (biorad). western blots were run on tgx gels using wet tank transfer to hybond-p polyvinylidene difluoride (pvdf) membranes (ge healthcare, little chalfont, uk). membranes were blocked with % bsa overnight at °c in tris/ . % tween (sigma-aldrich) followed by incubation with primary mab e diluted at : , , overnight at °c. secondary horse radish peroxidase (hrp) conjugated goat-α-mouse antibody (thermo fisher scientific) was diluted : , and incubated on the membrane for h at room temperature. detection of hrp was accomplished via pierce ecl western blotting substrate (thermo fisher scientific), hyperfilm ecl (ge healthcare), and kodak carestream film with x-omat processor (eastman kodak company, rochester, ny). were placed in trizol ls (thermo fisher scientific) at a ratio of : , mixed, incubated for min at room temperature, and transferred to fresh tubes. rna was isolated from sample mixtures using zymo research direct-zol rna min-prep (zymo research corp, irvine, ca), per manufacturers' instructions. rna was quantified using a nanodrop (thermo fisher scientific) and approximately ng total rna was used to create cdna using the superscript iii first-strand synthesis system (invitrogen) and a vsv-m, matrix protein gene ′ forward primer. sanger sequencing on the cdna was performed using vsv-m, vsv-l, and cchfv-gpc (codon optimized) open reading frame primer sets and accomplished by the utmb molecular genomics core using an abi prism xl dna sequencer (applied biosystems, foster city, ca). sequence analysis was performed using geneious r (biomattes, auckland, new zealand) based on consensus and plasmid maps. all cdna/sequencing primers and consensus/plasmid maps are available upon request. to analyze the stocks of cchfv or rvsv vaccine vectors used in this study we performed deep sequencing analysis of rna isolated from these virus stocks. briefly, viral rna was isolated from a trizol ls (invitrogen)/sample mixture using a direct-zol rna mini-prep (zymo research), per manufacturer's instructions. approximately ng of purified rna was used to make cdna using the ovation rna-seq. . kit (nugen) and this in turn was used for the preparation of the double-stranded dna library, using encore ion torrent library prep kit. sequencing was performed by the utmb molecular core on the ion torrent using -v deep sequencing chips. sequence analysis was performed using dna star seqman ngen software (dna star) based on unpaired analysis of base pair overlaps. ultrastructural analysis. viruses were propagated in multiple t- flasks of confluent bhk cells. viral supernatants were harvested and clarified as described above. clarified supernatants were concentrated by mixing with buffered x polyethylene glycol with incubation for hrs at °c, followed by centrifugation at , xg for mins at °c. concentrated pellets were re-suspended in pbs with protease inhibitor and overlaid atop optiprep (sigma-aldrich) continuous gradients of - % buffered iodixanol. viruses were banded by ultracentrifugation at www.nature.com/scientificreports www.nature.com/scientificreports/ [ems], hatfield, pa) for - mins, incubated with mab e at : dilutions. antibodies were absorbed for mins in wet chamber and washed with pbs containing % bsa, and incubated with the secondary antibody, goat-α-mouse conjugated to nm colloidal gold particles (ems), at a dilution of : for mins. grids were washed with pbs and % bsa, fixed using % (w/v) aqueous glutaraldehyde for mins, and finally stained with % (w/v) aqueous uranyl acetate. grids were examined at kv using a philips cm- transmission electron microscope. ethics of care, vaccination, and animal challenge. animal studies were approved by the utmb institutional animal care and use committee (iacuc). animal research was carried out in compliance with the animal welfare act and other federally regulated stipulations regarding animals and adherences to the guide for the care and use of laboratory animals, national research council, . the animal facilities where this research was conducted are accredited by the association for assessment and accreditation of laboratory animal care international. this study used female to weeks old s /svev-stat tm rds mice (stat- −/− ) (taconic, germantown, ny). after an acclimatization period in barrier conditions in environmentally enriched sterile housing, mice were anesthetized by isoflurane and implanted with subdermal transponders, which provide coded identifiers and permitted body temperature measurements (biomedic data solutions, seaford, de). vaccine preparations were diluted in hanks balanced salts medium with % fbs, along with pbs for control groups. after anesthesia by isoflurane, ul of each preparation was administered intraperitoneally (i.p.), with five stat- −/− mice per experimental group. clinical scoring, body temperature, and weight, were recorded daily. at days post vaccination (prime), mice were challenged with ul i.p. with either pfu of cchfv-ibar or cchfv-turkey (referred to as turkey ) (however, pfu calculated back titer of challenge preparation for the turkey administration and is thus reported as such in fig. ), respectively. all challenge doses were frozen for storage and verified by back-titrations by plaque assay on sw- -cdc cells as outlined above. after challenge, animals were observed for clinical scoring, temperature, and weight change. upon reaching institutionally approved endpoint score criteria, or study end-point, blood samples were collected into k edta containing collection tubes (granier, usa) and plasma was separated then frozen at − °c for storage and further analysis. euthanasia criteria was defined as: mouse displays severely hunched posture, inability or reluctance to move, appears weak (staggering when moving around cage), labored breathing, or weight loss of greater than % of starting body weight. anti-cchfv-gpc igg elisa development. iodixanol gradient purified Δgrvsv-cchfv-gpcΔ and rvsv-gfp were re-suspended in np- buffer and bca protein quantified (previously described above) and were used as whole virion antigens in coating immunosorbent -well plates (thermo fisher scientific). matrices of various antigen, blocking, primary antibodies (hyperimmune mouse ascetic fluid [hmaf kindly provided by t. ksiazek], a , and e ) to cchfv/cchfv-gpc, and secondary antibody (mab hrp-goat-α-mouse) concentrations were used to develop optimal detection conditions for the cchfv-gpc via elisa. per optimizations, one microgram of purified antigen per ml was suspended in sterile filtered sodium bicarb/carbonate buffer (ph . ) and allowed to incubate on immunosorbent plates overnight at °c. plates were washed with pbs containing a concentration . % tween- and . % thimerosal. blocking occurred with % milk dissolved in wash buffer, for hrs at room temperature. sera from stat- −/− mice was added : and diluted -fold by pipetting across plates and allowed to incubate for one hr at °c. plates were washed and a secondary anti-mouse antibody conjugated to hrp was added at : , dilution for one hr at °c. abts peroxidase substrate (kpl, seracare life sciences, milford, ma) was incubated for mins at room temperature prior to the addition of a % sds stop solution. plates were read with nine reads per well at nm with a plastic correction factor accounted for from a nm reading per well. test sera was evaluated using both purified Δgrvsv-cchfv-gpcΔ and rvsv-gfp antigen. plaque reduction neutralization assay. serial dilutions of sera from four mice per treatment group, were aliquoted into cluster tubes with d and allowed to incubate with pfu of cchfv, isolate turkey , for approximately hrs on ice. resulting sera plus virus mixture was then overlaid onto -well plates of confluent sw- -cdc cells and absorbed for hr at °c with % co with rocking at min intervals. plaque assays were carried out in a manner described above in previous methods section. resulting plaques were enumerated from virus + sera wells and compared to sera plus media only wells run for each sample, and a percent neutralization was calculated and reported for each dilution. hyperimmune mouse ascitic fluid [hmaf] raised against cchfv was additionally serially diluted and run as a positive control. immunohistochemistry of tissues. tissue sections were deparaffinized and rehydrated through xylene and graded ethanols. slides went through heat antigen retrieval in a steamer at °c for mins in sigma citrate buffer, ph . , × (sigma aldrich, st. louis, mo). to block endogenous peroxidase activity, slides were treated with a % hydrogen peroxide and rinsed in distilled water. the tissue sections were processed for ihc using the thermo autostainer (thermofisher, kalamazoo, mi). sequential min incubations with avidin d and biotin solutions (vector, burlingame, ca) were performed to block endogenous biotin reactivity. specific anti-cchfv immunoreactivity was detected using a primary polyclonal rabbit-α-cchfv-np antibody (ibt bioservices, rockville, md) at a : dilution for mins. a secondary biotinylated goat-α-rabbit-igg (vector laboratories, burlingame, ca) at : dilution for mins followed by vector horseradish peroxidase streptavidin, r.t.u (vector) for mins. slides were developed with dako dab chromagen (dako, carpenteria, ca) for mins and counterstained with harris hematoxylin for seconds. tissue sections from uninfected mice were used as negative controls. changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the role of ticks in the maintenance and transmission of crimean-congo hemorrhagic fever virus: a review of published field and laboratory studies crimean-congo hemorrhagic fever: 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single-dose attenuated vesiculovax vaccines protect primates against ebola makona virus statistical analysis. a kaplan-meier survival curve was constructed with graphpad prism software (graphpad software, inc., san diego, ca). a power analysis utilizing parameters that included survival, neutralization capacity, and elisa antibody titers from previous pilot studies was performed to assess adequate animal numbers for the current study to differentiate between surviving groups versus control groups to obtain a statistical significance of p < . with at least a % probability. the authors would like to thank the utmb molecular genomics core, assay development core (for deep sequencing support), animal resource center (for handling and husbandry of laboratory animals), electron microscopy laboratory core (specifically, vsevolod popov and julie wen for electron microscopy support), and natalie dobias for immunohistochemistry support. we gratefully acknowledge krystle agans, katharina schmitz, gordon wong, auja smith, angel padilla, emily mantlo, teresa sorvillo, courtney woolsey, corey fulton, stephanie foster, maria cajimat, benjamin satterfield, and daniel deer, viktoriya borisevich, joan geisbert, for various in vitro and in vivo technical guidance and support throughout the project. we thank thomas ksiazek, michael whitt, jeroen kortekaas, and Éric bergeron for the gifts of vector/insert plasmids, reagents, cell lines, and virus strains. additionally, we further thank Éric bergeron for fruitful discussions and unpublished data regarding cchfv-gpc reagents and thomas ksiazek for elisa development discussions. this research was conducted by s.e.r. in partial fulfillment of the requirements for a ph.d. from the university of texas medical branch, galveston, texas, usa. funding and support was provided by the utmb department of microbiology and immunology to t.w.g. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: s.e.r., r.w.c., c.e.m. and t.w.g. have filed a provisional patent application for replication competent vesicular stomatitis vector encoding crimean-congo hemorrhagic fever antigen.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -m fic g authors: horie, masayuki; kobayashi, yuki; honda, tomoyuki; fujino, kan; akasaka, takumi; kohl, claudia; wibbelt, gudrun; mühldorfer, kristin; kurth, andreas; müller, marcel a.; corman, victor m.; gillich, nadine; suzuki, yoshiyuki; schwemmle, martin; tomonaga, keizo title: an rna-dependent rna polymerase gene in bat genomes derived from an ancient negative-strand rna virus date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: m fic g endogenous bornavirus-like l (ebll) elements are inheritable sequences derived from ancient bornavirus l genes that encode a viral rna-dependent rna polymerase (rdrp) in many eukaryotic genomes. here, we demonstrate that bats of the genus eptesicus have preserved for more than . million years an ebll element named eebll- , which has an intact open reading frame of , codons. the eebll- coding sequence revealed that functional motifs essential for mononegaviral rdrp activity are well conserved in the ebll- genes. genetic analyses showed that natural selection operated on eebll- during the evolution of eptesicus. notably, we detected efficient transcription of eebll- in tissues from eptesicus bats. to the best of our knowledge, this study is the first report showing that the eukaryotic genome has gained a riboviral polymerase gene from an ancient virus that has the potential to encode a functional rdrp. scientific reports | : | doi: . /srep studied. notably, some of the ebln elements retain relatively long open reading frames (orfs) that are transcribed into rna , , leading us to speculate that they play functional roles in their host cells. indeed, it was reported that homo sapiens ebln- (hsebln- ) interacts with several cellular proteins . recently, we demonstrated that the epigenetic control of hsebln- expression affected the transcription efficiency of a neighbouring gene . additionally, we demonstrated that the expression of ictidomys tridecemlineatus ebln (itebln) inhibited the replication of a modern mammalian bornavirus (borna disease virus; bdv) . we also proposed that some ebln elements in rodent and primate genomes might function as non-coding rnas . these observations strongly suggest that rna viruses can also be a driving force of genome evolution in their hosts. ebll elements are widely distributed in many eukaryote species ranging from vertebrates to arthropods . a recent study demonstrated that the mosquito aedes aegypti has accumulated many eves in its genome and that an eve homologous to the l gene of rhabdovirus in the mononegavirales family has evolved under purifying selection . additionally, it was suggested that an eve from the parvovirus replicase gene have been exapted in degu (octodon degus) . because the l of bornavirus is also an enzymatic protein, the co-option of ebll elements may provide genetic innovations in the host species. however, little analysis has been performed for ebll elements. in this study, we analyzed ebll elements in mammals to gain insights into the biological roles of ebll elements in their hosts. surprisingly, we found that ebll elements in bats belonging to the genus eptesicus retain intact orfs consisting of , codons that have been maintained for at least . million years (my). furthermore, we showed that the eblls evolved under purifying selection and still possess functional motifs conserved among the mononegavirales rdrps. these results strongly suggest that the ebll elements encode functional proteins that may be rdrps. orf screening for these elements in several mammalian species and found an ebll element in the bat species eptesicus fuscus (designated efebll- ; accession number aleh ). efebll- retains a large and intact orf of , codons that is comparable in length to the l gene of exogenous bornaviruses with , codons (fig. a) . a reverse pblast analysis revealed that the best match to efebll- was the l protein of avian bornavirus (abv) isolate bil (accession number eu ; . % identity) (supp. table ). typically, the l proteins of bornaviruses are more similar to efebll- than other mononegaviral l proteins, including nyamiviruses . the topology of the phylogenetic tree based on the sequences of the efebll- and l proteins of bornaviruses and nyamiviruses was consistent with the results of the reverse blast analysis (supp. fig. ). these data suggest that efebll- is indeed derived from an ancient bornavirus. efebll- presents a typical transcription termination signal (t ) of exogenous bornaviruses followed by a short poly-a sequence (fig. b) . the distance between the stop codon and the t -like sequence of efebll- is similar to that of bdv (fig. b) . however, no viral transcription start signal (s )-like sequence is present upstream of the efebll- orf. notably, a -bp direct repeat sequence exists upstream of the orf and immediately downstream of the t and poly-a-like sequence (fig. b) ; direct repeat sequences can be a landmark of a long interspersed nuclear element (line- )-mediated insertion (so-called target site duplication or tsd) . therefore, it is highly likely that the mrna encoding the l gene of an ancient bornavirus integrated into the genome of the host via the line- reverse transcriptase as observed for anthropoid eblns , . the lack of an s -like sequence upstream of the efebll- orf may have resulted from a genomic mutation during evolution or an aberrant integration event that inserted a partial segment of the targeted mrna, as is often observed for line- -mediated integration . to elucidate whether efebll- was generated from an mrna species encoding a monocistronic l gene or polycistronic genes (i.e., m and g) (supp. fig. ), we surveyed sequences homologous to several bornavirus proteins in the contig containing the efebll- locus. to this end, we performed a tblastn search in the whole shotgun genome sequence of e. fuscus using the amino acid sequences of the n, x, p, m, g or l proteins of exogenous bornaviruses. although ebln and eblg elements were found in the genome of e. fuscus as previously reported , no ebl element was found closely located to the efebll- locus, which suggests that efebll- was generated from an mrna of transcription unit (supp. fig. ). we could not conclude more precisely regarding the source of the specific viral mrna transcript from which efebll- originated due to the lack of similarity between the upstream sequence of efebll- and the genomes of modern bornaviruses ( fig. and supp. fig. ). the genus eptesicus contains orthologous genomic efebll- sequences. to investigate whether orthologous efebll- sequences exist in the genomes of other bat species, we performed tblastn analysis using the deduced amino acid sequence of efebll- as a query. the tblastn analysis revealed homologous sequences of efebll- in bat species of the genus myotis (m. davidii and m. lucifugus) (supp. table ). some of the sequences were detected in a previous report . because sequence similarities among orthologous genes in different species are usually observed in both flanking regions and coding regions, we analyzed the upstream and downstream sequences of the orfs of the eblls detected by the tblastn analysis. however, we could not determine the gene orthology due to insertions of transposable elements and the recombination of the genomes of the genus myotis (supp. fig. ). because the database search did not provide any information for the efebll- orthology, we searched for orthologous efebll- sequences in vesper bat genome sequences that were not yet deposited in the database: e. serotinus, e. nilssonii, pipisterillus spec, nyctalus noctula and m. daubentonii. we performed pcr using primer sets that were designed based on the efebll- nucleotide sequence (supp. table ). the expected bands amplified and sequenced from the genomes of species of the genus eptesicus (e. serotinus and e. nilssonii) (supp. fig. ) revealed nucleotide sequences that were almost identical to efebll- (greater than % identity). to determine the gene orthology, we analyzed the entire sequences of the eptesicus eblls and their flanking regions by pcr and direct sequencing. notably, eblls in e. serotinus and e. nilssonii also retained huge intact orfs consisting of codons (accession numbers ab for e. nilssonii and ab for e. serotinus) with deduced amino acid sequences that were almost identical to efebll- (greater than % identity). in addition, the ′ and ′ flanking sequences are highly conserved among efebll- and the eblls in e. serotinus and e. nilssonii, indicating that these eblls are orthologous (supp. fig. ). therefore, we designated these hypothetical genes as eptesicus ebll- (eebll- ), with the eebll- genes in e. serotinus and e. nilssonii designated esebll- and enebll- , respectively. these observations suggest that the integration of an ancient bornavirus l gene occurred prior to the divergence of e. fuscus, e. serotinus and e. nilssonii. based on the timetree , the divergent time of e. fuscus and e. serotinus was . million years ago (mya). these data suggest that the endogenization of an l sequence of an ancient bornavirus occurred at least . mya (fig. ) . the putative tsds are also present in e. serotinus and e. nilssonii (supp. fig. ). analysis of the deduced amino acid sequence of eebll- . next, we investigated the deduced amino acid sequence of eebll- . the long-time conservation of the intact orf in eebll- allowed us to predict that eebll- might maintain functional motifs of a viral rdrp. therefore, we explored the functional domains in eebll- by a pfam search. the pfam search identified two pfam domains [pf (mononegavirales rdrp) and pf (mononegavirales mrna-capping region v)] in the bornavirus l and all eebll- sequences (fig. a , supp. fig. a and supp. table ), indicating that the eebll- s and bornavirus l proteins share similar properties. we also aligned the amino acid sequences of the eebll- s and l proteins of abv and bdv (fig. b ,c and supp. fig. b -d). six highly conserved blocks (block i -vi) were reported in the l proteins of mononegaviruses. the bornavirus l proteins lack block vi, which usually encodes a methyltransferase (mtase) domain , . five conserved stretches (motifs a, a, b, c and d) were reported to be present in the l proteins . motif "a" exists in block ii, and motifs a-d are located in block iii (fig. a) . the amino acid sequences in the motifs "a", a, b, c and d are well conserved among abv l, bdv l and eebll- s ( fig. b and supp. fig. b ). each motif was reported to contain an amino acid residue that was strictly conserved among known rna-dependent polymerases . the strictly conserved amino acid residues in mononegaviruses are also present in eebll- , with the exception of motif b because bornaviruses do not contain the conserved glycine residue in motif b ( fig. b and supp. fig. b ). the putative template-recognition site was reported to be composed of the keke [hydrophobic] k motif, followed by basic and hydrophobic amino acid residues spaced every four amino acids in motif "a" in block ii (fig. b) . the bornavirus l proteins do not possess strictly conserved motif sequences, and the eebll- s have stretches in the putative template-recognition sites that are similar to bornavirus l ( fig. b and supp. fig. b) . the gdn motif in motif b, which is essential for rdrp activity, exists in block iii of the eebll- s ( fig. b and supp. fig. b ). the putative guanosine ′ -triphosphatase and rna:gdp polyribonucleotidyltransferase (prntase) domain in block v, which consists of the [y/w]xg[s/t/a]xt motif (x represents any amino acid) and the hr motif , , are also present in all of the eebll- s ( fig. c and supp. fig. c ). furthermore, amino acid residues in block v (green letters in fig. c and supp. fig. c ) that are highly conserved among mononegaviruses were also observed in the eebll- s, although the function of these amino acid residues remains unclear ( fig. c and supp. fig. c ). the lack of an mtase in the eebll- s is consistent with the bornavirus l proteins. however, a putative nuclear localization signal (nls) sequence was not found in the eebll- s (supp. fig. d ). scientific reports | : | doi: . /srep notably, the nls sequence in bdv l is not conserved in abv l, and a report has shown that the nls of bdv l does not encode a functional sequence . detection of eebll- transcripts from eptesicus bats. we investigated whether the eebll- s are transcribed using rt-pcr with rna samples from five individuals of e. serotinus and e. nilssonii. notably, the expected bands were detected in the tissue samples of all ten eptesicus bats but not a myotis bat (fig. ) . therefore, the eebll- s are expressed as rnas in the eptesicus species. evidence for the exaptation of eebll- . the maintenance of the orfs for over . my and the presence of the transcripts suggested that eebll- might express a functional protein in the host species. to predict the evolutionary significance of eebll- s in the bats, we examined the natural selection of eebll- s. first, we simulated the numbers of premature stop codons acquired by the eebll- s under neutral evolution for . my. in the simulations, the probabilities of maintaining the orfs for . my were significantly low (p = . or < . ) (fig. a, supp. fig. ) , which indicated that the eebll- s evolved under selection pressure to keep the orfs after endogenization. next, we examined natural selection at each branch of the phylogenetic tree for the eebll- orfs by analyzing the non-synonymous to synonymous substitution ratios (d n /d s ) (fig. b) . purifying selection was detected at branches a and c of the phylogenetic tree in fig. b (branch a: d n /d s ratio = . , likelihood ratio test (lrt): p < . ; branch c: d n /d s ratio = . , lrt: p < . ). taken together, these results indicate that natural selection occurred during eebll- evolution, suggesting that eebll- was exapted as a functional protein in the bat species. phylogenetic analysis. to gain insights into the evolution of mononegaviral polymerases, we constructed a phylogenetic tree from the amino acid sequences of bat eblls and the representative l genes of the families bornaviridae and nyamiviridae. the tree revealed three major clusters: bat eblls, exogenous bornaviral l genes, and nyamiviral l genes (fig. ). together with fig. s , our data suggest that the bat eblls might be derived from ancient bornaviruses distantly related to known modern bornaviruses. in this study, we discovered an endogenous rna virus element that encodes a predicted rdrp gene of an ancient negative-strand rna virus in bats of the genus eptesicus. notably, this element (eebll- ) contains a , -amino acid orf that was conserved for more than . my and included almost all of the sequence motifs essential for the enzymatic activity of a rna virus rdrp. to the best of our knowledge, eebll- is the first example of an rna virus-derived rdrp encoded by the mammalian genome. together with the data of the transcription profile and the natural selection of eebll- , our observations strongly suggested that eebll- was exapted by the eptesicus genomes as a functional protein, rendering a survival advantage to the bats. however, at present its evolutionary significance for the host, if any, is unclear. based on the knowledge of eve-derived genes, we propose two hypotheses. one possible function of the eebll- s is to affect the replication of exogenous bornavirus-related viruses. eve-derived immunity (edi) is a well-known concept because eves act as anti-viral genes against genetically similar viruses . indeed, we have demonstrated that the expression of itebln inhibits bdv replication and that some eblns in rodent and primate genomes express pirnas that may interfere with bornavirus infection . the successful infection and replication of bornaviruses could be accomplished by the balanced activity of rdrp in infected cells. thus, eebll- might protect its hosts from related viruses by perturbing their replication balance via its rdrp activity. alternatively, enhanced viral replication may stimulate host immune responses. indeed, bdv antigen expression was reported to be enhanced by exogenous expression of the bdv l protein . therefore, excess rdrp activity might cause the dysregulation of transcription/replication, resulting in the disruption of infection. to investigate whether efebll- affects bdv polymerase activity, we conducted a minireplicon assay for bdv using recombinant eebll- . the result showed that the expression of esebll- did not have an effect on the polymerase activity of bdv (supp. fig. ). however, we cannot exclude the possibility that eebll- affects the replication of bornaviruses with l genes that are genetically similar to eebll- , although bornavirus has not yet been detected from bats. the phylogenetic analyses showed that bdv l is relatively distant from the eebll- s (fig. and supp. fig. ). bornavirus species may exist that are genetically much closer to eebll- in the bat species. another hypothesis is that eebll- might be involved in the rna interference (rnai) machinery of the host cells as an rdrp. at present, several eukaryotic proteins have been reported to act as rdrps [ ] [ ] [ ] . among these proteins, the rna-directed rna polymerases (rdrs) are well-studied, authentic rdrps that are encoded by several eukaryote species, such as plants, nematodes and fission yeasts . rdrs are essential for the rnai machinery, which is involved in anti-viral defence and gene regulation in these species. interestingly, although rdr genes are derived from common ancestral genes, higher eukaryotes have lost rdr genes during evolution , although they still retain the rnai machinery for anti-viral defense , . although the mononegaviral l proteins require the n and p proteins for their polymerase activities, the l protein of vesicular stomatitis virus has been reported to replicate rna from an rna template in the absence of the viral proteins n and p . thus, the eebll- s may function as rdrps in the absence of other viral proteins. the eebll- -mediated rnai machinery could restrict viral replication of not only bornaviruses but also other viruses as previously reported in other viruses , that may have conferred survival advantages to their host species. further studies are needed to assess our hypotheses. our findings provide novel insights into the co-evolution of rna viruses and mammalian species. tissue samples. in japan, the capture and handling of a bat were approved by the japanese ministry of environment (license no. - - to - - ), and conducted in accordance with the approved protocol. an e. nilssonii was captured in hokkaido, japan and euthanized; then, the liver was removed. the liver sample was stored in rnalater (thermo fisher scientific, waltham, ma, usa) prior to the isolation of genomic dna. bat muscle tissue (e. serotinus) and cell cultures from e. serotinus, m. daubentonii (mydauni/ ), m. nattereri (mylu/ ), nyctalus noctula (nynoni/ ) and pipistrellus pipistrellus (pipni/ ) were available from previous studies and originated from animals found dead in germany and delivered to centres for bat protection , , , . because all german bats are protected through the european commission (http://ec.europa.eu/environment/ nature/legislation/habitatsdirective) and the agreement on the conservation of populations of european bats (www.eurobats.org), investigative research requires special permission by local government bodies. as part of a study on diseases in european bats table ). the pcr conditions were as follows: denaturation at °c for min, cycles of °c for sec, °c for sec, and °c for sec, followed by a final extension at °c for min. amplified dna was analysed by agarose gel electrophoresis. to detect eebll- transcripts, rna samples were isolated from bat brain tissues preserved in rnalater (eptesicus nilssonii [n = ] and eptesicus serotinus [n = ]). the rna extraction was performed using the nucleospin rna extraction kit (macherey & nagel, düren, germany). for cdna synthesis, we utilized the taqman reverse transcription reagents kit (thermo fisher scientific) with an additional denaturing step of °c for min using oligo-dt primers. bat species were determined by amplification and sequencing of mitochondrial dna as described by sonntag et al. . pcr was performed using the premix ex taq hot start version (takara) in a final volume of μ l containing . μ m primers and μ l of cdna. the primer sets mh - , - and - were used for the detection of ebll sequences (primer sequences are available in supp. table ). the pcr conditions were as follows: denaturation at °c for min, cycles of °c for sec, °c for sec, and °c for sec, followed by a final extension at °c for min. the pcr products were analysed by electrophoresis. amino acid sequence analyses. amino acid sequences of bornaviral ls and eebll- s were aligned using the psi-coffee program . functional domain searches were conducted by pfam . the conservation of functional motifs among mononegaviral rdrps was determined by a manual search. phylogenetic analyses. most of the bat eblls identified by the tblastn search were fragmentary, probably due to the occurrence of point mutations, insertions/deletions, and genome rearrangements after the integration of the viral l genes (supp. fig. ). neighbouring fragments were considered to be derived from a single integration event and combined into a single ebll sequence if the number of overlapping amino acid positions between the regions in the efebll- sequence that were identified to be homologous to the fragments was smaller than . parts of fragments homologous to the overlapping positions were not included in the combined sequence. multiple alignments of (combined) amino acid sequences for bat eblls were constructed by mapping each ebll sequence onto the efebll- sequence according to the pairwise alignment of the sequences produced in the tblastn search described above. additionally, a multiple alignment of viral l protein sequences was generated by mafft . finally, multiple alignments of bat eblls and viral ls were joined according to the pairwise scientific reports | : | doi: . /srep alignment of efebll- (included in the bat eblls) and the bdv l of strain he/ /fr (included in the viral l gene) that were produced in the tblastn search, which identified efebll- as a homologue of the query he/ / fr l ; gaps were introduced at the same sites in all sequences in either alignment to maintain the relationship when joining the alignments. phylogenetic trees for the amino acid sequences of the bat eblls and viral ls were constructed by the maximum likelihood method with the pairwise deletion option in mega . the jtt + g and lg + g models were judged to be the fittest for the data because they had the lowest bayesian information criterion scores. the reliability of each internal branch in the phylogenetic tree was assessed by computing the bootstrap probability with resamplings. natural selection operating on eebll- . the d n /d s ratio at each branch of the phylogenetic tree for the eebll- s was estimated by the maximum likelihood method using the codon substitution model in paml ver. . . the topology was derived from the phylogenetic tree of bat eblls in fig. b . the equilibrium codon frequencies were treated as free parameters, and the d n /d s ratio was estimated under the free-ratio (selection) model and the branch-specific (null) model. the d n /d s ratio was allowed to vary among branches in the selection model, whereas in the null model the d n /d s ratio was fixed at at specified branches. the null hypothesis of no selection (d n /d s = ) at the specified branches was tested by the likelihood ratio test. simulation. the probability that efebll- and esebll- maintained the orf under selective neutrality after the divergence of e. fuscus and e. serotinus was determined by simulating the evolution of efebll- and esebll- using seq-gen . the nucleotide sequences of efebll- and esebll- were evolved according to the hky model after removing termination codons with an evolutionary rate of . × − per site per year, which was estimated from the evolutionary distance at the rd codon position of the rag exon and the divergence time of e. fuscus and e. serotinus ( . mya in timetree ). the transition/transversion rate ratio was assumed to be . , which was estimated from the rd codon position in the rag exon of e. fuscus, e. serotinus, and e. nilssonii with the tamura- parameter model by the maximum likelihood method. the distribution of the number of premature termination codons in the simulated sequences was obtained from , iterations. we developed a pol ii-driven firefly luciferase-based minireplicon assay for bdv (the construct is available upon request). for transfection, × t cells were seeded into each well of a -well plate. the next day, plasmids expressing the bdv n, p and l proteins, the minigenome and renilla luciferase (as a normalization control) with or without a plasmid expressing esebll- were transfected using lipofectamine (life technologies). after hours, the cells were lysed with passive lysis buffer (promega), and the luciferase activities were measured using the dual-luciferase reporter assay system (promega). the values of firefly luciferase were normalized by the renilla luciferase activity. effects of retroviruses on host genome function syncytin is a captive retroviral envelope protein involved in human placental morphogenesis genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin , a gene conserved on primate evolution syncytin-a and syncytin-b, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in muridae syncytin-a knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirusderived, envelope gene genomic organization of borna disease virus molecular and cellular biology of borna disease virus infection borna disease virus, a negative-strand rna virus, transcribes in the nucleus of infected cells unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/ marburgvirus sequences in vertebrate genomes endogenous non-retroviral rna virus elements in mammalian genomes endogenous viral elements in animal genomes comprehensive analysis of endogenous bornavirus-like elements in eukaryote genomes inhibition of borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome large-scale mapping of human protein-protein interactions by mass spectrometry transcription profiling demonstrates epigenetic control of non-retroviral rna virus-derived elements in the human genome pirnas derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals fossil rhabdoviral sequences integrated into arthropod genomes: ontogeny, evolution, and potential functionality parvovirus-derived endogenous viral elements in two south american rodent genomes nyamanini and midway viruses define a novel taxon of rna viruses in the order mononegavirales high frequency retrotransposition in cultured mammalian cells timetree: a public knowledge-base of divergence times among organisms sequence comparison of five polymerases (l proteins) of unsegmented negative-strand rna viruses: theoretical assignment of functional domains a. transfer to gdp for unique mrna capping by vesicular stomatitis virus rna polymerase an unconventional pathway of mrna cap formation by vesiculoviruses characterization of the nuclear localization signal of the borna disease virus polymerase active borna disease virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral x protein paleovirology and virally derived immunity molecular basis of rna-dependent rna polymerase ii activity an rna-dependent rna polymerase formed by tert and the rmrp rna evolution of the rna-dependent rna polymerase (rdrp) genes: duplications and possible losses before and after the divergence of major eukaryotic groups rna interference functions as an antiviral immunity mechanism in mammals antiviral rna interference in mammalian cells mechanism of rna synthesis initiation by the vesicular stomatitis virus polymerase amplification of emerging viruses in a bat colony bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses diseases and causes of death in european bats: dynamics in disease susceptibility and infection rates new adenovirus in bats censor-a program for identification and elimination of repetitive elements from dna sequences t-coffee: a web server for the multiple sequence alignment of protein and rna sequences using structural information and homology extension pfam: the protein families database mafft multiple sequence alignment software version : improvements in performance and usability molecular evolutionary genetics analysis version . paml : phylogenetic analysis by maximum likelihood seq-gen: an application for the monte carlo simulation of dna sequence evolution along phylogenetic trees a time-calibrated species-level phylogeny of bats (chiroptera, mammalia) we would like to thank to nicholas fredric parrish and christian louis volk murayama for helpful comments and discussion. we are grateful to angelika lander and annika brinkmann for technical assistance. all bat carcasses (rki) were kindly provided by bat researchers and bat rehabilitation centres in germany. this work was supported by the japan society for the promotion of science key: cord- - lnxb px authors: lyu, hongming; john, mathews; burkland, david; greet, brian; post, allison; babakhani, aydin; razavi, mehdi title: synchronized biventricular heart pacing in a closed-chest porcine model based on wirelessly powered leadless pacemakers date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: lnxb px about % of patients with impaired cardiac function have ventricular dyssynchrony and seek cardiac resynchronization therapy (crt). in this study, we demonstrate synchronized biventricular (biv) pacing in a leadless fashion by implementing miniaturized and wirelessly powered pacemakers. with their flexible form factors, two pacemakers were implanted epicardially on the right and left ventricles of a porcine model and were inductively powered at . mhz and . mhz industrial, scientific, and medical (ism) bands, respectively. the power consumption of these pacemakers is reduced to µw-level by a novel integrated circuit design, which considerably extends the maximum operating distance. leadless biv pacing is demonstrated for the first time in both open-chest and closed-chest porcine settings. the clinical outcomes associated with different interventricular delays are verified through electrophysiologic and hemodynamic responses. the closed-chest pacing only requires the external source power of . w and . w at . mhz and . mhz, respectively, which leads to specific absorption rates (sars) – orders of magnitude lower than the safety regulation limit. this work serves as a basis for future wirelessly powered leadless pacemakers that address various cardiac resynchronization challenges. miniaturized implantable medical devices (imds), overcoming the physical constraints of conventional devices, provide novel diagnostic and therapeutic solutions in the medical space. examples include implantable neural interface systems that achieve high spatiotemporal resolution and spatial coverage , microchips capable of wirelessly programmed scheduling of drug delivery for patients with osteoporosis , and a disposable endoscopic video capsule which permits visualization of the gastrointestinal tract by wireless transmission of images . while many remain battery-powered, wireless power transfer techniques can further reduce the form factor and invasiveness of these devices [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a conventional cardiac pacemaker consists of a pulse generator and pacing leads positioned using a transvenous approach. because it contains the battery, the pulse generator is relatively bulky. it is typically placed in the chest or abdomen, with the leads connecting to it on one end and to the myocardium on the other. approximately % of patients with chronic heart failure have electrical dyssynchrony in myocardial activation , and require cardiac resynchronization therapies (crts). existing crts rely on the use of multiple intracardiac leads to pace both ventricles in a synchronized sequence . while clinical improvements are generally achieved, six-month nonresponder rates have been reported to be as high as - % , , which is partially attributed to anatomic constraints of lead positioning . transvenous leads are also associated with various complications such as lead dislodgement , tricuspid valve dysfunction , and thromboembolism , etc., which would be solved with leadless pacemaker technologies [ ] [ ] [ ] . miniaturized and wirelessly powered pacemakers are envisioned to be directly implanted at the desired pacing sites. not only is the need for intravascular leads eliminated, but synchronized and leadless pacing across different chambers becomes feasible, which offers the flexibility in customizing patient-specific crts. prior studies have investigated wirelessly powered single-site cardiac pacing on rodent, rabbit and open-chest porcine models , , . this work takes a stride further to demonstrate the synchronization of multiple leadless pacemakers for the first time in a closed-chest porcine model. the improved clinical outcome is verified through both electrophysiologic and hemodynamic studies. local energy sources such as energy harvested from the cardiac and lung motions have been exploited as the replacement to batteries , , but is suspected to be insufficient in its power density . on the contrary, electromagnetic energy in near-and mid-field supplies sufficient power to medical implants , , as magnetic fields penetrate through tissue as in free space . utilization of such energy, however, poses two challenges: first, achieving resonant coupling in the low-frequency regime could often result in over-size of the implant due to the associated long wavelength. second, the magnetic field decays as the inverse cube of distance limiting the maximum operating range . this study seeks to mitigate the first challenge by achieving a relatively miniaturized, flexible and lightweight design to minimize the invasiveness for epicardial implantation , . for the second, the power consumption of the pacemaker is substantially reduced by customizing a low-power integrated circuit (ic), which alleviates the need for the incident power and, therefore, extends the distance of operation. in this work, we implement two inductively powered leadless pacemakers operating at . mhz and . mhz industrial, scientific, and medical (ism) bands, respectively. the pacemaker features a µw-level power consumption enabled by a custom low-power ic and is implemented on a flexible polyimide substrate with a dimension of mm × mm. to demonstrate wireless crt, two such pacemakers were epicardially implanted on the right ventricle (rv) and left ventricle (lv) in a porcine model for synchronized biventricular (biv) pacing with controllable interventricular offsets. biv pacing with zero offset achieves a qrs duration and hemodynamic response comparable to those of the intrinsic non-pathologic heartbeat. closed-chest biv pacing was further demonstrated with the specific absorption rate (sar) orders of magnitude lower than the safety regulation limit. this work is the first demonstration of the multisite crt based on batteryless and leadless pacemakers with improved clinical outcomes in both interventricular synchronization and cardiac stroke volumes. the wireless powering strategy can be applied to various other medical implants as well. system overview. biv pacing in this work is conceptually illustrated in fig. a . the pacemakers with the flexible design can be directly plunged into the cardiac epicardium. through resonant frequency selection, two pacemakers can be independently controlled in a biv pacing setting. the interventricular offsets can be programmed to optimize the clinical outcome according to established clinical practices . the as-fabricated pacemaker is displayed in fig. b . the device features an mm × mm dimension and resides on a µm thick flexible polyimide substrate housing the traces and the inductive coil. owing to this design, the pacemaker closely attaches to the surface of the heart as shown in fig. c , allowing the natural motion of the heart after implantation. the flexibility of the device is further demonstrated in fig. d ,e. pacemaker design. due to their excellent energy-efficiency , most existing implantable pulse generators (ipgs) rely on the voltage-controlled stimulation (vcs) scheme . the proposed pacemaker utilizes the vcs scheme with the core circuitry detailed in ref. . the block diagram schematic is shown in fig. a . most of the circuitry is integrated in a complimentary-metal-oxide-semiconductor (cmos) ic that includes the following main blocks: ( ) a rectifier that harvests the incident inductive energy and stores it on a storage capacitor, c sto . ( ) a voltage reference and an amplitude regulator that regulates the voltage of the stimulations. ( ) a demodulator that controls the timing and intensity of the stimulations. the operation scheme is illustrated in fig. b . the transmitting (tx) signal involves notches with programmable rates and widths. the ic detects and replicates these notches as the timing of the output stimulations . this method significantly simplifies the circuitry compared to prior arts , . the voltage of the pulses is regulated, and the pulse width is used to controls the stimulation intensity. as the notches in the tx signal only constitute a negligible portion timewise, they do not affect the wireless power transfer efficiency. the ic only consumes a static current consumption of na. as the supply voltage is about . v, the static power is merely µw. it also features a very small dimension, a pad-included area of µm × µm as shown in fig. c . the limited number of discrete components include an energy storage capacitor, c sto , a dc-block capacitor, c bck , for charge-neutralization of the stimulations, and a tuning capacitor, c tune , that adjusts the frontend resonant frequency. in addition, this work implements a green light-emitting diode (led) in the assembly, which helps to illustratively indicate the operation of the device. the pacemaker resides on a µm thick flexible polyimide substrate that incorporates a power receiving (rx) coil, as shown in fig. d . the rx coil features a double-sided design with six turns on both sides. the width and thickness of the copper trace are mils and mil, respectively. the inductance of the rx coils is about . µh and the self-resonance frequency (srf) is approximately at . mhz ism band. adding a c tune of µf in parallel with rx coil tunes the srf to be at around . mhz ism band. two pieces of awg stainless wire are used as the electrodes as well as the anchors for epicardial implantation. the front and back sides of the device are shown in fig supplementary fig. s a . the . mhz coil has a slightly larger dimension with six turns on both sides and an outer diameter of mm, as shown in fig. b . its backside is shown in supplementary fig. s b . the link efficiency is simulated with maxwell (ansys, inc) and simplorer (ansys, inc). figure c ,d demonstrate the d models for the . mhz and . mhz links, respectively. the link efficiency as a function of tx-rx distance is shown in fig. e . the . mhz shows a slower decay because of the slightly larger tx coil. both wireless power transfer links are validated in benchtop tests. with w tx power, . mhz and . mhz links render the maximum operating distance of . cm and cm as shown in fig. f ,g, respectively. it shows that while the efficiency of near-field inductive coupling systems decays according to the inverse-cubic relationship, µw-level wirelessly powered implants can still obtain a considerable range of operation. it is also worth noting that since the magnetic field penetrates through tissues equally as in the free air, biological layers in the link do not affect the coupling efficiency. the schematic in fig. h illustrates the setup for the synchronization of the two pacemakers in this study. an arbitrary waveform generator outputs two synchronized duty-cycled pulse signals that control two following signal sources via pulse modulation. the two signal sources generate rf signals at . mhz and . mhz ism bands, respectively, which inductively couple to the corresponding pacemakers. assuming tx-rx distance of cm, the isolation between the . mhz and . mhz channels is simulated as shown in fig. i . at . mhz, the selectivity of the . mhz pacemaker over the . mhz pacemaker is db ( x). likely, fig. a ,b, respectively, and fig. c shows biv pacing. pacing was performed at bpm with a pulse width of . ms in all cases. figure a ,b capture the blinking of the led and, in particular, fig. b shows that the . mhz pacemaker is powered under the porcine chest from the tx coil. the electrocardiogram (ecg) of the animal was monitored using the standard -lead setup. the ecg recordings of rv and lv single-site pacing are shown in fig. d ,e, respectively. rv pacing results in a qrs morphology with an initially positive vector, while lv pacing features a negative initial vector. most impressively, the ecg tracings of biv pacing in fig. f shows the transition from the intrinsic sinus rhythm to rv pacing and then to biv pacing. www.nature.com/scientificreports www.nature.com/scientificreports/ to test the impacts of different offsets in biv pacing, we programmed biv pacing at multiple offsets between the two ventricles, i.e., lv-lead ms before rv, lv-lead ms before rv, and rv-lead ms before lv. biv pacing with lv-lead ms and ms are demonstrated in supplementary videos s and s with -fold speed reduction. the qrs duration in the ecg tracing has been well used as a prognostic indicator of the clinical effectiveness of synchronized biv pacing , . the widening of qrs duration is clinically associated with worsening hemodynamics due to electrical blocks in the conduction system. in this study, the qrs durations corresponding to different single-site and biv pacing modalities are demonstrated in supplementary fig. s and the result is summarized in fig. g . the qrs duration of the intrinsic non-pathologic heartbeat is ms and biv pacing with zero offset renders a similar value. biv pacing with lv leading by ms and ms also outperforms single-site rv and lv pacing. in contrast, biv pacing with rv leading by ms shows a worsened result, suggesting a contradiction to the inherent conduction. diagnostic electrophysiology (ep) catheters (boston scientific, ma) inserted into the rv and coronary sinus (cs) successfully measured reproducible changes in myocardial wavefronts associated with each pacing modality as shown in supplementary fig. s a -d. distinct pacing artifacts with the programmed offsets are also evident. importantly, we further verified the acute benefits of biv pacing by analyzing the hemodynamic responses. pulse wave doppler images were used to calculate the velocity-time integrals (vti) that correspond to the stroke volumes (sv) in each pacing modality. aspiration was stopped for a brief period when the echocardiography data were collected as shown in fig. a . the hemodynamics of atrial pacing is first recorded, which renders an inherent vti of . cm (fig. b) . the vti during rv pacing, a condition classically associated with increased mortality, www.nature.com/scientificreports www.nature.com/scientificreports/ shows a significant reduction to . cm (fig. c) . in contrast, biv pacing with zero offset leads to a vti close to the inherent value (fig. d) , which is consistent with the qrs duration result. closed-chest pacing. the leadless pacing platform was then verified in a closed-chest setting. the porcine chest is about cm thick so that implanted pacemakers are about - cm from the body surface as shown in fig. a . the . mhz and . mhz pacemakers were implanted on the rv and lv, respectively, with their positions indicated in the fluorescent image in fig. b . tx coils were placed at approximately . - cm outside the sutured chest as shown in fig. c . closed-chest rv pacing and lv pacing were enabled with tx power as low as dbm ( . w) and dbm ( . w), respectively. biv pacing is also successfully performed in the closed-chest setting as shown in supplementary video s . figure d demonstrates the intrinsic ecgs in the closed-chest setting. the ecgs for rv pacing, lv pacing and biv pacing with lv-lead ms are shown in fig. e -g, respectively. a section of the qrs waveform during biv pacing as shown in fig. h evidently shows the pacing artifacts with a programed ms interval. the sars associated with the . mhz and . mhz links are investigated with the tx coils positioned at cm proximity to a male torso model. the two links generate the maximum sars of approximately . w/kg and . w/kg, respectively, as shown in fig. i ,j, which are significantly lower than the w/kg limit according to ieee std c . . it is remarkable that unlike prior arts operating at hundreds of mhz , , , the proposed inductive coupling links at . mhz and . mhz bands render sars - orders of magnitude lower than the safety regulation threshold. the magnetic fields penetrate through tissue, yet decay as the inverse cube of the distance. to harvest the inductive energy over sufficient distance for cardiac pacing, we designed a custom pacemaker ic with significantly reduced power consumption and dedicated power transfer links. the combination of efforts leads to the relative long-distance and reliable operation of the pacemaker. the power transfer links are implemented at . mhz and . mhz ism bands, rendering sars - orders of the magnitude below the safety threshold. this strategy can be applied to wirelessly powering many other minimal-power medical implants to realize future in-body sensor networks. in the pursuit of leadless crts, the wise crt system (ebr systems, inc.) has recently been launched in a clinical trial , . the current embodiment of this technology requires the use of a standard dual-chamber pacemaker with only the lv wirelessly paced via ultrasound power transfer, thus limiting the potential benefits of leadless pacing. the implantation of a hardware in the lv endocardium also necessitates a retroaortic access coursing through the arterial system. because the pacing lead is placed on the lv endocardium, antiplatelet www.nature.com/scientificreports www.nature.com/scientificreports/ therapy is warranted. our technology, however, does not have such limitations. the proposed work demonstrates synchronized leadless pacing over multiple sites for the first time, and this approach can potentially offer more flexibility and advantages in customizing crts for different individuals. in the comparison of the wireless powering strategies, magnetic fields in inductively coupling systems are not affected by biological tissues, while ultrasound power transfer essentially works through vibrations, which could deteriorate over air-filled viscera and obstructions, such as lungs and bones . the current form factor of the pacemaker is designed for epicardial pacing, which, for example, is a typical clinical approach for treating pediatric patients with congenital heart disease and requiring open-chest placement of epicardial pacing leads . our on-going research focuses on the miniaturization of the device and the associated venous delivery technologies, for example, epicardial implantation via venous tributaries that eliminates the necessity for anticoagulation. future iterations of the ic will also incorporate a unique identification code to avoid false-triggering. in summary, we report wirelessly powered pacemakers which feature µw-level power consumption and a miniaturized and flexible form factor that is suitable for epicardial implantation. two pacemakers were implemented at . mhz and . mhz ism bands, demonstrating the maximum operating distance of cm and . cm from w tx coils, 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future? cardiac pacing in paediatric patients with congenital heart defects: transvenous or epicardial? we thank dr. b. aazhang and dr. j. cavallaro for helpful discussions. aydin babakhani and mehdi razavi are co-founders of maxwell biomedical inc. supplementary information is available for this paper at https://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to a.b. or m.r. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - lvqpz authors: davies, patrick; yasin, samra; gates, simon; bird, david; silvestre, catarina title: clinical scenarios of the application of electrical impedance tomography in paediatric intensive care date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: lvqpz eit is a radiation-free functional modality that enables bedside imaging and monitoring of lung function and expansion. clinical interest in this method has been driven by the need for bedside monitoring of the dynamics of the lungs and the effects of ventilatory manoeuvres, including changes in ventilator settings, suctioning, chest drains, positioning and physiotherapy. we aimed to describe the use of electrical impedance tomography (eit) as a clinical tool in a tertiary paediatric intensive care unit. children requiring intensive care with a variety of clinical conditions had an electrode belt with electrodes wrapped around the chest, which sequentially applied a small alternating current from each electrode pair. the signal gives information on both real time, regional, global, and relative data. with the correct application, and understanding of the monitor, much clinical information can be gained, with potentially significant patient benefit. we present the clinical use of eit in six conditions: asthma, ventilation weaning and expansion recoil, sequential lobar collapse, targeted physiotherapy, pleural effusion assessment, and peep optimisation. screenshots and analyses are offered displaying the pragmatic use of this technology. electrical impedance tomography is a clinically useful tool on the paediatric intensive care unit. it allows monitoring of a patient’s respiratory function in ways which are not possible through any other means. an understanding of respiratory physiology will allow use of this information to improve patient outcomes. electrical impedance tomography (eit) is a radiation-free functional modality that enables bedside imaging and monitoring of lung function and expansion. it has been evaluated in a number of pulmonary conditions in humans and animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it has been used in various clinical settings including acute respiratory distress syndrome (ards), establishing the best positive end expiratory pressure (peep) [ ] [ ] [ ] [ ] [ ] , the response of the lungs to recruitment manoeuvres [ ] [ ] [ ] [ ] [ ] and trying to minimize areas of collapse and hyperinflation , . there are some studies that evaluate the application of high-flow nasal cannula therapy , and quantify the extent of pulmonary oedema in acute lung injury . eit however does not offer the same spatial resolution when compared with modalities such as computerised tomography (ct) scanning. it does however offer good temporal resolution and informs clinicians on real time regional ventilation distribution. the images produced can also be affected by body movement, changes in electrode contact, changes in posture , as well as interference with other medical devices . these need to be considered when interpreting the images. clinical interest in this method has been driven by the need for bedside monitoring of the dynamics of the lungs and the effects of ventilatory manoeuvres, including changes in ventilator settings, suctioning, chest drains, positioning and physiotherapy. our aim is to disseminate our experience of the use of electrical impedance tomography in the use of patients in paediatric intensive care, to act as a clinical primer for other clinicians and to communicate the pragmatic use of this technology. an electrode belt with electrodes is wrapped around the chest, which sequentially apply a small ac current from each electrode pair. the impedance between the electrodes sending the current and the receiving electrodes will change dependant on the makeup of the matter between the respective electrodes. air has a much higher impedance than, for instance, blood. with the application of tomography, the sequential signals can be built up in to a dimensional × pixel picture in real time with a frame rate of between and hz . the information is visualised on a screen as a "heat map", where areas of impedance change (equivalent to ventilation) are displayed. the signal gives information on both real time, regional, global, and relative data. with the correct application, and understanding of the monitor, much clinical information can be gained, with potentially significant patient benefit. the information can be categorised in to global chest expansion, regional ventilation, and ventilatory compliance (expansion over time), with both absolute and relative figures for all , . interpreting this data requires an understanding of ventilatory physiology and experience, however is easily learnt by interested parties. there are two main views: a live view which shows moving images of a cross section of the lung, with regional ventilation clearly seen, and an end expiratory lung impedance view, which gives breath by breath impedance of the whole lung. until now, this technology has mainly been clinically used, infrequently, in adults. we describe the clinical applications of the use of eit in children, with the physiological background and interpretation of the images. we used a draeger pulmovista eit monitor, with a variety of belts which allow monitoring of children above . kg in weight. the use of the monitor was explained to all parents and verbal consent obtained. the chair of the nottingham research ethics committee confirmed the united kingdom health research authority decision tool opinion, which states that this project is exempt from needing ethical approval. clinical scenarios. eit is used routinely in complex children on our picu. there was no specific case selection; we present six cases where the use of eit has been of direct clinical benefit to children being treated for a variety of conditions. we present six differing clinical scenarios; all commonly found on paediatric intensive care units, and demonstrate how eit can add important information which would not be otherwise available. we display the use of eit in: ventilatory changes were attempted with the aim of optimising his gas exchange and reducing air trapping. it was unknown whether air trapping was occurring, relative to baseline. the eit trace shows initially a change from a pip of cm h o to a pip of , with constant peep, and a change of rate from to ( fig. , first arrow). the global impedance curve immediately begins to fall, reaching steady state around two minutes after. this signifies an overall decrease in the chest expansion (or end-expiratory lung volume [eelv] ). this reduction in eelv is a strong indicator for a significant reduction in gas trapping, which should improve chest compliance. at minutes in to this recording (second arrow) the rate was increased to due to a significant reduction in the minute volume. there was a small, but insignificant rise in the global expansion. the expansion remains lower than at the initial settings. tidal volumes remained static, implying no change in the compliance (and no change in overdistension) after this rate increase. this allowed increased co clearance with no corresponding hyperinflation at reduced ventilatory settings. global chest impedance/end-expiratory lung impedance. a child with a diagnosis of pneumonitis whilst on volume ventilation was considered for weaning of ventilation as the ventilatory parameters had improved. however, when the peep was reduced by cm h o from to , the global chest expansion displayed a sequential loss of volume over time. the peep was returned to the previous settings. on suctioning and returning to the ventilator, his oxygen saturations fell, requiring ventilation by anaesthetic bag. analysis of the eit trace showed that on returning to his ventilation, his global expansion (eelv) took a long time to return to baseline (fig. ) . after another period of ventilation, a similar reduction in peep showed a reduction in volume (as would be expected with reduced pressures), but this was stable over time . he was suctioned for secretions and had not shown instability. analysis of his eit trace showed return to near baseline within a small number of breaths. eit was thus able to illustrate a lack of resilience to initial weaning, which later improved, providing added confidence to continue weaning towards extubation. www.nature.com/scientificreports www.nature.com/scientificreports/ sequential lobar collapse: effect of postural changes. eit parameter: live regional ventilation distribution/tidal image. a year old girl admitted with adenovirus and respiratory syncitial virus pneumonia with type respiratory failure, ventilated on pressure control with pip , peep , rr - , and fio . - . . she had frequent lobar collapse and she was monitored with eit which showed right sided lung collapse. she was nursed with the chest right side up, and within one hour her ventilation was balanced and equal, with expansion returning to the collapsed lung. however, after another hour the left (dependant) side lost ventilation. for the next hours, continuously monitored by eit, she was repositioned as soon as one lung showed signs of loss of ventilation, usually every - hours. with this technique, the position of the patient was optimised based on lung expansion, and allowed the patient to wean ventilation and be extubated days later. figure shows tidal images with a relative hypoventilation of the right lung, she was repositioned with the left side of the chest up and hours later, the right lung expansion had improved, at the cost of the left lung ventilation. she was then turned again. targeted physiotherapy: lobar or lung collapse: assessing progression and effect of physiotherapy. eit parameter: tidal image showing relative regional ventilation distribution. a year old boy was admitted with a background of brainstem glioma when he was years old, with significant sequelae including paraplegia and loss of brainstem function needing long term ventilation support hours a day though a tracheostomy he was admitted with right sided total lung collapse needing picu admission and escalation of his ventilation to a picu ventilator: pressure control with pip , peep , rr , and fio - %.eit monitoring was instigated. he received regular airway clearance using manual techniques and mechanical insufflation-exsufflation (mi:e). observing eit during physiotherapy treatment it was clear that the exsufflation component of mi:e, despite effectively clearing secretions, was causing temporary collapse of the right lung (fig. ) . this was not resolved by www.nature.com/scientificreports www.nature.com/scientificreports/ the re-inflation breaths available on the mi:e device. the physiotherapy team were able to adjust their treatment plan to include further re-recruitment following exsufflation. this prevented deterioration in objective parameters following airway clearance. following days of treatment, his right lung was clinically fully reinflated, confirmed by eit. he did not have any chest radiographs during this time. pleural effusion: quantifying respiratory compromise. eit parameter: relative regional ventilation distribution. a year old with a diagnosis of chronic myeloid leukemia present ed to the hospital with disseminated varicella zoster infection with multi organ failure needing respiratory, renal. and inotropic support. she had a pleural effusion of cm on the chest x-ray, which was measured at . cm on chest ultrasonography. her oxygen requirements were high, with an fio of . . %. a decision needed to be made whether insertion of a chest drain would be beneficial, with the heightened risk of an intrathoracic bleed due to the presence of disseminated intravascular coagulopathy. eit showed a very significant loss of expansion in the affected side, compared to the non affected side. on balance, a chest drain was felt to be in her best interests, and it was inserted without complication. the ventilation improved. eit pictures showed real time improvement of inflation, with aeration up to the chest wall (fig. ) . choosing the optimal peep level minimises overinflation and reduces collapse and makes physiological sense. analysis showed that the optimum peep (i.e. that which balances overdistension and collapse) was cm h o (fig. ) . the patient's peep was increased to cm h o. www.nature.com/scientificreports www.nature.com/scientificreports/ we have presented six clinical scenarios where the use of eit has materially benefitted our paediatric intensive care patients. the information obtained by using eit is difficult or impossible to be gathered by any other non-invasive technique. conventional monitoring modalities give some feedback, but this is often delayed, inaccurate, or with a low level of accuracy . the interpretation of the images is critical to the use of eit. our six scenarios can be interpreted as follows: . asthma: gas trapping is a difficult problem in such patients and quantifying the extent of the problem is impossible. it only becomes apparent when ventilation becomes compromised, and then it is difficult to know whether this change in ventilation is due to clinical deterioration, or the respiratory dynamics. use of www.nature.com/scientificreports www.nature.com/scientificreports/ eit gives instant, breath by breath feedback on the global chest expansion, allowing pre-emptive management of incipient gas trapping problems. . ventilation weaning and effects of disconnection. the patient will not display clinical signs soon after a ventilator change: it may take many hours for this to manifest. however, it can be anticipated that problems will arise if the patient is unable to maintain adequate lung expansion with the ventilatory settings which have been applied. if the global end-expiratory lung volume keeps dropping after the (expected) initial volume loss, then problems can be anticipated in the short to medium term. a patient who can hold a stable expansion after a ventilatory parameter change is likely to have a greater chance of medium term stability. current monitoring techniques are poor at predicting rapidity of atelectasis and lung collapse after weaning. knowledge of this may help to predict whether a patient is ready for extubation. the first trace shows a patient whose lungs cannot yet cope with a loss of peep, and is not ready for extubation. the second shows a patient who can rapidly restore lung volume and maintain adequate expansion making him suitable for extubation. . sequential lobar collapse. repositioning a patient with unilateral lobar collapse can cause respiratory instability, leading to a practice of minimal handling and reducing the frequency of unstable episodes. clinical examination is inaccurate in a small chest with many added sounds. chest radiography gives good knowledge of lobar collapse, but that may lead to worsening of gases and there is the added concern of radiation dosage. the use of eit allows for monitoring of lung inflation in real time and ensure that significant areas of dependent collapse are avoided limiting respiratory instability due to repositioning. direct visualisation identifies lung collapse rapidly making reinflation easier. . targeted physiotherapy. constant eit monitoring allowed us to monitor lung expansion without resorting to frequent chest radiographs. the physiotherapy team were able to quickly identify an adverse effect of treatment and adjust their plan accordingly. with standard monitoring it may not have been apparent why objective markers were deteriorating following airway clearance. . pleural effusion. chest x-ray, and sonography, show a depth of fluid but do not give a sense of how much this fluid affects the lung inflation. using live regional ventilation information allows precise visual quantification of this, meaning that clinical decisions can be based on more accurate information. in this case, the chest drain was high risk, but with the addition of eit data this risk was felt to be worth taking. cases of pneumothorax or foreign body would have a similar eit picture, with the ability to quantify which areas of lung are non-functional and to what degree. . peep optimisation. establishing the optimal peep in invasively ventilated patients is a challenge; the balance between collapse and over distension is difficult to assess and no method has been shown to prevent ventilator induced lung injury. eit can be used to evaluate the regional compliance changes at different levels of peep, measuring areas of collapse and over distention during a peep trial. both collapse and overdistension are detrimental to the patient. knowledge of the effects of the peep to the patient allows the clinician to titrate it to produce minimal detrimental effects. eit is often used for continuous monitoring. real time visualisation of chest expansion leads to knowledge of why an acute respiratory compromise has occurred. the treating team can be instantly aware of where the problem lies. we also use eit for progressing ventilation in the complex patient. no other non-invasive monitoring modality will give instant feedback over ventilatory changes. such patient's fragility means that delayed feedback from blood gas analysis or clinical change would be potentially harmful. the use of eit allows the treating team to make changes they would not have dared to otherwise make due to their fragility. by making quicker progress than anticipated we are able to minimise lung injury and maximise chances of recovery. a further clinical use on our unit is as a feedback spirometer. conscious patients of a suitable developmental age can respond well to visualisation of how to ensure that breathing is effective. there are multiple modalities of assessing a patient on mechanical ventilation (table ). there is, however, no perfect modality as they all have their disadvantages. clinical examination is a single event, low resolution method of understanding lung movement. the benefits of ct scanning are offset by the radiation exposure as well as the logistical difficulties involved in taking the patient to the scanner. x-rays have lower logistical www.nature.com/scientificreports www.nature.com/scientificreports/ difficulties and lower radiation, but they do not allow continuous investigation, or measurements of flow or dynamic expansion . ventilator spirometry feedback allows no regional data, but does have the benefit of allowing continuous data . lung ultrasound gives important and useful information, but is a non-continuous, low resolution modality. compared to these monitoring modalities, eit has significant benefits. it is a continuous, high resolution monitoring which allows assessment of dynamic, expansion, and flow data. with any novel monitoring technology, there is a learning curve for clinicians. although eit needs good education for treating staff, this is a very visual monitor which can give information even if the technology behind it is not well understood. our experience is that after some initial scepticism, the bedside team rapidly adopted it as the benefits were explained. there is presently little clinical evidence of the efficacy of eit in practice. good ventilatory practice, involving the avoidance of hyperinflation and/or collapse, is associated with better outcomes. the information from eit monitoring should allow better ventilatory practice, however the translation of this in to clinical outcomes is as yet unproven. suitably sized eit belts are not commercially available for paediatric patients and the use of individual electrodes is time-consuming and risks skin irritation. this severely limits the use in this population. its use in non-sedated patients is also limited due to patient anxiety and movement, causing potential image artefacts. studies have also shown a difference in skin impedance in neonates as compared to adults . reconstruction algorithms in current eit modalities also do not account for differences in chest shape or size for neonates . however, as this technology becomes more widely used, and the information gained from it better understood, there will be more commercial pressure to ensure that patients of all ages and sizes are catered for. as clinical experience and knowledge increases, this may become a routine part of clinical care in complex children. with all forms of highly individualised medicine, there are concerns as to its effectiveness. population evidence allows excellent assessment of a treatment's long-term effectiveness, for a population mean. however, population evidence cannot tell the clinician whether the treatment will work for the individual patient, merely that if they treated patients, it would be of overall benefit to give such a treatment. this monitor offers evidence to the clinician that the physiology has changed for the individual patient. it cannot tell the clinician whether this has long term, or outcome benefits. an excellent understanding of physiology is necessary to ensure that physiological benefits then translate to outcome benefits. electrical impedance tomography is a clinically useful tool on the paediatric intensive care unit. it allows monitoring of a patient's respiratory function in ways which are not possible through any other means. an understanding of respiratory physiology will allow use of this information to improve patient outcomes. absolute electrical impedance tomography (aeit) guided ventilation therapy in critical care patients: simulations and future trends effect of closed endotracheal suction in high-frequency ventilated premature infants measured with electrical impedance tomography assessment of lung ventilation in infants with respiratory distress syndrome using electrical impedance tomography electrical impedance tomography monitoring in acute respiratory distress syndrome patients with mechanical ventilation during prolonged positive end-expiratory pressure adjustments lung recruitment and endotracheal suction in ventilated preterm infants measured with electrical impedance tomography identification of regional overdistension, recruitment and cyclic alveolar collapse with electrical impedance tomography in an experimental ards model assessment of lung recruitment by electrical impedance tomography and oxygenation in ards patients positive end expiratory pressure titration after alveolar recruitment directed by electrical impedance tomography assessment of regional lung recruitment and derecruitment during a peep trial based on electrical impedance tomography bedside measurement of changes in lung impedance to monitor alveolar ventilation in dependent and nondependent parts by electrical impedance tomography during a positive end-expiratory pressure trial in mechanically ventilated intensive care unit patient assessment of respiratory system compliance with electrical impedance tomography using a positive endexpiratory pressure wave maneuver during pressure support ventilation: a pilot clinical study protective ventilation using electrical impedance tomography monitoring of recruitment and derecruitment by electrical impedance tomography in a model of acute lung injury end-expiratory lung impedance change enables bedside monitoring of end-expiratory lung volume change regional respiratory time constants during lung recruitment in high-frequency oscillatory ventilated preterm infants slow moderate pressure recruitment maneuver minimizes negative circulatory and lung mechanic side effects: evaluation of recruitment maneuvers using electric impedance tomography bedside selection of positive end-expiratory pressure by electrical impedance tomography in hypoxemic patients: a feasibility study oxygen delivery through high-flow nasal cannulae increase end-expiratory lung volume and reduce respiratory rate in post-cardiac surgical patients effect of high flow nasal cannula and body position on end-expiratory lung volume. a cohort study using electrical impedance tomography electrical impedance tomography (eit) for quantification of pulmonary edema in acute lung injury electrical impedance tomography: the realization of regional ventilation monitoring chest electrical impedance tomography examination, data analysis, terminology, clinical use and recommendations: consensus statement of the translational eit development study group positive end-expiratory pressure optimization using electric impedance tomography in morbidly obese patients during laparoscopic gastric bypass surgery bedside estimation of recruitable alveolar collapse and hyperdistension by electrical impedance tomography non-invasive radiation-free monitoring of regional lung ventilation in critically ill infants computed tomography-an increasing source of radiation exposure regional ventilation distribution in the first months of life p.d. conceived the paper and coordinated contributions from the clinical team. p.d., c.s., s.g., s.y. and d.b. shared their eit patients and knowledge. all authors wrote and edited the paper and figures. competing interests: draeger ag paid for the article processing fee. they were only contacted after final completion of the paper. draeger had no input in to the study design or patient selection, and did not edit any drafts or proofs of the paper. there are no other competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - a sgir authors: han, nayoung; oh, jung mi; kim, in-wha title: assessment of adverse events related to anti-influenza neuraminidase inhibitors using the fda adverse event reporting system and online patient reviews date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: a sgir the recommended antiviral drugs available for the treatment and prevention of influenza are neuraminidase inhibitors (nais). the aim of this study was to evaluate age-related clinical manifestations of adverse events (aes) related to nais. faers and webmd data were downloaded. the available nais selected for the analysis were oseltamivir, peramivir, zanamivir, and laninamivir. disproportionality was analyzed using the proportional reporting ratio (prr), the reporting odds ratio (ror), and the information component (ic) methods. in total, aes from patients and aes from patients in the faers and webmd, respectively, were included in the analysis. in the faers, aes were more common among those who were younger (< years) for zanamivir, while for those who were older (> years) for peramivir. a disproportionality analysis showed that signals for vomiting and hallucinations were detected in younger patients given oseltamivir, while an abnormal hepatic function, cardiac failure, shock, and cardio-respiratory arrest were detected in older patients given peramivir. psychiatric disorders were most common in younger and older patients, while gastrointestinal disorders were most common in adult given oseltamivir in the webmd. adverse symptoms related to nais varied and depended on the drugs used and the age of the patient. characteristics of the study population from the faers. each case patient has more than one preferred term for a specific drug reported, therefore resulting in more than one drug-ae pair for each ae report. in total, aes from patients from january of to december of were included in the analysis of the nais and aes from the faers data. the characteristics of the patients and the ae reports from the faers are presented in table . the mean age of the patients was . and . years for oseltamivir and peramivir, respectively. a mean of . for aes on average was reported per patient administered with oseltamivir, and these patients were co-administered with a mean of . for other drugs. aes were more common among younger patients ( out of , . %) for zanamivir, while they were more common for older patients ( out of aes, . %) for peramivir. aes by nais from the faers. the system organ classes of aes reported for nais are shown in table . aes were most frequently reported for oseltamivir ( , . %), followed by zanamivir ( , . %), peramivir ( , . %), and laninamivir ( , . %). psychiatric disorders ( , . %) were the most common ae clinical symptoms for oseltamivir. the occurrence rates of psychiatric disorders of zanamivir, laninamivir, and peramivir were . %, . %, and . %, respectively. cardiac and vascular disorders ( . % and . %, respectively) were the most common aes for peramivir, while general disorders and administration site conditions ( , . %) were the most common aes for zanamivir. a disproportionality analysis showed that the signals of vomiting, hallucination, headache, insomnia, fatigue, and dizziness were detected for oseltamivir (table ) . there was no ae signal defected for laninamivir, peramivir, and zanamivir. therefore, a further subgroup analysis was conducted with younger and elderly patients. the disproportionality analysis showed that signals for vomiting and hallucinations were detected in younger patients (< years) given oseltamivir, while an abnormal hepatic function, cardiac failure, shock and cardio-respiratory arrest were detected in older patients (> years) given peramivir (table ) . www.nature.com/scientificreports www.nature.com/scientificreports/ characteristics of the study population from the webmd. in total, review comments and subjects from reviewers from oct to may were included in the nai-associated aes analysis after excluding instances with no comments from the webmd data. the characteristics of the subjects from webmd are presented in table . these subjects consisted of younger ( . %), adult ( . %), and older subjects ( . %). review comments were most frequently reported for oseltamivir at ( . %), followed by zanamivir at ( . %). the content themes of the review comments contained mostly reasons why the medicines were being taken ( , . %) and aes ( , . %). aes by nais from the webmd. aes were most frequently reported for oseltamivir ( , . %), followed by zanamivir ( , . %). among those taking oseltamivir, psychiatric disorders ( , . %) were the most common symptoms, followed by gastrointestinal disorders ( , . %) and cardiac disorders ( , . %) ( table ). psychiatric disorders were most common in younger ( . %) and older ( . %) patients, while gastrointestinal disorders were most common in adult patients ( . %) given oseltamivir (table ) . nais remain a widely licensed class of antiviral drugs appropriate for the treatment and prophylaxis of seasonal influenza . however, there is still concern regarding the adverse effects of nais. this study analyzed the age-related aes associated with nais using data from faers and webmd. the results of this study demonstrated that the occurrence rate of aes and adverse symptoms varied and depended on the nais used and the age of the patient, despite the considerable degree of structural similarity. oseltamivir was the nai most commonly showing aes in the faers data, and the most common aes for this drug were psychiatric and gastrointestinal disorders, similar to the findings of previous studies , [ ] [ ] [ ] [ ] . for zanamivir, the most common aes were general disorders and administration site conditions, consistent with a previous report . the signal detection prr, ror, and ic methods were able to detect several aes associated with oseltamivir only in the faers data. the most likely cause is the extremely low number of ae cases for other nais. to support our results, sensitivity analyses were conducted using the disproportionality method stratified according to gender or type of reporter. similar trends were observed in other sensitivity analysis that limited the data further via certain gender or health professional reporters. additionally, ae signals for vomiting and hallucinations were detected in younger patients given oseltamivir, while an abnormal hepatic function, cardiac failure, shock and cardio-respiratory arrest were detected in older patients given peramivir. however, in the webmd data, we could not detect signals by these disproportionality analyses due to the small number of ae cases, although psychiatric and gastrointestinal disorders were the most common aes reported. the numbers of the younger and www.nature.com/scientificreports www.nature.com/scientificreports/ older subjects were quite low compared to the number of adults in the webmd data, possibly due to the low rate of accessibility to the internet or digital devices and/or the recognition of the need to report. oseltamivir phosphate is an oral prodrug which undergoes hydrolysis by hepatic esterases to convert an active metabolite, oseltamivir carboxylate . oseltamivir can induce neuropsychiatric adverse effects with either a sudden or delayed onset. sudden-onset reactions are due to the direct effects of oseltamivir on the central nervous system, whereas delayed-onset reactions are due to the effects of oseltamivir carboxylate [ ] [ ] [ ] . oseltamivir phosphate itself can cause the central depressant actions that may result in abnormal behavior, delirium, hallucinations, sleep, and respiratory depression . oseltamivir phosphate can inhibits nicotinic acetylcholine receptors and monoamine oxidase a. additionally, gamma-aminobutyric acid receptors and n-methyl-d-aspartate and their related receptors/channels are thought to be other candidates related to respiratory suppression . it has been shown that while oseltamivir carboxylate cannot pass through the blood-brain barrier (bbb) readily, it may do so when combined with other agents or when the bbb is immature or impaired . most oseltamivir-induced neuropsychiatric adverse effects have been reported in asian populations , , . li et al. hypothesized that a nonsynonymous single-nucleotide polymorphism rs , near the active site of human cytosolic sialidase which is a homolog of the virus neuraminidase and presents in asian populations ( . %), could increase the binding affinity of sialidase to oseltamivir carboxylate, thus reducing the sialidase activity and contributing to the occurrence of severe neuropsychiatric adverse effects . it has been reported that the most commonly reported events when using zanamivir as a treatment were gastrointestinal and respiratory, thoracic and mediastinal disorders, but the incidences were similar to those in a placebo group . the most frequently reported aes of laninamivir were gastrointestinal disorders ( . %), psychiatric disorders ( . %), and skin disorders ( . %) during early post-marketing phase vigilance . the aes of peramivir were a high mortality rate, the development of acute respiratory distress syndrome ( %), and renal table . demographic characteristics of the subjects from the webmd data. a total number of themes counted in each review. this is greater than the total number of reviewers because some reviews had more than one theme tied for the concern. scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ failure ( %) in the faers system . these results were similar to the results here. however, an abnormal hepatic function and cardiac failure were detected in older patients given peramivir in our results. to the best our knowledge, this is the first study that compares nai-related aes in senior citizen patients using faers data. because it is noteworthy that cardiovascular risks are high in older persons, care must be taken when administering these drugs in this population. a disproportionality analysis alone is not sufficient proof of the drug ae association nor a causality assessment of an individual report . there is no way to determine exactly how many people took a particular drug, nor is there any method by which to ascertain how many events occurred when they took the drug. therefore, in our study, we do not know actual real number of patients exposed to nais. accordingly, it is difficult to assess the relationship between their aes and incidence rates. it has been reported that there were , , prescriptions of oseltamivir capsules, , prescriptions of oseltamivir powder, and , prescriptions of zanamivir among , , outpatients from to in the sentinel system of the centers for disease control and prevention's influenza-like illness surveillance network system and national respiratory and enteric virus surveillance system . pharmacoepidemiological methods as presented in case-control or cohort studies are considered as the best sources of drug safety data. however, they are also associated with several unfavorable methodological issues, such as a limited sample size, reduced follow-up, and evaluations of surrogate markers . fortunately, a quantitative means of comparing the ae rates of one drug with those of all other drugs is now available. nevertheless, the outcomes of comparisons of ae rates through a disproportionality analysis can be influenced by many factors, including absolute report numbers, the presence of other aes associated with the same drug, significant heterogeneity, and potential bias issues such as physician preference for one drug over others and a patient's negative experience with a certain product along with reporting biases such as underreporting . additionally, other factors that can influence the association between the frequency of ae reporting and medicine include the length of time the medicine has been marketed and the extent of publicity about new safety concerns. furthermore, with this approach it is necessary for each ae report to be validated before any analysis in the context of the pharmacological and medical hypothesis of the study in order to prevent false results . another limitation of this study was that the reported numbers of adverse cases for nais were low, except for that associated with oseltamivir, possibly because the ae reports were submitted voluntarily, leading to their being underreported, or there may have been cases which lacked approval by the u.s. fda. however, the early detection of aes and risk evaluations in vulnerable populations are important. continued efforts with regard to www.nature.com/scientificreports www.nature.com/scientificreports/ the identification and evaluation of aes associated with nais as well as to understand their underlying susceptibility mechanisms, are needed to treat and manage aes more efficiently in patients with influenza. in conclusion, aes associated with nais were analyzed using data from the two databases. serious aes associated with nais may have a significant impact on younger or older patients. from the findings here, younger or older patients should be monitored carefully for aes when treated with nais. data collection. the study population consisted of patients reported to have aes in the faers and webmd datasets. reported ae cases related to nais from the faers database were used in this study. aes and medication errors are coded to terms in the medical dictionary for regulatory activities (meddra) terminology . the faers data from to were downloaded. duplicated reports were deleted according to the u.s. fda's recommendation of adopting the most recent case number. the drug lexicon was devised using both the generic and trade names in the faers database. the available nais selected for the analysis were oseltamivir, peramivir, zanamivir, and laninamivir. the preferred term and the system organ classes in the meddra were used for further analysis. two or more preferred terms reported in one patient were counted as different aes. instances of co-administration with nais were excluded from any further analysis. for text mining, subject comments pertaining to oseltamivir, peramivir, and zanamivir downloaded from webmd using the python-based library beautiful soup . the webmd data from oct to may were downloaded. each reported symptom was assigned the preferred terms in the meddra terminology manually. information about age, sex, condition, reviewer types, report date, and treatment duration was collected. statistical analysis. patients were classified into three age groups: children and adolescents (age < years), adults (age - years) and older people (age ≥ years). the meddra preferred term was used for a quantitative disproportionality analysis. disproportionality was analyzed using the proportional reporting ratio (prr) , the reporting odds ratio (ror) , and the information component (ic) methods . the prr is calculated according to the ratio of the proportion of all reported cases of the event of interest among people exposed to a particular drug to the corresponding proportion among people exposed to all or several other drugs . the ror is calculated according to ratio of the odds of the reporting of one specific event versus all other events for a given drug relative to the matching reporting odds for all other drugs . the ic measure can be considered as the calculation of the logarithm of the ratio of the observed rate of reporting of a specific drug-ae combination to the expected rate under the null hypothesis of no association between the drug and ae . for the prr, a given drug ae pair was defined as a signal if the event count was or more, while the prr was or more with an associated chi-square value of or more . for the ror, it was defined if the lower limit of the % two-sided confidence interval (ci) of ror exceeded . the information component (ic) algorithm performs signal detection via the ic metric, which is a lower bound of the % two-sided confidence interval of ic, with an ae signal indicated when the ic value exceeds . for the webmd data, the frequency of each theme of a patient medication concern was computed for each comment, with these outcomes then summed for all comments. data were analyzed with microsoft excel (microsoft, redmond, wa, usa) and sas version . 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spontaneous adverse drug reaction reports a comparison of measures of disproportionality for signal detection in spontaneous reporting systems for adverse drug reactions a bayesian neural network method for adverse drug reaction signal generation this study was supported by the national research foundation of korea grant funded by the korea government (msit) (no. r a b ). n.h. analyzed the data and prepared the manuscript. i.w.k. and j.m.o. contributed to the conception and design of the study. all authors were engaged in commenting on the manuscript, read, and approved the final manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to j.m.o. or i.-w.k.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -dmpoy b authors: rowe, john c.; attia, zayed; kim, eunsoo; cormet-boyaka, estelle; boyaka, prosper n. title: a novel supplementation approach to enhance host response to sublingual vaccination date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dmpoy b sublingual immunization is emerging as an alternative to nasal immunization and induction of mucosal iga responses. using bacillus anthracis edema toxin (edtx) as an adjuvant, we previously showed that innate responses triggered after sublingual immunization could limit generation of iga responses. we tested whether co-administration of a neutrophil elastase inhibitor (nei) could rescue the ability of edtx to induce broad antibody responses, including mucosal iga. nei supplementation of sublingual vaccines containing edtx promoted antigen-specific serum iga responses but also enhanced serum igg , and igg b responses. this enhancing effect of nei did not extend to all antibody isotypes and igg sublclasses, since nei reduced serum ige responses and did not affect igg a/c and igg responses. nei supplementation also promoted anti-bacillus anthracis protective antigen (pa) neutralizing antibodies and enhanced high affinity igg and iga antibodies. in addition to serum iga, nei supplementation stimulated antigen-specific mucosal iga responses in the gi tract, and enhanced antigen-specific igg responses in vaginal washes. analysis of cd (+) t helper cell responses revealed that co-administration of nei broadened the profile of cytokine responses, by stimulating th , th , th , and tfh cytokines. we also noted that nei had a higher stimulatory effect on il- , il- , il- responses. needle-free vaccines delivered via mucosal surface have the potential of being better-accepted by the most vulnerable and commonly vaccinated population of children. they also present higher likelihood to generate the necessary b and t cell responses for optimal protection at the portal of entry of infectious agents, in addition to promoting the levels of systemic immunity generally achieved by conventional injected vaccines . secretory iga (siga) represents the hallmark of immune responses at mucosal surfaces. the high resistance of these polymeric immunoglobulins to degradation in the harsh environment of mucosal surfaces, including the lumen of the gastrointestinal tract, allow them to provide frontline protection at the portal of entry of most infectious microbes . while mucosally delivered subunit-vaccines have the potential of stimulating broad mucosal and systemic immune responses, their ability to trigger mucosal iga relies on the addition of effective vaccine adjuvants. stimulation of inductive site immune responses in different mucosal sites (i.e., gastrointestinal tract, respiratory tract, rectal) imprints the expression of discrete mucosal homing receptors and adressins which allow effector b and t cells to home in distinct mucosal tissues. for example, intranasal delivery of vaccines containing appropriate mucosal adjuvants can promote specific immune responses in the airways. however, safety issues were reported following intranasal application of a ganglioside-binding toxin as adjuvant. sublingual immunization is now being considered as an alternative to the intranasal route of vaccination. nonetheless, a major challenge for the development of sublingual vaccines is the identification of appropriate antigen-adjuvant formulations , . we have previously shown that bacillus anthracis edema toxin (edtx) is an effective adjuvant capable of promoting both systemic immunity and mucosal siga responses against nasally co-administered vaccine antigens , . however, when edtx was tested as adjuvant for sublingual vaccination, it promoted antigen-specific igg responses in the bloodstream but failed to elicit iga responses in the serum or mucosal secretions . this lack of iga responses was not due to the route of immunization itself, since sublingual immunization could induce these responses when vaccines were administered with a range of adjuvants including bacterial enterotoxins, toll-like receptor ligands, and sting ligands [ ] [ ] [ ] [ ] [ ] . interestingly, the lack of iga response correlated with the recruitment of neutrophils after sublingual administration of edtx, and partial depletion of neutrophils before sublingual immunization restored the adjuvant activity of edtx for iga responses of myeloid cells in the bloodstream and characterize the initial response to inflammatory events through their own degranulation and cytokine production . unfortunately, depletion of neutrophils prior to immunization is not a feasible approach and thus, new strategies are needed to improve the efficacy of edtx-based, and possibly other, sublingual vaccines. neutrophils are recruited by inflammatory cytokines, including il- , il- β, and tnfα, and were more recently recognized to contribute to the chemotaxis of other myeloid cells through the products released after neutrophil degranulation , . neutrophil elastase inhibitors (nei) are a class of serine protease inhibitors that target the neutrophil granule protein elastase, commonly implicated in chronic lung inflammation [ ] [ ] [ ] . co-administration of a nei enhances serum igg and igg b responses to a sublingual vaccine. we first asked whether supplementation with a nei could affects igg responses induced by a model sublingual vaccine containing edtx as adjuvant. vaccines targeting two or more pathogens can reduce the schedule of vaccination. we choose to use ovalbumin (ova) plus bacillus anthracis protective antigen (pa) as a combinatorial antigen to test the ability of nei to regulate the immune response to two different antigens. ova is a well-studied model antigen which allow a more in-depth analysis of immune responses to vaccination thanks to the large number of reagents available to study ova-specific b and t cell responses. on the other hand, the use of pa as antigen allowed us to address the biological significance of the antibody responses through the assessment of anti-pa antibodies (abs) ability to neutralize anthrax lethal toxin (letx). analysis of ova-specific igg responses revealed that nei increased the magnitude of responses induced by edtx, and this effect was evident as early as day after the first immunization (fig. a) . we also found that the nei used in these studies had an intrinsic adjuvant activity and increased igg responses when co-administered with antigen in the absence of edtx (fig. a) . analysis of other igg subclasses showed no evidence that the nei enhanced igg a/c, or igg when co-administered with antigen in the absence of edtx (fig. b,c) . however, the nei significantly increased serum igg b responses induced by edtx as adjuvant (fig. b ,c). since the presence of nei differentially regulated igg subclass responses, we next addressed its potential effects on other immunoglobulin isotypes. sublingual immunization with edtx as adjuvant induced ova-specific ige responses which peaked weeks after the first immunization (day ) (fig. a) . co-administration of nei significantly reduced the magnitude of ige responses to levels barely at the detection threshold ( fig. a) . furthermore, no ige response was detected following sublingual application of nei and antigen alone ( fig. a ). as previously reported , sublingual immunization with edtx as adjuvant failed to induce ova-specific serum iga responses (fig. b) . mice co-administrated antigen and nei showed detectable but minimum levels of serum iga responses at day . minimum serum iga levels were also induced and could be measured on day in mice immunized with antigen (ova + pa) plus ef. interestingly, only mice immunized with antigen and ef in the presence of the nei developed significant serum iga responses (fig. b ). nei supplementation enhances the amount of high affinity abs and toxin neutralizing responses in mice immunized with edtx as adjuvant. results summarized above show that nei supplementation improves the kinetic of igg responses and allows mice immunized with edtx to reach maximum igg titers more quickly ( fig. ). they also show that nei broadens the antibody responses by enhancing igg b and promoting iga (figs and ). to determine the biological significance of the broader antibody responses measured in the blood of mice administered vaccines supplemented with nei, we first assess anti-pa neutralizing antibodies. serum of mice immunized with antigen alone (ova plus pa), antigen plus nei, or antigen plus nei contained low and comparable levels of anti-pa neutralizing antibodies (fig. a ). on the other hand, anti-pa neutralizing antibody titers were significantly increased in the group of mice that received edtx and nei (fig. a) . we also addressed whether the enhanced neutralization of letx toxicity was due to the increase in anti-pa antibody titers or whether it also resulted from an increase in high affinity antibodies. as with anti-ova responses above ( fig. ) , nei supplementation enhanced the titers of pa-specific igg and iga responses in mice with edtx as adjuvant (fig. b ). most importantly, the igg and iga promoted by nei supplementation were high affinity antibodies (fig. c ). our previous studies have shown that a major limitation of edtx as adjuvant for sublingual vaccines is its inability to promote iga responses in the bloodstream and in mucosal secretions . sublingual application of antigen alone or antigen plus nei did not stimulate antigen-specific iga responses in the gut. addition of nei to a vaccine formulation containing antigen and edtx resulted in the induction of fecal iga responses, which were detectable weeks after the first immunization (day ) and were further increased one week later (day ) (fig. a ). to determine if nei supplementation promoted generalized iga responses in other mucosal secretion, we analyzed these responses in vaginal washes. unlike mucosal secretions of the gastrointestinal tract, the vaginal washes of mice immunized with edtx and nei showed only non-significant levels of antigen-specific iga responses (fig. b ). however, nei supplementation was able to enhance the vaginal igg responses induced by edtx as adjuvant (fig. c) . these data suggest that nei might promote expression of gut homing receptors by iga producing cells, while the elevated igg antibodies found in the secretions of the genitourinary tract might be coming from diffusion of igg from the blood. it is important to indicate that anti-pa ab responses measured in mucosal we performed flow cytometry analysis of cytokines produced by cd + t cells upon in vitro restimulation with ova to elucidate the profile of t helper cytokines that supported the antibody responses induced by supplementation with nei. spleen cells from mice immunized with antigens plus nei exhibited the same frequencies of th cells (ifnγ + cd + t cells and tnfα + cd + t cells) as spleen cells from mice that received antigen plus edtx (fig. a ). the frequencies of th cells producing il- were also similar between these two groups, but mice immunized with antigens plus nei exhibited lower frequencies of il- + cd + t cells (fig. b ). these analyses also show that immunization with antigen plus nei promoted higher frequencies of il- + cd + t cells and il- + cd + t cells than immunization with antigen plus edtx (fig. c,d) . furthermore, nei and edtx promoted the same frequencies of antigen-specific il- + cd + t cells (fig. d) . consistent with the broad increase in antibody responses, co-administration of edtx and nei significantly increased all cd + t helper cytokine responses, including th , th , th and tfh responses (fig. ). the sublingual route is currently used for drug delivery and allergen specific immunotherapy (asit). sublingual vaccination is theoretically very simple as it involves application of liquid or rapidly dissolving tablets to the floor of the oral cavity. like vaccines delivered at other mucosal sites, sublingual vaccines have the potential of producing mucosal siga in addition to igg responses in the general bloodstream. because sublingually administered vaccines are less likely to reach central nervous system tissues, they are potentially safer than nasal vaccines. despite these advantages, identification of appropriate adjuvant formulations remains a requirement for the development of sublingual vaccines. here we show that supplementation of the edtx adjuvant with a chemical inhibitor of a neutrophil function broadens b and t cell responses induced after sublingual immunization and promotes antigen-specific siga abs. we have shown that the breath of antibody responses induced by edtx as a sublingual adjuvant is limited by the recruitment of neutrophils in mucosal inductive sites . while this work was the first to establish an inverse relationship between the presence of neutrophils and production of iga, the mechanisms underlying the suppressive effect of neutrophils on iga production remained elusive. it was previously shown that neutrophil peptide defensins enhance igg, but not iga responses against nasally co-administered vaccine antigen . the primary (or azurophilic) granules of neutrophils contain defensins, myeloperoxidase, lysozymes, and three serine proteases: neutrophil elastase, cathepsin g and protease , . we now show that inhibition of a single function of neutrophils can rescue the adjuvant activity of edtx and promote iga responses both in the blood stream and in mucosal tissues. this finding is significant because the use of a serine protease inhibitor might be a more realistic alternative to neutrophil depletion as a strategy for promoting iga responses. it also raises the question of the mechanism behind neutrophil elastase limiting iga production. neutrophil elastase and the related serine proteases cathepsin g and proteinase , were shown to regulate cytokine responses through activation or degradation of cytokines, cytokine receptors, or toll-like receptor , . our separate studies have shown that nei induce the expression of il- mrna but also aid and baff responses by spleen cells in vitro . because il- facilitates ig class switching for production of iga and regulate the expression of aid for induction of ig csr , this may be a mechanism for enhanced iga responses in mice sublingually immunized with edtx in the presence of nei. sublingual immunization of mice with sting ligands as adjuvant induces iga responses in mucosal tissues of the airways , and the genitourinary tract . however, neither cpg nor the sting ligand ′ -cgamp as sublingual adjuvant induced siga in the secretions of the gi tract (i.e., in fecal extracts) . we now show that when added to an edtx-based sublingual vaccine, nei primarily promoted siga responses in mucosal secretions of the gi tract. no significant levels of iga were measured in other mucosal secretions although igg responses were significantly increased in vaginal washes. the best-described factors that regulate homing of immune cells into the gut are ccr and gut homing receptors α β , . these factors are induced through signals provided by retinoid acid, a product of myeloid cells in mucosal tissues . to our knowledge, no study has examined whether neutrophils regulate expression of gut homing receptors. but it is worth noting that in conditions of normal homeostasis, neutrophils are not present in mucosal tissues where the concentration of iga is the highest in the body. the observation that nei primarily promotes siga in the gut suggests that it either directly stimulated retinoic acid production or stimulated cytokines such as il- , il- and il- , which promote or enhance expression of gut homing receptors , . iga was not the only immunoglobulin isotype affected by supplementation of the edtx-based sublingual vaccine with nei. in fact, nei also enhanced igg and igg b responses. this is consistent with the previous report that elastase inhibits the maturation and function of dendritic cells, including expression of the costimulatory molecules cd , cd and cd . thus, the presence of nei may have promoted the maturation of dendritic cells and their expression of baff and april, and perhaps costimulatory molecules (i.e., cd ), for enhanced antibody production. another important point revealed in the present study is that the presence of nei improved the quality of antibody responses. indeed, nei increased the titers of high affinity igg and iga antibodies, an effect that translated into higher titers of anti-pa neutralizing antibodies. our findings are in line with the recent report that neutrophils regulate germinal center formation in secondary lymphoid organs and production of autoreactive plasma cells in lupus . analysis of antigen-specific t helper cytokine responses provided some clues on how nei improved the breath, the magnitude and the quality of antibody responses in mice immunized via the sublingual route with edtx as adjuvant. we found that addition of nei resulted in higher frequencies of th cells producing ifn-γ, th cells producing il- and il- , th cells producing il- , and tfh cells producing il- . this broad profile of th cell cytokine responses has most likely contributed to increase b cell survival via signals from il- and il- [ ] [ ] [ ] [ ] [ ] . it is also consistent with the observation by others that neutrophils preferentially accumulate in proximity of t cells in secondary lymphoid organs during the early stage of lupus and that neutrophil depletion accelerate germinal center formation . in our study, the nei may have played the same role as neutrophil depletion and enhanced development of germinal centers and production of high affinity antibodies by facilitating differentiation of tfh producing il- . altogether, our results show that supplementation of sublingual vaccine by co-administration of a nei can improve the kinetic, breath and quality of antibody responses both in the bloodstream and in mucosal tissues. these findings raise the question of can nei display similar adjuvant activity for vaccines given through other mucosal routes and, possibly non-mucosal vaccines? our separate studies indicate that this is in fact the case and that co-administration of nei modulates immune responses induced by intranasal and injected vaccines (attia z and boyaka pn, manuscript in preparation). it is also worth noting that nei alone exhibited some adjuvant activity and enhanced igg and iga responses against the sublingually co-administered vaccine antigens. this effect does not appear to extend to other immunoglobulin isotypes/subclasses. previous studies have established that after oral administration, the plasma concentration of the nei alvelestat peaked by - . hrs and that it was eliminated thereafter . thus, the nei is believed to act quickly after sublingual immunization and to be eliminated within hrs. future studies will investigate the underlying mechanisms and whether nei can be used as a universal strategy for improving immune responses to sublingual vaccines regardless of the adjuvant or delivery system. ethics statement. all experiments were performed according to the guidelines for the care and use of laboratory animals adopted by the national institutes of health. the institutional animal care and use committee (iacuc) at the ohio state university approved all protocols. all efforts were made to avoid unnecessary pain and distress and to minimize animal suffering during the course of these studies. animals. female c bl/ j mice were obtained from the jackson laboratory (bar harbor, me). mice were maintained in a specific pathogen-free environment and used at ± wks of age. all the experiments were approved by and performed in accordance with the guidelines of nih and the ohio state university iacuc. sublingual immunization and samples collection. sublingual immunization was performed as previously described to address the effect of nei supplementation, groups of mice were given antigens plus μm of alvelestat (azd , c h f n o s) (selleckchem, houston, tx), or antigen plus ef plus alvelestat. mice that received the nei showed no change in their vitality, food consumption or body weight. all groups were immunized at weekly intervals for consecutive weeks (days , , and ). serum, fecal pellet and vaginal wash samples were collected weekly, and nasal washes were collected at day . to determine ova-specific and pa-specific antibody titers, elisa were performed with antigen-coated plates as described previously [ ] [ ] [ ] , briefly, microtiter plates were coated with ova ( mg/ml) or pa ( μg/ml). for detection of ova-or pa-specific igg and iga abs, serum or fecal material extracts were serially diluted in pbs % bsa, added to the plates and the binding antibodies were detected with hrp-conjugated anti-mouse γor α-heavy chain-specific antisera (southern biotech associates inc., birmingham, al). biotin-conjugated rat anti-mouse igg , igg a/c, igg b or igg monoclonal abs and hrp-conjugated streptavidin (bd bioscience, san jose, ca) were used to measure igg subclass responses. the reactions were revealed by addition of the water-soluble hrp substrate abts ( , ′-azinobis [ -ethylbenzothiazoline- -sulfonic acid]-diammonium salt, sigma-aldrich) and the ab titers were determined as the last dilutions of samples with an absorbance of > . above that of control samples from naïve mice. for assessment of iga levels in the intestinal secretions, freshly emitted fecal pellets were normalized by homogenization in pbs ( ml per . g feces). after centrifugation, dilutions of supernatants were used for evaluation of antigen-specific iga levels as described above. quantification of high affinity antibody responses. high affinity antibody responses were measured by elisa as described above with a minor modification. briefly, plates were coated with pa and incubated with dilutions of the samples. urea ( mm) was then added and the plates incubated for min at room temperature to remove antibodies that bound to the antigen with low affinity . after washing, detection antibodies were added and the remaining steps of the elisa conducted as described above. assessment of toxin neutralizing antibodies. toxin neutralization assay was performed as previously described [ ] [ ] [ ] . briefly, sample dilutions were added to j macrophages cultured in cultured in rpmi supplemented with % fetal calf serum. bacillus anthracis lethal toxin (letx) [i.e., pa plus bacillus anthracis lethal factor (lf, list biological, campbell, ca)] was then added to the plates. after overnight incubation, mtt ( -( , -dimethylthiazol- -yl)− , -diphenyl tetrazolium bromide; sigma-aldrich) was added to assess the viability of macrophages as a function of redox potential. toxin neutralizing antibody titers were determined as the lowest concentrations of sera that protect macrophages from the cytotoxicity of letx. mucosal immunity and vaccines inducing mucosal iga: a challenge for vaccine adjuvants and delivery systems topical immunization strategies contributions of edema factor and protective antigen to the induction of protective immunity by bacillus anthracis edema toxin as an intranasal adjuvant bacillus anthracis edema toxin acts as an adjuvant for mucosal immune responses to nasally administered vaccine antigens neutrophils negatively regulate induction of mucosal iga responses after sublingual immunization mucosal administration of cycle-di-nucleotide-adjuvanted virosomes efficiently induces protection against influenza h n in mice sublingual vaccination with sonicated salmonella proteins and mucosal adjuvant induces mucosal and systemic immunity and protects mice from lethal enteritis buccal and sublingual vaccine delivery sublingual targeting of sting with ′ ′-cgamp promotes systemic and mucosal immunity against anthrax toxins sublingual vaccination with influenza virus protects mice against lethal viral infection neutrophil function: from mechanisms to disease mechanisms underlying neutrophil-mediated monocyte recruitment the role of neutrophil elastase in chronic inflammation the potential of neutrophil elastase inhibitors as anti-inflammatory therapies azd : pharmacological characterization of a novel oral inhibitor of neutrophil elastase mechanisms for induction of acquired host immunity by neutrophil peptide defensins neutrophil granules: a library of innate immunity proteins neutrophil serine proteases: mediators of innate immune responses inhibitors of elastase stimulate murine b lymphocyte differentiation into igg-and iga-producing cells new concepts in the generation and functions of iga il- regulates aicda expression through mir- retinoic acid induces homing of protective t and b cells to the gut after subcutaneous immunization in mice differentiation and homing of iga-secreting cells interleukin (il)− promotes intestinal iga response to microbiota generation of gut-homing iga-secreting b cells by intestinal dendritic cells inflammatory lung secretions inhibit dendritic cell maturation and function via neutrophil elastase neutrophils slow disease progression in murine lupus via modulation of autoreactive germinal centers cyclooxygenase- orchestrates germinal center formation and antibody class-switch via regulation of il- plasticity of th cells in peyer's patches is responsible for the induction of t cell-dependent iga responses interleukin- regulates genes involved in b-cell terminal maturation il- induces igg isotype switch recombination in mouse cd -activated sigdpositive b lymphocytes transforming growth factor beta induces iga production and acts additively with interleukin for iga production pharmacokinetics and safety of azd , an oral neutrophil elastase inhibitor, in healthy volunteers and patients with copd ikkbeta in intestinal epithelial cells regulates allergen-specific iga and allergic inflammation at distant mucosal sites routes of allergic sensitization and myeloid cell ikkbeta differentially regulate antibody responses and allergic airway inflammation in male and female mice use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection analysis of antigen-specific t helper cell cytokines. antigen-specific t helper cell cytokine responses were analyzed by flow cytometry after in vitro restimulation and intracellular staining with cytokine-specific fluorescent antibodies. briefly, splenocytes and cervical lymph nodes were collected on day after the first immunization and restimulated with antigen (i.e., ova) in vitro as previously described - . after days culture, cells were subjected to extracellular staining with lineage-specific antibodies [i.e., anti-cd , and anti-cd (biolegend, san diego, ca)] and, after fixation, to intracellular staining with th , th , th , and tfh cytokine-specific antibodies (biolegend). labeled cells were then analyzed with an attune nxt flow cytometer (thermo fisher scientific, waltham, ma). results are expressed as mean ±one standard deviation. statistical significance was determined by one-way anova, followed by tukey post-hoc test. all statistical analyses were performed with the statase . software (statacorp llc, college station, tx) and prism software (graphpad software, la jolla, ca). this work was supported by national institutes of health grants dk and ai . competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -oqe gjcs authors: strano, emanuele; viana, matheus p.; sorichetta, alessandro; tatem, andrew j. title: mapping road network communities for guiding disease surveillance and control strategies date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: oqe gjcs human mobility is increasing in its volume, speed and reach, leading to the movement and introduction of pathogens through infected travelers. an understanding of how areas are connected, the strength of these connections and how this translates into disease spread is valuable for planning surveillance and designing control and elimination strategies. while analyses have been undertaken to identify and map connectivity in global air, shipping and migration networks, such analyses have yet to be undertaken on the road networks that carry the vast majority of travellers in low and middle income settings. here we present methods for identifying road connectivity communities, as well as mapping bridge areas between communities and key linkage routes. we apply these to africa, and show how many highly-connected communities straddle national borders and when integrating malaria prevalence and population data as an example, the communities change, highlighting regions most strongly connected to areas of high burden. the approaches and results presented provide a flexible tool for supporting the design of disease surveillance and control strategies through mapping areas of high connectivity that form coherent units of intervention and key link routes between communities for targeting surveillance. networks, the regular and planar nature of road networks precludes the formation of clear communities, i.e. roads that cluster together shaping areas that are more connected within their boundaries than with external roads. highly connected regional communities can promote rapid disease spread within them, but can be afforded protection from recolonization by surrounding regions of reduced connectivity, making them potentially useful intervention or surveillance units , , . for isolated areas, a focused control or elimination program is likely to stand a better chance of success than those highly connected to high-transmission or outbreak regions. for example, reaching a required childhood vaccination coverage target in one district is substantially more likely to result in disease control and elimination success if that district is not strongly connected to neighbouring districts where the target has not been met. the identification of 'bridge' routes between highly connected regions could also be of value in targeting limited resources for surveillance . moreover, progressive elimination of malaria from a region needs to ensure that parasites are not reintroduced into areas that have been successfully cleared, necessitating a planned strategy for phasing that should be informed by connectivity and mobility patterns . here we develop methods for identifying and mapping road connectivity communities in a flexible, hierarchical way. moreover, we map 'bridge' areas of low connectivity between communities and apply these new methods to the african continent. finally, we show how these can be weighted by data on disease prevalence to better understand pathogen connectivity, using p. falciparum malaria as an example. african road network data. data on the african road network (arn) were obtained from gps navigation and cartography as described in a previous study . the dataset maps primary and secondary roads across the continent, and while it does have commercial restrictions, it is a more complete and consistent dataset than alternative open road datasets (e.g. openstreetmap , groads ). visual inspection and comparison between the arn and other spatial road inventories validated the improved accuracy and consistency of arn, however a quantitative validation analysis was not possible due to the lack of consistent ground-truth data at continental scales. figure a shows the african road network data used in this analysis. the road network dataset is a commercial restricted product and requests for it can be directly addressed to garmin . plasmodium falciparum malaria prevalence and population maps. to demonstrate how geographically referenced data on disease occurrence or prevalence can be integrated into the approaches outlined, gridded data on plasmodium falciparum malaria prevalence were obtained from the malaria atlas project (http:// www.map.ox.ac.uk/). these represent modelled estimates of the prevalence of p. falciparum parasites in per × km grid square across africa . additionally, gridded data on estimated population totals per × km grid square across africa in were obtained from the worldpop program (http://www.worldpop.org/). the population data were aggregated to the same × km gridding as the malaria data, and then multiplied together to obtain estimates of total numbers of p. falciparum infections per × km grid square. detecting communities in the african road network. we modeled the arn as a'primal' road network, where roads are links and road junctions are nodes . spatial road networks have, as any network embedded in two dimensions, physical spatial constraints that impose on them a grid-like structure. in fact, the arn primal network is composed of , road segments that account for a total length of , , km, with an average road length of . km ± . km. such large standard deviations, as already observed elsewhere , , , are due to the long tailed distribution of road lengths, as illustrated in fig. c . another property of road network structure is the frequency distribution of the degree of nodes, defined as the number of links connected to each node. most networks in nature and society have a long tail distribution of node degree, implying the existence of hubs (nodes that connect to a large amount of other nodes) , with the majority of nodes connecting to very few others. for road networks, however, the degree distribution strongly peaks around , indicating that most of the roads are connected with two other roads. the long tail distribution of the length of road segments, coupled with the peaked degree distribution, indicates the presence of translational invariant grid-like structure, in which road density smoothly varies among regions while their connectivity and structure does not. within such gridlike structures it is very difficult to identify clustered communities, i.e. groups of roads that are more connected within themselves than to other groups. this observation is confirmed by the spatial distribution of betweenness centrality (bc), which measures the amount of time the shortest paths between each couple of nodes pass through a road. the probability distribution of bc is long tailed (fig. d) , while its spatial distribution spreads across the entire network, with a structural backbone form, as shown in fig. b. again, under such conditions and because of the absence of bottlenecks, any strategy to detect communities that employs pruning on bc values , will be minimally effective. to detect communities in road networks we follow the observation that human displacement in urban networks is guided by straight lines . therefore, geometry can be used to detect communities of roads by assuming that people tend to move more along streets than between between streets. we developed a community detection pipeline that converts a primal road network, where roads are links and roads junction are nodes , to a dual network representation, where link are nodes and street junction link between nodes , by mean of straightness and contiguity of roads. it is important to note here that the units of analysis are road segments, which here are typically short and straight between intersections, making the straightness assumption valid. community detection in the dual network is then performed using a modularity optimization algorithm . the communities found in the dual network are then mapped back to the original primal road network. these communities encode information about the geometry of road pattern but can also incorporate weights associated with a particular disease to guide the process of community detection. nodes in the dual network represent lines in the primal network. the conversion from primal to dual is done by using a modified version of the algorithm known as continuity negotiation . in brief, we assume that a pair of adjacent edges belongs to the same street if the angle θ between these edges is smaller than θ c = °. we also assume that the angle between two adjacent edges (i, j) and (j, p) is given by the dot product cos (θ) = r i, j r j,p /r i, j r j,p , where r i, j = r j r i . under these assumptions, the angle between two edges belonging to a perfect straight line is zero, while it assumes a value of ° for perpendicular edges. our algorithm starts searching for the edge that generates the longest road in the primal space, as can be seen in fig. a . then, a node is created in the dual space and assigned to this road. next, we search for the edge that generates the second longest road, and a new node is created in the dual space and assigned to this road. if there is at least one interception between the new road and the previous one, we connect the respective nodes in the dual space. the algorithm continues until all the edges in the primal space are assigned to a node in the dual space, as shown in fig. b . note that the conversion from primal to the dual road network has been used extensively to estimate human perception and movement along road networks (space syntax, see ) , which also supports our use of road geometry to detect communities. despite the regular structure of the network in the primal space, the topology of these networks in the dual space is very rich. for instance the degree distribution in dual space follows the power-law p(k) k −γ . this property has been previously identified in urban networks and it is strongly related to the long tailed distribution of road lengths in these networks (see fig. c ). since most of the roads are short, most of the nodes in dual space will have a small number of connections. on the other hand, there are a few long roads (fig. a ) that originate at hubs in the dual space (fig. b ). our approach for detecting communities in road networks consists then in performing classical community detection in the dual representation ( fig. c) and then bringing the result back to the primal representation, as shown in fig. d . the algorithm used to detect the communities is the modularity-based algorithm by clauset and newman . the hierarchical mapping of communities on the african road network, with outputs for , , and sets of communities, is shown in fig. . the maps highlight how connectivity rarely aligns with national borders, with the areas most strongly connected through dense road networks typically straddling two or more countries. the hierarchical nature of the approach is illustrated through the breakdown of the large regions in fig. a into further sub-regions in b, c and d, emphasizing the main structural divides within each region in mapped in a. some large regions appear consistently in each map, for example, a single community spans the entire north african coast, extending south into the sahara. south africa appears as wholly contained within a single community, while the horn of africa containing somalia and much of ethiopia and kenya in consistently mapped as one community. the four maps shown are example outputs, but any number of communities can be identified. the clustering that maximises modularity produces communities, and these are mapped in fig. . even with division into communities, the north africa region remains as a single community, strongly separated from sub-saharan africa by large bridge regions. south africa also remains as almost wholly within its own community, with somalia and namibia showing similar patterns. the countries with the largest numbers of communities tend to be those with the least dense infrastructure equating to poor connectivity, such as drc and angola, though west africa also shows many distinct clusters, especially within nigeria. apart from the sahara, the largest bridge regions of poor connectivity are located across the central belt of sub-saharan africa, where population densities are low and transport infrastructure is both sparse and often poor. the communities mapped in figs and align in many cases with recorded population and pathogen movements. for example, the broad southern and eastern community divides match well those seen in hiv- subtype analyses and community detection analyses based on migration data . at more regional scales, there also exist similarities with prior analyses based on human and pathogen movement patterns. for example, the western, coastal and northern communities within kenya in fig. b , identified previously through mobile phone and census derived movement data , . further, guinea, liberia and sierra leone typically remain mostly within a single community in fig. , with some divides evident in fig. c . this shows some strong similarities with the spread of ebola virus through genome analysis , particularly the multiple links between rural guinea and sierra leone, though fig. c highlights a divide between the regions containing conakry and freetown when africa is broken into the communities. figure highlights the connections between kinshasa in western drc and angola, with the recent yellow fever outbreak spreading within the communities mapped. figure d shows the'best' communities map for an area of southern africa, and the strong cross-border links between swaziland, southern mozambique and western south africa are mapped within a single community, as well as wider links highlighted in fig. , matching the travel patterns found from swaziland malaria surveillance data . integrating p. falciparum malaria prevalence and population data with road networks for weighted community detection. the previous section outlined methods for community detection on unweighted road networks. to integrate disease occurrence, prevalence or incidence data for the identification of areas of likely elevated movement of infections or for guiding the identification of operational control units, an adaptation to weighted networks is required. we demonstrate this through the integration of the data on estimated numbers of p. falciparum infections per × km grid square into the community detection pipeline. the final pipeline for community detection calculated a trade-off between form and function of roads in order to obtain a network partition. the form is related to the topology of the road network and is taken into account during the primal-dual conversion. the topological component guarantees that only neighbor and well connected locations could belong to the same community. the functional part, on the other hand, is calculated by the combination of estimated p. falciparum malaria prevalence multiplied by population to obtain estimated numbers of infections, as outlined above. the two factors were combined to form a weight to each edge of our primal network. the weight w i, j of edge (i, j) is defined as where m(r) is the p. falciparum malaria prevalence and p(r) is the population count, both at coordinate r. these values are obtained directly from the data. when the primal representation is converted into its dual version, the weights of primal edges, given by eq. , are converted into weights of dual nodes, which are defined as where i represents the i th dual node and Ω i represents the set of all the primal edges that were combined together to form the dual node i (see fig. a,b) . finally, weights for the dual edges are created from the weights of dual nodes, by simply assuming the dual network weighted by values of λ i,¯j was used as input for a weighted community detection algorithm. ultimately, when the communities detected in the dual space are translated back to primal space, we have that neighbor locations with similar values of estimated p. falciparum infections belong to the same communities. for the example of p. falciparum malaria used here, the max function was used, representing maximum numbers of infections on each road segment in . this was chosen to identify connectivity to the highest burden areas. areas with large numbers of infections are often 'sources' , with infected populations moving back and forward from them spreading parasites elsewhere , . therefore, mapping which regions are most strongly connected to them is of value. alternative metrics can be used however, depending on the aims of the analyses. the integration of p. falciparum malaria prevalence and population (fig. a ) through weighting road links by the maximum values across them produces a different pattern of communities (fig. b) to those based solely on network structure (fig. ) . the mapping of communities is shown here, as it identifies key regions of known malaria connectivity, as outlined below. the mapping shows areas of key interest in malaria elimination efforts connected across national borders, such as much of namibia linked to southern angola , but the zambezi region of namibia more strongly linked to the community encompassing neighbouring zambia, zimbabwe and botswana . in namibia, malaria movement communities identified through the integration of mobile phone-based movement data and case-based risk mapping show correspondence in mapping a northeast community. moreover, swaziland is shown as being central to a community covering, southern mozambique and the malaria endemic regions of south africa, matching closely the origin locations of the majority of internationally imported cases to swaziland and south africa , , . the movements of people and malaria between the highlands and southern and western regions of uganda, and into rwanda , also aligns with the community patterns shown in fig. b . finally, though quantifying different factors, the analyses show a similar east-west split to that found in analyses of malaria drug resistance mutations , and malaria movement community mapping . the emergence of new disease epidemics is becoming a regular occurrence, and drug and insecticide resistance are continuing to spread around the world. as global, regional and local efforts to eliminate a range of infectious diseases continue and are initiated, an improved understanding of how regions are connected through human transport can therefore be valuable. previous studies have shown how clusters of connectivity exist within the global air transport network , and shipping traffic network , but these represent primarily the sources of occasional long-distance disease or vector introductions , , rather than the mode of transport that the majority of the population uses regularly. the approaches presented here focused on road networks provide a tool for supporting the design of disease and resistance surveillance and control strategies through mapping (i) areas of high connectivity where pathogen circulation is likely to be high, forming coherent units of intervention; (ii) areas of low connectivity between communities that form likely natural borders of lower pathogen exchange; (iii) key link routes between communities for targetting surveillance efforts. the outputs of the analyses presented here highlight how highly connected areas consistently span national borders. with infectious disease control, surveillance, funding and strategies principally implemented country by country, this emphasises a mismatch in scales and the need for cross-border collaboration. such collaborations are being increasingly seen, for example with countries focused on malaria elimination (e.g. , ), but the outputs here show that the most efficient disease elimination strategies may need to reconsider units of intervention, moving beyond being constrained by national borders. results from the analysis of pathogen movements elsewhere confirm these international connections (e.g. , , , , building up additional evidence on how pathogen circulation can be substantially more prevalent in some regions than others. the approaches developed here provide a complement to other approaches for defining and mapping regional disease connectivity and mobility . previously, census-based migration data has been used to map blocks of countries of high and low connectivity , but these analyses are restricted to national-scales and cover only longer-term human mobility. efforts are being made to extend these to subnational scales , , but they remain limited to large administrative unit scales and the same long timescales. mobile phone call detail records (cdrs) have also been used to estimate and map pathogen connectivity , , but the nature of the data mean that they do not include cross-border movements, so remain limited to national-level studies. an increasing number of studies are uncovering patterns in human and pathogen movements and connectivity through travel history questionnaires (e.g. , , , ), resulting in valuable information, but typically limited to small areas and short time periods. there exist a number of limitations to the methods and outputs presented here that future work will aim to address. firstly, the hierarchies of road types are not currently taken into account in the network analyses, meaning that a major highway and small local roads contribute equally to community detection and epidemic spreading. the lack of reliable data on road typologies, and inconsistencies in classifications between countries, makes this challenging to incorporate however. moreover, the relative importance of a major road versus secondary, tertiary and tracks is exceptionally difficult to quantify within a country, let alone between countries and across africa. finally, data on seasonal variations in road access does not exist consistently across the continent. our focus has therefore been on connectivity, in terms of how well regions are connected based on existing road networks, irrespective of the ease of travel. a broader point that deserves future research is that while intuition suggests a correspondence in most places, connectivity may not always translate into human or pathogen movement. future directions for the work presented here include quantitative comparison and integration with other connectivity data, the integration of different pathogen weightings, and the extension to other regions of the world. qualitative comparisons outlined above show some good correspondence with analyses of alternative sources of connectivity and disease data. a future step will be to compare these different connections and communities quantitatively to examine the weight of evidence for delineating areas of strong and weak connectivity. this could potentially follow similar studies looking at community structure on weighted networks, such as in the us based on commuting data , or uk and belgium from mobile network data , . here, p. falciparum malaria was used to provide an example of the potential for weighting analyses by pathogen occurrence, prevalence, incidence or transmission suitability. moreover, future work will examine the integration of alternative pathogen weightings. the maximum difference method was used here to pick out regions well connected to areas high p. falciparum burden, but the potential exists to use different weighting methods depending on requirements, strategic needs, and the nature of the pathogen being studied. despite the rapid growth of air travel, shipping and rail in many parts of the world, roads continue to be the dominant route on which humans move on sub-national, national and regional scales. they form a powerful force in shaping the development of areas, facilitating trade and economic growth, but also bringing with them the exchange of pathogens. results here show that their connectivity is not equal however, with strong clusters of high connectivity separated by bridge regions of low network density. these structures can have a significant impact on how pathogens spread, and by mapping them, a valuable evidence base to guide disease surveillance as well as control and elimination planning can be built. results were produced through four main phases. phase : road network cleaning and weighted adjacency list production: the road cleaning operation aimed to produce a road network from the georeferenced vectorial network of roads infrastructure. this phase was conducted using esri arcmap . (http://desktop.arcgis.com/en/ arcmap/) through the use of the topological cleaning tool. the tool integrates contiguous roads, removes very short links and removes overlapping road segments. road junctions were created using the polyline to node conversion tool, while road-link association was computed using the spatial join tool. malaria prevalence values were assigned to each road using the spatial join tool. the adjacency matrix output, containing also the coordinates for each road junctions, was extracted in form of text file. phase : conversion from the primal to the dual network: the primal network created in phase was then used as input for a continuity negotiation-like algorithm. the goal of this algorithm was to translate the primal network into its dual representation (see fig. a,b) . the implementation of the negotiation-like algorithm used the igraph library in c++ (http://igraph.org/c/) on an octa-core imac. the conversion took around hours for a primal network with ~ k nodes running. the algorithm works by first identifying roads composed of many contiguous edges in the primal space. two primal-edges are assumed to be contiguous if the angle between them is not greater than ° degrees. because the dual representation generated by the algorithm strongly depends on the starting edge, we started by looking for the edge that produces the longest road. as soon as this edge was found, a dual-node was created to represent that road. next we proceeded to look for the edge that produced the second longest road and create a dual-node for that road. we continued this process until every primal-edge had been assigned to a road. finally, dual-nodes were connected to each other if their primal counterparts (roads) crossed each other in the primal space. phase : community detection: we used a traditional modularity optimization-based algorithm to identify communities in the dual representation of the road network. the modularity metrics were computed in r using the igraph library (http://igraph.org/r/). to incorporate the prevalence of malaria, we used the malaria prevalence values as edge weights for community detection. phase : mapping communities. detected communities were mapped back to the primal road network with the use of the spatial join tool in arcmap. all maps were produced in arcmap. global transport networks and infectious disease spread severe acute respiratory syndrome h n influenza-continuing evolution and spread geographic dependence, surveillance, and origins of the influenza a (h n ) virus the global tuberculosis situation and the inexorable rise of drug-resistant disease the transit phase of migration: circulation of malaria and its multidrug-resistant forms in africa population genomics studies identify signatures of global dispersal and drug resistance in plasmodium vivax air travel and vector-borne disease movement mapping population and pathogen movements unifying viral genetics and human transportation data to predict the 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transmission: a survey of movement patterns in four subsaharan african countries infection importation: a key challenge to malaria elimination on bioko island, equatorial guinea an economic geography of the united states: from commutes to megaregions redrawing the map of great britain from a network of human interactions uncovering space-independent communities in spatial networks e.s., m.p.v. and a.j.t. conceived and designed the analyses. e.s. and m.p.v. designed the road network community mapping methods and undertook the analyses. all authors contributed to writing and reviewing the manuscript. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -xna qve authors: zhou, yuqing; fu, xiaofang; liu, xiaoxiao; huang, chenyang; tian, guo; ding, cheng; wu, jie; lan, lei; yang, shigui title: use of corticosteroids in influenza-associated acute respiratory distress syndrome and severe pneumonia: a systemic review and meta-analysis date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xna qve influenza-related severe pneumonia and acute respiratory distress syndrome (ards) are severe threats to human health. the objective of this study was to assess the effects of systematic corticosteroid therapy in patients with pneumonia or ards. the pubmed, embase, web of science and scopus databases were searched up to july, . nineteen studies including individuals were identified, and fifteen studies ( patients) were included in the meta-analysis of mortality. eighteen were observational studies and one was a randomized controlled trial (rct). the meta-analysis results showed that corticosteroid therapy was associated with significantly higher mortality (or . , % ci [ . , . ]) and incidence of nosocomial infection (or . , % ci [ . , . ]). subgroup analysis showed that among patients with unadjusted estimates, the odds of mortality were higher in patients receiving corticosteroid treatment (or . , % ci [ . , . ]), however, among patients with adjusted estimates, the result showed no statistically significant difference between corticosteroid group and control group (or . , % ci [ . , . ]). current data do not support the routine use of corticosteroids in patients with influenza severe pneumonia or ards. rcts are needed to provide more robust evidence. influenza is a viral infection that attacks the respiratory system. rapidly progressing viral pneumonia and acute respiratory distress syndrome are pulmonary manifestations that are commonly observed in patients with influenza and are associated with considerable mortality - , representing a severe threat and imparting a substantial financial burden worldwide . individuals with community-acquired pneumonia may benefit from systematic corticosteroid therapy, which may block the inflammatory cascade reaction . corticosteroids could improve the lung tissue damage induced by influenza pneumonia and decrease the risk of mortality in animal models with influenza infections , . many clinicians administer corticosteroids as an anti-inflammatory treatment for patients with severe influenza-related pneumonia to stop disease progression and improve clinical outcomes. a large cohort study of patients admitted to icus in spain found that the frequency of corticosteroid treatment by study period was . % in , . % in , % in , and . % in . recently, some studies have shown that corticosteroids may not be beneficial for patients with severe influenza and may even increase mortality [ ] [ ] [ ] . however, there is considerable uncertainty regarding whether patients with influenza-related ards or severe pneumonia can benefit from adjuvant corticosteroid therapy. we aimed to systematically review all experimental and observational studies on corticosteroid use in patients with influenza-related ards and severe pneumonia. the effect of corticosteroid treatment on clinical outcomes was investigated. search strategy and study selection. we comprehensively searched the pubmed, embase, web of science and scopus databases from inception to july . the core search terms were defined as those related to influenza-related pneumonia, ards, acute respiratory failure and corticosteroid use (for details on the search strategy in embase, refer to supplementary table s ). the references of eligible studies were screened, and two authors independently reviewed all citations that met the inclusion criteria. study selection was performed in stages: first, study title and abstract screening; second, full text examination. data extraction and quality assessment. outcome data were independently extracted from the included studies by two investigators using a previously piloted standardized pro forma. we obtained the following data: (a) characteristics of studies (design, setting, country, period, methodological details for quality assessment); (b) characteristics of participants (demographics, co-morbid illnesses, disease severity, numbers in each group, influenza virus type); (c) characteristics of interventions (type, dose, timing and duration of corticosteroid use); and (d) outcomes. the quality of each study was independently assessed by two individuals according to the cochrane risk of bias tool for rcts and the newcastle-ottawa scale for nonrandomized trials and comparative observational studies. three domains are assessed on the nos for observational studies : ( ) "selection bias", ( ) "comparability bias", and ( ) "outcome bias". disagreements at any stage were resolved through discussion with the other authors until consensus was reached. sensitivity analysis. we performed sensitivity analysis to assess the effect of the study design on clinical outcomes using stratification if the number of studies was sufficient. data analysis. odds ratios (ors) and their corresponding % confidence intervals (cis) were generated during the analysis of dichotomous outcome data, and mean differences or standardized mean differences and their corresponding % cis were generated during the analysis of normally distributed continuous data. ors or hazard ratios (hrs) for adjusted outcome estimates and their corresponding % cis were obtained and are presented in the pooled analyses. medians and interquartile ranges were generated in the analysis of continuous data that were not normally distributed. the i² test for inconsistency was used to analyse heterogeneity. if i² > %, the heterogeneity across studies was significant, and a random-effects model was used in the meta-analysis; otherwise, a fixed-effects model was used. subgroup analysis was performed in the following areas where possible: adult population versus child population; seasonal influenza versus outbreak influenza or pandemic influenza; icu versus inpatient; adjusted estimates versus unadjusted estimates; and corticosteroid dose, timing and duration. all statistical analyses were performed using cochrane systematic review software review manager (revman; version . . ; the nordic cochrane centre, the cochrane collaboration, copenhagen, ). a total of relevant articles were identified during the initial search. after the removal of duplicates, articles remained. after screening the titles and abstracts of those articles, articles were excluded because of irrelevance. of the full-text articles reviewed, were excluded for various reasons, and articles remained. details regarding the reasons for the exclusion of these studies are shown in fig. and supplementary table s . ultimately, studies were included in the meta-analysis of mortality, while studies reported outcomes other than mortality in association with corticosteroid use. the characteristics of the participants in the included studies are summarized in table and supplementary table s . the studies were published between and . eighteen of the studies had an observational design, while one had a randomized controlled trial design . outcome data were reported in studies ( individuals, including in corticosteroid group and in the non-corticosteroid group), while mortality data were reported in studies ( individuals). eight studies (n = ) included only icu patients. fourteen studies assessed individuals with h n pdm virus infection, study assessed individuals with h n virus infection, and study assessed individuals with inter-pandemic influenza virus infection. eight studies ( individuals) had useable data related to patients with ards , , [ ] [ ] [ ] [ ] [ ] [ ] . fourteen studies (n = ) reported mortality associated with adults only. the median ages varied from . to . years in all patients included. the proportion of male participants was higher than that of females ( . % versus . %) and range varied from . % to . % ( studies, individuals). obesity (bmi ≥ kg/m ) was common ( . %, / ) in the included studies ( studies, individuals), and the proportion of obese individuals ranged from . % to . %. disease severity at baseline was reported in seven studies and in studies ( individuals), the baseline disease severity was higher in individuals in the corticosteroid group than in those in the non-corticosteroid group [ ] [ ] [ ] . methylprednisolone ( . %, / ) was the most common steroid used in the corticosteroid group ( studies), and the median duration varied from . to . days. almost all patients ( . %, / ) received antiviral therapy (ranging from . % to %, studies), and . % ( / ) of participants received antibiotic therapy ( studies). the details of the therapies are described in table . because all studies included in the meta-analysis of mortality were observational cohort studies, selection bias was inevitable. the risk of bias identified in the included studies is shown in supplementary tables s a,b. the studies' nos scores varied from to , indicating that the quality of the included studies was high . however, most included studies had substantial comparability bias because we could not adequately adjust for disease severity, and individuals with greater diseases severity tended to use corticosteroids. overall mortality in the included studies. mortality data were reported in studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the pooled analysis of the crude results of the included studies ( individuals) suggested that those who used corticosteroids had a significantly higher mortality rate (or . , % ci [ . , . ], p < . ), and a moderate level of heterogeneity was observed (i² = %; supplementary fig. s ). five studies , , , , reported adjusted effect estimates of -day or inhospital mortality (adjusted or or adjusted hazard ratio (ahr), table ). the pooled analysis of the crude results of ten included studies and five adjusted effect estimates suggested that those who used corticosteroids had a significantly higher mortality rate (or . , % ci [ . , . ], i = %, p < . , fig. ). the subgroup analysis of unadjusted effect estimates showed a similar result (or . , % ci [ . , . ], i = %, p = . ). however, the subgroup analysis of the five adjusted estimates showed no association between mortality and corticosteroid use (hr . , % ci [ . , . ], i = %, p = . ). the test for subgroup differences between adjusted and unadjusted mortality was not statistically significant (p = . ). there was no clear indication of publication bias in the funnel plot analysis ( supplementary fig. s ). eight studies reported the mortality of patients with ards due to influenza (n = , supplementary table s ). meta-analysis of unadjusted and adjusted estimates suggested that the difference in mortality between the corticosteroid and control groups was not statistically significant (or . , % ci [ . , . ], i = %, p = . , fig. ). li et al. reported that corticosteroid use was associated with a decreased risk of mortality in patients with ards (pao /fio < mmhg) (ahr . , % ci [ . , . ]) . however, brun-buisson et al. and chawla et al. suggested that the risk of mortality was higher in corticosteroid group in the patients with ards. cao et al. reported that a low-to-moderate dose of corticosteroids had no statistically significant association with the risk of mortality in patients with ards (or . , % ci [ . , . ]), whereas a high dose was associated with greater mortality (or . , % ci [ . , . ]) . the other four studies , , , showed that corticosteroid use had no association with mortality ( fig. ) . considering the different management strategies for paediatric and adult patients, one study investigating paediatric patients was excluded www.nature.com/scientificreports www.nature.com/scientificreports/ with low statistical heterogeneity (i = %), and a similar result was found for mixed patient groups (or . [ . , . ], i = %) (supplementary fig. s ). three studies (n = ) have reported the results of mortality excluding patients who had potential indications (e.g., asthma, copd exacerbation, pregnancy/post-partum, shock or immunosuppressive conditions) for corticosteroid treatment that may have skewed the results , , . a pooled analysis of these three studies showed no statistically significant association between corticosteroid use and mortality (or . , % ci [ . , . ]), with a high level of heterogeneity (i² = %, p = . ). the number of studies was insufficient to perform subgroup analysis according to the various reported regimens. two studies compared early versus later/no corticosteroid treatment; one defined early treatment as within three days of mechanical ventilation , and the result suggested that early treatment with corticosteroids was associated with greater mortality (ahr . , % ci [ . , . ]); the other defined early treatment as within h of nais , and the result showed no statistically significant difference in mortality between patients receiving corticosteroids within h of nais and those who did. three studies categorized corticosteroid dose as low/low-to-moderate and high , . a large retrospective cohort study reported that a low-to-moderate dose ( - . another study reporting the result of patients with acute respiratory failure due to influenza showed no statistically significant difference between low dose and high dose corticosteroid therapy ( / versus / , p > . ) . two of the included studies reported outcomes related to children, but only one reported the risk of mortality (or . , % ci [ . , . ]) related to corticosteroid use . however, in that study, all children who received corticosteroids had ards, while the patients in the non-corticosteroid group had less severe disease conditions. another retrospective cohort study of children with pneumonia caused by the h n influenza virus only reported length of hospital stay and duration of fever and found a shorter length of stay and duration of fever in corticosteroid group (table ) . in four of these studies, corticosteroid use was associated with an increased risk of developing a nosocomial infection , , , , while the remaining two studies did not show a statistically significantly increased odds of developing infection , . overall, the pooled results revealed that the odds of nosocomial infection were significantly higher in patients who were administered corticosteroids than in those who were not (or . , % ci [ . , . ], p < . ), but a high level of heterogeneity was observed (i² = %) (fig. ) . three studies reported the common pathogens isolated from patients with nosocomial infection. one study reported that the most common bacteria isolated was acinetobacter baumannii ( . %) . in a study of patients with severe influenza pneumonia , patients had nosocomial infection, and the most commonly isolated pathogens were acinetobacter baumannii ( . %), pseudomonas aeruginosa ( . %), and staphylococcus aureus ( . %), while in another cohort study of patients with severe influenza pneumonia , streptococcus pneumoniae ( . %), pseudomonas aeruginosa ( . %), and staphylococcus aureus ( . %) were the most frequently isolated microorganisms. www.nature.com/scientificreports www.nature.com/scientificreports/ length of stay and length of mv. seven studies reported length of stay according to corticosteroid use; all were unadjusted for disease severity (table ) . six studies found no statistically significant difference between the groups. one study showed a longer length of icu stay associated with corticosteroid use, while the total length of hospital stays was not significantly different between the groups. notably, one of the five studies analysed the duration of hospital stay in people with influenza pneumonia treated with corticosteroid versus those receiving placebo, and found no significant difference between the groups (adjusted difference − . days, % ci [− . , . ]) . linko et al. reported a longer duration of mechanical ventilation in the group treated with corticosteroid therapy while brun-buisson et al. and moreno et al. found no statistically significant difference between the groups. (table ) . two studies reported the time to fever alleviation according to corticosteroid use , . kudo et al. found no statistically significant difference between the groups, while another cohort study of children with severe influenza pneumonia reported a shorter time to fever alleviation. notably, two studies found a shorter time to clinical stability in the corticosteroid group. the study of influenza a/h n found a significantly longer duration of viral shedding associated with corticosteroid treatment . the details of these outcomes are described in table . the overall findings of this meta-analysis indicated that patients with pneumonia or acute respiratory distress syndrome who were administered corticosteroids had significantly higher mortality and incidence of nosocomial infection but the use of corticosteroids did not influence the length of hospital stay. our studies suggested a deleterious effect of steroids on mortality and nosocomial infection. several factors need to be accounted for in interpreting these findings. first, most studies did not adjust the clinical outcomes for potential confounding factors. clinically, more severe cases tended to be treated with corticosteroids, which may obscure the real value of this treatment regarding mortality , . therefore, in this study, we preferred the use of adjusted estimates of the effect to minimize potential confounding between the treatment groups. however, five studies reported adjusted estimates of mortality, and their inclusion in the meta-analysis still revealed a higher odds of mortality related to steroids use. good evidences indicated that secondary bacterial pneumonia is an www.nature.com/scientificreports www.nature.com/scientificreports/ important cause of mortality related to influenza , . therefore, increasing risk of nosocomial infection due to corticosteroid treatment may partly account for the potential harm from corticosteroid use. two included studies , found that secondary bacterial pneumonia such as due to acinetobacter baumannii, pseudomonas aeruginosa, streptococcus pneumoniae, staphylococcus aureus or invasive fungal infection, were more common in corticosteroid-treated patients. several studies showed that prolonged viral shedding and delayed viral clearance were noted in corticosteroid-treated patients , , whereas slower clearance of virus loads was associated with higher mortality in patients with ards due to h n pdm virus infection . thus, prolonged viral shedding and delayed viral clearance may also contribute to higher mortality. second, most of the included observational studies did not explain why some patients received systemic corticosteroid therapy and others did not. the initial intentions of corticosteroid therapy were unclear (was it used as a rescue therapy or due to copd/asthma exacerbation or due to pneumonia/ards?). different indication may easily confound the effect of the corticosteroid. some evidences supported the use of corticosteroids for asthma or copd or septic shock in the context of influenza infection [ ] [ ] [ ] . in order to minimize the influences of different indications, subgroup analysis of the mortality in three studies (n = ) was performed after excluding patients receiving corticosteroids as rescue therapy or due to copd/asthma exacerbation, and found no statistically significant difference between the steroid therapy groups and control groups and the heterogeneity was high (i² = %). however, the high level of statistical heterogeneity may result in unstable estimates of the meta-analysis. therefore, well-designed clinical trials should be conducted to decrease the heterogeneity of patients and to provide more robust evidence. the results from clinical studies of corticosteroid therapy in patients with influenza are conflicting. many studies have shown a significant association between corticosteroid treatment and mortality in patients with influenza; however, several studies have reported that corticosteroids can provide benefits to patients under certain conditions , , , . an rct included in this review noted an association between adjuvant corticosteroid therapy ( mg of prednisone given orally for days) and decreased time to clinical stability. low-to-moderate doses of corticosteroids are beneficial in people with hypoxia ((pao /fio ) < mmhg), whereas high doses of corticosteroids showed no benefit in this group; however, low-to-moderate doses of corticosteroids may increase the -day mortality rate in those with pao /fio > mmhg . kil et al. reported that rapid (methylprednisolone, mg/kg/d) and short-term (tapered off within a week) corticosteroid treatment for children with severe pneumonia halted clinical exacerbation and possibly prevented progression to ards. however, in another study, compared with no treatment, administration (steroid therapy was initiated at a median daily dose equivalent to (iqr, - ) mg of hydrocortisone, and a median duration of (iqr, - ) days within the first days of mv was more strongly associated with an increased risk of death, whereas when administration was beyond the first days of mv, the association was no longer significant . considering the findings of the aforementioned studies, the condition of the patients' respiratory system and the dose, timing and duration of corticosteroids could be contributing factors that affect the effects of corticosteroids. several recent systematic reviews and meta-analyses concluded that corticosteroid therapy is significantly associated with mortality [ ] [ ] [ ] . however, in these studies, there were no special limitations on subject inclusion criteria, which means that the patients were very diverse. additionally, there was no subgroup analysis for these patients under different disease conditions. compared to patients in those previous studies, we focused only on patients with pneumonia or ards, which is more specific and makes the outcomes more targeted. our study observed a different outcome according to corticosteroid use in patients with ards due to influenza. this study has some limitations, including the lack of sufficient data on the dose, duration, timing and rationales of corticosteroid administration and the timing and duration of antiviral therapy. in addition, only one study included in this meta-analysis was an rct, and were observational in nature. thus, it is possible that selection bias or comparability bias could have affected the quality of the analysed evidence. there is insufficient evidence in this meta-analysis to make a firm determination about the effectiveness of corticosteroids for people with influenza-related pneumonia or ards. the small number of included studies and the small number of patients in the included studies might also make the effect size of some outcome indicators insufficient, and we were unable to analyse the effect of some factors on the outcome indicators by meta-regression or subgroup analysis. current data do not support the routine use of corticosteroids in patients with influenza pneumonia or ards. however, the data assessed in this meta-analysis were extracted from observational studies and only one rct; therefore, the limitations associated with study design are important to consider. there is a need for more robust evidence on the role of corticosteroids in the treatment of influenza-related ards and severe pneumonia before a firm recommendation for clinical practice can be made. update on avian influenza a (h n ) virus infection in humans critically ill patients with influenza a(h n ) infection in canada global mortality estimates for the influenza pandemic from the glamor project: a modeling study influenza cost and cost-effectiveness studies globally-a review effect of corticosteroids on treatment failure among hospitalized patients with severe community-acquired pneumonia and high inflammatory response: a randomized clinical trial corticosteroid treatment ameliorates acute lung 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)pdm viral pneumonia. influenza and other respiratory viruses hospitalized adult patients with influenza a(h n ) in beijing, china: risk factors for hospital mortality effects of corticosteroid and neuraminidase inhibitors on survival in patients with respiratory distress induced by influenza virus predictors of mortality and length of stay in hospitalized cases of influenza a (h n ): experiences of a tertiary care center corticosteroid therapy in intensive care unit patients with pcr-confirmed influenza a(h n ) infection in finland effect of immunomodulatory therapies in patients with pandemic influenza a (h n ) complicated by pneumonia predictors and outcomes of respiratory failure among hospitalized pneumonia patients with h n influenza in taiwan critical evaluation of the newcastle-ottawa scale for the assessment of the quality of nonrandomized studies in metaanalyses experience in the management of the severe form of human influenza a h n pneumonia in an intensive care unit lung function and organ dysfunctions in patients requiring mechanical ventilation during the influenza a (h n ) pandemic clinical and prognostic features of patients with pandemic influenza a (h n ) virus in the intensive care unit neuraminidase inhibitors, superinfection and corticosteroids affect survival of influenza patients predictors of mortality in hospitalized children with pandemic h n influenza in pune early corticosteroid treatment for severe pneumonia caused by h n influenza virus systemic corticosteroids and early administration of antiviral agents for pneumonia with acute wheezing due to influenza a(h n )pdm in japan risk factors associated with death among influenza a (h n ) patients surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock deaths from bacterial pneumonia during - influenza pandemic predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness viral loads and duration of viral shedding in adult patients hospitalized with influenza delayed clearance of viral load and marked cytokine activation in severe cases of pandemic h n influenza virus infection differences between asthmatics and nonasthmatics hospitalised with influenza a infection. the european respiratory journal surviving sepsis campaign: international guidelines for management of sepsis and septic shock seasonal influenza vaccination in patients with copd: a systematic literature review h n influenza a virus-associated acute lung injury: response to combination oseltamivir and prolonged corticosteroid treatment multiphasic acute disseminated encephalomyelitis (adem) following influenza type a (swine specific h n ) the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and meta-analysis shigui yang designed the study. yuqing zhou, xiaofang fu, xiaoxiao liu, chenyang huang, guo tian, cheng ding, jie wu, lei lan collected the data. yuqing zhou, xiaofang fu, xiaoxiao liu, guo tian, cheng ding and jie wu analyzed the data. yuqing zhou and shigui yang interpreted the results. yuqing zhou wrote the manuscript. yuqing zhou and shigui yang revised the manuscript from preliminary draft to submission. shigui yang supervised the study. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to s.y.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -nl j s authors: nagaoka, hikaru; kanoi, bernard n.; ntege, edward h.; aoki, masamitsu; fukushima, akihisa; tsuboi, takafumi; takashima, eizo title: antibodies against a short region of pfripr inhibit plasmodium falciparum merozoite invasion and pfripr interaction with rh and sema a date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: nl j s plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. p. falciparum merozoite rh interacting protein (pfripr) forms a complex with rh and cyrpa in sequential molecular events leading to erythrocyte invasion. recently we described pfripr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. however, being a large and complex protein of amino acids (aa) with cysteine residues, pfripr is difficult to express in conventional expression systems towards vaccine development. in this study we sought to identify the most potent region of pfripr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-pfripr antibodies. using the wheat germ cell-free system, ecto- pfripr and truncates of approximately aa were expressed as soluble proteins. we demonstrate that antibodies against pfripr truncate (pfripr_ : c( )-d( )), a region within the pfripr c-terminal egf-like domains, potently inhibit merozoite invasion. furthermore, the antibodies strongly block pfripr/rh interaction, as well as that between pfripr and its erythrocyte-surface receptor, sema a. taken together, pfripr_ is a potential candidate for further development as a blood-stage malaria vaccine. antibodies against the c-terminal region of pfripr from residues c to d exhibit growth inhibitory activity. to characterize pfripr and to delineate the pfripr region that is essential for gia activity, as reported , we produced ecto-pfripr as a his-tagged recombinant protein using wgcfs (fig. a) . additionally, approximately equal and overlapping pfripr truncates were synthesized as n-terminal gstfused and c-terminal his-tagged proteins (fig. a) . the expressed recombinant proteins were ni + affinity purified as soluble proteins, resolved in . % sds-polyacrylamide gels, and stained with coomassie brilliant blue r- (cbb). the ecto-pfripr protein was visualized as a single band of about kda (fig. b, lane ) . the pfripr truncate regions resolved at approximately similar molecular weights (fig. b) . each purified recombinant pfripr protein was used to immunize and raise antibodies in six rats. sera from four rats which showed higher antibody titers were pooled and used in subsequent experiments. the specificity of rat antibodies to detect native pfripr was evaluated by western blot using p. falciparum schizont-rich parasite lysates. antibodies against the ecto-pfripr (fig. b) and each of the pfripr regions (fig. a) immunoprecipitated native pfripr, which could be detected as a double band of approximately and kda (supplemental fig. s ). to evaluate whether rat anti-pfripr antibodies could block parasite invasion in gia, the antibodies were tested for inhibition of parasite growth over one cycle of replication as determined by flow cytometry. at a final concentration of mg/ml (total igg), antibodies to pfripr c -d (pfripr_ ) showed the highest gia activity to the d strain by ± % (mean ± se); significantly higher than the negative control, anti-his-gst antibodies (p < . ; fig. c ). to further validate that antibodies against pfripr_ are responsible for the observed gia activity, anti-pfripr_ antibodies were purified from rabbit anti-pfripr k -d (fig. a) using recombinant pfripr_ immobilized on a hitrap nhs-activated hp column (ge healthcare). pfripr_ specific antibodies at . mg/ml induced a gia activity of up to ± % (mean ± se) (fig. d) , albeit lower than that reported for leading blood stage vaccine candidates; specifically, rh has an inhibition ic of µg/ml while cyrpa has an ic of . mg/ml of total igg . the anti-pfripr_ antibody-depleted flow-through fraction did not show detectable gia activity, indicating that pfripr_ contains all epitopes of invasion blocking antibodies against pfripr k -d antigen. pfripr_ is a relatively small protein with the potential of being an easily produced vaccine, and thus was synthesized as a soluble protein using a baculovirus protein expression system (bpes) amenable for downstream vaccine development. expression was confirmed by sds-page (fig. e ) and the recombinant protein was used to raise antibodies in rabbit. antibody specificity was confirmed by recognition of native pfripr at approximately kda in western blot analysis using a schizont-rich parasite lysate (fig. f) . to evaluate the gia activity of antibodies to bpes pfripr_ , we performed gia at two-fold serial dilutions ranging from to . mg/ml. we observed a dose dependent inhibition (fig. g) . indeed, at only mg/ml we observed an inhibitory activity of up to ± % and ± % (mean ± se) for . mg and . mg immunization doses, respectively (fig. g) . put together, these results confirmed the gia activity of anti-pfripr antibodies and identified that pfripr_ , spanning amino acids c -d , is the key target of the gia activity inducing antibodies. pfripr interacts directly with both rh and cyrpa. based on several studies , , , we sought to understand the nature of the complex formed between pfripr, rh , and cyrpa, and if this interaction was direct or indirect. to demonstrate and quantify the interaction between pfripr and rh , as well as with cyrpa, surface plasmon resonance (spr) analysis was performed. recombinant ecto regions of both rh and cyrpa were expressed as gst-fusion proteins using wgcfs and affinity purified by virtue of the gst tag. the sizes of affinity purified recombinant rh and cyrpa were as estimated (figs. a, s ). however, contrary to a recent study , we observed that pfripr directly interacts with rh at a k d value of . × − m (fig. b, table , fig. s a ). comparably, pfripr and cyrpa exhibited a k d value of . × − m (fig. b, table , fig. s a ). thus, we demonstrated for the first time that pfripr interacts directly with both rh and cyrpa. pfripr binds to human erythrocytes. to understand the mechanism of invasion inhibition by anti-pfripr antibodies, we sought to determine whether pfripr binds directly to the surface of host erythrocytes. first, we generated transgenic parasite lines expressing full-length pfripr (m -n ) with a c-terminal × ha tag (pfripr-ha). in vivo pfripr-ha expression was confirmed by immunofluorescence assay (ifa), showing co-localization with ama in the micronemes suggesting native pfripr localization (fig. a) . we further observed that anti ha-tag could immunoprecipitate basigin and rh (fig. s a ). since studies have indicated that culture supernatants can be a source of parasite proteins that bind host erythrocytes , we used culture supernatants of the transgenic parasites to further confirm successful expression and release of pfripr-ha into the culture supernatant (fig. b) . consistent with the reported size of native pfripr , , signals at approximately and kda were observed (fig. b) . although an earlier study had suggested that pfripr is processed, and that the - kda signal represents a processed c-terminal fragment, a recent study demonstrated that antibodies against the n-terminal and c-terminal egf like domains of pfripr detected the two signals, suggesting that the signals are derived from full-length pfripr , and the -kda signal might be due to multiple disulfide bonds in pfripr despite reducing conditions. the ratio of the signals varied in each preparation of the sds-page sample even from same culture supernatant or parasite lysate (data not shown). second, we performed erythrocyte binding assays (eba) to determine if pfripr-ha could bind to erythrocytes. neuraminidase treatment selectively removes sialic acid residues while trypsin and chymotrypsin treatments differentially cleave the peptide backbones of erythrocyte surface proteins [ ] [ ] [ ] . using culture supernatants, we observed that native pfripr-ha binds erythrocytes in a chymotrypsin, neuraminidase, and trypsin resistant www.nature.com/scientificreports www.nature.com/scientificreports/ manner (fig. c) indicating that binding is independent of enzyme-sensitive sites on the erythrocyte surface. as a control, binding of eba to erythrocytes was sensitive to neuraminidase and trypsin treatment but was unaffected by chymotrypsin treatment (fig. d) . to clarify if the observed interaction was a result of direct binding of pfripr-ha to the erythrocyte surface, we examined the erythrocyte binding ability of recombinant ecto-pfripr at a final concentration of nm. the results shown in fig. e indicated that n-terminal gst-fused recombinant ecto-pfripr bound erythrocytes with similar enzyme treatment profiles as native pfripr-ha (fig. c) , thus validating that native pfripr may directly bind to erythrocytes. recombinant his-gst was used as a negative control (fig. f) . to assess the physiological role of the erythrocyte surface binding of pfripr, we determined the gia activity of n-terminal gst-fused recombinant pfripr protein. the recombinant pfripr significantly inhibited parasite growth by ± % (mean ± se) at µm final protein concentration, which was significantly higher than that of his-gst, a negative control at the same concentration (p < . ; fig. g ). these findings (fig. g ) suggested at least two possibilities: ( ) the recombinant ecto-pfripr could inhibit pfripr interaction with parasite proteins, rh and cyrpa, and ( ) the recombinant ecto-pfripr inhibits interaction between parasite pfripr with human erythrocyte surface receptor(s) during merozoite invasion. we sought to validate these postulations in subsequent assays. to identify potential pfripr receptors on the host erythrocyte surface we used a systematic screening approach by first compiling a library of abundantly expressed erythrocyte surface proteins and subsequently synthesizing them as n-terminal gst and c-terminal his-tagged recombinant proteins using wgcfs. expression as soluble proteins was confirmed by sds-page (fig. a) . we then applied the alphascreen protein-protein interaction assay to screen for potential receptors binding pfripr. interaction between pfripr and rh , which is known to exist, was used to demonstrate positive interaction while pfripr interaction with his-gst was used as a negative control. three erythrocyte surface proteins had significantly higher signals compared to the negative control (kruskal-wallis test; p < . ) (fig. b) , suggesting interaction with pfripr; namely, adhesion molecule with igg-like domain (amigo , also referred to as ali ; dega), semaphorin- a (sema a; cd ), and neuroplastin (nptn). to validate the specificity of the interaction between pfripr and erythrocyte surface protein we systematically analyzed the strength of interaction between the three erythrocyte surface proteins and pfripr using surface plasmon resonance (spr). we established that recombinant pfripr (fig. b, table , fig. s b ). spr with cd , which ranked highest among proteins with non-significant values in alphascreen (fig. b) , did not show any interaction with pfripr ( fig. s c ). in summary, amigo , sema a, and nptn represent putative receptors of pfripr on the surface of host erythrocytes. using the recombinant ecto-pfripr protein and erythrocyte ghosts, immunoprecipitation experiments were conducted to confirm the above interactions. gst-fused ecto-pfripr and gst-fused ama (negative control) were incubated with erythrocyte ghosts, then sema a was pulled down with anti-sema a antibodies. anti-sema a antibody successfully immunoprecipitated recombinant gst-fused ecto-pfripr (fig. d , left panel) as well as native sema a (fig. d , right panel and fig. s b ). the data suggests that recombinant pfripr can bind native sema a. this data is consistent with the spr analysis. however, even though studies have suggested that amigo and nptn proteins exist on the surface of erythrocytes , multiple efforts were unsuccessful to detect the two proteins by western blot, or by immunoprecipitation with pfripr. we therefore focused only on characterization of pfripr/sema a interactions in further determinations of the potential mode of action of antibodies against pfripr_ . antibodies to pfripr_ block invasion by inhibiting interaction between pfripr/rh and pfripr/ sema a. having observed that pfripr interacts directly with rh , cyrpa, and human erythrocyte protein sema a, we sought to determine the mode of action of antibodies to pfripr_ . specifically, using spr we assessed whether antibodies against pfripr_ could inhibit protein-protein interactions between pfripr and either rh , cyrpa, or sema a. individual recombinant gst-fused proteins were used as ligands captured on spr sensor chips with a gst-capture kit, followed by addition of analyte consisting of recombinant ecto-pfripr protein and anti-pfripr_ antibodies. as shown in fig. a , anti-pfripr_ antibodies significantly inhibited interaction between both pfripr/rh and pfripr/sema a by ± % and ± % (mean ± se; student's t-test p < . ), respectively. anti-eba (iii-v) antibodies used as a negative control did not show significant inhibition. antibodies against pfripr_ had no observed effect on pfripr/cyrpa interaction (fig. a) . additionally, dose dependence assays revealed that antibodies to pfripr_ , at decreasing concentrations of , , , . and . nm, exerted an significant (kruskal-wallis test; p < . ) when compared against antibodies to his-gst. (e) expression of pfripr_ truncate protein using baculovirus protein expression system (pfripr_ -bpes). purified recombinant pfripr_ -bpes was resolved by sds-page under reducing conditions and stained with coomassie brilliant blue (cbb). m: all blue prestained protein molecular weight marker. (f) reactivity of anti-pfripr_ -bpes antibody on parasite pfripr. western blot analysis of pfripr in trophozoite and schizont-rich parasite lysate using rabbit anti-pfripr_ -bpes antibodies. m: molecular weight marker. (g) dose dependent gia activity of rabbit antibodies to pfripr_ . gia activities of anti-pfripr_ antibodies generated by bpes-expressed recombinant protein were measured at two-fold dilution; range to . mg/ml. solid red line, antibodies against pfripr_ -bpes immunized at . mg/dose; dashed red line, antibodies against pfripr_ -bpes immunized at . mg/dose. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ inhibitory effect on both pfripr/rh and pfripr/sema a interactions that decreased in a dose dependent manner (fig. b) . taken together, the data presented here suggest that antibodies to pfripr_ block invasion by potentially inhibiting pfripr/rh interaction, as well as that between pfripr and its novel erythrocyte-surface receptor sema a. pfripr_ therefore offers an ideal target for further evaluation as a blood-stage malaria vaccine. www.nature.com/scientificreports www.nature.com/scientificreports/ invasion of erythrocytes by p. falciparum merozoites is a fundamental step in malaria pathogenesis and therefore a primary target for vaccine development , . understanding biochemical interactions is key to identifying the role of proteins involved in the invasion process. one protein of particular interest is pfripr, a leading subunit vaccine candidate and a vital protein in erythrocyte invasion , , , . we recently determined that pfripr is highly conserved among african isolates, with the region spanning c -d (pfripr_ ) having only a single amino acid substitution at e q . the pfripr/cyrpa/rh -basigin complex is suggested to play a central role in the sequential molecular events leading to merozoite invasion . we and others have further demonstrated that specific antibodies raised against recombinant pfripr exhibit strain-transcending inhibition of p. falciparum in vitro growth , . however, it is not understood how rh and cyrpa interact with pfripr, and how anti-pfripr antibodies consistently achieve high gia activity in vitro. here, we investigated the mechanism of anti-pfripr antibodies in inhibiting merozoite invasion. pfripr, being a -kda protein with cysteine residues, is difficult to express as full-length protein in conventional expression systems. therefore, we utilized the eukaryotic wgcfs to express pfripr, which we subsequently subjected to systematic biochemical analyses. we demonstrated that antibodies to a short region of pfripr (pfripr_ : c -d ) in the c-terminal spanning five egf-like domains (fifth to ninth egf-like domains) inhibited merozoite invasion, as well as blocked interaction between pfripr and its erythrocyte receptors. the gia potency observed was lower than in a recent study , an aspect that should be given careful consideration in future studies. nevertheless, pfripr_ is a relatively small region that deserves further evaluation for its candidacy as a blood-stage vaccine antigen. previous studies indicated that pfripr localizes to merozoite micronemes and is released onto the parasite surface during invasion, forming a tripartite complex with rh and cyrpa . the complex is then anchored to the surface of erythrocyte via basigin , , . however, the data presented here is somewhat contradictory to those studies. specifically, using the avexis assay galaway et al. did not observe direct interaction between pfripr/ rh . similarly, the rh /cyrpa/pfripr complex structural analysis did not directly show pfripr/rh binding . nonetheless, they did observe that, after binding to basigin, the rh /cyrpa/pfripr complex disassembled to generate an erythrocyte membrane associated rh , and pfripr which migrated as a single band of high molecular weight (about kda), suggesting that they were in the same complex as oligomers. based on the latter observation, our report on a pfripr/rh direct interaction may offer insight to the situational path of the rh /cyrpa/ pfripr complex after binding to basigin. indeed, our eba studies with episomally expressed pfripr fused with an ha tag (fig. c) is consistent with previous results showing that pfripr was present within the erythrocyte membrane pellet . we further demonstrated that gst-pfripr binds to the erythrocyte surface and is resistant to chymotrypsin, trypsin, and neuraminidase treatment similar to pfripr-ha. in this non-traditional eba, we incubated recombinant gst-pfripr with erythrocytes, washed, and then hemolyzed the cell with tetanolysin. the erythrocyte ghost was further washed with buffer, mixed with sample buffer and applied onto an sds-page gel. thus, using alphascreen, we probed for interactions between biotinylated pfripr ectodomain as bait and gst-fused erythrocyte surface receptors (fig. a,b) . subsequently, analyses by spr confirmed that pfripr directly interacts with amigo , sema a, and nptn (fig. c ). in addition, anti-sema a antibody immunoprecipitated recombinant gst-pfripr suggesting that recombinant pfripr could bind native sema a (fig. d) . taken together, these findings suggest that pfripr can function as a parasite ligand. these data need to be further validated since it could mean that (i) the native pfripr indeed binds to erythrocyte sema a, or (ii) recombinant gst-ripr protein is structurally dissimilar from native pfripr, perhaps due to lacking a parasite specific post-translational modification, giving it a different erythrocyte surface binding phenotype. attempts to genetically disrupt pfripr have been unsuccessful, suggesting that it is essential for blood-stage parasite growth . in addition, using p. falciparum lines conditionally expressing pfripr, volz et al. demonstrated that loss of pfripr function blocks growth due to the inability of merozoites to invade erythrocytes . consistent with this, anti-pfripr antibodies have been known to induce high gia activity . the key question is therefore, what is the mechanism of anti-pfripr antibodies on parasite growth inhibition? in our study, anti-pfripr polyclonal antibodies blocked the interaction between pfripr and rh , as well as between pfripr and a host receptor sema a. it was therefore postulated that the antibody blockage of pfripr binding to its multiple partners could explain the observed growth inhibition activity. however, we were unable to show a correlation between interaction inhibition in spr and in vitro parasite gia. thus, in future studies we would like to address the mode of inhibition with monoclonal antibodies, as well as validate the in vitro spr findings by an in vivo protein-protein interaction detection system. sema a, the john-milton-hagen blood group antigen, was recently reported as the receptor for p. falciparum merozoite-specific trap homolog, mtrap . sema a is a gpi-anchored membrane bound protein expressed www.nature.com/scientificreports www.nature.com/scientificreports/ on several tissues with diverse functions; however, the functional role of the interaction remains unclear since antibodies to either mtrap or sema a did not cause inhibition in in vitro parasite invasion assays . similarly, our attempts to inhibit merozoite invasion with anti-sema a antibodies showed no gia activity, possibly due to insufficient antibody molecules which target sema a surfaces important for pfripr/sema a interactions (fig. s c) . another possibility is that the inhibition caused by the anti-pfripr_ antibodies may not have been due to inhibition of pfripr-sema a interaction, but by another unknown mechanism such as anti-pfripr antibodies inhibiting pfripr/rh interaction . our analysis showed that recombinant pfripr/sema a interacts at a nm level k d (fig. c) ; however, we could not immunoprecipitate a native pfripr/rh /sema a complex. this could suggest that the interaction is a "strong transient complex" and a trigger would be needed for its dissociation . indeed, anti-pfripr_ antibodies inhibit both pfripr/rh and pfripr/sema a interactions (fig. a,b) , suggesting that these pfripr interacting regions are close. recently, healer et al. reported that neutralizing anti-pfripr monoclonal antibody prevents merozoite invasion by not blocking rh /cyrpa/pfripr complex formation but by another mechanism . although our results may support an alternative mechanism of blocking ligand-receptor interaction, this model needs confirmation. still, the k d values determined by spr with recombinant pfripr and sama a proteins were low and suggested a high affinity interaction; however, this is generally not the case for natural ligand-receptor interactions which have low affinity adhesion . to understand the pfripr/sema a interaction in detail, we may need further analysis for k d values using other methods, for example radioligand binding assays. in conclusion, we show that a small region of pfripr (pfripr_ : c -d ) located within the c-terminal egf-like domains elicits antibodies that potently inhibit parasite growth and impede interaction between pfripr and its erythrocyte receptor. this is of considerable importance since the region was produced in a scalable and good manufacturing practice (gmp) compatible baculovirus-based vaccine production system allowing further vaccine development targeted adjustments. as a short and less complex protein that can be easily synthesized, pfripr_ is a potential candidate for further development as a blood-stage malaria vaccine. production of recombinant proteins and antisera. an ecto-pfripr (pf d _ ; d -n ) gene with a c-terminal his-tag was synthesized as a wheat-codon optimized nucleotide sequence by genscript (tokyo, japan) (table s ). pfripr truncates (regions - ; fig. a and table s ) were amplified from the wheat codon optimized ecto-pfripr dna sequence. all primer sequences used in this study are summarized in table s . the amplified dna fragments with c-terminal his-tag sequence were restricted by xhoi and noti, then ligated into a wgcfs plasmid (peu-e -gst-tev-n ; cellfree sciences, matsuyama, japan) for expression of gst-fusion proteins. the proteins were expressed with wgcfs (cellfree sciences) as n-terminal glutathione s-transferase (gst) fusion proteins with c-terminal his-tags as described . ecto-pfripr and pfripr truncate recombinant proteins were purified using a ni + sepharose affinity column (ge healthcare) as described . for gia studies, ecto-pfripr was also expressed with wgcfs after ligation of the synthetic ecto-pfripr gene (table s ) into the peu-e -mcs vector (cellfree sciences, matsuyama, japan). for further experiments, pfripr region (pfripr_ : c -d ) with his-tag at the c-terminus was synthesized as an insect-cell codon optimized gene (table s ). the gene was cloned into the pfastbac expression vector and expressed using a baculovirus protein expression system (genscript, piscataway, nj, usa). pfripr_ released into sf cell culture supernatant was purified by a ni-nta column followed by a superdex column (ge healthcare). parasite and erythrocyte surface proteins were expressed with wgcfs as n-terminal glutathione s-transferase (gst) fusion proteins with c-terminal his-tags. specifically, the parasite proteins rh (e gst-fused rh proteins were prepared for biocore single kinetics analysis by eluting gst-fused rh bound to a glutathione-sepharose b column using tobacco etch virus (tev) protease (invitrogen), which cleaves the tev recognition site located between the gst tag and the rh fragment, thereby releasing rh from the n-terminal gst tag. to generate antisera, each affinity purified recombinant protein or truncate was used to immunize animals. all rat immunizations were conducted at sumitomo dainippon pharma co., ltd. (osaka, japan); specifically, with ecto-pfripr (d -n ) and pfripr truncates (pfripr_ ) amino acid (aa), d -a ; , i -k ; , k -n ; , m -k ; , c -d ; , g -n ; , t -r ; , c -i ; , f -n ; , s -c ; and , s -i (based on the d isolate gene sequence; pf d _ ). six sprague-dawley rats (charles river laboratories japan, inc.) per group were immunized sub-cutaneously with each antigen ( µg) emulsified in freund's complete adjuvant, followed by booster immunizations in freund's incomplete adjuvant at three-week intervals. antisera were collected three weeks after the last immunization. rabbit anti-pfripr_ antiserum was purchased from kitayama labes. specifically, new zealand white rabbits were immunized twice with antigen in freund's complete adjuvant at four-week intervals. antisera were collected two weeks after the last immunization (kitayama labes co. ltd. ina, japan). marker. (g) gia activity of recombinant proteins. purified recombinant gst-fused pfripr and in vitro cultured p. falciparum d strain. his-gst protein was used as a negative control. error bar represents standard error of the mean. * indicates statistically significant (kruskal-wallis test; p < . ) when compared to his-gst protein. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ rabbit anti-pfripr antibodies were purified by affinity chromatography by applying rabbit sera against pfripr k -d to immobilized-recombinant pfripr_ coupled to a hitrap nhs activated hp column (ge healthcare) following the manufacturer's instructions. all fractions were assayed for protein concentration and used in gia. p. falciparum culture and transfection. asexual stage p. falciparum d strain was a kind gift from the national institute of allergy and infectious diseases (niaid), and was cultured as described . to generate a parasite line expressing pfripr-ha, full length pfripr was amplified by pcr with the primers indicated in table s (ripr-tran f and r). the dna fragments were digested with spei and psti, and ligated into pd ha plasmids with a pfef -alpha promoter for episomal expression , . for transfection, parasitized erythrocytes were resuspended in µl of cytomix ( mm kcl, . mm cacl , mm egta, mm mgcl , mm k hpo /kh po , mm hepes, ph . ) containing µg of plasmid dna. electroporations were performed in mm cuvettes using a gene pulser xcell electroporation system (bio-rad, hercules, ca) at the condition . kv, µf, and ∞ Ω. after electroporation the erythrocytes were immediately resuspended in complete medium. wr was added to nm in the culture medium one day post electroporation, and the culture maintained under the same drug pressure until drug-resistant parasites appeared. schizont-rich parasite pellets from p. falciparum d strain was conducted as described . additional details are included in the supplementary methods. imaging by indirect immunofluorescence assay (ifa). ifa was conducted as described with rabbit anti-ha antibody (abcam) at : ; mouse anti-ama antibody at : ; and secondary antibodies, alexa fluor -conjugated goat anti-rabbit igg and alexa fluor -conjugated goat anti-mouse igg (invitrogen), at : . erythrocyte binding assay (eba). erythrocyte binding assays were performed with native eba and pfripr shed into the culture supernatant of transfected parasites as described , . additional details are included in the supplementary methods. fresh human erythrocytes were washed three times in incomplete rpmi medium (irpmi; rpmi medium with l-glutamine, mm hepes buffer, and mg/l of hypoxanthine without sodium bicarbonate; invitrogen) before enzyme treatment. subsequently, µl of concentrated (× ) culture supernatant was incubated with µl of untreated and enzyme-treated human erythrocytes on a rotating wheel for min at rt. after incubation the tube was centrifuged at , × g for min at °c and the supernatant was removed. the pellet was incubated with µl of tetanolysin solution (final concentration of µg/ml in irpmi) for min at °c for hemolysis, and the reaction mixture was centrifuged at , × g for min to collect erythrocyte membranes. after repeating the centrifugation three times, the erythrocyte membranes were resuspended in µl of irpmi and transferred to a new tube. the tube was centrifuged at , × g for min and µl of reducing sds-page sample buffer added after the removal of the supernatant. the samples were incubated at °c for min and subsequently resolved by sds-page. following western blotting as described above, native pfripr and eba proteins were detected by the respective rabbit antibodies. for the eba with recombinant proteins (ecto-pfripr and his-gst), each recombinant protein was incubated in µl of untreated and enzyme-treated human erythrocytes with % hematocrit (final concentration of recombinant protein was . pm). protein bound to erythrocytes was determined as described above for the eba with native protein. growth inhibition assay (gia). the inhibitory activity of the total iggs from rat antisera against the recombinant proteins on merozoite invasion was determined over one cycle of d parasite replication. parasitemia was determined by flow cytometry . rat antibody to anti-his-gst was used as a negative control. gia experiments with n-terminal gst-fused recombinant pfripr protein were conducted similarly at a final protein concentration of µm. his-gst at the same concentration was used as the negative control. for each assay, three independent experiments were carried out in triplicate to confirm reproducibility. protein-protein interactions by alphascreen. interaction between pfripr and erythrocyte surface proteins was quantified by alphascreen as reported . briefly, reactions were carried out in µl of reaction volume per well in -well optiplate microtiter plates (perkinelmer). first, affinity-purified ecto-pfripr recombinant protein was biotinylated using a biotin labeling kit-nh (dojindo molecular technologies, kumamoto, japan) according to the manufacturer's instruction. secondly, µl of nm biotinylated protein was mixed with µl of nm for each erythrocyte surface protein in reaction buffer ( mm tris-hcl [ph . ], . % [v/v] tween- and . mg/ml [w/v] bovine serum albumin), and incubated for h at °c to form a protein-protein complex. subsequently, a µl suspension of streptavidin-coated donor-beads and anti-gst acceptor-beads (perkinelmer) mixture in : (v/v) in the reaction buffer was added to a final concentration of µg/ml of both beads. the mixture was incubated at °c for h in the dark to allow the donor-and acceptor-beads to optimally (fig. s ) . (d) western blot analysis: erythrocytes ghosts were first mixed with recombinant gst-pfripr, and sema a was subsequently immunoprecipitated with rabbit anti-sema a polyclonal antibodies. a parallel control experiment conducted with gst-ama was included. sema a pulldown; and immunoprecipitated sample, input. the samples were derived from erythrocyte ghosts mixed with recombinant gst-pfripr or gst-ama . the membrane was probed with mouse anti-gst antibodies (left panel) and rabbit anti-sema a antibodies (right panel) with membrane stripping, washing, and blocking in between. due to the low amount of sema a in the input lanes, no band was observed (right panel). arrowhead; sema a, arrow; heavy chain. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ bind to biotin and gst, respectively. upon illumination of this complex, a luminescence signal at nm was detected by the envision plate reader (perkinelmer) and the results were expressed as alphascreen counts. gst tagged rh , known to interact with pfripr, was included as a positive control and his-gst as a negative control. surface plasmon resonance (spr). all spr experiments were performed using a biacore x instrument (ge healthcare) according to the manufacturer's instructions and as reported . additional details are included in the supplementary methods. immunoprecipitation. erythrocyte ghosts were prepared as reported . recombinant proteins (either gst-pfripr or gst-ama , μm, μl) and erythrocyte ghosts ( μg) were incubated in lysis buffer ( mm tris-hcl, . m nacl, mm edta, . % np- , complete tm protease inhibitor) at °c for hour. protein g conjugated magnetic beads ( µl; thermo fisher scientific, san jose, ca) were then added and further incubated for min to remove nonspecific binding. the beads were precipitated with a magnetic apparatus and the supernatant sample ( µl) transferred to a new tube containing µl of magnetic beads preincubated with µl of rabbit anti-sema a polyclonal antibody (abcam, cambridge, uk). the mixture was incubated at °c for min. the beads were washed three times with µl of wash buffer ( mm tris-hcl, . m nacl, mm edta, . % np- ). finally, proteins were extracted from the protein g-conjugated beads by incubation with µl of × sds-page reducing loading buffer at °c for min. final supernatant was used for western blot analysis. similarly, pfripr-ha was immunoprecipitated using rabbit anti-ha antibody (abcam) from schizont-rich parasite lysates. ethical approval. human erythrocytes and plasma were procured from the japanese red cross society, and their use for parasite culture and in vitro experiments approved by the ethical review committee of ehime university hospital (aidaiibyourin ). animal experiments at sumitomo dainippon pharma co., ltd were approved by sumitomo dainippon pharma ethical review committee (approval number an ). all experiments were conducted in accordance with approved protocols and regulations. the datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. binding inhibition with anti-pfripr_ antibody. effect of anti-pfripr_ antibodies ( nm) to inhibit proteinprotein interaction between nm ecto-pfripr as analyte, and either sema a, rh , or cyrpa as ligand by spr. the sensor chip used was the same as in fig. b . each bar represents the average inhibition of three independent experiments. error bars represent se of the mean. * indicates statistically significant by student's t test, p < . . (b) dose dependent binding-inhibitory activity of anti-pfripr_ antibody on pfripr with sema a and rh . effects of antibodies to pfripr_ at a decreasing final concentration of (presented in a) above), , . , . and . nm were tested to inhibit protein-protein interaction between nm pfripr (analyte) and either rh or sema a by spr. red line, pfripr and sema a; black line, pfripr and rh . immune mechanisms in malaria: new insights in vaccine development extreme polymorphism in a vaccine antigen and risk of clinical malaria: implications for vaccine development identification of a potent combination of key plasmodium falciparum merozoite antigens that elicit straintranscending parasite-neutralizing antibodies multiprotein complex between the gpi-anchored cyrpa with pfrh and pfripr is crucial for plasmodium falciparum erythrocyte invasion neutralization of plasmodium falciparum merozoites by antibodies against pfrh the blood-stage malaria antigen pfrh is susceptible to vaccine-inducible cross-strain neutralizing antibody merozoite antigens of plasmodium falciparum elicit strain-transcending opsonizing immunity antibody titre as a surrogate of protection of the first malaria subunit vaccine, rts,s/as morphology and kinetics of the three distinct phases of red blood cell invasion by plasmodium falciparum merozoites revealing the sequence and resulting cellular morphology of receptor-ligand interactions during plasmodium falciparum invasion of erythrocytes the molecular basis of erythrocyte invasion by malaria parasites distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites functional analysis of the leading malaria vaccine candidate ama- reveals an essential role for the cytoplasmic domain in the invasion process binding of plasmodium merozoite proteins ron and ama triggers commitment to invasion the cellular and molecular basis for malaria parasite invasion of the human red blood cell overlaying molecular and temporal aspects of malaria parasite invasion localisation-based imaging of malarial antigens during erythrocyte entry reaffirms a role for ama but not mtrap in invasion erythrocyte and reticulocyte binding-like proteins of plasmodium falciparum reticulocyte-binding protein homologue -an essential adhesin involved in invasion of human erythrocytes by plasmodium falciparum phenotypic variation of plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes reticulocyte and erythrocyte binding-like proteins function cooperatively in invasion of human erythrocytes by malaria parasites rh -basigin interaction plays a major role in the host tropism of plasmodium falciparum an egf-like protein forms a complex with pfrh and is required for invasion of human erythrocytes by plasmodium falciparum essential role of the pfrh /pfripr/cyrpa complex during plasmodium falciparum invasion of erythrocytes passive immunoprotection of plasmodium falciparum-infected mice designates the cyrpa as candidate malaria vaccine antigen a full-length recombinant plasmodium falciparum pfrh protein induces inhibitory antibodies that are effective across common pfrh genetic variants a pfrh -based vaccine is efficacious against heterologous strain blood-stage plasmodium falciparum infection in aotus monkeys the plasmodium falciparum erythrocyte invasion ligand pfrh as a target of functional and protective human antibodies against malaria identification of plasmodium falciparum reticulocyte binding protein homologue -interacting protein, pfripr, as a highly conserved blood-stage malaria vaccine candidate enhancing blockade of plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against pfrh and other merozoite antigens synergistic malaria vaccine combinations identified by systematic antigen screening a molecular epidemiological study of var gene diversity to characterize the reservoir of plasmodium falciparum in humans in africa p is a merozoite surface protein that binds the n terminus of plasmodium falciparum rh pfrh : a novel reticulocyte-binding family homolog of plasmodium falciparum that binds to the erythrocyte, and an investigation of its receptor plasmodium falciparum erythrocyte invasion through glycophorin c and selection for gerbich negativity in human populations a novel erythrocyte binding antigen- paralogue from plasmodium falciparum defines a new trypsinresistant receptor on human erythrocytes erythrocyte-binding antigen mediates invasion in plasmodium falciparum utilizing sialic acid-dependent and -independent pathways identifying novel plasmodium falciparum erythrocyte invasion receptors using systematic extracellular protein interaction screens pfmsa is a novel plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (cd ) merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria structural basis for inhibition of erythrocyte invasion by antibodies to plasmodium falciparum protein neutralising antibodies block the function of rh /ripr/cyrpa complex during invasion of plasmodium falciparum into human erythrocytes basigin is a receptor essential for erythrocyte invasion by plasmodium falciparum structure of plasmodium falciparum rh -cyrpa-ripr invasion complex semaphorin- a is an erythrocyte receptor for p. falciparum merozoite-specific trap homolog transient protein-protein interactions: structural, functional, and network properties signal initiation in biological systems: the properties and detection of transient extracellular protein interactions the wheat germ cell-free protein synthesis system: a key tool for novel malaria vaccine candidate discovery ralp is a rhoptry neck erythrocyte-binding protein of plasmodium falciparum merozoites and a potential blood-stage vaccine candidate antigen human malaria parasites in continuous culture super-resolution dissection of coordinated events during malaria parasite invasion of the human erythrocyte pv , a novel plasmodium falciparum merozoite dense granule protein, interacts with exported protein in infected erythrocytes discovery of gama, a plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen electrophoretic analysis of the major polypeptides of the human erythrocyte membrane we thank t. j. templeton for critical reading of the manuscript. we also thank the japanese red cross society for providing human erythrocytes and plasma for culturing malaria parasites. this work was funded by jsps kakenhi (jp k , jp , jp , jp , jp k ) and research fund from sumitomo dainippon pharma co., ltd. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to e.t.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -z butywi authors: joyce, collin; burton, dennis r.; briney, bryan title: comparisons of the antibody repertoires of a humanized rodent and humans by high throughput sequencing date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: z butywi the humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. assessment of the baseline antibody repertoires of unimmunized model animals will be useful as a benchmark for future immunization experiments. we characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the omnirat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. multiple differences were found in both the heavy and kappa chain repertoires between omnirats and humans including gene segment usage, cdr length distributions, class switch recombination, somatic hypermutation levels and in features of v(d)j recombination. the inference and generation of repertoires (igor) software tool was used to model recombination in vh regions which allowed for the quantification of some of these differences. diversity estimates of the omnirat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. despite variation between the species repertoires, a high frequency of omnirat clonotypes were also found in the human repertoire. these data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies. rag , rag and artemis (among others). p and n nucleotides are added in the vh-dh and dh-jh junctions by artemis and tdt, dramatically increasing sequence diversity. after successful pairing of this newly formed heavy chain with surrogate light chain (slc), recombination of a light chain from v and j gene segments of the kappa or lambda loci occurs and the b cell swaps the slc for this new light chain. unless the immature b cell is autoreactive or anergic and undergoes receptor editing or clonal deletion, it matures into a naïve b cell and migrates to the periphery whereupon it can become activated by encountering antigen and form germinal centers with help from t-cells. sequence diversity is again enhanced in the germinal center by somatic hypermutation (shm) and/or class switch recombination (csr), two processes that depend on activation induced cytidine deaminase (aid). the omnirat was created by genomic integration of human immunoglobulin (ig) loci on a background of inactivated endogenous rat ig loci. it expresses chimeric heavy chains (i.e. human v, d, and j genes and rat constant genes) that pair with fully human light chains , . we sought to characterize the circulating antibody repertoire diversity in this animal and make comparisons to humans. high throughput antibody sequencing has been used to describe the circulating antibody repertoire of organisms, including more recently at unprecedented depth in humans . reverse transcription of antibody rna and combined tagging with unique molecular identifiers (umis) have allowed us and others , to correct for error and bias in antibody sequencing. using these methods to gain insight into the antibody repertoire of omnirats, we ask whether or not it accurately represents that of humans, and by extension allows for usefulness in the approximation of the human antibody response. we postulate that there are major differences in the repertoires due to distinctness in the ig loci genomic structure and genes that shape antibody diversity between species. here, we provide the most thorough description of humanized transgenic rodent antibody repertoires to date and leverage a novel extremely deep human dataset to make comparisons with implications of immediate use as a reference for omnirat immunization studies. we individually separated total rna from spleens and lymph nodes of three unimmunized omnirats and pcr amplified the heavy and kappa chain antibody v gene segments. the resulting amplicons were subjected to high throughput sequencing in conjunction with preprocessing and annotation by the abstar analysis pipeline (methods) which resulted in a mean of ~ × processed heavy chain sequences and ~ . × processed kappa chain sequences per transgenic animal (table s ). two previously published datasets , of the same humans which together contain a mean of ~ . × processed heavy chain sequences and ~ . × processed kappa chain sequences per individual were used for comparison. we started by making intra-animal comparisons, intra-species comparisons and inter-species comparisons of the immunoglobulin gene segment usage frequencies for each antibody repertoire by performing hierarchical clustering ( fig. ) and linear regression analysis (figs. s and s ). repertoires were found to cluster by species and tissue when variable heavy (vh) (fig. a) , diversity heavy (dh) (fig. b) , joining heavy (jh) (fig. c) and variable kappa (vk) (fig. s a ), but not joining kappa (jk) (fig. s b ) gene usage was examined. differences between the lymph node and spleens of individual omnirats were next investigated. vh gene, dh gene and jh gene usage frequencies between these tissues were highly correlated (fig. s ) , although a few vh gene segments were overrepresented in spleen as compared to lymph nodes including vh - , vh - and vh - (figs. a and s a). dh gene and jh gene usage remained highly correlated with minor differences in specific genes (figs. b,c, s and s b,c). inter-animal spleen gene usage was highly correlated for all three heavy chain gene segments (fig. s ). inter-human comparisons yielded similar, albeit slightly less correlated results (fig. s ). intra-species vh and dh usage comparisons made show much weaker correlations with lower r-squared values than any other previous comparison, while surprisingly jh gene usage was highly correlated (fig. s ). species specific gene usage biases were more predominant in variable genes (vh and vk) than in joining genes (jh and jk) (fig. s a-e) . we hypothesize that this may be due to species specific differences in variable gene order, but not joining gene order at the genomic loci , although no significant correlation between variable gene order and gene usage in the omnirat was found (data not shown). omnirats show a preference for dh gene families of shorter average length such as dh , dh , dh and dh as compared to humans which show a higher representation of longer dh genes from dh , dh , and dh families with the exception of dh - which appears at similar frequencies between each species (figs. b and s b). vk and jk gene usage frequencies were very similar for all comparisons made (figs. s - and s ). differences in cdr length distributions of each repertoire were next determined. the mean heavy chain cdr (cdrh ) length in humans is . amino acids, while in the omnirat we observed a mean cdrh length that is shorter with a mean length of . amino acids (fig. a) . there are minor differences in the kappa light chain (cdrl ) lengths between species with near identical average lengths of . and . for omnirats and humans respectively (fig. s c ). the frequency of light chains with a cdr of amino acids in length is an important consideration when choosing a model animal for vaccination experiments involving the germline targeting immunogen eod-gt which is in human clinical trials , , . this frequency of -amino acid cdrl s was lower in omnirats ( . %) than in humans ( . %) i.e. a factor of (fig. s c ). after observing a tendency for shorter cdrh lengths in the omnirat as compared to humans, we wanted to know if the number of n and p nucleotide additions in the heavy chain v-d and d-j junction sites were different. figure b ,c shows average v-d and d-j junction nucleotide addition lengths in the omnirat are indeed shorter as compared to humans. nucleotide additions in the v-j junctions of kappa chains are also shorter on average as compared to humans (fig. s e) . the longest dh gene segments are found in the dh family and the longest jh gene segments come from the jh gene family. gene segments from these families are important contributors to the generation of unusually long cdrh s in humans and are consistently found in certain broadly neutralizing antibodies (bnabs) that bind to the human immunodeficiency virus (hiv) envelope (env) glycoprotein, indicating the importance of these rearrangements in hiv vaccine studies . on average, the frequency of antibodies with d -j rearrangements in omnirats is . with little variation, while in humans the frequency of these antibody species is more variable between subjects with a higher mean of . (fig. d) . the preference of omnirats for shorter cdrh lengths and dh gene segments can be placed in the context of shorter dh gene lengths in the wild-type rat (rattus norvegicus) as compared to human dh genes (fig. e) , indicating a possible biologically intrinsic bias. we used igor to infer recombination models for each individual repertoire from , unmutated sequences allowing for the quantification of differences in features of heavy chain vdj recombination and generated , , synthetic sequences per model. cdrh length, vd insertion length and dj insertion length distributions from the synthetic sequence data (fig. s a-c) were found to be very similar to the observed data columns are antibody repertoires and rows are gene segments. data was scaled by calculating the z-score for each gene (row) and hierarchical clustering (euclidean distance metric) was done. a dendrogram representation of clustering is shown and indicates uniqueness in gene segment usage between the lymph node and spleen repertoires of the omnirat and between the omnirat and human repertoires. red and blue indicate high and low z-scores respectively (legend shown in a), and since it is calculated per gene it represents differences between repertoires and not the relative frequencies of gene segment usage in each repertoire. ( fig. a-c) . kullback-leibler (kl) divergence is a measure of how different two probability distributions are. kl divergence between models (fig. s d ) and model 'events' (fig. s e) were computed as previously described . kl divergence between pairs of omnirat models was found to be lower than kl divergence between pairs of human models for both complete and all event level calculations. the average pairwise omnirat model versus human model complete kl divergence calculation was found to be much greater than that of pairwise inter-animal calculations and more than twice that of pairwise inter-human calculations. "d-gene", "v-gene trim ( ')", and "d-gene trim ( ')" were among the events computed to have the mean highest kl divergence from pairwise inter-species event level model comparisons. class switch recombination and somatic hypermutation in omnirats. in supplementary fig. s a , the frequency of antibody isotypes is shown. the human repertoire contains average frequencies of . and . for igm and igg respectively as previously published , while in the omnirat antibody repertoire we observe mean frequencies of . and . for lymph node and spleen igg respectively and means of . and . for lymph node and spleen igm respectively. mean numbers of variable gene mutations in igm (fig. s b) , igg (fig. s c) and kappa (fig. s d) sequences of the omnirat were about half of those found in the human repertoire. the observed increase in shm of class-switched igg sequences as compared to igm sequences in the omnirat demonstrates the ability of the animal to generate memory b cells. we first examined clonotype (defined as identical vh gene, jh gene and cdrh amino acid sequence) diversity of the heavy chain repertoire for each individual animal. all sequences from lymph nodes and spleens were collapsed into unique clonotypes, separately for each tissue and animal. any clonotype found in both tissue compartments must have originated from different b cells, allowing for the measurement of repeatedly observed clonotypes. rarefaction curves for each animal were www.nature.com/scientificreports www.nature.com/scientificreports/ generated (fig. a) and indicate a low frequency of repeatedly observed clonotypes. we estimated diversity using chao , and recon , as previously described . diversity estimates were similar between the two estimators, ( . × - . × ) for chao and ( . × - . × ) for recon (fig. b) . we next estimated heavy chain sequence diversity for each animal (fig. c) and again found that both estimators broadly agreed, giving similar values of ( . × - . × ) for chao and ( . × - . × ) for recon. previously published estimates of both clonotype and sequence diversity in individual humans only exceed that in the omnirats by a maximum two orders of magnitude. this is surprising given that the omnirat is more restricted in cdrh length. for each combination of two or more animals, we computed the frequency of shared unique heavy chain clonotypes (fig. a) . there was on average . % of clonotypes shared between each combination of two omnirats. surprisingly, we found that . % of clonotypes were shared between all three of the animals. next, we pooled unique heavy chain clonotypes from all ten human subjects and measured the percentage of clonotypes in each animal that could be found in the total human pool (fig. b) . we found that ( . - . %) of each omnirat clonotype repertoire and . % of clonotypes combined from all animals could be found in the total human clonotype pool. shared clonotypes have shorter cdrh lengths than unshared clonotypes on average (fig. c) which is expected given that sequence diversity is expected to increase as the number of amino acids increases giving less of a chance for sharing. vh gene family usage between shared and unshared clonotypes indicates no major differences (fig. d) . sequence logos for amino acid long (fig. e) and amino acid long (fig. e) cdhr s from both shared and unshared fractions were made and indicate broad similarity between the two fractions. we set out to determine commonalities and differences between omnirat and human antibody repertoires to be used as a reference for vaccine studies. our results show that there exists substantial variation in gene usage frequencies and elements of recombination, indicating specific limitations of this animal model for predicting the human immune response. we found that by performing hierarchical clustering on gene segment usage, repertoires clustered together by both species and tissue. differences in gene segment usage between transgenic animal models and humans, as well as between tissues are expected. for example, multiple human ig loci transgenic rodents are reported to have gene usage profiles that slightly vary from that of humans , . furthermore, antibody repertoires from separate human tissues are known to deviate strongly enough to be clustered by hierarchical clustering . in our case, the lymph node repertoire from the omnirat was most likely able to be distinguished from that of the spleen due to the increased presence of antigen-experienced b cells in the latter as shown by somatic hypermutation and class-switched transcript frequencies. this indicates that tissue selection will affect the outcome of an antibody discovery campaign and reinforces evidence for normal b cell development by suggesting the existence of affinity maturation. investigation into cdr length distributions revealed that the omnirat prefers shorter cdrh s as compared to humans. interestingly, the mechanisms of this preference are due to decreased n additions, and a tendency to incorporate shorter dh gene segments. this result has also been seen in multiple other transgenic and wild-type rodents , , , . the specific reasons remain unclear, although the observation that wild-type rat germline d gene segment lengths are shorter suggests intrinsic species-specific mechanisms of selection as well as differences in tdt expression during bone marrow b cell development. we further speculate that another possible reason for intra-species variation can be attributed to distinct prior antigen exposure and divergent gut microbiomes , . the diversity of the omnirat heavy chain repertoire was shown to be slightly lower than that in humans. our results indicate biased gene usage and decreased junctional diversity are the primary reasons for the resulting repertoire diversity estimate comparisons. we also showed that there is a much higher frequency of 'public clonotypes' or clonotypes shared between members of this species than previously reported in humans , . lower sequence diversity combined with identical genetic background and highly similar gene usage are possible reasons for this result. in summary, we have determined specific differences between the omnirat and human antibody repertoires which must be taken into careful consideration when evaluating an antibody response in order to make predictions for human subjects. we have also shown that this animal's antibodies show signs of class switch recombination, somatic hypermutation and large diversity supporting its value for the discovery of monoclonal antibodies to targets that may not be immunogenic in other models. even though a high degree of variation exists, we still found many clonotypes to be shared between the species pools. finally, more studies will need to be done in order to characterize omnirat serum and memory b cell responses to immunogens. next-generation sequencing of omnirat antibody repertoires. total rna from spleens and lymph nodes was extracted (rneasy maxi kit, qiagen) from each unimmunized heavy chain and kappa chain only transgenic rat (omnirat, open monoclonal technology inc., palo alto, ca, usa) and antibody sequences were amplified as previously described except for different primers used during reverse transcription (table s ) . we chose to interrogate the antibody repertoires found in secondary lymphoid organs as opposed to peripheral blood due to the higher number of b cells. correct pcr product sizes were verified on an agarose gel (e-gel ex; invitrogen) and quantified with fluorometry (qubit; life technologies), pooled at approximately equimolar concentrations and each sample pool was re-quantified before sequencing on an illumina miseq (miseq reagent kit v , -cycle). all animal experiments were conducted in accordance with the institutional animal care and use committee of scripps research and approved by the institutional research boards of scripps research. (d) distribution of vh gene family usage for unshared clonotypes from the animal pool (black) or clonotypes shared by both species pool (red). (e,f) sequence logos of the cdrh s encoded by shared and unshared clonotypes of length (e) or (f). head-region amino acid coloring: polar amino acids (gstycqn) are green; basic amino acids (krh) are blue; acidic amino acids (de) are red; and hydrophobic amino acids (avlipwfm) are black. all torso residues are gray. murine antibody responses to cleaved soluble hiv- envelope trimers are highly restricted in specificity rational hiv immunogen design to target specific germline b cell receptors priming hiv- broadly neutralizing antibody precursors in human ig loci transgenic mice bacterially derived synthetic mimetics of mammalian oligomannose prime antibody responses that neutralize hiv infectivity a repertoire of monoclonal antibodies with human heavy chains from transgenic mice antigen-specific human monoclonal antibodies from mice engineered with human ig heavy and light chain yacs antigen-specific human antibodies from mice comprising four distinct genetic modifications human antibody production in transgenic animals transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies mechanisms of central tolerance for b cells high-affinity igg antibodies develop naturally in ig-knockout rats carrying germline human igh/igκ/igλ loci bearing the rat ch region human antibody expression in transgenic rats: comparison of chimeric igh loci with human vh, d and jh but bearing different rat c-gene regions commonality despite exceptional diversity in the baseline human antibody repertoire clonify: unseeded antibody lineage assignment from next-generation sequencing data genetic measurement of memory b-cell recall using antibody repertoire sequencing toward a more accurate view of human b-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding massively scalable genetic analysis of antibody repertoires rapid and focused maturation of a vrc -class hiv broadly neutralizing antibody lineage involves both binding and accommodation of the n -glycan hiv- vaccines. priming a broadly neutralizing antibody response to hiv- using a germline targeting immunogen identification of common features in prototype broadly neutralizing antibodies to hiv envelope v apex to facilitate vaccine design high-throughput immune repertoire analysis with igor estimating the population size for capture-recapture data with unequal catchability how many different clonotypes do immune repertoires contain? robust estimates of overall immune-repertoire diversity from high-throughput measurements on samples complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery mechanisms that shape human antibody repertoire development in mice transgenic for human ig h and l chain loci tissue-specific expressed antibody variable gene repertoires intrinsic bias and public rearrangements in the human immunoglobulin vλ light chain repertoire immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire microbial symbionts regulate the primary ig repertoire b cell superantigens in the human intestinal microbiota high frequency of shared clonotypes in human b cell receptor repertoires moderated estimation of fold change and dispersion for rna-seq data with deseq. the authors thank all the study subjects for their participation. c.j., d.r.b. and b.b. planned and designed the experiments. c.j. performed experiments. c.j. analyzed data. c.j. wrote the manuscript. all authors contributed to manuscript revisions. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to d.r.b. or b.b.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -hl fa af authors: matsuishi, yujiro; sakuramoto, hideaki; hoshino, haruhiko; shimojo, nobutake; enomoto, yuki; mathis, bryan j.; hiramatsu, yuji; inoue, yoshiaki title: down syndrome reduces the sedative effect of midazolam in pediatric cardiovascular surgical patients date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: hl fa af down syndrome (ds) is frequently comorbid with congenital heart disease and has recently been shown to reduce the sedative effect of benzodiazepine (bdz)-class anesthesia but this effect in a clinical setting has not been studied. therefore, this study compared midazolam sedation after heart surgery in ds and normal children. we retrospectively reviewed patient records in our pediatric intensive care unit (picu) of pediatric cardiovascular operations between march and march . we selected five days of continuous post-operative data just after termination of muscle relaxants. midazolam sedation was estimated by bayesian inference for generalized linear mixed models. we enrolled patients (average age weeks) of which ( %) had ds. ds patients had a high probability of receiving a higher midazolam dosage and dexmedetomidine dosage over the study period (probability = . , probability = . ) while depth of sedation was not different in ds patients (probability = . ). multi regression modeling included severity scores and demographic data showed ds decreases midazolam sedation compared with controls (posterior or = . , % cri = . – . ). in conclusion, midazolam dosages should be carefully adjusted as ds significantly decreases midazolam sedative effect in pediatric heart surgery patients. study design and participants. we retrospectively reviewed records of consecutive patients admitted to the university of tsukuba affiliated hospital pediatric intensive care unit (picu) who underwent cardiovascular operations between march and march . patients were excluded if they had other trisomy, stroke, epilepsy and/or picu stays of less than days after the end of muscle relaxant usage. we recorded patient information, including age, sex, surgical procedure, and daily severity data (including severity of organ dysfunction and sedative/muscle relaxant dosages) during picu stays for five days after the end of muscle relaxant usage. in our practice, we use muscle relaxants and sedation in cases of severe cardiac failure and pulmonary arterial hypertension. additional instances would be whenever careful control is needed for a stressed right ventricle (such as for the fontan procedure) and/or to prevent fighting the ventilator and reduce oxygen consumption. we also use muscle relaxants and sedation for high airway resistance patients, but all uses of relaxants and sedation are carefully monitored and weaning is judged on both a daily and case-by-case basis. the institutional review board of the university of tsukuba approved the study (approval #h - ). heart surgery (rachs- ) which classifies surgical procedures into six categories based on mortality risk. rachs- was previously validated by large multi-institutional data sets [ ] [ ] [ ] . sedation was assessed by using the state behavioral scale (sbs) , which scores sedation status over a range of − (unresponsive) to + (agitation), and is widely used in the pediatric critical care field as a sedation indicator . daily severity of organ dysfunction was evaluated by pediatric logistic organ dysfunction- score (pelod- ) . pelod- consists of ten variables corresponding to five organ dysfunctions and daily assessment allows for prediction of outcome in critically ill children . statistical analysis. model structure. the outcome of interest was sedation status measured by sbs and the dependent factor was mdz dosage. however, muscle relaxants are ordinarily used after cardiac operations to avoid negative hemodynamic effects and this may mask both sedation status and pharmaco-resistance to sedatives. however, excluding muscle relaxant usage days would cause lead time bias and possible overestimation of the outcome , . thus, we used muscle relaxant days as a covariate for the multi-regression modeling to mediate the bias in addition to a five-day post-usage period in our analysis. for adjustment of our model, additional covariates were chosen a priori: sex, age, rachs- , dose of dexmedetomidine, vecuronium dosage status (received/ not received) and pelod- (without the central nervous system component). to adjust patient demographic characteristics, we chose sex and age as covariates. rachs- was used to adjust operation severity and pelod- (without the central nervous system component) was used to adjust post-surgical daily severity. as our institute mainly uses dexmedetomidine as mdz alternatives for sedation, we chose dosages of this as covariate factors. interaction methodology. our modeling included considerations about interaction. as regression modeling assumes independence for each factor, we suspected that down syndrome and mdz dosage were not independent of each other and the magnitude (quantitative interaction) of mdz effect would change based on ds status. thus, we used a two-step system in which we calculated main effect (model ) then proceeded to interaction modeling (model ). we also applied an interaction methodology for dex (model ) as a control for midazolam pharmaco-resistance to sedation. in interaction models (model , ) the odds ratio of the main effect (down syndrome and midazolam; down syndrome and dexmedetomidine) was not significant, possibly due to the ability to capture only a segment of the main effect. statistical estimation. surveying for pharmaco-resistance assumes the inclusion of many outliers. a previous study already reported using robust methods in multivariate methods while bayesian methods , are also applicable for data that includes many outliers. to deal with population outliers in pharmaco-resistance studies, bayesian modeling , and bayesian inference for generalized linear mixed models (glmm) via markov chain monte carlo (mcmc) has been reported , . therefore, we applied hierarchical bayesian modeling, or bayesian inference for glmm using no-u-turn sampler (nuts), which is an extension of the hamiltonian monte carlo (hmc) algorithm of the markov chain monte carlo method . we used uninformative prior distribution as our prior distribution and all iterations were set to , , burn-in samples were set to , and the number of chains was set to . to check the modeling assumption, we used the value of rhat, monte carlo standard error (mcse)/ standard deviation (sd), and effective sample size (neff)/numbers (n). an mcse/sd less than %, a neff/n more than %, and a rhat for all parameters less than . indicated a good estimation for the model. we report the % percentile interval as a % credible interval (crl). we also report the probability for supporting the hypothesis as greater or less than another group in univariate analysis. ethics approval and consent to participate. we used an opt-out methodology coupled with informed consent for this study that was approved by the institutional review board of the university of tsukuba (approval # h - ). information about the study (study goals, methods, and the right to opt out at any point in the study) was available online and in printed form at the hospital. all procedures were approved under regulations of the university of tsukuba that equal or exceed the standards set by the declaration of helsinki. patient characteristics. two patients with another form of trisomy were excluded from this study. there were no instances of stroke and epilepsy but patients were excluded for a picu stay of less than days. we analyzed a total data points from patients for this study (fig. ) . table www.nature.com/scientificreports www.nature.com/scientificreports/ vs % in controls; probability of ds female prevalence was greater than normal = . ). average age of the total population was (± ) weeks and ds patients had a high probability to be younger than normal patients ( ± weeks vs. ± weeks, respectively; probability of the ds group mean was greater than normal = . ) while muscle relaxant days had a high probability to be longer for ds patients ( days in ds vs days in controls; probability of the ds group's days were greater than normal = . ). rachs- scores were almost identical in the ds patients compared with normal patients ( in ds vs in controls; probability of the ds group's severity was greater than normal = . ) and pelod- found that ds patients had a higher probability to be younger than normal patients ( . ± . in ds vs. . ± . in controls; probability of ds group severity was greater than www.nature.com/scientificreports www.nature.com/scientificreports/ normal = . ). depth of sedation was one area where ds patients did not have a high probability compared with normal patients over the study period (− in ds vs − in controls; probability of ds group mean was greater than normal = . ) but ds patients had a high probability of receiving significantly higher doses of midazolam, dexmedetomidine and fentanyl (midazolam: . mg/kg/day in ds vs. . mg/kg/day in control, probability of ds group mean was greater than normal = . ; dexmedetomidine: . μg/kg/day in ds vs. . μg/kg/day in control, probability of ds group mean was greater than normal = . ; fentanyl: . mg/kg/day in ds vs. . mg/kg/day in control, probability of ds group mean was greater than normal = . ). figure presents the relationship between operation risk score as measured by raches- and sedative dosage amounts. category operations tended to not differ in total amounts of sedative, but category and category had a high probability that ds patients would need a higher total amount of sedative (the probability of the ds group dosage of midazolam was greater than normal: category = . , category = . , category = . ; the probability of the ds group dosage of dexmedetoimidine was greater than normal: category = . , category = . ,category = . ). multi regression modeling. the mcse/sd was less than %, the neff/n more than %, and rhat for all parameters was less than . . therefore, our model was a good fit for estimation and did not violate any assumptions. (fig. ). this retrospective study aimed to evaluate factors that could contribute to differences in mdz sedative effect between ds and control patients. a total of pediatric patients in the picu after cardiac surgery were enrolled and evaluated using validated tools over consecutive days. we found that, overall, the amount of mdz administered was increased in ds versus controls after ending muscle relaxants and observed the reduced sedative effect of mdz for ds patients while dex was not different as estimated by bayesian inference modeling. these results are in line with previous research which showed higher requirements for mdz in neonatal cardiac surgery in ds patients . researching sedative effects in a minority population (such as in pediatric ds patients) is complicated from bias imparted by heterological prevalence and complications. the demographic data and operation risk of this study is also heterological between normal patients and ds patients. to adjust these biases, we used multivariate www.nature.com/scientificreports www.nature.com/scientificreports/ analysis with respect to these factors but we also "double checked" our results by using the demographic data propensity score for ds patients as a covariate in bayesian inference modeling. the result ( table ) also shows a reduced sedative effect of mdz while dex was not different. pediatric heart surgery is a complex process complicated by ds. although some studies showed no differences in mortality between ds and normal pediatric patients, it is in the recovery stage that ds complications arise , . recovery from heart surgery is difficult even in adult patients and for pediatric cases complicated by ds, recovery troubles are compounded by various developmental deficits . a retrospective study by nasser and colleagues found that almost % of ds patients recovering from heart surgery needed prolonged mechanical ventilation and almost half of these patients required medication for resultant hypertension . hematological abnormalities can also be present, complicating wound healing , . adding to this complex issue is the amount of sedative needed to prevent unnecessary suffering and anxiety during the healing process. in ds patients, there is clinical evidence that trisomy disorders increase the need for mdz and similar drugs due to alterations in the gabaergic transmission system , . on the translational side, many fundamental studies showed altered gabaergic transmission in murine models that mimic the brain morphology of ds and indicated that gaba a receptor-mediated synaptic transmission occurs in the hippocampus .the propensity of ds patients to more frequently suffer from epilepsy (mediated in the hippocampus), along with dysregulated gaba excitatory-inhibitory balance is hypothesized to be one of the main reasons for the reduced effect of bdz . with this hypothesis in mind, we sought to establish a more solid link between ds and bdz-class anesthesia midazolam requirements by a retrospective, single-center analysis. although we did not do genetic screens, we did have validated sedation and risk scales (sbs and pelod- ) to base our measurements on. we compensated for fluctuations in recovery inherent to cardiovascular surgery by using bayesian inference for glmm. we found that, in general, ds patients required more bdz over longer periods than normal patients and that daily cumulative doses of dexmedetomidine were increased to compensate for the lessened effect of mdz. although we did not observe any prevalence of sedation-related syndromes such as withdrawal and delirium over our study period, a longer duration, multi-centered study saw a prevalence of withdrawal syndrome of more than % after mechanical ventilation and sedative administration of more than days . as ds patients have more complex recoveries than normal, it is entirely possible that any recovery lengthened by ds complications will require more sedatives that, in turn, will increase withdrawal symptoms after prolonged usage. furthermore, a recent study showed that mdz usage is a risk factor for developing pediatric delirium . taken together, these results pair well with our results and indicate that a new approach to sedation in ds patients is required. unpredictable, variable and serious complications in respiration, clotting, drug metabolism, and slower healing after surgery require longer recoveries and more sedation to compensate for this. additionally, as ds patients may suffer from withdrawal symptoms after extended use of sedatives such as bdz, this could introduce additional suffering to patients already weakened from invasive surgery. to conclude, we conducted a retrospective study based on validated evaluative tools that indicated a need for higher doses of mdz with higher doses of compensating sedatives for the -day period immediately after muscle relaxant usage following pediatric heart surgery. our results indicate a need for careful monitoring of sedative effect as ds patients may not respond to mdz in a satisfactory manner. non-bdz sedatives (such as z-drugs) that reduce pain and avoid potential troubles such as withdrawal and delirium need to be evaluated with respect to trisomy disorders and pediatricians should work carefully with pain management specialists to ensure that patient needs are effectively met until alternate approaches are validated. there is some limitation that our retrospective design did not validate patient genetics but relied solely on recorded data. this could affect accuracy and there is variability in the evaluative tools we used due to the subjective nature of scales like the sbs. however, in spite of being a single-center study, we believe our patient pool was of sufficient size and power to maintain significance for our main result and our results were similar to other table . multiple regression model for sedation (sbs) using propensity score. a model estimated by bayesian inference for glmm using no-u-turn sampler (nuts) the mcse/sd was less than %, the neff/n more than %, and rhat for all parameters was less than . . b sex, age, rachs- was used for estimate propensity score for down syndrome. c pelod = pediatric logistic organ dysfunction , without the central nervous system component. studies in the field. moreover, as we used data points collected over days from patients and we adhered to a balanced design that ensured all participants had the same number of data points at each level, we are confident in the validity of our results. our statistical method was chosen based on a two-step process to take into account the magnitude of interaction with mdz but the original assumption of the independence of all factors may be correct. in this case, however, the results from our first modeling step would still be valid. in spite of the limitations inherent in a single-center, retrospective study, this report still shows a significant link between ds and altered bdz effect that could serve to bring insight into clinical practice and act as a basis for controlled, clinical studies. we revealed mdz's quantitative interaction for sedative effect with and without ds in clinical care settings and mdz dosages should be carefully adjusted in pediatric heart surgery patients. the datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request. the four ages of down syndrome neonatal characteristics, and first-year mortality of down syndrome: a national study congenital defects among liveborn infants with down syndrome live birth prevalence of down syndrome in tottori, japan, - population-based study of congenital heart defects in down syndrome upper airway obstruction in children with down syndrome dexmedetomidine is associated with an increased incidence of bradycardia in patients with trisomy after surgery for congenital heart disease dysfunctional hippocampal inhibition in the ts dn mouse model of down syndrome use of moderate sedation for a patient with down syndrome, intellectual disability, and eisenmenger syndrome: a case report. spec. care dent anaesthesia and postoperative analgesia in surgical neonates with or without downs syndrome: is it really different? consensus-based method for risk adjustment for surgery for congenital heart disease the rachs- risk categories reflect mortality and length of stay in a danish population of children operated for congenital heart disease the rachs- risk categories reflect mortality and length of hospital stay in a large german pediatric cardiac surgery population case complexity scores in congenital heart surgery: a comparative study of the aristotle basic complexity score and the risk adjustment in congenital heart surgery (rachs- ) system state behavioral scale: a sedation assessment instrument for infants and young children supported on mechanical ventilation protocolized sedation vs usual care in pediatric patients mechanically ventilated for acute respiratory failure pelod- : an update of the pediatric logistic organ dysfunction score daily estimation of the severity of organ dysfunctions in critically ill children by using the pelod- score breast-cancer tumor size, overdiagnosis, and mammography screening effectiveness the effects of early treatment, lead time and length bias on the mortality experienced by cases detected by screening a bayesian approach to some outlier problems linear models and spurious observations a bayesian population model with hierarchical mixture priors applied to blood count data mixture priors applied to blood count data nr nr the bayesian analysis of population pharmacokinetic models bayesian inference for generalized linear mixed models bayesian inference for generalized linear mixed model based on the multivariate t distribution in population pharmacokinetic study the no-u-turn sampler: adaptively setting path lengths in hamiltonian monte carlo bayesian data analysis syndrome patients: analysis of a national clinical down syndrome and postoperative complications after paediatric cardiac surgery: a propensity-matched analysis incidence and causes of prolonged mechanical ventilation in children with down syndrome undergoing cardiac surgery hematologic abnormalities in children with down syndrome the change of plasma endothelin- levels before and after surgery with or without down syndrome trisomy and early brain development developmentally altered inhibition in ts dn, a mouse model of down syndrome cerebal overinhibition could be the basis for the high prevalence of epilepsy in persons with down syndrome withdrawal assessment tool- monitoring in picu: a multicenter study on iatrogenic withdrawal syndrome delirium and benzodiazepines associated with prolonged icu stay in critically ill infants and young children we would like to thank all of the patients for participating in this study. the authors declare no competing interests. correspondence and requests for materials should be addressed to y.i.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -vclnb eh authors: de almeida, carlos podalirio borges; ziegelmann, patrícia klarmann; couban, rachel; wang, li; busse, jason walter; silva, denise rossato title: predictors of in-hospital mortality among patients with pulmonary tuberculosis: a systematic review and meta-analysis date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: vclnb eh background: there is uncertainty regarding which factors are associated with in-hospital mortality among patients with pulmonary tb (ptb). the aim of this systematic review and meta-analysis is to identify predictors of in-hospital mortality among patients with ptb. methods: we searched medline, embase, and global health, for cohort and case-control studies that reported risk factors for in-hospital mortality in ptb. we pooled all factors that were assessed for an association, and presented relative associations as pooled odds ratios (ors). results: we identified , records, of which we retrieved in full text; cohort studies that evaluated , patients proved eligible. moderate quality evidence suggested an association with co-morbid malignancy and in-hospital mortality (or . ; % ci . – . ). low quality evidence showed no association with positive sputum smear (or . ; % ci . – . ), or male sex (or . , % ci . – . ), and very low quality evidence showed no association with diabetes mellitus (or . , % ic . – . ), and previous tb infection (or . , % ci . – . ). conclusion: co-morbid malignancy was associated with increased risk of in-hospital death among pulmonary tb patients. there is insufficient evidence to confirm positive sputum smear, male sex, diabetes mellitus, and previous tb infection as predictors of in-hospital mortality in tb patients. mortality and worse outcomes compared with women , . previous tb with multiple treatments has also been associated with in-hospital mortality [ ] [ ] [ ] . furthermore, patients with malignant tumors are immunocompromised and can have unusual clinical presentations, both related to delayed diagnosis and high mortality [ ] [ ] [ ] . in tb program monitoring, tb deaths are crucial indicators of the impact of tb control measures [ ] [ ] [ ] [ ] [ ] , especially in areas with high hiv and tb prevalence. data on tb deaths should provide us with a better understanding of the factors associated with these deaths and help guide interventions to reduce mortality; however, there is uncertainty regarding which factors are associated with in-hospital mortality among patients with pulmonary tb . we therefore conducted a systematic review and meta-analysis to establish predictors of in-hospital mortality among patients with pulmonary tb. search strategy. we used a multimodal search strategy focused on bibliographical databases (medline, embase and global health). an experienced librarian (rc) used medical subject headings, adding terms and keywords from a preliminary search to develop the database search strategies. in each database, the librarian used an iterative process to refine the search strategy through testing several search terms and incorporating new search terms as new relevant citations were identified. there were no language restrictions. the search included the following databases from inception to november : medline, embase and global health. the search consisted of three concepts combined using the and operator : tuberculosis , hospitalization and mortality (appendix ). the protocol of this study was published elsewhere . study selection. eligibility criteria. eligible trials met the following criteria : cohort or case-control design ; explored risk factors for in-hospital mortality among patients with pulmonary tb in an adjusted analysis. assessment of study eligibility. two reviewers (cpba and drs) trained in health research methodology screened, independently and in duplicate, the titles and abstracts of all citations identified in our search. the same reviewers screened all full text articles for eligibility; disagreements were resolved by consensus, with consultation of a third investigator (jwb) when resolution could not be achieved. we measured agreement between reviewers with the kappa statistic to assess the reliability of full-text review using the guidelines proposed by landis and koch : < . as slight agreement, . - . as fair agreement, . - . as moderate agreement, . - . as substantial agreement and > . as almost perfect agreement. assessment of study quality. two reviewers (cpba and drs) assessed risk of bias for each eligible study, independently and in duplicate, using the newcastle-ottawa quality assessment scale (nos) for cohort studies . the scale consists of nine items that cover three dimensions : patient selection ( items) ; comparability of cohorts on the basis of the design or analysis ( items); and assessment of outcome ( items). a point is awarded for each item that is satisfied by the study. the total score therefore ranges from zero to nine, with higher scores indicating higher quality. a total score ≥ represents high quality. data extraction and analysis. two reviewers (cpba and drs) extracted data from each eligible study, including demographic information (e.g. sex, age, race), methodology, and all reported predictors. we performed meta-analysis for all predictors that were reported by more than one study. we used odds ratios (ors) with associated % ci to measure the association of binary predictors and in-hospital mortality. we used random effects models for all meta-analyses. if a study reported more than regression model, we used data from the most fully adjusted model presented. we also presented the results from the predictors explored by the studies but that were not eligible for meta-analysis. we evaluated heterogeneity for all pooled estimates through visual inspection of forest plots, because statistical tests of heterogeneity can be misleading when sample sizes are large and cis are therefore narrow . we used the software r. publication bias. for meta-analyses with at least studies, we assessed publication bias by visual assessment of asymmetry of the funnel plot and performed the begg rank correlation test . quality of evidence. we used the grading of recommendations assessment, development and evaluation (grade) approach to summarize the quality of evidence for all meta-analyses . we categorized the confidence in estimates (quality of evidence) as high, moderate, low or very low, on the basis of risk of bias , imprecision , indirectness, inconsistency and publication bias . we used grade evidence profiles to provide a succinct, easily digestible presentation of the quality of evidence and magnitude of associations . in case of doubt or missing details about the studies, authors were contacted for clarification. ethics and dissemination. this study is based on published data, and therefore ethical approval was not a requirement. this systematic review and meta-analysis is expected to serve as a basis for evidence to reduce in-hospital mortality in tb patients, and as a guide for future research based on identified knowledge gaps. it is anticipated that findings from this review will be useful for informing policy, practice and research priorities, improving the management of in-hospital tb patients. we also plan to update the review in the future to monitor changes and guide health services and policy solutions. search results and study characteristics. we identified , unique records, of which we retrieved english and non-english language articles in full text; cohort studies, published between and , that evaluated , patients proved eligible. figure shows the study selection flow diagram. there was substantial agreement (κ = . ) at the titles and abstract screening stage and perfect agreement (κ = . ) between reviewers at the full-text review stage. all eligible studies , , , - were single-center and there was one non-english (chinese) study included in our analysis. two studies , were conducted in japan, two , in taiwan, three , , in korea, one in germany, one in israel, one in iran and one in china. one study used tb-related mortality as defined by the world health organization (the number of tb patients who died during treatment, irrespective of cause) , two , used all-cause mortality, and eight , , , , , , , used tb-related mortality as judged by the investigators. the majority ( of ) , , , , - , , acquired data from medical records, with eight retrospective cohorts , , - and one prospective cohort study (table ) . overall, the quality, evaluated by the nos checklist for the outcome "mortality", was high (table ) . we did not have a sufficient number of studies in our meta-analyses to assess publication bias. a total of studies, involving a total of patients, reported the association of factors with in-hospital mortality , , , [ ] [ ] [ ] [ ] [ ] [ ] . on the basis of our criteria, we conducted meta-analyses for predictors of in-hospital mortality : acid-fast bacilli (afb) smear positive , diabetes mellitus , malignancy , history of previous tb, and . ; table ). table presents the associations with in-hospital mortality for the factors that were not amenable to meta-analysis. we found moderate quality evidence that co-morbid malignancy was associated with increased in-hospital mortality among tb patients. low quality evidence showed that sex and afb smear positive were not associated with in-hospital mortality, and very low quality evidence showed no association with previous tb infection and diabetes mellitus. our review has a number of strengths. our search, which had no language restrictions, was designed and implemented by a research librarian, and literature screening and data extraction were performed independently and in duplicate by two reviewers using pretested, standardized extraction forms. the main limitation of our review was the small numbers of events that contributed to our meta-analyses, resulting in wide estimates of precision for our pooled measures of association. other studies - also found that malignancy increases the risk of death in tb patients. patients with malignant tumors are immunocompromised due to the local or systemic effects of the disease itself, as well as to the treatment regimens, which can impair the immune system and make these patients particularly susceptible to developing tb . in addition, tb can have an unusual clinical presentation, making diagnosis more difficult in these patients, contributing to delay in diagnosis and high mortality rates , . while not significantly associated with mortality in our review, previous tb has been reported to be associated with in-hospital mortality in many studies , - . patients who undergo multiple treatment regimens for tb can develop resistance to drugs with the subsequent emergence of mdr-tb and xdr-tb, conditions highly associated with greater risk of death . further, in settings other than hospitals, studies , have demonstrated that smear positive patients have a better prognosis regarding mortality than smear negative patients. indeed, indicators of atypical manifestations, such as smear-negative sputum, were associated with delayed diagnosis and recently, a retrospective cohort study from brazil reported a high mortality rate during hospitalization ( . %), and negative sputum smear microscopy was an in-hospital mortality predictor in the population studied. however, patients with pulmonary and extrapulmonary tb were included in this study. we did not find a significant association between male sex and in-hospital mortality among pulmonary tb patients. worldwide tb notification data show that far more men than women have tb . some studies showed that mortality rates are higher in females during their reproductive years, but after that they are higher in men , . diabetes was also not associated with mortality in pulmonary tb patients in this study. only one study included in this meta-analysis showed that diabetes was a predictor of mortality in tb patients, possibly because they included a larger number of diabetes patients ( % of the enrolled individuals). some studies , - have found that diabetes increases risk of early mortality during tb treatment. this effect may be explained by impaired tb treatment response . in conclusion, the presence of malignancy was significantly associated with in-hospital death in pulmonary tb patients. other predictors were not associated with in-hospital mortality in tb patients, probably due to the small number of events. further research should explore promising predictors of in-hospital mortality in large prospective studies. table . unpooled predictors for in-hospital mortality among tb patients. factors associated with mortality in tuberculosis patients prognostic factors in tuberculosis related mortalities in hospitalized patients mortality of patients hospitalized for active tuberculosis in israel high mortality in adults hospitalized for active tuberculosis in a low hiv prevalence setting factors associated with mortality in hospitalized patients with newly diagnosed tuberculosis geneva: world health organization. available at: www.who tuberculosis in hospitalized patients: clinical characteristics of patients receiving treatment within the first h after admission time delays in diagnosis of pulmonary tuberculosis: a systematic review of literature hospitalizations for tuberculosis in the united states in : predictors of in-hospital mortality quality of life in tuberculosis: patient and provider perspectives the impact of comorbidity on mortality following in-hospital diagnosis of tuberculosis experience with a medical-psychosocial inpatient unit delay in diagnosis among hospitalized patients with active tuberculosis-predictors and outcomes the impact of nutritional deficit on mortality of in-patients with pulmonary tuberculosis diabetes is a strong predictor of mortality during tuberculosis treatment: a prospective cohort study among tuberculosis patients from mwanza, tanzania impact of diabetes and smoking on mortality in tuberculosis diabetes mellitus is associated with increased mortality during tuberculosis treatment: a prospective cohort study among tuberculosis patients in south-eastern amahra region a review of sex differences in the epidemiology of tuberculosis social and cultural dimensions of gender and tuberculosis mortality among mdr-tb cases: comparison with drug-susceptible tuberculosis and associated factors differences between risk factors associated with tuberculosis treatment abandonment and mortality independent predictors of tuberculosis mortality in a high hiv prevalence setting: a retrospective cohort study initial presentations predict mortality in pulmonary tuberculosis patients-a prospective observational study tuberculosis mortality: patient characteristics and causes excess mortality due to tuberculosis and factors associated to death in and annual cohort of patients diagnosed of tuberculosis predictors of in-hospital mortality among patients with pulmonary tuberculosis: a protocol of systematic review and meta-analysis of observational studies the measurement of observer agreement for categorical data the newcastle-ottawa scale (nos) for assessing the quality of non-randomised studies in meta-analyses. ottawa hospital research institute undue reliance on i( ) in assessing heterogeneity may mislead operating characteristics of a rank correlation test for publication bias grading quality of evidence and strength of recommendations grade guidelines: . rating the quality of evidence-study limitations (risk of bias) grade guidelines . rating the quality of evidence-imprecision grade guidelines: . rating the quality of evidence-inconsistency grade guidelines: . rating the quality of evidence-publication bias characteristics and outcome of patients with active pulmonary tuberculosis requiring intensive care development and validation of a tuberculosis prognostic score for smear-positive in-patients in japan risk factors related with mortality in patient with pulmonary tuberculosis patient mortality of active pulmonary tuberculosis requiring mechanical ventilation predictive factors for mortality among non-hiv-infected patients with pulmonary tuberculosis and respiratory failure hypoalbuminemia and lymphocytopenia are predictive risk factors for in-hospital mortality in patients with tuberculosis prognostic factors in pulmonary tuberculosis requiring mechanical ventilation for acute respiratory failure global tuberculosis programme. a framework for effective tuberculosis control the risk of tuberculosis in patients with cancer solid-organ malignancy as a risk factor for tuberculosis risk factors for and attributable mortality from tuberculosis in patients with hematologic malignances the directly observed therapy short-course (dots) strategy in samara oblast, russian federation risk factors for death during tuberculosis treatment in orel, russia deaths from pulmonary tuberculosis in a low-incidence country all authors made substantial contributions to conception and design. c.p.b.a. designed the study, collected data, and wrote the manuscript. r.c. designed the search strategy. l.w. designed the study and collected data. p.z. analyzed data and wrote the paper. j.b. designed the study and wrote the paper. d.r.s. designed the study, collected data, and wrote the paper. all authors provided final approval of the version to be published. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - evbeijx authors: pandey, rajan kumar; bhatt, tarun kumar; prajapati, vijay kumar title: novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: evbeijx malaria fever has been pervasive for quite a while in tropical developing regions causing high morbidity and mortality. the causal organism is a protozoan parasite of genus plasmodium which spreads to the human host by the bite of hitherto infected female anopheles mosquito. in the course of biting, a salivary protein of anopheles helps in blood feeding behavior and having the ability to elicit the host immune response. this study represents a series of immunoinformatics approaches to design multi-epitope subunit vaccine using anopheles mosquito salivary proteins. designed subunit vaccine was evaluated for its immunogenicity, allergenicity and physiochemical parameters. to enhance the stability of vaccine protein, disulfide engineering was performed in a region of high mobility. codon adaptation and in silico cloning was also performed to ensure the higher expression of designed subunit vaccine in e. coli k expression system. finally, molecular docking and simulation study was performed for the vaccine protein and tlr- receptor, to determine the binding free energy and complex stability. moreover, the designed subunit vaccine was found to induce anti-salivary immunity which may have the ability to prevent the entry of plasmodium sporozoites into the human host. increase the drug efficacy and kills the remaining parasites. the most widely used combination therapies are artemether-lumefantrine (commercial name: coartem) and amodiaquine-artesunate (commercial name: coarsucam) . while the recently approved combination therapies are artesunate-pyronaridine (commercial name: pyramax) and dihydroartemisinin-piperaquine (euartesim) . the recent emerging resistance against artemisinin urges to develop some new strategy to prevent the malaria diseases condition . therefore, in this study, we applied a novel immunoinformatics approach to design multi-epitope based subunit vaccine that may prevent the disease by maintaining the host hemostasis by the inhibition of anticoagulant and anti-inflammatory proteins present in mosquito saliva. it will also inhibit the entry of parasite within the host body by a similar mechanism. apart from this, if any way parasite enters into the host body, vaccine candidate will stop the salivary protein-mediated induction of parasitic growth. among different anopheline vectors, anopheles stephensi is a sub-tropical species that most abundantly present in the indian subcontinent and also distributed across the middle east and south asia region . a. stephensi transmit the malarial infection by injecting various plasmodium species to the human host typically via bites. salivary gland of mosquitos implement various functions for the survival of the vector and complement their feeding behavior by producing a large array of a biochemically active molecule that has immunomodulatory, anti-coagulant and anti-inflammatory properties that disable the host hemostatic response for successful blood feeding , . salivary proteins are antigenic and immunogenic in nature which helps the infectivity of parasite , . d protein and salivary apyrase are two different salivary proteins that help in binding, and inhibition of the platelets aggregation, respectively. salivary peroxidase helps in heme binding and peroxidase activity while putative til domain polypeptide functions as trypsin inhibitor . recently, it was reported that hamadarin is a kda protein present in anopheles stephensi saliva which inhibits the activation of plasma contact system and ultimately blood coagulation. another protein from anopheles gambie saliva namely gsg plays essential blood feeding , . recently, vijay s. et al. reported that salivary proteins might be utilized to develop novel antimalarial control strategies via innate immune protection against malaria . these proteins could likewise evoke a host igg response in natural conditions , . mosquito salivary gland surface (sgs) proteins are the prevalent immunogenic component present in saliva having ability to induce immunogenic responses . this is the reason why we chose the a. stephensi salivary proteins from the national center for biotechnology information (ncbi) and subjected to design multi-epitope subunit vaccine. allergenicity, antigenicity and physiochemical properties were also obtained for the vaccine protein. moreover, tertiary structure prediction followed by refinement was performed to get a refined d model having a higher number of residues in the favored region of ramachandran plot. molecular docking and molecular dynamics simulation of vaccine constructs with tlr were also performed to check the binding energy and complex stability. finally, disulfide engineering and in silico cloning was performed to increase the stability of vaccine construct and ensuring its effective expression in the microbial system, respectively. this study finally represents a novel approach to develop malaria vaccine using salivary protein instead of parasitic protein, which could be helpful to prevent the plasmodium infection to human host. sequence retrieval of salivary protein and assurance of antigenic conduct. in order to design an immunogenic multi-epitope subunit vaccine, the sum of a. stephensi salivary protein sequences was retrieved from the ncbi protein database. major proteins name is salivary lysozyme, a salivary protein precursor, salivary galectin, salivary lipase, anti-thrombin anopheline, salivary protein sg , salivary apyrase, salivary secreted serine protease inhibitor, salivary defensin and salivary cecropin. among salivary protein sequences, only proteins were found to be antigenic as predicted by antigenpro. these sequences were selected based on their score obtained for the probability of antigenicity and all these proteins having a score of ≥ . obtained score for antigenicity probability clearly denoting the antigenic nature of selected protein sequences which can be used for the subunit vaccine designing . subset of t-cell responses to kill those target cells having intracellular viral, bacterial or protozoan infection . during infection, whenever they encounter to the mhc-i mounted antigen specific to their receptor, they enter the cell cycle and perform several mitotic divisions followed differentiation into the effector cells . here, we tried to predict the ctl receptor specific immunogenic epitopes using the netctl . server and total ctl epitopes of mer length were obtained for the input of salivary protein sequences . in the next step, the immunogenicity of epitopes was determined and as per the instruction of iedb , higher score indicate greater probability to elicit an immune response; therefore total ctl epitopes with high immunogenicity score were selected and subjected to the vaccine designing (table ) . helper t-lymphocyte is the key player of both humoral and cell-mediated immune response . therefore, htl receptor specific epitopes are probably going to be a crucial part of the prophylactic and immunotherapeutic vaccine . all salivary protein sequences were subjected to iedb mhc-ii epitope prediction module and epitopes of mer length were obtained. in order to become highest immunogenic epitopes, they must have a lower percentile rank and ic value . only epitopes with lowest percentile rank ranging from . - . were selected for the vaccine designing (table ). their ic value ranging from - denoting that out of epitopes with lowest percentile rank, epitopes have intermediate affinity while remaining to have low affinity for the htl epitopes. on the other side, all epitopes were found to have ifn-γ inducing capability that was obtained from their positive score on the ifnepitope server output , (supplementary table ). all these epitopes were used for the vaccine construction. construction of multi-epitope subunit vaccine. a final vaccine construct of amino acid residues was designed using ctl and htl epitopes as described elsewhere , (supplementary figure ) . in order to attain maximum immune response tlr- agonist (rs ) was used as an adjuvant at the n-terminal site of the vaccine construct . each joint was occupied by the suitable linkers as described by nezafat et al. , for example, adjuvant and ctl epitopes were combined together by eaaak linker, intra-ctl and intra-htl epitopes joint by aay and gpgpg linker, respectively. finally, vaccine construct was obtained having adjuvant, linker, ctl, and htl epitopes in a sequence moving from n-terminal to c-terminal. as this designed subunit vaccine consisting of immunogenic ctl and ttl epitopes along with suitable adjuvant and linker, it may have the ability to inhibit the entry of malaria parasite within the human host body , . b-cell epitope mapping. b-cells are a key player of humoral immunity. an epitope corresponding to the b-cell receptor plays an important role in vaccine design following antibody production . therefore, bcpreds server was used to reliably predict the linear b-cell epitopes where bcpred was the selected prediction method . total b-cell epitopes of mer length were predicted among the primary input sequence of final vaccine construct. among them, only epitopes were selected and finalized because of their high score of . ( fig. a ). due to the selection of highest scoring b-cell epitope among designed subunit vaccine, our vaccine may have the ability to enhance humoral immunity as well as cell mediated immunity . discontinuous epitopes of amino acids long were also predicted from the final d model of vaccine construct with the probability scoring of . (fig. b) . the obtained probability score also confirming the immunogenic behavior of the designed subunit vaccine . antigenicity and allergenicity prediction of designed vaccine. a vaccine given to human host must be immunogenic in nature and capable to trigger significant humoral immune response which ultimately leads to the memory cell formation against the pathogenic epitopes. the antigenicity of designed vaccine construct was determined by using an alignment-free antigenpro server and found that it has the antigenicity probability of . , which represent the antigenic nature of vaccine construct . the antigenicity score obtained for this vaccine construct is comparable with the antigenicity of subunit vaccine reported elsewhere . allergy is an overreaction by our immune system to the previously encountered, ordinarily harmless substance that results in sneezing, wheezing, skin rash, and swelling of the mucous membrane . allergenicity of predicted vaccine construct was determined using allertop online server and found that the vaccine protein is nonallergic in nature and safe for the human use , . physiochemical properties assessment. the physiochemical properties of vaccine construct were characterized by using protparam server and evaluated for seven parameters. the molecular weight of vaccine protein was found to be kda which will favor the antigenicity of the vaccine construct . the theoretical pi was found to be . showing its slightly acidic nature while the total numbers of negative and positive charge residues were and , respectively . the estimated half-life in mammalian reticulocytes was . hours, in vitro; while and hours in yeast and e. coli, in vivo. the extinction coefficient was found to be m- cm- , at nm measured in water, assuming that all cysteine residues are reduced. the score obtained for instability index was . , showing the stable nature of vaccine construct. the value of the aliphatic index and grand average of hydropathicity (gravy) was . and − . , respectively. the estimated value of aliphatic index represents the thermostable nature of designed subunit vaccine because higher the value of aliphatic index, greater will be the thermo stability . while, negative value of gravy for the input subunit vaccine represents the hydrophilic nature of vaccine . conclusively, the designed vaccine is immunogenic, thermostable and hydrophilic in nature. tertiary structure prediction, refinement, and validation. the tertiary structure was predicted by using the raptorx server and d model was obtained as described elsewhere (fig. a) . the best template used for the homology modeling was crystal structure of a legionella phosphoinositide phosphatase (pdb id: fye). total amino acid residues were modeled as a single domain with % disorder. secondary structure information resulting in the presence of % helix, % beta sheet, and % coiled structure. p-value is a parameter of homology modeling where low p-value defines the good quality of modeled structure . the p-value obtained for the modeled structure was . e- which is low and significant. further, protein refinement using galaxyrefine leads to the increase in a number of residues in the favored region . initially, % of residues were in the rama-favored region while after refinement the number of residues in the rama-favored region reached to . %. the refinement output was also validated by plotting ramachandran plot and found the same that . % residue in rama-favored region, . % residues in allowed region and only . % residues in outlier region (fig. b ). disulfide engineering for vaccine stability. in order to stabilize the modeled structure of final vaccine constructs disulfide engineering was performed using disulfide by design v . and found that there are total pairs of residues that can be used for the purpose of disulfide engineering. but after evaluation on other parameters like energy and chi value, only four pairs of residues were finalized because their value comes under the allowed range i.e. the value of energy should be less than . and chi should be in between − and + degree . therefore, total mutations were created at the residues pairs namely ala -gln , tyr -gly , trp -glu , and gly -phe (fig. ) . codon adaptation and in silico cloning. the main purpose of in silico cloning was to express the vaccine protein epitope of anopheles mosquito origin into e. coli expression system . therefore, it was necessary to adapt the codon respective to subunit vaccine construct as per the codon usage of e. coli expression system. we adapted the codons as per e. coli k strain using jcat server and found that the gc-content of the improved sequence was . % while the value of codon adaptive index was . which is near to . that was satisfactory . later on, xhoi and ndei restriction sites were created and cloned into the pet a(+) vector (fig. ) . the target sequence in the clone is represented in blue color in between aforementioned restriction sites . the target sequence is also enclosed between -histidine residues on both ends that will be helpful to the purification purposes. the total length of the clone was . kbp. and tlr- receptor was performed using the cluspro . and total models were generated . among them, only that model was selected which properly occupied the receptor and having lowest energy score and found that model number . fulfill the desired criteria that's why selected as the best-docked complex (fig. ). the energy score obtained for the model . was found to be − which is lowest among all other predicted docked complex showing highest binding affinity. formed using gromacs . . using a gromos a force field . the potential energy obtained for the complex was − . kj/mol, while the value of temperature, pressure, and density was obtained as . k, . bar and . kg/m ( supplementary figure a-c) . the radius of gyration obtained for the docked complex showing that the distance in rotating complex from the center of mass is . nanometers that decreases up to . nanometers at the time duration of nanoseconds (supplementary figure d) . the rmsd value of protein backbone was . nanometers (fig. a) while rmsf score obtained for the protein side chain was found to be . nanometers (fig. b ). both these scores are satisfactory showing strong complex stability , . malaria is severe infection characterized by the high fever with irregularity but may also lead to brain injury and coma. it affects million people from countries, worldwide. lack of effective vaccine and emergence of resistance against artemisinin created a disastrous condition among the people living in the endemic zone. therefore, it's the need of time to search for the new options to tackle this severe problem. the vector for malaria is the anopheles stephensi in the indian subcontinent leads to the transfer of malaria parasite. literature survey reveals that salivary proteins of anopheles mosquito not only supports the pathogenesis but are also immunogenic in nature. therefore, this study was designed to reach a step ahead in the path of vaccine development. we used the primary amino acid sequence of anopheles mosquito salivary protein to design a subunit vaccine construct. the constructed vaccine has ctl, htl and bcl epitopes of varying length. it has antigenic properties in the absence of allergenic properties. it was stable and having a good binding affinity for the tlr- receptor. collectively, this study applied a series of immunoinformatics tools in a sequential manner to find an effective vaccine that may fight against the malaria infection. however, this study needs experimental validation to prove this computational work. the experimental work may include the synthesis of designed subunit vaccine followed by the in vitro and in vivo analysis to determine the immunogenicity and safety concern of the same. anopheles stephensi mosquito is the vector of malaria transmission to the human being in the indian subcontinent. while the salivary gland proteins of anopheles mosquito was reported for their role in parasite pathogenesis and having the ability to induce igg response in the natural host , . therefore, total salivary proteins of a. stephensi were obtained from the national center for biotechnology information (ncbi) protein database (retrieval date / / ) and subjected to multi-epitope vaccine designing. as the main purpose of vaccination is to induce an immunogenic response within the host body, all the retrieved protein sequences were subjected to their antigenicity prediction using antigenpro. based on the antigenicity result, only proteins were found to have an antigenic probability of ≥ . were selected and used in the next step. cytotoxic t-lymphocyte were shown to inhibit malaria parasitic growth and development, inside the hepatocytes cells . to get an immunogenic ctl epitopes having the ability to elicit cell-mediated immunity and form the memory cells, all salivary protein sequences were subjected to the netctl . server . netctl . is an online web server intended for predicting ctl epitopes among input protein sequences based on the training dataset. netctl was selected to predict the ctl epitopes because of its higher prescient execution on all execution parameters as compared to the recently developed servers namely mhc-pathway, mappp, and epijen. all the salivary protein sequences were submitted in the fasta format to predict the ctl epitope at the threshold score of . (default). those epitopes having a combined score of greater than . were selected as ctl epitope and further subjected to the immune epitope database (iedb) mhc class i immunogenicity prediction module. predicted ctl epitopes for each salivary protein was used as an input sequence and the result was obtained in the form of the score, where higher score determines that greater will be the probability of eliciting an immune response. helper t-lymphocyte (htl) epitope prediction. helper t-cell response is the major part of cell-mediated immunity and helps in pathogen clearance by the help of various cytokines and immune cells , . they have the ability to induce both ctl and humoral immune response by the secretion of lymphokines like il- , il- , il , granulocyte-macrophage colony-stimulating factor (gm-csf), and ifnγ. in view of that, we can say that htl epitopes mainly of th type are most likely going to be a crucial part of the prophylactic and immunotherapeutic vaccine. therefore, iedb mhc-ii epitope prediction module was used to predict the htl epitopes for all anopheles salivary protein sequences . the available parameters were kept default except for allele selection where the nominated alleles were h -iab, h -iad, and h -ied. output epitopes were ranked based on their percentile rank score where lower percentile rank representing that greater will be the binding affinity for htl receptor. secondly, to prove our work that the predicted htl epitopes will have ability to activate th type immune response followed by the ifn-γ production, top predicted htl epitopes were subjected to the ifn epitope server using predict option. all epitopes were submitted in the fasta format followed by the approach selection and model of prediction. motif and svm hybrid was selected as the approach and ifn-gamma versus other cytokine as model of prediction. designing of multi-epitope subunit vaccine. so as to plan an appropriate vaccine candidate, it must have the ability to induce ctl and htl immune response. in other words subunit vaccine must contain both ctl and htl epitopes along with suitable linkers. keeping in mind the end goal to effectively activate both innate and adaptive immune response, subunit vaccine must consist of a strong immunostimulatory adjuvant. in the previous decades, there is a huge headway in the adjuvant engineering, for instance, toll-like receptor (tlr) agonists have made its contribution as a part of peptide-based subunit vaccine as a functional option for present-day immunotherapy . recently, junqueira et al. have shown that cpgs oligodeoxynucleotides (cpg odns) and glycoinositolphospholipids (gipl) gotten from trypanosome cruzi having the ability to activate tlr- and tlr leads to actuate potent pro-inflammatory reaction . secondly, proteo-glycolipid complex (p glc) derived from leishmania parasite has shown its affinity for the tlr- receptor and recognized as ligand . moreover, tlrs having the capability to recognize the plasmodium ligands, for example, plasmodium falciparum primes the human tlr- response towards high proinflammatory cytokine profile . shanmugam a. et at. has reported that synthetic tlr- agonist namely rs- (sequence: apphals) can be used as a novel class of adjuvant , therefore, it was added as an adjuvant and linked with epitopes (ctl and htl) by using eaaak linker . linkers assume an imperative part in simulating the vaccine construct to work as an independent immunogen and producing higher antibody titer than that of single immunogen . total three linkers namely eaaak, aay, and gpgpg, were used to construct the final vaccine. aay and gpgpg linkers were added at the intra-epitope position to link the ctl and htl epitopes, respectively. b-cell epitope prediction. b lymphocytes, a type of white blood cells, are the key player of humoral immunity by antibody production. the identification of b-cell epitopes is an essential part in vaccine designing. bcpreds server was used to predict the linear b-cell epitopes of amino acids long. the amino acid sequence of final vaccine construct was used as an input sequence in plain format followed by the selection of fixed length epitope prediction method and length of the epitope. bcpreds (default method) was selected as the prediction method for the epitope of amino acids long . the specificity threshold was set to be by default at % to get the result in a user-friendly format. while conformational epitopes were predicted using ellipro server for the input of tertiary protein structure of vaccine construct. antigenicity and allergenicity prediction of designed vaccine. antigenicity determines the ability of an antigen to binds with the b-and t-cell receptor that may lead to the immune response and memory cell formation. therefore, the antigenic nature of predicted vaccine construct was determined to ensure its ability to interact with the immune receptor. antigenpro is a sequence based, pathogen independent and alignment-free prediction method that was used to check the antigenic behavior of vaccine protein. it uses svm classifier to summarize the probable antigenic or non-antigenic nature of proteins. antigenpro uses the existing protein antigenicity microarray files of eight feature sets for five pathogens to construct two-stage architecture; among them, the first one is multiple representations of the primary protein sequence and the second one is five machine learning algorithms. allergenicity is the potential of a material to cause sensitization and allergic reactions associated with the ige antibody response. therefore, the predicted vaccine construct must be free from the allergenic nature. allertop v. . was used to check the allergenicity of the vaccine construct based on the method that uses auto cross-covariance (acc) transformation of protein sequences into uniform equal-length vectors. input protein sequence of vaccine protein was classified by the k-nearest neighbor algorithm (knn, k = ) which is based on the training set of known allergen from different species and nonallergen from similar species. physiochemical properties assessment. the main purpose of vaccination is to induce an immune response after injecting the vaccine into the body. therefore, it is necessary to define the physical and chemical parameters associated with the vaccine. protparam web server, a part of expert protein analysis system (expasy), was used to define various physicochemical properties of predicted vaccine construct. the primary protein sequence of the vaccine was used to predict the various parameters including molecular weight (kda), estimated half-life, theoretical pi, aliphatic index, grand average of hydropathy (gravy) and so on. tertiary structure prediction. protein molecule achieves maximum stability in its lowest energy state by proper bending and twisting to form a tertiary structure. it is the interaction between the amino acids side chain residue which is responsible to stabilize the protein structure. the -dimensional structure of predicted vaccine construct was obtained by utilizing raptorx structure prediction server. raptorx is a pure ab initio method that can be used to build a d model in a template-free manner. it is the degree of likeness between the target and available template structure that determines the quality of protein model structure created by contemporary protein structure prediction techniques , . therefore, it was necessary to improve the template based predicted model beyond the accuracy by utilizing the template information. to fulfill this thought, output model of raptorx server was subjected to the galaxyrefine web server , which is based on the casp tested refinement method. galaxyrefine performs rehashed structure perturbation followed by overall structural relaxation by performing molecular dynamics simulation. disulfide engineering for vaccine stability. before proceeding to the next step, it was necessary to improve the stability of refined protein model. disulfide bonds are covalent interactions that emulate the stabilizing molecular interaction and provide a considerable stability to protein model by confirming precise geometric conformations. disulfide engineering is a novel approach for creating disulfide bonds into the target protein structure. therefore, the refined model of final vaccine construct was subjected to the disulfide by design . to perform disulfide engineering. initially, the refined protein model was uploaded and run for the residue pair search that can be used for the disulfide engineering purpose. total residue pairs were selected to mutate them with cysteine residue using create mutate function of the disulfide by design . server. in silico cloning. codon adaptation is a way to attain major expression rate of foreign genes in the host when the codon usage of the host differs from that of the organism where the gene stems from. unadapted codon may lead to the minor expression rate in the host. therefore, the primary sequence of vaccine protein was submitted to the java codon adaptation tool (jcat) to adapt their codon usage to most sequenced prokaryotic organisms (e. coli k ) . cai value and gc content of the adapted sequence was also obtained. later on, the adapted nucleotide sequence corresponding to the designed vaccine construct was cloned into the e. coli pet a(+) vector by using the restriction cloning module of snapgene tool . used to predict the preferred orientation of ligand molecule to the receptor molecule in their stable complex form . it can be also used to predict the binding affinity between these two molecules in terms of scoring function. as mentioned in the previous section, tlrs having the capability to recognize the plasmodium ligands and p. falciparum primes the human tlr- response towards high proinflammatory cytokine profile . therefore, tlr- was selected as receptor and its pdb file (pdb id: g a) was obtained from rcsb-protein data bank while the refined model of vaccine protein was used as a ligand. protein-protein docking was performed using the cluspro . : protein-protein docking server, to check the binding affinity between them . widely accepted computational approach which is used to determine the stability of protein-ligand complex at the microscopic level . the protein-protein docked complex output of cluspro was used as an input to perform the molecular dynamics simulation using gromacs v . . . initially, the crystal water of complex was removed followed by the topology generation using a gromos a force field. in the next step, protein complex was centered in a cubic boundary box and filled by water molecule using simple point charge (spc) water model and chloride ion was used for the charge neutralization of complex. moreover, energy minimization followed by canonical equilibration (nvt ensemble) and isothermal-isobaric (npt ensemble) was performed for a time duration of ps. finally, molecular dynamics simulation was executed for the time duration of ns . the root mean square deviation (rmds) for backbone and root mean square fluctuation (rmsf) for side chain was determined. cerebral malaria; mechanisms of brain injury and strategies for improved neuro-cognitive outcome exploring dual inhibitory role of febrifugine analogues against plasmodium utilizing structure-based virtual screening and molecular dynamic simulation antimalarial drug discovery -approaches and progress towards new medicines pre-travel malaria chemoprophylaxis counselling in a public travel medicine clinic in são paulo recent advances in malaria drug discovery a large focus of naturally acquired plasmodium knowlesi infections in human beings advances and challenges in malaria vaccine development the global pipeline of new medicines for the control and elimination of malaria recent clinical and molecular insights into emerging artemisinin resistance in plasmodium falciparum the biology and control of malaria vectors in india 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to design b and t cell multi-epitope subunit vaccine using immunoinformatics approach exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection regulation of cd (+) t cell responses to infection with parasitic protozoa the great balancing act: regulation and fate of antiviral t-cell interactions discovery of t-cell driven subunit vaccines from zika virus genome: an immunoinformatics approach properties of mhc class i presented peptides that enhance immunogenicity a novel multi-epitope peptide vaccine against cancer: an in silico approach designing of interferon-gamma inducing mhc class-ii binders intranasal vaccination against hiv- with adenoviral vector-based nanocomplex using synthetic tlr- agonist peptide as adjuvant the role of b cells and humoral immunity in mycobacterium tuberculosis infection epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases allertop -a server for in silico prediction of allergens disulfide by design . : a web-based tool for disulfide engineering in proteins a multi-subunit based, thermodynamically stable model vaccine using combined immunoinformatics and protein structure based approach developing imidazole analogues as potential inhibitor for leishmania donovani trypanothione reductase: virtual screening, molecular docking, dynamics and admet approach molecular docking and molecular dynamics simulation based approach to explore the dual inhibitor against hiv- reverse transcriptase and integrase t cells as mediators of protective immunity against liver stages of plasmodium large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction differential expression of mirna regulates t cell differentiation and plasticity during visceral leishmaniasis infection perturbed microrna expression by mycobacterium tuberculosis promotes macrophage polarization leading to pro-survival foam cell peptide binding predictions for hla dr, dp and dq molecules advances in the design and delivery of peptide subunit vaccines with a focus on toll-like receptor agonists trypanosoma cruzi adjuvants potentiate t cell-mediated immunity induced by a ny-eso- based antitumor vaccine mechanisms of immune evasion in leishmaniasis plasmodium falciparum infection causes proinflammatory priming of human tlr responses synthetic toll like receptor- (tlr- ) agonist peptides as a novel class of adjuvants a potential protein adjuvant derived from mycobacterium tuberculosis rv enhances dendritic cells-based tumor immunotherapy new directions in nicotine vaccine design and use predicting linear b-cell epitopes using string kernels protein identification and analysis tools in the expasy server molecular modeling and virtual screening approach to discover potential antileishmanial inhibitors against ornithine decarboxylase protein structure refinement driven by side-chain repacking febrifugine analogues as leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking, admet and molecular dynamics simulation the cluspro web server for protein-protein docking high-throughput virtual screening and quantum mechanics approach to develop imipramine analogues as leads against trypanothione reductase of leishmania structure-based virtual screening, molecular docking, admet and molecular simulations to develop benzoxaborole analogs as potential inhibitor against leishmania donovani trypanothione reductase r.k.p. is thankful to department of science and technology for providing inspire fellowship. v.k.p. is thankful to the central university of rajasthan for providing computational facility. we acknowledge the scientific help of ms. rani soni from department of biotechnology, central university of rajasthan for this manuscript. protocol designed by r.k.p., t.k.b., v.k.p. methodology performed by r.k.p., v.k.p. manuscript was written by r.k.p., t.k.b.,v.k.p. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -tqchfdmy authors: mikel, pavel; vasickova, petra; kralik, petr title: one-plasmid double-expression his-tag system for rapid production and easy purification of ms phage-like particles date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: tqchfdmy ms phage-like particles (ms plp) are artificially constructed pseudo-viral particles derived from bacteriophage ms . they are able to carry a specific single stranded rna (ssrna) sequence of choice inside their capsid, thus protecting it against the effects of ubiquitous nucleases. such particles are able to mimic ssrna viruses and, thus, may serve as the process control for molecular detection and quantification of such agents in several kinds of matrices, vaccines and vaccine candidates, drug delivery systems, and systems for the display of immunologically active peptides or nanomachines. currently, there are several different in vivo plasmid-driven packaging systems for production of ms plp. in order to combine all the advantages of the available systems and to upgrade and simplify the production and purification of ms plp, a one-plasmid double-expression his-tag system was designed. the described system utilizes a unique fusion insertional mutation enabling purification of particles using his-tag affinity. using this new production system, highly pure ms plp can be quickly produced and purified by a fast performance liquid chromatography (fplc) approach. the system can be easily adapted to produce other ms plp with different properties. . diagram of cloning experiments in this study. a specific control sequence derived from the mitochondrial dna (mtdna) sequences of two extinct species with one c-variant pac-site (tmpac) was cloned into the multiple cloning site (mcs ) of the pacycduet- vector (novagen, merck, germany) to construct the pacycduet- -tm vector. then, the fragment of maturase and single-chain version of the coat protein dimer containing the his-tag was cloned into the multiple cloning site (mcs ) of the pacycduet- -tm to create the pacycduet- -tm-coatdimer-his vector. scientific reports | : | https://doi.org/ . /s - - - induction of expression of his-tagged ms plp. the pacycduet- -tm-coatdimer-his plasmid was transformed into e. coli lb (de ) cells and the production of his-tagged ms plp was induced as described below. purification of his-tagged ms plp. his-tagged ms plp were purified as described below using an fplc approach. the integrity of his-tagged ms plp isolated using fplc was verified by tem (fig. ) . the tem images clearly show that the his-tagged ms plp were correctly assembled into intact capsids of approximately nm in diameter. the his-tagged ms plp were not damaged by the purification process, were isolated in a large quantity ( × particles/µl; rt-qpcr quantification according to ), and did not form any aggregates (fig. ) . characterization of the single-chain version of the coat protein dimer containing the his-tag. the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) showed that ms plp contained wild-type coat protein with an approximate molecular weight of kda (fig. ,a ) , whereas his-tagged ms plp incorporated a single-chain version of the coat protein dimer containing a his-tag with an approximate weight of kda (fig. ,a ) . while western blot analysis using the anti-enterobacterio phage ms coat protein polyclonal antibody identified both wild-type (fig. ,b ) and single-chain versions of the coat protein dimer containing the his-tag (fig. ,b ) , the anti-histag antibody specifically interacted only with the single-chain version of the coat protein dimer containing the his-tag (fig. ,c ) . to calculate the exact molecular weight of wild-type and single-chain versions of the coat protein dimer containing the his-tag, matrix assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof) analysis was performed. the measured molecular weight of the wild-type coat protein was , da (fig. d ). the measured molecular weight of the single-chain version of the coat protein dimer containing the his-tag was , da (fig. e ). testing of the stability of his-tagged ms plp against nucleases showed them to be resistant under conditions where naked dna and/or rna were rapidly degraded (fig. ) . schematic representation of de novo-synthesized sequence of maturase and single-chain version of the coat protein dimer containing the his-tag. de novo-synthesized sequence was used as a pcr template from which the fragment of maturase and single-chain version of the coat protein dimer containing the his-tag was amplified and subsequently cloned to multiple cloning site (mcs ) of the pacycduet- -tm. the sequence in green is the sequence of single-chain coat protein dimer containing his-tag (in blue) surrounded by two kpni restriction sites (in orange) which is localized between nucleotides - in de novo-synthesized sequence. full length de novo-synthesized sequence is presented in supplementary figure . , molecular weight of wild type ms bacteriophage coat protein was about kda (a ) whereas single chain version of the ms coat protein dimer containing the histag was about kda (a ); (b) western blot analysis using primary anti-enterobacterio phage ms coat protein polyclonal antibody (merck millipore, usa) and secondary goat anti-rabbit igg conjugated with hrp (jackson immunoresearch, uk) antibodies at wild type ms bacteriophage coat protein (b ) and single chain version of the ms coat protein dimer containing the his-tag (b ); (c) western blot analysis using primary anti-histag antibody (pierce, thermo scientific, usa) and secondary goat anti-mouse igg conjugated with hrp (jackson immunoresearch) antibodies at wild type ms bacteriophage coat protein (c ) and single chain version of the ms coat protein dimer containing the his-tag (c ); (d,e) laser desorption ionization-time of flight mass spectrometry (maldi-tof) analysis of wild-type bacteriophage ms coat protein and single-chain version of the ms coat protein dimer containing the his-tag; m, marker, spectra multicolor broad range protein ladder (fermentas); the marker values are in kda. the figures were cropped, full length gel and blots are presented in supplementary figures , determination of optimal method of thermal lysis of his-tagged ms plp and comparison of quantity and purity of ms plp and his-tagged ms plp. the rt-qpcr results showing the most suitable conditions for thermal lysis of his-tagged ms plp are summarized in table (table ) . based on these results, it was decided to include the step of particle lysis into the step of initial denaturation in rt protocol. the rt-qpcr results comparing the quantity and purity of ms plp and his-tagged ms plp are summarized in table (table ). it was shown previously that the newest and most advanced in vivo system for production of ms plp-the one-plasmid double-expression system-has great potential for the production of ms plp , . in comparison with other previously used systems, the system developed by zhan et al. has a good expression efficiency, produces ms plp containing only defined control ssrna sequences without any other ms bacteriophage sequences, and also allows packaging of long control ssrna sequences using multiple pac sites [ ] [ ] [ ] . however, the one-plasmid double-expression system does not enable rapid, simple, and effective purification of produced ms plp. this problem can be resolved by introduction of a his-tag into the structure of ms plp. this results in ms plp carrying his-tag epitopes, which allows their purification using affinity chromatography . unfortunately, at the beginning of this study it was found that introduction of a his-tag into the structure of ms plp according to cheng et al. and expression of his-tagged ms plp in a one-plasmid double-expression system does not result in the production of any his-tagged ms plp . it was subsequently discovered that another group also encountered this problem and failed in the production of his-tagged ms plp (harder, friedrich loeffler institute, germany, personal communication, march ). ponchon et al. also adopted the previously reported strategy of cheng et al. and tried to produce his-tagged ms plp , . however, ponchon et al. used a so-called two-plasmid system where the first plasmid carried the ms coat protein sequence under control of an inducible tac promoter and the second plasmid carried the sequence for packaging under the control of a constitutive lpp promoter , . this strategy allows successful co-expression of both the protein and ssrna in an appropriate ratio. but as a result, in all cases, it was observed that the ms coat protein with the his-tag only formed a dimer rather than intact his-tagged ms plp . it is known that peptide insertions into viral capsid proteins often result in protein misfolding that interferes with ms plp assembly . on the other hand, it is also known that fusion of two coat protein monomers (single-chain dimer) confers tolerance to peptide insertions in an ms coat protein dimer , . the basic idea of a using a single-chain version of the ms coat protein dimer with a his-tag in this study was inspired by these previously published results. the described plasmid-driven system is based on plasmid pacycduet- . plasmid pacycduet- was chosen as the basis for the new his-tagged ms plp production system because it was shown to be an optimal solution in previous studies that dealt with the construction of particles or production of ms coat protein , [ ] [ ] [ ] . zhan et al. used a one-plasmid double-expression system based on the pacycduet- plasmid for production of ms plp carrying a , -nt-long control sequence . to test the ability of such a system to also pack very short control sequences, our previously reported specific control sequence with a total length of nt was chosen for packaging . the final pacycduet- -tm-coatdimer-his expression vector was constructed with a view of easily allowing subsequent modifications. mcs of the vector containing the sequences of maturase and single-chain version of the coat protein dimer containing the his-tag was modified, so that the maturase sequence no longer contains the ndei restriction site. this modification opens the possibility for using the ndei restriction site for cloning into mcs , which increases the maximum length of packed ssrna control sequences, because ndei is the first restriction site in the mcs of the vector. furthermore, in the case that the ndei sequence will be present in the sequence of maturase, direct cloning of sequences into mcs of the vector using the ndei restriction site will not be possible. the inserted sequence for the his-tag is flanked by two kpni restriction sites. this flanking is a consequence of previously used methods of oligonucleotide-directed mutagenesis , . although the sequence of maturase and the single-chain version of coat protein dimer containing the his-tag was synthesized de novo, it was decided to maintain the presence of the kpni restriction sites, mainly due to the possibility of easy replacement of the his-tag sequence at this position. the single-chain version of the coat protein dimer is more tolerant of foreign peptide insertions than the wild-type coat protein monomer , . therefore, utilization of the described system should allow insertion of different peptides at this position without causing any problems for ms plp assembly. the possibility of producing ms plp carrying ssrnas of choice and presenting various peptides on the surface of the capsid is currently a very promising method for the production of rna vaccines based on recombinant ms plp - . the single-chain version of the ms coat protein dimer contains one wild-type coat protein fused with one mutagenized coat protein carrying the his-tag. therefore, the presented method results in the production of his-tagged ms plp containing his-tags in the capsid structure exposed outward from the particle and allowing chelated co + on the resin beads to access the his-tag. newly produced single-chain version coat protein dimer containing the his-tag was extensively characterized using sds-page, western blotting and maldi-tof analyses. the molecular weight of the wild-type coat protein measured using maldi-tof was , da, which is very close to its theoretical value of , . da. the measured molecular weight of the single-chain version of the coat protein dimer containing the his-tag was , da (fig. e) , which is also very close to its theoretical value of , da. the differences between theoretical and measured values of protein molecular weights are probably due to the sample preparation protocol and instrumentation used. these analyses clearly showed that the desired mutagenized coat protein with the expected properties was successfully obtained after induction. moreover, this single-chain version of the coat protein dimer containing the his-tag was able to create compact his-tagged ms plp, which were morphologically indistinguishable from previously produced ms plp , . the presence of the his-tag in the structure of the coat protein was used for purification of his-tagged ms plp through fplc. in comparison with the traditionally used method of ultracentrifugation , , , the fplc approach is a rapid purification technique enabling the isolation of huge quantities of his-tagged ms plp (~ particles/µl; rt-qpcr quantification according to ) in less than one hour. in fact, the quantity of isolated his-tagged ms plp purified through fplc is one order of magnitude higher than the quantity of ms plp purified through ultracentrifugation (~ particles/µl; rt-qpcr quantification according to ). the fplc purification procedure is also not as laborious as traditional methods of purification and does not have any negative impact on ms plp integrity. the integrity of his-tagged ms plp after fplc purification was tested by tem; the stability of particles against nucleases was also tested. in general, the ability of ms plp to withstand the action of nucleases is one of their most important properties, because only intact particles are able to protect the ssrna that they encapsulate. testing the stability of fplc-purified his-tagged ms plp against nucleases clearly showed that the particles were able to effectively protect the ssrna that they encapsidated against nuclease degradation. it was shown previously that ms plp consisting of a single-chain version of the coat protein dimer have lower temperature stability in comparison with ms plp consisting of a wild-type coat protein dimer , . our results indicate that lysis temperature is a function of quantity, time, and temperature. it is also interesting that the intensity of bands on an agarose gel was much higher in the case of ms plp in comparison with his-tagged ms plp, although the quantity of tested particles was the same- . µg per band, which corresponds approximately to particles . a possible explanation for this difference is that the lower intensity of his-tagged ms plp bands is related to a lower proportion of contaminating dna in comparison with ms plp (table ) . empirically, the lysis temperature of his-tagged ms plp was determined to be . °c/ min (fig. ) . this feature was utilized in our previously developed rt-qpcr system for quantification of ms plp, which uses a two-step format in which rt and qpcr reactions are separated . the step of particle lysis was included in the rt protocol and it was demonstrated that initial denaturation of °c/ minutes during the rt protocol is sufficient to lyse his-tagged ms plp. this approach not only saves time, but also represents the gentlest method of thermal lysis and provides the highest rt-qpcr yield. on the other hand, the results of this experiment also show that the temperature of lysis has no critical effect on ssrna stability, and, thus, the ste buffer in which the particles were diluted and lysed provided good protection to naked ssrna molecules. the purity of produced ms plp is essential, and, therefore, elimination of contaminating e. coli genomic dna from lysed bacterial culture prior to ms plp purification is a part of each production protocol; usually, a mixture of dnases and rnases are used , , , , , . it is generally accepted that this treatment is sufficient to eliminate contaminating dna. however, this may not be true. cheng et al. tested the purity of ms plp purified through ultracentrifugation and found that they exhibit significant dna contamination ms plp was only ~ particles/µl). based on these findings, the his-tagged ms plp produced in this study and previously produced ms plp were tested for the presence of contaminating dna. because some ms plp applications, e.g., use as a pcv in rt-qpcr detection and quantification of pathogenic ssrna viruses from a wide range of samples, require a greater quantity of ms plp (~ - particles/µl), the presence of contaminating dna was tested in range of - particles/µl. the results are consistent with those reported previously with the difference that his-tagged ms plp are also not absolutely free of contaminating dna, especially at higher concentrations. on the other hand, according to our findings his-tagged ms plp are always at least , × cleaner through all tested concentrations ( - particles/µl) than ms plp purified through ultracentrifugation (table ) . moreover, the proportion of contaminating dna decreases with progressive dilution, but only his-tagged ms plp can be diluted to a quantity where contaminating dna is not detectable in rt-qpcr. in general, the purity of isolated ms plp is very important because the presence of contaminating dna could affect quantification in experiments where particles are used as a pcv, or can confer toxic effects on ms plp serving as vaccines or drug delivery systems . thus, the one-plasmid double-expression his-tag system presented in this work is able to meet this demanding criterion, and can be used for the production of highly pure ms plp. on the other hand, the described purification method is dedicated mainly for using ms plp as a pcv and for using as a vaccines or drug delivery systems it is needed to perform additional laboratory tests. construction of specific control sequence. a specific control sequence derived from mtdna sequences of two extinct species-thylacine (thylacinus cynocephalus) and the moa bird (dinornis struthoides)-was constructed as described previously . construction of pacycduet- -tm. the specific control sequence was cloned into multiple cloning site (mcs ) of the pacycduet- vector (p a-type replication origin; novagen, merck, germany; fig. ). in brief, a -bp amplicon encoding the specific control sequence was obtained by pcr using a de novo template construct and the tm-ndei f and tm-avrii pac r primer pair, which included ndei and avrii restriction enzyme sites; the tm-avrii pac r primer also included one c-variant pac site ( table ). the reaction mixture was composed of . µl of faststart pcr master (roche molecular diagnostics, germany), . pmol of each primer and . pmol of template dna. the assay was run in a total volume of µl under the following conditions: °c for minutes, followed by cycles of °c for seconds, °c for seconds, and °c for seconds; final extension was at °c for minutes. the results of the pcr amplification were examined by agarose gel electrophoresis ( %). the pcr product was purified using the qiaquick pcr purification kit (qiagen, germany) and subsequently cleaved at °c for hours. the composition of the restriction mixture was ng of pcr product, µl of nebuffer (new england biolabs, uk; neb), u and u of ndei and avrii endonucleases, respectively (both neb), in a final volume of µl. the cleaved pcr product was also purified using the qiaquick pcr purification kit (qiagen). the pacycduet- vector was cleaved at °c for hours. the restriction mixture was composed of ng of plasmid dna, µl of nebuffer (neb) u and u of ndei and avrii endonucleases, respectively (both neb), in a final volume of µl. the cleaved vector was dephosphorylated with . u of calf-intestinal alkaline phosphatase (cip) (neb) at °c for hour. subsequently, vector dna was purified using the qiaquick pcr purification kit (qiagen). ligation of the specific control sequence to the mcs of the cleaved pacycduet- vector was performed using the quick-ligation kit (neb) according to the manufacturer's instructions. transformation of e. coli top (life technologies, usa) was also performed according to the manufacturer's recommendations. transformed cells were grown on luria-bertani (lb) (sigma-aldrich, czech republic) agar plates containing chloramphenicol ( µg/ml, sigma-aldrich) overnight. plasmid dna was isolated with the nucleospin plasmid kit (macherey-nagel, germany) according to the manufacturer's instructions. the presence of specific inserts was confirmed by vector-specific (duetup and t terminator) and insert-specific (tm f and tm r) primers ( construction of the expression vector with a single-chain version of the coat protein dimer containing the his-tag for production of his-tagged ms plp -pacycduet- -tm-coatdimer-his. the sequence of maturase and the single-chain version of the coat protein dimer containing the his-tag were synthesized de novo (life technologies) (fig. ) . the de novo construct also contained a transversion of adenine (a) at position to cytosine (c). this change does not influence the amino acid sequence of maturase but leads to deletion of the ndei restriction site (catatg −› cctatg). fusion of the two sequences encoding the coat protein was done as in the pct dl- construct , . the sequence (-ggtacccatcaccatcaccatcacggtacc-) encoding the his-tag flanked by two kpni restriction sites (underlined) was inserted between residues and of the second coat protein amino acid sequence (corresponding to the position between codons and of wild-type coat protein sequence) . the de novo construct was verified by sequencing (life technologies). the fragment encoding the maturase and single-chain version of the coat protein dimer with the his-tag modification was obtained by pcr with the kapa hifi hotstart pcr kit (kapa biosystems, usa) using the de novo template construct and the ms ncoi and ms noti primer pair, which included ncoi and noti restriction enzyme sites ( table ). the reaction mixture was composed of µl of × kapa hifi fidelity buffer, . mm dntp mix, . pmol of each primer, u of kapa hifi polymerase, and pmol of template dna. the assay was run in a total volume of µl under the following conditions: °c for minutes, followed by cycles of °c for seconds, °c for seconds, and °c for seconds; final extension was at °c for minutes. the results of the pcr amplification were examined by agarose gel electrophoresis ( %). the pcr product was purified using the qiaquick pcr purification kit (qiagen) and subsequently cleaved at °c for hours. the restriction mixture was composed of ng of pcr product, µl of nebuffer (neb), and u each of ncoi and noti endonucleases (both neb), in a final volume of µl. the cleaved pcr product was purified using the qiaquick pcr purification kit (qiagen). the pacycduet- -tm vector was cleaved at °c for hours. the restriction mixture was composed of ng of plasmid dna, µl of nebuffer (neb), and u each of ncoi and noti endonucleases (both neb), in a final volume of µl. cleaved vector was dephosphorylated using . u cip (neb) at °c for hour. subsequently, vector dna was purified using the qiaquick pcr purification kit (qiagen). ligation of the fragment encoding the maturase and single-chain version of the coat protein dimer with the his-tag modification to the mcs of the pacycduet- -tm vector was performed using the quick-ligation kit (neb) according to the manufacturer's instructions. transformation of e. coli top (life technologies) was also performed according to the manufacturer's recommendations. transformed cells were grown on lb (sigma-aldrich) agar plates containing chloramphenicol ( µg/ml) overnight. plasmid dna was isolated with the nucleospin plasmid kit (macherey-nagel) according to the manufacturer's instructions. the presence of specific inserts was confirmed by vector-specific (duetup and duetdown ) and insert-specific (ms ncoi and ms noti) primers (table ) in pcrs as well as by sequencing (eurofins mwg operon). the constructed pacycduet- -tm-coatdimer-his plasmid was transformed into e. coli bl (de ) cells (neb) according to the manufacturer's instructions and the bacterial cells were grown in lb broth (sigma-aldrich) containing chloramphenicol ( µg/ml) at °c until od = . . two ml of the bacterial culture were transferred to ml of terrific broth (tb) medium ( g/l yeast extract, g/l tryptone, ml/l glycerol, . m kh po , . m k hpo , all sigma-aldrich) containing chloramphenicol ( µg/ml) and cultivated at °c until od = . and centrifuged at × g for minutes at °c. subsequently, the pellet was resuspended in ml of fresh tb medium containing chloramphenicol ( µg/ml). protein expression was induced by addition of mm isopropyl-l-thio-d-galactopyranoside (iptg) (sigma-aldrich) to the culture, after minutes of culture growth at °c, rifampicin (sigma-aldrich) was added to the iptg induced culture to a final concentration of µg/ml and the culture was cultivated at °c for hours. the cell suspension was centrifuged at × g for minutes at °c and cells were washed and centrifuged ( × g for minutes at °c) twice in ml of pbs buffer (ph = . ). the pellet was resuspended in ml of sonication buffer ( mm tris, mm mgcl × h o, mm cacl , . m nacl, ph = . , all sigma-aldrich) and u of turbo dnase (ambion, thermofisher scientific, usa), u of rnase a (qiagen), and u of benzonase nuclease (sigma-aldrich) were added to eliminate e. coli genomic dna and rna. the cells were lysed by ultrasonic disruption (bandelin vw sonicator, probe ms , bandelin, germany) at amplitude %, using four pulses for a total length of minutes at °c. sonicated bacterial suspension was incubated at °c for hours. to eliminate cell debris, the lysed bacterial suspension was briefly centrifuged at × g for minutes at room temperature and the supernatant containing his-tagged ms plp was filtered through a . -µm syringe pes filter (tpp, switzerland). tris, nacl, mm edta, ph = . , all sigma-aldrich). his-tagged ms plp were verified by tem using a philips em electron microscope (fei, czech republic). the sample of supernatant containing his-tagged ms plp was applied to the grid, stained with % ammonium molybdate (ph = . ) for minute, and inspected at , × magnification and an accelerating voltage of kv. purified his-tagged ms plp were quantified by rt-qpcr as was described previously . the quantity of fplc-purified his-tagged ms plp was compared with previously produced non-his-tagged ms plp purified through ultracentrifugation . to confirm the production of the single-chain version of the coat protein dimer containing the his-tag, sds-page, western blot analysis, and maldi-tof analysis were applied. as a control material, previously produced ms plp without his-tag, composed of wild-type ms bacteriophage coat protein , were used. briefly, ms plp and his-tagged ms plp were resolved by sds-page using a % separating polyacrylamide gel and transferred to polyvinylidene difluoride membranes (pvdf; amersham, uk) which were subsequently blocked in % casein hydrolysate (imuna, czech republic) overnight at °c. protein bands resolved by sds-page were visualized by staining with coomassie brilliant blue r- . the membranes were incubated with anti-enterobacterio phage ms coat protein polyclonal antibody (merck millipore, usa) at a dilution of : for hour at room temperature, washed in pbs with . % tween- (pbs/t, serva, germany), and then incubated for hour at room temperature with goat anti-rabbit igg conjugated with hrp diluted : (jackson immunoresearch, uk). after a washing step, the protein bands were visualized with , ′-diaminobenzidine (sigma-aldrich). western blotting with an anti-histag antibody (pierce, thermo scientific, usa) diluted : as primary antibody and goat anti-mouse igg conjugated with hrp diluted : (jackson immunoresearch) as a secondary antibody was performed as described above. maldi-tof analysis was performed as follows: ms plp and his-tagged ms plp were treated with % β-mercaptoethanol (sigma-aldrich) at °c for minute and precipitated at °c for minutes. precipitated proteins were resuspended in % trifluoroacetic acid and desalted using kda and kda vivacon columns (sartorius). maldi-tof ms analysis was carried out using an ultraflextreme instrument (bruker daltonics, usa) operated in linear positive detection mode. ferulic acid (sigma-aldrich) ( . mg/ml in water:acetonitrile:formic acid, : : , v/v mixture) was used as the maldi matrix in combination with a stainless steel sample plate. stability of his-tagged ms plp against nucleases. stability of his-tagged ms plp against nucleases was verified by their incubation with the combination of u of turbo dnase (ambion) and/or u of rnase a (qiagen) at °c for hour. as controls for the reaction, dna ( ng of purified pcr fragment encoding the maturase and single-chain version of the coat protein dimer with his-tag modification) and rna ( ng of iac in vitro transcript ) , were added to the tested his-tagged ms plp. the ability of his-tagged ms plp to resist the impact of nucleases was determined using agarose gel electrophoresis ( . %). temperature stability of ms plp and his-tagged ms plp. ms plp and his-tagged ms plp (both µg, ~ particles) dissolved in ste buffer were incubated for minutes and minutes, respectively, at the selected temperatures. the biorad tetrad peltier thermal cycler (biorad, usa) temperature gradient program ( °c - °c) was used. immediately after heating, the samples were loaded onto % agarose gel and the particle position was visualized under uv illumination. determination of optimal method of thermal lysis of his-tagged ms plp and comparison of quantity and purity of ms plp and his-tagged ms plp. based on the results of temperature stability testing, his-tagged ms plp particles were diluted to an approximate concentration of particles/µl. the influence of lysis temperature on the exact quantity of his-tagged ms plp was tested using a previously described quantitative reverse transcription polymerase chain reaction (rt-qpcr) system with small modifications; non-treated and thermally treated ( °c, °c, °c and °c for minutes) his-tagged ms plp were added directly to the reverse transcription (rt) reaction. rt was carried out in octuplicates for each temperature and qpcr was done in duplicate for each sample. subsequently, the quantity and purity of his-tagged ms plp was compared with the quantity and purity of previously produced ms plp . ms plp were thermally lysed according to the original protocol ( °c for minutes), while his-tagged ms plp were added directly at the rt step without undergoing thermal lysis according to the results of determination of the optimal method of thermal lysis. the quantity of tested particles was in the range of - particles/µl. to determinate the precise amount of contaminating dna in the sample, rt-qpcr amplification of the specific control sequence was done using reactions with and without rt enzyme (the enzyme was replaced with rnase-free h o; top-bio, czech republic). rt was carried out in octuplicates for each set of samples and qpcr was done in duplicate for each sample. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. bacteriophage ms -another rna phage armored rna technology for production of ribonuclease-resistant viral rna controls and standards ribonuclease resistant viral rna standards encapsidation of heterologous rnas by bacteriophage-ms coat protein ribonuclease-resistant rna controls (armored rna) for reverse transcription-pcr, branched dna, and genotyping assays for hepatitis c virus highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-pcr with an armored rna internal control a novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis c viral rna with armored rna as internal control preparation of ms phage-like particles and their use as potential process control viruses for detection and quantification of enteric rna viruses in different matrices the true story and advantages of rna phage capsids as nanotools methods for preparation of ms phage-like particles and their utilization as process control viruses in rt-pcr and qrt-pcr detection of rna viruses from food matrices and clinical specimens armored long rna controls or standards for branched dna assay for detection of human immunodeficiency virus type fast preparation of a long chimeric armored rna as controls for external quality assessment for molecular detection of zika virus preparation of his-tagged armored rna phage particles as a control for real-time reverse transcription-pcr detection of severe acute respiratory syndrome coronavirus multiple presentation of foreign peptides on the surface of an rna-free spherical bacteriophage capsid simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed luminex-based assay co-expression of rna-protein complexes in escherichia coli and applications to rna biology affinity selection of epitope-based vaccines using a bacteriophage virus-like particle platform complementation of rna binding site mutations in ms coat protein heterodimers subunit fusion confers tolerance to peptide insertions in a virus coat protein a novel method to produce armored double-stranded dna by encapsulation of ms viral capsids external quality assessment for the detection of measles virus by reverse transcription-pcr using armored rna external quality assessment for avian influenza a (h n ) virus detection using armored rna ′- ′ exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis messenger rna vaccine based on recombinant ms virus-like particles against prostate cancer ms viruslike particles: a robust, semisynthetic targeted drug delivery platform a new rna vaccine platform based on ms virus-like particles produced in saccharomyces cerevisiae novel mir- delivery system based on ms virus like particle surface displaying cell-penetrating peptide tat for hepatocellular carcinoma a novel delivery platform based on bacteriophage ms virus-like particles structure and stability of icosahedral particles of a covalent coat protein dimer of bacteriophage ms thermal stability of rna phage virus-like particles displaying foreign peptides cleavage of structural proteins during assembly of head of bacteriophage-t the authors would like to thank neysan donnelly (clear science editing services, germany) for grammatical correction of the manuscript, radek tesarik (veterinary research institute, czech republic) for help with sds-page, western blot analysis and fplc, pavel kulich (veterinary research institute, czech republic) for help with tem and lenka cincarova (veterinary research institute, czech republic) for help with maldi-tof. the work was supported by the ma cr ro , mi cr vi and meys cr npu i program lo . conceived and designed the experiments: p.m., p.v., p.k. performed the experiments: p.m. data analysis: p.m. wrote and edited the paper: p.m., p.v., p.k. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -equnyib authors: kamath, kathy; reifert, jack; johnston, timothy; gable, cameron; pantazes, robert j.; rivera, hilda n.; mcauliffe, isabel; handali, sukwan; daugherty, patrick s. title: antibody epitope repertoire analysis enables rapid antigen discovery and multiplex serology date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: equnyib the detection of pathogen-specific antibodies remains a cornerstone of clinical diagnostics. yet, many test exhibit undesirable performance or are completely lacking. given this, we developed serum epitope repertoire analysis (sera), a method to rapidly discover conserved, pathogen-specific antigens and their epitopes, and applied it to develop an assay for chagas disease caused by the protozoan parasite trypanosoma cruzi. antibody binding peptide motifs were identified from chagas repertoires using a bacterial display random -mer peptide library and next-generation sequencing (ngs). thirty-three motifs were selected and mapped to candidate chagas antigens. in a blinded validation set (n = ), / chagas were positive, / non-chagas were negative, and / leishmania sp. was positive. after unblinding, a leishmania cross-reactive epitope was identified and removed from the panel. the chagas assay exhibited % sensitivity ( / ) and specificity ( / ) in a second blinded validation set including individuals with other parasitic infections. amongst additional epitope repertoires with unknown chagas serostatus, assay specificity was . % ( / ). thus, the chagas assay achieved a combined sensitivity and specificity equivalent or superior to diagnostic algorithms that rely on three separate tests to achieve high specificity. ngs-based serology via sera provides an effective approach to discover antigenic epitopes and develop high performance multiplex serological assays. the detection of antigen-specific antibodies in human specimens via serology remains an essential and fundamental necessity in laboratory medicine and therapeutic and vaccine development. antibody serology is used in the diagnosis of hundreds of infectious, allergic, and autoimmune diseases, and new biomarkers and tests continue to expand the utility of serology. nevertheless, antibody serologic tests frequently exhibit suboptimal performance characteristics as measured by clinical sensitivity and specificity, require subjective interpretation , and/or depend upon multi-step algorithms , . serologic assays using whole cell material and antigen mixtures can have high false positive rates, leading to unnecessary follow-up tests, inappropriate treatments, or misdiagnosis of patients. antibody serologic methods typically provide a narrow view of immunity towards a single, or small number of antigens or organisms, while an estimated pathogens may cause human disease . and, many of these diseases lack effective serology tests altogether. furthermore, many tests remain organism or antigen-based, even though knowledge of which antigen epitopes are targeted can be critical for inferring infection stage , reactivation , and immune protection . despite biomedical need, methods have not emerged to broadly analyze antibody repertoire composition to enable multiplex serology for infection, allergy, or autoimmunity. multiplex serology is typically limited to several organisms, and to only a small subset of their immunogenic epitopes , , even though infection or vaccination can give rise to hundreds of distinct antibody species . to expand the breadth and information content of parallel immunoassays, pathogen-proteome derived phage display libraries and planar peptide arrays have been developed. for example, a phage display library of , , -mer peptides spanning human virus proteomes was constructed , . following immunoprecipitation and dna sequencing, seropositivity towards each virus was inferred. however, comprehensive proteome tiling for many higher complexity organisms is currently impractical given their larger proteome size. similarly, the tiling of overlapping peptides from sets of a priori known antigens from eight tick-borne pathogens enabled construction of a planar peptide arrays with , features enabling detection of each infection . in principle, this method could be expanded to include a larger number of pathogens but requires known antigens or epitopes, and does not provide sufficient peptide diversity to mimic diverse structural epitopes. random peptide arrays of up to , members have proven effective to detect antibodies towards a range of organisms (i.e., viral, bacterial, fungal) . yet, they lack the diversity required to effectively mimic arbitrary protein antigens, and thereby detect the corresponding antibodies. thus, methods to analyze entire antibody repertoires to reveal the spectrum of antigenic epitopes are needed. to enable epitope resolution analysis of immune responses towards any organism, we applied parallel advancements in peptide display library technology , next-generation sequencing (ngs), and computational discovery algorithms . we applied serum epitope repertoire analysis (sera) to discover shared, but highly specific immunogenic epitope motifs associated with chagas disease caused by the protozoan parasite trypanosoma cruzi, and thereby develop a serological assay. chagas disease is estimated to impact more than , people in the united states and million in central and south america . confirmatory testing currently requires the use of three serology tests -two parallel, independent tests, and a third "tie-breaker" test to achieve a specificity of > %. thus, a single test with high specificity could streamline confirmatory testing and screening in blood donors and at-risk groups . our results indicate that ngs-based serology using sera provides an effective approach to antigen and epitope discovery, and an assay format capable of achieving exceptional diagnostic specificity without multiplexing limitations. to demonstrate the utility of sera in antigen discovery and multiplex serology we applied sera to discover conserved immunogenic epitopes of igg antibodies present in sera from individuals with chagas disease. the sera workflow consisted of the steps of (i) separation of antibody-binding peptide library members, (ii) preparation and next-generation sequencing (ngs) of amplicon libraries, (iii) computational discovery of disease-specific motifs and motif panel assembly , and (iv) experimental validation of panel performance (fig. ) . to effectively mimic the diverse linear, structural, and post-translationally modified epitopes from many different organisms, a random peptide library consisting of random -mers displayed on the outer surface of e. coli bacteria was used. as a source of diversity, we selected -mer random peptides since prior studies of antibody binding epitopes have reported that % of linear epitopes span fewer than amino acids . on the other hand, simple structural epitopes (e.g. alpha-helices, beta-hair-pin motifs) can benefit from longer candidate peptides. however, as peptide length grows library quality can deteriorate due to oligonucleotide synthesis errors, or expression and display bias introduced by the peptide display vector. furthermore, longer peptide sequences (e.g. > ) can contain a larger number of distinct epitopes, thereby increasing opportunities for peptide cross-reactivity with antibodies with divergent specificity. to maintain library stability and diversity during propagation, a tightly regulated expression vector was used for peptide display . table s ). specimens were from males and females, with a mean age of +/− years, and residing primarily in the southern united states. all disease specimens were seropositive for chagas disease using the cdc two-test algorithm requiring seropositivity on both the chagas antigen elisa, and a separate immunoblot. one of specimens exhibited discordant elisa/immunoblot results, and a second-tier ifa test was used to resolve the discordancy. additional presumed non-chagas specimens (n = ) were sourced from commercial vendors. each specimen was incubated with the peptide library and antibody binders were separated using protein a/g-conjugated magnetic beads. after amplifying the selected population through growth, plasmids were prepared and the peptide-encoding regions were amplified via pcr with a unique oligonucleotide bar-code for each specimen. the resulting amplicon libraries were pooled and the dna sequences of the peptide encoding regions were determined using ngs. on average, ~ million total sequence reads and ~ million unique reads were determined for each bar-code and corresponding specimen. the set of unique antibody binding peptides for each specimen, or epitope repertoire, was inferred by translation of the dna sequences into amino acid sequences, and unique sequences were archived for bioinformatic analysis. to identify antibody epitope motifs specific to chagas disease, we applied the imune motif discovery algorithm (see methods for parameters) to epitope repertoires from chagas specimens and non-chagas sera, yielding candidate motifs (supplementary table s ). we then down-selected the full set of imune output motifs by requiring a retained motif to be (i) significantly enriched (≥ standard deviations from mean of the controls) in at least % of chagas specimens (n = ) and (ii) absent from control repertoires (i.e., non-significantly enriched (< standard deviations from mean of controls) in % of control epitope repertoires (n = ). downselection yielded motifs with sensitivities ranging from - % (supplementary table s ). imune identified overlapping groups of motifs that mapped to the same t. cruzi epitopes and occurred in the same subsets of epitope repertoires. for example, [fw]kpwe and egxkxwe shared three identities, and both occurred within a metacaspase -mer epitope egfkpwe. motifs mapping to the same epitope were grouped and their equivalence was confirmed by clustering of motif seropositivity (supplementary table s ). thus, imune yielded more than chagas disease specific motifs (> % specificity) with varying sensitivity (fig. a) . individual motifs were remarkably specific to chagas disease, as determined by inspection of their corresponding enrichment values (# observations/# expected) within the discovery set of epitope repertoires (fig. b ). motifs within a given epitope group with the highest mean enrichment and sensitivity were selected for inclusion into a diagnostic motif panel. in two cases, two motifs from the same group were included in the panel because their combination increased sensitivity. chagas-specific motifs exhibited variable enrichments of up to -fold and each motif was present in a different subset of disease repertoires (figs. b, a). motif enrichment values were standardized using the mean and standard deviation of enrichments within non-chagas repertoires. individual chagas motif "z-scores" were then summed to obtain a composite sera score for each epitope repertoire. a composite score threshold of readily captured / serologically defined chagas specimens with % specificity ( / controls) (fig. b ). to evaluate the performance of chagas panel v . , a blinded and randomized set of biospecimens (table , supplementary table s ) was analyzed by sera and composite scores were calculated (fig. a,b) . the set contained leishmania seropositive ( ) negative controls ( ) leishmania ( ) blinded validation cdc na na chagas ( ) toxocara ( ) toxoplasma gondii ( ) cysticercosis ( ) www.nature.com/scientificreports www.nature.com/scientificreports/ specimens, known to cause false positives in chagas serology tests , due to the relatedness of these organisms. even so, the panel accurately classified all chagas as positive, as well as non-chagas controls and / leishmania as negative (fig. a,b) . a second leishmania seropositive epitope repertoire was similarly elevated on chagas panel v . but classified as negative (fig. b) . inspection of the leishmania motif enrichments revealed that the motif [adp]ggfg was enriched in these two leishmania specimens (fig. a) , and present in the proteomes of both t. cruzi and leishmania. removal of this motif from the panel, yielded chagas panel v . which resulted in a sensitivity and specificity of % within the first validation set (fig. c) . to further evaluate the performance of chagas panel v . , we processed an additional blinded and randomized validation sera from the cdc that contained sera from subjects with chagas disease and three other parasitic diseases (table ) . panel scores were again calculated for this set. the panel was % sensitive and specific www.nature.com/scientificreports www.nature.com/scientificreports/ within this second, blinded validation set (fig. d,c) . to further investigate the specificity sera chagas panel v . , epitope repertoires from individuals with unknown chagas serostatus were analyzed. remarkably, just / had composite scores above the threshold, thereby yielding an apparent lower bound of specificity of . % (fig. e) . in summary, chagas panel v . exhibited % sensitivity ( / ) and ≥ . % specificity within the validation cohorts. identification of candidate antigens from chagas-specific motifs. sera chagas panel v . motifs were queried against the trypanasoma cruzi proteome to identify candidate antigens and their antibody recognition epitopes. several chagas-specific motifs occurred as exact matches within established serological antigens, including trans-sialidase, masp, and ca- ( table ) . some motifs occurred multiple times within repeat regions of their corresponding candidate antigen. for example, there were instances of agxkpx[ae] within the established antigen trans-sialidase, copies of gxaaaxxk within surface antigen (ca- ), and copies of yx[ap] vxxx[as]y within microtubule-associated protein ( table ) . a few candidate antigens harboring chagas-specific motifs, including metacaspase, and kinetoplast dna-associated protein have not been described previously as antigens ( table ). these results demonstrate that motifs frequently contain sufficient information content to identify their corresponding antigens and that sera identifies bona fide pathogen antigens. here we present a general methodology for serum epitope repertoire analysis (sera) to rapidly discover the immunogenic epitopes within an organism proteome, and to arbitrarily multiplex the detection of epitope-specific antibodies to develop high performance ngs-based serology assays. within chagas disease sera, many conserved epitopes were identified as sequence motifs representing the shared amino acid preferences within established and putative candidate trypanosoma cruzi antigens. measurement of motif enrichments within the set of all ig-binding peptides for a given specimen (the epitope repertoire) provided a quantitative measure of epitope-specific ig binding activity that could be multiplexed as desired to detect any number of epitope-specific antibodies. a random peptide library of billion -mers provided sufficient diversity to represent all possible amino acid motifs with % confidence, and all possible -mers with % confidence. this diversity is thus about , - , -fold greater than that represented by pathogen proteome peptide arrays (e.g., representing a specific pathogen) or random peptide arrays . sera differs from reported phage display library immunoprecipitation (phip) or virscan approaches to antibody repertoire profiling, in that it makes no assumptions about which organisms, strains, or specific epitopes may be targeted by antibodies. consequently, sera provides a universal approach that can be applied to analyze the immune response to virtually any biological organism or protein antigen -including pathogens, commensals, allergens, biologics and autoantigens. the diagnostic accuracy of sera, measured by combined sensitivity and specificity, was superior to that reported for diagnostics in current use for chagas disease confirmatory testing. chagas disease resulting from infection with the protozoan parasite t. cruzi may be asymptomatic, and chronic, untreated infection can lead to heart failure and death . transfusions from asymptomatic, infected donors may transmit the parasite to blood recipients. consequently, serologic testing for chagas disease is included in routine blood bank testing of donors. for confirmatory testing and screening, no single serological test achieves adequate specificity, necessitating the use of two or three independent serology tests. using chagas disease seropositive specimens, a panel of peptide motifs was identified that exhibited % sensitivity ( / ) and % specificity ( / ) in independent validation sets. remarkably, the chagas sera assay exhibited . % specificity ( / ) amongst sera without known chagas serostatus, which compares favorably to the combined sensitivity and specificity of individual, fda-cleared tests . the exceptional specificity of sera could be expected to yield fewer false positive tests, a feature that may be useful in screening and surveillance studies , particularly when multiple tests are performed in parallel. six genetic lineages of t. cruzi or dtus (discrete typing units) have been described that infect humans. these dtus show different geographic distributions and may generate strain specific immune responses . the specimens in our study are from individuals receiving confirmatory testing at the cdc dpdrl, and are of undetermined dtu. however, the specimens used for discovery were from subjects who had lived or travelled in multiple countries where different dtus are prevalent including bolivia, brazil, el salvador, mexico, ecuador, costa rica, nicaragua and argentina (supplementary table s ). we were able to identify a set of epitope motifs that were conserved among these subjects, and independently validated with a sensitivity of % in a randomized, blinded set, demonstrating that the discovered motifs effectively captured the diversity in t. cruzi dtus among the tested population. nevertheless, our discovery and validation specimens may not represent all dtus, and thus, expanded validation amongst a larger cohort with known dtu may reveal whether all dtus are detected with equivalent sensitivity. additionally, we do not know whether these individuals were in the acute or chronic phase of infection. testing of subjects in which the phase of infection is known is necessary to determine whether our test has differential sensitivity based on phase of infection. there are at least two likely reasons for the improved specificity observed with sera. first, panels are constructed using exclusively specific epitopes within protein antigens and those epitopes that that cross-react can easily be identified and removed. unlike conventional serology in which whole antigens or organism lysates are often used as detection reagents , sera excludes a large number of potentially cross-reactive epitopes introduced by using whole protein antigens. second, computational discovery using epitope repertoires from other infectious diseases enabled optimization of motif combinations to minimize cross-reactivity in non-chagas sera. we discovered the motif [adp]ggfg to be enriched in the epitope repertoires from specimens seropositive for either t. cruzi or leishmania sp., (an organism known to generate false positive chagas test results ). identification and removal of the shared epitope did not adversely affect diagnostic panel performance. sera enabled rapid discovery and mapping of candidate proteome antigens targeted by antibodies associated with chagas disease. many t. cruzi strains within seven recognized discrete typing units are known to cause chagas disease . using a large library of billion random -mer peptides, and computational motif analysis, www.nature.com/scientificreports www.nature.com/scientificreports/ sera circumvents the challenge of identifying which particular species and strains should be represented for a given pathogen. strain level variation, a recurrent problem in infectious disease diagnostics , can be accommodated using motifs constructed from peptides enriched from a random library. here, chagas-specific motifs mapped to distinct candidate antigens. several of these antigens have been reported , while others have not been described previously. approaches to antigen discovery from biospecimens often require laborious antigen cloning, expression, purification, and reactivity testing . while feasible for small organisms such as viruses, this approach becomes difficult or impractical for organisms with large proteomes including bacteria, protozoans, fungi, and plants. sera could potentially be used without major modification within clinical testing laboratories. the primary methodological steps of incubation, magnetic bead pull-down/immuno-precipitation, pcr, and ngs are already performed in infectious disease testing laboratories. and, many laboratories are equipped for bacterial culture. nevertheless, we anticipate that the assay might be offered initially from a centralized clia-certified, cap-accredited clinical testing laboratory. longer term, commercial testing labs, and state and federal labs could develop laboratory-developed tests (ldts) within their existing clia labs using peptide library reagent plates, and published motifs and methods. in the authors' facilities, the assay is routinely performed in -well plates, using semi-automated commercial liquid-handling instrumentation, with a two day turn-around time. finally, the bacterial display peptide library used here could be manufactured economically by conventional fed-batch fermentation, and quality control of the library reagent can be performed using ngs to measure the composition/ diversity. we typically sequence - m library members to ensure maintenance of library diversity and lack of bias towards particular sequences. thus, we have not identified any barriers to routine use of sera in a clinical testing lab setting. because a single sera assay can detect an arbitrary number of motifs and thus an effectively unlimited number of pathogens, high-multiplex sera may be useful for syndromic testing to identify unsuspected underlying causes of disease. similarly, sera may be a useful tool for epidemiology studies of associations between organisms, antigens, or epitopes and diseases or syndromes. at present, % of cancers globally are associated with bacterial, viral, and parasitic infections and this number will likely rise as new associations are discovered . similarly, autoimmune and neurodegenerative diseases have long been suspected to be triggered or driven by infections or other environmental factors , . identification and removal of environmental drivers of these diseases, as with gluten in celiac disease, may lead to effective interventions . however, the ability to identify associations has been hampered by the limitations of available serologic approaches, including the requirement to predefine a small set of organisms/strains to investigate, and the inability to differentiate epitope-level immune responses. sera provides a potential means to overcome these serology platform limitations and provide a broad, high-resolution view of humoral immunity. biospecimens. de-identified specimens along with geographical and associated clinical testing data in this study are described (table , supplementary table s ). specimens from the cdc-dpdm reference lab and commercial vendors include the following cohorts: discovery chagas (n = ), confirmed negative discovery controls (n = ), specificity controls (n = ) from apparently healthy donors (n = ) and non-chagas controls from a similar geographical distribution as the chagas disease cohort (n = ), blinded validation (n = ), and blinded validation (n = ). epitope repertoire capture and ngs. specimens were incubated with a fully random -mer peptide library having estimated diversity of , expressed on the surface of e. coli in a -well, deep-well plate format. each well contained cells for a -fold oversampling of the library. library preparation, binder selection using magnetic-activated cell sorting (macs), sample-specific amplicon preparation, and next-generation sequencing (ngs) methods were performed as described (see supplementary methods). sequencing data from each sample was processed to generate a non-redundant peptide list of antibody binding epitopes using publicly available software as described . fastq dna sequencing files were deposited into the ncbi sequence read archive (sra) for public access. sequence processing occurred in the following steps: ( ) fastq dna sequencing data were read, filtered, and trimmed. filtering removed sequences greater than or less than lengths of bases, sequences with four or more mis-matches or base pair insertions in the regions flanking the peptide region, or sequences with in-frame stop codons. trimming removed the flanking sequences so that only the library peptide encoding sequences remained. ( ) the dna sequences were translated into peptide sequences of amino acids. ( ) peptide sequences were collapsed into a single sequence if they had nine or more shared identities. processing thus yielded a unique peptide list (each with a count of one) for each specimen representing an antibody epitope repertoire. chagas disease diagnostic motif panel creation. following ngs and processing of sequencing data, chagas specific motifs were discovered using the imune computational algorithm. motifs were down-selected based on sensitivity, specificity and mean enrichment in disease. the resulting chagas motif panel v . was then tested against the blinded validation cohort and refined to generate the final diagnostic panel v . . the methods used to select chagas-specific motifs for inclusion in the panel are described in detail below. chagas disease motif discovery using imune. chagas-specific motifs were discovered by comparing the epitope repertoires from the discovery chagas cohort (n = ) with those from discovery controls (n = ) using imune as previously described . imune identified patterns, defined as three to five amino acids interspersed with undefined residues ("x") within a -amino acid window, that were significantly (poisson p < . ) enriched in % of chagas samples but not enriched in any discovery control samples ( % specificity). pattern scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ enrichment was quantified as the ratio of pattern observations in a sample to the expected observations taking into account the amino acid frequencies in the peptide library and total number of sequences for that sample as described . pattern enrichment significance was evaluated via two statistics: poisson and standard deviation. the poisson statistic selected patterns if they were statistically enriched (p < . ) in disease repertoires but not enriched in controls. the standard deviation method identified patterns enriched in disease repertoires relative to those of controls. for the standard deviation method, patterns could be present or enriched in controls but were enriched by > - standard deviations above the controls in disease data sets. significantly enriched patterns were aligned and scored using a pam substitution matrix and imune , to generate motifs that could include positions where multiple amino acids are observed, indicated by brackets (e.g., [ivl] or [krq] . pattern clustering with imune generated motifs (supplementary table s ). to identify the most sensitive and specific motifs for inclusion into a chagas diagnostic panel, motif enrichment values were standardized using, where z i is the z-score of motif i, x is the motif enrichment within a repertoire, μ = average enrichment of control cohort, and σ is the standard deviation for motif i in the control cohort. a z-score of ≥ was considered positive. sensitivity and specificity in chagas and controls were calculated and motifs with a sensitivity < % or a specificity of < % were removed. motif grouping by similarity. after down-selection, the remaining motifs were grouped by amino acid similarity. motifs were grouped if they shared at least of amino acid identities, resulting in motifs being assigned into groups. clustering of motif positivity within chagas epitope repertoires confirmed motif redundancy with each group (supplementary table s ). the motif within an epitope group with the greatest sensitivity and mean enrichment was included in chagas panel v . . in two cases, two motifs were selected from the same group since their combination improved sensitivity. the remaining motifs that did not fall into a group were further down-selected based on a specificity of > . % (< / controls positive) and/or an average enrichment in the discovery chagas disease cohort of > , resulting in retained motifs (chagas panel v . ). imune generated motifs, down-selection steps and final selected motifs for panel v . are given in supplementary table s . a "composite score" for all motifs in panel v . was calculated as the sum of z i for each motif. if there was more than one motif in a group, the maximum z-score value amongst the motifs was used to calculate the composite score. collated enrichment, z-score, and composite score data for chagas panel v . tested against all samples used in discovery as well as the blinded validation cohort are provided (supplementary table s ). a composite score value for the motif panel > was used as the diagnostic criteria for chagas disease. after unblinding, a single motif enriched in one leishmania seropositive specimen was removed, resulting in chagas panel v . . collated enrichment, z-score, and composite score data for chagas panel v . tested in the second set of randomized and blinded samples are provided (supplementary table s ). identification of candidate antigens. panel motifs were queried against the t. cruzi proteome using scanprosite (uniprot/trembl) to generate a list of candidate antigens (strain cl brener -taxon identifier ). selected candidate antigens representing previously identified and potentially novel chagas antigens are provided along with sensitivity and specificity values calculated from the combined blinded validation cohorts and of chagas disease (n = ) and non-chagas controls (n = ) respectively ( table ) . motifs with multiple alignments to a single antigen (repeats) are indicated. advances in serodiagnostic testing for lyme disease are at hand next-generation elisa diagnostic assay for chagas disease based on the combination of short peptidic epitopes the past, present, and (possible) future of serologic testing for lyme disease host range and emerging and reemerging pathogens antigens to detect the acute phase of toxoplasmosis in pregnant women: standardized comparison reactivation of neutralized hiv- by dendritic cells is dependent on the epitope bound by the antibody a novel neutralizing human monoclonal antibody broadly abrogates hepatitis c virus infection in vitro and in vivo integrated serologic surveillance of population immunity and disease transmission development of a multiantigen panel for improved detection of borrelia burgdorferi infection in early lyme disease identification and characterization of the constituent human serum antibodies elicited by vaccination viral immunology. comprehensive serological profiling of human populations using a synthetic human virome a multiplex serologic platform for diagnosis of tick-borne diseases epitope identification from fixed-complexity random-sequence peptide microarrays directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides identification of disease-specific motifs in the antibody specificity repertoire via next-generation sequencing prevalence of chagas disease in the latin american-born population of los angeles chagas disease serological test performance in united states blood donor specimens high-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays evaluation of serological tests to identify trypanosoma cruzi infection in humans and determine cross-reactivity with trypanosoma rangeli and leishmania spp diagnosis and management of chagas cardiomyopathy in the united states enhanced classification of chagas serologic results and epidemiologic characteristics of seropositive donors at three large blood centers in brazil trypanosoma cruzi genetic diversity: something new for something known about chagas disease manifestations, serodiagnosis and drug sensitivity serological diagnosis of chronic chagas disease: is it time for a change? over six thousand trypanosoma cruzi strains classified into discrete typing units (dtus): attempt at an inventory immunogenomic screening approach to identify new antigens for the serological diagnosis of chronic chagas' disease development of a luminex bead based assay for diagnosis of toxocariasis using recombinant antigens tc-ctl- and tc-tes- global burden of cancers attributable to infections in : a synthetic analysis infection as a cause of type diabetes? triggers and drivers of autoimmunity: lessons from coeliac disease a general approach for predicting protein epitopes targeted by antibody repertoires using whole proteomes this work was supported in part by nih grants gm , and ai . k.k. and j.r. screened samples, developed the assay and panels, analyzed the data and contributed to the manuscript. t.j., c.g. and r.p. performed data analysis. s.h., h.r. and i.m. provided serum samples and disease expertise. p.d. designed experiments, analyzed data, and contributed to the manuscript. all authors reviewed the manuscript. authors h.r., s.h., i.m., r.p. declare no potential competing interests. authors k.k., j.r., t.j., c.g. and p.d. declare competing financials interests, as salaried employees of serimmune inc. that receive equity compensation. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to p.s.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -gkynxdz authors: rizzato, cosmeri; torres, javier; obazee, ofure; camorlinga-ponce, margarita; trujillo, esperanza; stein, angelika; mendez-tenorio, alfonso; bravo, maria mercedes; canzian, federico; kato, ikuko title: variations in cag pathogenicity island genes of helicobacter pylori from latin american groups may influence neoplastic progression to gastric cancer date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: gkynxdz helicobacter pylori (hp) colonizes the human stomach and induces acute gastritis, peptic ulcer disease, atrophic gastritis, and gastric adenocarcinoma. increased virulence in hp isolates derives from harboring the cag (cytotoxin-associated genes) pathogenicity island (cagpai). we analyzed the microvariants in cagpai genes with the hypothesis that they may play an important role in determining hp virulence. we tested dnas from caga positive patients hp isolates; a total of patients with chronic gastritis (cg, n = ), intestinal metaplasia (im, n = ) or gastric cancer (gc, n = ) from mexico and colombia. we selected non-synonymous variants with at least . % frequency in the original sequence outputs or with a minimum of isolates with minor allele. after adjustment for multiple comparisons, no variants were statistically significantly associated with im or gc. however, non-synonymous showed conventional p-values < . comparing the frequency of the alleles between the isolates from subjects with gastritis and isolates from subjects with im or gc; of these showed a significant correlation with the severity of the disease. the present study revealed that several cagpai genes from latin american western hp strains contains a number of non-synonymous variants in relatively high frequencies which could influence on the clinical outcome. however, none of the associations remained statistically significant after adjustment for multiple comparison. performance of genome-wide sequencing. we prepared sequence libraries and performed whole-genome sequencing on hp strains, including reference strain . details of the sequencing outcome are included in supplementary table . we obtained a total number of reads per samples between , and , , . genomes were assembled, and we found that the percentage of reads that aligned to the reference sequence ranged between % and . %. we excluded from further analysis genomes for which no reads could be aligned, where cagpai was absent, strains isolated from the same patient and reference strain that was analyzed only as quality control (data not shown). the coverage of the whole genome for the samples selected for further analysis was on average . ×(range . ×- . ×), and coverage for the cagpai was . x on average (range . ×- . ×). the number of reads that aligned to the cagpai per sample ranged between and . caga gene was missing in of sequenced strains and cagγ in one strain. variability by gene. we summarized the genetic variability detected in the twenty-six cagpai genes in table . for each gene we computed the degree of variability as the number of sites showing a different genotype compared with the reference strain out of the total number of nucleotides in the gene, both as synonymous and non-synonymous variations (causing therefore differences at amino acid level). we assessed a range of variability from . % in cagf to . % in caga at dna level calculated as ratio of polymorphic position to gene length, while the amino acid variability ranged from . % in the cage gene, which is the minimum variation that we found (with the exception of the analyzed region of cagy in which we did not find non-synonymous variations), to . % in cagc calculated as ratio of non-synonymous polymorphisms to number of amino acid in the translated protein. the number of polymorphisms identified for each gene are summarized in table . comparison of frequencies of polymorphisms showing a differential distribution between gastritis and im or gc cases. when comparing the frequency of the selected alleles between the isolates from subjects with gastritis and isolates from subjects with im or gc we found statistically significant differences in allele distribution for three polymorphisms in caga gene (q/k r, n g and v t), three in cagc gene (v i, v i, i v), one in the cage gene (k e), one in cagl gene (s f), one in cagx gene (g n), one in cags (g d), one in cagζ (s a), three in cagδ (v i, p l, n e) and one in cagβ (n a) ( table ) . furthermore polymorphisms in caga (v i, g r, s f and q/r k) showed a significant trend with grade of the disease. when im and gc were analyzed separately and compared with the non-atrophic gastritis, snps showed a marginally (p < . ) statistically significant association when comparing gastritis vs. im and when comparing gastritis vs. gc (table ). in the caga gene of these variants were associated with risk of im (v i, s f and q/r k) and one with gc (n g), as shown in table . in the cagc gene we detected three snps with a differential distribution between gastritis and gc (v i, v i, i v, see table ). in cagl gene polymorphism (s f) showed a differential allelic distribution between isolates derived from im cases and gastritis cases (or = . ; % ci . - . , p = . ) (fig. , table ). for the cagx gene polymorphism g n showed a differential distribution between cases of im and gastritis (or = . ; % ci . - . , p = . ) (fig. , table ). for cagζ one polymorphism (s a) showed a differential allelic distribution between isolates derived from im cases and gastritis cases (or = . ; % ci . - . , p = . ) (fig. , table ). in cagδ two adjacent polymorphisms www.nature.com/scientificreports www.nature.com/scientificreports/ showed a differential distribution between cases of cancer cases and gastritis in particular the association for p l showed an or = . , % ci . - . and for n e or = . % ci . - . . additionally, the allelic distributions of these two polymorphisms were significantly different between mexican and colombian samples (data not shown), namely the variant allele frequencies were extremely low in colombia, therefore the associations were driven by the mexican cancer cases. in the cagβ gene variant n a showed an inverse association with cancer with an or of . ( % ci . - . , p = . ) (fig. , table ). next, a multiple comparison analysis was performed by applying a bonferroni-corrected threshold, and none of the above described snps showed a p-value lower than the threshold adjusted for this type of analysis of p = . × − ( . / ). none of the snps reached this study-wise p-value. supplementary table lists all the polymorphisms observed in analyzed genes. epiya and cm motif analysis. we also analyzed epiya (a, b or c) and cm (cm) motifs distribution in caga positive sequenced strains [ ] [ ] [ ] , while two strains were cagpai positive but lacking the caga gene. we found a high degree of variability consisting of different patterns, all of the western type (supplementary figure ) . most of the strains ( ) presented the a/b/cm/c/cm pattern, followed by the pattern a/b/cm/c/cm/c/cm (in strains), and the pattern a/b/cm/a/b/cm/c/cm/ (in strains). other less frequent patterns are described in supplementary figure . there was no difference in the distribution of the various patterns when compared between the three different disease groups. this study was conducted in latin american hp strains, in order to identify specific cagpai micro-variants associated with high-grade gastric lesions. this effort is of high clinical and translational importance as the presence of caga gene does not predict outcomes of hp infection, particularly in high-risk populations where the majority of the strains carry cagpai. the present study not only confirmed the extremely high variability in the cagpai genes, but also pointed to several variants with potential clinical relevance in a few genes for future studies. other study have investigated the whole cagpai however none have performed an extensive analysis of polymorphic variant of each gene in the region. table . overview of genetic variability in genes in hp cagpai. a number of polymorphic positions differ from number of variants because we found that several indel variants span more than one nucleotide. b non-synonymous variants with at least . % frequencies when compared to the reference sequence. overall sequence variability derived from this study for the selected cagpai genes was consistent with that reported previously using different sequencing techniques , , and extend the study to a higher number of genes. these results support the signature of diversifying selection through bacterial evolution in the proteins that are surface-exposed and involved in interactions with host molecules . however, the frequencies of amino acid variants in caga, cagc and cagγ found in this study were substantially higher than those previously reported , . this may be partially due to a greater number of strains under investigation in our work compared to previous publications. while we cannot completely rule out artifacts from this high throughput platform, such artifacts should affect both synonymous and non-synonymous variants. in a previous study conducted with amplicon sequencing by for mexican and venezuelan samples we reported non-synonymous snps with differential allelic distribution between gastritis and gastric cancer at conventional p-values between . - . . in the present project that included equal numbers of mexican and colombian strains we did not see any disease association with these variants. although variant frequencies were not markedly different between mexico and colombia strains, particularly for variants showing a significant association with im + gc, we have previously reported important phylogenetic differences between strains of these two countries . these phylogenetic differences may partly explain discordant results between the previous and current studies. previous publications reported an association of gc with the presence of variants in position and of cagl protein; in two studies , the concurrent presence of tyrosine (y) in amino acid position and glutamic acid (e) in position (y e ) compared with the combination aspartic acid (d ) and lysine (k ), induced more efficiently a shift of gastric integrin a b in the corpus, which has been related with gastric carcinogenesis. in our previous publication we did not observe the y amino acid in position in any sample, although we did find that carriers of d at this position are at lower risk of gc in comparison with the asparagine (n) carriers . in the current work we confirmed the absence of polymorphism in y position and the presence of n d polymorphism. we also observed the e k polymorphism but we did not find association with either im or gc in our populations. it should be noted that there was a major difference between our current and previous studies in the composition of geographical origins of the samples. our former study included colombian while the current study included venezuelan strains; this is relevant considering our recent report where we document adaption of hp genome to different latin american populations . furthermore, gorrel and co-worker performed an expanded analysis of this region analyzing the sequence from amino acid to and found significant differences according to the geographical origin; in this sense, we confirmed the predominance of the dkmge aminoacid sequence. some of the variants may warrant further studies as the sift program predicts them to be damaging non-tolerant. in particular the cagl s f variant (located at codon ) changes from serine (polar) to phenylalanine (non-polar). interestingly, among the asian strains recently sequenced, no single s f variant was found, suggesting that this is a western-strain specific variant . thus, overall, the differences observed in these studies are likely to be driven by geographical origins of hp. the other variant that is considered to be detrimental non-tolerant is located at residue of cagx, exchanging glycine (non-polar) to asparagine (polar). cagx, vir homologous protein, has been found recently to be necessary for the formation of hp pilus , mediating the stabilization of cagt which is a t ss structural protein , . www.nature.com/scientificreports www.nature.com/scientificreports/ thus, cagx mutants prevent caga biological activities . in this context, our finding of a protective effect of the n allele is compatible with a lesser virulent behavior. one cagc variant, located at codon , replacing valine with isoleucine, was associated with risk of gastric cancer and predicted to be intolerant by sift. valine to isoleucine substitutions have been reported to result in changes in protein structure, kinetics and stability in both bacteria [ ] [ ] [ ] and humans [ ] [ ] [ ] . however, the possible function of this polymorphism remains unclear. the four polymorphisms in genes cagζ, cagδ and cagβ showing a different distribution in im or cancer cases were predicted to be tolerated by sift. there were several variants at the n-terminal of caga that showed rather strong (or > . ) associations with high grade lesions (p = < . ) ( table ) as well as a significant trend by type of lesions (p < . ) ( table ) . some involved significant changes in amino acid characteristics (e.g., q r, n g), although none were predicted to be detrimental by sift. caga n-terminal region (residue - ) has recently received intensive research interest owing to its ability to interact with exogenous molecules, including host tumor suppressors, adhesion molecules, inflammatory mediators as well as chemopreventive agents such as curcumin , . further characterization of caga n-terminal region may shed light on potential function of the variants found in this study. strengths of this work include the relatively large number of hp samples completely sequenced. this work adds significantly to the number of hp complete genomes publicly available, and it substantially increases the available number of hp genomes from latin america, spanning also different stages in the natural history of hp infection and progression to gastric cancer. these new data have already been used for an in-depth phylogenetic analysis of latin american hp genomes . additionally, the sequences used in the final analysis are of high quality, with an average coverage of about ×, which is more than enough for a thorough assessment of sequence variation. on the other hand, we acknowledge some limitations in this study, which was designed as a first stage to screen candidate cagpai variants to be validated in a larger study, and sample size felt short to obtain reliable risk estimate for high-grade gastric lesions, particularly when im and cancer were considered separately. in the same vein, the sample was too small to assess combinations of several potentially interesting variants. also, our study did not include asian strains that present marked differences in the caga epiya region, and thus our results do not apply to asian strains. furthermore, our sequencing platform hiseq was not suitable to analyze long repeat regions such as those in cagy, which are rather common in hp bacterial genome. that is a major reason why we limited cagy analysis to its conserved region (yc). finally, our data were exclusively based on cultivable hp from gastric biopsies. little is known as to whether bacteria that are easy to grow in vitro are genetically different from those that are difficult to grow in vitro but able to survive in the human stomach for extended periods of time. despite the several limitations discussed above, the present study revealed that several cagpai genes from latin american western hp strains contain a number of non-synonymous variants in relatively high frequencies. some of these variants warrant further investigation to better understand their clinical significance in larger association studies, as well as experimental studies to elucidate their biological functions, and bioinformatic analysis to gain structural insights of the sequence variants. study population. strains analyzed in this study were isolated from patients recruited in the context of a multi-centric study based in latin america , . sequences used in the present work are largely overlapping with those reported in a phylogenetic analysis on latin american hp strains . patients attended the gastroenterology or oncology services and were subjected to endoscopy for diagnostic purposes. we isolated hp in of these patients and sequenced them, however were dropped due to poor quality or because they did not carry the cagpai. samples included in the following analysis were therefore hp clinical isolates from individuals recruited in colombia (n = ) and mexico (n = , nine of which have been already sequenced with the technology for genes ). thirty-seven of these subjects had non-atrophic or atrophic gastritis, intestinal metaplasia (im) and distal gastric cancer (gc). table shows pertinent characteristics of the population. for mexican samples, all the patients signed an informed consent and the study was approved by ethical committees of the instituto mexicano del seguro social (imss) and general hospital of the secretaria de salud (ss), mexico city, mexico . for colombia, the clinical studies where patients were originally recruited were approved by the ethical and research committee of the instituto nacional de cancerología, and all the patients signed an informed consent. this study was approved by the ethical and research committee of the instituto nacional de cancerología . all research was performed in accordance with relevant guidelines and regulations. sample preparation. in order to isolate hp, mexican stomach biopsies were homogenated and inoculated on % sheep blood agar base (becton dickinson, new jersey, usa) supplemented with vancomycin, trimethoprim, polymyxin b (campylobacter-selective antibiotics, oxoid, ltd. england). colombian biopsies were homogenated and cultured on blood agar plates, supplemented with campylobacter-selective supplement (oxoid), % vitox (oxoid), % horse serum (invitrogen). plates, in both centers, were incubated at °c under a % co atmosphere, and genomic dna was extracted from hp colonies using the dneasy mini kit (qiagen, hilden germany). whole-genome hp sequencing. nextera xt sample preparation kit (illumina) was used for preparation of the sequencing libraries according the manufacturer's instructions. ng of dsdna libraries was quantified with picogreen and used as input for illumina sequencing. high sensitivity dna kit (agilent technologies) was used to verify the fragment length distribution of the libraries using the agilent bioanalyzer. sequencing was performed on an illumina hiseq. sequencer with v pe chemistry at the genomics and proteomics core facility of dkfz. bioinformatic and statistical methods. raw sequences with phred score > were analyzed with fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to further assess quality. the resulting sequencing output, in fastq format, was assembled with the "map by reference" function of the geneious software platform (http://www.geneious.com/), considering the sequence of the strain (nc_ . ) as reference. a consensus sequence was determined for caga (hp ), cagc (hp ), cage (hp ), cagf (hp ), cagi (hp ), cagl (hp ), c-terminal sequence of cagy (hp ) ( nucleotides at the ' end of the genes encoding for the last carboxyl terminal amino acids of cagy, cagyc) , cagx (hp ), cagγ (hp ), and single nucleotide polymorphisms and small insertions and deletions were identified for each gene. for the analysis of caga gene an additional strategy was used to better analyze the epiya and cm motifs (c-met motif mediate caga multimerization and membrane targeting) , . illumina reads of the caga gene were extracted and realigned by the "de novo assemble" option of the same software. to exclude potential artifacts in sequencing and to enrich variants with clinical relevance, we selected a total of non-synonymous variants with at least . % frequency in the hp isolates we included in the analysis or with a minimum of isolates with the variant allele. a bonferroni-corrected threshold (p = . / = . × − ) was used to adjust for multiple comparisons. the variant alleles were determined using strain as reference. when more than one variant resulted in substitution of the same amino acid, we also analyzed the frequencies of the combined amino acid variant. we used non-atrophic gastritis as the control group to compare frequencies of variants in im and gc groups or a combined group with the two pathologies, and genotypes at a given locus were dichotomized, a selected variant vs. all other genotypes. p-values for differences in allelic frequencies between the control and im/gc combined or separately were determined by the fisher's exact test ( -sided) . we also computed odds ratios (or) % confidence interval (ci) for these gastric pathologies using logit estimators to obtain the ci even for the variants with zero frequencies in any category. for the variants that showed an unadjusted fisher p-value of < . , we further tested linear trends of their frequencies across the three histological groups, control, im and gc, using mantel-haenszel chi-square test. these analyses were performed by sas version . the effect of dna polymorphisms on the predicted proteins was evaluated with a bioinformatics tool: sift (sorting intolerant from tolerant) http://sift.jcvi.org . all sequences from mexican strains are deposited at ddbj/ena/genbank under the bioprojects prjna and prjna . genome sequences from colombia are deposited under the bioproject prjna . genbank accession numbers for out of analyzed strains are reported in supplementary table , submission of the remaining strains to genbank is ongoing. in the meantime, the data are available upon request to the corresponding author. global cancer statistics cancer incidence and mortality worldwide: sources, methods and major patterns in globocan current issues and future perspectives of gastric cancer screening screening for gastric cancer in asia: 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in mexican mestizo populations to develop genomic medicine in mexico phylogenomics of colombian helicobacter pylori isolates predicting the effects of coding non-synonymous variants on protein function using the sift algorithm we thank the unit for high throughput sequencing of dkfz genomics and proteomics core facility led by dr. stephan wolf for their valuable services in providing the sequencing data used in this project. this work was supported by the national institutes of health, united states [ r ca to ik]. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to c.r.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - peyzc authors: lee, shwu-maan; hickey, john m.; miura, kazutoyo; joshi, sangeeta b.; volkin, david b.; king, c. richter; plieskatt, jordan l. title: a c-terminal pfs / malaria transmission-blocking vaccine candidate produced in the baculovirus expression system date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: peyzc the plasmodium falciparum gametocyte surface protein, pfs / , is a potential target for malaria transmission-blocking vaccines. however, due to its size and complexity, expression of the full-length protein has been difficult, leading to focus on the c-terminal six cysteine domain ( c) with the use of fusion proteins to facilitate expression and folding. in this study, we utilized the baculovirus system to evaluate the expression of three pfs / proteins including the full-length protein, the c domain fragment and the c domain mutant to prevent glycosylation. expression of the recombinant pfs / proteins was conducted in super sf cells combined with the use of tunicamycin to prevent n-glycosylation. the proteins were then evaluated as immunogens in mice to demonstrate the induction of functionally active polyclonal antibody responses as measured in the standard membrane feeding assay (smfa). only the c protein was found to exhibit significant transmission-reducing activity. further characterization of the biologically active c protein demonstrated it was homogeneous in terms of size, charge, conformation, absence of glycosylation, and containing proper disulfide bond pairings. this study presents an alternative expression system, without the need of a fusion protein partner, for the pfs / c protein fragment including further evaluation as a potential transmission-blocking vaccine candidate. baculovirus produces full-length pfs / and c fragments. the design of baculovirus expression constructs was based on previous work on the pfs c recombinant protein . a signal peptide (mkflvnvalvfmvvyisyiyad) to facilitate secretion through er pathway, and a six histidine c-terminal tag were included to facilitate protein expression and purification of the full-length pfs / (aa - ), c fragment (aa - ) and c mutant (aa - with n q and n d mutations). in this report, these three recombinant proteins are designated pfs / -fl, c and c-mut, respectively, as derived from baculovirus expression. the insect cells used in the baculovirus system have active glycosylation machinery while the evidence to date is that p. falciparum parasites do not . to address the glycosylation propensity of the pfs / molecule during insect cell production, two approaches were undertaken. in an effort to minimize modifications (e.g., mutations) to the native gene sequence, the primary approach was to include tunicamycin, an antibiotic, in the expression culture as it has been demonstrated to effectively inhibit n-glycosylation in the insect/baculovirus expression system , . the secondary approach to eliminate n-glycosylation in the c fragment was to mutate the two high probability n-glycosylation sites at positions n and n in the c construct (termed c-mut). to test the initial feasibility of expression in the presence of tunicamycin, ml culture was infected with baculovirus at moi (multiplicity of infection) and tunicamycin was added to a final concentration of , . or µg/ ml. it should be noted, as shown in supplementary table s , that tunicamycin significantly inhibited cell growth, and this inhibition may have impacted protein expression overall. the recombinant proteins were not expressed in the supernatant but were rather located in cell pellets as indicated by sds-page and anti-his western (fig. ). all three proteins were expressed at the expected molecular weight (fig. a) . however, the pfs / -fl and c protein both presented as double bands (fig. b) in the presence of or . µg/ml tunicamycin, likely the result of glycosylation. at tunicamycin concentrations of µg/ml, the glycosylation appeared completely inhibited as the proteins presented as a single band (fig. b) . tunicamycin did not impact the banding pattern of c-mut (fig. b) , which presented as a single band under all conditions, as n-glycosylation was already likely inhibited through the mutation of the two high probability n-glycosylation sites. to produce sufficient recombinant protein for further characterization and in vivo evaluation, the insect culture was scaled up to l, and µg/ml tunicamycin was selected for addition during infection, based on the results of small-scale experiments. the proteins were successfully extracted from homogenized cell pellets in the presence of % sarkosyl and purified by imac (immobilized metal affinity column) and size-exclusion chromatography into a formulation buffer of mm hepes, mm nacl, . % tween , ph . . the initial process, as presented here yielded < mg of purified protein per liter of culture for c and c-mut. these yields were considered low, but sufficient to conduct initial evaluations. the pfs / -fl was produced in even smaller quantities (< . mg/l culture). due to the much higher yield of the c fragment, we now prefer this as a candidate antigen, however, production of the full-length was sufficient to be used as a comparator in subsequent in vivo studies. efforts to improve expression were not explored further in the study reported here, and yield optimization would be required for further development as a tbv antigen. this however remains plausible given our past experience in process optimization , and with new focus on maximizing yield from the cell pellet, knowing that a homogeneous properly disulfide-paired protein can result. the c and c-mut proteins were > % pure by sds-page and densitometry and pfs / -fl was > % pure (fig. a) . to determine whether the important epitope i was preserved, the proteins were also analyzed via western blot with the rf . monoclonal antibody (fig. b) . native pfs / , present in nf parasite extract as well as the recombinant pfs / -fl and c proteins were successfully recognized by this reduction sensitive monoclonal, indicating this functional epitope was preserved. however, the c-mut protein was not recognized (fig. b ) by rf . , likely indicating that the mutation of two n-glycosylation sites disrupted epitope i conformation. www.nature.com/scientificreports www.nature.com/scientificreports/ baculovirus expressed c fragment elicits functional antibodies. to test if the recombinant proteins represented the native conformation and therefore could elicit functional antibodies, a mouse immunogenicity study was conducted on all three baculovirus pfs / proteins formulated with the water-in-oil emulsion adjuvant montanide isa . antibody responses against each immunogen were determined by elisa, and all recombinant proteins were immunogenic with median elisa titer of > , units and negative controls showing undetectable level of response (< elisa units) against all three proteins. biological activity of induced antibodies was then evaluated by smfa using purified igg derived from serum pool from all mice in each immunization group. in the initial assay ( fig. a and supplementary table s , feed # ), all purified iggs (one pooled sample per group of mice) were tested at μg/ml. the iggs from c ( µg) and c ( µg) groups were associated with significant reductions in oocyst density (> % reduction, p < . compared to adjuvant only negative control for both), while the iggs from pfs / -fl ( µg) and c-mut ( µg) groups showed < % reduction (p > . ). to confirm the functional activities of anti- c iggs, the two samples were further tested at three concentrations in an independent assay ( fig. a and supplementary table s , feed # ). significant reduction in oocyst density was reproduced by iggs tested at μg/ml (> % reduction, p < . ). although percentage reductions at lower concentrations ( and µg/ml) did not reach significant level for both iggs, a trend toward dose-dependent oocyst density reductions was observed. the antisera raised against pfs / -fl and c-mut was then tested for reactivity via elisa (fig. b) to the c protein alone, the only immunogen which elicited biologically active antibodies. the anti-pfs / -fl and anti- c-mut antisera both reacted to the c protein. to further investigate the mechanism of negative smfa for anti-pfs / -fl and anti- c-mut antibodies, immunofluorescence assay (ifa) was performed with fixed and permeabilized mature gametocytes, gametes/zygotes (fig. c ). as expected from the smfa results, anti- c iggs reacted to sexual-stage parasites, and negative control igg did not. corresponding to the elisa results, anti-pfs / -fl and anti- c-mut antibodies also recognized sexual-stage parasites judged by ifa. taken together, it is suggested that anti-pfs / -fl and anti- c-mut antibodies recognized epitope(s), which are likely to be non-functional, but presented both in the recombinant protein and parasites. baculovirus expressed c fragment is homogeneous. subsequent a significant difference among the four groups (p= . by a kruskal-wallis test) was determined, and following dunn's multiple comparison tests revealed that there were significant differences between c-mut ( µg) and c ( µg) groups (p = . ), and between c ( µg) and c ( µg) groups (p = . ). (c) immunofluorescence assay with fixed and permeabilized p. falciparum sexual-stage parasites. purified total iggs, which were used for smfa, were incubated at µg/ml with fixed mature gametocytes, gametes and zygotes. the antibody reactivity to the parasites are shown in green (alexa fluor ), and dna in blue (dapi; ′, -diamidino- -phenylindole). the scale bar represents µm. ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports www.nature.com/scientificreports/ (ms) and lc-ms peptide mapping analyses were performed. the intact ms results (fig. ) indicated that the observed mass of the c fragment was + da (dalton) higher than its theoretical value of molecular mass. this mass difference was likely due to the inclusion of an n-terminal asp residue from the last amino acid of the signal peptide, as reported previously , . after correcting for the asp addition at the n-terminal, the predominant intact mass of , ± da (n = ) matched the prediction for the non-reduced form of the c fragment. upon reduction, an increase of six daltons was observed, to , ± da (n = ), matching the theoretical reduced form (fig. a) , and confirming the formation of three intra-molecular disulfide bonds. lc-ms peptide mapping ( supplementary fig. s ) of the reduced and alkylated c fragment, utilizing gluc and trypsin for proteolysis, confirmed % of the c sequences and no post-translational modifications were identified. these results combined demonstrated that tunicamycin effectively inhibited glycosylation of the non-mutated c molecule. further, the intact mass of c-mut (fig. b) shows the oxidized and reduced form both matched the theoretical mass , ± da and , ± da (n = ) with an asp residue present at the n-terminus. in addition, these data demonstrate that the mutation of two sites effectively eliminated glycosylation of c-mut. chromatography analysis of c and c-mut proteins. size exclusion (se)-hplc indicated that c was homogeneous and monomeric (fig. a) eluting as a single peak at ~ min. this retention time was consistent with a monomer based on the elution of gel filtration molecular weight (mw) standards. in contrast, c-mut contained www.nature.com/scientificreports www.nature.com/scientificreports/ at least four species between ~ - kda with only % monomeric protein (fig. b) . it should also be noted that substantial absorbance was observed within . - min in the c sample, which was likely due to the high concentration of tween in the final formulation buffer. in the reversed phase (rp)-hplc analysis, the c protein was confirmed to be homogeneous with a single peak eluting under non-reducing or reducing conditions, at ~ . or ~ . min, respectively (fig. c) . consistent with the se-hplc results, the c-mut exhibited multiple species via rp-hplc. under non-reducing conditions, multiple peaks were observed for c-mut (between . - . min) in the chromatogram. however, under reducing conditions, a single peak (~ . min) was observed. these results strongly suggest that the non-reduced form of the c-mut contains multiple species with different disulfide linkages (fig. d) . isoelectric focusing. the biologically active c was evaluated for presence of isoforms. a single band was observed in the ief gel for non-reduced c ( supplementary fig. s ), which migrated between the . - . pi markers, a result consistent with the theoretical pi of . . this result further confirmed that the c protein was present in a singular form. given the importance of proper disulfide bond formation to ensure the native configuration of plasmodium proteins and subsequent induction of functional antibodies, the disulfide linkages were investigated by two methods. first, to simply confirm the formation of disulfide bonds, the number of thiol groups exposed on the protein surface was measured. the analysis showed that c contains little or no free thiol (< %) under both native-like and denaturing conditions (with and without m guanidine-hcl), suggesting that all six cysteines were fully oxidized and likely paired by disulfides. second, to discern the exact pairing of cysteines in the c protein, disulfide mapping was performed. the non-reduced and reduced c were subjected to an optimized thermolysin digestion method and the resulting peptides were analyzed by lc/ms peptide mapping. table shows the disulfide linked peptides that supported the pairing of the three disulfide bonds, primarily between cys (c ) and cys (c ), between cys (c ) and cys (c ) and between cys (c ) and cys (c ) along with the predicted theoretical www.nature.com/scientificreports www.nature.com/scientificreports/ peptide fragments (table ) . a schematic representation of the amino acid sequence and elucidated disulfide mapping is provided in fig. . this mapped pairing of cysteines for the baculovirus c matched the predicted disulfide bonding pattern of the cysteine-rich motif . in these studies, we report for the first time the expression of pfs / proteins using the baculovirus system and their biological and biochemical evaluation. after expression, all three proteins were found in the cell pellet. this result suggests that in insect cells the inclusion of a signal peptide in the construct is not sufficient to direct secretion for the pfs / derived sequences. further, no post-translational modifications were observed, indicating that n-glycosylation was successfully inhibited by the incorporation of tunicamycin during baculovirus expression. while produced in low yields, the pfs / full-length protein was expressed and reacted to conformational monoclonal antibodies, though it did not induce functional antibodies as determined by smfa. production of the c protein resulted in an un-optimized yield of mg/l which allowed for detailed analysis of the biochemical properties. we attempted to produce pfs / proteins in the non-glycosylated state presumably present in the parasite. in both the c and c-mut proteins, n-glycosylation was successfully prevented by two independent approaches. the addition of µg/ml tunicamycin resulted in undetectable glycosylation for the c protein as evaluated by intact mass spectrometric analysis and lc-ms peptide mapping. further, the mutation of the two high probability glycosylation sites was also successful in preventing n-glycosylation for the c-mut protein, as indicated by no detectable post translational modification based on intact mass. however, the mutation strategy did not produce a properly folded protein and multiple species were present as observed by se and rp-hplc. c-mut was also not recognized by the conformational monoclonal antibody rf . , and it did not induce transmission reducing activity in mice. tunicamycin was previously used by tonkin et al. to produce a secreted, soluble, non-glycosylated malaria antigen, pf , in the high five cell/baculovirus expression system . in those studies, the concentration of tunicamycin was . µg/ml and served as the basis for the concentrations tested here. they also reported in those studies that protein expression was severely attenuated with the addition of tunicamycin, consistent to our observations (supplementary table s ).protein translocation through the er, including its mechanisms and attenuation, while not fully known -have been proposed and described . in the case of pfs / proteins produced here, even though the signal sequence was recognized by signal recognition particle and later cleaved, there was no guarantee of secretion. similarly, klaus et al. also faced an inefficient secretion of a recombinant vascular endothelial growth factor c (vegf-c) to baculovirus/insect culture medium and was unable to improve secretion by altering culture conditions or signal peptides . the lysate of the baculovirus infected insect cells, however, still constituted a valuable source of biologically active proteins . although the proteins reported here were successfully extracted from cell pellets using sarkosyl, a systematic and thorough evaluation of various extraction reagents and stabilization excipients were not explored, which merit further evaluation as part of future work. in recent studies, kundu . after endoh treatment, the n-acetylglucosamine attached to n forms h-bonds with d and d of the c molecule. on the other hand, n was found not to be glycosylated with the side chain of n involved in h bonding with s . these two n-glycosylation sites (n and n ) are not directly involved in the binding of c to rf . , but the results reported here, together with kundu et al, suggest that amino acid sequence at these sites can play important roles in the proper folding of c . while the glycosylation at these sites are not expected to occur in the parasite, our results also suggest that mutation of the primary sequence may also disturb higher order structure. in this work, the c protein was the only protein that elicited transmission-reducing antibodies in mice. the c was also found to be properly folded and homogeneous in terms of charge, size and conformation. in contrast, the c-mut was likely not in a native conformation, supported by a lack of rf . binding and the presence of multiple chromatographic forms. therefore, it is not surprising that no sera with transmission-reducing activity were identified for the c-mut. it is not clear why the full-length version did not induce transmission reducing activity; perhaps potent epitope in the c domain was masked or out competed by other regions of the protein. www.nature.com/scientificreports www.nature.com/scientificreports/ our results on expression of full-length pfs / is consistent with the many previous attempts to generate this recombinant protein. while some of the pfs / -fl protein was likely correctly folded in our analyses, additional strategies are needed to study this protein in greater detail before it may be considered as a viable vaccine candidate. given the presence of a pure, homogeneous protein for c fragment, coupled with the ability to elicit transmission reducing antibodies, we further confirmed the disulfide pairing of the recombinant protein. our initial effort in mapping disulfides using gluc and trypsin digestion of non-reduced and reduced, alkylated c was not successful due to proximity of cys and cys (data not shown). subsequently, improved methods were developed using thermolysin digestion which cleaves at the n-terminal of the hydrophobic amino acid residues . here, thermolysin cleaved multiple positions between cys and cys , including cys and cys at the amino acid residue (isoleucine). as shown in table and fig. , cys (c ) and cys (c ) are isolated to different peptides after thermolysis digestion, allowing us to accurately map the c and c connection and c and c connection unequivocally. these results coupled with the biochemical analysis (se-hplc, rp-hplc, ief) indicate the c protein is expressed in baculovirus as a homogeneous and properly folded molecule. in this report, we demonstrated that baculovirus can express a properly folded c fragment without a fusion partner and tunicamycin was successful in producing a homogeneous, non-glycosylated c. the reported assessment of baculovirus-produced c represents a first step towards developing a tbv vaccine that targets the pfs / protein. baculovirus expression constructs. three constructs of the pfs / protein were prepared for expression in baculovirus. " c," the c-terminal sequence of the gametocyte surface protein pfs / of d strain (accession q t ), containing six cysteines as part of a predicted cysteine-rich domain (aa - ); " c-mut, " which has two mutations of n-glycosylation sites, n q and n d, incorporated in the c construct (aa - ); and "pfs / -fl, " a full-length pfs / without the signal peptide and gpi anchor (aa - ). codon optimization for baculovirus expression was performed by dna . (now atum) with an additional n-terminal secretion signal (mkflvnvalvfmvvyisyiyad from honeybee melittin) and a c-terminal hexa-histidine tag. genes were cloned into pfastbac donor vector (invitrogen) with bamhi ( ′) and ecori ( ′) sites. the resulting plasmid pfastbac- / constructs were sequence verified. production of recombinant viruses followed the same procedure as described earlier . expression and purification of pfs / baculovirus proteins. super sf cells were seeded at × cells/ml in esf medium (expression systems, ca). moi of one was used to infect a l super sf wave culture and tunicamycin was added at a final concentration of µg/ml. at h post infection, culture was harvested, and the cell pellet was resuspended in lysis buffer ( mm tris-hcl, % sarkosyl, protease inhibitor tablets, ph . ) and processed with a high-pressure homogenizer (panda plus). the soluble fraction was recovered by centrifugation, diluted with mm tris-hcl, mm nacl, mm imidazole, ph . (buffer a) and clarified with . μm filtration. the clarified supernatant was loaded on a ml ni-nta column ( . × cm, his- , clontech). the wash steps were performed with ten column volume (cv) of buffer a with mm imidazole and . % tween- . the protein was eluted with a to % gradient of buffer b ( mm tris-hcl, mm nacl, mm imidazole, . % tween- , ph . ). pooled eluents from ni-nta column were loaded onto a superdex column (ge healthcare, . × cm, ml) and buffer exchanged into mm hepes, mm nacl containing . % tween- (ph . ). for the pfs / -fl protein, a superdex was used after his- column. the eluents were collected, analyzed with sds-page, and final pool selected based on purity. (qiagen) were performed as described earleir . www.nature.com/scientificreports www.nature.com/scientificreports/ western blotting (mab rf . ). following sds-page, proteins were transferred onto pvdf membrane and blocked in % skim milk in tris buffered saline containing . % tween- (tbst) at room temperature for one hour. primary antibody at a : , dilution of mab rf . ( mg/ml) in tbst was added and incubated for h at room temperature. membranes were washed with tbst ( x for min) and secondary antibody, : , dilution of goat anti-rat igg-alkaline phosphatase (bio-rad) in tbst was incubated at room temperature for h. membranes were washed with tbst ( x for min), developed using alkaline phosphatase substrate kit (bio-rad), followed by a water wash to stop the reaction and air-dried. protein concentration determination. recombinant pfs / proteins contain no tryptophan residue and uv at a was not ideal for protein determination. the bca assay kit (thermo) was therefore used according to manufacturer's instructions. mouse immunization. all animal experiments were performed in accordance with the animal study protocol (lmvr e) which was reviewed and approved by the niaid animal care and use committee. cd- mice (n = per group) were immunized intramuscularly with pfs / -fl ( μg per dose), c-mut ( μg) or c ( or μg) recombinant protein formulated with montanide isa (seppic inc., fairfield, nj) on days and . serum samples were collected on day . as a negative control, a group of mice (n = ) were immunized with isa alone on the same schedule. igg purification, smfa and elisa. the biological activity of anti-pfs / iggs was tested by smfa at μg/ml with complement in the first assay, and the two positive iggs (anti- c iggs) from the first assay were further evaluated at , and μg/ml with complement in a second assay. in each assay, the anti-adjuvant igg was tested at μg/ml as a negative control. the standardized methodology for performing the smfa has been described previously . in brief, - -day old gametocyte cultures of the p. falciparum nf line were mixed with test iggs at indicated concentrations, and fed to anopheles stephensi mosquitoes. all feeding experiments were performed with human complement and n = mosquitoes per group were examined days after feeding experiment for oocyst count. basic methodology of regular elisa has been described elsewhere . for pfs / -fl and c-mut groups, individual antisera were tested against both the corresponding immunogen and c protein. for c ( µg) and c ( µg) groups, elisa units were determined against the c protein alone. the antisera from isa control group were tested against all three proteins. statistics. percent inhibition in mean oocyst intensity (%tra) of a test sample was calculated against a control sample examined in the same feeding experiment. the best estimate and % confidence intervals ( %cis) of %tra, and p-value (whether the inhibition is significantly different from no inhibition) for each test condition was calculated using a negative binomial model with zero inflation model, as described previously . all statistical analysis was performed in r (version . . ) or prism (graphpad software), and p-values < . were considered significant. immunofluorescence assay (ifa). the ifa was performed as described previously with minor modifications. in this study, mature gametocytes were incubated with % human serum at °c for minutes to induce gametes and zygotes, and ifa slides prepared using the mixture of gametocytes, gametes and zygotes. the cells were fixed and permeabilized with a : mixture of methanol-acetone, and the slides were blocked by pbs with % skim milk. the primary antibodies (purified igg from each group, which were used for smfa) were incubated at µg/ml for hour at °c, then stained with secondary antibody and dapi ( ′, -diamidino- -phenylindole) as described previously. all images were captured on a lecia sp confocal microscope with las x software version . . . . images were deconvolved using huygens essential software version . . p b. intact mass spectrometry. the c or c-mut protein was incubated in either water (non-reduced) or mm dtt (reduced) for min at °c and desalted on a c column (waters corp.) prior to ms analysis. the intact mass of the protein was measured using a model b time-of-flight (tof) mass spectrometer (agilent technologies). ms spectra were acquired over a mass range of - m/z, with a scan rate of one spectra/sec. protein deconvolution was performed using masshunter (agilent technologies). lc-ms peptide mapping. recombinant c was bound to a strong anion exchange spin filter (pierce), washed with phosphate and eluted with m nacl. the sample was reduced with mm dtt for min at °c and alkylated with mm iodoacetamide at °c for min in the dark. the reduced and alkylated c was digested with : mixture of gluc and trypsin at °c overnight and subjected to lc/ms for peptide identification using an ultimate uhplc system (thermo scientific) with a . µm c column ( . × mm, thermo) at a flow rate of . ml/min. c column and autosampler temperatures were set at °c and °c, respectively. mobile phase (a) consisted of water with . % trifluoroacetic acid (tfa) and mobile phase (b) consisted of acetonitrile with . % tfa. ms range of - m/z was used. peptides were identified using ms and ms data and pepfinder (thermo scientific). se-hplc. se-hplc used the same method as described earlier with the exception of the mobile phase, which was mm hepes, mm nacl, ph . . proteins were incubated with either % water (non-reduced) or mm dtt (reduced) for min at °c, centrifuged for min at , × g, and then subjected to rp-uhplc using the same method as described earlier . triplicate experiments were performed. ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports www.nature.com/scientificreports/ isoelectric focusing. precast cleangel ief with pharmalyte - . (ge healthcare) was used with µg of non-reduced c sample and electrophoresis conducted at v, ma, w for min. the gel was fixed with % tfa for min, stained with coomassie r for min, and then detained with % methanol and % acetic acid. free thiol determination. free thiol (number of free cysteine residues) was measured using ellman's reagent (thermo scientific) following manufacturer's instructions. the c protein was concentrated to . mg/ml (~ µm) prior to thiol assay. a standard curve was constructed using known concentrations of reduced glutathione. absorbance was measured at nm. disulfide mapping. the c protein was buffer exchanged into mm tris buffer containing mm cacl , ph without denaturation or alkylation. samples were digested with thermolysin (promega) at a : enzyme to protein ratio overnight at °c. a portion of the digest was reduced using mm tecp [tris ( -carboethyl) phosphine] at °c for min. the digested peptides were then subjected to lc/ms using a c column ( . × mm, . µm, waters beh) set to °c. mobile phases were a (water + . % tfa) and b (acetonitrile + . % tfa) and the peptides were eluted using a - % b gradient over min at . ml/min flowrate. differences between the reduced and non-reduced samples were used to locate the disulfide peptides. peptides were identified using a waters synapt g or qtof premier mass spectrometer in positive-ion mode and a mass range of - da. the identities of the disulfide peptides observed in the thermolysin digest were confirmed with ms e data. peptides were considered confirmed if at least two a, b or y ions were observed for each individual peptide in the disulfide peptide pair. a control protein, r . c was used to develop and optimize the mapping method. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. antimalarial transmission-blocking interventions: past, present, and future progress toward a transmission-blocking vaccine against malaria. the lancet. infectious diseases transmission-blocking vaccines: old friends and new prospects development of a pfs -epa malaria transmission blocking vaccine as a chemically conjugated nanoparticle safety and immunogenicity of pfs -epa/alhydrogel(r), a transmission blocking vaccine against plasmodium falciparum: an open label study in malaria naive adults safety and immunogenicity of pfs h-epa/alhydrogel, a transmission-blocking vaccine against plasmodium falciparum: a randomised, double-blind, comparator-controlled, dose-escalation study in healthy malian adults. the lancet. infectious diseases antibody responses to two new lactococcus lactis-produced recombinant pfs / and pfs proteins increase with age in malaria patients living in the central region of ghana unravelling the immune signature of plasmodium falciparum transmission-reducing immunity an n-terminal pfs domain produced in baculovirus as a biological active transmission-blocking vaccine candidate. clinical and 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function of recombinant efflux drug transporters in polarized epithelial cell monolayers. drug metabolism and disposition: the biological fate of chemicals expression and characterization of ha protein of highly pathogenic h n avian influenza virus for use in a serodiagnostic assay structural and biochemical characterization of plasmodium falciparum (pf ) reveals a unique interdomain organization and the potential for an antiparallel arrangement with pf the concept of translocational regulation overcoming inefficient secretion of recombinant vegf-c in baculovirus expression vector system by simple purification of the protein from cell lysate effect of proline residue on the hydrolysis of substrates by thermolysin qualification of standard membrane-feeding assay with plasmodium falciparum malaria and potential improvements for future assays development and characterization of a standardized elisa including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines transmission-blocking activity is determined by transmission-reducing activity and number of control oocysts in plasmodium falciparum standard membrane-feeding assay publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank ashley birkett for his support and assistance in conceptualization of the work presented here and in the review of this manuscript. in addition, the authors thank several contributors to the analyses presented here. yimin wu for scientific review of the manuscript. bingbing deng and carole long for mouse immunization and ex vivo studies. luwen zhou, thao p. pham and ababacar diouf for smfa support. david mcadams for performing sds-page and western on the purified proteins. robert sauerwein for providing the rf . monoclonal antibody. ppd laboratories for performing disulfide mapping. lastly, the authors thank the project team at syngene international, a biocon company, in bangalore, india for the cloning, production, and preliminary analysis of the recombinant pfs / proteins described here. the work presented here was funded by a grant from the bill & melinda gates foundation. the views expressed herein are solely those of the authors and do not necessarily reflect the views of the foundation. the mouse immunization and smfa activities were supported in part by the intramural research program of niaid, n.i.h. s.m.l. conceived study design, oversaw protein projects/analysis, and manuscript preparation. j.m.h. conducted the biochemical analysis of the protein and assisted in manuscript review. k.m. conducted animal immunization study including elisas and smfa. s.b.j. and d.b.v. designed and provided oversight of the biochemical analysis and reviewed the manuscript. c.r.k. provided oversight for the overall study effort and in writing and reviewing the manuscript. j.l.p. led the overall study effort, data analysis, and manuscript preparation. all authors read and approved the final manuscript. the authors declare no competing interests. key: cord- - uok wb authors: lin, chun-yu; lu, cheng-hui; lee, hsiu-an; see, lai-chu; wu, meng-yu; han, yi; tseng, chi-nan; su, i-li; li, han-yan; tsai, feng-chun title: elderly versus non-elderly patients undergoing surgery for left-sided native valve infective endocarditis: a -year institutional experience date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: uok wb this retrospective study aimed to clarify the short- and mid-term outcomes of elderly patients who underwent surgery to treat left-sided native valve infective endocarditis (lsnie). between july and september , patients underwent surgical treatment for active lsnie at a single institution. patients were classified into two groups: ≥ years (elderly group) and < years (non-elderly group). clinical features, surgical information, postoperative complications, and three-year survival rates were compared. the average ages were . ± . and . ± . years in the elderly and non-elderly groups, respectively. the elderly group had a higher predicted mortality rate and a lower incidence of preoperative septic emboli-related complications. echocardiographic assessments of infected valves were generally homogenous between the groups. the elderly patients had a higher in-hospital mortality rate than the non-elderly patients ( . % vs. . %, p = . ). for patients who survived to discharge, the three-year cumulative survival rates were . % ± . % and . % ± . % in the elderly and non-elderly groups, respectively (p = . ). in conclusion, elderly patients are at a higher risk of in-hospital mortality after surgery for lsnie. however, once elderly patients are stabilized by surgical treatment and survive to discharge, the mid-term outcomes are promising. each patient fulfilled the modified duke criteria for ie , and surgical treatment was indicated based on published guidelines prior to , . general cardiac function, valvular destruction severity, and vegetation size/location were assessed by an experienced cardiovascular physician using transthoracic or transoesophageal echocardiography. active endocarditis was defined as definitive ie requiring intravenous antibiotic therapy until surgery . after excluding patients with sub-acute ie that resolved after a complete antibiotic treatment course and underwent delayed surgery due to residual valvular insufficiency found by follow-up echocardiography, and with prosthetic valve ie, patients were included. the patients were classified into two age groups: ≥ years (elderly group, n = , . %) and < years (non-elderly group, n = , . %) according to previous definitions of elderly , . preoperative septic emboli-related complications were diagnosed according to clinical symptoms and image studies, including computed tomography and magnetic resonance imaging, which were interpreted by experienced radiologists. if the vegetations were located in the aortic valve, preoperative coronary angiography was avoided to prevent new systemic embolic events. for patients with unstable haemodynamics or cardiopulmonary failure, depending on the inotropic agents and ventilator support, surgery was performed on an emergency basis. surgical management. intraoperative transoesophageal echocardiography was performed by specialized cardiovascular anaesthesiologists to confirm the preoperative diagnosis and extent of the valvular lesion. using a standard sternotomy approach, cardiopulmonary bypass (cpb) was established via cannulation of the ascending aorta and right atrium or the venae cavae. cardiac arrest was induced by a single dose of histidine-tryptophanketoglutarate solution or intermittent cold-blood cardioplegic solution through the coronary orifice. the diseased valves were carefully inspected, and the feasibility of valvular repair was considered after complete debridement of the infected tissues. the decision of valve repair or replacement was dependent on the surgeon's discretion regarding the degree of valvular destruction and patient characteristics, including heart function, comorbidities, and preoperative haemodynamics. for infected mitral valves that were repairable, the carpentier principle was applied , and ring annuloplasty was used to ensure the long-term durability of repair. in regards to infected aortic valves that were repairable, a pericardium patch was usually used for reconstructing the valvular defects following debridement. if valve replacement was necessary due to limited healthy tissue, the prosthetic choice was based on the patient's individual preference after a detailed discussion before surgery. before patients were weaned off cpb, haemostasis and good competency of the treated valve were confirmed, and a global assessment of cardiac function was performed. postoperative care and interventions. after undergoing surgery for endocarditis, all patients were transferred to a specialized cardiovascular intensive care unit (icu) for further treatment and observation. at hours post-surgery, a ventilator-weaning protocol was initiated if there was no active bleeding, unstable haemodynamics, persistent arrhythmia, or signs of organ malperfusion. renal replacement therapy was applied if acute renal failure developed after surgery, according to the acute kidney injury network criteria . further survey imaging, endovascular intervention, and surgical exploration for bleeding or malperfusion were performed, if indicated. statistical analyses. statistical analyses were performed using spss for windows (version . , ibm corp., armonk, ny). the shapiro-wilk test was used to test whether the continuous variables were normally distributed. data were presented as means ± standard deviation for normally distributed variables, whereas medians and interquartile ranges were used to describe non-normally distributed data. the independent t test was performed for comparison of normally distributed data and the mann-whitney u-test was used for non-normally distributed data between the two study groups. dichotomous variables are presented as numbers (n) and percentages (%). univariate analyses of categorical variables were performed using the chi-square test, or fisher's exact test to determine inter-group differences, where appropriate. preoperative and surgical variables found to be significant in the univariate analysis of in-hospital mortality were included in a multivariate logistic regression analysis to identify the independent predictors of in-hospital mortality. numbers of events (in-hospital mortality) per covariate fell below four or five in the present study; therefore, we used penalization through data augmentation to perform multivariate logistic regression to avoid sparse data bias , . the hosmer-lemeshow test was used to evaluate the goodness-of-fit for the multivariate logistic regression model . the kaplan-meier method was used to estimate -year cumulative survival, which was compared using the log-rank test. for all analyses, statistical significance was set at p < . . patient demographics. table shows the clinical demographics, comorbidities, preoperative conditions, and clinical presentation of the elderly and non-elderly groups. the average ages were . ± . and . ± . years in the elderly and non-elderly groups, respectively. overall, . % of patients were female, and diabetes mellitus was the most prevalent comorbidity, accounting for > % of cases in both age groups, followed by hypertension and end-stage renal disease (esrd). the average european system for cardiac operative risk evaluation (euroscore) ii estimated that mortality risk was higher in the elderly group ( . % [ . - . %] vs. . % [ . - . %]; p = . ) than in the non-elderly group. bacteraemia was the most prevalent clinical finding, followed by fever and septic emboli-related complications in both the elderly and non-elderly groups. the elderly group had a lower incidence of septic emboli-related complications ( . % vs. . %; p = . ). there were no differences in the antibiotic courses and length of hospital stays before surgery between the elderly and non-elderly groups. the epidemiology of pathogens was generally similar, and streptococci accounted for > % in both groups (fig. ). preoperative echocardiographic assessment and surgical information. table provides detailed information regarding preoperative echocardiographic assessment and surgical variables. in general, there were no differences in the location of the diseased valves, size/number of vegetations, and occurrence of periannular abscess between the groups. overall, . % of the patients underwent emergency surgery. regarding the valvular procedures, the rates of repair and replacement were similar in the elderly and non-elderly groups. however, more patients in the non-elderly group underwent mechanical valve replacement for both the aortic and mitral valve. the time span of cpb, aortic cross-clamp, and rate of histidine-tryptophan-ketoglutarate cardioplegic solution usage did not differ between the two groups. in total, . % of patients underwent extracorporeal membrane oxygenation installation in the operating room due to intraoperative myocardial failure. postoperative complications. as shown in table , a significantly higher in-hospital mortality rate was observed in the elderly group compared to the non-elderly group ( . % vs. . %; p = . ). moreover, the elderly group showed a higher incidence of complications, including new cerebral infarction, prolonged ventilator dependence, and prolonged icu course. the median hospital stays were . ( . - . ) and . ( . - . ) days in the elderly and non-elderly groups, respectively. an increasing number of elderly patients suffer from cardiovascular diseases, including ie, which can be life-threatening [ ] [ ] [ ] . however, elderly patients tend to receive a more conservative treatment course due to concerns regarding surgical complications , . in this single-centre study, a comparative cohort of patients who underwent surgical treatment for active lsnie is presented, which includes elderly patients aged > years. in-hospital mortality rates and postoperative complication rates were higher in this population than in non-elderly patients. however, the mid-term outcomes were still satisfactory for elderly patients who survived to hospital discharge. due to the increase in average life expectancy and the higher incidence of cardiovascular disease with advancing age, more elderly patients nowadays present for cardiac surgery . however, advanced age is considered a predictor of adverse outcomes following cardiac surgery , . an increased but acceptable surgical mortality rate has been previously observed among elderly patients with ie , , which was also observed in our study. therefore, surgery may be justified as a treatment option for elderly patients with lsnie and should be considered in selected patients. thorough consultation between the medical and surgical teams is essential to make this a reliable strategy. traditional surgery with cpb remains the standard treatment option for ie. however, the aging process is associated with structural and functional changes in various organ systems, possibly reducing the tolerance for haemodynamic fluctuation, and increasing coagulopathy and the systemic inflammatory response induced by cpb. therefore, higher in-hospital mortality and complication rates are expected in this population. in the present study, the elderly group demonstrated a higher euroscore ii-estimated risk, which also addresses the inferior short-term outcome. the aging process is associated with numerous ionic, molecular and biochemical www.nature.com/scientificreports www.nature.com/scientificreports/ changes in the heart , . these age-related changes can affect cardiac morphology and reduce the physiologic function. therefore, cardiac aging results in decreased mechanical and contractile efficiency, and may increase the rate of cardiomyocyte apoptosis after cardiac surgery. in our study, approximately % of elderly patients required mechanical support following surgery, and > % of the mortality in the elderly group was associated with postoperative myocardial failure. although the rates of postoperative mechanical support were similar in the two groups, the aging heart may exhibit inferior recovery compared with younger hearts. septic emboli-related complications occur in - % of patients with ie, especially when the diseased valve is on the left side , . the central nervous system is the most common destination of embolism, followed by the spleen, kidneys, lungs, and liver. in our study, the elderly group had a relatively lower incidence of preoperative septic emboli-related complications than the non-elderly group. we suggest several reasons for this finding. first, contrast-enhanced imaging may have been arranged more conservatively for patients in the elderly group due to the lower reserved renal function. second, the symptoms and presentations of emboli-related complications in the elderly may not be apparent and, therefore, could have been overlooked. when the emboli-related complications have progressed to a serious stage, the timing of surgical treatment may be delayed, and the outcome is compromised. finally, no patients in the elderly group experienced preoperative intracranial haemorrhage or cerebral mycotic aneurysm. these two cerebral complications could increase the surgical risk and induce permanent neurologic deficits. according to previous studies , , % of hospital mortality and % of postoperative cardiac and cerebrovascular complications were reported in this high-risk subgroup. as these complications are extremely serious and life-threatening, elderly patients with these conditions may be reluctant to undergo surgery. the elderly population is associated with a high incidence of comorbidities, including chronic kidney disease (ckd), and relative risk factors, including hypertension and diabetes, which predispose them to ckd . according to a previous multicentre study , patients with ckd had an increased perioperative mortality rate and reduced long-term survival after cardiac surgery. in our study, the elderly group showed worse renal function compared to the non-elderly group before surgery ( . ( . - . ) vs. . ( . - . ) ml/min/ . m ; p = . ). furthermore, esrd was one of the independent predictors of in-hospital mortality for the elderly www.nature.com/scientificreports www.nature.com/scientificreports/ these influences can be magnified. therefore, we suggest that elderly patients with ckd who present with clinical signs of ie should undergo early assessments and receive careful surgical management to optimize the outcomes. elderly patients who underwent surgery for ie were associated with higher in-hospital mortality and morbidity rates owing to their comorbid conditions and delayed timing of surgery. however, the late surgical outcomes are rarely reported. similar short-term results were presented in our study. furthermore, elderly patients who survived to discharge could have a comparable -year survival to that of the non-elderly group. therefore, we suggest that a guideline-directed surgical strategy according to the presence of complications, which include embolism events, large vegetation, heart failure, or uncontrolled infection, would be beneficial to improve the mid-term outcomes of elderly patients with lsnie. limitations of this study. our study had several important limitations. first, because the study used a retrospective and nonrandomized control design with a small sample size, bias might exist that influenced the homogeneity of the study groups and the stability of the multivariate logistic regression model. second, as this cohort spanned a period of > years, the technology of cpb and myocardial protection, as well as strategies for epidemiology, characteristics, and outcome of infective endocarditis in italy: the italian study on endocarditis contemporary epidemiology and prognosis of septic shock in infective endocarditis esc guidelines for the management of infective endocarditis: the task force for the management of infective endocarditis of the european society of cardiology (esc) early surgery versus conventional treatment for infective endocarditis early operation for endocarditis complicated by preoperative cerebral emboli is not associated with worsened outcomes ageing populations: the challenges ahead influence of referral bias on the apparent clinical spectrum of infective endocarditis changing epidemiology of infective endocarditis: a retrospective survey of cases, - . eur infective endocarditis: an analysis based on strict case definitions current features of infective endocarditis in elderly patients: results of the international collaboration on endocarditis prospective cohort study age-dependent profile of left-sided infective endocarditis: a -center experience proposed modifications to the duke criteria for the diagnosis of infective endocarditis infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the committee on rheumatic fever, endocarditis, and kawasaki disease, council on cardiovascular disease in the young, and the councils on clinical cardiology, stroke, and cardiovascular surgery and anesthesia aha/acc guideline for the management of patients with valvular heart disease: executive summary: a report of the american college of cardiology/american heart association task force on practice guidelines valve surgery in active endocarditis patients complicated by intracranial haemorrhage: the influence of the timing of surgery on neurological outcomes native valve infective endocarditis in elderly and younger patients: comparison of clinical features and outcomes with use of duke criteria and the duke endocarditis database carpentier's reonstructive valve surgery acute kidney injury network: report of an initiative to improve outcomes in acute kidney injury a simulation study of the number of events per variable in logistic regression analysis sparse data bias: a problem hiding in plain sight a goodness-of-fit test for the multiple logistic regression model the evolution of cardiovascular surgery in elderly patient: a review of current options and outcomes early and late predictors of mortality following on-pump coronary artery bypass graft surgery in the elderly as compared to a younger population outcomes in nonagenarians after heart valve replacement operation infective endocarditis in elderly patients: clinical characteristics and outcome physiology of cardiovascular aging the changing clinical aspects of infective endocarditis: descriptive review of episodes in a french teaching hospital and risk factors for death risk of embolism and death in infective endocarditis: prognostic value of echocardiography, a prospective multicenter study optimal timing of surgery for active infective endocarditis with cerebral complications: a japanese multicentre study chronic kidney disease in the elderly: evaluation and management outcomes of cardiac surgery in chronic kidney disease we thank tao-hsin tung, department of medical research and education, cheng-hsin general hospital, for statistical assistance. c.y. lin had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. study concept and design: the authors declare no competing interests. correspondence and requests for materials should be addressed to c.-y.l.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -xb r axf authors: heng, kevin; althaus, christian l. title: the approximately universal shapes of epidemic curves in the susceptible-exposed-infectious-recovered (seir) model date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xb r axf compartmental transmission models have become an invaluable tool to study the dynamics of infectious diseases. the susceptible-infectious-recovered (sir) model is known to have an exact semi-analytical solution. in the current study, the approach of harko et al. (appl. math. comput. : – , ) is generalised to obtain an approximate semi-analytical solution of the susceptible-exposed-infectious-recovered (seir) model. the seir model curves have nearly the same shapes as the sir ones, but with a stretch factor applied to them across time that is related to the ratio of the incubation to infectious periods. this finding implies an approximate characteristic timescale, scaled by this stretch factor, that is universal to all seir models, which only depends on the basic reproduction number and initial fraction of the population that is infectious. compartmental models provide a key tool in infectious disease epidemiology for studying the transmission dynamics of various pathogens [ ] [ ] [ ] . the susceptible-infectious-recovered (sir) model is known to have an exact semi-analytical solution [ ] [ ] [ ] . no such solution exists for the susceptible-exposed-infectious-recovered (seir) model, although some of its properties have been examined using an approximate analytical approach . in the current study, the approach of is generalised to demonstrate that, while no exact semi-analytical solution is possible, an approximate one does exist. it will be demonstrated that this approximate solution of the seir model implies the curves of all seir models are simply stretched or compressed relative to one another by the factor, where the incubation period is /σ , the infectious period is /γ and the generation time is /σ + /γ . the sir model is a special case with α = . this property implies the time taken for the infectious curve to peak is approximately universal for the seir model when scaled by α. in "the sir model" section, the sir model is concisely reviewed and extended. in "the seir model" section, approximate solutions of the seir model and their implications are elucidated. a concise summary is provided in "summary" section. in the sir model, the fraction of the population that is susceptible (s) becomes infected at a rate β = r γ , where r is the basic reproduction number. there is no incubation period. the fraction of the population that is infected is immediately infectious (i) for a period of /γ , after which a fraction of the population recovers (r). the sir model is described by the following set of coupled ordinary differential equations , , review of harko et al. . as a starting point, the derivation of is made more compact and cast in the mathematical notation of the current study. by taking the derivative of the first equation of ( ) with respect to time, one obtains equation ( ) of , where for convenience one uses shorthand notation for the derivatives with respect to time, one obtains from eq. ( ) an expression that is equivalent, but not identical, to equation ( ) of , because one has chosen to work directly with s (and not s/s ) as the independent variable. the preceding expression is recognised as a bernoulli differential equation, which may be solved to obtain an expression that is equivalent, but not identical, to equation ( ) of , where the initial value of s is denoted as s . the constant of integration is set by demanding that s + i + r = . recalling the definition of φ , an expression that is equivalent to equation ( ) of where t is the initial time. the preceding integral has no exact analytical (closed-form) solution and needs to be evaluated numerically, which is why it is strictly speaking an exact semi-analytical solution of the sir model. the first and third equation of ( ) may be combined to obtain where the initial fraction of the population that has recovered is chosen to be r = , which in turn implies that the initial fraction of the population that is infectious is extension of harko et al. . by setting i ′ = in eq. ( ), one realizes that the infectious curve i peaks at s = γ /β = /r . thus, eq. ( ) may be used to express the time taken for i to peak, where one assumes i ≪ . the quantity γ �t is the time interval expressed in terms of the infectious period and depends only on two parameters: r and i . variations in i shift the s, i and r curves back and forth in time without changing their shapes. we emphasize a subtle choice of notation: r is the initial fraction of the www.nature.com/scientificreports/ population that has recovered (and is always set to zero in the current study), whereas r is the basic reproduction number. when the infectious curve i first starts to rise from its initial value, the logarithm term in the integral of eq. ( ) may be approximated as ln (s/s ) ≈ s/s − , which allows the integral to be evaluated analytically. it follows that where we have defined the epidemic growth rate as from which one obtains the known relationship between the basic reproduction number and the growth rate , , where d ≡ /γ is the infectious period. seeking an approximate semi-analytical solution. the seir model builds on the sir model by considering an additional compartment for the fraction of the population that is exposed (e): infected but not yet infectious. the incubation period is /σ . the seir model is described by the following set of coupled ordinary differential equations , since this set of equations does not consider births or deaths, we have s + e + i + r = . the first and fourth equations may be combined to obtain which is identical to the sir model. again, the choice of r = is made with no loss of generality. by combining all four equations, one obtains the approximation is taken that the rate of change of the acceleration of r is vanishingly small, retaining the r ′′′ term in eq. ( ) would lead to a second-order, non-linear ordinary differential equation of φ(s) with no known analytical solution. ( ) www.nature.com/scientificreports/ solving for φ as in "review of harko et al. " section yields where φ is the initial value of φ . the preceding expression leads to an expression for i, in terms of s, with a yet unspecified constant of integration ( φ ), let the initial fraction of the population that is exposed be e . demanding that s + e + i + r = yields expressions for e and i, in terms of s, are obtained finally, s can be expressed in terms of t via the following integral, since i ≪ , the time taken for i to peak is the preceding expression is identical to eq. ( ) of the sir model, except for the extra factor of α . it should be noted that the upper limit of the integral ( /r ) assumes the approximation i ′ = e ′ = . however, eq. ( ) is not used to compute the peak times in fig. . its only purpose is to demonstrate that one may factor out αγ from the integral. stating the upper limit of the integral in eq. ( ) more accurately does not alter the main conclusion of the current study. the relationship between the growth rate and the basic reproduction number can again be derived. using the same series expansion of the logarithm term in the integral of eq. ( ), one obtains albeit with a different definition of the growth rate, it follows that where d ′ ≡ /σ is the incubation period. when α = , the expression for the sir model in eq. ( ) is recovered. if s ≈ and i ≪ , then one obtains r ≈ + �(d + d ′ ). the exact relationship between the growth rate and r has been derived in various ways (and references therein) and is given by r = ( + �d ′ )( + �d) . this equation accounts for the characteristic generation time distribution of seir models, which is a convolution of the exponentially distributed incubation and infectious periods with mean durations of d ′ and d, respectively. the approximate solution of eq. ( ) lacks the term d ′ d . hence, it corresponds to the case of an exponentially distributed generation time with mean duration d ′ + d , which is the same as the solution for the sir model assuming an infectious period of d ′ + d. ( ) has non-trivial implications. it suggests that the susceptible, exposed, infectious and recovered curves of seir models, with different values of d ′ and d, follow approximately universal shapes that are stretched by a factor of /α = + d ′ /d relative to one another. to demonstrate this property, the full set of coupled equations in ( ) is solved numerically using the solve_ivp routine of the python programming ( ) www.nature.com/scientificreports/ language suite . for illustration, one assumes r = and i = − . figure shows the solution curves of seir models, where the values of the incubation ( d ′ ≡ /σ ) and infectious ( d ≡ /γ ) periods are randomly drawn from an interval between and days. when time is scaled by the factor αγ , the susceptible, exposed, infectious and recovered curves lie approximately on top of one another. the second implication is that the time taken for the infectious curve to peak is approximately universal for all seir models when scaled by α and expressed in terms of the infectious period. in other words, αγ �t should only depend on r and i . to demonstrate this property, the full set of equations in ( ) is again solved numerically for , random draws of /σ and /γ and for r = to . for each seir model, the time taken for the infectious curve to peak ( t ) is calculated numerically. all , values of t are multiplied by αγ ; two sets of curves with different i values are shown in fig. for illustration. for all , seir models, the αγ �t values lie approximately on the same curve across r for a given value of i , demonstrating that αγ �t is a dimensionless (with no physical units), approximately universal timescale of the seir model. in the current study, approximate semi-analytical solutions of the seir model are found by generalising a previous approach for deriving an exact solution of the sir model. this finding implies that the entire family of susceptible, exposed, infectious and recovered curves of the seir model follow approximately universal shapes that are stretched or compressed, relative to one another, by a factor consisting of the incubation and infectious periods. the time taken for the infectious curve to peak is the characteristic timescale of the system and depends only on the basic reproduction number and the initial fraction of the population that is infectious when scaled by the infectious period and this stretch factor. for illustration, the basic reproduction number has been set to r = and the initial fraction of the population that is infectious has been set to i = − . each set of curves is generated using random realisations of the incubation and infectious periods, each drawn from an interval between and days for illustration. figure . time until the infectious curve (i) peaks as a function of the basic reproduction number r . in the seir model, the time to the epidemic peak ( t ) scales approximately with α and γ . for illustration, two values of the initial fraction of population that is infectious ( i ) are considered. each set of curves is generated using , random draws of the incubation and infectious periods from an interval between and days. an introduction to infectious disease modelling message passing approach for general epidemic models impact of the infectious period on epidemics a contribution to the mathematical theory of epidemics exact analytical solutions of the susceptible-infected-recovered (sir) epidemic model and of the sir model with equal death and birth rates mathematical models of sir disease spread with combined non-sexual and sexual transmission route analytical solution of seir model describing the free spread of the covid- pandemic how generation intervals shape the relationship between growth rates and reproductive numbers scipy . : fundamental algorithms for scientific computing in python k.h. formulated the problem, derived the equations, performed the numerical calculations and wrote the manuscript. c.l.a. made the link between the reproduction number and growth rate, checked the equations and edited the manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.h. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -acdfvjm authors: vila-vicent, susana; gozalbo-rovira, roberto; rubio-del-campo, antonio; santiso-bellón, cristina; navarro-lleó, noemí; muñoz, carlos; buesa, javier; rodríguez-díaz, jesús title: sero-epidemiological study of the rotavirus vp * protein from different p genotypes in valencia, spain date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: acdfvjm the aims of the present work were to determine the prevalence and titer of serum antibodies against several rotavirus vp * proteins from different p genotypes in children and adults in valencia, spain; and to determine the role of the secretor status (fut (g a) polymorphism) in the antibody response. the vp * protein from the p[ ], p[ ], p[ ], p[ ], p[ ], p[ ] and p[ ] genotypes were produced in e. coli. these proteins were tested with serum samples from children (n = , . years old in average) and from adults (n = , years old in average) by elisa. a subset of samples were genotyped for the fut (g a) polymorphism and the antibody titers compared. the same subset of samples was also analysed by elisa using whole rotavirus wa particles (g p[ ]) as antigen. ninety-three per cent of the samples were positive for at least one of the vp * antigens. differences in the igg seroprevalence were found between children and adults for the p[ ], p[ ] and p[ ] genotypes. similarly, significant differences were found between adults and children in their antibody titers against the p[ ], p[ ], and p[ ] vp * genotypes, having the children higher antibody titers than adults. interestingly, positive samples against rare genotypes such as p[ ] (only in children), p[ ] and p[ ] were found. while no statistical differences in the antibody titers between secretors and non-secretors were found for any of the tested p genotypes studied, a higher statistic significant prevalence for the p[ ] genotype was found in secretors compared to non-secretors. significant differences in the antibody titers between secretors and non-secretors were found when the whole viral particles from the wa rotavirus strain (g p[ ]) were used as the antigen. the spike protein vp is processed by proteolytic cleavage into two subunits, vp * and vp *. vp * is mainly involved in the virus -cell attachment process, while vp * is involved in the translocation of the virus into the cytoplasm of host cells. for more than years sialic acid was considered the glycoside receptor for animal rotaviruses . interestingly, human strains were considered sialic acid independent. the discovery that the vp * from human rotavirus strains also interacts with sugars, components of the histo-blood group antigens (hbgas), broke the paradigm of the understanding of rotavirus biology, showing that the first steps of infection of human rotaviruses are mediated by specific carbohydrate-virus interactions , . hbgas synthesis occurs by a sequential addition of monosaccharides to a disaccharide precursor. the precursor contains the sugars galactose and n-acetylglucosamine (glcnac) linked by a β- , or β- , linkage. depending on the linkage the sugars are classified as type i containing β- , linkage whereas type ii includes β- , linkage. to synthesize the h antigen is needed the addition of a fucose in the α- , position by the enzyme fut . lewis antigens are synthesised by the addition of a fucose residue in the position α- , or α- , to the terminal glcnac or h type i and ii precursors respectively to create the different lewis a, b, x and y antigens . the secretor status is defined by the fut gene, non-secretor individuals are those that lack functionality in both fut- alleles and subsequently do not express h-antigen structures (fig. ) . several studies suggested that non-secretor status reduces the susceptibility to rv infections mostly related to the p [ ] , p [ ] and p [ ] rotavirus genotypes [ ] [ ] [ ] . this is true in our area where the non-secretor infants are less prone to suffer symptomatic rotavirus infections . sero-epidemiological studies are of importance to elucidate the different viral agents and genotypes that are circulating in a particular area, for this reason the main aim of this work was to determine the serum antibody prevalence and titer to a panel of vp * proteins from different rotavirus genotypes (p [ ] , p [ ] , p [ ] , p [ ] , p [ ] , p [ ] and p [ ] ) in children and adults. the second aim was to determine if the secretor status (fut g a polymorphism) was related to differential antibody response as a measure of susceptibility to the different rotavirus genotypes utilised here. expression and purification of the rotavirus vp * protein from several genotypes. the vp * protein from p [ ] , p [ ] , p [ ] , p [ ] , p [ ] , p [ ] and p [ ] genotypes were produced in e. coli as described in the material and methods section. the purified proteins were analysed by sds-page in a % polyacrylamide gel (fig. ) and confirmed by western blot using an anti-gst antibody. the purity of each protein reached % and their sizes were as expected. prevalence of serum anti-rotavirus vp * antibodies. the results of the present study show that ninety-three per cent of the serum samples were positive for at least one of the vp * antigens ( / ). different prevalence was found for each of the genotypes both in adults and children ( table ). the higher prevalence was found against the p [ ] and p [ ] genotypes, while the lower prevalence was against the p [ ] genotype. considering the total prevalence, no differences were found among children % ( / ) and adults % ( / ) ( table ) . most interesting, the prevalence to p [ ] , p [ ] and p [ ] genotypes was higher in children than in adults (p < . ) (table ) . contrarily, not significant differences were found for any of the other tested genotypes. the number of different genotypes recognised by a single serum sample was also studied. the results show that children serum samples recognize a higher number of genotypes ( . ± . ) compared to adults ( . ± . ) (p < . ) (fig. ) . www.nature.com/scientificreports www.nature.com/scientificreports/ serum antibody titers to rotavirus vp * proteins. the serum samples from the present study had different antibody titers against each of the different vp * genotype studied (fig. a ). the higher antibody titers were detected against the p [ ] and p [ ] genotypes and the lower antibody titers towards the p[ ] genotype (p < . ). these differences were observed both among children and adults (fig. b ,c respectively). the comparison of the antibody titers between adults and children ( fig. ) , showed that the children have higher titers against p [ ] , p [ ] and p [ ] genotypes compared to adults (p = . , p = . and p < . respectively). the antibody titers to p [ ] , p [ ] and p [ ] genotypes were similar in both groups, and the titer against p [ ] genotype was higher in the adults group, but not significative. also, no differences were found between children and adults when the complete rotavirus wa viral particle was used as antigen (fig. h ). secretor status and antibody response to the rotavirus vp * proteins. genomic dna was extracted directly from serum samples and the fut genotype could be determined in of them. the remaining samples did not present enough dna or it was too damaged to perform the genotyping. the supplementary table s shows a compilation of the antibody response and secretor status of each of the analysed samples. of the samples were secretor positive ( %) and were secretor negative ( %); these percentages are similar to those previously published by us for the general population in the study area , . many comparations were stablished to study the differences in prevalence (table ), in antibody titers (fig. ) and in the number of different . statistical analyses (one way anova) were performed and show that in the three populations the antibodies against p [ ] , p [ ] , p [ ] and p [ ] are significantly lower than against p [ ] and p [ ] (p < . a ). the lower antibody titers were found to the p [ ] genotype compared to any of the other genotypes (p < . b ). comparison of the rotavirus vp * antibody titers in children and adults. from a to g panels, the igg titer against each vp * protein is shown. y axis represents the mean titer of igg reached in the serum samples from adults or children against the vp * protein. the error bars represent the standard error of the mean (sem). only for p [ ] , p [ ] and p [ ] the differences obtained between adults and children were statistically significant (p < . ). the h panel shows the results using the whole rotavirus particle from the wa strain, a p [ ] strain. ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ genotypes recognised between secretors and non-secretors (fig. ) . interestingly, p [ ] genotype showed a higher prevalence in secretors compared to non-secretors (p = . ). the other genotypes showed similar frequencies between these two populations ( table ). the comparison of the antibodies titers between secretors and non-secretors did not showed any difference between both groups (fig. ) . moreover, the genotypes recognised by each serum sample in secretor ( . ± . ) and non-secretor ( . ± . ) population did not show significant differences (p = . ) (fig. ) . interestingly, when the whole viral particle of the rotavirus wa strain, a p [ ] strain, was used as antigen in the elisa experiments, significant differences were found between secretors and non-secretors (p = . ) (fig. h ). rotaviruses are a major cause of acute gastroenteritis in children under years of age worldwide and rotavirus infections are responsible of approximately . deaths per year . the prevalence is similar in developed and developing countries, but the majority of deaths occur in developing countries, where rotavirus vaccines are not available or are less efficient than in developed countries . in the present study the reactivity of serum samples from adults and children was analysed against seven different rotavirus vp * proteins from different p genotypes, some from common rotavirus genotypes, p [ ] , p [ ] , p [ ] and p [ ] but others from uncommon such as p [ ] , p [ ] and p [ ] . to our knowledge, this is the first time that seven different gst::vp * protein have been used in a sero-epidemiological study, providing a high capacity of screening. our study reveals the presence of serum igg antibodies against all the genotypes assessed, indicating that the exposure to rotavirus is habitual in all age stages. the most common g genotypes distributed worldwide are represented by g , g , g , g , g and g in combination with mostly p [ ] and p [ ] (approximately %) . these genotypes are responsible for almost % of the rotavirus infections globally and also in our study area . in two recent publications addressing the molecular epidemiology of human rotavirus in valencia (spain), it was shown that . % of the samples were from the p [ ] genotype and only . % belonged to the p[ ] genotype , . none of the other genotypes assayed on the present study were found in clinical samples (p [ ] , p [ ] , p [ ] , p [ ] , p [ ] ). accordingly, the results of the present study show a higher prevalence and titers to the p [ ] and p [ ] genotypes. interestingly, the p [ ] genotype was only recognised by the serum samples from children, reinforcing the evidence that p [ ] genotype has a higher age restriction than other genotypes, probably due to the ability of this genotype to recognize non fucosylated receptors . interestingly, antibodies against all the tested genotypes were found, indicating that even if they are not found in clinical samples these viruses are also circulating in the population at a subclinical level. some of the less frequent genotypes including p [ ] , p [ ] and p [ ] are considered reassortant strains between animal and human rotaviruses or even transmitted directly from animals to humans . this fact might explain a lower viral fitness in the human host causing less severe or asymptomatic infections, in fact the rotateq vaccine is a bovine -human reassortant. histo-blood group antigens (hbgas) are found in the gastric mucine and in the surface of the epithelial cells in the gut. many investigations have shown that vp * from rotavirus are able to recognize different hbgas in a genotype depending manner . the p [ ] and p [ ] genotypes belong to genogroup iii and are able to recognize the a blood group antigen . p [ ] genotype (a genotype that infects pigs and humans), belongs to the genogroup ii and binds to the h antigen and to the mucin core glycan . in the same manner, p [ ] , p [ ] and p [ ] that also belong to the genogroup ii, are able to recognize h type related antigens and the type i precursor , . finally the p [ ] genotype interacts with the h type ii precursor molecule . several studies have demonstrated a different susceptibility to rotavirus infections depending in the ability to produce the h antigen. in a recent epidemiological study performed in the same geographic region it was shown that % of the clinical samples belonged to secretor positive children indicating that rotaviruses from the p [ ] and p [ ] genotypes infect more severally these children . the serology presented here shows that the antibody titers against all the vp * from the different p types do not depend on the secretor status. interestingly, when the whole rotavirus wa particle was used, significant higher titers against rotavirus was found in secretors, compared to non-secretors. these data confirm a previous report of higher anti-rotavirus titers in secretor individuals in sweden . it is important to mention that structural and non-structural proteins such as vp ,vp and nsp are immunodominant , while vp * is poorly recognised by serum antibodies . these differences in immunodominance might explain why the differences between secretors and non-secretors can only be observed when the whole viral particle containing vp and vp (plus vp and table . seroprevalence of anti-rotavirus vp * proteins in secretors and non-secretors in valencia, spain. the significance levels are indicated for each genogroup. p < . is considered significative (*). vp ) are used as the antigen to determine antibody titers. these results are highly relevant, in the context of vaccine development, since a vp * subunit vaccine is under development . common vaccines such as rotarix (g p [ ] ) and rotateq (g -g p [ ] ) are protective against a broad range of rotavirus g and p genotypes, even against those not included in the vaccines . this might be due to the cross immunity raised to the immunodominant vp and vp proteins that are more conserved in group a rotaviruses, independently of the g and p genotypes . this might explain, as well, the level of protection achieved with the rotavac vaccine which is composed of a g p [ ] genotype . in regard to the vp * subunit vaccine, it has been demonstrated that the vp * protein can elicit neutralising and protective antibodies [ ] [ ] [ ] . however it has also been demonstrated that the protection is homotypic , indicating that this vaccine might only protect against the rotavirus carrying the genotypes included in the formulation. on the other hand, one of the advantages of the vp * subunit vaccine is that it is administered by the parenteral route, shortcutting the differences in the vaccine uptake observed between secretors and non-secretors . figure . relationship between the secretor status and the antibody titer against each vp * genotype, panels a to g. the data analysed include secretor and non-secretor individuals. statistical analyses were performed (u mann whitney to unpaired samples) to determine the differences between the antibodies titers. the h panel shows the results using the whole rotavirus particle from the wa strain, a p [ ] strain. the error bars represent the standard error of the mean (sem). the samples did not show any significant differences between these two populations for the vp * proteins (p > . ). significant differences (p = . ) were found when the complete rotavirus particle was used (panel h). number of genotypes recognised by serum antibodies. in secretor individuals (n = ) the data show that these serum samples contain antibodies against . genotypes as a mean. besides, non-secretor population serum samples (n = ) were able to recognize . genotypes. these data were analysed statistically (nonparametric test from unpaired samples) to confirm that there are non-differences between secretor and nonsecretor (p > . ). error bars represent the standard mean error (sem). scientific reports | ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ our research group has recently elucidated the molecular bases of p [ ] ::h antigen interactions and we demonstrated that rotavirus can bind both the h antigen and its precursor (h-type i, lacto-n-biose) but this interaction is weaker when the fucose is lacking reinforcing the idea that the secretor status is a key host factor in rotavirus infections. furthermore, when the vp * binding pocket was compared between different genotypes and lineages it was found that the vp * from the rotarix vaccine possesses a mutation (l f) that disrupts the binding pocket providing an explanation for the attenuation of this strain . more surprisingly, we found significative differences in the prevalence of antibodies against the p [ ] genotype in secretors and non-secretors. this genotype is known to bind to the blood group a antigen . the presence of the a group antigen depends on the a enzyme coded in the abo locus. the a enzyme utilizes the h antigen as substrate, this means that secretor negative individuals are unable to synthesize the a blood group antigen in their mucosae, giving an explanation to the lower susceptibility of non-secretors to a virus that binds to the a antigen instead of the h antigen. as a conclusion we show here the usefulness of sero-epidemiological studies to understand the circulation of common (p [ ] and p [ ] ) and uncommon (p [ ] , p [ ] , p [ ] , p [ ] , p [ ] ) rotavirus genotypes in a given population and showed for the first time a relationship between the secretor status and the susceptibility to the rotaviruses of the p [ ] genotype. expression and purification of recombinant vp *. the vp * protein from p [ ] , p [ ] , p [ ] , p [ ] , p [ ] , p [ ] and p [ ] genotypes were produced as described previously . briefly, the different e.coli clones were cultured in lb medium with ampiciline and kanamycine (final concentration μg/ml and μg/ml respectively) at °c with vigorous shacking until the culture reached an o.d. = . , then the cultures were induced with iptg to a final concentration of . mm. the cultures were maintained at °c overnight. after induction, the cells were recovered by centrifugation at , × g for minutes. the cells from l of culture medium were lysed in ml of lysis buffer (pbs with . % of protease inhibitor cocktail (sigma), lysozyme mg/ml, u dnasei (thermo fischer scientific) and . mm dtt). the lysates were centrifuged at , × g for minutes and the supernatants were filtered through . μm syringe filters (millipore). the proteins were purified in an Äkta prime fplc system (ge, healthcare) carrying a gst-trap (ge, healthcare) column in pbs. the elution buffer consisted in mm tris-hcl, mm glutathione, ph . . gst::vp * proteins were desalted using a hitrap desalting column (ge, healthcare) in the same Äkta prime fplc system according to manufacturer's protocol. sample collection. serum samples were obtained from hospital clínico universitario de valencia. a total of serum samples were collected from adults and children from general population. samples were from . years old kids in average and from years old adults in average. the information about the samples is shown in supplementary table . ethic statement. this study was conducted with the approval of the ethics committee of the university of valencia (code h ). the ethic committee waived the need of informed consent since human blood samples from hospital clínico universitario de valencia were anonymised previously to their inclusion in the present study. the study was performed following the declaration of helsinki on ethical principles for medical research involving human subjects. determination of anti-vp * igg antibodies in serum samples. igg antibodies from serum samples were analysed by elisa against seven different vp * genotypes, p [ ] , p [ ] , p [ ] , p [ ] , p [ ] , p [ ] and p [ ] as previously described with modifications . elisa plates were coated by each gst::vp * proteins (p [ ] , p [ ] , p [ ] , p [ ] , p [ ] , p [ ] and p [ ] ). the purified proteins were diluted in carbonate/bicarbonate buffer ph . at μg/ml. one-hundred μl of solution was added to each well and incubated overnight at °c. in parallel, wells in the same plates were coated with the gst protein ( μg/ml) as reference protein to establish the cut-off for each sample. the plates were blocked by adding μl pbs containing . % tween (pbs-t) and % bsa for hour at °c. after blocking, the plates were washed three times with pbs-t. the serum samples were serially diluted two-fold from / to / , and were added to the plates in triplicate and incubated for hour at °c. after three washes a goat anti-human-igg antibody ( : , ) conjugated with hrp was added for hour at °c. one hundred μl of the o-phenylenediamine (opd-fast reagent. sigma) were added to each well and the reaction was stopped adding μl of m of h so and incubating for minutes. the absorbance was recorded at nm. the dilution was considered positive when the absorbance in the gst::vp * well was higher than the same dilution against gst plus three standard deviations. determination of anti-wa rotavirus igg antibodies in serum samples. igg antibodies from the serum samples that could be fut -genotyped were analysed by elisa against the whole rotavirus from the wa strain, a human g p [ ] genotype. rotavirus particles were purified as previously described and elisa plates were coated with μl per well of a solution containing . μg/ml of purified virus in carbonate/bicarbonate buffer ph . . the elisa was performed as described above but, in this case, the cut-off was calculated in wells coated with μl of bsa at μg/ml in carbonate/bicarbonate buffer ph . . the dilution was considered positive when the absorbance in the rotavirus wa well was higher than the same dilution against bsa plus three standard deviations. genotyping secretor status. genomic dna was extracted from serum samples with a commercial kit (jetflex genomic dna purification kit, genomed, vilnius, lithuania) following the manufacturer's instructions. the fut g a polymorphism was determined by pcr and rflp as previously described , . advisory committee on immunization practices (acip), c. for d. c. & (cdc), p. prevention of rotavirus gastroenteritis among infants and children. recommendations of the advisory committee on immunization practices (acip) the molecular virology of enteric viruses uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg unraveling the role of the secretor antigen in human rotavirus attachment to histo-blood group antigens cell attachment protein vp * of a human rotavirus specifically interacts with a-type histo-blood group antigen the interactions between host glycobiology, bacterial microbiota, and viruses in the gut the vp * domain of neonatal rotavirus strain g p[ ] binds to type ii precursor glycans rotavirus vp *: phylogeny, host range, and interaction with histo-blood group antigens both lewis and secretor status mediate susceptibility to rotavirus infections in a rotavirus genotype-dependent manner histo-blood group antigens in children with symptomatic rotavirus infection relevance of secretor status genotype and microbiota composition in susceptibility to rotavirus and norovirus infections in humans rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than years effectiveness of rotavirus vaccination: a systematic review of the first decade of global postlicensure data rotavirus: genetics, pathogenesis and vaccine advances web-enabled reporting and real-time analysis of genotyping and epidemiological data rotavirus symptomatic infection among unvaccinated and poly-lacnac as an age-specific ligand for rotavirus p[ ] in neonates and infants global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine fut secretor genotype and susceptibility to infections and chronic conditions in the alspac cohort structural basis of glycan specificity of p[ ] vp *: implications for rotavirus zoonosis and evolution glycan recognition in globally dominant human rotaviruses association of elevated rotavirus-specific antibody titers with hbga secretor status in swedish individuals: the fut gene as a putative susceptibility determinant for infection viral proteins vp , vp , and nsp are strongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection serum antibody responses to individual viral polypeptides in human rotavirus infections safety and immunogenicity of a parenteral p -vp -p[ ] subunit rotavirus vaccine in toddlers and infants in south africa: a randomised, double-blind, placebo-controlled trial global impact of rotavirus vaccination on diarrhea hospitalizations and deaths among children &lt; years old: - a randomized, open-labelled, non-inferiority phase clinical trial to evaluate the immunogenicity and safety of the live, attenuated, oral rotavirus vaccine, rotavac ® in comparison with a licensed rotavirus vaccine in healthy infants human vp * mabs neutralize rotavirus selectively in human intestinal epithelial cells antibodies to the trypsin cleavage peptide vp neutralize rotavirus by inhibiting binding of virions to target cells in culture homotypic protection against rotavirus-induced diarrhea in infant mice breast-fed by dams immunized with the recombinant vp * subunit of the vp capsid protein humoral immune response to rotavirus nsp enterotoxin in spanish children secretor genotyping for a t, g a, c t, c t, deltgg, g a, and other mutations from a single pcr influence of the combined abo, fut , and fut polymorphism on susceptibility to norwalk virus attachment this work was supported by spanish government (ministerio de economía y competitividad) grants agl - -c - -r and ryc- - to jrd. this work was also supported by valencian government grant idifeder/ / . rgr is the recipient of a postdoctoral grant from the valencian government apost/ / . csb is the recipient of a predoctoral grant fpi from the spanish government re - . svv is the recipient of a predoctoral grant from the valencian government acif/ / the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -x.correspondence and requests for materials should be addressed to j.r.-d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -c nlie authors: coleman, kristen k.; sigler, william v. title: airborne influenza a virus exposure in an elementary school date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: c nlie influenza contributes significantly to childhood morbidity and mortality. given the magnitude of the school-aged child population, a sizeable proportion of influenza virus transmission events are expected to occur within school settings. however, influenza virus activity in schools is not well-understood, likely due to our limited ability to accurately monitor for respiratory viruses without disrupting the school environment. in this study, we evaluated the use of a bioaerosol sampling method to noninvasively detect and quantify airborne influenza a virus (iav) densities in a public elementary school. air samples were collected from multiple locations in the school, two days per week, throughout an eight-week sampling period during influenza season. real-time rt-pcr targeting the iav m gene revealed detectable iav on five occasions in densities ranging from . × (− ) to . × ( ). no significant differences in iav densities were related to student presence/absence. the majority of iav-associated particles were ≤ μm in diameter, and theoretical calculations indicate infectious thresholds after minutes of exposure. our study represents the first identification and quantification of airborne influenza virus in an elementary school, and the results suggest that airborne iav has the potential to circulate in schools during influenza season, in large enough doses known to cause infection. a total of air samples were collected over an eight-week period (february-march), followed by qrt-pcr targeting the influenza a virus (iav) m gene, which revealed detectable iav in % ( / ) of the air samples collected indoors, in densities of . × − , . × , . × , . × and . × m gene copies m − air (table ) . iav was not detected in any of the outdoor reference samples. an r value of . for each standard curve was achieved for the qrt-pcr assays and the detection limit for iav m gene was rna copy per reaction volume ( μl) when the cut-off for positive result was set at cycles. because each influenza virus particle packages one copy of the m gene , our results can also directly reflect the number of virus particles m − air. however, our results are reported in copy numbers m − air to remain consistent with other quantitative studies measuring airborne iav densities. significantly different (p = . ) airborne iav densities were detected between all three indoor locations (i.e., gymnasium, classroom, and corridor) and all positive samples were collected during the last two weeks of , and a - % relative humidity level; descriptive of an average elementary school student in the usa weighing ~ - kg with an assumed tidal volume (v t ) of ml per kg of body mass. † irrespective of iav target gene; based on the assumption that one tcid is equivalent to ~ pcr-detectable iav rna copies [ ] [ ] [ ] [ ] . the study (month of march, table ). sample location was strongly correlated with iav density (r = . ), with the highest density of iav detected in the corridor. two ( %) of the air samples collected from the gymnasium were positive for iav, as well as two ( %) from the corridor, and one ( %) from the classroom. no significant difference in iav densities was observed between samples collected during and after school (p = . ). indoor relative humidity (rh) and temperatures remained relatively consistent ( - % rh and - °c) throughout the study. no significant difference in iav densities was observed between samples collected at different rh levels (p = . ) or temperatures (p = . ). significantly different (p = . ) airborne iav densities were detected among particle size fractions. particle size was strongly correlated with iav density (r = . ), with % of virus-laden particles detected in respirable size fractions (≤ μm in diameter). the largest virus-laden particles (> μm, % of total particles) were detected only in the gymnasium, while the smallest (< μm; % of total particles) were detected only in the classroom. no significant difference in virus-laden particle size was observed among samples collected at different rh levels (p = . ) or temperatures (p = . ), nor during and after school hours (p = . ). theoretical exposure threshold calculations accounting for body mass, tidal volume, and breathing rate predict that exposure to an equivalent of . × - . × iav rna copies m − air for one minute is sufficient to induce infection in a student ( table ). based on the airborne iav densities detected in the school, we estimated that students in the classroom (day ), gymnasium (day ), and corridor (day and ) were at risk of infection following , and minute(s) of breathing, respectively (table ). aerosolization is an important mechanism for spread of iav . since children are significantly burdened by influenza and play a key role in transmission , , , , we established a protocol through which iav densities could be monitored in a school setting. viral densities were compared to theoretical iav exposure thresholds, above which students are expected to become infected. student illness and absenteeism caused by respiratory diseases is thought to occur, in part, because of prolonged time spent in school buildings, % of which experience impaired indoor air quality . therefore, understanding how the student environment influences disease transmission can not only help to better address student health, but also improve sanitation/cleaning practices and inform predictive efforts. to our knowledge, our work represents the first identification and quantification of airborne iav in an elementary school, showing that schools can not only harbour airborne iav densities similar to those found in clinical settings , , but also in infectious doses. furthermore, the detection and quantification of airborne iav in the school airshed warrants future investigations to determine the relationship between iav densities and student illness. three locations in an elementary school were sampled for airborne iav, including the main corridor, gymnasium, and a classroom. because each location featured different iav densities and particle sizes, the exposures of students to iav differed according to sample location. for example, students were likely at an elevated risk of iav infection by breathing in the classroom on day , especially since the particles were < μm in diameter. infection could have also arisen from breathing for minutes in the gymnasium on day , but is less likely, as particles were > μm in diameter. while no specific activity can explain elevated iav densities in the classroom, two defined activities in the gymnasium throughout the school day are thought to promote elevated iav densities. first, the gymnasium is used by approximately students during each class period for physical education instruction. physical activity/exercise can result in aerosolized iav through increased respiratory rates, and increased respiratory distress due to bronchoconstriction , which has been demonstrated to affect up to % of children . second, the gymnasium hosts the student lunch period, which lasts for minutes, during which two cohorts (approximately students each) eat for minutes. the entire student body is not only represented in the gymnasium during the lunch period, but is also moving, en masse, as each cohort enters and exits the space, theoretically creating turbulence to maintain suspension of iav (if present) throughout the period, and likely into the post-lunch hours. airborne iav densities were highest in the school corridor, which was expected, and can be partially explained by two factors. first, student lockers are situated along the walls of the corridor which encourages air turbulence as students pass through the corridor and open and shut locker doors. second, the corridor represents the only available passage between classrooms and therefore each student passes through the corridor multiple times per day, which dually increases the probability that an infectious student will shed virus in the area, and that other students will be exposed. in a college student office setting, zhang and li ( ) demonstrated that the frequency of close contact (within m) is . contacts per hour per student, which contributed to % of reported iav infections. additionally, previous studies have suggested that influenza illness and death rates could be decreased by as much as % by reducing the contact rates of infected persons . given the high airborne iav densities detected in the school corridor, along with elevated student contact rates, it is plausible to conclude that the school corridor is a "hotspot" for influenza virus transmission. studies focusing on clinical environments have demonstrated that a considerable proportion ( - %) of total airborne iav-laden particles are respirable , . in the current study, the majority ( %) of airborne particles associated with iav were respirable (≤ μm in diameter), representing the most infectious fraction based on size. particles > μm in diameter are deposited predominantly in the nasal cavity or trachea, and are subject to mucociliary clearance before initiating infection . in contrast, particles ≤ μm in diameter can be deposited deep into the lungs, and are more likely to result in lower respiratory tract infections, which disproportionately impact children during influenza pandemics . therefore, identifying the particle size distribution of airborne iav is critical for understanding the potential transmission and infectious impact of the virus. although we successfully detected airborne iav in the school with appropriate sensitivity to accurately quantify iav densities, environmental factors created sample processing challenges and difficulty interpreting the data. airborne pathogen densities in nonclinical environments can be several orders of magnitude lower than ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ those detected in clinical environments , , . furthermore, desiccation stress is known to limit the stability of airborne iav . therefore, in the school, lower iav densities and high variability were anticipated, and sampling parameters were chosen to maximize detection, including short sampling durations to preserve the integrity of captured iav rna. we chose a flow rate of . l/min for four hours at a time, which was previously demonstrated to efficiently capture and preserve airborne iav rna for rt-pcr detection , . next, iav packages a single copy of our rna target, the m gene , and therefore a : relationship between the number of gene copies detected and number of virions is a valid assumption. however, brown et al. ( ) indicated that iav densities can be overestimated when quantifying gene copies , and studies have assessed the relationship between rna copy number and the number of viable viruses, suggesting that one tcid is equivalent to ~ pcr-detectable iav rna copies [ ] [ ] [ ] [ ] . while iav viability was not directly assessed in the current study, our quantitative results do provide an estimate of the potential for iav transmission in the school environment, consistent with the prevailing public health proposals for conservative estimates of disease transmission risk . lower densities of airborne iav were expected in the absence of students from the building. however, no significant difference in airborne iav density, as a function of student presence or absence, was observed. this result was unexpected, as children are thought to be key vehicles of iav transmission and are viewed here as the major factor contributing iav to the school airshed. we did not collect individual student health data during the study, and therefore could not definitively link the prevalence of influenza among the student population with virus detections. however, the detection of iav during the absence of all students from the building was nonetheless an important observation, indicating persistence of the virus in the airshed. airborne iav can remain viable for up to hours , and is likely facilitated by two factors. first, it has been demonstrated that temperature and rh influence the viability and transmission of influenza viruses [ ] [ ] [ ] [ ] [ ] . however, research has recently demonstrated that rh does not influence influenza virus viability , but rather the rate of aerosol deposition, which influences the concentration of virus particles in the air. in the current study, the environmental conditions during the school day ( - % rh and - °c) were optimal for iav persistence. second, children shed iav for a longer duration than adults shed the virus , encouraging prolonged iav aerosolization in a school setting where children predominate. overall, our findings demonstrate that on the short term, iav is not fully cleared from the school environment upon removal of students, and support the assertion that schools should be considered an iav transmission hotspot, even in the absence of students. although airborne influenza virus has been detected in select indoor settings , , , [ ] [ ] [ ] [ ] [ ] , the ability to consistently detect the virus in the airshed remains limited in environments featuring low iav densities. we now have the first molecular evidence of airborne iav in an elementary school, during a portion of the influenza season when students were exposed for appropriate durations to densities of influenza-laden particles that could facilitate infection. furthermore, given the magnitude of the school-aged population, our data provide justification for considering schools as influenza hotspots, warranting further study to determine the relationship between airborne iav densities and student health to improve influenza management in the greater community. bioaerosol sampling. air samplings were performed four times per week during an eight-week sampling period (february-march) in an elementary school (toledo, oh area) that enrolls approximately students, in grade levels k- . airborne iav was sampled in the school gymnasium, a classroom, main corridor, and an outdoor reference, using two-stage bioaerosol cyclone samplers provided by the national institute for occupational safety and health (niosh) and chosen for their portability, durability, minimal preparation time, and efficiency that equals that of commercial samplers . each sampler was placed . m from the ground, simulating the average elementary school student's breathing level, and connected to an skc airchek xr pump (skc, eighty four, pennsylvania) with . -mm tygon tubing, operating at a flow rate of . l of air min − , collecting a total of l of air for each sample. the pump flow rate and sampling duration was based on previous studies that demonstrated efficient capture of airborne influenza virus rna for rt-pcr detection , , . each sampler collects particles > μm in diameter into a ml centrifuge tube, particles - μm in diameter into a . ml centrifuge tube, while particles < μm in diameter are collected onto a -mm diameter, polytetrafluoroethylene filter with -μm pores. the influence of student presence/absence on airborne influenza virus detection was determined by collecting samples early in the school day ( : am- : pm, students present), and in the afternoon/evening ( : - : pm, students absent). after sampling, collection tubes and filter cassettes were transported to the laboratory on ice and stored at − °c, if not immediately processed. samplers were washed with isopropanol and air dried between sample collections. temperature and rh were continuously recorded inside the school building near each sampler using hobo dataloggers (onset; bourne, ma, usa). sample rna was extracted and purified using the magmax viral rna isolation kit (ambion) following the manufacturer's instructions with slight modifications, including the addition of lysis/binding solution directly to (i) sampler tubes, and (ii) -ml falcon tubes containing the ptfe filters. xeno rna control (ambion), a synthetic rna transcript, was added to the sample lysis solution to act as an internal, positive control target for assessing the efficiency of rna recovery. purified rna was eluted in μl of elution buffer. all analysis materials were purchased rnase-and pyrogen-free, if possible, and otherwise depyrogenated by autoclaving at °c for minutes. one-step, real-time, rt-pcr targeting the influenza a virus m gene was performed in an applied biosystems step-one real-time pcr system with commercial taqman aiv-matrix reagents (ambion) in a total reaction volume of μl. reverse transcription was performed at °c for min, °c for min, and °c for min, followed by cycles of qpcr analysis at °c for s, and annealing/elongation at °c for min. three negative control reactions (no template) were included in each qrt-pcr assay. to quantify the viral load present in scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ each sample, the ct value from each reaction was compared to those of a standard curve derived from a dilution series of known quantities of iav m gene copies. iav exposure threshold calculations. based on observed densities of airborne iav, we estimated the breathing time, above which a student could theoretically become infected. to calculate the time, we used a known range of . - . tcid (tcid is the number of iav particles that induce infection in % of inoculated tissue culture cells) , which has previously been used to estimate the risk of airborne iav infection after exposures consistent with a one-hour clinical visit, an eight-hour workday, and after hours indoors . since approximately rna copies is equivalent to one tcid - , the resulting threshold iav density capable of initiating an infection is theoretically equivalent to - rna copies. assuming the average elementary school student weighs - kg, has a respiratory rate of breaths min − , and a tidal volume (air volume displaced in a single breath) of ml per kg of body mass, we calculated an inhalation volume range of - ml air min − for a typical student, which was then compared with our estimated iav densities to determine the number of minutes of breathing necessary to cause an infection. statistical analysis. data were imported into stata version . (statacorp, college station, tx, usa) and a two-sample t-test or one-way analysis of variance was performed to test for significant differences in iav densities and particle sizes between indoor sample locations (gym, classroom, corridor) and environmental conditions (temperature, rh, and student presence). regression analyses were then performed to test for correlations between variables demonstrated to be statistically significant. datasets generated during this study are available from the corresponding author upon reasonable request. outbreak of pandemic influenza a (h n ) at a new york city school epidemiological and clinical characteristics of the outbreak of pandemic influenza a (h n ) at a middle school in luoyang effectiveness of non-pharmaceutical interventions in controlling an influenza a outbreak in a school pandemic (h n ) virus outbreak in a school in london influenza and school-based influenza-like illness surveillance: a pilot initiative in maryland interrupting the 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inhalation this study was approved by the university of toledo irb (ref no: ). we thank the ottawa hills elementary school principal, kori kawczynski, and superintendent, dr. kevin miller, for graciously allowing us access to their school; dr. bill lindsley (national institute for occupational safety and health, niosh) for loaning the aerosol samplers used in this project and guiding us in their use; drs. april ames, daryl dwyer, sheryl milz, and daryl moorhead for their helpful insight and support throughout the study; and former ms student, marcus keller, and undergraduate student jessica dilworth for volunteering their time in the laboratory and out in the field. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.k.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ujzrqzv authors: lu, xiaying; wang, juan; chen, xiaohuan; jiang, yong; pan, zhixing k. title: rolipram protects mice from gram-negative bacterium escherichia coli-induced inflammation and septic shock date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ujzrqzv sepsis is typically triggered by an overwhelming systemic inflammatory response to pathogens, and may lead to severe organ dysfunction and/or death. sepsis consequently has a high mortality rate and a high rate of complications for survivors, despite modern medical advances. therefore, drug identification and validation for the treatment of sepsis is of the utmost importance. as a selective phosphodiesterase- inhibitor, rolipram also exhibits the abilities of inhibiting multiple pro-inflammatory cytokines production in macrophages and toxin-induced inflammation in mice. however, this drug has never been studied as a sepsis treatment method. we found that rolipram significantly improves survival in mice challenged with gram-negative bacterium e. coli, clp, or e. coli derived lipopolysaccharide. we have also found that rolipram inhibits organ damage, pro-inflammatory cytokine production, and intracellular migration of early-stage inflammatory elements. our results also show that rolipram increases anti-inflammatory cytokine production. the protective effects of rolipram on septic mice may result from inhibition of the map kinase and nf-κb signaling pathways. rolipram may therefore be a potential novel sepsis treatment, one that would bypass the time-consuming and costly drug-discovery process. rolipram significantly reduces the mortality rates in multiple mice septic models. to assess the protective effect of rolipram on sepsis induced by e. coli, clp, or e. coli derived lps, we investigated the effect of the drug on survival rate. first, mice were injected intraperitoneally (i.p.) with rolipram or vehicle hr before e. coli injection. in the absence of rolipram, % of infected mice died within hr of e. coli injection (fig. a) . in contrast, injection with rolipram resulted in % mortality over days, suggesting that rolipram pretreatment can prevent e. coli -induced septic shock in mice. pretreat the clp model mice can also significant improve the mice survival rate, from % to % (fig. b) . to confirm the protective effect of rolipram both in e. coli-induced and clp-induced septic mice, we assessed the role of rolipram in mouse sepsis induced by lipopolysaccharide (lps) derived from e. coli. mice were i.p. injected with rolipram hr before lps injection. in the absence of rolipram, % of endotoxic mice died within hr of lps injection, but % mice survival in the rolipram pretreated group (fig. c) . these results suggest that rolipram may have a protective effect in sepsis. the survival dose-response curve for rolipram indicates that the mice receiving the highest dose, mg/kg rolipram, experienced the most benefit (fig. d ). taking into account the differential survival rates and occurrence of first mortality, mg/kg rolipram showed the highest efficacy of all tested concentrations. rolipram at mg/kg significantly improved the survival rate to % as opposed to % (p < . ). mg/kg rolipram did not significantly improve the survival rate ( %) compared to the lps-only group. rolipram significantly reduces e. coli derived lipopolysaccharide-induced release of serum pro-inflammatory cytokines in mice. the overwhelming release of pro-inflammatory cytokines plays an important role in the pathology of sepsis. therefore, the serum levels of multiple pro-inflammatory cytokines male c bl/ mice were injected with different doses of rolipram ( mg/kg, mg/kg, and mg/kg, i.p.) hr before lps injection. survival of mice was monitored for days. kaplan-meier analysis, followed by a log-rank test, was used for survival time analysis. *represents p < . in treatments vs. lps groups. were examined. results show that the concentrations of il- β, il- , il- , il- (p ), tnf-α, mcp- , mip- , eotaxin, kc, mig, lif, and vegf, as well as the anti-inflammatory cytokine il- , were all significantly elevated in serum after hr and hr of lps challenge (fig. ) . in contrast, administration of rolipram effectively reduced the production of pro-inflammatory cytokines and chemokines, and further increased the levels of il- . rolipram prevents e. coli derived lipopolysaccharide-induced lung injury in mice. lps-induced endotoxic shock is known to cause a number of effects in the murine host, including severe lung injury. histopathological analysis, consisting of hematoxylin and eosin (h&e) staining of lung sections from lps-only mice, revealed signs of extreme inflammation. edema, neutrophil recruitment, and hemorrhage in the lung samples were also seen ( fig. a ). lung injury in the lps group was visibly increased in comparison with the control group. rolipram treatment, however, markedly attenuated lps-induced lung injury. in bronchiolar lavage fluid, total protein ( rolipram alleviates e. coli derived lipopolysaccharide-induced liver and kidney damage. h&e staining of liver tissue from control mice showed that the liver plate was radially arranged around the central vein in normal hepatic lobules. the livers of lps-only mice sacrificed at the -hr time point showed structural disorder of the hepatic lobule, narrowing or even disappearance of the hepatic sinusoidal space, vacuolar degeneration of hepatocytes, and nuclear pyknosis. however, rolipram pre-treatment for hr significantly reduced these changes (fig. a ). renal tissue h&e staining (fig. b ) clearly showed normal structure and morphology in glomeruli, renal tubules, and renal tubular epithelial cells in control mice. the renal lumen was regular, and no inflammatory cells infiltrated the renal interstitial cells. the kidneys of lps-only mice at the -hr time point showed inflammatory infiltrate in the renal interstitial cells, swelling of renal tubular epithelial cells, and an unclear cell gap. rolipram pretreatment for hr significantly reduced these changes. we also examined biochemical markers for liver and kidney injury in serum from each group of mice treated with lps for hr. rolipram inhibited levels of aspartate and alanine aminotransferases (ast and alt), creatinine (cr), and brain natriuretic peptide (bun) in serum. this indicates that rolipram can ameliorate liver and kidney injury induced by lps ( fig. c-f ). mapk signaling pathways in mouse lungs. immunofluorescence microscopy using anti-nf-κb/p antibody, revealed that lps induces nf-κb/p nuclear translocation in the mouse lung hr after injection. however, rolipram markedly inhibited this translocation (fig. a ). three hours after lps treatment, phosphorylation of nf-κb/p significantly increased in the mouse lung as compared to controls. rolipram markedly suppressed phosphorylation of this molecule (fig. b ). western blot results showed that lps treatment induced we demonstrate that rolipram may protect against lps-induced inflammation and shock in mice, likely through the inhibition of the nf-κb and mapk signaling pathways. we found that rolipram significantly improved animal survival, decreased inflammatory cytokines essential to the process of shock, alleviated organ injury, and dephosphorylated critical inflammatory pathways. these results indicate that rolipram suppresses inflammatory responses that are essential to the process of sepsis, and thus may be protective against sepsis. therefore, rolipram may serve as a novel drug treatment for inflammatory disease, such as septic shock. sepsis is induced by a dysregulated innate immune reaction, leading to a harmful host response to pathogens. this is includes excessive amounts of pro-inflammatory cytokines, such as tnf-α and il- β , , as well as other inflammatory mediators. these molecules can in turn trigger secondary inflammatory processes, leading to inflammatory pathology and life-threatening organ damage. proteins that induce the migration of inflammatory cells into tissue are also upregulated. we have found that rolipram inhibits pro-inflammatory cytokines and chemokines released by the administration of lps, leading to the suppression of excessive inflammatory responses, cell adhesion and migration, and the further sequelae of shock and multiple organ failure in the mouse host. sepsis-induced multi-organ dysfunction and injury are the main mechanisms of patient shock and death. therefore, sepsis treatment guidelines consider multi-organ dysfunction a key point of attack in sepsis treatment . it has previously been reported that rolipram significantly decreased hyperoxia-induced neutrophil numbers in balf and inhibits il- and mcp- transcription in rat lungs . rolipram also alleviates pulmonary edema and reduces neutrophil numbers in balf during chlorine-induced mouse shock . our results go further in showing that rolipram inhibits pulmonary edema, neutrophil infiltration in alveoli, and protein concentrations and neutrophil numbers in balf. liver and kidney function is also improved, indicating that rolipram can prevent lps-induced multi-organ failure. the exact mechanism by which rolipram controls the inflammatory cascade, thereby preventing shock and multiple organ failure in the host, is unknown. however, it is known that the nf-κb pathway is crucial in the regulation of inflammatory gene expression. nuclear translocation activates the transcription and expression of a variety of cytokines and adhesion molecules, all of which are closely associated www.nature.com/scientificreports www.nature.com/scientificreports/ with inflammation and the immune response . the nf-κb family, including p (rela), p /p (nf-κb ), p /p (nf-κb ), relb, and c-rel, exist in the cytoplasm in homo-or heterodimers that can bind with iκb. during conditions of stimulation by lps or other pro-inflammatory factors, iκb kinase (ikk) phosphorylates iκb, leading to nf-κb nuclear translocation and further activation of inflammatory genes . it has been confirmed that toll-like receptor -nfκb signaling plays a key role in acute lung injury . our studies show that lps activates nf-κb and its signaling pathways, while rolipram restrains activation. this indicates that the nf-κb may be part of the mechanism by which rolipram exerts its effects. the mapk signaling pathway also plays a critical role in regulating inflammation and the immune response. as a conserved signaling cascade, the mapk pathway exists in almost all eukaryotic cells. mapks can regulate target protein function through phosphorylation, thus participating in cell proliferation, growth, differentiation, and function. mapk signaling is activated through three levels, including map k, mapkk, and mapk. at present, four mapk signaling pathways have been identified: erk / , erk , p mapk, and jnk . it has been found that rolipram promotes the maturation of glial progenitor cells and regeneration of myelin through enhancement of erk phosphorylation . studies have also found that rolipram restrains bone cancer pain through inhibition of the jnk signaling pathway in the bone marrow, suppressing neuron-stellate cell activation . in addition, rolipram can inhibit lps-induced p mapk phosphorylation in j cells . however, the role of rolipram on phosphorylation of the mapk downstream signaling pathways has not yet been reported. our results indicated that rolipram suppresses lps-induced erk, jnk, and p mapk phosphorylation in lung tissue, suggesting that the mapk signaling pathway -as well as nf-κb -may mediate the anti-inflammatory actions of rolipram. in conclusion, we have found that rolipram protects mice from massive inflammatory responses and endotoxic shock through inhibition of nf-κb and mapk signaling pathway activation. rolipram may thus serve as a novel drug treatment for inflammatory disease, namely sepsis and septic shock. as rolipram is an approved drug in the united states for chronic obstructive pulmonary disorder, this drug repositioning strategy may circumvent the lengthy and expensive drug discovery process, and allow faster and better treatment for sepsis patients. animals. male c bl/ mice, weighing - g, all - weeks old, were used for experiments. the mice were purchased from the department of laboratory animal science of southern medical university (guangzhou, china). all animals were housed with free access to food and water under conditions of optimal light, temperature, and humidity ( : -hr light-dark cycle, approximately degrees celsius, - % humidity). all experimental procedures were approved by the ethics committee of animal research at the college of medicine, southern medical university, and were conducted in accordance with the international guidelines for care and use of laboratory animals. animal model. lipopolysaccharide was dissolved in phosphate-buffered saline (pbs) and stored at − degrees celsius. rolipram was dissolved in dimethyl sulfoxide (dmso) and further diluted in pbs (final dmso concentration: < . %). first, male c bl/ mice were intraperitoneally (i.p.) injected with µl rolipram ( mg/kg) hr before, hr after, or at the same time as lps ( mg/kg in µl). survival rate was monitored for days. the experiments were repeated using variable doses of rolipram ( , , and mg/kg). at , , , , and hr after lps injection, mice were anesthetized and sacrificed. blood was collected for analysis of serum cytokines. for histological analysis, normal saline ( ml) was perfused through the right heart ventricle. lung, liver, and kidney samples were collected and processed as described later. mouse blood samples were left at room temperature for hr, then spun at × g for minutes at degrees c. serum was obtained and stored at − degrees c until required. the chemiluminescent signal was quantified; phosphorylated erk, jnk, and p mapk were normalized against total erk, jnk, and p mapk. phosphorylated protein levels are expressed in arbitrary units. lpsstimulated cells were set at %, and other values relate to that setting. data represent the mean and sd of at least three independent experiments, performed in triplicate. significant differences between more than two groups were performed using anova. comparisons between two groups were performed using two-tailed unpaired student's t-tests. *represents p < . vs. control, # represents p < . vs. lps alone. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ cytokine assays. cytokines from mouse serum were measured using the milliplex ® map mouse cytokine/chemokine magnetic bead panel kit for -well plate assay, run on a luminex platform. cytokines include the three primary cytokines of interest: tumor necrosis factor-α (tnf-α), interleukin- -β (il- β), and il- . multiple cytokines were also explored: il- α, il- , il- , il- , il- , il- , il- (p ), il- (p ), il- , il- , il- , ifn-γ inducible protein (ip- ), eotaxin, interferon-γ (ifn-γ), monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein- α (mip- α), mip- β, mip- , migration-inducing protein (mig), keratinocyte chemoattractant (kc), leukemia inhibitory factor (lif), lipopolysaccharide-induced cxc chemokine (lix), lipopolysaccharide-induced cxc chemokine (rantges), granulocyte-macrophage colony-stimulating factor (gm-csf), g-csf, m-csf, and vascular endothelial growth factor (vegf). for quality assurance, each sample was run twice. liver and renal function tests. serum activity of aspartate aminotransferase (ast, a nonspecific marker for hepatic injury), alanine aminotransferase (alt, a specific marker for hepatic parenchymal injury), blood urea nitrogen (bun, a marker for glomerular renal function), and creatinine (cr, a marker for glomerular filtration rate and renal failure) were measured by fengrui biotechnology company kits (hunan, china). hr of lps challenge. lungs were lavaged with pbs ( . ml × ). the recovery rate of bal fluid (balf) was approximately %. cells were isolated from the balf by centrifugation of x g for minutes, and the cell pellets were resuspended. the total cells in the balf were counted using a hemocytometer. subsequently, the cell suspension was cytospin-centrifuged onto microscope slides and stained using liu stain (baso, china). the percentage of neutrophils was determined by counting cells. the balf supernatant was analysed for total protein concentration by using bicinchonic acid (bca) assay, according to the manufacturer's instructions. histology. lungs, livers, and kidneys were fixed in % paraformaldehyde (ph . ). the organs were then dehydrated and embedded in paraffin. sections µm thick were cut. the slides were then stained with hematoxylin and eosin (h&e), and examined by a light microscope. extraction reagent (pierce, il, usa). protein concentrations were determined using a bca assay kit (keygen, china). equal amounts of lung tissue protein ( µg per animal) were run on a % sds-page gel and transferred onto polyvinylidene difluoride (pvdf) membranes. the membranes were blocked with % bovine serum albumin (bsa) in tbst at room temperature for hr, and then incubated with primary antibody against mouse erk / , p-erk / , jnk, p-jnk, p , p-p , or p (all at a : antibody:bsa ratio) at degrees c overnight. after washes with tbst, the membranes were incubated in secondary hrp-conjugated anti-rabbit igg at room temperature for hr. the membranes were then washed with tbst, processed with an ecl detection kit (pierce, il, usa), and measured on film in a darkroom. immunofluorescence. all tissue stainings were performed on frozen µm-thick oct-embedded mouse lung tissue sections. sections were blocked to eliminate non-specific binding with . % bsa in pbs for hr, then incubated with primary antibody against mouse phosphorylated p ( : ) at degrees overnight. after subsequent washes with pbs, the sections were incubated with alexa fluor ® goat anti-rabbit igg (h + l) for hr at degrees c, then washed with pbs. the slides were stained with dapi (santa cruz biotechnology, ca, usa) for min. slides were then washed in pbs. coverslips were mounted onto the slides using anti-fade reagent (beyotime biotechnology, china). the images were acquired using a zeiss lsm confocal microscope (zeiss, germany). data are expressed as the mean plus or minus standard deviations. statistical evaluation was performed with spss software (version . ). significant differences between more than two groups were performed using anova. comparisons between two groups were performed using two-tailed unpaired student's t-tests. kaplan-meier analysis, followed by a log-rank test, was used for survival time analysis. p < . was taken as the maximum significant difference. epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care epidemiology of severe sepsis long-term cognitive impairment and functional disability among survivors of severe sepsis coordinated molecular cross-talk between staphylococcus aureus, endothelial cells and platelets in bloodstream infection how bacterial pathogens colonize their hosts and invade deeper tissues the response of cd d-restricted invariant nkt cells to microbial pathogens and their products anti-endotoxin vaccines: back to the future nf-kappab at the crossroads of life and death tnf-alpha induction by lps is regulated posttranscriptionally via a tpl /erk-dependent pathway map kinase targeted by endotoxin and hyperosmolarity in mammalian cells a protein kinase involved in the regulation of inflammatory cytokine biosynthesis cytokine storm in a phase trial of the anti-cd monoclonal antibody tgn attenuation of tnf production and experimentally induced inflammation by pde inhibitor rolipram is mediated by mapk phosphatase- rolipram versus imipramine in inpatients with major, "minor" or atypical depressive disorder: a double-blind double-dummy study aimed at testing a novel therapeutic approach potential antidepressant activity of rolipram and other selective cyclic adenosine ′, ′-monophosphate phosphodiesterase inhibitors the immunopathogenesis of sepsis pathogenetic mechanisms of septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock effects of phosphodiesterase inhibition on alveolarization and hyperoxia toxicity in newborn rats inhibition of chlorine-induced lung injury by the type phosphodiesterase inhibitor rolipram shaping the nuclear action of nf-kappab signaling to nf-kappab identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury mitogen-activated protein kinase phosphatase as an inflammatory factor and drug target rolipram promotes remyelination possibly via mek-erk signal pathway in cuprizoneinduced demyelination mouse the analgesic effect of rolipram is associated with the inhibition of the activation of the spinal astrocytic jnk/ccl pathway in bone cancer pain we would like to acknowledge ms. hallie hanna dolin at the university of toledo for helping us modify the manuscript. this work was supported by national natural science foundation of china, grant number . z.k.p. and y.j. designed the experiments; x.y.l. and j.w. performed the experiments and collected the samples; x.y.l., j.w., and x.h.c. analyzed the results; x.y.l. and z.k.p. prepared and wrote the manuscript, z.k.p., y.j. and x.h.c. modified the manuscript. all authors read and approved the final manuscript for publication. the authors declare no competing interests. correspondence and requests for materials should be addressed to y.j. or z.k.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -qw tusd authors: krishna, smriti m.; omer, safraz mohamed; li, jiaze; morton, susan k.; jose, roby j.; golledge, jonathan title: development of a two-stage limb ischemia model to better simulate human peripheral artery disease date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: qw tusd peripheral arterial disease (pad) develops due to the narrowing or blockage of arteries supplying blood to the lower limbs. surgical and endovascular interventions are the main treatments for advanced pad but alternative and adjunctive medical therapies are needed. currently the main preclinical experimental model employed in pad research is based on induction of acute hind limb ischemia (hli) by a -stage procedure. since there are concerns regarding the ability to translate findings from this animal model to patients, we aimed to develop a novel clinically relevant animal model of pad. hli was induced in male apolipoprotein e (apoe(−/−)) deficient mice by a -stage procedure of initial gradual femoral artery occlusion by ameroid constrictors for days and subsequent excision of the femoral artery. this -stage hli model was compared to the classical -stage hli model and sham controls. ischemia severity was assessed using laser doppler perfusion imaging (ldpi). ambulatory ability was assessed using an open field test, a treadmill test and using established scoring scales. molecular markers of angiogenesis and shear stress were assessed within gastrocnemius muscle tissue samples using quantitative polymerase chain reaction. hli was more severe in mice receiving the -stage compared to the -stage ischemia induction procedure as assessed by ldpi (p = . ), and reflected in a higher ischemic score (p = . ) and lower average distance travelled on a treadmill test (p = . ). mice undergoing the -stage hli also had lower expression of angiogenesis markers (vascular endothelial growth factor, p = . ; vascular endothelial growth factor- receptor , p = . ) and shear stress response mechano-transducer transient receptor potential vanilloid (p = . ) within gastrocnemius muscle samples, compared to animals having the -stage hli procedure. mice subjected to the -stage hli receiving an exercise program showed significantly greater improvement in their ambulatory ability on a treadmill test than a sedentary control group. this study describes a novel model of hli which leads to more severe and sustained ischemia than the conventionally used model. exercise therapy, which has established efficacy in pad patients, was also effective in this new model. this new model maybe useful in the evaluation of potential novel pad therapies. major amputation, renal failure and death) and poor long-term durability [ ] [ ] [ ] [ ] . there is great interest in developing novel medical therapies for the leg symptoms of pad. recent efforts have focused on stimulating development of new blood vessels within the leg through angiogenesis or by encouraging the remodelling of existing small vessels into improved collateral channels (arteriogenesis). promising results for novel treatments, such as viral vectors carrying angiogenesis promoting agents and stem cells, in pre-clinical models of pad have not been consistently replicated in large clinical trials - . in patients that have pad, atherosclerosis-associated arterial narrowing develops gradually over many years allowing the legs to adjust to the gradual decrease in blood flow through compensatory mechanisms within the blood vessels and muscle fibres . in contrast, the most commonly used animal model for initial testing of novel therapies for pad is a model of acute blood supply interruption through ligation or excision of the femoral artery (referred to here as the -stage hind limb ischemia (hli) model) , . previous studies report that the ligation and excision of the femoral artery in the -stage model leads to increased fluid shear stress within the limb collateral arteries resulting in altered gene expression patterns through shear stress responsive elements which promote arterio-and angio-genesis [ ] [ ] [ ] . hind limb blood supply in this -stage model therefore usually naturally recovers over a period of approximately weeks . this model does not therefore simulate the clinical presentation of pad. patients typically present with a history of acute exacerbation of chronic symptoms of leg pain on walking and have ongoing ischemic symptoms. the -stage hli model may therefore not be an ideal model to study therapeutic angiogenesis and arteriogenesis , . another approach to inducing hli is the placement of an ameroid constrictor around the femoral artery to induce gradual occlusion , , . previous studies suggest that this approach leads to mild ischemia and that blood flow recovery occurs within - weeks , . furthermore, previous pre-clinical pad research has mainly focused on assessing hind limb blood supply with limited assessment of ambulatory ability , . on the other hand, the assessment of novel treatments in pad patients usually involves measures of walking ability using treadmill or corridor walking tests , . there is therefore a need for an increased focus on functional tests of the limb within clinically-relevant rodent models. we hypothesised that the limb ischemia produced by the current -stage hli model would be more severe and sustained if the model was modified to include an initial more slowly progressive arterial narrowing over days prior to the induction of acute ischemia (i.e. a -stage model). our overall aim was to develop a more clinically relevant rodent model that could incorporates stable on-going limb ischemia in order to test therapeutic interventions. mice. male apolipoprotein e deficient (apoe −/− ) mice (n = , obtained from animal resources centre, western australia) were used for the experiments. mice were housed in a temperature-controlled room ( ± °c) with an automatic : -h light/dark cycle ( : to : hours). mice were singly housed in a clear individually-ventilated, temperature and humidity-controlled cage system (aero ivc green line; tecniplast) with enrichment. all experiments were performed during the light phase ( : - : hours) and mice were fed with standard rodent chow and water ad libitum during the course of these experiments. approval for the animal studies was obtained from the institutional ethics committee (animal ethics committee, james cook university) and experimental work performed in accordance with the institutional and ethical guidelines of james cook university, australia, and conforming to the guide for the care and use of laboratory animals (national institutes of health, usa). hli models. the first phase of the study utilised two hli models: the most commonly used unilateral acute hli model ( -stage hli) , , and the newly developed -stage hli model. male apoe −/− mice aged months were randomly divided into groups as follows: group = -stage hli model (n = ), group = -stage sham (n = ), group = -stage hli model (n = ) and group = -stage sham (n = ). body weight and primary outcome measures were recorded at regular intervals as illustrated in fig. a . all functional assessments were performed in a subset of mice randomly selected from each experimental group (n = - ). the creation of the -stage hli model involved exposure of the left femoral artery through a vertical . - cm skin incision under a stereotactic microscope (leica). the femoral artery and its side branches were then ligated with - silk sutures (ethicon) immediately distal to the inguinal ligament and proximal to the popliteal bifurcation before being excised ( supplementary fig. s a,b) . femoral nerves were carefully preserved. the wound was irrigated with sterile saline and then the overlying skin was closed using - vicryl sutures (ethicon). post-operative pain was reduced using lignocaine (troy laboratories). a similar surgery without ligation or excision of the femoral artery was performed on the -stage sham controls. the -stage hli model was performed using a -stage surgical procedure. the left femoral artery was exposed as described above and custom made miniature ameroid constrictors of . mm internal diameter (research instruments sw) were positioned on the artery. one was placed on the femoral artery immediately distal to the inguinal ligament and one was positioned proximal to the sapheno-popliteal bifurcation ( supplementary fig. s c ). after days, a new incision was made and the femoral artery ligated and excised, as described for the -stage hli model. a similar two-stage surgery was performed without placement of ameroids, nor ligation and excision of the femoral artery for the -stage sham controls. assessment of the effect of exercise training in the -stage hli model. during the second phase of the study, the effect of an exercise program on male apoe −/− mice aged months undergoing the -stage hli model (male apoe −/− mice, n = ) was tested. mice subjected to -stage hli were randomly allocated to an exercise training or control group (n = per group). mice in the exercise group received to m (between - mins of running wheel access) of exercise each day on a running wheel ( station home cage running the movement of animals were monitored and functional scores of the various groups were assessed according to the scoring criteria detailed in the materials and methods section. the -stage hli model showed reduced function throughout the experimental period compared to the -stage hli model. data shown as mean ± sem and analysed by repeated measures -way anova, and p value significant at ≤ . . (e) graph showing modified ischemia scores. the animals were monitored and scored for signs of ischemia according to previously published criteria detailed in the materials and methods. the -stage hli model showed a higher level of ischemia compared to the -stage hli depicted by a lower scoring throughout the study period. data shown as median ±sem and analysed by repeated measures -way anova, and p value significance set at ≤ . . suggested that the hli typically resolved over the course of days, with hind limb blood supply reaching a plateau between and days after surgery . the ldpi measurements were therefore performed at the following time points for -stage hli model: day prior to surgery, immediately after surgery, days , , , and after surgery. for the -stage hli model, the ldpi measurements were performed at the following time points: day prior to the first operation (ameroid placement), immediately after the first operation, days , , and after the first operation, immediately after the second operation (femoral artery excision), and , , and days after the second operation (i.e. , , , and days after the first operation). a schematic illustration of the experimental design is shown in fig. a . body mass was also measured on the same day as the ldpi measurements. in clinical practice pad patients are treated to improve pain free walking capacity and resolve rest pain and tissue loss (critical limb ischemia, cli). in clinical trials, these are usually investigated by walking tests and assessment of pain. similar to clinical trials, in the current study ambulatory ability was assessed with a treadmill test, voluntary physical activity examined through an open field test and foot pain estimated through a mechanical allodynia test. all outcomes were assessed by an assessor blinded to mice group. semi-quantitative assessments of limb function and ischemia were performed at the same time points as the blood flow measurements ( fig. a ; supplementary tables s , s ). limb function was assessed using the clinical use score (tarlov scale) as: = no movement; = barely perceptible movement, no weight bearing; = frequent and vigorous movement, no weight bearing; = supports weight, may take or steps; = walks with only mild deficit; = normal but slow walking and = full and fast walking , . limb ischemia was scored using the ischemia scoring scale as previously reported: = auto-amputation of leg; = leg necrosis; = foot necrosis; = two or more toe discoloration; = one toe discoloration; = two or more nail discolorations; = one nail discoloration and = no necrosis . all scoring was performed by two independent observers and found to be identical. treadmill test. mice were run on a six lane excer / treadmill (columbus instruments) without incline. mice were acclimatised to the treadmill by ambulating on it at m/min for min once daily on three consecutive days prior to any testing. before each treadmill test, mice were fasted for h. the speed of the treadmill was controlled using the software and calibrated using an inbuilt speedometer mounted on the treadmill platform. a treadmill test involved an initial warm up at m/min for min followed by a progressive speed increase from to m/min, accelerated at m/min. following this the treadmill speed remained at m/min for up to a total running time of min. during the test a stimulus grid of hz was kept on until mouse exhaustion as previously reported . exhaustion of the mouse was defined if the mouse returned to the stimulus grid times despite a hz electrical stimulus to encourage walking on the belt. the treadmill software recorded the total distance walked by a mouse until exhaustion. a blinded observer supervised the experiment to assess outcomes. the treadmill belt and lanes were cleaned with water and % alcohol and dried with paper towel after each test to remove any body scent. treadmill testing was carried out before ameroid placement, days after ameroid placement, and and weeks after completion of the -stage hli (fig. a) . for the -stage hli model, treadmill testing was performed before ligation and excision of the femoral artery and and weeks after ischemia induction. voluntary physical activity test. the open field test is a common measure of voluntary physical activity in rodents suggested to be similar to a -min walk test used in humans . to ensure consistency prior to the test, mice were brought to the testing room in their home cages at least hr prior to the start of behavioural testing. the mice were fasted during the acclimatisation period and given free access to water under normal lighting. the open field box was made of opaque plastic ( × × cm), divided into an outer field (periphery) and a central field ( × cm) which was primarily used for analysis. mice were individually placed in the centre of the arena and movements of the mice were recorded using a video camera (logitech) supported with acquisition software (capture star ver. ; cleversys inc) and analysed by the topscan lite software (high throughput version . ; cleversys inc). the test protocol used was identical for each mouse assessed. after each test the open field box was cleaned with water and % alcohol and dried with a paper towel to remove the body scent, which could be a cue to movement of the mice. room lighting, temperature, and noise levels were kept consistent for all tests. the mouse movements were recorded for min, to mimic the short timed nature of the -min walk test. rest time was recorded as motion measure score < . in the software and average speed was calculated only for motion measure score > . . total distance travelled (m), frequency of movement, time spent in the arena (s) and average velocity in the arena (mm/s) were measured. mechanical allodynia test. the paw pressure transducer and the pressure application measurement device (pam; ugo basile) is a non-invasive tool for measuring mechanical allodynia threshold and hypersensitivity in rodents. the pam device allows an accurate measurement of primary mechanical hypersensitivity in rodents , . a gradually increasing squeeze force is applied across the joint at a rate of approximately gms/sec until a behavioural response (paw withdrawal, freezing of whisker movement, wriggling or vocalization) is observed with a cut-off of sec. the peak gram force (gf) applied immediately prior to limb withdrawal was recorded by the base unit, and this value was designated the limb withdrawal threshold (lwt). lwt was measured twice in both the ipsilateral and contralateral limbs by two independent observers. the measurements were averaged and presented as a ratio of operated left limb to the un-operated right limb. blood tests. blood was collected by cardiac puncture at the completion of the experiments. platelet poor plasma was separated as described previously , . the plasma concentrations of interleukin (il)- , interferon (ifn)-γ, monocyte chemoattractant protein- (mcp- ) and tumour necrosis factor (tnf)-α were determined scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ using a cytometric bead array kit (cba, bd biosciences). the inflammatory markers were assessed in samples (n = /group) selected from each group using a random number generator. briefly, μl of mixed capture beads and μl of serially diluted standard or plasma sample and μl of phycoerythrin (pe) detection reagent, were incubated in the dark for h in sample assay tubes. samples were then washed twice with ml of the wash buffer, resuspended, and acquired on the cyan adp flow cytometer (beckman coulter). results were analysed and quantified by fcap array ™ software (v , bd biosciences). we previously reported this method to have good reproducibility with an inter-assay coefficient of variation of - % (n = - ) . total nitrate was measured in plasma samples by a nitrate/nitrite colorimetric assay kit following the manufacturer's protocol (inter-assay coefficient of variation . %; cayman chemicals) as reported previously . briefly, nitrate was converted to nitrite using nitrate reductase. subsequently, addition of the griess reagents converted nitrite into a deep purple azo compound and the absorbance was measured at nm using an omega plate reader. histological assessments. low capillary density has been reported in the gastrocnemius muscle of pad patients and animal hli models and associated with functional impairment , . hence at the end of experiments gastrocnemius muscle samples were collected from the mice and stored in optimal cutting compound (oct, proscitech) which was progressively frozen in isopentane (sigma) suspended in liquid nitrogen. sections ( µm-thick) were obtained on poly l-lysine coated slides (proscitech) from each sample with muscle fibres oriented in the transverse direction. all histological assessments were performed on sections that were examined in a blinded fashion at x magnification. muscle fibre structure. fixed cryostat sections were stained with hematoxylin and eosin (h&e, proscitech), examined at magnifications of x or x to assess the integrity of the tissues. degenerating muscle fibres were identified in the h&e stained sections by morphological assessment. assessors looked for the presence of mature skeletal muscle fibres (small peripheral nuclei) versus immature skeletal muscle myoblasts (large lobulated central nuclei) . muscle fibre number and size were examined in separate fields in distinct areas in each specimen. muscle fibrosis. the extent of skeletal muscle fibrosis was assessed by staining the cryostat sections ( µm-thick) with picrosirius (proscitech). briefly, tissue sections were stained and examined under x power light microscope and skeletal muscle fibrosis was analysed using the image analysis software (zeiss axio imager z ). quantification of fibrosis was expressed as the percentage of fibrotic tissue present within the section ( mm tissue area) using a previously published protocol . immunohistochemistry and morphometric analysis of capillary and arteriolar density. these were performed as previously reported , . the gastrocnemius muscles from ischaemic and non-ischaemic hind-limbs were collected and embedded in oct compound (proscitech), frozen, and cut into µm-thick sections. the slides were fixed at − °c in % ethanol for hr. slides were washed three times in cold pbs with % horse serum ( min/ wash) and blocked overnight with % horse serum in pbs at °c. immunohistochemistry was performed using primary antibodies against cd ( : dilution; abcam) and smooth muscle α-actin (α-sma, : dilution; abcam). bound primary antibodies were detected by using appropriate secondary antibodies (biotinylated anti-goat igg and biotinylated anti-rat igg, all at : dilutions, vector labs) using avidin-biotin-peroxidase (vector labs) as described previously . pictures from four random areas of each section and three sections per mouse were taken by using a digital camera (nikon eclipse sci epifluorescence microscope, nikon corporation) at × magnification. capillary density were quantified by measuring the percentage of cd and α-sma staining out of the total area as previously described. western blotting. gastrocnemius muscles were mainly harvested at the end of studies (i.e. weeks after full ischemia induction). gastrocnemius muscles were also harvested from a subset of mice (n = ) subjected to the -stage hli (n = /time-point) prior to and days and after ameroid placement. samples were frozen in liquid nitrogen, and stored in oct compound (proscitech) at − °c. tissues were pulverised in ripa buffer ( mm sodium chloride, . % np- or triton x- , . % sodium deoxycholate, . % sodium dodecyl sulfate, mm tris, ph . ) with protease inhibitors (roche diagnostics, australia) and phostop (roche diagnostics, australia) to extract proteins and quantitated using the bradford protein assay kit (biorad, usa). samples ( μg of protein/lane) were loaded onto a % sds-polyacrylamide electrophoresis gel. after electrophoresis ( v, min), the separated proteins were transferred ( ma, min) to a polyvinylidene difluoride membrane (biorad, usa). non-specific sites were blocked with % non-fat dry milk for min, and the blots were then incubated with following antibodies: anti-vascular endothelial growth factor (anti-vegf www.nature.com/scientificreports www.nature.com/scientificreports/ mrna analysis by quantitative real-time pcr. at the end of the study gastrocnemius muscle samples were harvested, placed in rna later (qiagen) and stored at − °c. samples (n = /group) were selected from each group using a random number generator and were processed for gene expression analysis. total rna was isolated using an rneasy mini kit (qiagen) according to manufacturer's instructions and quantified spectrophotometrically using nanodrop . rna samples ( ng) were subjected to quantitative real time pcr (qrt-pcr) analysis of genes of interest using the quantitect sybr green one-step rt-pcr assay (qiagen). qrt-pcr was performed using primers for mouse vegf-r (ppm f), vegf-r (ppm a), trpv (ppm a), klf (ppm b) and gapdh (qt ). the relative expression of these genes were calculated by using the concentration-ct-standard curve method and normalized using the average expression of mouse gapdh for each sample using the rotor-gene q operating software (version . . ) as previously reported , . statistical analyses. all data were tested for normality using the d' agostino-pearson normality test. data with normal distribution were expressed as mean ± standard error of mean (sem) and analysed using parametric tests. non-normally distributed data were expressed as median and interquartile ranges (iqr) and analysed using non-parametric tests. statistical significance was determined using the unpaired student t test for comparison between two groups or analysis of variance followed by student-newman-keuls post-hoc analysis for comparison between multiple groups. comparison of the time course of ldpi indices, clinical scores, open field tests and treadmill exercise tests were done by -way anova for repeated measures, followed by bonferroni post hoc analysis or by linear mixed effect method using r studio software. difference in the clinical ischemia score were determined by fisher's exact test. analyses were performed using prism (graphpad software, san diego, ca) or r software. a p value of ≤ . was considered to be statistically significant. mice undergoing -stage hli had more severe ischemia than those undergoing -stage hli. immediately after femoral artery excision, limb perfusion assessed by ldpi was similarly reduced in both hli models by approximately %. mice subjected to the -stage hli had more rapid recovery of hind limb perfusion than those subjected to the -stage procedure (p = . , fig. b,c, supplementary fig. s ). by days after ischemia induction limb perfusion was similar in mice subjected to the -stage procedure and sham controls (p = . ) but still reduced in mice subjected to the -stage procedure by comparison to sham controls (p < . ). there was no change in overall body mass after ischemia induction (supplementary fig. s ). mice subjected to -stage hli showed more severely impaired hind limb use than those undergoing -stage hli. after ischemia induction, mice subjected to both methods of hli developed limb oedema, paleness of skin and occasional muscle necrosis. mice in all experimental groups exhibited a severe functional deficit after surgery (fig. d) . functional score was significantly worse in mice subjected to the -stage hli than those subjected to the -stage hli (p = . ). both the hli models showed increased ischemia compared to the respective shams. there were no cases of auto-amputation or foot or limb necrosis (supplementary fig. s ). mice subjected to the -stage hli showed a significantly worse ischemic score compared to those subjected to the -stage hli (repeated measures way anova, p = . , fig. e ). mice subjected to -stage hli had reduced treadmill performance. mice subjected to the -stage hli showed no significant difference in total distance travelled during the study period on a treadmill test when compared to shams (repeated measures way anova, p = . ; fig. a ). after the first procedure of the -stage model (ameroid placement) the treadmill ambulatory performance of mice was not significantly affected ( supplementary fig. s a ). in contrast mice subjected to -stage hli had a significant reduction in total distance travelled on the treadmill compared to their sham controls (p = . ) and mice subjected to -stage hli (p = . ; fig. a ). supplementary fig. s b-d) . this reduction in physical activity was maintained after completing the -stage hli by comparison to sham controls and also mice subjected to -stage hli (fig. b) . the reduction in physical activity of mice subjected to -stage hli was reflected in less distance travelled, less total time in motion and lower velocity of the movement compared to sham controls (fig. c-e) . when compared to the -stage hli, the stage-hli model showed a reduction in the total distance travelled in the open field arena (linear mixed effect model, p = . ). mice subjected to hli had enhanced mechanical allodynia. mice subjected to both -stage and -stage hli showed significantly increased sensitivity to pressure compared their respective sham controls and there was no significant difference in pressure sensitivity between the two models (fig. f ). hli induces systemic inflammation. the plasma concentrations of the cytokines assessed were below the detectable ranges in both the sham control groups (table ) . mice subjected to hli, irrespective of model, had plasma cytokine concentration significantly higher than the sham controls although levels were not significantly different between models ( table ). the plasma concentrations of nitric oxide (no) metabolites were higher in mice undergoing -stage hli than the sham control group (p < . ) and mice undergoing -stage hli (p = . , table ). myofibers which are healthy and functionally active are characterised by peripheral nuclei, while myofibrils with central nuclei are immature and do not show optimal contraction . in gastrocnemius muscle samples removed from sham controls, myocytes were angular with peripheral nucleus (fig. a) . at day after ischemia induction, mice subjected to -stage hli showed microscopic changes such as cellular swelling, focal necrosis and interstitial oedema. there were also numerous infiltrating inflammatory cells. gastrocnemius muscle samples from mice subjected to -stage hli showed more homogenous appearance with all myocytes showing peripheral nuclei and limited inflammatory cell infiltration (fig. a) . histological evaluation revealed that tissues of mice subjected to -stage hli had fewer immature myofibers cells than the tissues from -stage hli model. furthermore, muscle samples from mice subjected to -stage had prominent oedema, myofibre separation and multifocal neutrophilic infiltration. neutrophils were observed throughout the tissue sections. necrotic muscle fibres were prominent and formed confluent areas (fig. a) . muscle fibrosis was assessed by picrosirius red staining which suggested that -stage hli led to increased skeletal muscle fibrosis compared to -stage hli (p = . ) or sham controls (p = . ; fig. b ,e). mice subjected to -stage hli had fewer hind limb collaterals. consistent with the reduced perfusion as assessed by ldpi, both arteriogenesis and angiogenesis was inhibited in the ischemic gastrocnemius muscles of the -stage hli model (fig. c,d) . measurement of angiogenesis by cd immunostaining showed that the presence of arterioles was significantly reduced in samples from the -stage hli compared to the -stage hli (p = . ) or sham controls (p = . ; fig. ). the arteriolar density was also significantly less in samples from the -stage hli model compared to the -stage hli (p = . ) or sham control (p = . ; fig. ). protein concentrations of angiogenesis and shear stress response markers were downregulated in the gastrocnemius muscles of mice undergoing -stage hli. the relative total vegf and vegfr- (but not vegfr- , p-enos/enos and hif-α) protein levels in gastrocnemius tissue collected from mice weeks after -stage hli were significantly less than in sham controls and mice undergoing -stage hli (fig. a-d, supplementary fig. s ). analysis of tissues from the -stage hli model prior to ameroid placement and day and day after ameroid placement suggested no significant changes in concentrations of trpv or vegf in response to ameroid constriction ( supplementary fig. s ). at the end of the experiment, protein concentrations of trpv and klf were significantly less in the gastrocnemius muscle samples from mice undergoing -stage hli compared to sham controls and mice undergoing -stage hli (fig. e,f) . qrt-pcr showed that the relative expressions of vegf-r , trpv and klf in gastrocnemius muscle of mice subjected to -stage hli were significantly lower than within the gastrocnemius muscle of mice undergoing -stage hli or in sham controls (fig. g-j) . exercise training improved functional capacity but not limb perfusion in mice subjected to -stage hli. supervised exercise training is an established method to improve functional capacity in pad patients , and previous studies show that mice respond positively to exercise training , . in order to examine whether an established clinically effective therapy was effective in the novel animal model, the effect of exercise training (using a running wheel) on functional capacity of mice subjected to -stage hli was assessed. exercise training was commenced days after ischemia induction. exercise training did not affect limb perfusion as shows the data from the ischemic limbs from the -stage and -stage hli models and the respective sham controls (n = /group, scale bars in all images = µm). (e-g) quantitative bar graphs showing the effect of hli on (e) skeletal muscle fibrosis by picrosirius red staining, (f) angiogenesis by immunohistochemical staining against cd and (g) arteriogenesis by immunohistochemical staining against α-sma. all values are median and interquartile ranges (n = /group) and p value significance set at ≤ . . ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ assessed by ldpi (linear mixed effect test, p = . ; fig. b ,c). mice subjected to exercise training showed a significant increase in average treadmill walking capacity compared to sedentary controls (p = . ; fig. d ). exercise training upregulated gastrocnemius muscle vegf and trpv levels. since improvement in ambulatory performance as a result of exercise training could be due to enhanced angiogenesis, the expression of angiogenesis and shear stress responsive proteins vegf, vegf-r , vegf-r , trpv and klf were assessed in the gastrocnemius muscles (fig. ). vegf (p = . , fig. b ) and trpv (p = . , fig. e ) but not vegf-r , vegf-r and klf , were highly upregulated following exercise training (fig. c,d,f ). this report describes the development of a novel model of hli which results in more severe and prolonged ischemia than the traditional model. mice subjected to the -stage hli had functional and ambulatory impairment and a positive response to exercise training as has been reported for pad patients . previous rodent studies suggest that placing of ameroid constrictors alone without manipulating the femoral artery results in mild ischemia and blood flow recovers within weeks . hence, the novel hli model was based on placement of two ameroid constrictors on the femoral artery to promote gradual occlusion followed by excision of intervening segment after days to induce severe ischemia. a previous report suggests that recovery of hind limb blood flow is reduced in apoe −/− compared to c bl/ j mice due to their limited collateral arteries and hence apoe −/− mice were used in the current study . pad patients are generally older and exhibit metabolic derangements that limit angio-and arterio-genesis and previous studies suggest that apoe −/− mice have delayed skeletal muscle healing , reduced angiogenesis responses and impaired functional recovery after hli further supporting the rationale for choosing this mice species , , . mice subjected to -stage hli had rapid recovery of limb blood flow and reached a perfusion level similar to the sham controls within days as has been reported by other investigators , , . ligation and sudden excision of the femoral artery is believed to generate a pressure gradient between the proximal and distal ends of the occluded vessel, resulting in increased shear stress and a redirection of blood flow towards the collaterals and through numerous branches arising from the internal iliac artery, resulting in rapid improvement in blood flow , . ameroid constrictors have been shown to cause luminal occlusion within days, however, the blood flow slowly increases in the next - weeks , . hence, in the new model, we superimposed a secondary acute event by excising the intervening femoral artery segment along with the ameroid constrictors. in contrast to the -stage hli, mice subjected to -stage hli had on-going limb ischemia and a prolonged functional deficit on both forced and voluntary ambulation tests. these findings support the value of the novel model for the testing of interventions aimed at achieving clinical improvements in pad patients. since angiogenesis is an inflammation-driven process , the concentrations of circulating cytokines were measured. these markers of systemic inflammation increased in response to hli in both models examined. twenty eight days after hli induction, the concentrations of circulating cytokines were similar in the two models studied. gastrocnemius muscle samples obtained from the -stage hli model had marked neutrophilic infiltration. it has been previously reported that inflammatory cells accumulate in hypoxic tissues and promote angiogenesis . it is possible that the systemic concentrations of cytokines were not reflective of the level of inflammation within the hind limb. markers of angiogenesis and arteriogenesis in gastrocnemius tissue, such as cd and α-smc, were found to be less evident in the -stage than the -stage hli model. furthermore, gastrocnemius vegf and vegf-r protein levels and amount of total plasma no metabolites were significantly lower in the -stage than -stage hli model. vegf promotes angiogenesis through binding to vegf-r expressed on endothelial cells . vegf induces the release of no thereby promoting microvascular perfusion and endothelial progenitor cell mobilization [ ] [ ] [ ] . endothelial cell derived microrna, such as mir- , have also been implicated in controlling angiogenesis through inhibiting rho gdp dissociation inhibitor (rhogdi)-α, an important regulator of enos phosphorylation . it appears likely that the low levels of pro-angiogenic markers in the -stage hli model reflect less activation of endothelium-dependent pro-angiogenesis signalling pathways which were stimulated by collateral flow within the -stage model. shear stress promotes arteriogenesis by stimulating remodelling of collaterals , . endothelial cells transduce changes in shear stress into intracellular signals which promote expression of a distinct set of genes which can control the response to ischemia [ ] [ ] [ ] . previous studies suggest that increased shear stress promotes phosphorylation and upregulation of mechano-sensors, such as trpv [ ] [ ] [ ] [ ] . it was postulated that the distinct ways of inducing hli in the two models studied might be reflected in different trpv expression. mice subjected to -stage hli had lower expression of trpv compared to those undergoing -stage hli. furthermore, in the -stage hli model, there was no change in trpv protein levels after ameroid placement, suggesting that ameroid constriction is a gradual process that does not lead to shear stress changes capable of stimulating mechano-sensors like trpv . these findings also suggest that -stage hli results in more limited collateral flow than the -stage approach. the acute reduction in arterial pressure gradient following femoral artery excision in the -stage hli model it thought to be registered by endothelial shear stress response elements resulting in upregulation of angiogenesis and arteriogenesis promoting genes . simulation of trpv , for example by α-phorbol , of vegf-r (g), vegf-r (h), trpv (i) and klf (j) assessed in the gastrocnemius muscles of mice undergoing hli. quantitative real time pcr (qrt pcr) was performed on extracted total mrna using specific primers and normalised to glyceraldehyde phosphate dehydrogenase (gapdh) expression. data analysed by mann-whitney u test (n = samples/group). ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ -didecanoate, has been reported to promote increase no release and increased hind limb blood flow . trpv deficient rodents have impaired vasodilatation [ ] [ ] [ ] [ ] . this supports the important role of trpv in promoting adaptation to acute limb ischemia. four weeks of exercise training led to an approximate -fold increase in treadmill ambulatory distance in the mice subjected to -stage hli, paralleling findings from pad patients [ ] [ ] [ ] [ ] . these findings suggest the novel -stage hli mouse model simulates the walking impairment experienced by patients. in support of the relevance of this model to patients, we found that exercise therapy improved treadmill performance without improving limb perfusion, a finding similar to that described in pad patients , , . since exercise training increases shear stress in pre-existing collaterals we examined the expression of trpv and angiogenesis markers . compared to sedentary controls, mice undergoing exercise training showed increased total protein levels of vegf and trpv , which was in accordance with previous reports showing enhanced expression of pro-angiogenesis markers after exercise training [ ] [ ] [ ] . overall these findings suggest flow-mediated upregulation of shear stress responsive genes is important in stimulating angiogenesis responses in the new model. this study have several strengths and weaknesses. the -stage hli model which is usually utilised for pad research has many limitations including its disparate pathophysiological mechanisms compared to patient presentations, the temporary nature of the ischemia and its relative responsiveness to a variety of therapies which are not effective in patients . this study suggests the novel -stage model has clear advantages over the -stage model since ischemia is more severe and prolonged and does not naturally recover making it suitable to access the 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arteriesendorsed by: the european stroke organization (eso)the task force for the diagnosis and treatment of peripheral arterial diseases of the european society of cardiology (esc) and of the european society for vascular surgery (esvs) cilostazol for intermittent claudication a systematic review of the uptake and adherence rates to supervised exercise programs in patients with intermittent claudication exercise for intermittent claudication a systematic review of treatment of intermittent claudication in the lower extremities cardiovascular effects of exercise: role of endothelial shear stress exercise-induced expression of angiogenesis-related transcription and growth factors in human skeletal muscle the influence of physical training on the angiopoietin and vegf-a systems in human skeletal muscle exercise linked to transient increase in expression and activity of cation channels in newly formed hind-limb collaterals this research was funded by a faculty administered grant and a research infrastructure block grant from james cook university and funding from the queensland government. jg holds a practitioner fellowship from the national health and medical research council, australia ( ) and a senior clinical research fellowship from the queensland government. smo was supported by funding from the graduate research school and college of medicine, james cook university. we would like to acknowledge with thanks the help of prof. zoltan sarnyai (james cook university) who provided access to open field test assessment facility, dr. joseph moxon (james cook university) who assisted with the lme used to analyse data from the exercise study and dr. pacific huynh (james cook university) who provided the mouse images in figs. a and a. s.m.k. lead the design of the research, undertaking of experiments, and interpretation of data and writing of the manuscript. s.m.o., j.l., s.m. and r.j.j. performed parts of the experiments, contributed to interpretation of the data and gave critical comments on the manuscript. j.g. contributed rationales for the studies, led funding applications, co-wrote the manuscript and contributed to project supervision and data interpretation. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -b zlig r authors: mukherjee, shradha title: quiescent stem cell marker genes in glioma gene networks are sufficient to distinguish between normal and glioblastoma (gbm) samples date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: b zlig r grade glioma or gbm has poor prognosis and is the most aggressive grade of glioma. accurate diagnosis and classification of tumor grade is a critical determinant for development of treatment pathway. extensive genomic sequencing of gliomas, different cell types, brain tissue regions and advances in bioinformatics algorithms, have presented an opportunity to identify molecular markers that can complement existing histology and imaging methods used to diagnose and classify gliomas. ‘cancer stem cell theory’ purports that a minor population of stem cells among the heterogeneous population of different cell types in the tumor, drive tumor growth and resistance to therapies. however, characterization of stem cell states in gbm and ability of stem cell state signature genes to serve as diagnostic or prognostic molecular markers are unknown. in this work, two different network construction algorithms, weighted correlation network analysis (wgcna) and multiscale clustering of geometric network (megena), were applied on publicly available glioma, control brain and stem cell gene expression rna-seq datasets, to identify gene network regulatory modules associated with gbm. both gene network algorithms identified consensus or equivalent modules, huagegbsplit_ (wgcna) and c _huagegbsplit_ / (megena), significantly associated with gbm. characterization of huagegbsplit_ (wgcna) and c _huagegbsplit_ / (megena) modules showed significant enrichment of rodent quiescent stem cell marker genes (gse _qnpbytap). a logistic regression model built with eight of these quiescent stem cell marker genes (gse _qnpbytap) was sufficient to distinguish between control and gbm samples. this study demonstrates that gbm associated gene regulatory modules are characterized by diagnostic quiescent stem cell marker genes, which may potentially be used clinically as diagnostic markers and therapeutic targets in gbm. sva + lm adjustment the resultant normalized log tpm + gene expression was used to build a glioma gene network with subnetworks or modules using wgcna_ . and megena_ . . r packages , , , . in wgcna, default parameters and a minimum module size of was used to calculate a topology overlap matrix (tom) based on gene expression correlations. hierarchical clustering was then used to build a glioma gene network consisting of interconnected subnetworks or modules , . in megena, default parameters and a minimum module size of was used to calculate a planar filtered network (pfn) from gene expression correlations. multiscale clustering method was applied to build a glioma gene network consisting of interconnected subnetworks or modules . wgcna computes scale-free or single scale networks, while megena computes multi-scale networks to include different possible variations of gene-gene interactions. therefore, in megena a given gene or node can be part of multiple modules representing different possible interactions, while in wgcna a given gene or node is assigned to only a single module. to determine module trait correlations, module eigengenes were computed with moduleeigengenes r function and correlations were calculated . to compare wgcna and megena modules, previously published module preservation analysis and hypergeometric enrichment tests were used , . briefly, hypergeometric test was implemented with userlistenichment r function widely used to compare wgcna gene network modules with each other and with user supplied gene lists , , . both module preservation and userlistenrichment r functions are from wgcna , . after module preservation analysis between wgcna and megena modules, significant overlap of genes between wgcna and megena modules was done with userlistenrichment r function for all wgcna and megena modules i.e. each wgcna module was compared with each megena module. wgcna and megena modules that significantly overlapped and were significantly associated with gbm were retained. stem cell differential gene expression analysis with limma, edger and simple comparison of means. differentially expressed genes (degs) between proliferative and quiescent stem cell states were identified using r packages limma_ . . , edger_ . . and simple comparison expression means , , . to calculate genes enriched in proliferative stem cells, gene expression of samples annotated to proliferative stem cells were compared with gene expression of samples annotated to quiescent stem cells for each of the five datasets (gse , gse , gse , gse and gse ). similarly, to calculate genes enriched in quiescent stem cells, gene expression of samples annotated to quiescent stem cells were compared with gene expression of samples annotated to proliferative stem cells for each of the five datasets (gse , gse , gse , gse and gse ). in simple comparison of means method, mean expression of genes were simply compared between proliferative and quiescent stem cell states to determine degs. in limma and edger model design included variables stem cell state, study or batch, gender, age and tissue, and stem cell states were contrasted to determine degs, while all other variables were held constant in the model. in limma and edger methods benjamini and hochberf (bh) corrections for multiple testing is included as a large number of genes were included in analysis . degs with bh corrected adjp-values < . and fold change > . were considered significant degs. to visualize gene expression values of degs volcano plots, barplots and density plots were made using ggplot _ . . r package . consensus degs were obtained by overlapping deg lists produced by limma, edger and simple comparison of means with a significance of overlap p-value < . as calculated with geneoverlap_ . . and visualized by venndiagram_ . . r packages , . consensus degs that belonged to atleast two of the three deg lists produced by limma, edger and simple comparison of means were designated significantly enriched genes or degs in proliferative and quiescent stem cell states-simply referred to as ( ) proliferative stem cell marker genes and ( ) quiescent stem cell marker genes. following is a detailed description of all deg analysis contrasts, sample size for each dataset and abbreviations used to represent proliferative and quiescent stem cell marker genes: (a) adult proliferative neural progenitor cells (pnpcs) vs adult quiescent neural stem cells (qnscs) deg analysis to identify genes enriched in pnpcs relative to qnscs in mouse dataset with series number gse and sample size of each, abbreviated as gse _pnpcbyqnsc (b) adult quiescent neural stem cells (qnscs) vs adult proliferative neural progenitor cells (pnpcs) deg analysis to identify genes enriched in qnscs relative to pnpcs in mouse dataset with series number gse and sample size of each, abbreviated as gse _qnscbypnpc (c) adult hippocampal stem cells in proliferative condition or transient amplifying progenitor cells (taps) vs adult hippocampal stem cells in quiescent condition or quiescent progenitor cells (qnps) deg analysis to identify genes enriched in taps relative to qnps in rat dataset with series number gse and sample size of each, abbreviated as gse _tapbyqnp (d) adult hippocampal stem cells in quiescent condition or quiescent progenitor cells (qnps) vs adult hippocampal stem cells in proliferative condition or transient amplifying progenitor cells (taps) deg analysis to identify genes enriched in qnps relative to taps in rat dataset with series number gse and sample size of each, abbreviated as gse _qnpbytap (e) adult proliferative sub-ventricular zone stem cells (psvzscs) vs adult quiescent sub-ventricular zone stem cells (qsvzscs) deg analysis to identify genes enriched in psvzscs relative to qsvzscs in mouse microarray dataset with series number gse and sample size of each, abbreviated as gse _psvzscbyqsvzsc (f) adult quiescent sub-ventricular zone stem cells (qsvzscs) vs adult proliferative sub-ventricular zone stem cells (psvzscs) deg analysis to identify genes enriched in qsvzscs relative to psvzscs in mouse microarray dataset with series number gse and sample size of each, abbreviated as gse _qsvzscbypsvzsc (g) gbm cells cultured in proliferative condition or proliferative gbm cells (pgbcs) vs gbm cells cultured in quiescent condition or quiescent gbm cells (qgbcs) deg analysis to identify genes enriched in pgbcs relative to qgbcs in human dataset with series number gse and sample size of and , respectively, abbreviated as gse _pgbcbyqgbc (h) gbm cells cultured in quiescent condition or quiescent gbm cells (qgbcs) vs gbm cells cultured in proliferative condition or proliferative gbm cells (pgbcs) deg analysis to identify genes enriched in qgbcs relative to pgbcs in human dataset with series number gse and sample size of and scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ , respectively, abbreviated as gse _qgbcbypgbc (i) gbm organoids cultured in proliferative condition or proliferative gbm organoids (pgbos) vs gbm organoids cultured in quiescent condition or quiescent gbm cells (qgbos) deg analysis to identify genes enriched in pgbos relative to qgbos in human dataset with series number gse and sample size , abbreviated as gse _pgbobyqgbo and (j) gbm organoids cultured in quiescent condition or quiescent gbm organoids (qgbos) vs gbm organoids cultured in proliferative condition or proliferative gbm organoids (pgbos) deg analysis to identify genes enriched in qgbos relative to pgbos in human dataset with series number gse and sample size of , abbreviated as gse _qgbobypgbo . enrichment of proliferative and quiescent stem cell marker genes in glioma modules. enrichment of proliferative and quiescent stem cell marker genes identified by differential gene expression analysis above, in wgcna and megena modules was determined using uselistenrichment r function , , . additionally, a supplementary table containing a set of genes potentially involved in transition of gbm from stem-like state to differentiation identified by swim tool were downloaded directly from the published paper , . enrichment of swim gbm list in wgcna and megena modules was also determined using uselistenrichment r function , , to determine proliferative and quiescent stem cell marker genes common between all five stem cell datasets (gse , gse , gse , gse and gse ), the following consensus significantly enriched genes or degs in proliferative and quiescent states were overlapped, respectively, using online tool https ://www.molbi otool s.com/listc ompar e.html : (a) for proliferation comparison of (gse _pnpcbyqnsc + gse _ tapbyqnp + gse _psvzscbyqsvzsc + gse _pgbcbyqgbc + gse _pgbobyqgbo) and (b) for quiescence comparison of (gse _qnscbypnpc + gse _qnpbytap + gse _ qsvzscbypsvzsc + gse _qgbcbypgbc + gse _qgbobypgbo). to determine proliferative and quiescent stem cell marker genes common to both normal stem cells and gbm in culture, following consensus significantly enriched genes or deg lists in proliferative and quiescent states were overlapped, respectively, using online tool https ://www.molbi otool s.com/listc ompar e.html (a) comparison of normal stem cells (gse _pnpcbyqnsc + gse _tapbyqnp + gse _ psvzscbyqsvzsc) and gbm cell cultures (gse _pgbcbyqgbc + gse _pgbobyqgbo) deg sva + lm approach reduces batch effects in rna-seq datasets. to identify gbm specific transcriptome features, a meta-analysis was performed with glioma and control brain human rna-seq samples. sva + lm normalization reduced variability due to batch effects as indicated by greater overlap of expression data in density plots after sva + lm normalization (fig. a) . box-whiskers plots showed a slightly skewed mean expression in gse dataset that was fixed by sva + lm normalization (fig. b) . pca plots showed that sva + lm normalization reduces dispersion of samples from same study (fig. c) . correlation plots to evaluate correlation of gene expression values among different studies, showed an increase in positive correlation after sva + lm normalization (fig. d) . thus, sva + lm normalization may be used to achieve reduction in batch effects in global gene expression when rna-seq studies are combined. network analysis reveals gbm associated modules within glioma network. glioma gene coexpression networks were constructed with wgcna and megena to uncover underlying molecular mechanisms. wgcna identified modules with largest module huagegbsplit_ comprising of , genes and smallest module huagegbsplit_ comprising of genes (table ) . megena identified modules with largest module c _huagegbsplit_ comprising of , genes and smallest modules c _huagegb-split_ / / comprising of genes each ( table ) . to identify gbm associated modules, a module eigengene was used to represent overall expression pattern of each module produced by wgcna and megena. spearman correlations were calculated for gbm and other clinical traits, such as batch, age, and gender. eight modules in wgcna, with module size ranging from genes in huagegbsplit_ to genes in hu_agegbsplit_ , were found to be significantly associated with gbm ( fig. a,b) . all wgcna modules, including gbm associated modules, were preserved with megena modules (fig. c) . comparison of gbm specific wgcna modules with megena modules showed a significant overlap of all wgcna gbm modules with megena modules (fig. d ). all megena modules that significantly overlapped with gbm wgcna modules, with module size ranging from genes in c _huagegbsplit_ to genes in c _huagegbsplit_ , were also significantly associated with gbm ( fig. e,f) . thus, wgcna and megena complemented each other and helped identify gbm specific modules in largely preserved glioma wgcna and megena gene networks. differential gene expression analysis reveals proliferative and quiescent stem cell marker genes. to identify genes specific to quiescent and proliferative states of stem cells, differential gene expression analysis was performed on different stem cell datasets as described under methods. in mouse adult hippocampal stem cell dataset, , ( , human gene symbols) and , ( , human gene symbols) degs with a fold change of . (p-value < . ) were identified in proliferation (gse _pnpcbyqnsc) and quiescence (gse _pnpcbyqnsc), respectively (fig. a,b) . in rat adult hippocampal stem cell dataset, , ( , human gene symbols) and , ( , human gene symbols) degs with a fold change of . (p-value < . ) were identified in proliferation (gse _tapbyqnp) and quiescence (gse _qnpbytap), respectively ( fig. c,d) . in mouse adult subventricular zone stem cell dataset, , ( , human gene symbols) and , ( , human gene symbols) degs with a fold change of . (p-value < . ) were identified in proliferation (gse _psvzscbyqsvzsc) and quiescence (gse _qsvzscbypsvzsc), respectively (fig. e,f) . in human gbm cell culture dataset, , and , (human gene symbols) degs with a fold change of . (p-value < . ) were identified in proliferation (gse _pgbcbyqgbc) and quiescence (gse _qgb-cbypgbc), respectively (fig. g,h) . in human gbm organoid culture dataset, and , (human gene symbols) degs with a fold change of . (p-value < . ) were identified in proliferation (gse _pgbobyqgbo) and quiescence (gse _qgbobypgbo), respectively (fig. i ,j). stem cell marker genes common between all normal stem cells and gbm culture datasets (gse , gse , gse , gse and gse ), consisted of proliferation genes or degs (acyp , akap , lrp , mysm , slc a , tert, tspan ) and quiescence genes or degs (ethe , fzd , ninj , p rx , ptp a ) ( tables , ). overlapping stem cell marker genes obtained from normal stem cell datasets (gse , gse and gse ), with gbm culture datasets (gse and gse ), showed an overlap of , proliferation genes ( . % of gbm proliferation degs) and only quiescence genes ( . % of gbm quiescence degs (supplementary tables s a,b ). showed that huagegbsplit_ wgcna gbm module and its equivalent c _huagegbsplit_ / megena gbm modules, were the only gbm modules significantly enriched with proliferative and quiescent stem cell marker genes (fig. a,b) . comparison of genes in c _huagegbsplit_ and c _huagegbsplit_ megena modules, revealed that all genes in c _huagegbsplit_ were also present in c _huagegbsplit_ ( grade_ grade_ grade_ con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con con concon con con con con con con con con con con a different color is used for each study logistic regression model built with quiescent stem cell marker genes in gbm modules. a logistic regression model was built with select quiescent stem cell marker genes (gse _qnpbytap) to diagnostic between control and gbm samples. from a total of genes from gse _qnpbytap enriched in gbm wgcna module huagegbsplit_ , genes that were atleast -fold upregulated in qnp relative to tap were selected (cd , cend , dchs , smpd , tpp , gatd , rnh and smcr ) for logistic regression. effects plots showed that probability of gbm relative to control increases with increased expression of cend , dchs , tpp , gatd , rnh and smcr (fig. b ,c,e-h) and decreases with increased expression of cd and smpd (fig. a,d) . logistic regression model with these genes without gene-gene interaction term was not significant (chi-square p-value = . ), while with gene-gene interactions the model was a significant (chi-square p-value = . ) predictor for gbm (fig. i ,j). hosmer-lemeshow gof test on without gene-gene interaction logistic regression model showed a large difference between observed and expected probabilities, and there was significant evidence of poor fit (p-value = . , less than . ) (fig. i , table ). therefore, in logistic regression model without gene-gene interaction, ho is rejected and ha is accepted, and the model is rejected for being a poor fit for the data. hosmer-lemeshow gof test on with gene-gene interaction logistic regression model showed a small difference between observed and expected probabilities, and there was no significant evidence of poor fit (p-value = , greater than . ) (fig. j, table ). therefore, in logistic regression model with gene-gene interaction, ho is accepted and the model is accepted as a good fit for the data. tumors are commonly treated by surgical removal, chemotherapy, radiotherapy and immunotherapy . safe surgical removal of glioma is challenging due to its critical location in brain. additionally, high grade glioma or gbm is highly resistant to chemotherapy and radiotherapy. immunotherapy treatments are fda approved for certain blood cancers, but for solid tumors such as gbm immunotherapy ineffective due to incomplete infiltration of immunotherapeutic agent and immune suppression by tumor microenvironment , . diagnosis is the first c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ , c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ ii c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ www.nature.com/scientificreports/ step in development of an effective treatment plan for any disease, including glioma. gbm is the most aggressive form of glioma that advances quickly giving healthcare providers limited time for diagnosis and treatment . therefore, to gain deeper understanding of glioma, especially gbm, with hope to develop early accurate diagnosis tools and novel therapies, molecular profiling and network medicine have emerged as research forerunners. molecular profile or gene based classification and diagnosis help physicians plan treatment and predict clinical outcome. for example, idh gene mutation is highly correlated with glioma survival and is therefore used for glioma classification . idh mutation is lowest in grade glioma that correlates with slow tumor growth and good survival . on the other hand, idh mutation is highest in grade glioma or gbm that correlates with fast tumor growth and poor survival . improvement in technology, reduced cost of high-throughput sequencing, extensive collaborations and data sharing have made a plethora of glioma molecular profiling datasets such as rna-seq available to research community , . with the explosion of molecular profiling genomics big data, research focus has now shifted from big data mining to big data analysis to prioritize a set of genes with diagnostic value that would eliminate need to profile all k protein coding genes from glioma samples. however, prioritization of a subset of genes for glioma diagnosis and classification has been challenging due to high cellular heterogeneity across and within tumor samples of glioma . stem cell-like cells in glioma are thought to be responsible for tumor initiation, progression and recurrence . chemotherapy and radiotherapy kill proliferative stem cells, but are unable to kill quiescent stem cells in the tumor. quiescent stem cells left in the tumor at end of treatment enter a proliferative state and reconstitute tumor, which leads to tumor recurrence . therefore, here the goal was to identify distinct sets of proliferative and quiescent stem cell marker genes in gbm that can be used for diagnosis and can serve as potential drug targets. a meta-analysis was performed on publicly available high-throughput gene expression datasets from human glioma samples, control human brains, normal stem cells and gbm cells in quiescent and proliferative states , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . degs specific to stem cell states were identified from normal stem cell and glioblastoma cell culture datasets in quiescent and proliferative states. interestingly, only . % and . % of genes from gbm cell cultures in proliferative and quiescent states were common with normal stem cells in proliferative and quiescent states, respectively (supplementary table s a,b). this suggests that cancer stem cells, especially those in quiescent state are distinctly different from normal stem cells. network analysis facilitates grouping of genes with highly correlated gene expression patterns into modules. it is assumed that modules corelate with distinct biological and cellular states, such as diseases and cell types, respectively. presently, network analysis identified wgcna modules and megena modules that were highly correlated with gbm (fig. b,f) . one of these wgcna modules, huagegbsplit_ wgcna module (equivalent c _huagegbsplit_ / megena modules) was also significantly enriched with adult hippocampal rodent quiescent stem cell genes (gse _qnpbytap) (fig. b) . interestingly, though this quiescent stem cell marker enriched huagegbsplit_ wgcna module (equivalent c _huagegbsplit_ / megena c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ c _huagegbsplit_ www.nature.com/scientificreports/ modules) had a significant correlation with gbm (p-value . ) it had a small correlation value of . with gbm (fig. b) . possible reasons for this small but significant correlation are discussed here: (a) the result is consistent between wgcna and megena, two completely different network analysis algorithms. this supports that the results are biologically robust and not a computational artifact that would alter based on alterations in default algorithm settings. (b) sva + lm normalization was used in this study to retain effects of glioma on gene expression and remove effects of all other covariants. it is possible that effects of covariants such as batch effects are not completely removed by this normalization, which is confounding glioma gene expression effects. (c) it is possible there are other covariants that significantly effect gene expression, such as patients' comorbidities. however, as this information was not available, it could not be included in sva + lm normalization and therefore glioma effects could not be effectively retained. (d) controls used in this study comprise of rna-seq datasets from different parts of the brain derived from humans other than the patients themselves. ideally control tissue should be derived from the same patient who has the glioma, but presently such patient matched controls were not available for analysis. lack of patient matched controls is a common challenge in the field of glioma and human disease research. quiescent stem cells exist in non-proliferative g cell cycle phase, but retain ability to reversibly enter cell cycle in response to stimuli. depletion of surrounding proliferative stem cells and differentiated cells stimulate quiescent stem cells, which are multipotent and have self-renewal potential, to enter cell cycle and replenish the tissue . quiescent stem cell properties of rodent hippocampal stem cells have been extensively experimentally characterized , . though proliferative stem cell marker genes of gbm origin (pgbcbyqgbc) were significantly enriched in huagegbsplit_ wgcna gbm module (equivalent c _huagegbsplit_ / megena gbm modules) with p-value . e- , it was normal quiescent stem cell marker genes from rodent hippocampal stem cells (gse _qnpbytap) that were most significantly enriched with p-value . e- (fig. b) . normal stem cells transition from quiescent state to proliferative state and further to differentiated state. recently, a set of genes were identified in gbm with swim network analysis method, which are potentially involved in transition from stem-like state to differentiated state , . interestingly, no significant enrichment (p-value . , overlap of genes) of swim glioblastoma gene list was found in huagegbsplit_ gbm wgcna module (equivalent c _huagegbsplit_ / megena gbm modules). this supports that quiescent stem cell marker genes (gse _qnpbytap) enriched in huagegbsplit_ wgcna gbm module (equivalent c _huagegbsplit_ / megena gbm modules) represent an undifferentiated quiescent stem cell state distinct from differentiating stem cells. gene ontology (go) analysis of gse _qnpbytap enriched huagegbsplit_ wgcna gbm module (equivalent c _huagegbsplit_ / megena gbm modules) revealed enrichment of biomolecule synthesis table . common stem cell markers of proliferation. common to degs listed below from normal stem cells and glioblastoma in proliferative conditions www.nature.com/scientificreports/ go terms, such as lipid metabolism, dna modification, protein post-translational modification, ribosome rna processing, cell cycle go terms (g /m transition, mitotic cell cycle) and signaling pathways such as wnt signaling ( fig. c-e) . this is consistent with cell cycle and ribosome biogenesis go terms, previously reported in the rodent hippocampal stem cell dataset from which gse _qnpbytap signature genes were identified . as quiescent stem cells can replenish tumor after proliferative stem cells are killed by chemotherapy and radiotherapy, a combinatorial therapy that targets both proliferative and quiescent stem cells could be more effective in gbm treatment. quiescent stem cell marker genes (gse _qnpbytap) enriched in huagegbsplit_ wgcna gbm module (equivalent c _huagegbsplit_ / megena gbm modules) reported here, could serve as potential gbm quiescent stem cell drug targets. small molecule dyrk b inhibitors were recently shown to target quiescent stem cells and potentiate treatment benefits of chemotherapy . this supports development of treatment strategies to target both proliferative and quiescent stem cells in gbm. gene expression of eight genes (cd , cend , dchs , smpd , tpp , gatd , rnh and smcr ) from gse _qnpbytap enriched in huagegbsplit_ wgcna gbm module (equivalent c _huageg-bsplit_ / megena gbm modules), were sufficient to build a logistic regression diagnostic model that could distinguish between gbm and control samples (fig. j) . four of the eight genes (cd , cend , smpd and rnh ) used in the model have previously been reported to be important in gbm, other types of cancer or in development [ ] [ ] [ ] [ ] . cd is a member of tetraspanins scaffolding protein family that is involved in cell-cell adhesion, integrin interaction, cell signaling, cancer progression and metastasis . in gbm, cd associates with α β integrin to potentiate egfr signaling, drive cancer cell motility and tumor aggressiveness . cend or bm protein acts as a cell-cycle inhibitor to negatively regulated proliferation and promote differentiation in spinal cord development . in cell membrane lipid bilayer, apoptosis and cellular growth is regulated by balance between sphingosine- -phosphate and ceramide molecules . smpd gene that regulates 'ceramide sphingosine- -phosphate rheostat' drives tumor growth and immune escape in non-small cell lung cancer . rnh repairs dna in response to dna damage and is a known diagnostic and prognostic marker of glioma . taken together, this provides support for biological relevance of the eight genes (cd , cend , dchs , smpd , tpp , gatd , rnh and smcr ) used here to build a logistic regression diagnostic model for gbm. a molecular table . hosmer and lemeshow goodness of fit (gof) test for model without gene-gene interaction shows significant difference between observed and expected probabilities (p-value = . , < . ). model without gene-gene interaction: diseasegrade_ ~ cd + cend + dchs + smpd + tpp + gatd + rnh + smcr . www.nature.com/scientificreports/ screening kit could be developed with these eight genes for faster and accurate screening of gbm. however, as results of present study were obtained by using computational meta-analysis alone, further experimental validation is required. overall, this study deconvolutes highly heterogeneous glioma molecular profiles and provides a new perspective to diagnose and develop therapeutic strategies using a small number of quiescent stem cell markers in gbm. glioblastoma rna-seq datasets, srp and srp , were obtained from ncbi sra. other rnaseq datasets for control brains (gse , gse , gse and gse ) and stem cells (gse , gse , gse , gse and gse ), were obtained from ncbi geo. the author is most grateful to ncbi sra and ncbi geo for storing and making these datasets open access. the author also appreciates the organizations and investigators who generously submitted these datasets for sharing to ncbi geo and ncbi sra. received: december ; accepted: june literature review of spinal cord glioblastoma survival in glioblastoma: a review on the impact of treatment modalities overview of disease and treatment the world health organization classification of tumors of the central nervous system: a summary the who classification of tumours of the central nervous system hallmarks of glioblastoma: a systematic review. esmo open , e current challenges and opportunities in treating glioblastoma glioblastoma treatments: an account of recent industrial developments the cancer stem cell gamble secretion-mediated stat activation promotes self-renewal of glioma stem-like cells during hypoxia 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cooperate with egfr to drive tumor cell motility and invasion cd in cancer progression and metastasis: a complex scenario ceramide/sphingosine/sphingosine -phosphate metabolism on the cell surface and in the extracellular space this work was conducted at the author's home using personal resources at a) sarat ghosh garden road, calcutta, west bengal, india and b) garland avenue, downtown los angeles, los angeles, u.s., on personal laptops (macbook pro and windows os) from to . the author would like to express deepest gratitude to springer nature waivers team and scientific reports team for the generous discount on publication cost. other open source and free resources utilized for this work are acknowledged below. author contributions s.m. ideated and managed the work presented in this paper. s.m. also performed experimental design, wrote the manuscript, prepared the figures/tables and submitted the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to s.m.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - dljap authors: young, ginger; bohning, kelly j.; zahralban-steele, melissa; hather, greg; tadepalli, sambasivarao; mickey, kristen; godin, c. steven; sanisetty, srisowmya; sonnberg, stephanie; patel, hetal k.; dean, hansi j. title: complete protection in macaques conferred by purified inactivated zika vaccine: defining a correlate of protection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dljap a critical global health need exists for a zika vaccine capable of mitigating the effects of future zika epidemics. in this study we evaluated the antibody responses and efficacy of an aluminum hydroxide adjuvanted purified inactivated zika vaccine (pizv) against challenge with zika virus (zikv) strain prvabc . indian rhesus macaques received two doses of pizv at varying concentrations ranging from . µg − µg and were subsequently challenged with zikv six weeks or one year following the second immunization. pizv induced a dose-dependent immune response that was boosted by a second immunization. complete protection against zikv infection was achieved with the higher pizv doses of . µg, µg, and µg at weeks and with ug pizv at year following vaccination. partial protection was achieved with the lower pizv doses of . µg and . µg. based on these data, a neutralizing antibody response above . log( ) ec was determined as a correlate of protection in macaques. pizv elicited a dose-dependent neutralizing antibody response which is protective for at least year following vaccination. in and , large outbreaks of zika virus (zikv) occurred in the americas. these outbreaks were associated with clusters of congenital microencephaly and other severe neurological sequelae in infections in approximately of infants born to pregnant women with laboratory confirmed zika in the us and us territories . incidence of zikv infections subsequently declined in most of the americas throughout and . with the sporadic nature of zikv outbreaks and a very low incidence of symptomatic disease in both endemic and non-endemic areas, conducting phase clinical efficacy trials is not feasible. still, the risk of re-emergence and the severe consequences of infection in pregnant women demonstrate that the need for an effective zika vaccine remains. in such circumstances, alternative regulatory strategies such as animal rule approval or accelerated approval pathway may be relevant for licensure . non-human primate studies have contributed to the development of zikv vaccines by demonstrating protective efficacy and identifying biomarkers of protection against zikv. results to date have supported neutralizing antibodies as an immune marker that is reasonably likely to predict clinical benefit of several zikv vaccines , . indian rhesus macaques (macaca mulatta) are susceptible to zikv infection and have been used extensively as a model to study efficacy of zikv vaccines and pathogenesis of multiple zikv isolates [ ] [ ] [ ] [ ] [ ] . zikv infection can be performed by subcutaneous injection, which mimics infection via mosquito bite and causes consistent viremia [ ] [ ] [ ] [ ] [ ] . the kinetics of zikv infection are similar in rhesus macaques and humans where serum or plasma viremia typically peaks within the first six days of infection and resolves within - days , , . the purified inactivated zika vaccine (pizv) has previously been evaluated in mouse models and was immunogenic in ag and cd mice and protected ag mice against lethal zikv challenge . in those studies, baldwin et al. demonstrated that neutralizing antibodies correlate with protection in ag mice. pizv is currently being evaluated for safety and immunogenicity in phase trials (clinicaltrials.gov nct ). pizv elicits a dose dependent neutralizing antibody response. rhesus macaques were vaccinated with pizv on days and and challenged on day . serum neutralizing antibodies were tested using a zika reporter virus particle (rvp) assay. all macaques were seronegative on day and control macaques remained seronegative prior to zikv challenge (fig. ) . twenty-eight days following the first vaccine dose (study day ), little or no seroconversion was observed for the . and . µg pizv doses, while / macaques vaccinated with the . µg pizv dose seroconverted and / macaques receiving the and µg pizv dose seroconverted. immune responses were boosted in all groups after the second dose (all p-values ≤ . ). twenty-eight days after the second pizv dose (study day ), % of vaccinated macaques seroconverted. the magnitude of neutralizing antibody titers on day was similar after the second dose of . , , or µg, with no statistically significant difference detected among these groups. the titer on day was lower in the . (p = . ) and . µg groups www.nature.com/scientificreports www.nature.com/scientificreports/ (p = . ) compared to the . µg dose group. titers prior to zikv challenge on day were slightly decreased compared to day . following challenge, neutralizing antibody titers increased significantly in the placebo, . and . µg groups (all p-values ≤ . ), but not in the . , , and µg groups. neutralizing antibody levels after two pizv doses of . , , or µg were similar to post-challenge neutralizing antibody titers in the placebo group. in conclusion, we observed a dose-dependent neutralizing antibody response to one or two doses of pizv. after two pizv doses, the neutralizing antibody levels reached a plateau for vaccine doses above . µg, and the level of neutralizing antibody was comparable to infection with zikv challenge in the placebo group. the lack of anamnestic antibody responses after challenge in the . , , and µg pizv dose groups suggests that infection by zikv challenge virus may have been prevented by vaccination. in addition to evaluating neutralizing antibodies, we also quantified anti-zika-specific igg using a luminex-based assay. as with neutralizing antibodies, all pizv doses were immunogenic and no anti-zika igg was detected in the control group prior to zikv challenge. pizv elicited dose-dependent anti-zika igg responses (fig. ) in the vaccinated groups. anti-zika igg responses were significantly boosted after a second pizv dose for all vaccinated groups (all p-values ≤ . ). following zikv challenge, the anti-zika igg responses increased significantly in the placebo, . and . µg groups (all p-values ≤ . ), but not in the . , , and µg groups. similar to the neutralizing antibody response, the anti-zika igg titers following vaccination with the . , , or µg doses were similar to post-challenge anti-zika igg titers in the placebo group. pizv protects against zikv challenge. rhesus macaques were challenged subcutaneously on day with ffu prvabc . all macaques receiving the . , , or µg pizv dose were protected against zikv challenge, as no zika vrna could be quantified in any of the macaques in these groups. zika vrna was quantified in / macaques receiving the . µg dose, / macaques receiving the . µg dose, and in all macaques in the control group (fig. ) . the peak post-challenge zika vrna level decreased with increasing vaccine dose level. the peak zika vrna occurred on day ( days post-challenge), with a geometric mean level of . log copies/ ml in the control group (range . - . log copies/ml), . log copies/ml in the . µg dose group (range . - . log copies/ml) and . log copies/ml in the . µg dose group (range . - . log copies/ml). no clinical signs to the vaccine or challenge virus were seen throughout the studies. correlate of protection. pizv elicited a dose dependent neutralizing antibody immune response and an anti-zika igg response which correlated with a reduction in zikv vrna post-challenge (table ). an immune correlate analysis was subsequently performed to correlate zika vrna with neutralizing antibody titers (fig. ) and anti-zika igg (fig. ) , which demonstrated that an increase in both neutralization antibody and anti-zika igg titers correlates with a decrease in vrna concentration. correlation with protection was observed with both neutralizing antibodies ( . log ec ) and anti-zika igg ( . log u/ml). we chose the functional assay, neutralizing antibodies, to establish a correlate of protection in macaques as a neutralizing antibody titer of > . log ec , which conferred protection against zikv serum viremia in indian rhesus macaques. due to overlap among the distributions of neutralizing antibody titers between the protected and infected animals, titers in some protected animals were below the correlate of protection. to determine the kinetics of neutralizing antibodies over a year long period, a separate set of four male indian rhesus macaques were vaccinated with µg pizv on study days and and challenged with zikv prvabc on study day . zika neutralizing antibody levels were measured every days through day (except for days , , and ), as well as on day ( days post-zikv challenge). all macaques seroconverted following the first vaccine dose, with a significant boost in antibody titers following the second dose (p < . ; fig. a ). neutralizing antibody titers peaked on day , days following the second immunization, declined from day to day , and then remained stable from day www.nature.com/scientificreports www.nature.com/scientificreports/ to day . anti-zika igg levels also peaked on day and subsequently declined through study day (fig. b) . igg titers increased following zikv challenge. no zika vrna was detected in serum from any of the macaques following zikv challenge year following the second dose. in addition, no statistically significant increase in neutralizing antibodies was observed post-zikv challenge on day (log ec range of . - . on day compared to log ec range of . - . on day ). the combined results suggest that two doses of pizv prevented zikv infection year post-vaccination. in conclusion, two µg pizv vaccinations elicits persistent neutralizing antibodies and long-term protection in rhesus macaques. we demonstrated that pizv elicits a dose-dependent response of both zika neutralizing and anti-zika igg antibodies. pizv elicited neutralizing antibodies that persisted for at least year and protected against zikv challenge. vaccinating with a broad range of pizv dose levels enabled us to correlate both neutralizing and anti-zika igg antibody titers to protection against zikv infection. we determined a neutralizing antibody correlate of protection of . log ec , which we define as the maximum ec among the unprotected animals in the study. we chose the neutralizing antibody assay to establish a correlate of protection as the literature supports using functional neutralizing antibodies as a correlate of protection for zika and other flaviviruses. table . neutralizing antibody titers on day of zikv challenge, zika vrna levels post-challenge. summary of mean neutralizing antibody titers (log ec ), range of peak vrna post-zikv challenge (log copies/ml), and percent of macaques with quantifiable zika vrna post-zikv challenge. all reported and analyzed data is above the rt-qpcr lloq. < lloq = detected vrna was below the assay lower limit of quantitation. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ neutralizing antibodies directed against the envelope (e) protein have been identified as correlates of protection for vaccines japanese encephalitis virus (jev), yellow fever virus, and tickborne encephalitis viruses . other laboratories using different vaccine platforms (dna, rna, inactivated virus, protein subunit, adenovirus-vectored, vlp) have reported induction of zikv-specific neutralizing antibodies that conferred protection against live zikv challenge in animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these data support zikv e-specific neutralizing antibodies as a mechanism of protection against zikv infection. in flavivirus vaccine development, neutralizing antibody titers that can confer protection against viremia have be reported in animal models using several different assay methods: microneutralization (mnt), rvp, and plaque reduction neutralization (prnt). studies of other candidate zikv vaccines have demonstrated that a neutralizing antibody titer as low as , using a mnt assay, or a titer of , using an rvp assay can confer protection against viremia in animal models , , . these results are qualitatively similar to those of licensed vaccines against flaviviruses such as jev, where a neutralizing antibody titer of > (as measured by prnt) is considered to determine the correlate of protection, the neutralizing antibody titers (log ec ) of infected (positive for vrna at any given timepoint) and protected (negative for vrna) macaques were plotted. the correlate of protection was defined as the maximum neutralizing antibody titer across all unprotected macaques in this study. based on this definition, the correlate of protection was established as > . log ec . cop = correlate of protection. www.nature.com/scientificreports www.nature.com/scientificreports/ to correlate with protection [ ] [ ] [ ] . further supporting this correlate of protection, it has been reported that passive transfer of zika neutralizing monoclonal antibodies or antisera from vaccinated humans and animals to mice is sufficient to protect against zikv challenge , , , . adoptive transfer of serum from immunized mice fully protected against viremia, while splenocytes from the same donors provided marginal protection demonstrating that the mechanism of protection against zikv infection is antibody mediated. while zikv exists as three genotypes (west african, east african and asian), they all behave as a single serotype , . our previous studies demonstrated that pizv is capable of eliciting antibodies that neutralize both african and asian zikv isolates in vitro . others have shown that the magnitude or duration of viremia in unvaccinated macaques challenged with zikv isolates from brazil or puerto rico is similar , and that vaccinated macaques are protected against challenge with heterologous zikv strains , , . protection of animal models against heterologous challenge and cross-neutralization capabilities of antisera from multiple vaccine platforms , , , suggest that data from a single challenge strain may be sufficient to show cross protection against zikv strains from other lineages. several groups have developed rhesus macaque challenge models using zika strain prvabc , , , , which is a well-documented and characterized isolate from human serum and is representative of viruses that were circulating in the americas during the - outbreak . we therefore selected prvabc as the challenge strain for our studies. in this study, we extended our observation of pizv efficacy in mice to demonstrate efficacy in prevention of zikv vrna and to evaluate anamnestic antibody responses after zikv challenge in non-human primates. we did not assess presence of vrna in tissues. our working hypothesis is that prevention of serum zikv vrna may be a surrogate for prevention of the most serious sequelae of zikv infection in humans -fetal infection. by employing a pizv dose titration study design and a zikv rvp assay, we have established a minimum protective vaccine dose of . µg in rhesus macaques and established a neutralizing antibody correlate of protection against zikv challenge of . log ec . finally, we demonstrated that zikv neutralizing antibodies persist and are capable of preventing zikv infection for at least year post-vaccination. in the event that human phase efficacy studies are not feasible, identifying a correlate of protection in an appropriate non-human primate challenge model may be important to support licensure of a zika vaccine , , . altogether, these data support neutralizing antibodies as an immune marker that is associated with efficacy in a relevant animal model and that may predict a reasonable likelihood of clinical benefit in humans. vaccine. pizv is an aluminum hydroxide adjuvanted whole purified inactivated virus vaccine based on zikv strain prvabc which was originally isolated from serum from a human infected in puerto rico (genbank accession number ku ). pizv has been previously described and characterized by baldwin et al. . the same lot of pizv used in this study was also used in clinical trial zik (clinicaltrials.gov nct ). rhesus macaque challenge studies. thirty-five indian rhesus macaques were separated into groups. three male and three female macaques per group were vaccinated intramuscularly with either . , . . . , or µg pizv on study days and . to achieve statistical significance comparing vaccine doses, six animals/ group were selected to obtain > % power if the true infection rate among controls is ≥ % and the true infection rate among vaccinated animals is ≤ % based on a one-sided fisher's exact test with % type error rate . two male and three female macaques were vaccinated with placebo control (pbs) on study days and . one female from the control group was euthanized on the day of zikv challenge due to reasons unrelated to the study, resulting in macaques in the control group. macaques were challenged subcutaneously with ffu/ . ml zikv prvabc on study day . serum samples were collected and tested for antibody titers on study days , , , and , and , and for zikv rna on study days - , and study day . www.nature.com/scientificreports www.nature.com/scientificreports/ a second study was conducted to assess long-term pizv immunogenicity and efficacy. flavivirus seronegative male rhesus macaques (n = ) were vaccinated with the µg pizv dose on days and and challenged subcutaneously with ffu/ . ml zikv prvabc on day . serum samples were collected and tested for antibody titers monthly up to year post-vaccination and days following zikv challenge (study day ). to assess replication of zika vrna following zikv challenge, serum samples were collected on study days - , and . challenge virus. zika virus puerto rico strain prvabc was used for the challenge ( . ml containing a nominal dose of ffu). the seed stock was passaged three times on vero cells at cdc, fort collins, colorado. virus stocks were prepared by passaging two times on c / mosquito cells. stocks were from a master working virus bank and tested for sterility, mycoplasma and endotoxin prior to use. screening. to select flavivirus naïve macaques, baseline igg serostatus was determined using a multiplex luminex kit (ampersand biosciences flavivirus serological panel). briefly, macaque serum was diluted : , in sample diluent and added to multiplexed magnetic beads coupled with zika, dengue, yellow fever, japanese encephalitis, west nile, usutu, saint louis encephalitis, and chikungunya virus antigens. serum and beads were incubated on a plate shaker for hour at room temperature and then washed three times with assay buffer. detection antibody, anti-igg pe, was added to each sample and incubated on a plate shaker for minutes at room temperature. following another three wash cycles, beads were resuspended in assay buffer and read on the bio-plex magpix to measure median fluorescent intensity (mfi) for each bead set. macaques were considered flavivirus naïve and selected for the study if the mfi was below pre-defined assay cutoff criteria for seronegativity for all antigens. neutralizing antibodies. a zika rvp assay (sonnberg et al., manuscript in preparation) was used to determine neutralizing antibody titers in serum following the administration of pizv (study days , , , and ), and days post-zikv challenge (day ). briefly, macaque serum was heat inactivated at °c for minutes and then serially diluted -fold in assay media for an -point dilution series. diluted serum and rvp were plated in duplicate in a -well assay plate and incubated for hour at °c. vero cells were added to each well and incubated at °c for hours. renilla-glo substrate (promega, wi, usa) was then added to the plate and incubated for minutes at room temperature. finally, plates were analyzed by a luminometer. the effective concentration at % (ec ), was determined by a non-linear regression curve fit with the lower asymptote constrained to in graphpad prism. the lloq for the nhp assay is . log ec , below which the serum matrix interfered with the measurement. the upper limit of quantitation (uloq) of the standard assay is . log ec . any samples returning titers >uloq were retested with a higher initial pre-dilution. anti-zika igg binding antibodies. anti-zika igg antibody levels were measured for study days , , , and (prior to zikv challenge) using a luminex based anti-zika igg assay. heat-inactivated serum samples were diluted : in assay buffer and then serially diluted -fold for an -point dilution curve. magnetic beads covalently coupled with pizv antigen were added to each sample dilution in a -well plate and each sample dilution was tested in duplicate. plates were incubated on a plate shaker for minutes at room temperature and then washed two times with assay buffer. diluted anti-ig-pe detection antibody was then added to each sample and incubated on a plate shaker for hour at room temperature. after two wash cycles, beads were resuspended in assay buffer and read on the luminex flexmap d to measure the mfi. the igg antibody concentration was quantified using a reference standard serum with an assigned igg concentration in units/ml (u/ml). the lloq for the assay is . log u/ml. total rna from serum samples was extracted using the qiagen biorobot universal instrument and qiaamp virus biorobot mdx kit (qiagen; valencia, ca). extracted and purified rna was evaluated in two separate rt-pcr assays. primarily, zika vrna was detected and quantified using rt-qpcr with primers and probe specific to known zikv genotypes as described in lanciotti et al. and a standard curve generated from synthetic reference zika vrna (atcc). a qualitative extraction control rt-pcr was also performed. the extraction control rt-pcr utilized a primer/probe set, specific to macaca mulatta c galt c l mrna, confirmed adequate nucleic acid extraction from serum samples independent of zika vrna detection. the geometric mean, range of peak zika vrna detection, and the respective percentage of protected macaques (as defined by absence of vrna) was calculated using quantities greater than the assay lower limit of quantitation of . log copies/ml. samples with vrna concentration lower than the assay limit of detection of . log copies/ml were considered negative. correlate of protection. the mean neutralizing antibody titers (log ec ) determined by zika rvp assay was computed for each dose group at day (day of zikv challenge). zika vrna copies/ml determined by rt-qpcr assay were computed for each dose and timepoint post-challenge (study days - and ). peak vrna for each macaque was defined as the highest observed vrna concentration across all timepoints tested. macaques were considered protected if vrna was not detected or was below the assay lloq for all timepoints tested. the correlate of protection was defined as the maximum neutralizing antibody titer across all unprotected macaques in this study. this definition was conservative in that some protected macaques could have neutralizing antibody titer levels below the correlate of protection due to overlap between the distributions of protected and unprotected macaques. since the correlate of protection was not a statistical estimate, no confidence intervals were reported. statistical analysis. statistical tests were performed using the r software version . . .; log neutralization titers were compared for selected pairs of days for each dose group using a paired two-sided t-test. at day , tukey's test was used to compare all dose groups with each other. a two-sided spearman's test was applied to the unprotected macaques to check for an association between day neutralizing antibodies titers and peak vrna. comparisons between selected pairs of days were also performed with the log igg antibody levels in the main study and with the log neutralization titers from the long-term immunogenicity study using paired two-sided t-tests. a p-value threshold of . was used to determine statistical significance. the data that support the findings of this study are available from the corresponding author upon reasonable request and with permission of takeda vaccines, inc. vital signs: zika-associated birth defects and neurodevelopmental abnormalities possibly associated with congenital zika virus infection -u.s. territories and freely associated states who region of the americas/pan american health organization. plisa health information platform for the americas: cases of zika virus disease clinical development strategies and considerations for zika vaccine licensure correlates of protection induced by vaccination demonstrating vaccine effectiveness during a waning epidemic: a who/nih meeting report on approaches to development and licensure of zika vaccine candidates protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys rapid development of a dna vaccine for zika virus modified mrna vaccines protect against zika virus infection comparative pathogenesis of asian and african-lineage zika virus in indian rhesus macaque's and development of a non-human primate model suitable for the evaluation of new drugs and vaccines nonhuman primate models of zika virus infection, immunity, and therapeutic development macaque monkeys in zika virus research: -present characterization of a clinical isolate of zika virus in non-human heterologous protection against asian zika virus challenge in rhesus macaques a rhesus macaque model of asian-lineage zika virus infection zika virus infection of rhesus macaques leads to viral persistence in multiple tissues purified inactivated zika vaccine candidates afford protection against lethal challenge in mice protective efficacy of zika vaccine in ag mouse model zika virus protection by a single low-dose nucleoside-modified mrna vaccination in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunisation with a prmenv dna vaccine vaccine protection against zika virus from brazil durability and correlates of vaccine protection against zika virus in rhesus monkeys passive transfer of immune sera induced by a zika virus-like particle vaccine protects ag mice against lethal zika virus challenge report on a who consultation on immunological endpoints for evaluation of new japanese encephalitis vaccines yellow fever vaccine: direct challenge of monkeys given graded doses of d vaccine neutralizing antibodies protect against lethal flavivirus challenge but allow for the development of active humoral immunity to a nonstructural virus protein preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase , randomised, double-blind, placebo-controlled clinical trials a 'furry-tale' of zika virus infection: what have we learned from animal models? phylogeny of zika virus in western hemisphere a tale of two viruses: does heterologous flavivirus immunity enhance zika disease? zika viral dynamics and shedding in rhesus and cynomolgus macaques what is the predictive value of animal models for vaccine efficacy in humans? the importance of bridging studies and species-independent correlates of protection genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia correspondence and requests for materials should be addressed to g.y.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -kbwz xop authors: toubanaki, dimitra k.; margaroni, maritsa; prapas, athanasios; karagouni, evdokia title: development of a nanoparticle-based lateral flow strip biosensor for visual detection of whole nervous necrosis virus particles date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: kbwz xop effective analysis of pathogens causing human and veterinary diseases demands rapid, specific and sensitive detection methods which can be applied in research laboratory setups and in field for routine diagnosis. paper lateral flow biosensors (lfbs) have been established as attractive tools for such analytical applications. in the present study a prototype lfb was designed for whole particles (virions) detection of nodavirus or fish nervous necrosis virus. nodavirus is an important threat in the aquaculture industry, causing severe economic losses and environmental problems. the lfb was based on polyclonal antibodies conjugated on gold nanoparticles for signal visualization. brain and retinas from fish samples were homogenized, centrifuged and the supernatant was directly applied on the lfb. formation of a red test line was indicative of nodavirus virions presence. nodavirus visual detection was completed in short time ( min). key factors of the lfb development influencing the assays’ detection limit were characterized and the optimum parameters were determined, enabling increased efficiency, excluding non-specific interactions. therefore, the proposed lfb assay consists a robust, simple, low cost and accurate method for detection of nodavirus virions in fish samples. the proposed biosensor is ideal for development of a commercial kit to be used on aquaculture facilities by fish farmers. it is anticipated that disease monitoring and environmental safety will benefit from the simplification of time consuming and costly procedures. instrumentation. the thin layer chromatography (tlc) applicator linomat and wincats software were from camag (muttenz, switzerland). the desktop scanner hp scanjet g was from hp (california, usa) . the nanodrop spectrophotometer was purchased from thermo fisher scientific (delaware, usa) and the ultraviolet-visible spectroscopy plate reader was the mrx model from dynatech laboratories (chantilly, va, usa). fish samples. european sea bass (dicentrarchus (d.) labrax) samples with or without vnn clinical signs were collected from sea-cage fish farms in cyprus, saronikos, korinthian and amvrakikos gulfs. dissected brain and retinas were isolated aseptically and stored in sterile polypropylene tubes at − °c until use . ethical statement. the collection of biological samples from fish farms was conducted by licensed personnel of the respective aquaculture facility. all qualified personnel were previously informed of the purpose of the study, the confidentiality of the data, and their voluntary participation. the present study was approved by the hellenic pasteur institute animal bioethics committee regulations according to greek (pd / ) and eu (directive / ) legislation for the protection, care and use of animals used for scientific purposes and all samples used were euthanized in the sites of fish farming according to the ethical principles and other requirements of the law . virus preparation and titration. nodavirus was isolated from naturally infected d. labrax fish. all infected samples were verified by rt-pcr . the virus was propagated in ssn- cells as follows: fish brains were homogenized either in emem or leibovitz l -medium containing % fbs. centrifugation ( , × g, min, °c) of the homogenates ( % w/v) resulted in supernatant, which was filtered through . μm filters and was inoculated on cell cultures. following inoculation, the ssn- cells were cultured at °c, in falcon primaria cell culture flasks containing leibovitz's l -medium, supplemented with μg/ml streptomycin, u/ml penicillin, mm glutamine and % fbs. virus titration was performed on monolayers of ssn- cells grown in a -well plate. viral suspensions were prepared with -fold serial dilutions in emem supplemented with % fbs. quadruplicates of µl of each dilution were added in a -well plate seeded with ssn- cells. cultures were incubated at °c for days. during this period, the cell monolayers were observed for the appearance of cytopathic effect and the final titer, expressed as tcid /ml, was estimated by the end-point titration method . rabbit anti-nodavirus polyclonal antibodies preparation. female new zealand rabbits were used for anti-nodavirus polyclonal antibodies production with prior approval by the animal bioethics committee of the hellenic pasteur institute (athens, greece) according to the ec directive / and the national law / . rabbits were reared in institutional facilities under specific pathogen-free conditions, receiving a diet of commercial food pellets and water ad libitum . for immunization, the rabbits were inoculated with culture supernatant of nodavirus infected ssn- cells. in immunization scheme , one subcutaneous injection containing μg of antigen emulsified with cfa was administered in final volume of μl. three weeks later, a subcutaneous booster [ μg of antigen emulsified with ifa] was injected. a final booster of μg antigen without any adjuvant was given days later. blood was collected from rabbit marginal ear vein days after the final booster, the serum was isolated by centrifugation at × g for min and kept in − °c. three weeks later, another boost ( μg of antigen) was injected subcutaneously to the same rabbit. fifteen days later, blood was collected from the marginal ear vein. centrifugation was performed as above for serum isolation. in scheme , two weeks interval between immunizations was applied instead of three weeks interval used in scheme . the anti-nodavirus igg was purified using the nab tm spin columns, following the manufacturer's instructions. briefly, the samples were diluted : in binding buffer ( . m phosphate buffer, . m nacl). the columns were centrifuged ( × g, min, room temperature (rt)), the flow through was discarded and were equilibrated with ml binding buffer, followed by centrifugation ( × g, min, rt). the procedure was repeated once. subsequently, the bottom ends of the columns were closed, the samples were added and the columns were stirred end-over-end ( min, rt). after centrifugation, the flow through containing non-bound serum components was collected and the columns were washed times with ml binding buffer. one ml of elution buffer (glycine . m, ph = - , × times) was added to the columns, the fractions were collected in tubes containing μl of neutralization buffer (tris m, ph = - ) and stored in − °c. finally, the columns were regenerated with ml of elution buffer (× times) and stored with ml of storage buffer ( . % nan in pbs). to avoid possible denaturation of purified antibodies due to sensitivity to elution buffer, purified antibodies were desalted using zeba tm spin desalting columns. briefly, the columns were centrifuged at xg for min, rt. ultrapure water was added in each column and centrifugation was repeated twice. samples were placed in each column and columns were centrifuged in order to collect the purified samples. the antibody concentrations were measured using the micro bca protocol. a specific elisa was utilized for anti-nodavirus polyclonal antibodies determination in rabbit serum. elisa plates ( -well flat bottom) were coated with μl of , % poly-l-lysine in bicarbonate/ carbonate buffer, ph . , (incubation h, rt). the plate was washed times with lswb and μl of nodavirus suspension were added, followed by incubation for h (rt). subsequently, μl of mm glutaraldehyde in pbs were added in each well and the incubation was continued for min (rt). the plate was washed with lswb ( times) and then blocked with μl % bsa in lswb, for h, at °c. following plate washing ( times with hswb), μl of serially diluted rabbit anti-nodavirus serum ( : , : , : and : in pbs/ ml/l tween- ), were added (incubation h, rt). the plate was washed with hswb and μl of anti-rabbit igg-hrp antibody was www.nature.com/scientificreports www.nature.com/scientificreports/ added; diluted in blocking buffer ( / , / , / and / ), and incubated for h at rt. the plate was washed with hswb and μl of chromogen (tmb) was added for min. the reaction was stopped using μl of . m h so . the optical density was measured at nm in spectrophotometer. nps. an aliquot of nm au nps solution (i.e. ml, . × particles/ml) was adjusted to ph by adding the appropriate amount of mm borax solution. in parallel, µg of polyclonal anti-nodavirus antibody ( . µg/µl) were diluted in µl of mm borax solution and added to the nanoparticles solution, gradually by stirring ( times × µl). the mixture was incubated for min at rt, and µl of % bsa in mm borax solution were added. the final solution was incubated for min at rt, and the excess of reagents were removed by centrifugation at , × g for min. the supernatant was discarded and the pellet was re-dispersed in washing solution ( ml % bsa in a mm borax solution), followed by centrifugation at , × g for min. the supernatant was discarded once again, and the red pellet was re-dispersed in µl of an aqueous solution containing . % bsa and . % nan in mm borax. all incubation steps were performed in darkness. finally, the functionalized antibody au nanoparticles were stored at °c . flocculation studies. gold colloid suspension ( . pmol/ ml) adjusted to ph . , was pipetted into a series of . ml siliconized tubes. colloidal gold and antibody ( - ng/µl) solutions were mixed and the au functionalization protocol was applied as described above. determination of the optimal antibody amount for au nanoparticles conjugation, was based on light absorption measurements. for that reason each tube received % nacl and was shaken for min. absorption of each tube at and nm was determined min later. preparation of the nodavirus lateral flow biosensors. the dry reagent lateral flow biosensors ( × mm) consisted of a cellulose immersion pad, a glass-fiber conjugate pad, a nitrocellulose diagnostic membrane, and a cellulose absorbent pad. the parts were assembled on a plastic adhesive backing as follows: the diagnostic membrane was placed on center of a laminated card by the manufacturer; the conjugate pad was placed below the membrane, the immersion pad was placed below the conjugate pad, and the absorbent pad was positioned above the membrane. each part overlapped mm to ensure that the solution could migrate through the biosensor. the tlc applicator was employed to construct the test zone (tz) and the control zone (cz) by loading polyclonal anti-nodavirus antibody (anti-pn) and anti-rabbit igg antibody (anti-r) on the membrane, respectively. the anti-pn and anti-r zones were constructed at distances of and mm from the edge of the membrane, respectively. in details, for the anti-pn zone, a solution consisting of mg/l purified polyclonal anti-nodavirus antibody, ml/l methanol, and g/l sucrose in freshly prepared mm nahco buffer (ph . ) was loaded at a density of ng per lfb, with dispensing velocity nl/s. for anti-r zone, a solution containing mg/l anti-rabbit igg antibody, ml/l methanol, and g/l sucrose in mm nahco buffer (ph . ) was loaded at a density of ng per -mm membrane, with dispensing velocity nl/s. the membrane was dried in an oven for h at °c, and the sensors were assembled as described before. lfbs with a -mm width were cut using a guillotine cutter and stored dry, at ambient temperature , . lateral flow biosensor detection assay of nodavirus virions. for detection of nodavirus virions, brain tissue samples were homogenized and centrifuged as described in section "virus preparation and titration". alternatively, aliquots of ssn- cell culture supernatant infected with nodavirus were used. the samples were directly applied to the conjugation pad next to . µl of polyclonal anti-nodavirus conjugated au nps. the immersion pad of the biosensor was then dipped into μl of the developing solution. the visual detection was completed within min. longer times did not affect the assay results . after completion of the assay, the lfbs were scanned with a desktop scanner and the band densities were quantified with imagej software . assay principle. the principle of viral particles detection based on lateral flow biosensor with functionalized nanoparticles is presented in fig. . nodavirus intact particles (virions), obtained either from ssn- cell culture supernatants or homogenized tissue samples, are directly applied on the lfb. the biosensor is dipped in a developing solution, which is transferred through the lfb by capillary action. the solution rehydrates the anti-nodavirus conjugated gold nanoparticles, and the virion -anti-nodavirus conjugated nanoparticles complex is captured from immobilized anti-nodavirus antibody at the lfb test zone. the resulted accumulation of au nps generates a characteristic red line. proper function of the biosensor is confirmed by immobilization of excessive au nps in captured anti-rabbit igg (control zone) forming a distinct red line. absence of virus particles in the sample results in the formation of the control zone only. the proposed lfb is based on the well-established immune reaction between antibody functionalized gold-nanoparticles and virions, which serve as antigens. since each virion surface i.e. the virus coat protein, contains multiple antigenic sites (e.g. b-cell epitopes) , a sandwich format was implemented for virion detection assay. it has been suggested that a sandwich elisa resulted in optimum quantitation results, with higher reproducibility and greater detectability range when it was compared with an antigen-immobilized elisa for nodavirus particles detection , therefore the sandwich format was chosen in the present project. moreover, the sandwich format has been successfully implemented in various other formats for low cost portable devices, e.g. nanoparticle-mediated photothermal assays with common thermometer as the signal reader , indicating its appropriateness for methodologies with on-site applications. therefore, polyclonal antibodies to nnv were immobilized on the test zone of the biosensor as well as on the gold nanoparticles. production and reactivity of rabbit anti-nodavirus polyclonal antibodies. the anti-rabbit igg concentration for the elisa assay was tested in the range of : , : , : and : dilution. the serum ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports www.nature.com/scientificreports/ dilution range was : , : , : and : . the results are presented in fig. s a . to optimize the polyclonal antibodies efficiency, the rabbit serum was purified with the appropriate columns and specificity of the purified and desalted antibodies was tested with elisa. optical densities (od ) for a representative fraction was . (elute ) and . (elute ) versus . of the unpurified serum. the use of : dilution of the anti-rabbit igg antibodies was selected since it results in similar signal intensity with higher amount of the antibody, for the same serum dilution. the immunization scheme consisted of two parts: times of antigen administration with weeks intervals between immunizations and a th immunization followed weeks later. antibody levels were quantified with a specific elisa, and the final immunization was performed to raise higher anti-nodavirus antibodies titer (od = . for immunizations vs od = . , for immunizations). in parallel, another female rabbit was immunized with scheme which allowed a weeks interval between the immunizations. as shown in fig. s b , scheme raised even higher titer of igg anti-nodavirus antibodies compared to scheme . therefore it was used for antibody production. colloidal gold nanoparticles were selected since they are the most common used nanosized labels for lateral flow biosensors, occupying more than % of the respective market in immunochromatographic assays, since they can be biolabeled with simple procedures, have excellent optoelectronic properties, have very low toxicity and are easily synthesized with low cost . conjugation of the anti-nodavirus antibody on the au nps was optimized in terms of: usage of a monoclonal versus the polyclonal anti-nodavirus antibody, the size of the used particles, the anti-nodavirus antibody amount on the nanoparticle conjugates and the amount of the conjugates that was applied on the lateral flow biosensors. effect of the use of polyclonal instead of a monoclonal antibody for au nps functionalization. the conjugation of a monoclonal anti-nodavirus antibody on the nanoparticles was tested in order to increase specificity. as shown in fig. a , the monoclonal antibody failed to provide functionalized au nps since not even the cz was visible. on the contrary, implication of a polyclonal anti-nodavirus antibody in the conjugation reaction resulted in a discrete signal in the test zone of the lfb and a strong signal in cz. effect of the gold nanoparticles size. the au nps size has important effects on the particles optical properties, the affinity of the conjugates with the used antibodies, their valency and the antigen binding kinetics . large sized particles develop steric hindrance in a high degree, affecting interactions of the target with a labelled antibody, which could impede the fluidity in the strip. on the other hand, small nanoparticles, are not optimal for visual signals formation . to determine the size where the signal would be optimal, commercially available nanoparticles were studied. gold nanoparticles stabilized either in citrate ( nm) or in pbs buffer ( , , , , nm) were conjugated with the polyclonal anti-nodavirus antibody. formation and intensity of tz and cz on the lfb was assessed for evaluation of the particles optimum size for the nodavirus lfb. figure b proves that all pbs stabilized particles were successfully conjugated with antibodies while no conjugation occurred on the citrate nm nanoparticles. therefore, au nps stabilized with pbs buffer facilitates proteins (antibodies) conjugation while the citrate buffer is more efficient on conjugation of sh-tailed oligonucleotides . use of and nm nps resulted in faint control zones. the and nm formulations formed strong control zones but the test zones were faint possibly due to difficulty in interaction with the larger nodavirus particle. the optimum signal was achieved with nm au nps, a size which is similar to the virus size. that size seems to be optimum for conjugation with different antibodies for different analytes intended for use on lfbs, e.g. anti-e.coli monoclonal antibody or anti-biotin antibody , indicating that nanoparticles in the range of - nm are better for antibody conjugation than smaller or bigger particles. effect of anti-nodavirus antibody amount on au nps conjugates. the antibody amount used for nanoparticles functionalization is a crucial factor for optimal lfb performance. variation in the antibody amount in the surface of the nanoparticles will affect its density on the conjugate, affecting the binding degree between the antigen and the conjugate. thus, the assays' detection limit can be changed based on the various binding efficiency. the minimum antibody concentration which inhibits nanoparticles aggregation in the presence of excess salt ( % nacl), ap: absorbent pad. a test zone (polyclonal anti-nodavirus antibody (tz) and a control zone (anti-rabbit igg antibody (cz)) have been immobilized on the biosensors' diagnostic membrane. the sample contains the nodavirus particles (virions) and is applied on the conjugation pad next to functionalized gold nanoparticles (au) with polyclonal anti-nodavirus antibody. the biosensor is dipped in the developing buffer, the sample and the nanoparticles are captured on the test zone and the positive signal is visualized, as a red zone. the excess nanoparticles bind to the control zone of the biosensor. the assay components are not in scale. is usually chosen as the optimum amount for au nps conjugation . several antibody amounts were studied and the optimal antibody concentration for the particle functionalization was determined by comparing the absorption between and nm (a -a ), in the presence of % nacl. the resulted conjugates are presented in fig. c , where all nanoparticles appear to be of the same color. the gold nanoparticles stability in the presence of concentrated nacl is indicated by the flocculation curve, which is an indirect measure of the immobilized protein surface coverage degree. a hydrated charged corona of proteins on the nanoparticles is formed by adsorption on their surface, which inhibits the particles aggregation, mostly due to steric hindrance and electrostatic repulsive forces. addition of a concentrated nacl solution inverts the au nps protection by uncovering their surface, leading to aggregation . the resulted flocculation curves indicated that ng/µl was the optimal antibody amount, which was therefore used throughout the project (fig. d) . application of that conjugate on the lfb resulted in a strong signal in the control zone, confirming the successful conjugation of anti-nodavirus antibody to au nanoparticles (fig. e) . the au nps amount on the conjugate pad was evaluated, in order to achieve satisfactory cz and tz intensity of the biosensor, combined with minimum background membrane coloration . the nanoparticles amount was optimized in the range of . - . fmol per lfb. the use of . fmol of au nanoparticles per lfb resulted in the optimum signal (fig. f) . less conjugate amount resulted in lower band intensities possibly because low conjugate content is inadequate for binding of specific epitopes amount on highly concentrated antigens. use of higher amounts of particles had a slight signal decrease, as expected since higher conjugate cause epitopes blocking on the antigen surface, and inhibits their binding in the lfb test and control zones . based on these observations, . fmol ( . μl of the prepared solution) of gold nanoparticles conjugates, corresponding to . μl of the prepared solution, were used in all subsequent experiments. important since a large number of parameters would affect the sensitivity and specificity of the assay. several factors were studied including the developing solution composition, the type of the used diagnostic membrane, the dispensing velocity, the use of a polyclonal instead of monoclonal antibody and the immobilized antibody amount for tz construction. effect of the composition of the developing solution. the developing solution composition has great impact on the antibody-antigen interaction on the biosensor. ideally, the developing solution facilitates the nanoparticles and sample mixture movement along the biosensor with flow rates that allows test completion in short time providing, in parallel, sufficient time for virion interaction with the modified nps. moreover, the developing solution should also prevent the non-specific nanoparticles absorption by the lfb membrane . glycerol increases the mixture viscosity causing a slower flow rate. addition of several surfactants (sds, tween- , sds, etc) or proteins (e.g. www.nature.com/scientificreports www.nature.com/scientificreports/ bsa) enables the particles movement from the conjugate pad. eleven different formulations were studied and their detailed composition is described in table , while the respective results are presented in fig. s . the use of high ph buffer or bsa did not result in any specific signal formation (fig. s a) . two formulations were optimal for non-specific signal elimination: i) g/ s/p, and ii) g/ s/ . t/p. the formulation that contained % glycerol resulted in prolonged running times (∼ min); therefore it was excluded from further studies. it was observed that tween- concentration seemed decisive for the biosensor specificity. for that reason, increasing tween- concentrations were tested in the range of . - % and the optimum results were obtained with . % tween- in developing solution (fig. s b) . concluding, the optimum developing solution for background elimination, rapid assay times, and specific bands was obtained using % glycerol, . % tween- and % sds in ×pbs, ph . . effect of the antibody dispensing velocity. the dispensing velocity for construction of the tz based on the polyclonal anti-nodavirus antibody was subsequently tested (fig. a) . the antibody dispensing velocity is an essential www.nature.com/scientificreports www.nature.com/scientificreports/ parameter for production of a uniform immobilized test zone. in general, high volatile solutions (e.g. acetone) are dispensed with high velocity (> nl/s) whereas low volatile solutions (water, ethanol) are sprayed with low velocity (< nl/s). based on previous studies for antibody immobilization on nitrocellulose membranes , a methanol containing solution ( ml/l) was used. methanol results in medium volatile solutions and the use of nl/s dispensing velocity was optimum for the uniform formation of the anti-rabbit igg control zone. however, the same dispensing velocity resulted in the formation of two 'pseudozones' when the anti-nodavirus antibody solution was sprayed on the membrane. it seems that high dispensing velocity creates a gap in the middle of the spraying zone, which doesn't allow proper absorption of the antibody solution from the membrane, perhaps due to non-uniform evaporation. the use of lower dispensing velocity resulted in a single uniform test zone and the velocity of nl/s was chosen since it resulted in the optimum signal. effect of the use of polyclonal instead of monoclonal anti-nodavirus antibody. in order to enhance the lfbs' specificity, a monoclonal anti-nodavirus antibody which was commercially available was used instead of the produced polyclonal antibody. it is obvious in fig. b , that the monoclonal antibody did not produce visual signal whereas use of the polyclonal antibodies led to high signal formation, since more antigenic epitopes interacted with higher antigen amounts and were subsequently immobilized. effect of the type of the diagnostic membrane. the porosity of nitrocellulose membranes plays a critical role for the specificity and the sensitivity of a lateral flow biosensor because it influences the capillary flow rate. two membranes, the hf and hf (millipore) were tested for the nodavirus lfb development, with corresponding migration times of and min, respectively. as shown in fig. c , the signals of the lfbs which were based on the hf membrane were more intense than the signals obtained with the hf membrane. thus, longer migration time is favorable for the sandwich-type complexes on the lfb, in accordance with independent observations . effect of the immobilized antibody amount. the anti-nodavirus antibody amount immobilized on the biosensor test zone was examined. ng of antibody per -mm lfb were used to obtain the optimum signal (fig. d) . the use of ng/µl for antibody immobilization resulted in slightly decreased signal, which was significantly lower when . ng/µl of the antibody were applied. interestingly, the signal decreased as the antibody concentration increased. the use of high amounts of antibodies on lateral flow biosensors could cause the so-called "hook effect". the prozone or hook effect is basically a false negative result of a particular analyte, when an antibody has very high concentrations. if too many antibodies that can bind to the antigen are present, then the antigenic sites are coated by these antibodies, and few or no conjugated au nanoparticles directed toward the analyte are able to bind more than one antigenic particle. since the antibodies do not bind to antigens, no signal is formed. therefore, the result is a false negative . lfb detectability was assessed by samples consisting of decreasing concentrations of nodavirus infected ssn- cell culture supernatants. all samples were studied by applying the optimized experimental conditions. in details, supernatant volumes ranging from . to . µl of supernatants were applied on lfbs. the respective lfbs are shown in fig. a ,b. the lowest detectable amount of nodavirus containing supernatant was . µl. to define a positive result and discriminate from any background signal, the signal/noise (s/n) ratio (s/n = ) was used. the signal definition refers to the tz optical intensity of each detected sample, whereas noise is the produced tz intensity when using culture medium alone. the lowest amount corresponded to . tcid (supernatant titer was . tcid /ml) which is the measure of infectious virus titer. high amounts of virus supernatant seem to result in stable densities. nodavirus lfb reproducibility. reproducibility is one of the most crucial parameters of the lfb evaluation procedure. for that reason, it was assessed with a constant amount of nodavirus containing ssn- cell culture supernatant ( µl). five biosensors were tested with the nodavirus supernatant for studying test and control zones. all lfbs were prepared in variable batches. the lfbs are shown in fig. s , and analysis by densitometry resulted in coefficient of variation (cv) of . % for the test zones and . % cv for the control zones. nodavirus lfb specificity. specificity of nodavirus biosensor was assessed by studying the assay cross-reactivity with non-infected ssn- cell culture supernatant and homogenized tissues of non-infected fish (fig. ) . only the samples which were characterized positive by cell culture resulted in detectable signal. developing solution-only negative controls were also tested in each run. concluding, the analytical performance of the proposed assay can be summarized as follows: × − tcid of cell culture supernatant was detectable, by using a standard centrifugation step and the proposed biosensor. the assay is specific for infected fish and highly reproducible. in table , the assay sensitivity of previously reported methods for nodavirus virion detection by sandwich/ capture elisa, as well as nodavirus detection with lateral flow assays in general, are summarized. even though comparison of these methods' sensitivity cannot be highly accurate, since the limit of detection is determined by slightly different way in each case, the present assay seems to have higher sensitivity for virion detection in comparison to elisa methodologies and the mab/ lfia www.nature.com/scientificreports www.nature.com/scientificreports/ brain homogenization and separation of the insoluble fraction by centrifugation or ssn- cell culture supernatant). the samples supernatants were applied directly to the nodavirus biosensors, which were run and scanned. eleven infected (positive; n = ) and non-infected (negative; n = ) d. labrax samples were applied on the lfb (fig. ) . all results were visualized within minutes. all tested samples were verified with ssn- cell culturing. five samples were originated from saronikos gulf area (s , , , , ), samples were from korinthian gulf region (s - ), sample was collected from amvrakikos gulf (s ) and samples were originated from aquacultures in cyprus (s , , ). a negative control, containing only developing solution, and a positive control, containing . µl of nodavirus containing cell culture supernatant (corresponding to the limit of detection), were included in each run. the use of the cell culture supernatant in an amount corresponding to the assays detection limit was intended to be used as a visual "yardstick", a comparison measure to indicate which signal could be positive. if the positive control was intended to be used just to confirm the lfbs' proper operation more supernatant could be applied to provide more intense signal. we observe that in the lfbs with positive signal the gold nanoparticle conjugates appear to bind non-specifically between the test and the control zones. in any case that non-specific binding is not crucial for the nodavirus presence assessment, since the test zone is clearly positive or negative. most of the non-infected samples (s - ) did not show any signal in the tz. the infected samples resulted in positive signals with varying intensities above the positive control signal, which correlates with different amounts of nodavirus in the sample. one negative and one positive sample (s and s , as characterized by ssn- cell culturing) resulted in similar faint zones. in order to decide the disease state of each sample, the test zones were quantified with the publicly available, easy to use imagej software, based on scanned lfb images. quantification confirmed that s was negative and s positive with very low virus load (fig. b) . in the case of such ambiguous results on the field, the lfbs images could be photographed and quantified with imagej with a smartphone. the scanned images of the lfbs were independently evaluated by a second person, and were compared to lfb images with known outcomes (positive and negative). cost-effective and labor-saving methods for fish infectious diseases identification have attracted an increasing interest to aquaculture industry in recent years, due to their high economic impact. the development of a highly promised robust and rapid lateral flow biosensor has been reported in the present study. the proposed lfb can be used for simple and accurate screening of nodavirus detection in various fish species. to enable on site analysis of nodavirus, we developed and optimized a paper lfb based on gold nanoparticles for easy, specific and sensitive visual detection of nodavirus viral particles (virions) in biological samples. the target virions can be simply detected by the red colored signal formation in the lfbs test zone. the lfb permits the visual detection of nodavirus particles within minutes without using any instrumentation. the total assay starting including tissue homogenization, centrifugation and virion detection with the biosensor was accomplished in less than min. the total cost of the assay is less than €. undergoing studies by our research group include complete validation of the nodavirus biosensor on fish culture facilities, and efforts to improve the biosensors' sensitivity of the lfb utilizing dual nanoparticles labels , and signal enhancement systems in general. moreover, the elimination of centrifugation from the sample preparation step is among our future goals to further simplify nodavirus detection on-site. concluding, the proposed biosensor could become a reliable tool for the rapid screening of questionable samples in aquaculture facilities. as a screening tool it can be used to decide whether a sample is positive or negative, and indicate which samples have ambiguous state of the disease and need further analysis by other methodologies in a laboratory setup. such a procedure could lower the cost and the analysis time to 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leishmaniasis. vaccine image processing with imagej identification of b-cell epitopes on the betanodavirus capsid protein reproducibility of antigen-immobilized enzyme-linked immunosorbent assay (elisa) and sandwich elisa for quantitative detection of nnv particles nanoparticle-mediated photothermal effect enables a new method for quantitative biochemical analysis using a thermometer effect of different-sized spherical gold nanoparticles grown layer by layer on the sensitivity of an immunochromatographic assay a remarkable sensitivity enhancement in a gold nanoparticle-based lateral flow immunoassay for the detection of escherichia coli o :h . rsc adv oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for dna analysis by hybridization identification of single-nucleotide polymorphisms by the oligonucleotide ligation reaction: a dna biosensor for simultaneous visual detection of both alleles visual detection of microrna with lateral flow nucleic acid biosensor studies of the 'hook' effect in the one-step sandwich immunoassay development of a lateral flow immuno-chromatic strip assay for the detection of nervous necrosis virus (nnv, rgnnv genotype) double-enhanced lateral flow immunoassay for potato virus x based on a combination of magnetic and gold nanoparticles production of monoclonal antibodies against grouper nervous necrosis virus (gnnv) and development of an antigen capture elisa detection of betanodavirus in juvenile barramundi, lates calcarifer (bloch), by antigen capture elisa quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass dicentrarchus labrax rapid and sensitive detection of macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (cpa-lfd) for rapid and specific detection of red-spotted grouper nervous necrosis virus reagents. ssn- cells were purchased from the european collection of animal cell cultures (salisbury, uk). fetal bovine serum (fbs) and leibovitz l -medium were from biochrom (berlin, germany), and whatman filters (ptfe, . μm) were from ge healthcare (buckinghamshire, england). penicillin, streptomycin and l-glutamine were from gibco (paisley, uk). cell culture flasks falcon primaria were purchased from becton dickinson labware (franklin lakes, usa) and flat bottom -well elisa plates were obtained from greiner (kremsmunster, austria). micro bicinchoninic acid (bca) protocol for protein quantification, nab tm spin columns and zeba tm desalting columns were from pierce (thermo fisher scientific, delaware, usa). borax, dimethyl-sulfoxide, bovine serum albumin (bsa) and glutaraldehyde, were purchased from applichem (darmstadt, germany). eagle minimum essential medium (emem), complete and incomplete freund's adjuvant (cfa and ifa, respectively), anti-rabbit igg-horseradish peroxidase (hrp), poly-l-lysine, anti-rabbit igg (whole molecule) antibodies, , ′, , ′-tetramethylbenzidine (tmb) substrate, au nps stabilized in pbs ( , , , , nm), or citrate buffer ( nm), siliconized tubes and tween- were obtained from sigma-aldrich (steinhem, germany). monoclonal anti-nodavirus antibody was from aquatic diagnostics (stirling, uk). nitrocellulose membranes on laminated cards (hf mc and hf mc ), glass (gfcp ) and cellulose fiber pads (cfspoo ) were all from millipore (billerica, ma, usa). applichem or sigma provided all common reagents.low the research project was implemented within the framework of the action «supporting postdoctoral researchers» of the operational program "education and lifelong learning" (action's beneficiary: general secretariat for research and technology, ls _ ), and was co-financed by the european social fund (esf) and the greek state. d.k.t. conceived and designed the experiments; d.k.t. and m.m. performed the experiments; a.p. provided d. labrax samples; d.k.t. and e.k. analyzed the data; d.k.t. wrote the paper; a.p. and e.k. proof-read the manuscript; d.k.t. and e.k. supervised the work. all authors have approved the present article. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to d.k.t.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -uxpedpz authors: ciencewicki, jonathan m.; schouest, katherine r.; gierman, todd m.; vandeberg, peter j.; gooch, barry d. title: plasma donors in the southwestern united states positively contribute to the diverse therapeutic antibody profile of immune globulin products date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: uxpedpz human-plasma-derived immune globulin (ig) is used in augmentation therapy to provide protective levels of antibodies to patients with primary immune deficiency diseases (pidd) and for prophylaxis against infectious diseases. to maintain the breadth of antibodies necessary for clinical protection, it is important to understand regional patterns of antibody seroprevalence in source plasma from which ig products are manufactured. in this study, source plasma from donation centers in various locations of the southwestern quarter of the united states was surveyed for antibody titers to hepatitis a virus (hav), measles virus (mev), and cytomegalovirus (cmv). a broad range of anti-hav ig plasma titers was observed among these centers, with some centers exhibiting – times the titers of the others. minor to no differences were observed for levels of anti-mev and anti-cmv, respectively. importantly, elevated anti-hav ig titers were broadly observed across plasma units obtained from the centers exhibiting high titers, indicative of a potential regional phenomenon among donors as opposed to few donors with singularly high titers. plasma from these high-titer centers conferred significantly greater neutralization against hav in vitro. the outcomes of this study give a glimpse of the antibody diversity inherent in human plasma used to manufacture ig products.. observed among these centers, with some centers exhibiting - times the titers of the others. minor to no differences were observed for levels of anti-mev and anti-cmv, respectively. importantly, elevated anti-hav ig titers were broadly observed across plasma units obtained from the centers exhibiting high titers, indicative of a potential regional phenomenon among donors as opposed to few donors with singularly high titers. plasma from these high-titer centers conferred significantly greater neutralization against hav in vitro. the outcomes of this study give a glimpse of the antibody diversity inherent in human plasma used to manufacture ig products.. immune globulin (ig) products made from human plasma are used in augmentation therapy to provide antibodies critical for the protection of persons with primary immune deficiency diseases (pidd), as well as for prophylaxis of various infectious diseases. the broadly-protective properties of the antibodies in ig products stem from their human origin and because they are purified from pools made up of large numbers of plasma donations. ig products manufactured in the united states (us), as well as some ig products manufactured in other countries, are derived from plasma collected exclusively from donation centers in the us, which can span the entire geography of the country. safety measures implemented nearly two decades ago-collaborative, industry-wide epidemiological surveillance of the donor population, judicious selection of donors, rigorous testing of plasma by serological and molecular (e.g., nucleic acid testing) methods and validation of manufacturing methods for virus clearance capacity-provide a high degree of confidence that ig products, regardless of plasma origin, safely deliver a diverse, natural combination of human polyclonal antibodies , . seroprevalence of antibodies to infectious diseases in the human population is influenced by a number of factors, including management of food and water resources, sanitation, vaccination, climate, sociodemographics, and the emergence of infectious agents, previously unknown or familiar . since the status of factors such as these can vary over time and across geographic regions, the profiles of antibodies among donors whose plasma is used to manufacture ig products are dynamic. for example, changes in viral antigen exposure in recent years have had a real impact on ig products' antibody levels against a number of viruses, among which are hepatitis a virus (hav) and measles virus (mev). in the case of hav, declining overall rates of hepatitis a infection, due particularly to increased vaccine coverage beginning in the mid- s, have had the effect of reducing naturally-acquired anti-hav titers among adult plasma donors [ ] [ ] [ ] [ ] [ ] [ ] . consequently, available ig preparations have been found to fall short of potency expectations . this development has prompted grifols, manufacturer of the lone ig product (gamastan ® ) approved in the us for hav prophylaxis, to update dosage regimens to ensure that adequate circulating anti-hav immunoglobulin levels are reached in patients , . the use of widespread measles vaccination has had a similar effect on mev-neutralizing antibodies in ig products for intravenous administration . nevertheless, human plasma ig augmentation therapy still offers the unique advantage of a diverse, polyvalent therapeutic antibody repertoire that cannot be obtained by any means other than purification of ig from large numbers of plasma donations collected across a wide geographic spectrum. a better understanding of the antibody diversity with respect to geography could help to mitigate the decline in antimicrobial potencies observed in ig preparations. for example, it is known that anti-hav seroprevalence in the us is greatest among populations residing in the southwestern us , , a phenomenon not necessarily due to expanded immunization coverage, but more likely indicative of more potent, longer-lived naturally-acquired immunity. the present study was designed primarily to examine the anti-hav content, and potential benefit, of plasma obtained from donors in the southwestern quarter of the us. in addition, since it is possible this plasma possesses elevated antibody levels to other infectious agents , we examined levels of antibodies to mev and human herpesvirus , also known as cytomegalovirus (cmv). human plasma. plasma was obtained from donation collection centers located in el paso, texas (tx); mcallen, texas (tx); san diego, california (ca); midwest city, oklahoma (ok); and provo, utah (ut), all of which are associated with the southwestern us. plasma was also obtained from one center, clarksville, tennessee (tn), outside of this region. the primary selection criterion for donation centers was proximity to regions of high hav incidence and seroprevalence. while texas and california have some of the highest incidence and seroprevalence rates in the us, oklahoma and utah-which are loosely associated with the southwest-and tennessee have relatively low incidence rates. texas and california also have a higher foreign born population than the other states, which may contribute to the higher seroprevalence. beyond the selection of states, criteria were: ( ) centers that perform testing at the facility to allow for quick access to samples; ( ) centers that have sufficient unique donors over a short time-span to produce an adequate sample size. donors are allowed to donate two times per week, and since unique donors were desired, it was prudent to limit the collection period of samples to a few days in order to avoid duplicate donors. all plasma samples used in this study were residuals of samples processed as part of grifols' standard routine for the qualification of donors and material for further manufacture. each donor provides informed consent that allows grifols to conduct research on samples collected at its plasma collection centers. the informed consent documentation has been reviewed by institutional review boards (irbs) as part of several prospective clinical studies. all plasma units from which samples for this study were pulled were non-reactive for all serological and nucleic acid tests performed, including hav ribonucleic acid (rna) by real-time quantitative reverse transcription (qrt) polymerase chain reaction (pcr) testing. for the purpose of this study, to allow for higher throughput antibody testing, plasma pools were created by combining aliquots of virus propagation. b-sc- cells (seeded h prior) were infected with hav (hm / f; atcc) at a multiplicity of infection (moi) of . tcid per cell. after incubation at °c for d, a cell lysate was prepared and stored frozen at − °c. thawed lysate was treated with trixton-x and lithium dodecyl sulfate and concentrated via tangential flow filtration (tff). the tff retentate was centrifuged at approximately × g for . h at °c in order to force the virus into a pellet, after which the pellet was suspended in buffer and the buffer was exchanged by ultrafiltration. the virus preparation was stored at − °c until use. a strain of cmv (ad- ; atcc) frequently used in laboratories has been passaged extensively in mrc- fibroblasts such that it no longer exhibits tropism for naturally permissive epithelial/endothelial cells. in order to obtain a more clinically-relevant system, endothelial arpe- cells were infected with ad- at an moi of . - . tcid per cell, and the cells were serially passaged post infection until a desirable cmv titer was achieved. adaptation of the virus did not impact its ability to infect mrc- fibroblasts (data not shown). the supernatant from infected cells was used without further purification in neutralization experiments. enzyme-linked immunosorbent assay. a commercially-available enzyme-linked immunosorbent assay (elisa) (bio-rad monolisa ™ anti-hav eia kit, cat# ) was used to estimate the anti-hav ig (igg and igm) titer in a plasma sample. an in-house protocol was followed to convert the elisa to a semi-quantitative format. with each assay plate was included a series of dilutions of the kit's calibrator, from which a standard curve was generated. a plasma sample was diluted such that its absorbance value was within the range of the standard curve, and an ig titer was estimated from a linear fit of the standard curve. the ig titer was then corrected according to the dilution factor. commercially-available elisas (abcam anti-cmv igg human elisa kit cat#ab or abcam anti-mev virus igg human elisa kit cat# ab ) were used to semi-quantitatively determine the amount of anti-cmv or anti-mev igg in a plasma sample. the assay was performed according to the manufacturer's instructions. a single plasma sample, neat or diluted in hank's buffered salt solution (hbss), was added to the first well of each row of a -well microtiter plate. an equal volume of hbss was added to the remaining wells of the entire plate. the plasma in these first wells was serially diluted across the succeeding nine wells of a respective row. an equal volume of a virus preparation was added to the same wells such that the concentration of virus in each well was . to . tcid ·ml − . a positive control was included on each plate by adding the virus preparation to the wells of a single column containing only hbss. a negative control was also included in which hbss alone was left in the wells of a single column. the plate was then incubated for min at °c. following the incubation, µl from each well were used to inoculate a corresponding well on a microtiter plate that had been seeded with recipient cells (frhk for hav and arpe- for cmv; mev were not evaluated). the recipient cells were incubated with the inoculum for h at °c. following infection, the inoculum was removed, maintenance medium was added, and the cells were incubated at °c for hav or °c for cmv. twenty-one days post inoculation, the cells were microscopically examined for the visual appearance of a cytopathic effect (cpe). the virus neutralization factor of a given plasma sample was calculated as the reciprocal of the largest dilution at which not less than % of its inoculated wells were negative for cpe. in this case, each dilution had eight replicates (the eight wells comprising a column of a microtiter plate); therefore, the last dilution in the series that exhibited at least four cpe-negative wells dictated the neutralization factor for the plasma sample on test. statistical analyses. statistical analyses of data were performed using sas jmp (v ) and graphpad prism (v ) software. for analyses of grouped data sets, an unpaired two-tail t test was used to determine if means differed significantly. for the analysis of collection centers, a one-way analysis of variance (anova) was used to determine if the means differed significantly, and tukey's honestly significant difference (hsd) post hoc test was used to directly compare means of each center. anti-hav, -mev, and -cmv immune globulin seroprevalence in source plasma from us southwest. all plasma pools were screened by an elisa for anti-hav ig (igg and igm). when analyzed per center (fig. ) , the mean anti-hav ig titers of the plasma pools from el paso-tx ( , ± , miu·ml − ) and mcallen-tx ( , ± , miu·ml − ) were comparable to each other and significantly (p < . ) higher than the mean titers of all other centers. the mean anti-hav ig titers of pools from the san diego-ca ( , ± , miu·ml − ) and midwest city-ok ( , ± , miu·ml − ) centers were comparable; the mean titer of san diego-ca pools was significantly (p < . ) higher than those of provo-ut and clarksville-tn. the plasma pools from el paso-tx and the pools from midwest city-ok were analyzed for anti-mev and anti-cmv igg content (fig. ) . the level of anti-mev igg calculated for midwest city-ok pools was found to be slightly higher compared to that of pools from el paso-tx (p < . ). there was no significant difference in levels of anti-cmv igg between the two sets of pools. titers were due to one or a few donors in a given pooled sample or due to an overall higher prevalence in the population, samples from the individual donations comprising twelve of the plasma pools were tested for anti-hav ig titers. it was necessary to choose a subset of the pools in order to maintain a feasible laboratory workload. three pools each from the el paso-tx, mcallen-tx, midwest city-ok, and clarksville-tn centers were arbitrarily selected from within the second and third quartiles of the anti-hav ig titers for their respective centers. for samples of individual donations that had composed the pools from el paso-tx and mcallen-tx, a broad range of anti-hav ig titers were observed, but less than % of the donations exhibited values above , miu·ml − . the mean and median titers of the donations making up each pool never differed by more than approximately . -fold (fig. ) . a much lower mean anti-hav ig titer and a narrower range of titers were observed for the samples of individual donations pooled from centers in the other locations. assessment of antiviral activity. the same twelve pools identified for analysis of their component donations, as described in the prior section, were also evaluated for their capacity to inhibit hav and cmv infection in vitro (fig. a,b) . neutralization of hav infectivity resulting from plasma pools obtained from the el paso-tx center (mean neutralization factor of ) was significantly (p < . ) higher than what was observed for plasma pools from the midwest city-ok center (mean neutralization factor of ). no significant difference was observed in the capacity to neutralize cmv. neutralization of mev was assayed and activity was detected, but the results were inconclusive. the method had been optimized for concentrated ig samples, not for raw plasma, and did not possess sufficient sensitivity to distinguish differences in titers between plasma samples. further evaluation was not deemed necessary. the use of pooled, plasma-derived human ig has become a critical therapy in clinical medicine [ ] [ ] [ ] . while originally indicated as a plasma protein augmentation therapy for patients with pidd and some secondary immunodeficiency diseases, ig has also been shown to exhibit other clinical benefits, many stemming from its anti-inflammatory and immunomodulatory effects , . it is the diverse, polyclonal nature of ig that has endowed it with its broad clinical range. in order to maintain the therapeutic diversity of ig products, it is critical to understand patterns of antibody seroprevalence in source plasma. to this end, we tested plasma obtained from donor centers within the southwestern quarter of the us. the data confirm that the notable anti-hav ig seroprevalence in particular areas of the us southwest translates into elevated anti-hav ig titers in plasma collected at donation centers in those areas. clearly, elevated antibody levels specific for hav imply a higher incidence of infection. however, in areas where hav is endemic, most infections occur during childhood and resolve without any lasting impact, except a robust anti-hav response . fortunately, as suggested by the present study, healthy plasma donors emerge from such an area with elevated anti-hav ig titers. in fact, all plasma units from which samples for this study were pulled were negative for hav rna, an early marker of viral infection, by real-time qrt-pcr testing. it is important to note that the manufacture of ig products is globally regulated and that industry practices over the past few decades have resulted in ig products with strong pathogen safety records irrespective of the geographical region within the us from which the plasma originates. such measures include medical screening of donors, testing of plasma for disease-causing agents, and igg purification processes that incorporate segments with validated capacities to inactivate and/or remove blood-borne pathogens, in the event they were present. in the present study, plasma from six donation centers-five of which are in various locations of the us southwest-were surveyed for anti-hav ig titers. we observed a wide range of titers among the six centers, yet three obvious groups coalesced: high-titer, mid-titer, and low-titer groups. the el paso-tx and mcallen-tx centers yielded high-titer plasma with significantly higher anti-hav ig levels than those observed for all other centers surveyed. the san diego-ca and midwest city-ok centers produced mid-titer plasma, while the provo-ut center and the lone non-southwest center, clarksville-tn, exhibited low titers. we also examined if plasma from a high-titer center might also be enriched with other antiviral antibodies. similar to hav, mev and cmv are viruses for which suitable antibody levels in ig products are clinically important , . when the full sets of plasma pools from el paso-tx and midwest city-ok were compared, there was no significant difference in anti-cmv igg levels. but the anti-mev igg level observed in midwest city-ok plasma was approximately % of that of el paso-tx. while the result is subtle and inverted from that of anti-hav ig, the result is interesting nonetheless. it supports the premise that regional differences in seroprevalence helps to maintain efficacious antibody levels in ig products. despite the recent decline in anti-mev titers observed for some products, current ig preparations and doses are still sufficient to provide protection against mev infection for pidd patients . the analyses of individual units originating from select pools served to demonstrate that a number of centers in the us southwest are capable of consistently generating plasma with elevated anti-hav ig titers. the elevated mean titers are not singularly driven by a few donors with extraordinarily high levels; rather, the majority of individual donations exhibit values that are not more than one standard deviation above the pool mean. a similar pattern is evident in the individual donations making up pools from centers with lower anti-hav ig titers. www.nature.com/scientificreports www.nature.com/scientificreports/ furthermore, anti-hav ig titers in more than two-thirds of the individual donations from the high-titer centers were greater than the aggregate mean of plasma sample titers from the centers with lower titers. there does, however, seem to be a consistent, albeit small, number of individual donations in each high-titer pool that exhibit titers above , miu·ml − , which is most certainly an assuring contribution. in all, the high-titer centers consistently enrich plasma pools for anti-hav ig content. the twelve pools described in the preceding paragraph were also used to determine how differences in anti-hav ig titers relate to the capacity to neutralize hav in vitro. high-titer plasma pools from the el paso-tx center demonstrated a significantly greater capacity to neutralize hav than the plasma pools from the mid-titer midwest city-ok center, confirming that elevated anti-hav ig titers associate with greater inhibition of hav infection in vitro, which is consistent with previous observations . more importantly, the neutralization result underscores the clinical benefit plasma from these high-titer centers provides. this work is timely, because, despite declining overall rates of hepatitis a infection, a spike in outbreaks of late has highlighted the need for consistent ig augmentation therapy , . moreover, mev is reemerging as a serious public health threat with outbreaks occurring even in highly developed countries , . notably in , at the end of april the us had already seen its greatest number of reported cases since measles was said to be eliminated in . meanwhile, anti-mev ig titers in the general population have also been on the decline, which has caused some ig manufacturers to have had difficulty meeting the us food and drug administration (fda) product specification for anti-mev content. to prevent shortages in product, the us fda has lowered the specification . however, as a precondition for lowering the specification, the agency recommended that firms add labeling for dosing of patients needing measles pre-or post-exposure prevention. herein we report the levels of anti-hav antibodies in plasma collected from various donor centers in the southwestern quarter of the us, and we confirmed that some locales known to harbor an elevated seroprevalence produce donors with significantly higher levels of anti-hav antibodies (el paso-tx and mcallen-tx) than plasma acquired from other locations loosely associated with the us southwest (san diego-ca, midwest city-ok, and provo-ut), as well as a locale outside of the region entirely (clarksville-tn). these results are one example of how a diverse plasma supply ensures that the variety of antibodies needed for treatment of pidd is maintained. but a broader strategy deserves consideration. perhaps the production of some hyperimmune ig augmentation therapies could be made more efficient and cost-effective by a rational approach that combines epidemiologic surveillance and strategic management of the plasma supply chain rather than blind screening or immunization methods. at the very least, manufacturers of ig products could attempt to understand regional differences in antibody potency stemming from natural immunity versus immunization. on a global scale, regional sourcing has the potential to be an inherent first-line defense against emerging infectious diseases in other parts of the world. regardless of the specifics, the power of such a strategy will derive from the dynamic antibody repertoire of a diverse plasma collection platform. data reported in this manuscript are available within the article. the datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request. the epidemiology of virus transmission by plasma derivatives: clinical studies verifying the lack of transmission of hepatitis b and c viruses and hiv type ensuring the biologic safety of plasma-derived therapeutic proteins effects of social, environmental and economic factors on current and future patterns of infectious diseases hepatitis a virus antibodies in immunoglobulin preparations incidence of hepatitis a in the united states in the era of vaccination hepatitis a in the era of vaccination progress toward eliminating hepatitis a disease in the united states hepatitis a antibody titres after infection and immunization: implications for passive and active immunization seroprevalence of hepatitis a virus antibodies in the u.s.: results from the national health and nutrition examination survey evaluation of potencies of immune globulin products against hepatitis a solution for intramuscular injection prescribing information, grifols therapeutics, llc human) supporting documents, new recommendations to increase the dose of gamastan s/d (immune globulin [human]) when used for prophylaxis for hepatitis a measles-virus-neutralizing antibodies in intravenous immunoglobulins prevention of hepatitis a through active or passive immunization: recommendations of the advisory committee on immunization practices (acip) rising hepatitis a immunity in u.s. military recruits infectious disease morbidity in the us region bordering mexico use of intravenous immunoglobulins for prophylaxis or treatment of infectious diseases history of immunoglobulin replacement an update on the use of immunoglobulin for the treatment of immunodeficiency disorders use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology update on the use of immunoglobulin in human disease: a review of evidence seroprevalence of measles, mumps, rubella and varicella antibodies in the united states population official cdc health advisory distributed via the cdc health alert network (han): outbreak of hepatitis a virus (hav) infections among persons who use drugs and persons experiencing homelessness measles antibody trough levels after treatment with immunoglobulin products and predicted levels assuming lower measles antibody specifications increase in hepatitis a virus infections-united states the re-emergence of measles in developed countries: time to develop the next-generation measles vaccines? vaccine measles-united states letter to immune globulin (human) licensed manufacturers: option to lower lot release specification for required measles antibody potency testing the authors would like to thank laneisha farrar and jennifer carter for their tireless efforts to conduct the hundreds of elisas and neutralization assays necessary to complete this study. we also thank joann hotta and shih-fong chao for their expertise involving cmv propagation and neutralization. lastly, we thank mangkey bounpheng for her insightful contributions to our discussion of donor center locations. editorial assistance was provided by maryjane silvey, writemonitor, llc, durham, nc, usa, which was funded by grifols. j.c. -contributed to the study conception, design, and writing of the manuscript; was primarily responsible for data collection and analysis, as well as creation of the figures for the manuscript. k.s. -contributed to the study conception, design, and writing of the manuscript; was primarily responsible for material preparation. t.g. -contributed to the study conception, design, and writing of the manuscript. p.v. -contributed to the study conception, design, and writing of the manuscript. b.g. -contributed to the study conception, design, and analysis; was primarily responsible for writing of the manuscript. all authors are employed by grifols, the manufacturer of immunoglobulin products including gamunex ® -c, flebogamma ® dif, xembify ™ and gamastan ® . funding was provided by grifols, research triangle park, nc correspondence and requests for materials should be addressed to b.d.g.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -uxhpufnv authors: nusshag, christian; stütz, alisa; hägele, stefan; speer, claudius; kälble, florian; eckert, christoph; brenner, thorsten; weigand, markus a.; morath, christian; reiser, jochen; zeier, martin; krautkrämer, ellen title: glomerular filtration barrier dysfunction in a self-limiting, rna virus-induced glomerulopathy resembles findings in idiopathic nephrotic syndromes date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: uxhpufnv podocyte injury has recently been described as unifying feature in idiopathic nephrotic syndromes (ins). puumala hantavirus (puuv) infection represents a unique rna virus-induced renal disease with significant proteinuria. the underlying pathomechanism is unclear. we hypothesized that puuv infection results in podocyte injury, similar to findings in ins. we therefore analyzed standard markers of glomerular proteinuria (e.g. immunoglobulin g [igg]), urinary nephrin excretion (podocyte injury) and serum levels of the soluble urokinase plasminogen activator receptor (supar), a proposed pathomechanically involved molecule in ins, in puuv-infected patients. hantavirus patients showed significantly increased urinary nephrin, igg and serum supar concentrations compared to healthy controls. nephrin and igg levels were significantly higher in patients with severe proteinuria than with mild proteinuria, and nephrin correlated strongly with biomarkers of glomerular proteinuria over time. congruently, electron microcopy analyses showed a focal podocyte foot process effacement. supar correlated significantly with urinary nephrin, igg and albumin levels, suggesting supar as a pathophysiological mediator in podocyte dysfunction. in contrast to ins, proteinuria recovered autonomously in hantavirus patients. this study reveals podocyte injury as main cause of proteinuria in hantavirus patients. a better understanding of the regenerative nature of hantavirus-induced glomerulopathy may generate new therapeutic approaches for ins. . characteristics of patients with acute hantavirus infection. acr = albumin-to-creatinine ratio, crp = c-reactive protein, dpo = days post onset of first symptoms, gcr = gram creatinine, los = length of hospital stay, max = maximum, min = minimum, pcr = protein-to-creatinine ratio, scr = serum creatinine. bold values are statistically significant for p < . . (fig. ), but the characteristic picture of tubular interstitial nephritis in the renal medulla (fig. ) . electron microscopy of glomeruli revealed enlarged visceral podocytes, a focal foot process effacement together with a mild thickening of the glomerular basement membrane (gbm) and vacuolization of podocytes in hantavirus patients (fig. , figure s ). immune deposits or further ultrastructural changes were not present. mean gbm widths were . nm (± . ), . nm (± . ), . nm (± . ) and . nm (± . ) for the control and hantavirus biopsy samples i, ii and iii, respectively. maximum podocyte foot process width was highest in proteinuric patients i and iii with . nm and . nm and significantly lower in patient ii and control with . nm and . , respectively. however, due to the focal nature of the foot process effacement, mean podocyte width did not differ between patients and control (table s ) . compared to the control, em analysis of proximal tubular cells showed relevant subcellular lesions indicated by severe apical cytoplasmic vacuolization (fig. ) . interestingly, these changes were predominantly observed in the two patients with severe proteinuria at the time of biopsy (hantavirus patient i and iii). biomarker levels on admission. on admission, patients suffering from hantavirus infection showed significantly increased urinary nephrin, igg, α -mg and serum supar levels compared to healthy controls (fig. a ). when further dividing hantavirus patients according to the severity of pcr on admission, patients with severe pcr showed significantly higher median nephrin and igg levels compared to patients with moder- www.nature.com/scientificreports/ ate pcr. remarkably, an almost dichotomous distribution of urinary biomarkers of a defective gfb (nephrin and igg) was observed when moderate and severe pcr were compared on admission, indicating substantial differences in permeability of the glomerular slit diaphragm at that specific time point. a trend towards higher supar levels was also seen in patients with severe proteinuria. correlation analyses between serum supar levels, maximum scr and scr levels within the first h showed no significant correlations, whereas a significant positive correlation was found for serum supar levels and levels of urinary nephrin, pcr, acr and igg (table s ). the normalization of nephrin and igg levels to urinary creatinine excretion led to similar results ( figure s ). in contrast, the previous significant difference of absolute α -mg levels between patients with moderate and severe pcr disappeared after creatinine normalization ( figure s ). though, urinary biomarker levels decreased in both groups over time, patients with severe pcr showed significantly higher levels of nephrin, igg, acr and pcr during the first h after admission ( table ). the greatest absolute differences were seen for urinary biomarkers that indicate a defective glomerular barrier such as nephrin, igg, acr. at the same time, only minor absolute differences were observed for the tubular proteinuria marker α -mg. when analyzing urinary nephrin concentrations over time in individual patients and in relation to the dpo instead of time after admission, urinary nephrin and pcr levels decreased almost in parallel. interestingly, the start of normalization of urinary nephrin and pcr levels preceded the first decline of scr by - h. furthermore, in patients with an available urine sample at the time of pcr normalization, the normalization of urinary nephrin levels tended to precede the normalization of pcr levels. figure b shows two exemplary biomarker courses of patients with acute hantavirus infection. in a next step, we analyzed the course of dpo-synchronized pcr values between patients with moderate and severe pcr in the entire cohort (table ) . patients with severe pcr showed significantly higher pcr levels up to dpo compared to patients with moderate pcr, indicating a higher renal disease severity between both groups at the same dpo. due to the self-limiting character of the hantavirus disease and subsequent autonomous recovery of the gfb in both groups, no differences in pcr levels were seen beyond dpo ( table ). the morphology and time course of pcr slopes within both groups www.nature.com/scientificreports/ was comparable, but the origin of the slope started at higher pcr levels in patients with severe proteinuria, especially before dpo . analyses showed a strong positive correlation between urinary nephrin levels and pcr, acr, igg, α -mg and c-reactive protein (crp) levels ( table ). the highest correlation coefficients (r) were thereby achieved between nephrin levels on admission and biomarkers of (non-selective) proteinuria. in contrast, weaker correlations where seen for scr, especially on admission and for maximum levels. furthermore, nephrin levels on admission showed a moderate positive association with the length of hospital stay (los) and a moderate negative association with platelet count and the time of admission in terms of dpo. hemoglobin and leukocytes values showed no relevant correlation with urinary nephrin levels. to our knowledge, this is the first comprehensive study to investigate the role of direct podocyte damage in acute puuv infection in vivo. our data show a strong association between urinary nephrin levels and the extent of (non-selective) glomerular proteinuria, suggesting that hantavirus infection causes a pronounced podocyte damage and subsequent impairment of the gfb. the significant findings in electron microscopy analyses were a focal foot process effacement, podocyte vacuolization and apical tubular vacuolization (indicating massive proteinuria) which all are known as typical histopathological features in ins , , [ ] [ ] [ ] [ ] [ ] . however, the pathomechanical role and underlying mechanisms of the observed podocyte vacuolization need further clarification. while differences between patients with moderate and severe proteinuria were preserved for urinary nephrin and igg levels after normalization to urinary creatinine excretion, differences in α -mg levels were no longer present. this further supports the idea that proteinuria in hfrs is predominantly from glomerular origin. furthermore, when individual patients were analyzed, the normalization of urinary nephrin levels tended to precede the normalization of proteinuria. the highest correlation coefficients (r) were achieved between urinary nephrin levels on admission and pcr, acr and igg levels within h. both observations suggest urinary nephrin levels as an early indicator of gfb dysfunction and a direct pathophysiological connection between the current impairment of gfb integrity by hantavirus infection and the subsequent extent and clinical course of proteinuria. as a further similarity to ins , , hantavirus patients showed significantly elevated serum supar levels in comparison to healthy controls. supar levels correlated significantly with urinary nephrin, pcr, acr and igg levels, but not with scr. it was generally believed that hantaviruses predominantly infect endothelial cells, leading to capillary permeability due to a loss of cell-to-cell contacts, but without a direct cytopathic effect . the deregulation of systemic angiopoietin levels and a vascular endothelial growth factor a (vegfa)-induced downregulation of ve-cadherins accompanied by a β integrin-mediated dysregulation of vegf receptor (vegf ) are suggested mechanisms , [ ] [ ] [ ] . though, these findings may explain major symptoms and complications of hfrs such as capillary leakage and pulmonary edema, the exact cause of (nephrotic) proteinuria is still poorly understood. vascular endothelial barrier dysfunction or tubular damage, as discussed by some authors , , does not adequately explain the extent of proteinuria. we have recently shown in vitro that hantaviruses additionally infect tubular epithelial cells, glomerular endothelial cells and especially podocytes, leading to disruption of cell-to-cell contacts and impaired intra-cellular integrity with rearrangements of the podocyte cytoskeleton , , . here, we show for the first time in vivo an interdependent relationship between ultrastructural and functional impairments of the gfb and a direct podocyte damage as indicated by increased nephrin excretion and electron microscopy. in addition, urinary nephrin, pcr, acr and igg levels correlated significantly with serum supar values. to date, one other study showed significantly elevated blood supar levels and their association with hantavirus disease severity but did not include nephrinuria and the extent of proteinuria in their analysis . supar is suggested to interfere with the cross-directional signaling between the glomerular basement membrane (gbm) and podocytes, thereby affecting gfb integrity . in addition, alfano et al. recently showed that supar downmodulates nephrin expression in podocytes in vitro and in vivo . together, these mechanisms may disturb table . comparison of proteinuria in relation to the days post onset of first symptoms. dpo = days post onset of first symptoms, gcr = gram creatinine, pcr = protein-to-creatinine ratio. bold values are statistically significant for p < . . www.nature.com/scientificreports/ table . spearman's correlation of urinary nephrin levels on admission with parameters of hantavirus disease activity. acr = albumin to creatinine ratio, α -mg = α -microglobulin, ci = confidence interval, crp = c-reactive protein, dpo = days post onset of first symptoms, igg = immunoglobulin g, r = correlation coefficient, s-alb = serum albumin, scr = serum creatinine. bold values are statistically significant for p < . . www.nature.com/scientificreports/ podocyte-gbm-interaction, where rearrangements of the podocytic cytoskeleton may result in podocyte damage, subsequent foot process effacement and gfb dysfunction with release of intercellular gfb components such as nephrin into the urine , , , . further studies are required to clarify whether supar is a directly involved pathomechanically mediator in hantavirus-induced podocyte injury. nevertheless, a very unique feature in puuv infection is that proteinuria and kidney function usually recover autonomously in all patients , while therapeutic outcomes in ins are variable and autonomous recovery is rare , . hantavirus induced proteinuria usually peaks around dpo and normalizes within - weeks . but, due to a varying hantavirus disease severity, the actual extent of proteinuria differs in patients . the same observation applies to our cohort. patients with severe pcr on admission still showed significantly higher pcr levels compared to patients with moderate pcr, when pcr values were matched based on the dpo instead of the time of hospital admission. scr in turn peaks - days after the peak of proteinuria . this tempts authors to claim that proteinuria predicts the severity of emerging aki, by showing an association between glomerular proteinuria and maximum scr levels . however, it is generally accepted that scr reflects rapid changes in renal function with a latency of at least - h (time until a new steady state is reached after a single renal insult) . we therefore hypothesize that the extent of maximum renal impairment is at least in part already present at the time of maximum proteinuria but is indicated with a delay by scr. our results support this hypothesis by showing a stronger correlation between pcr values on admission and scr concentrations after - h ( h: r = . , p < . ; h: r = . , p < . ) than for maximum scr levels or scr levels on admission (adm: r = . , p = . ; max: r = . , p = . ). other accepted markers of hantavirus disease severity such as leukocyte and platelet count, crp and hemoglobin levels, showed weaker correlations with nephrin. this suggest gfb dysfunction once more as an independent, subcellular disease manifestation of hantavirus infection in addition to capillary leakage and aki. both impairment of renal function and proteinuria must probably be seen as two individual disease manifestations, the extent of each depending on the current hantavirus disease activity. there are study limitations, which need to be addressed. first, kidney biopsy material was only available for three hantavirus patients in our database who were initially suspected to have kidney diseases other than hantavirus infection. routine kidney biopsies could not be justified in terms of a risk-benefit analysis, due to the mostly reliable hantavirus diagnostics by serological tests and the self-limiting disease character with exclusively symptomatic therapy. second, the use of a creatinine-normalized description of proteinuria/albuminuria in hantavirus patients may overestimate total protein excretion relative to -h volume measurements because urinary creatinine excretion decreases with aki. however, we are confident that this does not affect the results of our study, as our results were extremely consistent when absolute or creatinine-normalized proteinuria parameters were used in the analyses of glomerular proteinuria. third, the reported minimum or maximum biomarker levels in our study may differ from the actual absolute minimum or maximum values that may have occurred prior to hospitalization. in summary, puuv hantavirus infection shows clinical and ultrastructural similarities to ins, but with the unique feature of an autonomous recovery. this special feature highlights the potential of further comparative studies with ins and other rna-virus induced glomerulopathies in order to improve our understanding of regenerative mechanisms in the context of gfb dysfunctions and potential future therapeutic approaches. study design and patient population. this retrospective study was conducted at the department of nephrology at heidelberg university hospital and approved by the local ethics committee of the medical faculty of heidelberg. all patients with acute hantavirus infection hospitalized in were included for further analyses. in total, patients without pre-existing kidney diseases and with serologically proven, acute puuv infection were analyzed. age and gender matched volunteers served as controls (table s ). written informed consent was obtained from all participants. all methods or experiments were performed in accordance with relevant guidelines and regulations. the overall median creatinine-normalized proteinuria (protein-to-creatinine ratio, pcr) at the time of admission was used to categorize patients in two disease severity groups (moderate pcr vs. severe pcr): (a) moderate pcr (≤ mg/g creatinine, gcr) and (b) severe pcr (> mg/gcr). baseline serum creatinine (scr) was defined as the lowest value between months before or after an acute hantavirus infection. maximum pcr or scr values were defined by the highest value measured during hospitalization or recorded in medical reports immediately prior to hospitalization. data collection and laboratory methods. patient characteristics and presented laboratory parameters such as scr, urinary albumin-to-creatinine ratio (acr) and pcr were obtained from medical records. urine nephrin, igg, α -mg and serum supar levels were measured retrospectively. nephrin and supar levels were quantified by enzyme-linked immunosorbent assay (elisa) as instructed by the manufacturer (human nphn antibody elisa kit, elabscience biotech co. ltd, wuhan, china; supar elisa kit, r&d systems, minneapolis, mn, usa). urinary igg and α -mg concentrations were measured in the accredited central laboratory of the heidelberg university hospital. light and electron microscopy. three biopsy samples of patients with acute hantavirus infection were analyzed. biopsy samples (heidelberg biopsy register) were fixed in glutaraldehyde and embedded in epon-araldite after post-fixation with osmium tetroxide and analyzed by transmission electron microscopy (jem- , jeol, freising, germany). biopsy material from a time-zero biopsy of a living kidney allograft served as control. all analyses were performed at the institute of pathology (heidelberg university hospital, germany). the gbm width was evaluated using representative measurements per patient. to account for the focal nature scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ acute kidney injury in critically ill patients with covid- acute kidney injury in patients hospitalized with covid- multiorgan and renal tropism of sars-cov- renal histopathological analysis of postmortem findings of patients with covid- in china kidney disease is associated with in-hospital death of patients with covid- uncovering the mysteries of hantavirus infections virus-and cell type-specific effects in orthohantavirus infection clinical course and long-term outcome of hantavirus-associated nephropathia epidemica deregulation of levels of angiopoietin- and angiopoietin- is associated with severe courses of hantavirus infection cardiopulmonary 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vegf, while angiopoietin and sphingosine -phosphate inhibit hantavirus-directed permeability motility of human renal cells is disturbed by infection with pathogenic hantaviruses pathogenic old world hantaviruses infect renal glomerular and tubular cells and induce disassembling of cell-to-cell contacts full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes issues of acute kidney injury staging and management in sepsis and critical illness: a narrative review christian nusshag was funded by the physician scientist programme of heidelberg faculty of medicine. author contributions n.c. conceived the study design, was responsible for acquisition, analysis and interpretation of data and drafted the article. s.a. assisted with acquisition, analysis and interpretation of data and assisted in drafting the article. h.s., s.c., k.f., e.c., b.t., w.m.a. and m.c. contributed to the acquisition and interpretation of data and revised the article critically. z.m., r.j. and k.e. contributed to the conception of the study, assisted with analysis and interpretation of data and revised the article critically. all authors approved the final version of the article for publication. open access funding enabled and organized by projekt deal. reiser j. is a co-founder and shareholder of trisaq, a biotechnology company developing therapies for renal diseases. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to c.n. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -lpk r i authors: ferreira-ramos, ana sofia; sulzenbacher, gerlind; canard, bruno; coutard, bruno title: snapshots of adp-ribose bound to getah virus macro domain reveal an intriguing choreography date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: lpk r i alphaviruses are (re-)emerging arboviruses of public health concern. the nsp gene product is one of the key players during viral replication. nsp comprises three domains: a macro domain, a zinc-binding domain and a hypervariable region. the macro domain is essential at both early and late stages of the replication cycle through adp-ribose (adpr) binding and de-adp-ribosylation of host proteins. however, both its specific role and the precise molecular mechanism of de-adp-ribosylation across specific viral families remains to be elucidated. here we investigate by x-ray crystallography the mechanism of adpr reactivity in the active site of getah virus macro domain, which displays a peculiar substitution of one of the conserved residues in the catalytic loop. adpr adopts distinct poses including a covalent bond between the c′′ of the adpr and a conserved togaviridae-specific cysteine. these different poses observed for adpr may represent snapshots of the de-adp-ribosylation mechanism, highlighting residues to be further characterised. protein production and purification. the dna coding sequence of the nsp macro domain (aminoacids to ) of getah virus (strain m , genbank: abk . , amino-acids , to , ) was optimized for expression in escherichia coli and synthesized by thermo fischer scientific. the coding sequence was cloned into a pdest vector using the "gateway" cloning procedure (thermo fischer scientific). a hexahistidine ( -his) coding sequence was added at the ′ end to produce the getv macro domain in fusion with a c-terminal -his tag. a short sequence of two codons coding for methionine and lysine were added to the ′ end in order to allow the translation in e. coli. the bacterial growth conditions for optimal expression of getv macro domain were obtained from an incomplete factorial expression screen . getv macro domain was produced in e. coli rosetta (de ) plyss cells (novagen). bacteria were grown at °c with shaking ( rpm) in terrific broth (tb) medium (sigma-aldrich) containing µg·ml − chloramphenicol and µg·ml − ampicillin. the expression of the recombinant gene was induced with . mm isopropyl β-d- thiogalactopyranoside (iptg) when the bacterial cell density reached the exponential phase at od nm of . . after induction, the incubation temperature was dropped to °c for overnight expression. subsequently, the bacterial cells were harvested by centrifugation at , g for min and pellets were resuspended in ml of lysis buffer made of mm tris-hcl ph . , mm nacl, mm imidazole, % glycerol, . % triton x- and one tablet of complete edta-free protease inhibitor cocktail (roche). the resuspended pellets were stored at − °c until the time of use. pellets were thawed and . mg·ml − of lysozyme (sigma-aldrich), µg·ml − of dnase i (sigma-aldrich) and mm phenylmethylsulfonyl fluoride (pmsf) (sigma-aldrich) were added. the samples were incubated at °c for min and then sonicated. samples were then centrifuged at , g for h. the supernatant was loaded onto a ml his prep column (ge healthcare) for immobilized metal affinity chromatography (imac) using an Äkta xpress system (ge healthcare). after loading the supernatant, the column was washed with a buffer containing mm tris-hcl, mm nacl, mm imidazole, ph . . the recombinant protein was eluted with mm tris-hcl, mm nacl, and mm imidazole, ph . . size exclusion chromatography was next performed on a superdex hiload / column (ge healthcare) equilibrated in mm hepes, mm nacl, ph . . the eluted protein was concentrated up to mg·ml − using an ultracel regenerated cellulose membrane with a kda molecular weight cut-off (amicon ultra- centrifugal filter, merck millipore). purity and stability assessment. the purity of the protein was assessed on sds-page gels stained with coomassie blue. besides, protein stabilization by adp-ribose was additionality checked by thermal shift assay (tsa) in a -well thin-wall pcr plate (bio-rad) using a quantitative pcr machine icycler iq (bio-rad). briefly, the protein at a final concentration of . mg·ml − was mixed with a sypro orange solution at concentrations recommended by the manufacturer (thermo fisher scientific) in a final volume of µl and tested with the following concentrations of adp-ribose: . mm, . mm, . mm, . mm, . mm, . mm and mm. accumulative steps of temperature from to °c were applied to the samples. the increase of the fluorescence emitted by the probe that binds the exposed hydrophobic regions of the denatured protein was used to monitor the denaturation of the protein. a melting temperature (t m ) was calculated as the mid-log of the transition phase from the native to the denatured protein using a boltzmann model (origin software). crystallization, co-crystallization and crystal soaking. initial crystallization trials were carried out with getv macro domain at mg·ml − using the commercial screens wizard classic & ht , stru ctu re & ht , and stura footprint screen (molecular dimensions limited) in -well sitting-drop swissci crystallization plates (tpp labtech). nl of crystallization solutions were added to , or nl of getv macro domain solution using a mosquito robot (ttp labtech). the conditions where crystal hits were obtained were then optimized using either -well swissci crystallization plates or -well hanging drop linbro plates. co-crystallization experiments were set-up with getv macro domain complemented with ( ) adp-ribose, ( ) adp-ribose and glutamic acid or ( ) adp-ribose and aspartic acid, with final concentrations of mm for adp-ribose, mm for glutamic acid and mm or mm for aspartic acid. in general, crystals appeared after one day and were grown for approximately two weeks. soaking experi-scientific reports | ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports/ ments were performed with crystals of getv macro domain co-crystallized with adp-ribose by adding mother liquor supplemented with aspartic acid to the crystallization droplet followed by - h or overnight incubation at °c. afterwards, crystals were flash-cooled in liquid nitrogen using % of glycerol in the mother liquid as cryo-protectant. data collection and structure determination. diffraction www.nature.com/scientificreports/ the resolution limit, were set aside for freer cross-validation purposes. where data-sets of ligand complexes were in the same space group as the native data set, the composition of cross-validation data sets was systematically taken over from the parent data set. the structure of native getv macro domain was determined by molecular replacement with molrep using the macro domain of chikungunya virus (chikv, pdb gpg) as a search model. the structures of getv macro domain in complex with ligands were determined either by molecular replacement using the native getv macro domain as a search model or by difference fourier synthesis. refinement was performed using refmac , interspersed with cycles of manual model adjustments with coot . ligands were fitted into unbiased fo-fc difference electron density maps calculated after cycles of rigid-body refinement. hydrogens were added in the riding position. coordinates and restraints for adp-ribose in the close conformation were retrieved from the ccp ligand dictionary and a model and restraints for adpribose in the open conformation were generated with the ccp monomer library sketcher. model quality was assessed with internal modules of coot and with the molprobity server . crystallographic models are of good quality with . - % of residues in favoured regions of the ramachandran plot and no outliers. data collection and refinement statistics are summarized in table protein production, crystallization and structure determination. the sequence corresponding to the putative macro domain on the getv nsp sequence (genbank reference abk . ) is encompassing aa , to , (positions in the polyprotein; hereafter numbered - for convenience). its sequence identity with alphavirus homologues with known structures such as chikv and veev macro domains is % and %, respectively ( fig. ). macro domains harbour the consensus motif g(d/e/g)gv . a focused analysis of these residues (amino acids to of the nsp sequence) of macro domains revealed that the consensus motif is followed by a togaviridae-specific cysteine. it can also be noticed that the getv macro domain is an exception regarding the consensus motif, as its sequence harbours a serine instead of a glycine at position . we thus first verified that this macro domain is a bona fide adp-ribose binding module. the recombinant getv macro domain was produced in e. coli and purified under non-denaturing conditions. after purification, the integrity of the protein was assessed by thermal shift assay (tsa). tsa experiments showed that the protein could be denatured by heat in a classical folded-to-denatured transition phase (data not shown) with a melting temperature (t m ) of . ± . °c. addition of . mm adp-ribose to the protein solution led to a °c increase of the t m suggesting that the recombinant getv macro domain indeed binds adp-ribose (fig. ). the structure of the getv macro domain was determined at . Å resolution by molecular replacement using the chikv macro domain ( gpg) as a template. the crystals of the getv macro domain belong to the space group p and two molecules are present in the asymmetric unit. all residues from ala to thr are well defined in the electron density and the model has excellent stereochemistry with . % of side-chain rotamers in the favoured conformation and . % of residues in the favoured ramachandran plot regions ( table ). the structure of getv macro domain consists of a central twisted six-stranded β sheet (strand order β , β , β , β , β , β ) sandwiched between one α-helix (α ) at one side and three α-helices (α , α and α ) at the opposite site (fig. a) . the two molecules present in the asymmetric unit are virtually identical, with an r.m.s.d. of . Å between the two chains. a structural homology search with the dali server structures of the getv macro domain in complex with adp-ribose. the early evaluation of the integrity of getv macro domain by monitoring the thermostability using thermal shift assay revealed an increase of t m by °c induced by the presence of small amounts of adp-ribose, advocating that similar to other alphavirus macro domains, getv macro domain can bind adp-ribose. therefore, we produced crystals of the getv macro domain in the presence of mm adp-ribose. the study of the structure of the dimanganese mono-adp-ribosylhydrolase drag with a trapped amino acid-adp-ribose reaction intermediate, lys-adp-ribose , inspired us to add glutamic or aspartic acids to the crystallization trials, based on the rational that in this way we could reverse the de-adp-ribosylation reaction mediated by alphavirus macro domain, documented to de-adp-ribosylate carboxylic amino-acid side-chains , , . thus, we added glutamic or aspartic acids to co-crystallization or soaking solutions at equal or five to fifteen-times higher concentrations than that of adp-ribose. all the optimal crystallization and co-crystallization solutions converged to the following buffer composition: . m imidazole/malate ph . ± . and ± % of peg k. the crystallization and soaking procedures that led to the structures discussed in this study are summarize in table , and data collection and refinement statistics are presented in table . no clear electron density could be detected for aspartic or glutamic acid in any of the crystal structures. however, structural analyses revealed that the addition of the amino acids to the crystallization medium was associated with conformational changes both in the catalytic loop and of adpribose. five different conformations could be observed for adp-ribose bound to the getv macro domain, documented here below. (figs. , a ). the interaction of n- with aspartate residues structurally equivalent to asp is well conserved in most macro domain/adp-ribose complexes and has been shown to be crucial for adp-ribose binding in the archaeoglobus fulgidus macro domain . at the other hand, arg is not conserved, not even within the alphavirus macro domain family, and its side-chain is rather disordered in all the structures described in this work, indicating that the stacking interaction with the adenine ring makes probably only weak contributions to the binding energy. the proximal ribose interacts with getv macro domain through a single hydrogen bond between ′-oh and the side-chain of thr . it is noteworthy that trp , conserved in alphavirus macro domains, protrudes into the adp-ribose binding site and makes a steric clash with the ′-oh. in the other alphavirus macro domain structures, the side-chains of the equivalent trp residues are shifted about . to Å away from the adp-ribose binding site with respect to the position of trp in the getv macro domain. here, a val residue pointing from the back towards the trp indole ring, val , impedes an equivalent rearrangement of the trp side-chain, which appears to be highly dynamic and could not be modelled in a satisfactory fashion in all the structures described in this work. similarly to what had been observed in other macro domain/adp-ribose complexes, the phosphate groups of adp-ribose are lined by the catalytic loop β α and loop β α and tightly coordinated by hydrogen bonds with main-chain atoms of residues val , ser , gly , thr and phe and the side-chain of thr . finally, the distal ribose, observed in the anomeric α-configuration, interacts with getv macro domain through hydrogen bonds contracted between ″-oh and the side-chain of asn , between ″-oh and the side-chains of asn and ser , and between ″-oh and the side-chain ser and the main-chain of asp , and establishes a stacking interaction with the side-chain of phe . the ser of the catalytic β α loop is unique to getv macro the tight interactions established between the distal ribose and ser point towards a role in substrate binding or catalysis for this residue. not surprisingly, we found that the most important structural changes occurring in getv macro domain upon adp-ribose binding could be observed in the catalytic loop β α and loop β α , which close-up over the ligand to establish tight binding interactions. adp-ribose presenting the distal ribose in an unusual conformation, "pose ". by adding mm adp-ribose and mm glutamic acid to the crystallization medium of getv macro domain we obtained a second crystal for a getv macro domain/ adp-ribose complex for which diffraction data extending to . Å resolution were collected. the structure was determined by difference fourier synthesis and the final model has excellent stereochemistry. thr in chain a could not be modelled in satisfactory fashion and in chain b electron density revealed the presence of lys , originating from the cloning strategy that includes an aaa triplet (lys codon) to promote efficient translation initiation . in this second getv macro domain/adp-ribose structure the interactions between the getv macro domain and the adenine moiety, the proximal ribose and the phosphate groups are virtually identically to the interactions described above. however, the distal ribose is tilted by approximately ° with respect to the "classical" pose described above and observed in all other macro domain/adp-ribose complexes described so far (fig. b) , with exception of the crystal structure of human histone macroh a . in complex with adp-ribose in form b ( iif) . however, in this latter crystal structure the distal ribose is tilted in the opposite direction with respect to the distal ribose observed in the present getv/adp-ribose structure. in the novel and unusual pose the distal ribose, here in the b-anomeric configuration, establishes hydrogen bonds with ala and asn main-chain atoms via ″-oh, with the side-chains of asn and ser via ″-oh and with the ser side-chain and the asp main-chain mediated by the ″-oh hydroxyl group. the ribose ring lines up in perfectly parallel fashion with the side-chain of phe . interestingly, residual difference electron density was observed close to the distal ribose in chain a and was modelled as an acetate molecule, assuming that it represented the ordered www.nature.com/scientificreports/ part of glutamic acid added to the crystallization medium. this carboxylic moiety coordinates the ″-oh and ″-oh hydroxyl groups of adp-ribose and is further stabilized by an h-bond interaction with ser . we dare to speculate that the position of the acetate molecule relative to the distal ribose ring is reminiscent of a putative adp-ribose-glutamate conjugate, a possible substrate for getv macro domain as inferred from the function of other alphavirus macro domains , , . and yet again, ser appears to play a crucial role in substrate binding. we further obtained two additional getv macro domain/adp-ribose complexes by co-crystallizing the protein in the presence of mm adp-ribose and mm aspartic acid. diffraction data were recorded to . and . Å, respectively. again, in these two crystal structures the interactions between getv macro domain and the adenine moiety, the proximal ribose and the phosphate groups are comparable to those seen in the above-described models. conversely, preliminary electron density maps, calculated before the incorporation of the ligand, clearly indicated that the distal ribose was present in the open conformation ( supplementary fig. s ). in the complex structure at resolution of . Å the distal ribose is present in a single conformation, whereas the best way to account for difference electron density in the . Å data set was to model the distal adp-ribose in a double open conformation (fig. c,d) . both structures were determined by difference fourier synthesis and the final models present excellent stereochemistry. in both structures no clear electron density could be observed for thr in chains a and the additional lys is present in chains b. in one of the conformations of adp-ribose in the double open conformation, conformation a (fig. c) , the carbon atoms of the distal ribose superpose with the carbon atoms of the distal ribose ring as seen in the complex with adp-ribose in pose and the hydrogen-bonding interactions are to a large extent comparable. however, due to the ring opening the ′′-oh is now free to make hydrogen-bonding interactions with the side-chain of thr and the main-chain of asp . the acetate molecule, as observed in the complex with adp-ribose in pose , could not be spotted in this structure. the conformations of the distal ribose in conformation b and the one in the complex with open adp-ribose in single conformation are virtually identical (fig. d ). here the linear ribose chain underwent a ~ ° rotation around the bond connecting carbon atoms ″-c and ″-c, and consequently the hydrogen-bonding pattern between adp-ribose and the getv macro domain changed drastically. in this new conformation the ″-oh binds the side-chain of thr and the main-chain of asp , ″-oh interacts with the side-chains of asn and ser , the ″-oh hydroxyl group contacts the side-chains of ser and cys and ″-oh is coordinated by the main-chain atoms of ala and cys and hydrogen bonds the α-phosphate. an acetate molecule, reminiscent of aspartic acid added to the crystallization solution, is isosteric to the one observed in the complex with the distal ribose in pose and interacts with the ″-oh hydroxyl of adp-ribose and the side-chain of ser . structure of the getv macro domain/adp-ribose complex with the distal ribose covalently bound to cys . finally, we obtained a last getv macro domain/adp-ribose complex by co-crystallizing the protein with mm adp-ribose and mm aspartic acid and diffraction data were collected to . Å resolution. for no apparent reason the space group in this complex changed from p (as observed in all the previously described structures) to c and only one molecule is present in the asymmetric unit. we verified cautiously whether the position of adp-ribose in this novel complex could have influenced the crystal packing and could not retrieve any plausible explanation for the change of space group. the structure was solved by molecular replacement, using the native getv macro domain structure as a search model and the final model presents very good stereochemistry. the interactions between getv macro domain and the adenine moiety, the proximal ribose and the phosphate groups of adp-ribose are specular to the interactions observed in all the getv macro domain/adp-ribose complexes described so far. to our surprise, adp-ribose in this structure is found in the open conformation and a covalent bond is established between ″-c and cys sg ( fig. e and sup. fig. s ). it is noteworthy that in all . finally, a substrate-assisted mechanism for cleavage of the adp-ribose/protein linkage had been proposed for human macrod where the α-phosphate of the conformationally strained adp-ribose activates an ideally positioned water molecule for nucleophilic attack on the c ′′ atom . seemingly the ubiquitous macro domains are built on a common globular scaffold prone to accommodate adp-ribose. still, subtle variations in loop structures elicit profound mechanistic diversity, which calls for further functional and structural dissection of this interesting class of molecules. intrigued by the substitution of a highly conserved glycine residue for serine and the presence of a togaviridae-specific cysteine in the catalytic loop of getv macro domain, we set out for a comprehensive structural investigation deemed to unravel the functional role of these substitutions. inspired by the structural study of the mono-adp-ribosylhydrolase drag in which an amino-acid/adp-ribose intermediate was trapped , we cocrystallized or soaked getv macro domains with adp-ribose and with increasing concentrations of aspartic or glutamic acid, which could mimic host protein adp-ribosylated side-chains, thereby reversing the de-adpribosylation reaction. in a first adp-ribose complex, obtained from a crystal where getv macro domain had been co-crystallized with adp-ribose and subsequently soaked in a solution containing aspartic acid, adp-ribose had been found in the binding pocket with the distal ribose in the low energy ′′-endo twist conformation and in a position commonly observed in other macro domain/adp-ribose complexes (pose ). however, when adpribose and glutamic acid were added to the co-crystallization solution, in the resulting crystal structure the distal ribose (pose ) was tilted with respect to the one observed in the previously determined adp-ribose complex and adopted the energetically less favourable o- ′′-endo envelop configuration. though, this strained ribose conformation is stabilized by a tighter network of hydrogen bonds contracted with the getv macro domain when compared to the pose complex structure. furthermore, in one of the two molecules in the asymmetric unit an acetate molecule, reminiscent of glutamic acid added to the crystallization solution, coordinates the ′′-oh and ′′-oh hydroxyl groups, evoking the position of the leaving group after the de-adp-ribosylation reaction. apparently, it was the crystallization method deployed, soaking versus co-crystallization with carboxylic acids, which induced the conformational change between pose and pose . when getv macro domain was co-crystallized with adp-ribose and aspartic acid, the distal ribose was found in two different open conformations, one of them closely resembling the closed-ring ribose of pose , the other resulting from a rotation around c ′′ and c ′′, which positioned the aldehyde function close to the α-phosphate, buried in a pocket surrounded by cys , val , asn and ala . open conformations of an integral ribose in the open conformation bound to macro domains have so far not been observed, but dehydrated open adp-ribose adducts have been observed in the crystal structures of targ and drag . lastly, co-crystallization of getv macro domain with low concentrations of adp-ribose and aspartic acid ( mm each) led to a structure where the distal ribose is covalently attached to cys . this covalent thio-hemiacetal adduct is reminiscent of the keto-amine transition states involving a lysine residue described for drag and targ . it is interesting to notice that an aspartate, asp , proposed to be the catalytic acid/base catalyst in human macrod , is isosteric to cys , as well as asp of human macrod and glu of t. curvata parg , two residues proposed to play crucial roles in catalysis. noteworthily, drag, targ , macrod and macrod are among the closest structural homologues of getv macro domain. the above mentioned structural similarities advocate for cys being the catalytic residue in the getv macro domain, and maybe as well in macro domains of other alphaviruses. cys-dependent hydrolases found in nature are mainly represented by cys-proteases, operating through a catalytic mechanism relying on a cys-his-asp catalytic triad. getv macro domain exhibits no overall structural similarity with any known cys-protease and the only his and asp residues found in the vicinity of cys , namely his and asp , though pretty well conserved throughout alphavirus macro domains, are too far apart to form a functional catalytic triad. the role of the peculiar ser substituting a glycine in the catalytic loop of the getv macro domain could ultimately not be resolved by this study. the serine residue might contribute to the trapping of adpr in different poses that were to date not observed in the other alphavirus macro domains having a glycine in position . still, the tight interactions of this residue with the distal ribose observed in the different complex structures described herein plead for a function in substrate binding or eventually a direct role in catalysis in interplay with cys . altogether the five different conformations of adp-ribose presented in this study could picture a conformational itinerary (fig. ) representing the second part of the de-adp-ribosylation reaction, leading from the covalent reaction intermediate through different conformational arrangements of the open ring conformations to the final products, represented by pose , strained in an energetically unfavourable conformation in the presence of the leaving group, and finally collapsed into the energetically favoured ′′-endo twist conformation (pose ) as observed in most macro domain/adp-ribose complexes documented so far. we cannot ascertain whether the covalent cys -adp-ribose adduct portrays the true interaction intermediate, or whether it represents just an artefact standing for an abortive complex. in any case, the conservation in alphavirus macro domain sequences and the proximity of cys to the catalytic centre argues for an important role of this residue in the catalytic mechanism. in this respect, mutational studies, which go beyond the purpose of this report, will be necessary to ascertain the true identity of the catalytic residue of the getv macro domain. in this study, we describe by means of crystallographic structures different poses adopted by a molecule of adp-ribose in the binding site of getv macro domain. in addition to the pose of adp-ribose found in other structures of alphavirus macro domain, this work reveals original features such as the opening of the distal scientific reports | ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports/ ribose, and its stabilization by ser , representing a peculiar getv specific substitution in the catalytic loop. in addition, we were able to identify a covalent link between adp-ribose and a cysteine, cys , located in the catalytic loop, as well as several poses of adp-ribose susceptible to provide clues about the catalytic mechanism. since cys is conserved in alphaviruses, this finding would deserve to be further explored by enzymatic assays and reverse genetics. the atomic coordinates and structure factors for the structures of getv macro domains and complexes thereof have been deposited in the protein data bank with accession numbers qzu, r f, r g, r p, r r and r t. all the other data are available from the corresponding authors on request. received: may ; accepted: july understanding the alphaviruses: recent research on important emerging pathogens and progress towards their control a comparison of the nucleotide sequences of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related rna viruses the enigmatic alphavirus non-structural protein (nsp ) revealing its secrets at last. viruses multiple host factors interact with hypervariable domain of chikungunya virus nsp and determine viral replication in cell-specific mode macrodomains: structure, function, evolution, and catalytic activities viral macrodomains: unique mediators of viral replication and pathogenesis viral macro domains reverse protein adp-ribosylation adp-ribosyl-binding and hydrolase activities of the alphavirus nsp macrodomain are critical for initiation of virus replication macrodomain adp-ribosylhydrolase and the pathogenesis of infectious diseases the crystal structures of chikungunya and venezuelan equine encephalitis virus nsp macro domains define a conserved adenosine binding pocket structural and functional insights into alphavirus polyprotein processing and pathogenesis nmr study of non-structural proteins-part i: ( )h, ( )c, ( )n backbone and side-chain resonance assignment of macro domain from mayaro virus (mayv) identification of protease and adp-ribose ''-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein adp-ribosylhydrolase activity of chikungunya virus macrodomain is critical for virus replication and virulence the conserved macrodomains of the non-structural proteins of chikungunya virus and other pathogenic positive strand rna viruses function as mono-adp-ribosylhydrolases getah virus as an equine pathogen recombinant protein expression and solubility screening in escherichia coli: a comparative study the impact of protein characterization in structural proteomics imosflm: a new graphical interface for diffraction-image processing with mosflm overview of the ccp suite and current developments scaling and assessment of data quality how good are my data and what is the resolution? on the treatment of negative intensity observations molecular replacement with molrep refinement of macromolecular structures by the maximum-likelihood method features and development of coot molprobity: all-atom structure validation for macromolecular crystallography clustal omega deciphering key features in protein structures with the new endscript server protein structure comparison by alignment of distance matrices mechanism of adp-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-adp-ribosylhydrolase drag the macro domain is an adp-ribose binding module the translation of recombinant proteins in e. coli can be improved by in silico generating and screening random libraries of a - /+ mrna region with respect to the translation initiation codon a macrodomain-containing histone rearranges chromatin upon sensing parp activation structural characterization of the histone variant macroh a the recognition and removal of cellular poly(adpribose) signals deficiency of terminal adp-ribose protein glycohydrolase targ /c orf in neurodegenerative disease a family of macrodomain proteins reverses cellular mono-adp-ribosylation identification of macrodomain proteins as novel o-acetyl-adp-ribose deacetylases the structure and catalytic mechanism of a poly(adp-ribose) glycohydrolase we thank the european synchrotron radiation facility (esrf) and synchrotron soleil for beamtime allocation, and the staff of beamlines proxima , id - and id a- for assistance with data collection. this work was supported by the european union horizon marie skłodowska-curie etn "antivirals" (grant agreement the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - -w.correspondence and requests for materials should be addressed to g.s. or b.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -qhlatg authors: verma, anukriti; sharda, shivani; rathi, bhawna; somvanshi, pallavi; pandey, bimlesh dhar title: elucidating potential molecular signatures through host-microbe interactions for reactive arthritis and inflammatory bowel disease using combinatorial approach date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: qhlatg reactive arthritis (rea), a rare seronegative inflammatory arthritis, lacks exquisite classification under rheumatic autoimmunity. rea is solely established using differential clinical diagnosis of the patient cohorts, where pathogenic triggers linked to enteric and urogenital microorganisms e.g. salmonella, shigella, yersinia, campylobacter, chlamydia have been reported. inflammatory bowel disease (ibd), an idiopathic enteric disorder co-evolved and attuned to present gut microbiome dysbiosis, can be correlated to the genesis of enteropathic arthropathies like rea. gut microbes symbolically modulate immune system homeostasis and are elementary for varied disease patterns in autoimmune disorders. the gut-microbiota axis structured on the core host-microbe interactions execute an imperative role in discerning the etiopathogenesis of rea and ibd. this study predicts the molecular signatures for rea with co-evolved ibd through the enveloped host-microbe interactions and microbe-microbe ‘interspecies communication’, using synonymous gene expression data for selective microbes. we have utilized a combinatorial approach that have concomitant in-silico work-pipeline and experimental validation to corroborate the findings. in-silico analysis involving text mining, metabolic network reconstruction, simulation, filtering, host-microbe interaction, docking and molecular mimicry studies results in robust drug target/s and biomarker/s for co-evolved ibd and rea. cross validation of the target/s or biomarker/s was done by targeted gene expression analysis following a non-probabilistic convenience sampling. studies were performed to substantiate the host-microbe disease network consisting of protein-marker-symptom/disease-pathway-drug associations resulting in possible identification of vital drug targets, biomarkers, pathways and inhibitors for ibd and rea. our study identified na((+))/h((+)) anti-porter (nhaa) and kynureninase (kynu) to be robust early and essential host-microbe interacting targets for ibd co-evolved rea. other vital host-microbe interacting genes, proteins, pathways and drugs include adenosine deaminase (ada), superoxide dismutase (sod ), catalase (cat), angiotensin i converting enzyme (ace), carbon metabolism (folate biosynthesis) and methotrexate. these can serve as potential prognostic/theranostic biomarkers and signatures that can be extrapolated to stratify rea and related autoimmunity patient cohorts for further pilot studies. www.nature.com/scientificreports/ approach has advantages over the traditional approach for network analysis that can help to simultaneously characterize several protein interaction modules and has the potential to study complex diseases. the vital information obtained in our study from in-silico analysis is cross-validated through targeted gene expression experimental analysis on patient cohorts. this study will help us to obtain clinico-molecular informatics-based outcomes and expand our knowledge regarding the understanding of biological functions for ibd co-existent rea. text mining: data screening and selection. systematic data search and organization was carried out incorporating data identification, data screening and data selection to find target microorganisms involved in inflammatory bowel disease (ibd) and reactive arthritis (rea). data identification was carried out to obtain records through data sources utilising keywords (e.g. "microorganism and inflammatory bowel disease and reactive arthritis") incorporating boolean operators (and/or/not). data screening and selection were carried as part of the manual curation through primary and secondary screening scrutinizing collected data records to obtain organized records relevant for the autoimmune and enteric disorders triggered by microorganisms, especially ibd and rea and the microbial triggers implicated in ibd and rea that were utilised for further metabolic network reconstruction. bottom-up approach consisting of draft reconstruction and manual reconstruction refinement was followed to create metabolic networks of obtained target microorganisms. genome-scale metabolic models simulation, reconstruction and visualization (gemsirv) software that includes reciprocal basic local alignment search tool (blast) of target microorganisms against a template metabolic network of its phylogenetic neighbour and incorporates information from national center for biotechnology information (ncbi), kyoto encyclopedia of genes and genomes (kegg) and transport db was used for creating draft reconstructs. the manual curation of missing links or gaps in the draft reconstruct was done by mapping the incomplete information to other databases such as expert protein analysis system (expasy) and integrated relational enzyme database (intenz) . this fully connected and annotated network was used for further simulation studies . the metabolic networks thus obtained were visualized using celldesigner, a tool for modelling and editing biochemical and gene-regulatory networks. simulation analysis was carried by converting the metabolic networks obtained into a mathematical model and performing the gene deletion analysis to retrieve essential genes. model conversion was through generation of stoichiometric based matrixes consisting of reactions (columns) and metabolites (rows) corresponding to respective genes. upper boundary and lower boundary fluxes i.e. movement of matter across a system were generated for the gene associated reactions and metabolites that was extracted in systems biology markup language (sbml) format. the next step was gene deletion analysis done using the constraint based reconstruction and analysis toolbox (cobra) that runs in matrix laboratory (matlab) for finding the essential genes based upon the gene-reaction matrix and boolean relationship between genes and reactions . the purpose of data filtering is to remove repeats and homologs from essential genes of target microorganisms associated with ibd co-existent rea. the non-homologous protein sequences corresponding to the essential genes of target microorganisms were extracted from pathosystems resource integration (patric) database . refinement of protein sequences was further done using cluster database at high identity with tolerance (cd-hit) suite so as to have % identity non-repeat sequence tolerance stringency. blast-p was further used to remove the homologs from such non-repeats against human database at e-value of - to obtain nonhomologous protein sequences used for further in-silico analysis. essential host-microbe and microbe-microbe interactions. the host-microbe interactions of the non-homologous proteins for the selected target microorganisms were obtained using host-pathogen interaction database (hpidb) , . the host-microbe interactions were visualised using cytoscape. simulation analysis (gene essentiality) was done to obtain the essential host proteins interacting with common microbe proteins of microorganisms triggering ibd and rea utilising the human metabolic model hmr , a cobra compliant metabolic model of human consisting of around , genes, , reactions and , metabolites . this led to profiling of the common host-microbe and microbe-microbe interactions comprehending the complex 'interspecies communication' as complex interaction maps, executed using search tool for the retrieval of interacting genes/proteins (string) , . host-microbe disease network and molecular mimicry studies. the host-microbe disease network is a multilayered archetype that connects the protein-marker-symptom/disease-drug-pathway associations. the contributions of the microorganisms in the co-evolved ibd and rea as part of the disease network was created through the interactive maps of the essential host interaction proteins (verified using literature survey) and the information processed through gene expression data analysis . the information patronised here is mostly scored through the available non-specific protein diagnostic markers of both ibd and rea e.g. c-reactive protein (crp), interleukin (il ) and toll like receptor (tlr ), major histocompatibility complex, class i, b (hla-b) and major histocompatibility complex, class ii, dr beta (hla-drb ) with the essential host proteins determined using string . database genecards was used to assess the role of these interacting partners aka proteins further with symptoms/diseases associated with ibd and rea. the pathways of the above host interacting proteins were found out using kegg database that provides ontologies for proteins related to biological processes www.nature.com/scientificreports/ subsequently, the role of drugs or inhibitors used to suppress the effect of ibd and rea such as indomethacin, prednisone, ciprofloxacin, sulfasalazine, azathioprine, methotrexate and hydroxychloroquine was scored in the disease network through their docking studies against the potential targets (both host as well microbial targets) as per published methodologies , . the host-microbe disease network which is an amalgamation of all the above patterned associations was visualized using cytoscape software . molecular mimicry analysis between the vital targets triggering ibd co-evolved rea, essential human proteins including hla-b , hla-b and hla-drb was done using data repository expasy. this led to retrieval of microbe relayed protein sequences that have been implicated in disease development after sequence alignment performed using emboss . experimental evidences to identify the signature molecules in patient samples. the cross-validation of vital in-silico targets was done in rea patient cohort cases via targeted gene expression analysis. scientific and ethical clearance was taken from amity university ethics committee and institutional ethics committee, fortis noida for handling the patient samples. all experiments were performed in accordance with indian council of medical research (icmr) guidelines constituting the ethics committees. the study was carried out for months on the rare disorder rea patients, with the inclusion criteria as patients having rea according to european spondyloarthropathy study group (essg) and exclusion criteria as patients undergoing treatment from last - months and healthy controls (hc). the participants were inducted in the study design with an informed consent form along with a questionnaire containing information regarding symptomatic and diagnostic history of patient and linked disorders. blood ( ml) was drawn from participants in ethylenediaminetetraacetic acid (edta) vacutainers. these were transported to the laboratory for further analysis. the processing of the samples was done within - h of procurement . peripheral blood mononuclear cells (pbmc's) were isolated from blood using density gradient centrifugation . rna was isolated from pbmc's using trizol method . the quantification of rna was done using nano-drop . the high capacity cdna reverse transcription kit (applied biosystems™) was used for conversion of rna to single-stranded cdna as per the standard protocol . quantitative pcr analysis of target gene was executed using biorad cfx real time-pcr taking human housekeeping gene, gapdh as a reference. previously reported primers for qpcr analysis of target and reference gene were selected for this study , following the standard protocol . relative gene expression analysis from qpcr data was performed using the relative expression software tool (rest® ) that utilises the expression of reference genes to normalize expression of target genes in different samples. the schematic representation of methodology involved in our combinatorial analysis is provided in fig. . text mining: data screening and selection. a systematic literature mining and curation for our thematic connecting autoimmune disorders, inflammatory bowel disease (ibd) and reactive arthritis (rea) was carried out. data identification extracted , records (articles in journals, book chapters, conference papers etc.) corresponding to autoimmune and enteric disorders. data screening extracted records of autoimmune and enteric disorders triggered by microorganisms that belong to class of bacteria, fungi, protozoan, mites, virus, yeast and nematode. data selection yielded ibd, rea and ibd co-evolved rea records. data selection was directed towards the microbial contenders implicated here resulting in target microorganisms namely campylobacter jejuni, escherichia coli o :h , klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica, whose genome information was available. the etiopathogenesis in the co-evolved disorders have been documented through gut microbiome associated host-pathogen interactions studies, perpetuating where pathogen microorganisms involve in dysbiosis leading to autoimmunity. the results of text mining are provided in fig. . the list of microorganisms is provided in supplementary table s online. ing of genes along with their corresponding proteins, reactions and metabolites for the selected microorganisms serve as primary set of partial metabolic network information. the missing data persistent in the draft reconstruct obtained through genome-scale metabolic models simulation, reconstruction and visualization (gem-sirv) was manually refined. entirely associated metabolic networks of target microorganisms were obtained (genes, proteins and reactions). the essential genes of microorganisms (vital for survival. sustenance and growth) were obtained after performing simulation on mathematical models consisting of gene associated reactions and metabolites (metabolites, inner cell reactions, exchange reactions and essential genes). due to lack of availability of exchange reactions for campylobacter jejuni, simulation analysis on the partial metabolic network could not be carried out and essential genes could not be retrieved. an alternative approach for finding essential genes of campylobacter jejuni was carried out. the essential genes of campylobacter jejuni were taken from our previous published report and were found out to be . table portrays the results of metabolic network reconstruction and simulation of target microorganisms. the metabolic network and simulation analysis data of target microorganisms is provided in supplementary table s online. the proteins corresponding to essential genes, non-repeats and non-homologs were obtained as stated below according to the parenthesis {proteins corresponding to essential genes, non-repeats, non-homologs}. the essential genes, their corresponding proteins, reactions and metabolites from the curated dataset were refined to create a list of most relevant molecular indicators to assess their coveted role in disease establishment. the non-redundant filtered proteins were utilised further in the computational work-pipeline canvassing the drug targets and signatures in the interspecies communication. essential host-microbe and microbe-microbe interactions. the central mechanism of hostmicrobe/microbe interface conferred through gut microbiome was correlated for the selected microbial species and processed to obtain the common signatures so as to follow the core system of metabolic changes affecting the host harbouring them as either commensal or pathogenic loads. the interactors between human and target microorganisms were obtained. the interactors of escherichia coli o :h were ; klebsiella oxytoca were ; salmonella typhimurium were ; shigella dysenteriae were and yersinia enterocolitica were . there were no interactors for campylobacter jejuni (supplementary table s -s online). table shows the results of filtering and host-microbe interactions of protein sequences corresponding to essential genes of target microorganisms. www.nature.com/scientificreports/ the host-microbe interactors were analysed for all the target microbial species and processed to obtain the common signatures. proteins were found between all target microorganisms having interaction among themselves and with human proteins. the essential host correlative targets to the microbial gene targets were followed by obtaining host essential genes and corresponding proteins from human metabolic model hmr . there were , essential proteins (supplementary table s online) the essential human protein was found out to be kynu having interaction with essential microbial protein nhaa (fig. ) . nhaa was also having interactions with non-essential hcls associated protein x- (hax ), prolyl endopeptidase-like (ppcel), biogenesis of lysosomal organelles complex subunit (hps ) and eukaryotic translation initiation factor alpha kinase (e ak ) proteins of human host. kynu was further mapped with host proteins (direct and indirect) resulting in interactions. out of these the single connected essential protein interactions were and protein interactors were ( fig. and see supplementary table s online). the research design here followed to assess the interaction map of essential proteins in human host to indicate the clinical insights in pathophysiological trends in the autoimmune development. host-microbe disease network and molecular mimicry. the human essential proteome complement with its interacting proteins were analysed further as part of the disease network. human essential protein interactors were found to be associated with ibd and similarly essential protein interactors namely adenosine supplementary table s online) . these proteins can be postulated as probable contenders transcending their role in the simulated network as important regulators in the co-existent disorders. the composite associations of the above proteins with non-specific protein diagnostic markers of ibd and rea were obtained (see supplementary table s online) . this gave rise to a single connected protein network consisting of proteins and , interactions. the association of above with symptoms and diseases linked with ibd and rea were obtained (see supplementary table s online) . apart from non-specific diagnostic markers, the major protein linked with majority of symptoms/diseases is angiotensin i converting enzyme (ace). pathways of the proteins were obtained (see supplementary table s online) in total out of which the pathway associated with majority of proteins was carbon metabolism. another layer of disease network substantiates the role of therapeutic regime followed in the studied autoimmune diseases, so the docking analysis of drugs used to suppress the effect of ibd and rea against nhaa of target microorganisms and kynu of human host was done. the docking analysis resulted in docking scores that represent binding of drugs with host kynu and microbial nhaa of all microorganisms selected in our study. higher the negative docking score more is the binding . escherichia coli o :h nhaa shows highest and lowest docking score with methotrexate (− . ) and azathioprine (− . ); klebsiella oxytoca nhaa with methotrexate (− . ) and azathioprine (− . ); salmonella typhimurium nhaa with ciprofloxacin (− . ) and hydroxychloroquine (− . ); shigella dysenteriae nhaa with methotrexate (− . ) and azathioprine (− . ); yersinia enterocolitica nhaa with hydroxychloroquine (− . ) and azathioprine (− . ) and human kynu with hydroxychloroquine (− . ) and indomethacin ( . ). our results portray methotrexate to have highest docking scores with maximum proteins and therefore can be considered as a vital drug for ibd associated rea. the resultant docking scores are provided in fig. . the extensive interaction pattern of nhaa with kynu along with proteins, markers, symptoms/ diseases, pathways and drugs give rise to a host-microbe disease network of ibd co-existent rea (fig. and see supplementary table s online) . the final league of information processed in this study design was to accommodate the concept of molecular mimicry between the essential host proteins and selected microorganisms. nhaa protein of target microorganisms shows homology with human hla-b , hla-b and hla-drb (fig. ) . peptides homologous to hla-b : peptides homologous to hla-drb : experimental evidences to identify the signature molecules in patients. the in-silico analysis followed for the molecular signature identification till far through gene expression datasets and curated metabolic reconstructs strongly indicate the host protein, kynu being the singular common predictive markers for all pathogenic microbes. kynu has also been indicated in the expression data of inflammatory linked disorder, www.nature.com/scientificreports/ ibd. there is lack of data available regarding kynu differential expression in rea, therefore the experimental evaluation of kynu through targeted expression analysis in rea patients was carried out. a non-probabilistic convenience sampling was followed for our single blind study. this study encompassed individuals: % male with mean age of . and % female with mean age of ( males and females). out of these cases were: with rea and controls were: currently undergoing treatment, with poncet's disease (pd) and healthy control (hc). the clinical characteristics of the patients recruited in the study included inflammatory back pain in %, fatigue in %, fever in %, swollen joint in %, ankylosing spondylitis (as) that affects spine in %, dactylitis that is inflammation in finger or toe in % and poncet's disease (pd) in % of participants. the clinical characteristics of the recruits are provided in table . the expression of kynu in peripheral blood mononuclear cells (pbmc's) of rea cases vs controls was evaluated using relative expression software tool (rest) software that estimated a sample's relative expression ratio in relation to the control housekeeping gene (here gapdh) by calculating an intermediate absolute concentration value: where cp = point at which fluorescence escalates considerably above the background fluorescence. here the cp values for reference and target genes are collectively redistributed to control and sample groups and the expression ratios are calculated based on the mean value. a pair wise fixed reallocation randomisation test is followed for normalisation of the target genes with a reference gene and for calculating the statistical difference of variation between groups . it utilises a bootstrapping technique providing a % confidence interval for expression ratios. it uses a p(h ) test for testing the significance between the samples and controls. according to our analysis, kynu sample group is different to control group where p(h ) = . . kynu was found to be downregulated in sample group (in comparison to control group) by a mean factor of . (standard error range is . - . ) as depicted in the whisker-box plot (fig. ) . kynu expression showed a ~ ninefold decline in rea cases as compared to controls. gut microbiome is pitched to be the central theme housing enormous diversity of microbial species, characterizing the fine balance between healthy and diseased states. the physiological drifts from healthy to diseased and vice-versa is tuned to sophisticated interactive networks of human host and the microbial flora residing the gut. the autoimmune conditions reactive arthritis (rea) and inflammatory bowel disease (ibd) have been linked to prevalent dysbiosis of the gut, where disease development occurs as a perceptive reaction due invading population of microbes. to find out the basal networks of interactions at the host-microbe interface, common microbes affecting the co-evolved diseases with shared characteristics were studied. these involved comprehensive analysis of the bimolecular functional networks including the gene, protein, metabolite molecular signatures engraved at the host-microbe and microbe-microbe interface. this 'interspecies communication' have been linked now with immuno-pathogenesis of most human autoimmune disorders , . www.nature.com/scientificreports/ the etiopathology of these interactions have remained elusive leading to non-specific diagnostic criteria and therapeutic regimes. it is suggested that microbial dysbiosis, pathogenic infection and host-microbe interactions cause incidence of rea. in this study, utilising the combinatorial approach we have compiled a repertoire of microorganisms, biomolecules and pathways that are possibly involved in triggering co-evolved autoimmune disorders ibd and rea. in our study, text mining results convey the presence of microorganisms namely campylobacter jejuni, escherichia coli o :h , klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica implicated in both the disorders. the thematic concepts for microbe contribution in host immunity have been explored in our previous analysis of metabolic reconstruction and simulation of campylobacter jejuni and salmonella enterica , . in our current study, we used a designated work-pipeline for metabolic network reconstruction and simulation of target microorganisms. the analysis conducted extracted the information via constraint-based bottom-up approach that was filtered and utilised for further computational analysis. the essential genes, proteins and metabolites of microorganisms represent the promising drug targets as these are speculated to contribute towards infection triggered host physiological drifts leading to development of the co-evolved pattern of autoimmunity in ibd and rea. a thorough curation pattern followed led to provide robust molecular cues in terms of essential proteins and biological networks that are correlated to the 'interspecies communication' using the host-microbe and microbemicrobe interaction profiling. the most closely associated common protein observed in all the selected common microbial species involved in both ibd and rea is na (+) /h (+) antiporter (nhaa), microbial integral membrane protein, catalyzing the exchange of h (+) per na (+) and involved in processes crucial for cell viability. similarly, the common host interacting protein with nhaa is kynureninase (kynu), involved in tryptophan metabolism and whose differential expression (upregulation and downregulation based on the control samples) have been followed in ibd patient cohorts [ ] [ ] [ ] . as per the scientific discourse presented in the studied disorders, the pathological mechanism hypothesizes that after bacterial infection, antigen-presenting cells transport bacterial antigens/peptides into the synovial membrane, where the bacterial components persist causing inflammation. it is suggested that in host-microbe interactions, bacterial proteins entering host cells interact with host proteins and inject their effector components, but has not been proven in rea and ibd. so, this formed a basis of one of the parameters in our study design where we found the physical interactions between nhaa and kynu and predicted that these might be the early host-microbe interactors for establishing pathogenesis in ibd associated rea. this could assist to comprehend the very few reports indicated in the rare autoimmune rea, where gene expression datasets of the co-evolved disorder ibd can serve to incorporate the larger theme of gut-microbiome associations. the theme of gut-microbiome paradigm shifts thus contemplates the vital cues in triggering autoimmunity with indirect linkages to diet and environmental triggers. this is indicative of the identified target molecular signature, kynu, found to be differentially regulated in the patient cohorts with history of infection triggered or ibd co-evolved rea. kynu and nhaa could serve as the robust early and essential host-microbe interacting targets and molecular indicators involved in interspecies communication in ibd associated rea. the investigations further were targeted for parallel analysis of other host-essential protein partners enmeshed to have interaction with host protein kynu indicating the intricate details of host-microbe interaction information. the disease network constructed through our approach consists of single connected essential protein interactors of kynu, where human essential protein interactors are found to be associated with ibd, while of them (adenosine deaminase (ada), catalase (cat) and superoxide dismutase (sod )) are associated with both ibd and rea. ada protein has been reported in juvenile idiopathic arthritis and rea patient cohorts in serum samples . similarly, cat and manganese superoxide dismutase (sod) genes polymorphisms were observed in rea patient cohorts , . these become part of the host-microbe disease network where such molecular elements and co-regulatory pathways represent the intricate biological cross-talk followed during disease development. pathological conditions can also trigger immune cells such as il's and tlr's and various cytokines leading to immune cell infiltration in host and higher levels of inflammation. genetic factors such as hla alleles encode susceptibility, contribute to bacterial persistence and increase risk in rea cases. based on this we also found the interactions of important targets in our study with immunogenic and genetic factors. the host harboured assorted essential proteins were further probed for their association with non-specific protein diagnostic markers as well as with symptoms/diseases linked with ibd and rea, accruing towards a single connected network consisting of interdependent proteins. the reciprocation of these integrated protein indicators to the disease development is conveyed through metabolite monitoring as in the study, angiotensin i converting enzyme (ace) was found to be linked with maximum symptoms/diseases. ace is involved in catalyzing the conversion of angiotensin i into angiotensin ii that is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance . this could be the indicator of involvement of microbe triggered host physiological drifts. subsequently, the pathways associated with the proteins ramified into pathways of human host speculated to give details of metabolic regulatory checkpoints where carbon metabolism is found to be associated with majority of deduced proteins. carbon metabolism pathway implicated here as the vitally generic pathway for ibd co-related rea confers how diet, balance of gut microbiome, antibiotic exposures can have layered impact on autoimmune disease progression and remissions. kynu is found to be downregulated in rea patients as compared to controls through our targeted gene expression analysis. collectively, the disease network followed here confers interaction of microbial nhaa with host kynu, that is further correlated to proteins, markers, symptoms/diseases, pathways and drugs. docking analysis of drugs used to suppress the effect of ibd and rea predicts methotrexate as an important drug that could be useful for early treatment of ibd co-evolved rea. www.nature.com/scientificreports/ genetic factors found common in both rea and ibd are hla-b , hla-b and hla-drb . the most important mechanism of susceptibility of hla in rea is molecular mimicry that is microbial peptides mimicking hla autopeptides of human host leading to autoimmunity. this mechanism has been observed in rea where reports have predicted microorganism peptides such as chlamydial proteins (clpc, nqra and dnap) and yersinia pseudotuberculosis peptides (yoph) showing homology with human hla-b via bioinformatic analysis . similarly, molecular mimicry has also been observed in ibd cases having extraintestinal manifestations. we performed targeted molecular mimicry analysis in our study using our robust microbial protein (nhaa) with hla-b , hla-b and hla-drb , enhancing the importance of nhaa acting as a trigger for generating ibd associated rea. we generate a putative hypothesis amalgamating key findings with literature. we state that the initial hostmicrobe triggers for ibd associated rea is when pathogenic microbial protein nhaa interacts with host protein kynu that further interacts with human proteins ada, sod , cat and ace and carbon metabolism involving the above host proteins is hampered. methotrexate regulates carbon metabolism and the associated host-microbe proteins reducing effect of ibd associated rea. since carbon metabolism is the most basic aspect of life and therefore an extensive network consisting of sub-pathways, we narrowed down our findings towards a consequentially central and a significant pathway that embrace the carbon metabolism pathway involving the molecular signatures kynu, ada, sod , cat and ace, further is also effectuated by potential drug methotrexate and is associated with ibd/ rea/ ibd and rea cohorts. it is reported that methotrexate is incorporated intracellularly interfering with adenosine concentrations and affecting proinflammatory cytokines in ibd reducing inflammation . in inflammatory arthritis, the mechanisms reported by which methotrexate reduces inflammation include enhanced adenosine release, de novo synthesis of purines and pyrimidines, inhibition of transmethylation reactions, diminished accumulation of polyamines and nitric oxide synthase uncoupling. most of the mechanisms are associated with folate biosynthesis, a type of carbon metabolism . kynu, ada, sod , cat and ace are also found to be involved in folate biosynthesis and metabolism from genecards. apart from the above targets, parallel interactors, pathways and drugs for ibd co-evolved rea obtained in our host-microbe disease network can be utilised further as disease determinants. the experimental validation of these targets in patient cohorts need to be performed on a pilot scale in future to increase the robustness of this network. the intertwined information processed through the knowledge-base created for the linked disorders have given the most elaborate layout of patterns observed in disease diagnosis and analysis. the major information after processing the gene expression profiles, protein markers, molecular networks and metabolic networks involved here have led to chalk out as well as connect the strings for robust gut microbiome paradigm shifts. the current work on host-microbe interactions provides a starting point for researchers and clinicians to investigate inflammatory bowel disease (ibd) associated reactive arthritis (rea). in this study a combinatorial approach is utilised to reveal the interactions of gut microbes with human host extensively sketched through the work-pipeline providing the vital insights for the drug targets, biomarkers, pathways and inhibitors for etiology, prognosis, diagnosis and treatment attributes of pathogenic rheumatic autoimmunity. 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advanced studies, new delhi for providing the facility and technical support during the preparation of the manuscript. we also thank fortis hospital, noida for providing the patient samples. s.s. and b.r. conceived the study concept; s.s., b.r. and p.s. jointly designed and supervised the work; b.d.p. supervised the clinical setting and recruitment of participants; b.d.p. and a.v. recruited the participants and contributed to the sample collection and preparation; a.v. performed the experiments; s.s., b.r., p.s. and a.v. contributed to the analysis and interpretation of data; a.v. generated all figures and tables; a.v. wrote the first draft of the manuscript; s.s., b.r., p.s. and b.d.p. critically reviewed and edited the manuscript; all authors reviewed and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to s.s.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -s oqphvn authors: baral, prabin; pavadai, elumalai; gerstman, bernard s.; chapagain, prem p. title: in-silico identification of the vaccine candidate epitopes against the lassa virus hemorrhagic fever date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: s oqphvn lassa virus (lasv), a member of the arenaviridae, is an ambisense rna virus that causes severe hemorrhagic fever with a high fatality rate in humans in west and central africa. currently, no fda approved drugs or vaccines are available for the treatment of lasv fever. the lasv glycoprotein complex (gp) is a promising target for vaccine or drug development. it is situated on the virion envelope and plays key roles in lasv growth, cell tropism, host range, and pathogenicity. in an effort to discover new lasv vaccines, we employ several sequence-based computational prediction tools to identify lasv gp major histocompatibility complex (mhc) class i and ii t-cell epitopes. in addition, many sequence- and structure-based computational prediction tools were used to identify lasv gp b-cell epitopes. the predicted t- and b-cell epitopes were further filtered based on the consensus approach that resulted in the identification of thirty new epitopes that have not been previously tested experimentally. epitope-allele complexes were obtained for selected strongly binding alleles to the mhc-i t-cell epitopes using molecular docking and the complexes were relaxed with molecular dynamics simulations to investigate the interaction and dynamics of the epitope-allele complexes. these predictions provide guidance to the experimental investigations and validation of the epitopes with the potential for stimulating t-cell responses and b-cell antibodies against lasv and allow the design and development of lasv vaccines. to in the t-loop (residues - ) and hr (residues - ) regions of gp . site b contains residues to of the fusion peptide and residues to of hr (residues - ) of gp , . although the antibody predominantly binds to gp , gp is required to maintain the proper prefusion conformation of gp for antibody binding . identification of epitopes is an essential step for understanding disease etiology, immunotherapy, immunodiagnostics, and the discovery and development of epitope based-vaccines. an epitope-based vaccine has fewer side effects compared to conventional vaccines. experimental identification of a promiscuous epitope involves many expensive and time-consuming steps, including the production of antibodies to map antigenic regions on a target protein, animal models, and determination of the crystal structure of antigen-antibody complexes using x-ray crystallography. computational identification of epitopes is often employed as a powerful and fast approach to facilitate the identification of potential epitope candidates that can decrease the number of validation experiments and time , . multi-epitope based vaccine development has already proven effective against several viral infections and cancer , . in this study, we have identified and characterized t and b-cell epitopes for the lasv gp using different sequence and structure-based computational epitope prediction methods. we then selected potential b and t-cell epitopes for the lasv gp based on a consensus approach, and the novelty of the epitopes was examined with the immune epitope database (iedb) tools. subsequently, we identified strongly binding alleles to the mhc-i t-cell epitopes and modeled the allele structures and performed docking to understand the interaction between alleles and epitopes. we further investigated the stability and dynamics of the epitope-allele complexes using molecular dynamics simulations. analyses of root-mean square deviations, hydrogen bond, interaction energy, and solvent accessibility showed that epitope-allele complexes are stable, indicating that the epitopes strongly bind to the alleles. the identified b and t-cell epitopes of lasv gp in the study can be useful for the development of effective vaccines against lassa hemorrhagic fever. selection of lasv gp sequence and d structure. the sequence of gp for different lasv strains was obtained from the niaid virus pathogen database and analysis resource (vipr) . subsequently, multiple sequence alignments were performed between the sequences using clustal omega to select a conserved lasv gp for sequence-based epitope prediction. the corresponding x-ray crystal structure of the mouse/sierra leone/josiah/ lasv gp was obtained from the protein data bank (pdb id: vk ) , for structure-based b-cell epitope prediction. the missing residues were modeled using the charmm-gui - . prediction of b-cell epitopes. sequence-based b-cell epitope prediction was performed with the use of bepipred . , bcpreds and bcepred servers separately. these servers predict epitopes based on physico-chemical properties of amino acids, and these servers accept the primary sequence of lasv gp as an input. . each gp has a gp subunit and a gp subunit (zoomed view). each monomer is colored differently in the gp trimer. in the zoomed view, the gp subunit is lightly shaded to differentiate from the gp subunit, and some of the antibody binding sites (site a, site b) are highlighted (figure generated from the crystal structure of the lasv gp in the protein data bank , pdb id: vk ). structure-based b-cell epitope prediction for the lasv gp (pdb id: vk ) was carried out using three different programs separately: ellipro , epitopia and discotope . these servers predict epitopes regions based on the geometrical and solvent surface-accessibility of a protein structure, and these servers accept the d structure of a protein as input. the consensus epitopes from both sequence and structure-based predictions were selected as potential epitopes for further analysis. sequence-based mhc-i t-cell epitope predictions for lasv gp were carried out by using three different servers, propred-i , ctlpred and netctl . . to predict their alleles, the consensus epitopes among these three prediction methods were analyzed using iedb . the epitopes that strongly bind to the alleles (lowest ic ) were selected for further analysis. sequence-based mhc-ii t-cell epitope predictions for lasv gp were performed with the use of three different servers: propred , netmhcii . ] were obtained from the pdb. the experimental structure for the hla-a* : (a ) allele is not available, and thus, the sequence of this allele was obtained from the uniprot database (uniprotkb id: p ), and subsequently its structure was modeled using swiss-model [ ] [ ] [ ] . the selected consensus mhc- epitopes were extracted from the crystal structure of lasv gp (pdb: vk ). the epitopes and the alleles were prepared for docking using autodock tool version . . . autodock vina . . was used for peptide docking with a grid space that covered the entire allele. the best peptide-allele complexes were selected for further investigation based upon visual inspection of peptide-allele interactions and the autodock vina criteria. the stability and dynamics of the selected peptide-allele complexes were further studied using molecular dynamics simulations. all-atom, explicit-solvent molecular dynamics (md) simulations were performed to investigate the stability and dynamics of the mhc- t-cell epitope-allele complexes using the charmm m force field with the namd . software package . the systems were minimized for , steps followed by ps of equilibration. this was followed by md production runs for ns at a temperature of k using a fs time-step. the long-range ionic interactions were calculated using the particle mesh ewald (pme) method while the covalent hydrogen atoms were constrained by using a shake algorithm . the temperature was controlled by using the langevin temperature coupling with a friction coefficient of ps − and pressure was controlled using the nose-hoover langevin-piston method . visualization, and rendering of trajectories and pictures were performed using vmd . the multiple sequence alignment of the lasv gp sequences resulted in the lasv gp mouse/sierra leone/ josiah/ ) [uniprotkb id: p ] as a highly conserved strain, and we thus selected this strain for the sequence-based mhc-i and mhc-ii t-cell epitope predictions and for both structure and sequence-based b-cell epitope predictions. in addition, a search of this strain with the experimentally determined structure available in the pdb displayed the . Å resolution crystal structure of the prefusion gp trimer of lasv in complex with the human neutralizing antibody . h. [pdb id: vk ] as shown in fig. . this structure was used for the structure-based b-cell epitope prediction. a schematic representation of the epitope prediction cascade is shown in fig. . we have adopted multiple methods to predict and rank the epitopes as they use different criteria for their predictions. some approaches may incorporate some properties that are similar such as solvent accessible surface area, but the predicted epitopes are different. previous studies , have suggested that the consensus approach would improve the specificity and accuracy of the epitope prediction as it can reduce the false positives. therefore, we employ a consensus approach; for example, an epitope can be considered if it overlaps with even a single residue by at least two prediction methods. our consensus approach selected several nanomer epitopes for mhc-i (table s ). although the predicted epitopes for mhc-ii t-cell vary in length, the consensus core region between predicted mhc-ii epitopes is a nanomer (table s ) which is considered an optimal length for the hla. prediction of t-cell epitopes. mhc-i t-cell epitope prediction with the lasv gp sequence was performed using three different methods separately: propred- , ctlpred, and netctl . , and the results are shown in supplementary table s . the epitopes listed by at least two of the methods are listed in table along with their binding affinity (ic ), antigenicity, and allele. among these four consensus epitopes, the nanomer e epitope fatcglvgl shows the lowest average ic value of nm against the a allele as predicted by the iedb, and it has also a reasonable antigenicity score of . . this was followed by the e epitope fsrpspigy, which has an average ic value of nm against the a allele, and also has a better antigenicity score of . compared to the fatcglvgl epitope. interestingly, the e epitope rrgtftwtl is predicted by all three methods though its ic and antigenicity scores are not as good as the other epitopes (table ). all four of these consensus epitopes were docked to the alleles and we performed the md simulations to investigate the stability and dynamics of the allele-epitope complex as discussed later. mhc-ii t-cell epitope prediction with the lasv gp sequence was performed using three different methods separately: propred, netmhcii . , and epitop . , and the results are shown in supplementary table s . propred uses a quantitative matrix approach and netmhcii . uses ann , while epitop . uses quantitative structure-activity relationship models (qsar) to predict the mhc-ii t-cell epitopes. the epitopes that were predicted by at least two methods are listed in table . among these consensus mhc-ii t-cell epitope predictions, the e and e epitopes were predicted by all three methods and have a reasonable antigenicity score of . , indicating that these two epitopes can be potential candidates for the design of mhc-ii t-cell based vaccines. propred and epitop . predict most epitopes as nanomers whereas netmhcii . predicts varying lengths of epitopes (table ) . interestingly, the -mer epitopes predicted by netmhcii have the consensus core nanomer epitopes, suggesting that the core region is responsible for strong binding of the epitope into the mhc-ii binding site - . prediction of b-cell epitopes. in addition to the t-cell epitope predictions, we also predicted the linear b-cell epitopes for the lasv gp using sequence-based methods bepipred . , bcpreds , and bcepred . the bepipred predicts the epitopes based on a random forest algorithm trained on epitopes annotated from antibody-antigen structures. bcpreds predicts epitopes by using svm combined with a different kernel method, including string kernels, radial basis kernels, and subsequence kernels. the bcepred locates b-cell epitopes using four physicochemical properties like hydrophilicity, polarity, exposed surface and beta-turns . the epitope e containing residues was predicted by all three of these sequence methods (table ) but with a negative antigenicity score. we also performed structure-based b-cell epitope prediction using three representative structural and geometrical properties-based methods: ellipro, epitopia and discotope. for this, the experimental d structure lasv gp (pdb id: vk ) with the modeled missing residues was used. ellipro predicts linear and conformational epitopes by incorporating the antigenicity, solvent accessibility, and flexibility of protein structures . epitopia uses a machine learning algorithm to analyze the antigenic features on protein structure and predicts the probable conformational epitope regions . discotope uses amino acid statistics, spatial information, and surface accessibility on the protein d structure to predict residue-by-residue conformational epitopes . the e , e , e and e structure-based epitopes in table are especially interesting as potential candidates as they were predicted by all three methods. in table , we also ranked each epitope based upon how many of the sequence and structure-based methods predicted each epitope, which do not always correlate with the highest antigenicity scores of e , e , e , e and e . robinson et al. have recently reported the cloning of many human monoclonal antibodies derived from memory b cells of lassa fever survivors in west africa. these antibodies specifically bind to both gp and gp epitopes of lasv. the comparison of our predicted b-cell epitopes with those epitopes shows that there are five consensus epitopes ( table ) that share similarity with robinson et al. (table s ) , and another five epitopes that do not share similarity, indicating that our consensus epitope prediction strategy has identified new epitopes. epitope surface mapping. for efficacy of vaccines, the epitopes should be located on an accessible region of the protein so that the epitope will be able to bind with antibodies . this is especially important for the six epitopes that we list in the tables above that do not share any part of their sequence with known epitopes: e , www.nature.com/scientificreports www.nature.com/scientificreports/ e , e , e , e , e . in fig. , we highlight the positions of these epitopes on lasv gp. we also highlight the positions of e and e because the four mhc-i t-cell epitopes have ic information readily available. figure shows that the e , e , e , e , e , e and e epitopes are well located on the exposed regions and thus can interact well with the alleles. www.nature.com/scientificreports www.nature.com/scientificreports/ crystal structure of the hla class i antigen (pdb id: ei ) as the best template for constructing models. the sequence identity between a and the template was %. the best model was then selected based on multiple validation methods, including gmqe (global model quality estimation) and qmean. the gmqe and qmean values , of the model are . , and . , respectively. in addition to these analyses, ramachandran plots and errat were also used for the model validation. analysis of ramachandran plot of the model shows . % of residues are either in favored or in allowed regions ( supplementary fig. s ), indicating that backbone torsion angles of these models are acceptable. the errat overall quality factor score was computed as , which is greater than the normally accepted score range for a high quality model of . these analyses show that the model is within a high quality range and can be used for further analysis. docking of the four consensus mhc-i epitopes (table ) was performed using autodock vina, which enabled the docking of epitopes obtained from the sequence-based mhc- t-cell prediction into the promising allele structures. the autodock vina docking protocol has been previously demonstrated to successfully dock epitopes into allele structures . however, we validated the capability of the docking protocol before docking the epitopes by redocking the epitopes into the allele crystal structure (pdb id: ox ) to see whether the crystal bound conformation of the peptide could be reproduced or not. the docked allele-epitope complex showed the same residue-epitope interactions observed in the epitope bound crystal structure, indicating that the autodock vina docking protocol was capable of reproducing the experimentally observed binding mode of the epitope. we applied autodock vina to each of the four mhc-i allele-epitope complexes. autodock vina found that the highest ranked docking structure had the following binding affinities: − . kcal/mol for a ::e − . kcal/mol table . prediction of the b-cell epitopes. the epitopes predicted by either all three sequence-or structurebased methods are highlighted by boldface. conformational epitopes chosen by all three structure-based methods are indicated in italics. www.nature.com/scientificreports www.nature.com/scientificreports/ for a ::e , − . kcal/mol for a ::e , and − . for a ::e . these epitopes-alleles docking complexes are shown in fig. . in order to investigate the dynamics and stability of the four mhc-i allele-epitope complexes, we performed ns all-atom, explicit solvent md simulations. to quantitatively understand the stability of the allele-epitope complex, we calculated the root mean square deviations (rmsd) of the backbone atoms of the allele-epitope complexes as a function of simulation time as shown in www.nature.com/scientificreports www.nature.com/scientificreports/ figure also includes curves of the rmsd of the backbone atoms of just the allele, and separately, just the backbone atoms of the epitope. all alleles have an rmsd compared to their initial structures of approximately Å, whereas the allele-epitope complexes have a bit higher rmsd of approximately . Å, indicating that the epitopes make the complexes more flexible. interestingly, in the case of a ::e , the allele and the complex show almost the same rmsd, suggesting that the complex is especially stable. to pinpoint why the complexes show a higher rmsd, we further computed the rmsd of only the backbone atoms of the epitope in each the complex. figure shows that the initial configuration of epitopes e and e is compact, and that both of these epitopes rearrange their configuration in the binding site and elongate during the ns md simulation. this elongated configuration is consistent with the investigations of antunes et al. on mhc-i epitopes. since the interactions between protein and epitope peptide are mostly influenced by non-covalent interactions, we computed the number of hydrogen bonds and the interaction energy between the allele and epitope as a function of the md simulation time. the hydrogen bond was calculated between the protein interface atoms with a distance cut-off of . Å and angle cut-off of o between the donor and acceptor heavy atoms. as shown in fig. , the number of h-bonds fluctuates during the md simulations for all the complexes. the a complex has the largest number of h-bonds. table shows that during the last ns of the md simulation trajectory, the a complex averages . h-bonds. additional analysis of the hydrogen bonding between allele and epitope are listed in supplementary table s . figure b shows the interaction energy (electrostatic interaction + van der waals contacts) throughout the entire md simulation and table lists the average over the last ns. the a ::e and a ::e display relatively stronger interaction energies than the a :e and a ::e complexes. the comparison of rmsd, hydrogen bond, and interaction energy information indicates that the e epitope is an especially promising epitope candidate. novelty analysis. the novelty of the four mhc i t-cell epitopes in table , the nineteen mhc ii t-cell epitopes in table , and the ten b-cell epitopes in table identified in this study were analyzed using iedb . the iedb database contains the epitopes that are annotated based on scientific literature. the iedb showed that the e , e , e , e , e , e epitopes, which bind to solvent exposed regions on the protein (fig. ) , have not been previously reported as lasv epitopes or vaccine candidates. in addition, this analysis further indicates that other epitopes (e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e ) have partial segments of their sequence reported as subsets of other epitopes, whereas e , e , e are exact match to previously reported sequences. for these epitopes, a comparison showing the overlap between the predicted epitopes in this study and previously known epitopes documented in iedb is given in table s . in addition to the epitopes in the iedb, we compared our consensus predicted epitopes with the previously reported predictions [ ] [ ] [ ] [ ] [ ] [ ] in table s . this comparison shows a varying degree of overlap in the predicted sequences. the www.nature.com/scientificreports www.nature.com/scientificreports/ novelty results confirm that thirty epitopes have not been previously tested experimentally as lasv epitopes, suggesting that their therapeutic potentials in designing vaccines against lasv can be further explored. lasv hemorrhagic fever is endemic in west africa, and no approved effective therapeutics are currently available. therefore, there is an urgent need for the discovery and development of potential antiviral therapeutics. the lasv gp spike has emerged as a promising selective target for the development of novel vaccines as it plays an essential role in the virus-host interaction. several in-silico studies [ ] [ ] [ ] [ ] [ ] [ ] were performed to predict lasv gp epitopes with the use of a single prediction tool for each type of epitope. we have identified new t and b-cell epitopes using a variety of computational approaches, including twelve epitope prediction methods, protein-peptide docking, and md simulations. the mhc i and ii t-cell epitopes were separately predicted with the lasv gp sequence using well-known prediction methods. the predicted mhc i t-cell epitopes then were prioritized based on the consensus score, binding affinity, and antigenicity, while mhc ii t and b-cell epitopes were prioritized based on the consensus score. novelty analysis of the consensus-selected epitopes showed that thirty of these predicted epitopes have either no overlap or only a partial overlap to previously reported sequences. within this list of new epitopes, six sequences have no overlap with any known experimentally tested epitopes in the iedb. in addition, docking and md simulations were performed to further validate the mhc i t-cell epitopes. the simulation results show that the allele-mhc-i epitopes are stable, with favorable hydrogen-bond and interaction energy. of these, epitope e ( fsrpspigy ) segment was found to be especially stable. this study demonstrates that the adopted consensus epitope prediction strategy is valuable for in-silico investigations of known epitopes and the identification of new epitopes. experimental validation of these epitopes may lead to the design and development of effective lasv vaccines. table . allele-epitope interaction parameters calculated by averaging over the last ns of the md simulation trajectory. ictv virus taxonomy profile: arenaviridae lassa fever, a new virus disease of man from west africa. i. clinical description and pathological findings understanding the cryptic nature of lassa fever in west africa structural basis for antibody-mediated neutralization of lassa virus lassa fever, a new virus disease of man from west africa. . isolation and characterization of the virus a case-control study of the clinical diagnosis and course of lassa fever protein structure of lymphocytic choriomeningitis virus: evidence for a cell-associated precursor of the virion glycopeptides lassa fever in west africa: evidence for an expanded region of endemicity genome sequence of lassa virus isolated from the first domestically acquired case in germany passive antibody therapy of lassa fever in cynomolgus monkeys: importance of neutralizing antibody and lassa virus strain acidic ph-induced conformations and lamp binding of the lassa virus glycoprotein spike arenavirus stable signal peptide is the keystone subunit for glycoprotein complex organization the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles most neutralizing human monoclonal antibodies target novel epitopes requiring both lassa virus glycoprotein subunits prediction of mhc class ii binding affinity using smm-align, a novel stabilization matrix alignment method an introduction to epitope prediction methods and software multi-epitope vaccines: a promising strategy against tumors and viral infections in-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases virus pathogen database and analysis resource (vipr): a comprehensive bioinformatics database and analysis resource for the coronavirus research community fast, scalable generation of high-quality protein multiple sequence alignments using clustal the protein data bank charmm-gui: a web-based graphical user interface for charmm charmm: the biomolecular simulation program simulations using the charmm additive force field bepipred- . : improving sequence-based b-cell epitope prediction using conformational epitopes predicting linear b-cell epitopes using string kernels international conference on artificial immune systems ellipro: a new structure-based tool for the prediction of antibody epitopes epitopia: a web-server for predicting b-cell epitopes reliable b cell epitope predictions: impacts of method development and improved benchmarking propred : prediction of promiscuous mhc class-i binding sites prediction of ctl epitopes using qm. svm and ann techniques large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction the immune epitope database (iedb) . propred: prediction of hla-dr binding sites improved methods for predicting peptide binding affinity to mhc class ii molecules epitop-a proteochemometric tool for mhc class ii binding prediction vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines structural insights into the binding of hepatitis b virus core peptide to hla-a alleles: towards designing better vaccines the structure of the human allo-ligand hla-b* in complex with a cytochrome p peptide: steric hindrance influences tcr allo-recognition swiss-model: homology modelling of protein structures and complexes the swiss-model repository-new features and functionality automated comparative protein structure modeling with swiss-model and swiss-pdbviewer: a historical perspective autodock and autodocktools : automated docking with selective receptor flexibility autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading charmm all-atom additive protein force field: validation based on comparison to nmr data scalable molecular dynamics with namd a smooth particle mesh ewald method numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes a simulation study used to design the sequential monitoring plan for a clinical trial vmd: visual molecular dynamics evaluation and use of in-silico structure-based epitope prediction with footand-mouth disease virus mapping putative b-cell zika virus ns epitopes provides molecular basis for anti-ns antibody discrimination between zika and dengue peptide selection by mhc class i molecules mhc class ii epitope predictive algorithms fundamentals and methods for tand b-cell epitope prediction conserved b and t cell epitopes prediction of ebola virus glycoprotein for vaccine development: an immunoinformatics approach toward the estimation of the absolute quality of individual protein structure models stereochemistry of polypeptide chain configurations verification of protein structures: patterns of nonbonded atomic interactions structural allele-specific patterns adopted by epitopes in the mhc-i cleft and reconstruction of mhc: peptide complexes to cross-reactivity assessment in silico prediction of b-and t-cell epitope on lassa virus proteins for peptide based subunit vaccine design computer aided epitope design as a peptide vaccine component against lassa virus hla-c-restricted viral epitopes are associated with an escape mechanism from kir dl (+) nk cells in lassa virus infection design of peptide-based epitope vaccine and further binding site scrutiny led to groundswell in drug discovery against lassa virus identification of protective lassa virus epitopes that are restricted by hla-a lassa fever virus peptides predicted by computational analysis induce epitope-specific cytotoxic-t-lymphocyte responses in hla-a . transgenic mice p.c. conceived the idea. p.b. and e.p. performed and analyzed the computations and simulations, and prepared figures. p.b., e.p., b.g., and p.c. interpreted the results and wrote the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to p.p.c. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -v h yro authors: han, ki-cheol; park, daechan; ju, shinyeong; lee, young eun; heo, sun-hee; kim, young-ae; lee, ji eun; lee, yuna; park, kyong hwa; park, se-ho; lee, hee jin; lee, cheolju; jang, mihue title: streamlined selection of cancer antigens for vaccine development through integrative multi-omics and high-content cell imaging date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: v h yro identification of tumor antigens that induce cytotoxic t lymphocytes (ctls) is crucial for cancer-vaccine development. despite their predictive ability, current algorithmic approaches and human leukocyte antigen (hla)-peptidomic analysis allow limited selectivity. here, we optimized a method to rapidly screen and identify highly immunogenic epitopes that trigger ctl responses. we used a combined application of this method involving immune-specific signature analysis and hla-associated peptidomics using samples from six patients with triple-negative breast cancer (tnbc) in order to select immunogenic hla epitopes for in vitro testing. additionally, we applied high-throughput imaging at the single-cell level in order to confirm the immunoreactivity of the selected peptides. the results indicated that this method enabled identification of promising ctl peptides capable of inducing antitumor immunity. this platform combining high-resolution computational analysis, hla-peptidomics, and high-throughput immunogenicity testing allowed rapid and robust identification of highly immunogenic epitopes and represents a powerful technique for cancer-vaccine development. . scheme of the rapid high-throughput approach for discovering natural ctl epitopes. preselected til-resident tnbc tumors underwent hla-peptidomic analysis to identify hla-bound peptides. integrated wts data revealed a higher priority to select promising hla-peptides via high-resolution bioinformatics analysis, showing immune-cell-specific signatures and tcr-repertoire diversity in tumors. combined ngs analysis and the use of predictive algorithms for mhc-binding affinity enabled selection of highly immunogenic hla-peptide candidates. analysis of ifnγ-producing cd + t cell response using the highcontent imaging system in a -well format at the single-cell level for discovery of immunogenic hla epitopes eliciting a ctl response. filtrating lymphocytes) play a significant role in tumor-sites. additionally, large amounts of tils correlate with improved tumor survival ; therefore, we preselected til-resident tnbc tissues for histologic analysis to identify potentially promising cancer epitopes ( fig. a and supplementary fig. s ) and scored til density by measuring the proportion of the stromal area infiltrated by lymphocytes, as previously described . to select highly immunogenic hla epitopes, we analyzed the intratumoral heterogeneity of the tcr repertoires in til-resident cancers from six patients with tnbc (fig. b,c) . the tcr repertoires comprise somatic recombination of the tcrα and β chains, allowing the specificity of each t cell clone to be determined by rearrangement of the v, d, and j segments of the tcrβ chain during generation of the highly variable complementary determining region , . to evaluate tcr diversity of tils, we assembled cdr sequences using the sequence reads of rna-seq data. a unique cdr sequence of tcrα and tcrβ, respectively, was defined as a clone, and the number of clonotypes represents the number of unique clones per sample after normalization with the corresponding rna-seq depth (fig. b) . the t cell clonal fraction was defined as the frequency of the top % of tcrα or tcrβ clones among total tcr clones (fig. c) . the top most abundant tcrα and tcrβ sequences in each patient are shown in supplementary fig. s , and expression of the three hla-class i genes (hla-a/b/c) is shown in fig. d . we found a linear positive correlation between the number of tcrβ clonotypes and the expression of mhc-class i genes according to pearson's correlation coefficient (r = . ) (fig. e) . til immunoprofiles and immune-specific signatures. several recent studies demonstrated the strong relationship between significant overall survival (os) and cancer patients harboring a high number of cd + t cells and a low number of foxp + t cells . in particular, the abundance of regulatory t (treg) cells and macrophages correlated with worse outcomes, whereas the abundance of intratumoral cd + t cells and cd + t-helper (th) cells correlated with better prognosis . additionally, immune-specific signatures in tils are of potential clinical significance; therefore, we estimated the distribution of til types in each patient according to wts data using cibersort computational analysis (fig. and supplementary fig. s ). the relative proportion of each infiltrated immune cell was evaluated in each patient by quantifying immune composition from bulk-tissue gene-expression profiles, as enrichment of cd /cd ro and th cells are considered positive prognostic factors . the results indicated that cd + t cells were highly infiltrated in both patients tnbc# and tnbc# , whereas a large proportion of treg cells was observed in patients tnbc# and tnbc# (fig. a-c) . patient tnbc# displayed a highly enriched frequency of cd + t cells, as well as treg cells, suggesting increased accumulation of tils. interestingly, we found a significantly increased proportion of cd + t cells relative to treg cells in patient tnbc# as compared with that observed in other patients (fig. d) , suggesting the emergence the relationship between the number of tcrβ clonotypes and the expression of mhc class i genes. pearson's correlation was calculated between two groups. of promising tumor-associated antigens. on the other hand, we found elevated levels of immune-suppressive macrophages in patients tnbc# , tnbc# , and tnbc# , which is predictive of a negative outcome (fig. e ). to identify naturally existing mhc class i -restricted ligands, we used an immunoproteomic approach involving tissue from six patients with tnbcs and an mhc-І antibody specific for the hla-a, b, and c molecules ( fig. and supplementary fig. s ). after immunoprecipitation, high-resolution lc-ms/ms analysis identified and quantified the hla peptide sequences with a % false discovery rate (fdr) (supplementary fig. s ) . notably, the number of eluted peptides from each of the six patients were substantially different ( supplementary fig. s ), although > % of the hla peptides analyzed by lc-ms/ ms showed typical properties associated with epitope length. as expected, most of the peptides were nine amino acids long, with only a few having to amino acids, suggesting a high level of consistency ( fig. a and supplementary fig. s ). clustering of the -mer hla peptides showed predominant enrichment of residues at peptide positions and and consistent with the anchor motifs required by the binding groove of each hla molecule (fig. b and supplementary fig. s ). additionally, we found high numbers of cd + t and cd + th cells infiltrating into the tumor sites of patient tnbc# and relative to treg cells, with patient tnbc# showing a -fold higher number of cd + t cells as compared with that in patient tnbc# and accompanied by the lowest expression of mhc class i genes, suggesting a higher accumulation of antigen-specific ctls in patient tnbc# (supplementary figs. s ,s ). a total of peptides were identified from the tissue of patient tnbc# along with elevated expression of hla genes, whereas only five peptides were received from tissue from patient tnbc# and all showing the lowest expression of mhc class i genes ( fig. c and supplementary fig. s ). moreover, we observed a positive correlation between the number of eluted peptides relative to input lysate and the expression of mhc class i genes (fig. c) . we then performed kyoto encyclopedia of genes and genomes (kegg) pathway enrichment analysis to investigate the genes associated with the hla-binding peptides in patient tnbc# and the homotypic hla-a* : allele (fig. d) . interestingly, the high-count genes (n > ) were significantly enriched in kegg pathways related to cancer, protein processing in the endoplasmic reticulum (er), viral carcinogenesis, and www.nature.com/scientificreports www.nature.com/scientificreports/ antigen processing and presentation. numerous cancer-related genes overexpressed in cancer tissues contribute to cancer-specific or associated epitopes , and hla epitopes require proteasomal digestion and translocation into the er to bind mhc class-І molecules . it would be expected that the expression of genes encoding machinery responsible for antigen processing would be elevated under these circumstances. these results suggested that the eluted hla peptides identified were naturally presented by hla molecules. we further investigated the levels of the eluted peptides based on rna-seq analysis of corresponding mrna from the same sample (fig. e ,f). compared with normal breast tissues, of peptides corresponding to proteins from the same sample showed elevated abundances in cancer tissues accompanied by significant differences in mrna expression (≥ log fold change). subsequent in silico prediction of the hla-binding affinities to the hla peptides and calculation of their respective binding affinity to specific alleles (predicted ic ) yielded a list of the top highest ranking peptides derived from patient tnbc# (table ) . a rapid imaging-based screening method to determine antigen-specific t cell response at the single-cell level. to determine whether the experimentally identified peptides can functionally elicit an immune response, we evaluated cytokine production by the cd + t cells. currently, intracellular cytokine staining (ics)-based detection methods for monitoring ex vivo ifnγ response show low throughput relative to the number of candidate antigens being tested. moreover, an individual antigen test requires large amounts of immune cells . therefore, we developed an efficient and comprehensive screening system to test ctl response based on a high-content, high-throughput imaging approach ( supplementary fig. s ). this fluorescence-imaging-based screening system allows the use of lower numbers of viable cells up-scaled performance , . development of a -well format capable of screening mixed populations of t cells for their response against large number of www.nature.com/scientificreports www.nature.com/scientificreports/ peptides enables a cost-effective approach to phenotype analysis. notably, cancer-associated antigens are highly attractive targets for determining their efficacy in triggering a t cell response; however, numerous clinical trials targeting taas for vaccine development have failed to demonstrate clinical efficacy due to immune self-tolerance. to identify highly immunogenic peptides incapable of eliciting self-tolerance, we tested the antigen-specific t cell response in pbmcs from a healthy donor. monitoring ex vivo ifnγ-producing pbmc reactivity using our fluorescence-labelled cell-based screening system ( fig. a -c, supplementary figs. s ,s ) revealed significant ifnγ responses to two individual epitopes (eif a -p and tcp -p) in cd + t cells labelled with a fitc conjugated anti-human cd antibody (fig. b) . additionally, treatment with the hla-a* : -specific epitopes allowed detection of apc-conjugated ifnγ released from cd + t cells (fig. a,b) , with pma and ionomycin co-treatment used to trigger t cell activation as a positive control. similarly, pbmcs from two of the three healthy donors were reactive against same two epitopes (fig. c) . to further analyze the peptide-induced cd + t cells, we generated hla-a* : tetramers targeting specific peptide-reactive cd + t cells. facs analysis revealed that . % and . % of t cells were targeted by eif a -p and tcp -p, respectively, and detectable on day of ex vivo t cell expansion (fig. d) . these findings suggested the efficacy of our method to screen highly immunogenic ctl epitopes using an imaging system on the detection of intracellular ifnγ levels following peptide stimulation. the two genes associated with the peptide epitopes, the translation initiation factor eif a and tcp , a member of the chaperonin-containing complex tcp -containing ring complex (tric), are involved in tumor proliferation and survival. eif a controls translation initiation and is a critical checkpoint protein involved in cell proliferation and tumorigenesis . additionally, tcp , as a tric member, is involved in tumor survival and growth and an oncogene driver . moreover, these two genes are significantly overexpressed in tumor tissues according to wts data and data from the genotype-tissue expression project public proteomic database, suggesting their potential as therapeutic targets. these results identified two epitopes derived from eif a and tcp as potential promising immunogenic antigens to boost t cell response. patients with tnbc, which is defined by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor , have a higher tendency for recurrence at ~ years after diagnosis , . there are currently no known therapeutic targets for tbnc patients due to the molecular heterogeneity of the disease . recent accumulation of massive and comprehensive bioinformatics data allowed identification of potential therapeutic targets associated with clinical survival . interestingly, a tnbc subtype characterized by high levels of immune genes involved in t cell function, immune response, and antigen processing, was found to be associated with favorable prognosis, suggesting a close correlation between immune-gene signatures and better clinical outcomes . therefore, it is possible that tils controlling clinical cancer progression represent key factors for preselection of tumors prior to hla-immunopeptidomics. in this study, til-resident tissues were pre-selected comprehensively to investigate a diversity of tcr repertoires and immune profile as predictors of clinical outcome. we then found a positive correlation between tcr diversity, reflecting clonal composition, and the expression of mhc-class І molecules, suggesting that active tumor-antigen presentation promotes the generation of antigen-specific tils. additionally, immune-specific signature analysis can discriminate specific immune-cell types in each patient and thus enhance the efficiency of selective hla-peptidomic approaches. notably, the expressional comparison of cd + t cells relative to immune-suppressive treg cells is extremely crucial to select the high antigenicity of antigens reflecting the therapeutic efficacy. thus, the extreme increase in the number of cd + t cells relative to that of treg cells in patient tnbc# presented a major analytical reason for further in vitro t cell response testing. furthermore, our results indicated a positive correlation between the number of peptides identified via hla-peptidomics and the amount of hla molecules expressed on the surface of cancer cells. it is also possible that the observed difference in the number of eluted peptides might have been influenced by hla-expression levels resulting from active induction of antigen presentation on mhc molecules, which subsequently elicited a strong immune response. thus, these findings suggested that several factors should be considered for successful hla-peptidomic approaches influenced by tcr diversity and elevated expression of hla genes. www.nature.com/scientificreports www.nature.com/scientificreports/ additionally, we showed that integrating hla-peptidomics with imaging-based immunogenicity screening is applicable for the discovery of highly immunogenic ctl epitopes. characterization of antigen-specific cellular immune response is essential to confirm vaccine-related effects specific to a cancer antigen. currently, there are www.nature.com/scientificreports www.nature.com/scientificreports/ only few assays (e.g., the enzyme-linked immunosorbent spot assay) capable of quantifying t cell responses , , and the choice of which assay to use depends on the experimental scale, cost, equipment, reproducibility, and required detection sensitivity. a high-throughput imaging system provides an optimal platform for highly sensitive and quantitative analysis of individual t cells at the single-cell level. moreover, ics-based cytokine detection allows identification of specific cell subpopulations, even when using a small number of cells. the immunopeptidome approach serves massive hla-associated peptides as the collection of cancer epitopes; however, there are obstacles to rapidly determining the optimal set of promising epitopes by testing an enormous pool of peptide candidates in a single measurement. in this study, the hla-peptidomics approach combined with comprehensive analysis of immune-specific signatures and tcr repertories showed high selectivity to determine the immunogenic t-cell epitopes. sequentially, the high-content imaging system allowed high-resolution analysis for t cell reactivity. despite the need for discovery of tumor-derived antigens for effective cancer vaccine development, selection of antigens that elicit robust immune response remains challenging. here, we report a smart strategy for streamlined selection of cancer antigens in vaccine development. through integrative multi-omics and high-content cell imaging, we identified highly immunogenic epitopes from patients with tnbc. identification of potential vaccine epitopes coupled with immune-specific signature analysis, hla-peptidomics, and single-cell-based immunogenicity testing offers a discriminative and powerful tool for cancer-vaccine development. analysis of high-throughput sequencing. dna and rna were simultaneously extracted from cryo-pulverized tnbc tissue powder using an allprep kit (qiagen, hilden, germany), and libraries for whole-exome sequencing and total rna sequencing, respectively, were prepared using truseq library prep kits (illumina, san diego, ca, usa). the libraries were sequenced using the hiseq platform (illumina), and raw data were mapped onto hg using the bwa mem algorithm (http://bio-bwa.sourceforge.net/). variant calling was performed using the genome analysis toolkit (https://software.broadinstitute.org/gatk/) as previously described . a custom proteogenomic search database was generated for the variants using the proteomics tool quilts (http://www.fenyolab.org/tools/tools.html). for wts data, contaminating adapters and low-quality bases were removed with trimmomatic , and the trimmed data were mapped onto hg using star (version . . a) . gene expression, including that of hla genes, was calculated by rsem (version . . ) , and hla presentation on multiple cancer cells was evaluated using the expression atlas (https://www.proteinatlas.org/humanproteome/ tissue/cancer) . for identification and quantification of tils from wts data, we used mixcr (v. . . ; https:// mixcr.readthedocs.io/en/master/) with the alignment parameter -p rna-seq. . hla typing at -digit resolution was performed using the hlascan method and wes data (synthekabio, korea). purification of hla-class i peptides. the lc-ms/ms analysis was performed as previously described . hla-class i peptides were purified from tnbc tissues, and frozen tissues were pulverized with a cp cry-oprep automated dry pulverizer (covaris, woburn, ma, usa), followed by incubation at °c for h with lysis buffer containing . % sodium deoxycholate, . mm iodoacetamide, mm edta, mm pmsf, % octyl-β-d-glucopyranoside (sigma-aldrich, st. louis, mo, usa), and a protease-inhibitor cocktail (roche, mannheim, germany) in phosphate-buffered saline (pbs). the lysates were cleared by centrifugation for min at , g and °c. hla-class i molecules were purified using the w / monoclonal antibody bound to amino-link beads (thermo scientific, waltham, ma, usa), as previously described . the anti-hla-class i antibody was purchased from abcam (cambridge, uk). hla-peptide complexes were eluted from the affinity column using five column volumes of . n acetic acid. the eluted hla-class i proteins and the released peptides were loaded on sep-pak tc columns (waters, milford, ma, usa), and the peptide fraction was eluted with % acetonitrile in . % trifluoroacetic acid, followed by drying by vacuum centrifugation. the lc-ms/ms analysis was performed as previously described . dried peptide samples were reconstituted in µl of . % formic acid, and an aliquot containing ~ μl was injected from a cooled ( °c) autosampler into a reversed-phase magic c aq column ( cm × μm (packed in-house); michrom bioresources, auburn, ca, usa) on an eksigent nanolc d system at a flow rate of nl/min. prior to use, the column was equilibrated with % buffer a ( . % formic acid in water) and % scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports www.nature.com/scientificreports/ buffer b ( . % formic acid in acetonitrile). the peptides were eluted with a linear gradient from % to % buffer b over min and % to % buffer b over min, followed by an organic wash and aqueous re-equilibration at a flow rate of nl/min, with a total run time of min. the high-performance liquid chromatography system was coupled to an ltq-orbitrap xl mass spectrometer (thermo fisher scientific, bremen, germany) operated in data-dependent acquisition mode. full scans (m/z - ) were acquired at a resolution of , using an automatic gain-control (agc) target value of e and a maximum ion-injection time of ms. tandem mass spectra were generated for up to precursors by collision-induced dissociation in the ion-trap using a normalized collision energy of %. the dynamic exclusion was set to s, and fragment ions were detected at a normal scan mode using an agc target value of e and a maximum ion-injection time of ms. source ionization parameters were as follows: spray voltage, . kv; and capillary temperature, °c. data analysis of the hla-class i peptidome. ms data were analyzed using maxquant software (v. . . . ) against the uniprot database (april , ; https://www.uniprot.org/), and personalized human database from patient-derived wes data. n-terminal acetylation and methionine oxidation were set as variable modifications, enzyme specificity was set as "unspecific, " and fdrs for peptides and proteins were set at . and , respectively. possible sequence matches were restricted to eight to amino acids, a maximum peptide mass of da, and a maximum charge state of three. main search peptide tolerance was set at , and the box for "use ms centroids" was checked. hits to the reverse database and contaminants were removed from the "peptide. txt" output file produced by maxquant. peptides were subjected to hla-class i binding and immunogenicity prediction analyses using netmhc (http://www.cbs.dtu.dk/services/netmhc/) and the mhc i immunogenicity portion of the iedb analysis resource (http://tools.iedb.org/immunogenicity/), respectively. the logo-plots were constructed using a seq. logo method . pbmcs isolated from three hla-a* : -positive healthy donors. t cell responses to treatment with individual peptides were monitored after days of in vitro culture, as previously described . briefly, pbmcs were cultured in rpmi- supplemented with l-glutamine, non-essential amino acids, hepes, β-mercaptoethanol, sodium pyruvate, penicillin/streptomycin (gibco; thermo fisher scientific), and % human ab serum (gibco), with a total of × pbmcs used in each well. for antigen-specific t cell expansion, individual peptides ( µg/ml) were incubated with pbmcs in the presence of interleukin (il)- ( ng/ml; peprotech, rocky hill, nj, usa) for days. each peptide with > % purity was synthesized by synpeptide (shanghai, china). for non-specific t cell expansion, t cells were stimulated using a t cell activation/expansion kit (miltenyi biotec, bergisch gladbach, germany), and cells were cultured every days by replacing the medium with fresh half-medium containing il- ( ng/ml) and il- ( ng/ml; peprotech). on day , for ex vivo intracellular ifnγ detection, each peptide ( µg/ml) or phytohemagglutinin (pma) ( ng/ml; sigma-aldrich) plus ionomycin ( µg/ml; sigma-aldrich) for the positive control was administered in the presence of brefeldin a ( : ; biolegend, san diego, ca, usa) for overnight incubation according to the ics protocol. after intracellular fixation using bd cytofix/cytoperm fixation/permeabilization kit (bd biosciences, san jose, ca, usa), cells were stained with antibodies against surface markers and ifnγ at °c for either h or overnight. for visualization of ifnγ-producing t cells, a fluorescein isothiocyanate (fitc)-conjugated anti-human cd α antibody (r&d systems, minneapolis, mn, usa), an alexa -conjuated anti-human cd antibody (biolegend), and an allophycocyanin (apc)-conjugated anti-human ifnγ antibody (biolegend) were specifically labeled for cd + t, cd + t, and intracellular ifnγ capture, respectively. cells were then washed with pbs buffer, and high-throughput imaging was performed using the operetta cls high-content analysis system equipped with harmony software (perkinelmer, waltham, ma, usa). to generate the p/mhc tetramer, a biotinylated hla-a* : monomer complexed with each peptide was obtained from immunomax co., ltd. 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visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion we would like to express my special appreciation and thank dr. supplementary information is available for this paper at https://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to k.-c.h., c.l. or m.j.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - g y i authors: cheng, chao-min title: small-volume point-of-care analytical methods date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: g y i detecting clinically relevant diagnostic biomarkers, suitable for point-of-care detection, may facilitate rapid treatment and prevention of disease. however, medical diagnosis for which there are limited quantities of testable tissue or fluid, require point-of-care technologies with superior sensitivity and specificity. the purpose of this editorial is to provide an overview of the collection’s content, comprising original research into fluidics-based diagnostic platforms—specifically those made of paper or other low-cost materials, that can suitably operate with tiny sample volumes from ml to nl. in particular, we will focus on the clinical applications of such research, with potential uses in a number of fields including combating the covid- pandemic for instance. d esigning diagnostic procedures, and improving their accuracy, has been a long-standing research focus that is constantly evolving as it incorporates recent pursuits and technologies from a range of areas, including analytical chemistry, bioanalytical chemistry, translational medicine, and medicine. with recent technological advances in multiple research fields-such as materials science, micro-/nano-technology, cellular and molecular biology, and bioengineering-attention is shifting toward the development of new diagnostic tools that not only address needs for high sensitivity and specificity, but that fulfill economic, environmental, and rapid point-of-care (poc) needs, for groups and individuals with constrained resources and, possibly, limited training. tang et al. refined the capacity of nuclear magnetic resonance relaxometry, successfully examining a tiny sample (below μl), with a miniaturized nmr platform, at °c and bar pressure by integrating electronics, microfabrication, and novel chemistry . zhao et al. demonstrated a simple, universal approach for nucleic-acidbased pathogen diagnostics-requiring no electricity, or special equipment. the approach used a hand-powered syringe for the pump, with inexpensive amine-functionalized diatomaceous earth and a μm teflon filter as a reaction matrix in two stages of their process, which also used homobifunctional imidoesters. for a large sample volume (< ml) they demonstrated a -fold better capture efficiency than that of a common commercial nucleic acid isolation kit . lode et al. reported on the development of a clinically based compounding approach, using silicone oil-free syringes and a g × mm low dead space hub injection needle. they have evaluated this approach for three anti-vegf biologics commonly used in ophthalmology: aflibercept, ranibizumab (lucentis), and bevacizumab (avastin). their results indicated that compounding and storage for week did not compromise the functional activity of the biologics, and allowed for safe and cost-effective compounding of anti-vegf biologics for intravitreal injections in prefilled silicone oil-free syringes . sypabekova et al. presented clinically relevant results regarding the detection of mycobacterium tuberculosis-secreted mpt protein . they used an interdigitated electrode as a platform to capture the immunogenic protein, and electrochemical impedance spectroscopy as a detection approach. their assay involved a special receptor, a single stranded dna aptamer that specifically recognized the mpt protein. an evaluation of this approach demonstrated % specificity for clinical serum and sputum samples, and a sensitivity of . % and . %, respectively . microfluidic technologies, in particular, are considered very powerful tools for diagnosing and monitoring human diseases, and miniaturized fluidics-based platforms can precisely manipulate tiny body fluid volumes for rapid and accurate medical diagnoses. yen et al. demonstrated a low-power microfluidic pump, based on travelling-wave electroosmosis, that could be operated at . v driving voltage, with a power consumption of . mw. this in-situ cmos-based microfluidic pump is the first to drive a clinically diluted serum sample, driving a diluted ( : , dilution ratio) serum sample with a flow rate of μm/s . behrens et al. reported on a low-cost, open-source peristaltic pump, constructed using d-printed parts and common hardware that could be used with microfluidic devices for poc diagnostics. this pump accepted commonly available silicone rubber tubing in a range of sizes, from . to mm, and was capable of producing flow rates up to . ml/min www.nature.com/scientificreports/ microfluidic technologies may be used for a variety of poc diagnostic applications (e.g., infectious diseases, covid- diagnosis) because they are disposable, inexpensive, portable, and easy to use, especially when they are manufactured using low-cost materials such as paper (and other materials such as cotton or bamboo , ). one of the purposes of this collection in scientific reports is to bridge microfluidic technologies (polydimethylsiloxane-based and paper-based microfluidics) and biology with medicine, with a focus on applications of microfluidics for poc diagnostics. tsai et al. have developed a multi-target lateral flow immunoassay strip, with a stacking pad design, to obtain on-site, rapid diagnosis of periprosthetic joint infection. the multi-target design of this device increases specificity, and the stacking pad enhances detection sensitivity . schwenke et al. developed a user-friendly lateral flow test that could detect very low amounts of free chlorine and demonstrated a times higher sensitivity than a commercial dip test. this test could detect chlorine quantities as low as . ppm when oxidation stable flow test substrates were used, and results could be obtained within - min, with no additional action required by the operator . noh et al. developed a pipetting-based immunoassay using a removable magnetic ring-coupled pipette tip. their "magnetic bead-capture antibody-targeted protein complex" was purified by pipetting and quantified by enzymatic color development, and could be integrated with a lateral flow system. this pipetting-based immunoassay was applied to detect the nucleoprotein (np) of the influenza a virus. when this assay was applied exclusively for antigen capture in a lateral flow system, the limit of detection improved -fold and displayed greater sensitivity than the lateral flow system alone . it is noted that this pipetting-based immunoassay may be used to detect sars-cov- infection. mcardle, h. et al. demonstrated an electrochemically based direct detection method using electrocatalytic platinum nanoparticles to detect specific trfs: ′alatgc, ′glygcc, and ′gluctc. using synthetic trf mimics, this system was linear over orders of magnitude, with sub-attomolar limits of detection. specificity was tested using naturally occurring mismatched trf mimics. further, mcardle et al. quantified trf levels in patient plasma and showed that their detection system recapitulates results obtained by qpcr. they have also designed a trf detection system with high sensitivity and specificity capable of quantifying trfs in low plasma volumes using benchtop apparatus as well . the collection has now been open for submissions since early , and new submissions are still being welcomed. here, we would like to show our deep appreciation to all authors and reviewers. without their invaluable support and contributions, this collection could not be published on schedule. this collection may not cover all topics in this emerging scientific field-the development of practical tools for poc diagnostics. however, we firmly believe that our efforts would promote highly impactful innovations and challenging discussions in relevant academic and commercial communities. a miniaturized spectrometer for nmr relaxometry under extreme conditions a robust, hand-powered, instrument-free sample preparation system for point-of-care pathogen detection a new method for pharmaceutical compounding and storage of anti-vegf biologics for intravitreal use in silicone oil-free prefilled plastic syringes an aptasensor for the detection of mycobacterium tuberculosis secreted immunogenic protein mpt in clinical samples towards tuberculosis detection a low-power cmos microfluidic pump based on travelling-wave electroosmosis for diluted serum pumping open-source, d-printed peristaltic pumps for small volume point-of-care liquid handling cotton-based diagnostic devices lignocellulose-based analytical devices: bamboo as an analytical platform for chemical detection development of a multiplex and sensitive lateral flow immunoassay for the diagnosis of periprosthetic joint infection analysis of free chlorine in aqueous solution at very low concentration with lateral flow tests pipetting-based immunoassay for point-of-care testing: application for detection of the influenza a virus quantification of trna fragments by electrochemical direct detection in small volume biofluid samples this work was supported by the ministry of science and technology, taiwan (most - -e- - -cc to c.-m.c.). the author declares no competing interests.correspondence and requests for materials should be addressed to c.-m.c.reprints and permissions information is available at www.nature.com/reprints. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -nk iyg authors: chin, wei chien benny; bouffanais, roland title: spatial super-spreaders and super-susceptibles in human movement networks date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: nk iyg as lockdowns and stay-at-home orders start to be lifted across the globe, governments are struggling to establish effective and practical guidelines to reopen their economies. in dense urban environments with people returning to work and public transportation resuming full capacity, enforcing strict social distancing measures will be extremely challenging, if not practically impossible. governments are thus paying close attention to particular locations that may become the next cluster of disease spreading. indeed, certain places, like some people, can be “super-spreaders”. is a bustling train station in a central business district more or less susceptible and vulnerable as compared to teeming bus interchanges in the suburbs? here, we propose a quantitative and systematic framework to identify spatial super-spreaders and the novel concept of super-susceptibles, i.e. respectively, places most likely to contribute to disease spread or to people contracting it. our proposed data-analytic framework is based on the daily-aggregated ridership data of public transport in singapore. by constructing the directed and weighted human movement networks and integrating human flow intensity with two neighborhood diversity metrics, we are able to pinpoint super-spreader and super-susceptible locations. our results reveal that most super-spreaders are also super-susceptibles and that counterintuitively, busy peripheral bus interchanges are riskier places than crowded central train stations. our analysis is based on data from singapore, but can be readily adapted and extended for any other major urban center. it therefore serves as a useful framework for devising targeted and cost-effective preventive measures for urban planning and epidemiological preparedness. | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ is the low-high outliers, that is any location with low density of disease cases which is surrounded by high density locations, thereby making it more vulnerable as it has a higher probability to report a higher number of cases in the following time period . in the field of network science, the concepts of 'spreaders' and 'receivers' first appeared in the hyperlink-induced topic search (hits) algorithm as hubs and authorities, respectively. in the hits algorithm, hubs describe highly influential nodes, while authorities represent highly popular destination nodes. to sum-up, the spatial super-susceptibles correspond to the susceptible locations in a spatial diffusion network. these locations are identified as being more vulnerable within the network as they are the destination of more people, hence generating a higher probability of being visited by infected agents. the spatial super-susceptibles are vulnerable locations as they are prone to disease infection thereby having the potential to become hotbeds for disease spreading to the rest of a city or region. note that if a place is both spatial super-spreader and spatial super-susceptible, it would require particular attention since it would pose the risk of simultaneously being a hotbed of infection and disease spreader. identifying these places would therefore be critical in the fight with infectious diseases such as in this article, we report a study aimed at systematically identifying the spatial super-spreaders and spatial super-susceptibles in the spatial human network of the city-state of the republic of singapore. the particular choice of singapore stems from it having: ( ) been hit early in the first wave of infection directly from wuhan and with a systematic tracking and mapping of infected people , ( ) one of the highest population densities in southeast asia, ( ) a dense and highly interconnected human mobility and transportation networks , , and ( ) detailed and reliable data for the construction of spatial networks . as mentioned earlier, a spatial super-spreader is a locus with a high outflow of people-i.e. a place where a lot of people are originated from and those people are moving to a high variety of places. in the same vein, a spatial super-susceptible is a destination for a large number of individuals originating from different places. hence, this work proposes a systematic data-centric framework enabling the identification of spatial locations, which should be targeted by public health agencies in the event of an epidemic such as covid- . with these critical places identified, policy makers would then be able to implement cost-effective targeted responses with prevention and intervention measures directly connected to the level of vulnerability of a given location. this section is divided into three parts: descriptions of (a) the study area, (b) the flow data, and (c) the metrics and indexes. study area. this study focuses on the public transportation flow network in singapore. the city-state primarily occupies an island located in southeast asia with a total surface area of about . km . as of , the total population of singapore is about . million people (with a population density of about , . per km ), in which . % are residents (citizens and permanent residents) and . % are non-residents (foreigners with long-term passes). according to the general household survey , about . % students and . % working person relies on bus or rail transport services to travel to schools or work places, thereby making public transportation the primary mode of transportation in singapore. as a result, the density of people using the public transports during the morning and evening peaks are high, and the distance between people at the stations and vehicles is short. hence, a direct consequence of the high population density combined with a high rate of people using public transportation is that physical distancing is extremely challenging if not impossible during regular operations. this issue is a serious concern when facing the spreading of a highly contagious disease such as to analyze the data, we consider the administrative subzone level spatial boundaries (from the singapore master plan ) as the analysis unit. the residential population density (from the general household survey ) are shown in fig. . there are five regions (central, west, north, north east, and east), with planning areas, and subzones . some of the subzones contain no residential population (white areas), which include airports and airbases (e.g. changi airport in the east region) and industrial parks or ports (e.g. jurong island and bukom at the south of the west region, and simpang north and south at the north region). although these places lack residential population, they are the workplaces (destinations) of a large number of individuals. the darker color areas indicate the home for a large number of people; in other words, a large number of journeys starting from and ending at these locations. weekday and weekend flow networks. we used the origin-destination (od) ridership data of bus and train to generate the public transport flow networks. the od ridership data is systematically collected by the singapore land transport authority (lta is a government statutory board under the ministry of transport) through api calls . in this study, we used the ridership from november to january . in terms of temporal resolution, the od ridership data provides hourly passenger flows between each pair of bus stops or train stations (including mass rapid transit and light rail transit). the raw data are then aggregated into weekdays (a total of days in november , days in december and days in january ) or weekends ( days in both november and december and days in january). the total number of trips for trains and buses starting from november to june are shown in fig. s (see supplementary material). as the raw data records the flow between od pairs of bus stops or train stations, we spatially aggregate the data into flows between subzones, according to the bus stop or train station locations. a total of subzones (out of a total of ) contained at least one bus stop or one train station. these subzones then form the nodes ( nodes) of the weighted direct network, with flows between nodes corresponding to the weight of directed edges. a total of , edges were found, with a vast majority ( , edges or %) being edges across subzones, | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ and less than % (exactly edges) were within-subzone flows (i.e. corresponding to self-loops from the network perspective). given that very limited number of such intra-subzone flows, they were ignored in this study. to carry out this study, we introduce two indexes, namely the spreader index (spi) and the susceptible index (sui) to search for the spatial super-spreaders (ssp) and spatial super-susceptibles (sss). both indexes sui and spi are quantitatively determined and calculated using two key elements: ( ) the local strength of human in-and outflows, and ( ) the diversity of their respective neighborhoods . the local strength of in-and outflows for a given location is the number of people coming to or leaving from the location, i.e. respectively the weighted in-degree and weighted out-degree of the corresponding node. the neighborhood diversity is captured and quantified by two types of concepts: ( ) the diversity of zones and ( ) the diversity of coreness. the diversity of zones , refers to people that are coming from different parts of the city. as for the diversity of coreness [ ] [ ] [ ] , it refers to people either coming from the core or from the periphery of the country. more details about what constitutes core and periphery is given in step below. we applied this analysis framework to the singapore public transport flow network, and identified the ssp and sss using the sui and spi indexes. the population flow patterns are expected to be different for weekdays and weekends. thus, the flow data were separated into weekday and weekend ones. the calculation flow of the spatial spreader and spatial susceptible indexes is detailed in fig. . the first part consists in aggregating the bus and train od flow data to subzones as mentioned earlier. that top layer provides the main data for the calculation, i.e. two weighted and directed networks: weekday and weekend flow networks. these networks are subsequently used to compute three network characteristic measurements, including degree centrality (step ), community detection (step ), and k-shell decomposition (step ), which are described in full details in the following subsections. the degree centrality is used as a proxy for the intensity of the local out-and inflows, whereas the community detection and k-shell decomposition results enable the computation of neighborhood diversity, including zone-entropy and coreness-entropy as introduced below. finally, in the last step (step ), the three network characteristics are used to calculate the sui and spi. step : degree centrality. the degree centrality in this study includes both the non-weighted and weighted inand out-degrees. the non-weighted and weighted versions of the degree centrality represent different concepts in terms of network characteristics. the non-weighted in-degree and out-degree are the number of links (or edges) that are pointed to and from a subzone, respectively. this non-weighted degree centrality measures the number of relationships that a particular subzone has. as for the weighted in-degree and out-degree, they correspond to the summation of incoming/outgoing flows for a given subzone, respectively. this weighted version of degree centrality indicates the total strength of a node in terms of gathering flows or spreading flows without accounting for the actual number of (incoming or outgoing) edges. in this study, the weighted degree centrality is used to represent the local intensity of nodes for the calculation of the sui and spi. the weighted degree centrality is scaled within the unit interval (see eq. ( ) for weighted outdegree and eq. ( ) for weighted in-degree), where outdegree(i) and indegree(i) are respectively the weighted out-degree and weighted in-degree of node i, o is the set of all out-degree nodes, and i is the set of all in-degree nodes. on the other hand, both non-weighted and weighted degree centralities are used in the weighted k-shell decomposition analysis performed as step . ( ) www.nature.com/scientificreports/ step : zone-entropy. this study uses a community detection method (mapequation algorithm ) to identify the zones from the flow network, instead of using the administrative spatial boundaries (i.e. the boundaries of planning areas and regions as defined by the singapore government in its master plan ) that were designed and selected for governance and political purposes. the communities from this flow network analysis capture both the strength and direction of flows, which reflect the spatial activity of people derived from their daily commuting/mobility behaviors . as the community distribution is identified for weekday and weekend networks, similarly the distribution should be differentiated between weekdays and weekends. mapequation is used to identify the communities in the flow networks . this algorithm considers the direction and weight of edges to identify the strongly connected nodes in a directed and weighted network. this particular algorithm is different from modularity-based community detection methods since mapequation's calculation concept emphasizes the strength of flows in community, i.e. higher flow intensities within a community than between communities (flows cycling within communities). mapequation captures the effect of direction while ensuring large amount of flows are kept within the community. moreover, the communities obtained with mapequation are used as the zones that contain strong human flows cycle, which is quantified with the concept of zone-entropy. note that to maintain the spatial continuous properties of the community, we integrate a distance decay effect in the flow intensity calculation (see eq. ( )) before running mapequation: where f(o, d) is the number of people moving from the origin subzone o to the destination subzone d, distance (o, d) is the distance between the two subzones, and f ′ (o, d) is the actual flow intensity incorporating the distance decay effect. first, we run the mapequation algorithm on the two networks (weekdays & weekends), and identify the zone (set of communities z = {z , z , . . . , z max } with z j = {n| ∀ n belongs to community j} ) in which each subzone (node) belongs to. then, for each subzone, the incoming/outgoing neighbors' zones are retrieved from the results together with the weights of incoming/outgoing edges (w(j, i) or w(i, j)). the neighbors' zone information and flow weights are used to calculate the normalized entropy ( h zone neigh (i) ) using eqs. ( )- ( ) . the entropy is normalized using the total number of zones in the network to enable a comparison between nodes. note that the zone-entropy value ranges between and as a consequence of this normalization. www.nature.com/scientificreports/ step : coreness-entropy. the k-shell decomposition is a method to label the coreness (k-shell levels) of nodes in a network based on the connectivity structure . because the edges of the flow networks were weighted, we use the weighted k-shell decomposition , which is an extended version that consider both the number of links (degree) and the weights of links while labeling coreness. the coreness of a location indicates the position of the location in the range from periphery (low k-shell levels) to core (high k-shell levels). in a population flow network, the core locations indicate the common origins or destinations for a large number of passengers. in this study, we first run the weighed k-shell decomposition using the non-weighted and weighted in-/ out-degree (from step ) to calculate the in/out-k-shell levels for each subzone. then, the k-shell levels are grouped into core (in-/out-core) or periphery (in/out-non-core) using the median value as a cutoff. finally, for each node, its incoming/outgoing neighbors' core/non-core information is integrated with the flow weights to calculate the so-called coreness-entropy ( h core neigh (i) ) as defined in eqs. ( )- ( ) . the entropy is normalized using the total number of coreness levels (binary levels here, i.e. c = {core, periphery} ), to facilitate the comparison of the results between nodes. note that the coreness-entropy value ranges between and after this normalization. step : spatial spreader and susceptible indexes. the spatial spreader index (spi) and spatial susceptible index (sui) are base on the general concepts of the framework proposed by fu et al. and zhang et al. . however, the exact indices are largely modified to account for the specificities of our study. specifically, the spi and sui calculations are based on a geometric average of three key network metrics. the spi (see eq. ( )) is the geometric average of the local normalized weighted out-degree ( nwoutdegree(i) ), the zone-entropy of outgoing neighbors ( h zone outneigh (i) ), and the out-coreness-entropy of the outgoing neighbors ( h core outneigh (i) ). to understand this particular definition, one may for instance consider the case for which a node's spi is high: this node has a high volume of outgoing flows (high local intensity), half of the flows are directed to the core area and the other half to the non-core area (periphery); these flows are equally divided into different zones (high out-neighbors' zoneentropy). in other words, a high spi subzone has a large number of travelers originating from there, and these individuals are on their way to both core and periphery places, which are located in various zones. therefore, with such a high spi index value, the disease spreading would be facilitated within a short period of time. the flow intensity and diversity measurements are all normalized in the unit interval, and consequently the geometric average also varies between zero and one. the spatial susceptible index sui (see eq. ( )) is constructed in a completely similar way as the spi, with the exception that we are considering all incoming components as opposed to outgoing ones in the spi: e.g. local normalized weighted in-degree ( nwindegree(i) ), the zone-entropy of incoming neighbors ( h zone inneigh (i) ), and the in-coreness-entropy of incoming neighbors ( h core inneigh (i) ). again, the concept associated with the sui is better understood when considering a subzone with large incoming flows: half of the flows are coming from the core area and the other half from the non-core area, and these flows are equally coming from different zones. in other words, this subzone is a destination for a large number of travelers originating from various zones and their origins of movement contain both core and periphery areas. therefore, a high sui subzone is expected to be a place where travelers would be more vulnerable and sensitive to being infected. like the spi, the sui varies in the unit interval. both spi and sui are calculated as the geometric average of the three components, including normalized weighted degree, zone-entropy and coreness-entropy. we have also tested the arithmetic average of these three , if neigh = inneigh. ln |core(all)| , scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ components (see fig. s in supplementary material). we chose the geometric average method because the two proposed indexes are meant to be used for identifying super-spreader and super-susceptible locales. thus, only when all the three components are high, the spreading effectiveness of a subzone shall be considered high. local intensity of human movement flows. the spatial distribution of the non-weighted/weighted in-degree and out-degree for weekdays are shown in fig . to observe the spatial distribution of the in-and out-degree, the townships are separated into four groups using the %, % and % percentile of the corresponding degree values as cutoffs, thereby giving the "low", "mid-low", "mid-high" and "high" intensities. it appears that the patterns for the non-weighted and weighted in-degrees (top row) are similar to those of their out-degree counterparts (bottom row). this points to the fact that inflows and outflows are fairly balanced, which is expected for daily aggregated data associated with steady human movements. for the non-weighted degree measurements (left column), the high in-and out-degree subzones appear to be mainly concentrated at the east, north east and central regions, whereas the west and north have a higher number of lower degree subzones. these results are correlated with the distribution of human density in singapore, namely high to very high in the east, north east and central regions, and lower in the west and north of the island. for weighted degree measurements (right column), the east region has higher degree subzones; the number of high degree subzones drop in the central region; north, north east, and west regions have relatively more higher degree subzones when compared with their non-weighted counterparts. the distribution of the non-weighted measurements for weekends are essentially the same as the results for weekdays. figure displays the differences in weighted in-and out-degree between weekdays and weekends. most subzones are in the lightest green or purple colors, thereby indicating that their degree measurements are only very slightly larger than each other (the differences are less than . times). these subzones have a similar number of people using public transportation during weekdays and weekends. only a few subzones are in dark colors indicating larger changes as compared to weekdays. these subzones reveal a notably different usage of public transportation at these locations between weekdays and weekends; the changes of usage for weekdays are twice larger than weekends (dark purple), or the other way round (dark green). community detection. as discussed in the "materials and methods" section, a critical component of our network analysis is based on community detection. figure shows the spatial distribution of communities for both weekdays and weekends. the mapequation algorithm with the provided data reveals different commu- figure . spatial distribution of the degree centralities for the weekday dataset. left column (a,c) shows the distribution for non-weighted measurements and the right column (b,d) shows the distribution for weighted measurements of the degree. the top row (a,b) displays the in-degree, while the bottom row (c,d) refers to the out-degree. the townships are separated into four groups using the %, % and % percentile as breaks, thereby giving the "low", "mid-low", "mid-high" and "high" intensities. generated with python ( . . ), matplotlib ( . . ) and geopandas ( . . ). | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ nities for both weekday flow network and weekend flow network. most communities are spatially continuous as the flow data is integrated with the inverse of the distance. however, some exceptions exist in both weekday and weekend communities (e.g. weekday and weekend community # ). the spatially-continuous patterns are expected given the spatial embedding of our networks and it indicates, as expected, that interactions between closer subzones are effectively stronger. on the other hand, the few spatially-split communities appear to be the by-product of a strong flow of human movement between two spatially-distant locations with sparser spaces between them. although weekday communities and weekend ones are different-some are split and others have different boundaries-overall, they show some notable similarities (e.g. weekday community # and weekend community # ). this observation can be attributed to two particular features of singapore: ( ) given the limited available land, singapore has a dense and compact urban landscape with a high level of mixed-use areas, be them residential, industrial and/or commercial, ( ) a non-negligible fraction of the working population is active on saturdays, which creates a high flow of travelers with the same commuting patterns as during weekdays. for instance, in the western region, weekday communities # and # are extremely similar with weekend communities # and # . these particular communities are fairly large with a heavy mixed-use of residential and industrial areas, where people have similar daily activities within a week. the north east region (ner) contains three similar communities during weekdays and weekends (community # (upper part), # , and part of # during weekdays, and the similar patterns of # (upper part), # , and # during weekends). the north region (nr) is split into multiple communities (community # (lower part), # , # , # , # , # during weekdays, and # (lower part), # , # , # , # during weekends). the identified communities # , # , # , # , # , # during weekdays, and communities # , # , # , # , # during weekends are similar and fit well with the central region (cr), which is the central business district of singapore. the community detection results show that the boundaries of human activity can be changed between weekdays and weekends. community # in weekends appears to be an area resulting from the merger of communities # , # and part of # during weekdays. this indicates that the area has stronger human movement interactions during weekends than weekdays, probably because the area is mostly residential with few shopping places providing daily needs products and necessities. in summary, the human movement boundaries are not fixed to a static pattern, and it is usually smaller than the shape of the known regional/administrative boundaries. coreness. the spatial distribution of the core area is shown in fig. . as detailed in the "materials and methods" section, the calculation of coreness is separated into two parts for each network, one of which uses the (weighted or unweighted) in-degree, and the other the (weighted or unweighted) out-degree. hence, two sets of coreness results (outgoing core area and incoming core area) are obtained for each network. some areas are identified as core in both incoming and outgoing directions (red subzones in fig. ), some are core for either incoming (pink subzones in fig. ) or outgoing (purple subzones in fig. ) but not both. however, the vast majority of areas are core ones from both the incoming and outgoing flows perspective. these red areas happen to have a notable overlap with residential areas with a high population density, thereby indicating that places where people live would always have high incoming and outgoing flow: a core area of human movement and commuting. spreader and susceptible indexes. the calculation of spreader and susceptible indexes require access to the local normalized in-degree and out-degree centrality, as well as the incoming and outgoing neighborhood zone-entropy (eqs. ( )-( )) and coreness-entropy (eqs. ( )-( )). note that these three key indicators (local weighted degree, zone-entropy and coreness-entropy) are in the unit interval, i.e. with variations between zero and one. figure shows the local out-and in-degree (left column), the outgoing and incoming neighborhood zone-entropy (central column) and coreness-entropy (right column) of the weekday (first two rows) and weekend (bottom two rows) flow networks. for observation purposes, the subzones were grouped into "low", "midlow", "mid-high" and "high" categories using the %, % and % percentile of each variables as cutoffs. the www.nature.com/scientificreports/ spatial distribution shows notable differences between centrality, zone-entropy and coreness-entropy. in addition, high levels of local weighted out-and in-degree are mostly concentrated in the east, north east, and central regions. as for the zone-entropy, these high levels are primarily located in the north and central regions, while high levels of coreness-entropy are mostly found in subzones in the north region. essentially, most of the subzones have high levels of one, two or even three of these key indicators. however, only subzones with high levels of all three indicators are ssp or sss. the distribution of the spreader index (spi) and susceptible index (sui) of each subzone for weekdays and weekends are shown in fig. . all four distributions suggest a similar poisson-like type of distribution, with a mean value between . and . (solid vertical lines). a detailed comparison between the two indexes and the three components is given in fig. s (see supplementary material). the fact that these mean values are very close for both indexes on weekdays and weekends is in line with our previous comment related to an expected balance between incoming and outgoing flows of human movement. however, for our analysis the locations of interest are those that are outliers corresponding to large spi and/or sui values. using the interquartile range (iqr) method, the outliers are identified as the subzones located above the q + . www.nature.com/scientificreports/ quartile ( q ) is also shown in fig. as a reference level (dotted vertical lines). the subzones that lay between q and q + . × iqr are categorized as secondary-spreaders or secondary-susceptibles. for comparison purposes, we tested the arithmetic average of the three components and compared it with the geometric average results (fig. s in the supplementary material); we also present fig. s (see supplementary material) to highlight the differences between the spi or sui, and the geometric average of either two of the three components (degree, zone-entropy, and coreness-entropy). this analysis reveals that a non-negligible number of locations exhibit large spi and/or sui values, thereby contributing to our identification process of spatial super spreaders and spatial super susceptibles. comparison with population density. the spatial distribution of population can naturally be considered to be key to understanding the spreadability and susceptibility of a given place. indeed, places with higher population density could be suspected to have higher spreadability of and susceptibility to an infectious disease. to test this hypothesis, we compare the subzones' spi and sui with the residential population density (see fig. ). the low correlation coefficients (including the pearson coefficient and the kendall tau are below . ) indicate that the spi and sui are not correlated with population density. interestingly, some low population figure . distribution of core/non-core areas from the weighted k-shell decomposition. the coreness in (a) refers to weekday flow data, while in (b) it is for weekend flow data. red-colored areas are for subzones identified as both incoming and outgoing core areas, purple-colored areas refer to solely outgoing core subzones, and pink-colored subzones highlight solely incoming core subzones. generated with python ( . . ), matplotlib www.nature.com/scientificreports/ density subzones have a high spi or sui. these subzones may be categorized as business or commercial land use areas, hence the low or zero residential population. on the other hand, some of the high residential population density subzones (i.e. above , ) have a low spi or sui. this may because their public transport flow structure is relatively simple, i.e. the outgoing or incoming flows are connecting with places with similar features (in the same zone or with the same coreness level). since residential population distribution represents the spatial patterns of where people live, it is only one type of the destination of population movements, and it therefore lacks the capability to capture places where people work and interact with each other, e.g. commercial area, business area and transport hubs. susceptibility vs. spreadability. while both spi and sui are calculated based on the same spatial flow network, they capture fundamentally different concepts in relation with disease spreading interactions as compared to classical influential node concept in network science . the key differences between the calculation of spi and sui is the flow direction, i.e. incoming flow to a node or outgoing flow from a node. in fig. , we compare the outgoing and incoming measurements for the three components (weighted degree, zone entropy, and coreness entropy), and the two indexes (spi and sui). strong correlations exist between the normalized weighted in-and out-degree (nw in-degree and nw out-degree, in fig. a for weekdays and (e) for weekends, both correlation coefficients are above . ). this is expected as we consider daily average flows to compute the weighted in-and out-degree, i.e. the people who leave their home area in the morning will eventually go back to their home area at some point during the day. differences between the zone entropy of incoming and outgoing neighbors can be observed in fig. b ,f (for weekdays and weekends, respectively, with pearson coefficient figure . spatial distribution of the three key indicators: weighted degree, zone-entropy and coreness-entropy. left column: local weighted in-and out-degrees; central column: outgoing or incoming zone-entropy; right column: outgoing or incoming coreness-entropy. first two rows: weekdays; bottom two rows: weekends. the subzones are separated into four groups using the %, % and % percentile as cutoffs, thereby giving the "low", "mid-low", "mid-high" and "high" categories. generated with python ( . . ), matplotlib ( . . ) and geopandas ( . . ). (fig. d for weekdays and (h) for weekends) show the relationship between sui (horizontal axis) and spi (vertical axis). since both indexes are geometric averages of the previous three directed components, in overall it shows correlated patterns because of the degree and zone-entropy, and the subzones with larger indexes (i.e. q ≤ spi ≤ q + . iqr and q ≤ sui ≤ q + . iqr ) tends to scattered within the box that is composed by the dashed ( q ) and dotted ( q + . iqr ) reference lines. this study focuses on the disease spreading process, thus emphasizing the differences between the outgoing and incoming directions. a subzone with high spi indicates that it has a stronger capability to affect other subzones due to the fact that a lot of people are leaving from this subzone, and they are moving to a high variety of places. on the other hand, a subzone with high sui indicates that it is easily affected by other subzones owing to an intense flow of people coming from a large variety of places. figure shows that the number of people leaving from a subzone is highly correlated to the number of people going to the subzone; but the diversity in terms of zone-and coreness-entropy shows differences between the subzones' incoming and outgoing neighbors. for instance, the coreness-entropy of a subzone's incoming neighbor can be high while its outgoing neighbor's coreness-entropy is low. although the correlation between spi and sui is high, we can still observe some deviations between them, especially beyond q and below q + . iqr. and super-susceptible (sss) is shown in fig. for weekdays and in fig. for weekends. for weekday flow movement (see fig. ), subzones are identified as ssp (red-colored zones in fig. a ) corresponding to www.nature.com/scientificreports/ spi ≥ q + . × iqr ; subzones are identified as sss (red-colored zones in fig. b ) corresponding to sui ≥ q + . × iqr . it is worth noting that subzones overlap in both figures, thereby corresponding to both spatial super-spreaders and super-susceptibles (subzones a-i in both figures, shown as red-colored subzones with a purple border). this indicates that most of the subzones with the highest spi values would also have the highest sui values, and vice versa. in fig. a , all identified ssp are also identified as sss. in fig. b , two subzones-j khatib, and k tampines east-are identified as sss only, with a lower spi ( q ≤ spi < q + . × iqr). the weekend distributions exhibit slightly different patterns. there are subzones identified as ssp on weekends, with of them also being identified as ssp on weekdays (subzones a to h in fig. a) ; none of which are less than q in the previous figure. similarly, all weekend sss are either super-or secondary-susceptibles on weekdays, and vice versa. a total of sss are found with the weekend human movement network (fig. b) ; of them (subzones a to i) are also weekend ssp; of which overlap with those of the weekday sss results, the other two subzones-j boulevard, and k bukit batok central-are promoted from weekday secondary-susceptibles subzones (pink subzones in fig. b ). this result further confirms that the spi and sui are not dramatically different between weekdays and weekends. there are eight subzones (a to i except h in fig. ), and (a to h in fig. ) , including three at the west region (choa chu kang central, jurong gateway, and jurong west central), two at the central region (maritime square and toa payoh central), and three at the north region (sembawang central, woodlands regional centre and yishun west) are identified as both ssp and sss (in red) in both weekdays and weekends. during weekdays, most of the identified ssp or sss areas belong to the regional core that contained a higher density of human activity. the eight ssp and sss can be separated into two types. the first type consists of five subzones (a, c, e, f, and i in fig. ), which contain high population density; the second type consists of the other three subzones (b jurong gateway, d maritime square, and h woodlands regional centre in fig. ) associated with a lower population density. the subzones in the first type are typical residential area, where the intensity of human activity are high due to the extensive need to travel out during the day time and travel back in the evening. on the other hand, the subzones in the second type are regional hubs of public transportation, which naturally attract a large population flows. for example, maritime square contains harbourfront mrt, which is the terminal station of two one counter-intuitive observation can be made from figs. and : the cbd contains less ssp and sss as one could expect. the cbd of singapore is located at the southern central part of the central region. high intensity of human activity exists within the cbd area. as shown in fig. , most of the subzones in the cbd have either a low weighted degree or a low neighborhood coreness-entropy. the low weighted degree probably finds its origin in the smallness of the area itself, which limits the catchment of incoming and outgoing flows. as for the low coreness-entropy, we trace it to the fact that a majority of the people are circulating within the cbd, which are mainly composed by the core area (fig. ). this result indicates that the cbd workplaces are less influential in terms of quickly spreading the disease to the rest of the city/island, but a contagious disease would quickly spread inside the cbd area as a consequence of its strong internal flows. in summary, the key influential areas are clearly identified as being the regional transport hubs, which connect the residential areas with the rest of the country. the concept of super-spreader was originally introduced in the field of social network analysis to identify the most influential persons or nodes within a given social network. these persons could be opinion leaders, trend setters, public figures within a group of people , . furthermore, this concept of super-spreader individual has been borrowed by epidemiologists to identify and study the abnormally high spreading activity of a small group of individuals , in large populations during an epidemic outbreak. while previous studies focused on the identification of super-spreaders within a social network-nodes are individuals and edges represent the existence of interactions between two persons (binary edge)-this study focused instead on spatial networks of population flow with nodes representing physical locations and weighted/ directed edges representing flows of human movement. this study sought to extend the concept of super-spreader to spatial interaction networks, with the objective of identifying possible spatial super-spreader locations-a set of locations that have the most influential effects in terms of disease spreading. the concept and calculation method were also reversed to uncover another group of critical locations: the most vulnerable places defined as super-susceptibles. our results based on large-scale data analytics show that most of the ssp are also sss. this is reasonable and somehow expected given the nature of the daily population flow network. specifically, since we are considering daily-aggregated data, the number of people who are leaving from a place can be expected to be of the same order as the number of people who are going to this place, i.e. we are in the presence of balanced commuting flows and the larger the outgoing flow intensity, the larger the incoming flow intensity. based on the results, the places with intense flows have higher potential to be both ssp and sss, and this is captured by the directed nature of the www.nature.com/scientificreports/ networks and the incorporation of the weighted in-degree or out-degree in our calculations. it is worth noting that our results are in good agreement with previous studies based on the k-shell decomposition method: the core nodes of a social group tend to be, in general, the most influential ones , . besides the local incoming and outgoing flow intensities, this study also considers two critical neighborhood diversities of these networks: the zone-entropy and coreness-entropy. the diversity of neighborhood is especially important while identifying multiple super-spreaders from a network , . the zone-entropy is used to measure if the outgoing flows are directed towards more zones within the city-state. for instance, if the outgoing flows from a given place are converging to one zone only, this place can only affect one of the zones among all throughout singapore, thus its influential power is clearly weak. conversely, if human movement originating from one place flows to many zones across the country, its influential power is relatively high. in addition, coreness-entropy captures the diversity of flows to or from core or periphery areas. if the flows are all directed towards one of the periphery or core, its influential power is somehow limited to this particular type of areas. conversely, if human movement flows to both core and periphery areas, this clearly indicates that whenever an outbreak happens at this place, it could quickly affect and spread to both core and periphery areas. these two diversity metrics complement one another and are combined in the calculation framework for differentiating places with high density of flows into strong and weak influential places (see materials & methods) . this study enables us to establish a list of subzones, which have a strong capability in terms of diseases spreading, as well as a list of subzones, which are more vulnerable in terms of being a place of high risk of contagion. in summary, the identified subzones are found to be mainly in the core area of residential and transportation figure . the spatial distribution of (a) spreader index (spi), and (b) susceptible index (sui) for weekdays. the subzones with purple border in (a,b) respectively indicate the super-susceptible ( sui ≥ q + . × iqr ) and super-spreader ( spi ≥ q + . × iqr ). generated with python ( . . ), matplotlib ( . . ) and geopandas ( . . ). | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ hubs. these places have high population density and activity, such as transportation hubs or community hubs. therefore, these places should be targeted by public health agencies, with higher resource allocations and disease monitoring aimed at prevention and intervention purposes. for example, public health agencies could consider these locations while planning to setup body temperature checkpoints, or to provide personal hygiene toolkits, or also setting up advertisements related to appropriate behaviors to counteract the ongoing epidemics. moreover, since these locations are more vulnerable and more influential, they should get more attention while setting up differentiated policies such as the temporary closure of some businesses or restrictions on large-scale human activities as opposed to a blanket lockdown across the country. the proposed network analysis framework rests upon the integration of the local flow intensity with neighborhood diversity measures-zone and coreness-to assess the effective spreading ability of particular locations. from the theoretical perspective, the proposed framework considers weighted and directed interactions between nodes (places) to identify super-spreaders and super-susceptibles. from the practical perspective, this study presents a quantitative and systematic framework to identify the key influential and vulnerable locations based on public transport flow data usually available by most transportation agencies in metropolitan areas. it is worth noting that there are several limitations to this study. first, our analysis is limited to human flow associated with the use of public transportation, which is high in places like singapore or other continental european cities but could be much lower in other urban areas with far less developed public transportation networks, such as in the united states for instance. in addition, our data only includes ridership of buses and trains and misses out on other important means of public transportation, including taxis, private-for-hire automobiles www.nature.com/scientificreports/ (cars, motorcycles, shuttle buses or vans), and active transportation (by walking, bicycle, skateboard, scooter, personal mobility devices, etc.). some of the subzones currently do not have bus stops or train stations. however, as mentioned previously, public transportation by bus and train in singapore is fairly high-more than % of daily commuting-thereby confirming the importance of the obtained results, as being representative of key human movement patterns. second, singapore is an island country with its northern national border connected to malaysia through two land checkpoints. unfortunately, these cross-border flows are not included in this study. many workers and students commute daily between singapore and the state of johor in malaysia. there are some dedicated bus services directly connecting stations in johor bahru, malaysia and various places across singapore, including woodlands at the north region, jurong east at the west region, and bugis at the central region, etc. since these data were ignored, the in/out-flows of these places in singapore are certainly underestimated. third, inter-mode trip transfers and bus transfers are not captured in the dataset used to carry out our study. trip transfers between mass rapid transit (mrt) lines are captured from the tap-in and tap-out records, i.e. passengers changing lines at some interchanges. but the od data for buses only records the direct flow between bus stops, i.e. the records present only the tap-in and tap-out bus information, the records of the exchange of bus services are not shown/captured in the data. on the other hand, the data about changing from bus to train and vice versa is also unfortunately not available. therefore, we can only capture direct bus services and this naturally limits the movement of travelers to the existing direct bus/train services. fourth, the short-time scale dynamics throughout a day is ignored. indeed, we considered daily-aggregated data. however, a higher temporal resolution could be considered (say on an hourly basis), which could reveal different patterns of ssp and sss. the temporal evolution of the sui and spi indexes would be the topic of a future study. on the other hand, a long-term analysis may also provide insights into the evolution of spi and sui or spatial super-spreaders and super-susceptibles over time. the bi-monthly analysis of spi and sui (fig. s -s ) , and identified ssp and sss (fig. s -s ) are given in supplementary material. however, the distribution pattern for the long-term analysis requires a more in depth investigation and discussion owing to a possibly large number of unidentified factors that may affect the overall human movement structure. in summary, we have developed for the first time a framework allowing the identification of spatial superspreader and super-susceptible locations. we believe that our results and analysis could be extended in two key directions. first, our analysis would benefit from being complemented by working with epidemiologists specialized in simulations of disease spreading through human contact networks. this would integrate our results with differential spreading across more or less vulnerable places. specifically, the dynamic patterns of disease propagation could be observed from the simulation models, and thus the effects of the ssp and sss could be quantified in terms of its actual contamination rate in the population. second, the geography, demography, and social-economic of the spatial super-spreaders and super-susceptibles could be accounted for and included in our analysis using some statistical models, to identify the potential social and physical environmental factors that made these locations super-spreaders and super-susceptibles. in conclusion, it is well known that dealing with the reopening of economies and cities after a blanket lockdown requires a finely calibrated approach from governments. although, here we used the singapore public transport flow data to build these networks as a case study, similar analyses can readily be carried out using the exact same process in order to uncover the ssp and sss in any large urban center. our data-driven methodology, analysis and results offer an effective way of devising targeted and localized preventive measures when lifting stay-at-home orders. such targeted measures for vulnerable locations are also critical in order to optimize government resources in the face of economic decline. the datasets-generated from the singapore lta database -used for this study are available from the following spatial_spreader_susceptible_data repository: https ://githu b.com/wcchi n/spati al_sprea der_susce ptibl e_data. coronavirus 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precautionary measures transportation and territorial development in the singapore extended metropolitan region republic of singapore. passenger volume by origin destination bus stops & passenger volume by origin destination train stations general household survey master plan subzone boundary (no sea the map equation a k -shell decomposition method for weighted networks a model of internet topology using k-shell decomposition identifying and ranking influential spreaders in complex networks by neighborhood coreness a computer movie simulating urban growth in the detroit region searching for superspreaders of information in real-world social media the effect of superspreading on epidemic outbreak size distributions this research was supported by an sutd grant (cities sector: pie-sgp-ctrs- ). w.c.b.c. conceived and conducted the experiment and the data analysis. w.c.b.c. and r.b. analyzed the results and wrote the manuscript. all authors reviewed the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to r.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - atfsivf authors: liu, hong yan; gao, xiaohu title: a universal protein tag for delivery of sirna-aptamer chimeras date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: atfsivf sirna-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of sirna due to the modularity of their diblock rna structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. however, additional challenges must be addressed for efficient rna interference (rnai), in particular, endosomal escape. currently, vast majority of sirna delivery vehicles are based on cationic materials, which form complexes with negatively charged sirna. unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsrna binding domain (dsrbd) for sirna docking and a ph-dependent polyhistidine to disrupt endosomal membrane. the protein selectively tags along the sirna block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition. more interestingly, we found that extending the c-terminal polyhistidine segment in the protein tag to amino acids completely abolishes the rna binding function of dsrbd. final large complexes (typically s nanometers) and the chemical composition of the nanocarriers can drastically change chimera's targeting profile, in vivo biodistribution, and clearance . furthermore, it is ideal to make the aptamer loop structure exposed and the sirna block hidden for specific binding, but electrostatic condensation with cationic nanocarriers does not warrant that selectivity. as demonstrated previously, immobilizing sirna-aptamer chimeras onto cationic nanoparticles via the sirna end offers significantly improved silencing effect compared to condensing chimeras onto cationic nanoparticles through random sites . this is understandable since ( ) exposure of the sirna end would only increase the chances of non-specific binding and reduce the stability sirna against enzymatic degradation; and ( ) interaction between cationic nanocarriers with anionic aptamers could alter aptamers' conformation and targeting capability . therefore, it is of critical importance to design a delivery system that is simple for potential regulatory approval and mass production, universal for all sirnaaptamer chimera, neutral and sirna-binding specific to ensure aptamer targeting, and small to avoid major alteration of chimera's biodistribution profile. a system simultaneously achieving these features could expedite clinically translation of the highly promising sirna-aptamer chimera technology. here, we report the development of a small protein tag for efficient delivery of sirna-aptamer chimeras. as shown in figure , the protein tag is composed of two functional domains: a dsrbd used as a sirna docking module and a ph-dependent polyhistidine to help disrupt the endosomal membrane. the dsrbd is the n-terminal region ( kda) of human protein kinase that binds dsrna in a sequence-independent fashion , . because aptamers are typically ssrna with complex secondary structures, dsrbd does not bind with them (dsrbd only tolerates small bulges) and thus will selectively bind chimera through the sirna end, leaving the aptamer end accessible. to add endosomal escape functionality, a short histidine (his) oligomer is added to the c-terminus of the dsrbd. his has been incorporated into a number gene carriers because its endosomal buffering capacity promoting drug cytoplasmic release , . his molecules have a pka value of approximately . at neutral ph (such as in circulation), they are mainly deprotonated (uncharged), which is desirable over positively charged counterparts due to reduced accumulation within the res (reticuloendothelial system). in acidic compartments such as endosome, his becomes protonated and facilitates osmotic swelling that leads to cargo release, a mechanism proposed as the proton sponge effect . overall, this protein tag is equally small, simple, and biodegradable as sirna-aptamer chimera, while perfectly complementing chimera's functionalities. when complexed together, they remain small in size, discrete and stable in solution, low positive charge for circulation, and simultaneously achieve therapeutic, targeting, and endosomal escaping capabilities. expression and characterization of dsbrd-his protein tag. to add endosomal escape capability, a short polyhistidine peptide was added to dsrbd. the dsrbd domain comes from the first amino acids of human protein kinase r (hpkr), and has two double-strand rna binding motifs (dsrbm and dsrbm ) for cooperative and dsrna-specific binding . because dsrbm towards the n terminal dominates the binding with dsrna , we introduced the histidine peptide towards the c terminal ( figure ) to minimize impact on dsrbd's biological activity. in theory, the endosomal escape capability should increase with longer his chain; on the other hand, long his chain could potentially interfere with dsrbd protein folding and binding. to achieve a balance, dsrbd with cterminal histidines of various lengths (his n , n , , , and ) were cloned into the pet a ( ) vector. bamh and xho restriction enzyme sites were introduced to the -and -flanking region by pcr, respectively. because all the genetic constructs contain his at the n-terminal from the cloning vector (this nterminal his has been previously proved to have no impact on dsrbd binding) , the total numbers of his encoded by the final constructs are , , , and , respectively (sequences see methods). post expression and purification, the resulted protein tags were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page, figure a ). the sizes of four protein tags show in excellent agreement with theoretical values (figure ). to assess their dsrna binding activity, sirna-aptamer chimera labeled with fluorophore fam were incubated with the protein tags and probed with gel electrophoresis ( % agarose). as shown in figure b , the dsrna binding capability of dsrbd with his at the c terminus (total his ) is well preserved compared with dsrbd without a cterminus histag insertion. the minimum rna length for high affinity binding with dsrbd has been determined to be base-pairs . at the current rna length, the sirna segment and the adjacent short stem in the aptamer structure can bind with - copies of dsrbd. however, it has been well documented that only the first dsrbd binds to rna stably, while, at high dsrbd/rna ratio, a second copy of dsrbd can bind, but at significantly lower affinity , . using unmodified dsrbd and sirna alone, similar dsrbd-sirna binding profiles have been observed previously by kim and coworkers, who also show that the enzymatic stability of sirna is significantly enhanced upon binding with dsrbd . it is important to mention that a key difference of our technology compared to these prior works utilizing dsrbd for sirna , is that we do not introduce highly positively charged peptides. although positively charged nanocarriers promote sirna cell entry, it is well known that they are also quickly cleared by the res, increase non-specific binding with cells and cytotoxicity . furthermore, as aforementioned, avoiding positive charges in carrier design is particularly important for sirna-aptamer chimera because excessive positive charges could non-specifically interact with aptamer and affect its targeting capability. more interestingly, the gel electrophoresis experiments also reveal that extending the c-terminal his by another or amino acids completely abolish dsrbd's binding activity. therefore, for the following gene expression regulation studies we chose the dsrbd with a total of his due to its balanced dsrna binding and endosomal escape functionalities, in comparison with the original dsrbd with no c-terminus his as a control. design, synthesis, and characterization of sirna-aptamer chimera. to evaluate the universal protein tag for sirna-aptamer chimera, we first designed and made a chimera based on the protocols described by dassie and coworkers, taking advantage of the shortened aptamer sequence for specific targeting of psma as well as the optimized sirna strands with enhanced therapeutic potency . the psma targeting aptamer was kept in our chimera, because psma has been identified as one of the most attractive cell surface markers for both prostate epithelial cells and neovascular endothelial cells . accumulation and retention of psma targeting probes at the site of tumor growth is the basis of radioimmunoscintigraphic scanning (e.g., prostascint scan) and targeted therapy for human prostate cancer metastasis. we replaced their sirna sequence with a sirna silencing gfp expression, because gfp is the best model for quantitative assessment of the silencing effect using optical imaging and flow cytometry. the long ssrna composed of psma aptamer and sirna antisense strand ( figure ) was prepared by in vitro transcription with the presence of fluoro-modified pyrimidies for improved resistance to ribonucleases. it has been shown previously that -f modification is compatible with dsrbd binding unlike -h or -och substitutes , . the transcript was annealed to chemically synthesized sirna sense strand. before combining the chimera with our small protein tag, we first tested the activities of the chimera. to test the targeting function of the aptamer block, psma-positive lncap and psma-negative pc prostate tumor cells were incubated with dye-labeled chimera. as shown in figure c , the chimera selectively binds and enters lncap cells indicating targeting specificity. to test the silencing effect separately, the chimera was transfected into gfp-expressing c - prostate tumor cells (a derivative of lncap) using conventional transfection agents, lipofectamine. as shown in figure d , the silencing effect is indistinguishable with the positive control using sirna only, proving that chimera can be enzymatically processed intracellularly to generate functional sirna. targeting delivery and silencing in cells. with the biological activities of our protein tag and sirna-aptamer chimera separately characterized, we proceeded to evaluate the gene silencing effect of this simple yet functionally highly complementary protein tag in sirna-aptamer chimera delivery. gfp-expressing c - cell line was used as a model because of the advantages of fluorescence imaging techniques such as microscopy and quantitative flow cytometry. figure a -f shows confocal images of the c - cells without treatment, treated with gfp-sirna alone, chimera alone, a random sequenced sirna with the protein tag (his ), chimera with protein tag (his ), and chimera with protein tag (his ). qualitatively, only the experimental treatment, chimera with protein tag (his ), clearly shows gfp silencing, whereas none of the five control treatments leads to significant suppression of gfp expression. quantitative flow cytometry studies further confirm this result (figure g-l) . at the current gate value set for gfp fluorescence intensity, the original untreated cells showed a gfp-negative population of . %. treating the cells with a random sequenced sirna with protein tag (his ) shows virtually no change in this population (difference: . % of total cell population, within error range) proving sequence-specific silencing of rnai. for cells treated with gfp sirna and chimera, the gfp negative cells only increase by . % and . % of the total cell population respectively. even by increasing the chimera concentration by ten times ( mm), the total gfpnegative cell population only increase by , % (supplementary figure s ) , strongly suggesting the need of carrier materials. direct comparison of the chimera tagged by dsrbd-his and dsrbd-his shows major difference in silencing efficiency, too ( . % and . % change). taken together, these results clearly indicate that ( ) chimera alone at concentration commonly used in rnai experiments does not lead to effective silencing, and ( ) his is remarkably more effective than his in endosomal destabilization since the dsrbd block is identical in structure and function. to put the silencing efficiency of dsrbd-his in the context of those of conventional rna delivery vehicles such as lipofectamine, quantitative flow cytometry was also conducted. in agreement with the microscopy results shown in figure d , lipofectamine reduces gfp-negative cells from the original . % to . % ( . % change, supplementary figure s ), which is slightly more efficient than the protein tag. however, it is important to note that lipofectamine delivers chimera into cells mainly via electrostatic interactions (positively charged lipofectamine and negatively charged cell surface, non-targeted delivery), whereas our protein tag delivers chimera by cell type-specific molecular recognition (targeted delivery). it is also worth mentioning that the molar ratio of mixing chimera with protein tag is because the sirna block can bind up to copies of dsrbd, although the second copy has very weak binding affinity. indeed, changing the binding ratio to or does not affect the rnai efficiency (supplementary figure s ) . to further confirm the difference in endosomal escape capability between the two protein tags (dsrbd-his and dsrbd-his ), we performed a dual color imaging assay using non-fluorescence lncap cells. in this experiment, chimera was labeled with cy and endosome/lysosome was marked with a lysotracker (spectrally distinguishable green fluorescence). direct contrast in chimera distribution and intracellular density of endosome/lysosome was observed between the two protein tags. as shown in figure , cy labeled chimera evenly distributes inside cells when tagged by dsrbd-his , whereas dsrbd-his treated cells show much higher density of endosomes and lysosomes and lower level of cy fluorescence. this confocal imaging comparison directly explains the difference between the two protein tags in rnai efficiency, and unambiguously demonstrates the superior endosome escape capability of dsrbd-his over dsrbd-his . cytotoxicity. lastly, we probed the cytotoxicity of the best performing protein tag dsrbd-his using a standard cell viability assay (celltiter-blueh). the assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin). nonviable cells lose metabolic capacity and thus do not generate fluorescent signals. as illustrated in figure , virtually no toxicity was detected up to a concentration four times as high as the one used in the delivery work in reference to the untreated control. this is perhaps not too surprising due to the biocompatibility of dsrbd, a small protein of human origin. more importantly, for future in vivo applications, we envision that the small protein tag would have improved clearance capability compared with synthetic polymers and inorganic nanoparticles used for sirna delivery. discussion sirna-aptamer chimera is one of the most promising approaches for cell type-specific rnai, owing to its low immunogenicity, ease of chemical synthesis and modification, small size, and the modularity of both the targeting aptamer block and the therapeutic sirna segment. more importantly, employing only rna molecules, the simple formulation of chimera-based targeted sirna therapy leads to outstanding clinical translation , . due to the incapability of chimera to efficiently escape endosome, delivery nanocarriers are needed. however, almost all current targeted sirna delivery formulations involve cationic nanocarriers such as polymers, inorganic nanoparticles, peptides, and proteins , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . unfortunately, these conventional sirna nanocarriers are unsuitable for chimera delivery, and, in fact, reverse the signature property of chimera, simple formulation for regulatory approval and clinical translation , . this is because the charge induced complex formation is basically an aggregation process, which lacks control over aggregate size, shape, stoichiometry, chimera orientation, aptamer functionality, and reproducibility during scale-up production. in addition, the final complexes often carriers positive charges as well, which is unfavorable for systemic uses . as a result, first clinical trials of sirna duplexes are mainly limited to local administrations [ ] [ ] [ ] [ ] . our protein tag does not rely on high positive charge to interact with rna molecules. in fact, it only recognizes relatively long dsrnas (. bp) such as the sirna segment and the short stem region of the aptamer in our chimera molecule. extensive biochemistry investigations have shown that for the current length of the chimera, maximum two copies of dsrbd can bind to it with differential affinity (the first copy binds much stronger than the second copy). the gene silencing experiments conducted here reflect this effect since mixing chimera with or protein tags does not affect the silencing efficiency. considering the molecular weights of the chimera ( . kda) and the protein tag ( . kda), molecular weight of the final complex at binding will become . kda. based on well-documented size effect for in vivo drug delivery , this size is sufficiently large to reduce premature renal clearance while still small enough for deep tissue penetration. for example, by tagging sirna-aptamer chimera with a kda peg, its in vivo circulating half-life has been shown to increase from approximately min to hours ; whereas large nanoparticles (. nm) have been shown to be ineffective in tumor treatment except for some hyperpermeable tumors . in conclusion, to solve the endosome escape problem of the highly promising sirna-aptamer chimera based therapy, we have designed a dual-block small protein by combining dsrbd and polyhistidine and identified the optimal length of polyhistidine. the resulted protein tag shares the simplicity feature of sirna-aptamer chimera, yet offers exactly complementary functionalities. the dsrbd selectively binds to the sirna block, leaving the targeting aptamer accessible. in terms of size, different from conventional cationic delivery vehicles, the dsrbd-his tagged chimera remains discrete in solution rather than forming large aggregates. in terms of functionalities, chimera and dsrbd-his are highly complementary to each other, and thus offer the complete set of features necessary for targeted sirna delivery (e.g., targeting, therapeutic, sirna protection, and endosomal escape). this platform is also universal, able to chaperone any chimera sequences for cell type-specific delivery. largely based on natural proteins, dsrbd-his is an excellent candidate for potential clinical translation because of its simple structure and biodegradability. further development of this small protein tag with in vivo testing should raise exciting opportunities for sirna clinical translation and personalized medicine. materials. vendors for specific chemicals are listed below. in general, restriction enzymes were obtained from new england biolabs, and cell culture products were purchased from gibco/invitrogen. chimera composed of aptamer targeting psma and sirna targeting gfp. ssdna of the psma aptamer ( nucleotides, -gggaggacgatgcggatca-gccatgtttacgtcactcct- ) was chemically synthesized by integrated dna technologies (idt) and used as the template to generate one strand of the sirna-aptamer chimera. for amplification, pcr was performed with primer containing the anti-sense strand of gfp sirna (underlined) and primer containing t rna polymerase promoter site (bolded). the pcr primer sequences are: primer: -ggcaagctgaccctgaagttcttttaggagtgacgta-aac- primer: -taatacgactcactatagggaggacgatgcgg- the bp pcr product was put into t-a cloning pcr . vector (invitrogen). after sequencing, positive plasmids were selected and used as the template for pcr. the resulting pcr product was separated with % agarose gel and recovered with qiaex ii gel extraction kit (qiagen). the purified pcr product was used as the template for in vitro transcription with megascriptt kit (ambion) according to manufacturer's instruction. fluoro-modified pyrimidines (trilink, san diego) were added to replace ctp and utp. rna molecules generated by the transcription reaction were annealed with the sense strand of gfp sirna (chemically synthesized with or without -cy or fam by idt). the sequence is -(cy or fam)-caagcugacccugaaguucuu- . for annealing, the transcripted rna and the synthetic sirna sense strand were mixed at molar ratio in duplex buffer (idt) and incubated at uc for min followed by slow cooling to uc in hour. the final chimera was store at uc. the constructs were cloned into pet a ( ) expression vector (novagen). the constructs for dsrbd-his and dsrbd-his were obtained using full-length pkr gene (clone id ) as pcr template, and the dsrbd-his and dsrbd-his constructs were made by grafting additional histidines to the dsrbd-his plasmid using pcr. the restriction enzyme sites for bamh and xho were introduced in the pcr primers for cloning. dsrbd-his construct was introduced with two stop codons (taa and tga) before the xho site. for the other three constructs, the reading frames cover the his sequence in the vector at the c-terminal end before the stop codon. the pcr products and pet a ( ) single colonies were selected and grown at uc for h in circlegrow medium containing mg/ml kanamycin. overnight cultures were diluted at (v/v) into fresh medium and incubated at uc until the od values reach . - . . expression was induced by addition of isopropyl-b-d-thiogalactopyranoside (iptg, mm), and cell growth was continued for another - hour at uc. cells were harvested by centrifugation (beckman ja- rotor) at , g for min and stored at uc. cells were suspended in bug-buster mix (novagen) with ml reagent per gram of wet cell paste. bug buster mix was added with protease inhibitor edta-free cocktail (pierce), % glycerol, and . mm thp (novagen). the cell suspensions were incubated on a shaker platform for min at room temperature. insoluble cell debris was removed by centrifugation (beckman tl ) at , g for min at uc. the soluble extracts were loaded onto affinity columns with ni-charged his bind resin (novagen). following washing with binding buffer and washing buffer, the desired proteins were eluted with volume elution buffer (novagen). the eluted proteins were dialyzed with pbs containing % glycerol and . % (v/v) b-mercaptoethanol for hours. purified proteins were probed using % sds-page and stained with coomassie brilliant blue g- (bio-rad). protein concentrations were determined with the bio-rad protein assay with bovine serum albumin as the standard. functional characterization of sirna-aptamer chimera. to test the functionality of the sirna block, the chimera described above and gfp sirna control (qiagen) at a final concentration of nm were transfected into c - prostate cancer cells stably expressing gfp using lipofectamine rnai max (invitrogen) following the instructions provided by the manufacturer. to evaluate the targeting specificity of the aptamer block, psma-positive lncap cells and psma-negative pc cells were treated with complex of chimera and dsrbd-his (chimera/protein tag molar ratio at , nm chimera) in serum free medium for hours, followed by incubation in complete medium for another h. dapi ( nm) was added to stain cell nuclei. fluorescent images were captured on an olympus ix- inverted microscope equipped with long-pass filters and a colored ccd camera. characterization of rna binding capability of the four protein tags. the binding capabilities of the four polyhistidine modified dsrbd proteins were evaluated by native agarose gel. the chimera was labeled with fam at the end of sirna's sense strand (idt). to prepare chimera/dsrbd complex, chimera ( mm, ml) was incubated with the protein tags at protein/chimera molar ratios of , , or for h at uc. bound chimera and unbound chimera were quantified on % agarose gel using a macro imaging system (lightools research, ca). evaluation of endosomal escape. psma-expressing lncap cells were seeded on mm glass-bottom petri dishes (matteck corp) at a density of cells/well for hours in rpmi supplemented with % fcs. complexes of chimera labeled with cy (idt) and protein tags (his and his ) were added to lncap cells in serum-free medium for hours, followed by incubation in complete medium for hours. lysotrackerh green dnd- ( nm, invitrogen) was then added for hours at uc. images were captured on a confocal laser scanning microscope (lsm , carl zeiss, germany). microscopy and flow cytometry studies of gene knockdown efficacy. c - prostate cancer cells expressing gfp were seeded into mm glass-bottom petri dishes for confocal imaging or -well plates for flow cytometry. cells were treated with chimera & dsrbd-his and compared with five control groups including no treatment, treated with gfp-sirna alone, chimera alone, a random sequenced sirna with the protein tag (his ), and chimera with protein tag (his ) for h in serum free media and then incubated in complete media for h. confocal images were again obtained with lsm confocal microscope equipped with argon ( nm) and hene ( nm) lasers; and quantitative flow cytometry investigation was done on a bd facscantoii flow cytometer. cytotoxicity assay. lncap cells were seeded in -well plate at /well for hours, and then treated with different concentrations of dsrbd-his protein tag for hours. celltiter-blue reagent ( ml) was added into each well. after h incubation at uc, cell viability was assessed by fluorescence intensity at nm (excitation nm) on a tecan infinite m microplate reader. killing the messenger: short rnas that silence gene expression approaches for the sequence-specific knockdown of mrna knocking down barriers: advances in sirna delivery visualizing a correlation between sirna localization, cellular uptake, and rnai in living cells sirna conjugate delivery systems lung delivery studies using sirna conjugated to tat( - ) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity efficient in vivo delivery of sirna to the liver by conjugation of 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chitosan/sirna nanoparticle formulation and gene silencing quantum dot-amphipol nanocomplex for intracellular delivery and real-time imaging of sirna emerging application of quantum dots for drug delivery and therapy quantum dots as a platform for nanoparticle drug delivery vehicle design inhibition of respiratory viruses by nasally administered sirna evaluation of the safety, tolerability and pharmacokinetics of aln-rsv , a novel rnai antiviral therapeutic directed against respiratory syncytial virus (rsv) using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque small interfering rna (sirna) targeting vegf effectively inhibits ocular neovascularization in a mouse model clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats accumulation of sub- nm polymeric micelles in poorly permeable tumours depends on size this work was supported in part by nih (r ca , ca ), the department of bioengineering, and the office of research at university of washington. we are also grateful to drs. eva corey and bob vessella for their prostate tumor cell lines and fruitful discussions. key: cord- -ko y m s authors: scalabrin, matteo; frasson, ilaria; ruggiero, emanuela; perrone, rosalba; tosoni, elena; lago, sara; tassinari, martina; palù, giorgio; richter, sara n. title: the cellular protein hnrnp a /b enhances hiv- transcription by unfolding ltr promoter g-quadruplexes date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ko y m s g-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. a dynamic g-quadruplex forming region in the hiv- ltr promoter represses hiv- transcription when in the folded conformation. this activity is enhanced by nucleolin, which induces and stabilizes the hiv- ltr g-quadruplexes. in this work by a combined pull-down/mass spectrometry approach, we consistently found hnrnp a /b as an additional ltr-g-quadruplex interacting protein. surface plasmon resonance confirmed g-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnrnp a /b is able to efficiently unfold the ltr g-quadruplexes. evaluation of the thermal stability of the ltr g-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the ltr full-length environment. when hnrnp a /b was silenced in cells, ltr activity decreased, indicating that the protein acts as a hiv- transcription activator. our data highlight a tightly regulated control of transcription based on g-quadruplex folding/unfolding, which depends on interacting cellular proteins. these findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated hiv- , present both in actively and latently infected cells. . (a) ltr-ii + iii + iv sequence of the g-rich ltr region comprising tracts that fold into three mutually exclusive ltr g s, i.e. ltr-ii, ltr-iii and ltr-iv. these three-stacked tetrads g s are indicated in brackets. g bases in g-tracts or involved in g-quadruplexes are shown in bold. (b) ms/ms spectrum of the precursor ion observed at m/z . in the sample mixture from digestion of ltr-ii + iii + iv g sample (see table ). only characteristic y ions are indicated . the data match the sequence of peptide n -r of hnrnp a /b , which is reported on top with the observed fragments. c) pull-down assay of nuclear extract proteins with wt, mutant g ltr-ii + iii + iv (m) and random (r) sequences, immobilized on agarose beads. shown is the western blot analysis with an anti-hnrnp a /b antibody. proteins complexed to the beadsbound ltrs were washed with augmented stringency by increasing the ionic strength of the wash buffer ( . and m). the final elution was obtained in denaturing buffer at °c. scientific reports | : | doi: . /srep mutant ltr-ii + iii + iv m + was bound with a lower affinity (k d . ± . nm). chi and u-values were < and < , respectively, indicating optimal data fitting , . in contrast, binding to a random oligonucleotide of the same length as ltr-ii + iii + iv was so low that in these conditions it was not possible to obtain a meaningful k d value (fig. ). hnrnp a unfolds the ltr g s. we next set out to investigate the effect of hnrnp a binding to the ltr g s. a taq polymerase stop assay was performed on the ltr-ii + iii + iv template. in the presence of mm k + , stop sites corresponding to formation of ltr-iii and ltr-ii were visible (compare lanes and , fig. a ). no stop corresponding to ltr-iv was detected, as expected, since ltr-iv has been previously reported to form upon induction by g ligands . upon addition of hnrnp a , both ltr-iii and ltr-ii stop sites showed an insignificant decrease (compare lanes and , fig. a and b) . similarly, no effect was induced by addition of k + and hnrnp a in the control ltr-ii + iii + iv m + template lacking the possibility to form g (lanes - , fig. a) . as suggested by xodo , the apparent lack of effect in the presence of an unfolding protein could be due to the protein binding to the template sequence which would stimulate polymerase stop and mask g release at the same binding site. we thus switched to a dual-labelled system where oligonucleotide folding could be monitored by changes in fluorescence. fluorescence resonance energy transfer (fret) is a spectroscopic technique that provides information about structure and dynamics of nucleic acids folding. it involves a donor fluorophore in an excited state, the excitation energy of which can be transferred to a proximal acceptor chromophore. given that the major determinant of fret efficiency is the distance between the acceptor and the donor, fluorescence intensities depict nucleic acids folding states. consequently, when annealed to its complementary sequence to form a double-stranded structure, the tested oligonucleotide would yield the maximum fluorescence intensity, while the g folded conformation would be the least fluorescent. in these conditions the measured fluorescence intensity allows to calculate the energy transfer (e) and the end-to-end distance (r) between the two fluorophores, and therefore the unfolding degree. the g folded ltr-ii + iii + iv sequence was characterized by r of ~ Ǻ, with e ~ . . when the g structure was converted into the duplex conformation by addition of the complementary c-rich strand, the fluorophores were separated by ~ Ǻ, and fret was approximately . treatment of ltr-ii + iii + iv g with hnrnp a increased fluorescence by -folds with respect to the free g , and e decreased to . ( fig. a and b, supplementary table s ). considering that the complete unfolding of the g structure required Δ e = . , the Δ e = . induced by hnrnp a reflected a % unfolding (fig. c) . the negative control protein bsa showed negligible unfolding activity in these conditions ( fig. c and supplementary table s ). to test the statistical significance of the effect observed in the presence of the protein, we applied a model comparison approach, which allows to test the statistical role of an independent variable (in our case the hnrnp a protein) through the comparison of two models differing only in that variable. the test yielded f ( , ) = . and p < . , indicating that the difference observed in the presence and absence of the protein was statistically significant. analysis of the activity of hnrnp a was extended to shorter ltr sequences, i.e. ltr-iii + iv, ltr-iii and ltr-iv g s, folded in mm k + . the unfolding of ltr-iii + iv and ltr-iv was similar but lower than that on the full-length sequence ( %); unfolding of ltr-iii was very low ( . %) (fig. c is the probability that the observed match is not a random event. the score is reported as − x log (p) where p is the absolute probability. the mass of the putative peptides and of the most intense fragment ions were used for the data base search. significant mascot hits were accepted as positive matches and their ion score reported. the highest score obtained in the three experiments is reported. unfolding up to % was obtained in the ltr-ii + iii + iv oligonucleotide (fig. c , supplementary table s and supplementary fig. s ). because g s are less stable at lower k + concentrations, we proposed that the diverse unfolding efficiency towards the different-length ltr g s depended on the stability of the ltr g s. to test this hypothesis, melting temperatures (t m ) of the dual-labelled oligonucleotides were thus measured by fret-monitored thermal unfolding ( table ). the most stable sequence was ltr-iii (t m . ± . °c), followed by ltr-iv and ltr-iii + iv (t m . ± . °c and . ± . °c, respectively); ltr-ii + iii + iv was the least stable sequence (t m . ± . ), confirming that the observed unfolding scale depended on the oligonucleotide stability in the fret system. to note that in the unlabelled oligonucleotides measured by circular dichroism (cd), ltr-iv was the least stable sequence (table ) , bias which likely derives by the absence of fluorophores at the oligonucleotides' ′ -and ′ -end, which affect t m values, as previously noted . in addition, the longest sequences, i.e. ltr-ii + iii + iv and ltr-iii + iv, where multiple g s may form, display t m values that represent the average stability, which depends on the effective formation of the possible g s. we thus performed a taq polymerase stop assay to assess the g mutual formation and stability at increasing k + concentration in the full-length ltr. ltr-iii was used as a control sequence where only one g could form. we observed that in the full-length ltr-ii + iii + iv sequence, both ltr-ii and ltr-iii g s formed upon addition of k + (lanes - , fig. a ); of these, ltr-iii g induced a slightly more intense stop than ltr-ii, suggesting a preferred formation of ltr-iii in the full-length sequence (fig. b) . ltr-iii was also the only g forming in the ltr-iii + iv template (lanes - , fig. a ). interestingly, the ltr-iii that formed in the full-length sequence was less intense than ltr-iii forming in the shortest oligonucleotides (compare lanes - , with - and - , fig. a and b). these data indicate that i) ltr-iii is the most prominent g among the mutually exclusive ltr g s and ii) when ltr-iii forms in the full-length ltr promoter, its stability maintains a level that is fully susceptible to hnrnp a processing. we have shown that ltr g folding inhibits viral transcription, whereas when point mutations that disrupt g s are introduced in the ltr promoter, an increase in promoter activity is evidenced . silencing of the ltr g folding/stabilizing protein nucleolin significantly increased promoter activity, indicating the inhibitory effect of nucleolin on ltr-driven transcription . to assess the effect of hnrnp a /b binding/unfolding of the full-length ltr g s, a set of anti-hnrnp a /b sirnas was used, which effectively silenced hnrnp a /b expression in t cells (fig. a) . in these conditions, ltr-driven transcription decreased by % at the highest sirna concentration in the wt ltr sequence (fig. b) . in contrast, the ltr sequence with two point mutations that disrupt g folding was only marginally affected by hnrnp a /b depletion (fig. b) , indicating that the effect was g -specific. in these conditions, hnrnp a / b -silenced t cells showed no reduction in cell viability at h with respect to the controls, thus confirming the consistency of the inhibitory effects on ltr promoter activity. silencing of hnrnp a /b was also performed in the tzm-bl reporter cell line, which contains stably integrated copies of the luciferase gene under control of the ltr wt promoter and thus closely resembles the integrated provirus. the ltr-driven reporter transcription decreased up to % in hnrnp a /b -silenced cells ( supplementary fig. s ). these data further confirm the unfolding activity of hnrnp a /b on the ltr g s. we have shown that hnrnp a /b selectively binds the hiv- ltr g s and unfolds them. while hnrnps are rna/protein complexes that bind newly synthesized rna (pre-mrna) in the cell nucleus during gene transcription and post-transcriptional modifications , two of the most abundant of them, hnrnp a and hnrnp a /b , have been reported to bind also dna in the quadruplex conformation. initially hnrnp a was found to unfold the telomeric g s and promote telomerase activity [ ] [ ] [ ] , to unfold the g in the promoter of the kras oncogene and regulate transcription and to destabilize the tetraplex forms of the fragile x expanded sequence d(cgg)n . hnrnp a has been shown to unfold the fragile x repeats and the shorter hnrnp a splice variant to promote telomere extension in mammalian cells . our finding that hnrnp a binds and unfolds the g s in the promoter of hiv- not only confirms the dna g unfolding activity of this protein, but also indicates for the first time that this type of activity is exploited by a virus. in hiv- , hnrnp a /b has been reported to play with rev an important accessory role in promoting nuclear viral rna retention and nucleocytoplasmic viral rna transport . in this context, the lower virus production observed upon sirna-mediated depletion of hnrnp a /b in infected cells was ascribed to accumulation of viral genomic rna in cytoplasmic compartments or in the nucleus . our results showing decreased transcriptional activity upon depletion of hnrnp a /b indicate a possible additional mechanism of inhibition of virus production mediated by increased g folding in the hiv- ltr promoter in the absence of hnrnp a /b . surprisingly, overexpression of this protein and other hnrnp members induced similar effects, i.e. inhibition of transcription and reduction of virus production . we suggest that upon stimuli that boost viral transcription (i.e. integral release of ltr g s upon hnrnp a /b massive overexpression) the virus (or the cell) activates counteracting mechanisms to avoid excessive exploitation of the cell by the virus that would lead to fast cell death, in the end impairing virus production. therefore, the reported transcription inhibition might be mediated by other factors triggered by hnrnp a /b overexpression. the activity of hnrnp a /b is fit to unfold the g s that actually form in the hiv- ltr region. in fact, g s folded in short oligonucleotides, such as ltr-iii and ltr-iv, which displayed higher thermal stability, were only partially unfolded by the protein. in contrast, the same g structures folding in a longer context (ltr-ii + iii + iv) were less stable and were effectively processed by hnrnp a /b . obviously, the longer oligonucleotide better mimes the actual g condition in the full-length ltr, as also proved by transcription inhibition obtained by hnrnp a /b depletion in cells that contained the entire hiv- ltr. the ltr g system displays regulating features similar to those described for the c-myc , , hras and kras oncogene-promoters , [ ] [ ] [ ] . as in these eukaryotic g -modulated promoters, the hiv- ltr promoter is processed by g stabilizing (nucleolin) and destabilizing proteins (hnrnp a /b ). because the ltr promoter has been suggested to be the region where viral latency is regulated , , the g switch may play a role not only in activation of effective viral transcription, but also in its shift to latency. currently, only the actively transcribed virus is targeted by antiviral drugs, while eradication of the hiv- infection has been made impossible by the existence of reservoirs of the latent virus . therefore, our findings not only advance our understanding on the mechanism of viral transcription but may also constitute a progress from a therapeutic point of view. , where r (förster distance) is the distance at which energy transfer is % of the maximum value. between fam and tamra fluorophores, r is assumed to be Ǻ . f-test (f) and the probability (p) values were calculated using r statistical environment (v. . . ) . a conventional alpha = . was considered to evaluate the test significance. taq polymerase stop assay. taq polymerase stop assay was carried out as previously described , . briefly, the ′ -end labelled primer was annealed to its template (table s ) in lithium cacodylate buffer in the presence or absence of kcl ( - mm) and by heating at °c for min and gradually cooling to room temperature. where specified, samples were incubated with ng of human recombinant hnrnp a (origene technologies, rockville, usa) at °c for min. primer extension was conducted with u of amplitaq gold dna polymerase (applied biosystem, carlsbad, california, usa) at °c for min. reactions were stopped by ethanol precipitation, primer extension products were separated on a % denaturing gel, and finally visualized by phosphorimaging (typhoon fla ). circular dichroism spectroscopy. dna oligonucleotides were diluted to a final concentration ( μ m) in lithium cacodylate buffer ( mm, ph . ) and kcl mm. samples were annealed by heating at °c for min and gradually cooled to room temperature overnight. cd spectra were recorded on a chirascan-plus (applied photophysisics, leatherhead, uk) equipped with a peltier temperature controller using a quartz cell of mm optical path length, over a wavelength range of - nm. for the determination of t m , spectra were recorded over a temperature range of - °c, with temperature increase of °c. the reported spectra are baseline-corrected for signal contributions due to the buffer. observed ellipticities were converted to mean residue ellipticity (θ ) = deg × cm × dmol − (mol ellip). t m values were calculated according to the van't hoff equation, applied for a two-state transition from a folded to unfolded state, assuming that the heat capacity of the folded and unfolded states are equal . immunoblot analysis. immunoblot analysis was performed on cell protein extracts obtained in ripa buffer ( mm tris-hcl ph . ; mm nacl, mm edta, % np- , % sodium deoxycholate, mm na vo , x protease inhibitors). protein concentrations were quantified using the pierce ® bca protein assay kit (thermo scientific, rockford, il, usa). each sample was electrophoresed on % sds-page and transferred to a nitrocellulose blotting membrane (amersham tm protan tm, ge healtcare life science, milan, italy) by using trans-blot sd semi-dry transfer cell (bio-rad laboratories, milan, italy). the membranes were blocked with . % skim milk in pbst ( . % tween in pbs). membranes were incubated with the respective primary antibody directed against hnrnp a /b (mouse monoclonal; santa cruz biotechnology, dallas, tx, usa), alpha-tubulin (mouse monoclonal; sigma-aldrich, milan, italy). after three washes in pbst, membranes were incubated with ecl plex goat-α -mouse igg-cy (ge healthcare life sciences, milan, italy). images were captured on the typhoon fla . sirna and luciferase reporter assay. gene-specific pooled sirna trilencer targeting human hnrnp a / b and a scrambled negative control duplex were purchased from origene (human hnrnp a /b trilencer- human sirna, origene technologies, rockville, md, usa). t cells or tzm-bl reporter cells were transfected with increasing concentrations of sirna ( , , and nm for t and , , nm for tzm-bl cells) and scrambled sirna by using lipofectamine rnaimax (invitrogen, thermo fisher scientific, waltham, ma, g -associated human diseases g-quadruplexes and their regulatory roles in biology topology of a g-quadruplex dna formed by c orf hexanucleotide repeats associated with als and ftd quantitative visualization of dna g-quadruplex structures in human cells detection of g-quadruplex dna in mammalian cells g-quadruplexes in viruses: function and potential therapeutic applications the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes human papillomavirus g-quadruplexes a highly conserved g-rich consensus sequence in hepatitis c virus core gene represents a new anti-hepatitis c target. science advances , e zika virus genomic rna possesses conserved g-quadruplexes characteristic of the flaviviridae family chemical targeting of a g-quadruplex rna in the ebola virus l gene g-quadruplexes regulate epstein-barr virus-encoded nuclear antigen mrna translation role for g-quadruplex rna binding by epstein-barr virus nuclear antigen in dna replication and metaphase chromosome attachment visualization of dna g-quadruplexes in herpes simplex virus -infected cells the herpes simplex virus- genome contains multiple clusters of repeated g-quadruplex: implications for the antiviral activity of a g-quadruplex ligand formation of a unique cluster of g-quadruplex structures in the hiv- nef coding region: implications for antiviral activity topology of a dna g-quadruplex structure formed in the hiv- promoter: a potential target for anti-hiv drug development a dynamic g-quadruplex region regulates the hiv- long terminal repeat promoter u region in the hiv- genome adopts a g-quadruplex structure in its rna and dna sequence anti-hiv- activity of the g-quadruplex ligand braco- binding and antiviral properties of potent core-extended naphthalene diimides targeting the hiv- long terminal repeat promoter g-quadruplexes nucleolin stabilizes g-quadruplex structures folded by the ltr promoter and silences hiv- viral transcription the evolving world of protein-g-quadruplex recognition: a medicinal chemist's perspective how shelterin protects mammalian telomeres the kras promoter responds to myc-associated zinc finger and poly(adp-ribose) polymerase proteins, which recognize a critical quadruplex-forming ga-element hras is silenced by two neighboring g-quadruplexes and activated by maz, a zincfinger transcription factor with dna unfolding property nuclear functions of heterogeneous nuclear ribonucleoproteins a/b. cellular and molecular life sciences: cmls label-free interaction analysis as a tool to demonstrate biosimilarity of therapeutic monoclonal antibodies use of surface plasmon resonance for the measurement of low affinity binding interactions between hsp and measles virus nucleocapsid protein protein hnrnp a and its derivative up unfold quadruplex dna in the human kras promoter: implications for transcription vienna r foundation for statistical computing analysis of multidimensional g-quadruplex melting curves function of conserved domains of hnrnp a and other hnrnp a/b proteins recognition and binding of human telomeric g-quadruplex dna by unfolding protein structure-based incorporation of -methyl- -( -deoxy-beta-ribofuranosyl) isoxanthopteridine into the human telomeric repeat dna as a probe for up binding and destabilization of g-tetrad structures hnrnp a associates with telomere ends and stimulates telomerase activity distinct domains in the carg-box binding factor a destabilize tetraplex forms of the fragile x expanded sequence d(cgg)n. nucleic acids research destabilization of tetraplex structures of the fragile x repeat sequence (cgg)n is mediated by homolog-conserved domains in three members of the hnrnp family telomere-and telomerase-interacting protein that unfolds telomere g-quadruplex and promotes telomere extension in mammalian cells depletion of hnrnp a /b overrides the nuclear retention of the hiv- genomic rna trafficking of hiv- rna is mediated by heterogeneous nuclear ribonucleoprotein a expression and impacts on viral assembly role of cellular rna processing factors in human immunodeficiency virus type mrna metabolism, replication, and infectivity targeting myc expression through g-quadruplexes nm -h may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element iii( ) making sense of g-quadruplex and i-motif functions in oncogene promoters regulation of oncogenic kras signaling via a novel kras-integrin-linked kinase-hnrnpa regulatory loop in human pancreatic cancer cells g dna in ras genes and its potential in cancer therapy an ap- binding site in the enhancer/core element of the hiv- promoter controls the ability of hiv- to establish latent infection establishment and molecular mechanisms of hiv- latency in t cells identification of a reservoir for hiv- in patients on highly active antiretroviral therapy probability-based protein identification by searching sequence databases using mass spectrometry data using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions proposal for a common nomenclature for sequence ions in mass spectra of peptides m.s. performed the mass spectrometry analysis and protein identification; i.f. provided silencing, reporter and viability data in cells, e.r. performed the fret analysis, r.p. performed the taq polymerase stop assays and silencing in tzm-bl cells, e.t. performed the pull-down assay and western blot, s.l. provided the spr data, m.t. performed cd analysis, g.p. commented on the manuscript, s.n.r. conceived of the work and wrote the manuscript. all authors analyzed the data and reviewed the manuscript. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- -jlhht l authors: cruz-flores, roberto; mai, hung n.; kanrar, siddhartha; aranguren caro, luis fernando; dhar, arun k. title: genome reconstruction of white spot syndrome virus (wssv) from archival davidson’s-fixed paraffin embedded shrimp (penaeus vannamei) tissue date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: jlhht l formalin-fixed paraffin-embedded (ffpe) tissues are a priceless resource for diagnostic laboratories worldwide. however, dna extracted from these tissues is often not optimal for most downstream molecular analysis due to fragmentation and chemical modification. in this study, the complete genome of white spot syndrome virus (wssv) was reconstructed from ~ -year-old archived davidson’s-fixed paraffin-embedded (dfpe) shrimp tissue using next generation sequencing (ngs). a histological analysis was performed on archived dfpe shrimp tissue and a sample showing a high level of wssv infection was selected for molecular analysis. the viral infection was further confirmed by molecular methods. dna isolated from dfpe and fresh frozen (ff) tissues were sequenced by ngs. the complete genome reconstruction of wssv (~ kbp) was achieved from both dfpe and ff tissue. single nucleotide polymorphisms, insertion and deletions were compared between the genomes. thirty-eight mutations were identified in the wssv genomes from the dfpe and ff that differed from the reference genome. this is the first study that has successfully sequenced the complete genome of a virus of over kbp from archival dfpe tissue. these findings demonstrate that dfpe shrimp tissue represents an invaluable resource for prospective and retrospective studies, evolutionary studies and opens avenues for pathogen discovery. scientific reports | ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports/ in human clinical research, many researchers have successfully extracted nucleic acids from ffpe tissue for pcr-based amplification work despite the degradation of nucleic acids and thereby giving them access to a previously untapped resource. however, it must be clarified that in most cases the successful use of ffpe tissue for molecular analysis depends largely on how the sample was fixed (temperature, time, ph). in cancer research ffpe tissues have been extensively used for genome sequencing of tumor tissues for copy-number and mutation-analysis, expression profiles, screening for mutational hotspots, single-cell sequencing and genome sequencing from laser capture microdissected cells [ ] [ ] [ ] [ ] [ ] [ ] . ffpe tissues have been used in pathogen discovery and uncovering novel genetic features in pathogen genomes. for example, the spanish influenza pandemic virus was reconstructed from ffpe tissue from . the virus species involved in pandemics showed large differences to the contemporary human influenza h n strain . recently, ffpe tissue has been used for the detection and discovery of a novel rotavirus . in another recently published retrospective study, ffpe tissue was used to sequence the rna genome of ~ kb length of the newcastle disease virus (ndv) that naturally infects many avian species . the study revealed the continuous evolution and previously unrecognized genetic diversity in ndv . the first study of the use of ffpe material in aquatic organisms dates back to when krafft et al. used fixed tissue to detect morbillivirus in lung tissue of bottlenose dolphins. in finfish, mollusk and crustacean pathology, so far no attempt has been made to explore the feasibility of using ffpe/dfpe tissues for any retrospective genetic studies or pathogen discovery. in shrimp aquaculture, existing as well as emerging diseases are a threat to a sustainable growth of the industry worldwide. outbreaks of diseases in shrimp aquaculture cause major economic losses to shrimp farmers directly, and indirectly impact the lives and livelihood of those who depend of shrimp farming especially in developing nations with large coastal boundaries. there is an urgent need to understand the origin and evolution of pathogens in shrimp aquaculture to prevent epizootics that are becoming more common than ever. archived davidson's-fixed paraffin-embedded (dfpe) tissues in the aquaculture pathology laboratory of the university of arizona are an untapped invaluable resource for pathogen discovery, metagenomic and evolutionary studies to understand the origin, evolution and spread of shrimp pathogens worldwide. in this study, we demonstrated the feasibility of using dfpe tissue in pathogen discovery by reconstructing the complete genome of a large dsdna-containing virus, white spot syndrome virus (wssv), with a genome size of ~ kbp from dfpe tissues. to our knowledge, this is the first report of genome reconstruction of such a large genome from archived tissue for any virus known to infect humans, animals or plants. this study shows the utility of dfpe tissues in shrimp pathology research, opens avenues for novel pathogen discovery and enables us to address questions related to the origin and evolution of shrimp viral pathogens that continue to cause catastrophic losses to farmers globally. selection of davidson's-fixed paraffin embedded blocks. histopathological evaluation of the experimentally infected penaeus vannamei shrimp revealed a severe infection graded as g -g in all tissues examined. a representative histology section showing the intranuclear inclusions in the gut epithelial cells of p. vannamei that are pathognomonic of wssv infection is shown in fig. fig. ) . sequence analysis of the wssv genome from dfpe shrimp tissue and annotation. a total of , , million reads were generated from the second round of sequencing in a pe × bp format. from the total of reads, , , unique reads (approximately . % reads) were mapped to the wssv china reference genome and generated complete coverage of the entire genome with a mean coverage of , . . the maximum and minimum coverage obtained were , and , respectively. the reconstructed wssv genome was , bp and presented a pairwise identity of . % with the genome of the china reference strain. a total of dna coding sequences were annotated using rast and geneious prime (fig. ) . additionally, a histogram of read size distribution is presented in supplementary fig. . the wssv genome sequence obtained from dfpe tissue sample were aligned with genbank sequence of the wssv china reference strain and the snps were identified. a total of sequences variations that include snps, deletions or insertions were detected. the genomic regions that contained the snps and sequence variations were amplified by pcr, sequenced and aligned with the reference wssv genome and the wssv genome reconstructed from dfpe tissue ( table , fig. ). the snps and sequence variations were confirmed when the sanger sequence and the dfpe derived sequence matched. all of the mutations found in the wssv genome reconstructed from dpfe were confirmed by sanger sequencing. detection and characterization of novel viruses is frequently hampered by the lack of properly stored materials . for the retrospective identification of viruses associated with disease outbreaks, often only formalin-fixed paraffin-embedded (ffpe) tissue samples are available . retrieving genomic information from ffpe has always been a challenge but the availability of genome sequencing technologies such as ngs has now made it possible to reconstruct genetic information from archived samples. in the case of viral diseases of shrimp, often the etiologic agent was identified long after the disease was initially reported and spread within and across countries worldwide. while samples originally collected during disease outbreaks were used for histopathological and ultrastructural studies to elucidate possible etiologic agent, those samples were almost never used for genetic characterization of the pathogen associated with the disease. as a result, it remains unknown how the pathogen evolved during the time frame from when the disease outbreaks were initially reported vs. when the genomic characterization of the pathogen was accomplished. for example, the infectious hypodermal and hematopoietic necrosis virus (ihhnv) was initially detected in by lightner et al. however, the genetic characterization of the virus was carried out years later by shike et al. . ihhnv was the first shrimp virus for which the genome sequence was determined . considering a high rate of nucleotide substitution ( . × - substitutions/site/year) of ihnnv, it is possible that the strain that caused massive mortalities of blue shrimp (p. stylirostris) in the early 's in mexico and later in the rest of the americas is different from the strains that have been characterized later. another interesting fact that supports this hypothesis is that the current strains of ihnnv do not cause mortalities or major histological alterations in p. vannamei and p. stylirostris shrimp and no major epizootics have been attributed to ihhnv in recent years , . this could be due to accumulation of mutations in the ihhnv genome and/or development of host resistant/tolerance over time. upon reconstructing the wssv genome from dfpe tissues, we have shown that the genetic characterization of pathogens containing large genomes is possible from archived fixed tissue (dfpe) and this opens the door for future retrospective studies to better understand the genomic properties of pathogens from the past that once caused mass mortalities but causes little to no mass mortality anymore. these studies would enable to better understand the evolution of host-pathogen interactions not only in shrimp but also in viruses infecting other animals and humans. in this study, the dna extracted from the dfpe tissue were used for pathogen detection via pcr and qpcr. wssv was successfully detected by qpcr and nested pcr following oie-recommended (paris, france) protocols and wssv genomic fragments ranging bp (for real-time pcr) to , bp (for st step of the nested pcr) were amplified. however, it is important to mention that for the nested pcr protocol the increase in the amount of template dna was key to obtaining amplification in all samples (n = ). in preliminary assays, amplification was only obtained in two samples ( - a and - a ) when using > ng of dna per reaction. all the fragments designed to amplify the areas where the snp where located ranged between - bp and thus fell within the size range of qpcr and nested pcr diagnostics. a previous published study involving human housekeeping genes by ludyga et al. have shown that products between - bp can be reliably amplified from ffpe. the study by these authors also showed that the amplifiable fragment size decreases with storage time with the maximum amplifiable fragment decreasing from bp from samples from the year to bp from samples from . our results show that a relative short storage time (~ years) of dfpe shrimp tissue, as used in this study, can provide dna of sufficient quality that can be used to amplify dna fragments of almost , bp. in addition, these results suggest that davidson's fixative is not as damaging to nucleic acids as previously postulated by hasson et al. and that dna from dfpe shrimp tissue does not undergo sever degradation in short storage times (~ years). however, we should underscore that some degradation does occur, as observed from the percentage of virus mapping reads where the ff sample had a high percentage (~ %) in comparison to the dfpe samples (~ - . %) and our inability to amplify large pcr products at lower dna concentrations. single-nucleotide polymorphism changes in bacterial genomes can cause significant changes in phenotype, including antibiotic resistance and virulence, therefore detecting them within metagenomes is vital www.nature.com/scientificreports/ replication and pathogenicity. in taura syndrome virus (tsv) of shrimp, it was shown that a single nucleotide mutation changed the predicted tertiary structure of the rna-dependent-rna polymerase in a highly virulent strain compared to less virulent strain . the error rates for some ngs data sets generated by illumina technologies are very low: a rate . (errors per base) . despite the low error rate, it was critical to confirm that the detected snps, insertions and deletions were present in the wssv genome reconstructed from dfpe and were not sequencing errors. by amplifying, and sequencing the regions where the variations were located, we were able to confirm all the snps, insertions and deletions. this results further highlight the robustness of this methodology since it proves that small sequence variations can be efficiently detected from dfpe tissue and it underscores its value for retrospective phylogenetic analysis. www.nature.com/scientificreports/ in recent years, the availability of novel genome sequencing technologies have increased the chance and speed of detection of unknown viruses in samples collected from humans and animals. in particular ngs played an important role in the discovery and characterization of many novel viruses , - . next generation sequencing using dna isolated from ffpe tissue enabled pathogen detection, identification of endogenous viral elements, genome sequencing, exome and transcriptome sequencing in animals and humans [ ] [ ] [ ] [ ] [ ] , , , , , . although ffpe tissues have been used to detect known viral sequences, the application of ffpe tissues for detection of novel viruses is very limited. recently, bodewes et al. showed that sequence-independent amplification in combination with ngs can be used to detect sequences of known and unknown viruses in herring gull and ferrets, although with relatively low sensitivity. the findings of bodewes et al. indicate that ngs from ffpe is a viable approach to detect known dna (adenovirus) and rna (influenza a/h n ) viruses, and unknown rna viruses (novel herring gull rotavirus). our results confirm that ngs from dna extracted from dfpe tissue is also a viable approach to detect know viral sequences. however, our results show this approach is robust and can generate enough data to sequence very large viral genomes with a very high coverage. furthermore, our results suggest that with sufficient data even the sequencing of complete bacterial genomes from this type of samples might be possible. unlike plant and human virology, shrimp virology is a relatively newly emerged field of virology. the first shrimp viral disease was reported only about years ago (baculovirus penaei) and the first shrimp virus was sequenced about years ago (ihhnv) . however, as shrimp aquaculture has evolved from a subsistence level of farming to a major industry providing jobs to millions of people around the world directly and indirectly especially in countries with large coastal boundaries, viral diseases poses a serious threat to the sustainable growth of this nascent industry. as of now, viral disease prevention through biosecurity and early disease diagnosis remain as corner stones to mitigate losses in shrimp aquaculture. since these diseases primarily spread through the movement of infected broodstock and post-larvae across countries and continents, it is critical to understand how these pathogens evolve in new environment as virus-infected animals are moved across continents and how naïve host adapt to new pathogens. the ability to reconstruct dna viral genomes as large as kbp size from dfpe tissues shows the feasibility to generate baseline genetic data from archived tissue and determine how pathogens have evolved over time. to our knowledge, this is the first study that shows the feasibility of using ngs as a viable option for genetic characterization of shrimp pathogens and potentially discovering novel pathogens from samples stored in pathology laboratories worldwide. dna extraction, quantification and pcr. dna was extracted using the commercial kit ffpe dna purification kit (norgen biotek corp) in accordance with the manufacturer's recommendations with some modifications. during the deparaffinization step, the xylene washes were doubled, and the pellet was air dried for min. finally, during the lysate preparation step, the incubation at °c was increased from to h min. two elution's were obtained from each sample. additionally, from the samples fixed in liquid nitrogen dna was extracted using the genomic dna isolation kit (norgen biotek corp) following the manufacturer's instructions. the quantity and quality of the dna was determined using a nanodrop . the presence of wssv was further confirmed by qpcr and nested-pcr following published protocols , . for the nested-pcr protocol published by lo et al. one modification was made, the input volume of dna was increased to . µl ( . - . ng/per reaction). to test the feasibility of performing ngs using dna isolated from dfpe shrimp tissue, we conducted two rounds of ngs. the first sample was sequenced using an illumina miseq system (pe × bp) (illumina). once we determined it was possible to efficiently sequence dna from archived dfpe tissues using an illumina miseq system, we sequenced two additional samples. the second round of sequencing was done using an illumina hiseq system (pe x bp) (illumina) to generate a more robust data set. dna extracted from both dfpe tissue and ff tissue were sent for ngs at omegabioservices, norcross, ga. library for the dna samples were generated at omegabioservices using the library kit, kapa hyper prep for wgs (roche). for the dna extracted from the dfpe tissue the fragmentation step prior to library generation was avoided since the isolated dna was already fragmented. mapping and annotation. the dna reads were paired and duplicate reads were removed using the dedupe plugin in geneious prime . dna reads from the wssv shrimp tissue (dfpe and ff) were checked for quality and trimmed before being mapped to the china wssv reference genome (genbank accession number: af ) using geneious prime (biomatters) . the geneious mapper was used for the mapping analysis and the setting were set to detect structural varients . the wssv isolate of the apl originated from china and hence the china wssv reference genome was utilized, however, it is unknown if they represent the same strain. the mean coverage of each based was calculated. the contigs generated from the mapping were annotated using the rast server and geneious prime , . the wssv complete genome reconstructed from dfpe (genbank accession: mn ) was submitted to genbank. the mauve software was used to perform whole genome alignments and comparisons . the genomes of wssv obtained from the dfpe tissue, ff tissue and the wssv reference genome were compared to identify differences among the sequences. to confirm the sequence variations observed between the wssv china reference genome and the wssv genome reconstructed from the dfpe tissues, primers were designed to amplify the regions flanking the snps, deletions and insertions. snps located in the repeat regions were not analyzed. primers were designed using geneious prime. the nucleotide sequence of primers designed to amplify the regions showing variations are presented in table f-wssv gac cac acc agc cct aaa gg snp , - , -r-wssv ttc gat ttg ggt cct ccg tc , - , -f-wssv caa tgg gca taa cct tgt tgga snp , - , -r-wssv agc gtt ctt caa gat caa tag gag a , - , -f-wssv atg ctg gct ctc gat tcg tt snp , - , -r-wssv aag gcc cac tta atc cag f-wssv tct gta tca gca gca gca gc snp , - , -r-wssv aat gtt ggg ccg tat ccg tt , - , -f-wssv aac cct aac aat ggt gtg cc insertion , - , -r-wssv aca cat atc tca tcg cac gtct , - , -f-wssv gct gca tgt cta tct tgt gttt insertion , - , -r-wssv acg acc atg ggc tgt aga aa table . the nucleotide sequence and location of the primers flanking the single nucleotide polymorphism (snps), deletions and insertion regions in wssv genome constructed from the davidson's-fixed paraffinembedded shrimp tissue. genome-wide massively parallel sequencing of formaldehyde fixed-paraffin embedded (ffpe) tumor tissues for copy-number-and mutation-analysis next-generation sequencing of rna and dna isolated from paired fresh-frozen and formalin-fixed paraffinembedded samples of human cancer and normal tissue clinical validation of a next-generation sequencing screen for mutational hotspots in cancer-related genes exome enrichment and solid sequencing of formalin fixed paraffin embedded (ffpe) prostate cancer tissue virus characterization and discovery in formalin-fixed paraffin-embedded tissues effect of fixatives and tissue processing on the content and integrity of nucleic acids targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics nucleic acids from long-term preserved ffpe tissues are suitable for downstream analyses distribution and the impact on international trade of two penaeid shrimp viruses in the evaluation of the preservation of shrimp samples with davidson's afa fixative for infectious myonecrosis virus (imnv) in situ hybridization a new rna-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cdna genomic probes clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the , genomes project next-generation sequencing of ultra-low copy samples: from clinical ffpe samples to single-cell sequencing mate pair sequencing of whole-genome-amplified dna following laser capture microdissection of prostate cancer characterization of the reconstructed spanish influenza pandemic virus. science ( -) whole-genome sequencing of genotype vi newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the postmortem diagnosis of morbillivirus infection in bottlenose dolphins (tursiops truncatus) in the atlantic and gulf of mexico epizootics by polymerase chain reaction-based assay detection of ihhn virus in penaeys stylirostris and p. vannamei imported into hawaii infectious hypodermal and hematopoietic necrosis virus of shrimp is related to mosquito brevidensoviruses genetic signature of rapid ihhnv (infectious hypodermal and hematopoietic necrosis virus) expansion in wild penaeus shrimp populations diversity of single-stranded dna containing viruses in shrimp. virusdisease illumina error profiles: resolving fine-scale variation in metagenomic sequencing data characterization of a taura syndrome virus isolate originating from the texas epizootic in cultured shrimp genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans a novel rhabdovirus associated with acute hemorrhagic fever in central africa viral surveillance and discovery detection of viral pathogens in high grade gliomas from unmapped next-generation sequencing data deep clonal profiling of formalin fixed paraffin embedded clinical samples an enzootic nuclear polyhedrosis virus of pink shrimp: ultrastructure, prevalence, and enhancement white spot syndrome baculovirus (wsbv) detected in cultured and captured shrimp, crabs and other arthropods quantitative real time pcr for the measurement of white spot syndrome virus in shrimp geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data the rast server: rapid annotations using subsystems technology mauve: multiple alignment of conserved genomic sequence with rearrangements funding for this research was provided by the college of agriculture & life sciences in the university of arizona to arun k. dhar. we would like to thank jasmine millabas for her aid in the histological processing. a special thanks to sara heather lynn for assistance with the dna extractions. r.c.f. and a.k.d. designed the experiments. r.c.f. wrote the manuscript, analyzed the ngs data and carried out the snp analysis. h.n.m. analyzed the ngs data and edited the manuscript. s.k. analized the ngs data. l.f.a.c. and r.c.f. selected the histological slides and archived blocks for study. l.f.a.c. edited the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - -x.correspondence and requests for materials should be addressed to a.k.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -x tnweih authors: yang, szu-chi; lin, huan-chun; liu, tzu-ming; lu, jen-tang; hung, wan-ting; huang, yu-ru; tsai, yi-chun; kao, chuan-liang; chen, shih-yuan; sun, chi-kuang title: efficient structure resonance energy transfer from microwaves to confined acoustic vibrations in viruses date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: x tnweih virus is known to resonate in the confined-acoustic dipolar mode with microwave of the same frequency. however this effect was not considered in previous virus-microwave interaction studies and microwave-based virus epidemic prevention. here we show that this structure-resonant energy transfer effect from microwaves to virus can be efficient enough so that airborne virus was inactivated with reasonable microwave power density safe for the open public. we demonstrate this effect by measuring the residual viral infectivity of influenza a virus after illuminating microwaves with different frequencies and powers. we also established a theoretical model to estimate the microwaves power threshold for virus inactivation and good agreement with experiments was obtained. such structure-resonant energy transfer induced inactivation is mainly through physically fracturing the virus structure, which was confirmed by real-time reverse transcription polymerase chain reaction. these results provide a pathway toward establishing a new epidemic prevention strategy in open public for airborne virus. in the past few decades, tremendous efforts have been made to kill airborne viruses such as severe acute respiratory syndrome (sars) coronavirus or influenza viruses, which have caused catastrophic illness worldwide. current airborne virus epidemic prevention to be used in public space includes strong chemical inactivation, uv irradiation, and microwave thermal heating. all these methods affect the open public. in s, robach et al. and cerf demonstrated that ultrasonic energy can be absorbed by viruses. in , babincová et al. hypothesized that viruses can be inactivated by generating the corresponding resonance ultrasound vibrations of viruses, which is in the ghz region. based on this hypothesis, several groups started investigating the vibrational modes of viruses in this frequency range [ ] [ ] [ ] [ ] . recently we demonstrated that dipolar mode of the confined acoustic vibrations (cavs) inside viruses can be resonantly excited by microwaves of the same frequency with a resonant microwave absorption effect . the observed microwave resonance absorption phenomenon indicates a possible structure-resonant energy transfer (sret) effect from electromagnetic waves (em waves) to cavs of viruses. theoretically this sret process is an efficient way to excite the vibrational mode of the whole virus structure due to a % energy conversion of a photon into a phonon of the same frequency, but the overall sret efficiency is also related to the mechanical properties of the surrounding environment , which influences the quality factor of the oscillator (virus). a study on the sret efficiency to inactive virus is thus highly desired and it will determine if this sret phenomenon provides a solution to inactivate airborne viruses in open public for epidemic prevention. in this article, we show that sret from microwave to virus can be efficient enough so that airborne virus was inactivated with reasonable microwave power density safe for the open public. to investigate the sret efficiency from em waves to cavs in viruses, we first developed a theoretical model to describe the relation between the induced stress and the field magnitude of the illuminating microwave. since the viruses could be inactivated when the induced stress fractures the structure of viruses, we propose to explore the sret efficiency from microwaves to viruses through measuring the virus inactivation threshold. based on the proposed model, we studied the inactivation ratio of influenza a (h n ) virus at dipolar-mode-resonance and off-resonance microwave frequencies as well as with different microwave powers. plaque assay was then applied to calculate the titer of virus samples before and after the microwave illumination. our results indicate efficient sret from microwave to viruses, which resulted in higher inactivation ratio of viruses at the dipolar resonant frequency. at the resonant frequency, the microwave power density threshold for h n inactivation was found to be below the ieee safety standard, also agreeing well with our developed theoretical model. the real-time reverse transcription polymerase chain reaction (real-time rt-pcr) method was further performed to confirm that the main inactivation mechanism is through physically fracturing the viruses while the rna genome was not degraded by the microwave illumination, supporting the fact that our studied sret mechanism is fundamentally different from the microwave thermal heating effect. these results provide a pathway toward establishing a new epidemic prevention strategy in open public for airborne virus. from the transmission electron microscope images, people knew that the virions of influenza viruses are basically spherical balls packing genomes inside. since the protein and genome have similar mechanical properties , for the estimation of dipolar vibration frequencies, we treat the virion as a homogenous sphere. dipolar mode of a homogeneous sphere. due to the spatial confinement, not only electronic but also acoustic energy quantization has been observed in low dimensional systems such as quantum dots and nano-wires. in , lamb studied the torsional (tor) and spheroidal (sph) modes of a homogeneous free sphere by considering the stress-free boundary condition on the surface . among these modes, the sph mode with =  allows dipolar coupling and the corresponding eigenvalue equation can be expressed as , : j is the spherical bessel function of the first kind, ω is the angular frequency of the vibrational mode, r is the radius of the nano-sphere, c l and c t are longitudinal and transverse sound velocities respectively. a comparison between the commonly observed =  breathing mode and the =  dipolar mode can be found in supplementary online. the since the =  dipolar mode of a nano-sphere cannot be detected by the light scattering experiments , it was not observed until a previous study of the resonant excitation of dipolar mode through thz wave or microwave excitations , when the core and shell of the nano-sphere have permanent charge separation. once the resonantly oscillating electric field was applied to the nano-sphere, opposite displacement between core and shell was generated, thus excited the dipolar mode vibrations. compared with the breathing ( =  ) and quadrapolar ( =  ) modes, dipolar mode ( =  ) is the only sph mode to directly interact with the em waves whose wavelength is much longer than the particle's size. due to the permanent charge separation nature of viruses, in , dipolar coupling with =  cavs is confirmed to be the mechanisms responsible for microwave resonant absorption in viruses by treating spherical viruses as free homogeneous nanoparticles , . figure shows the simulated displacement field of the dipolar mode (calculated by the finite element method, comsol multiphysics, comsol, inc.) of a homogeneous sphere (mass density and viscoelastic properties are constant throughout the sphere). we define the relative displacement direction of the dipolar mode as the z-direction, which will also be the field direction of the applied em waves discussed in the next section. by plotting the displacement field of the x-z plane (y = ) of the sphere, the opposite displacement between the core and shell regions can be clearly observed in fig. (b) . meanwhile fig. (c) shows the side view of the distortion of the x-y plane of the sphere at different z locations, which concludes that the maximum distortion occurs on the equatorial plane (z = ) of the sphere. figure (d) shows the top view of the displacement field of the equatorial plane (z = ). it is interesting to find out that the magnitude of averaged positive displacement (inner region) is . times the magnitude of the averaged negative displacement (outer region), while positive and negative displacements occupy % and % area, respectively. furthermore one can find that the maximum magnitude of the displacement, occurring either at the very center or the outer surface of the equatorial plane, is approximately twice of the averaged magnitude of the displacement. a damped mass-spring model. in this work, microwaves were applied to excite the dipolar resonance of the whole virus structure. by exciting the dipolar mode of the nanosphere, core and shell with opposite charge distributions would move in opposite directions and will resonate like a damped mass-spring system . our following analysis is similar to the drude-lorentz model describing the light-atom interaction, which connects a damped mass-spring system to the quantum-mechanical electronic resonant transitions. in the damped mass-spring system by adopting the reduced mass (m*) of core and shell in the analysis, the relative motion of the displacement can be shown in the following equation: where z is the relative displacement between the core and shell; b is the damping coefficient, which is related to the surrounding environment; k is the effective spring constant of this system. by assuming z(t) proportional to exp(iω t), one can solve the complex angular frequency of the resonator as: therefore the decay rate of the oscillation equals to the imaginary part of the frequency (b/ m*), which corresponds to ω / q : the intrinsic resonance angular frequency (ω ) of this system is (k/ ⁎ m ) . . q is the quality factor of the resonator. from equation ( ) , stronger damping increased the energy transfer between the resonator and its surrounding environment, which decreases the confinement of the vibration and leads the lower q. now we approximate that a spherical virus is like a homogeneous sphere but with opposite and equal charges in the core and shell regions. when the oscillating electric field ( = e e o cosωt) of microwaves is applied to the system, forced displacements would be induced with the same frequency as the applied microwaves. the equation of motion now needs to include the force induced by the applied electric field (qe), where q is the total amount of charge distributed in the core and shell region of a virus: we describe the forced displacement as where a is the amplitude of the forced displacement and θ is the phase delay between the displacement and the applied electric field. by solving the particular solution of this differential equation, one can obtain the phase delay and the amplitude of the forced oscillating displacement as the instantaneous power absorption of this system is then described as the following equation, where v is the velocity of the oscillating motion : by integrating over one full cycle, one can obtain the average power absorption from the system: threshold to fracture a virus. with oscillating dipolar vibration to inactivate a virus, the most possible mechanism is to fracture the most outer surface of the equatorial plane (z = ) due to the location of the maximum distortions, as illustrated in fig. (c,d). for influenza viruses, this corresponds to the lipid membrane of the envelope. to estimate the maximum induced stress ( ) p stress max on the equatorial plane, we divide the maximum induced force by the area of the shell region (defined by the moving direction in the approximate model) on the equatorial plane. following above discussion, we found that the maximum induced stress is twice the average value and the shell region covers % of the equatorial plane: if the required stress threshold p stress t to fracture a virus can be obtained, the threshold electric field magnitude ( ) e t of the incident microwaves can also be obtained by using: figure shows the threshold magnitude of the incident electric field at different frequencies with different q based on equation ( ) with a fixed p stress t threshold value. one can observe that the minimum of the threshold electric field magnitude occurs when the applied frequency is closed to the intrinsic resonant frequency. moreover cavity quality factor q plays a major role. by changing the ph value of the solution, charge status of viral surface can be modulated, which affects the q of the vibration. for example, previous studies indicated that the cavity q of spherical viruses ranges between - by raising the ph value of the solution from . to . . with increased q, more energy can be confined inside the resonator, which leads to much lower microwave field threshold magnitude at the resonant frequency. to experimentally study the efficiency of the sret from microwaves to cavs of spherical viruses, influenza a virus subtype h n was used. h n is a subtype of influenza a virus that causes flu. such viruses can infect birds and mammals and are increasingly abundant in seasonal influenza, which kills an estimated people in the united states each year, including pneumonia and influenza causes . based on previous studies, the averaged mass and the diameter of the h n virus are mda and nm . here we approximate the structure of the virus as a nanosphere with a core-shell structure of opposite charge distribution. the shell ( % of the total mass) contains lipid, neuraminidase (na), hemagglutinin (ha), and m-protein. the core ( % of the total mass) includes rna and rnp. the reduced mass (m*) of virus is thus . mda. from the literature , force with pn applied on the afm tip can fracture the lipid envelope. since the radius of the tip was nm , the threshold stress to fracture the shell was . mpa (p stress t ). in order to calculate the threshold magnitude of the electric field to fracture h n virus following equation ( ), some important parameters such as q, q and ω of the studied h n virus has to be obtained by measuring the microwave absorption spectrum of viruses. as shown in fig. (a) we covered the structure of the coplanar waveguide (cpw) by the microfluidic channel with a . mm-long sensing zone (l) in order to measure the microwave absorption spectrum of viruses. this microwave microfluidic channel can provide a microwave bandwidth over ghz. the measured results were summarized in fig. (b) . as the figure shows, the power absorption ratio (α ) by the virus at the resonant frequency from equation ( ), the theoretical absorption cross-section of the virus at the resonant frequency is: by setting  r of the pbs at . ghz as . , q = . × e can be obtained by comparing equation ( ) and equation ( ) . so far, all parameters for estimating electric field threshold in equation ( ) are obtained. by substituting threshold p stress = . mpa, q = . × e , m* = . mda, q = . and ω = π × . ghz into equation ( ) , threshold magnitude of electric field to fracture virus at different frequencies of microwave can be calculated. the result is shown in fig. (c) . in order to compare with the following inactivation experiments, our estimated threshold magnitude of electric field at , and ghz are . , . and . v/m, respectively. the minimum threshold occurs close to ghz due to resonance, and sufficient internal stress to fracture virus can be expected to be more efficiently generated by weaker electric field. based on the ieee microwave safety standard, the spatial averaged value of the power density in air in open public space shall not exceed the equivalent power density of (f/ ) / w/m at frequencies between and ghz (f is in ghz) . this corresponds to w/m at ghz, w/m at ghz, and w/m at ghz for averaged values of the power densities in air. assuming all the microwave power in air % transmitted into a specimen, and by taking the dielectric constant of water . ( ghz), . ( ghz), and . ( ghz) for calculation, this safety standard then corresponds to the average electric field magnitude of v/m ( ghz), v/m ( ghz), v/m ( ghz) inside the water-based specimens. it is interesting to notice that the required threshold electric field magnitudes at the resonant frequency ( . v/m) to fracture h n viruses as shown in fig. (c) are within the ieee microwave safety standard ( v/m), indicating high sret efficiency, even though the quality factor of the h n virus is low. to investigate the resonant effect, we first measured the residual viral infectivity of influenza a virus after illuminating microwave of different frequencies. as shown in fig. (a) , viral samples were placed below the horn antenna. the sponge under the sample was used to decrease the reflection of the microwave. to check the inactivation ratio, illuminated viruses were then analyzed by plaque assay to measure the residual infectivity of viruses. we compared the titer of illuminated viruses (n test ) and the titer of unilluminated control sets (n control ) to calculate the inactivation ratio ( − n test /n control ) at different frequencies between - ghz. based on fig. (c) , the field intensity threshold for inactivating h n virus ranges between . - . v/m, which corresponds to . - w/m , for microwaves between and ghz. since the aperture size of our horn antenna was . cm × . cm. the required threshold power input ranges from . w to . w for - ghz microwaves. we thus first applied . w ( dbm) fixed microwave power, which is higher than all the threshold power input, into the horn antenna for the frequency dependency studies. after considering the transmission coefficient of our horn antenna, this experimental condition corresponded to - w/m average illuminated power density on the sample surface, corresponding to the field intensity inside the specimen of - v/m respectively. for - . ghz microwave at the resonant frequency, the average illuminated power density was about w/m , equivalent to v/m effective field intensity inside the sample. we thus expect to observe the inactivation effect throughout the studied spectral range. as been summarized in fig. (b) , a frequency dependent inactivation ratio can be observed in our experiments, with a peak located at the resonant frequency of the dipolar mode while higher than % inactivation ratio can be observed throughout the studied frequency range. at . ghz, the measured titer count was zero, indicating % inactivation ratio, which means that the remaining active viral concentration was smaller than the system sensitivity of pfu/ml. this result indicates at least a three-order of magnitudes attenuation on the virus titer, when the microwave frequency was tuned to the dipolar mode resonant frequency with the electric field intensity times higher than the threshold. the illuminated average power density was roughly . times higher than the ieee safety standard for the - . ghz cases. it is important to notice that the power density is proportional to the square of the field intensity. to further investigate the efficiency of this sret effect from microwave to virus and the threshold effect, we further measured the inactivation ratio of h n virus with different power densities at the resonant frequency ~ ghz of the confined acoustic dipolar mode. our theoretical model predicted an inactivation threshold field intensity of . v/m, corresponding to an average microwave power density of . w/m in specimen. since we assume all power can transmit from air to specimen, power density in air is also . w/m , which is . times lower than the ieee safety standard. figure (b) summarized the measured inactivation ratio for different average microwave power densities of , , , and w/m in air, corresponding to an effective field intensity inside samples of , , , and v/m, respectively. it is noted that the experiment with w/m in air was performed in a different experimental setup, as shown in fig. (a) . a significant threshold effect can be observed when the effective field intensity inside samples started to be on the order of or exceed the estimated threshold. a % inactivation ratio can be observed with a field intensity of v/m, while the inactivation ratio dropped drastically to only negligible % with a slightly lower v/m field intensity. with a times higher field intensity than the threshold, the inactivation ratio saturated at a % value. discussions compared with the simple theoretical model for threshold estimation as summarized in fig. (c) , our result agrees qualitatively and surprisingly quantitatively. first, in our experiments, we observed a strong resonant effect on the virus inactivation ratio at the dipolar oscillation frequency of . ghz, thus indicating that the observed virus inactivation after microwave illumination was due to the proposed sret from microwave to virus through dipolar coupling. second, at the resonant frequency, we do observe h n virus inactivation by illuminating w/m (lower than the ieee safety standard in public space) ghz microwaves on our viral solution, corresponding to an average v/m electric field intensity inside the solution, confirming that our proposed simple model to estimate the field threshold ( . v/m) to structurally fracture the virus is quantitatively correct, especially combining the observed threshold effect as discussed above. with a low resonator quality factor (around and less than for h n ), we also observed virus inactivation in off-resonant frequencies ( - ghz), following a trend predicted with our model. however for off-resonant frequencies, the simple harmonic oscillator model seems to always estimate a lower threshold in the stoke-side (lower-frequency-side) of the resonant frequency than the anti-stoke-side (high-frequency-side). for example, at dc ( frequency) it still predicts a relatively low threshold field magnitude to fracture the virus. this is different from our observation. we observe that the anti-stoke-side is with a better inactivation ratio than the stoke-side, and the source for disagreement should be the over-simplification of the adopted model. our result regarding the efficient sret to inactivate virus with a low microwave power has a profound meaning. as we introduced, in the past few decades, tremendous efforts have been made to kill airborne viruses such as sars or influenza a, which have caused catastrophic illness worldwide. active airborne viruses are always transported inside tiny water droplets, thus similar to our experimental condition. a strategy for airborne virus epidemic prevention in open public is thus highly desired. our finding represents the first possible mechanism to inactivate airborne viruses without affecting the open public, since the required microwave power could be within the ieee safety standard. comparing this work with traditional microwave thermal inactivation, previous works , used a microwaves oven with more than w to heat the phage suspension. the inactivation ratio could reach almost % by increasing the temperature of the phage suspension to °c. in our case, % inactivation were achieved with . w ( dbm) as the input power into the horn antenna at . ghz. a power reduction by more than a factor of is achieved. it is however not possible to directly compare the irradiating power density, since the irradiating area was not provided in previous literatures. nevertheless our work still shows sharp contrast to current methodologies, including strong chemical inactivation, uv irradiation, and microwave thermal heating with w microwave power , , which are not safe for the open public. a previous study has shown that to inactivate human h n viruses through thermal heating, the temperature need to be higher than °c. compared with the w/m radiated microwave power density ( . w required power input) in our resonant inactivation case, the current microwave thermal heating method to inactivate virus usually requires more than w microwave power at . ghz , , which is way beyond the safety standard, in order to raise the sample temperature to be higher than °c for protein denature. it is known that the microwave thermal heating has a weak frequency dependency between - ghz, and this is not the case for our frequency dependent result as shown in fig. (b) . to confirm that our observation is not due to the microwave thermal heating effect, we had monitored the sample temperature change during the microwave illumination experiments with a radiated power density of w/m at a frequency of ghz by using an infrared thermal imaging camera with a temperature accuracy of . °c (chct, p - ). the temperature rise after minutes radiation was °c, from . °c up to . °c. we thus exclude the possible contribution of microwave thermal heating effect to inactivation h n viruses under our experimental condition. to double-confirm our proposed mechanism that the inactivation was through physically fracturing the structure of viruses, we established a fractured virus model by freezing the virus samples with liquid nitrogen and thawed immediately and repeated several times. we then preformed real-time rt-pcr (reverse transcription polymerase chain reaction) experiment for rna amplification in order to compare the results after microwave illumination with the established fractured-virus model. without protein denature, the established fractured-virus model allowed the viral rna content to be extracted after fracturing. we then performed the rt-pcr experiments to amplify the extracted viral rna after fracturing either through the frozen fracturing model or after microwave resonant irradiation. figure (a) summarized our results. the applied average microwave power density was w/m , the microwave frequency was tuned to the dipolar mode resonant frequency of . ghz, and the illumination time was minutes. to avoid possible existing viral rna in solution before the microwave illumination, we pretreated the virus samples by adding rnase to degrade the existing rna outside the viral particles. as can be revealed in fig. (a) , the control fractured-virus model (shown as "control") showed the same rna amplification trend with excellent quantitative agreement with the rnase-pretreated samples after microwave resonant illumination (shown as "pretreat"). obvious increase of copies in pcr can be observed after cycle . we have also performed two post-treat groups to double confirm the effect of the rnase. for post-treat groups, rnase was added right after the microwave resonant illumination. even if the virus particles were fractured, the released rna would be degraded by rnase, we did not expect to detect the viral rna. as been also confirmed by our rt-pcr experiment as shown in fig. (a) , we were not able to detect the signal of rna for the post-treat groups even after cycles. since all the rnas outside the virus surface in the solution were eliminated before the microwave illumination, for the pretreat samples the only way to detect the rna signal after the illumination was to fracture the virus so that the rna can be released. this result was also in good agreement on the amount of amplified rna in the control cases. these facts confirmed that the inactivation of viruses by illuminating microwaves at the dipolar mode resonant frequency was through physically fracturing the lipid-envelope of the influenza virus without denaturing the viral rna. to show that the investigated sret effect can be applied to deactivate viruses other than h n , we have also performed the real-time rt-pcr experiment on h n virus. the result was shown in fig. (b) . the applied microwave frequency was ghz, while the applied average microwave power density was w/m , corresponding to an effective electric field of v/m inside the specimens. the rest of the experimental conditions were the same as that of fig. (a) . similar results can be observed, indicating that the same sret induced inactivation effect can also occurs in virus other than h n . in summary, we investigated the structure resonance energy transfer from microwave to cavs of h n virus in water-based solution. the efficiency of such energy transfer was investigated through exploring the virus inactivation ratio. based on the proposed damped mass-spring model and the experimentally measured microwave absorption cross-section of a single virus, threshold magnitude of electric field to fracture viruses at different illuminated frequencies can be estimated. after the illumination by the microwave, the plaque assay experiment indicated that the inactivation ratio reaches its maximum at the resonant frequency of the dipolar resonance. the real-time rt-pcr experiment double confirmed that the main inactivation mechanism was through physically fracturing viruses without degrading viral rna genome. this work not only theoretically and experimentally demonstrates a new energy transfer mechanism between em waves and viruses, but also indicates an efficient sret effect. our results have important implications for the interaction between microwaves and biological tissues, which is a highly concerned public issue. with an observed inactivation threshold with a microwave power density within the ieee safety standard, the demonstrated sret mechanism also provides a pathway toward establishing a new epidemic prevention strategy in open public for airborne viruses. microwave absorption spectrum measurement. the microwave absorption spectrum measurement was performed by combining the coplanar waveguide (cpw) circuit with a microfluidic channel. as shown in fig. (a) , we covered the structure of the cpw by the microfluidic channel with a . mm-long sensing zone (l). the gap between the signal electrode and the ground electrode of the cpw was μ m. in order to decrease microwave loss on the electrodes, the gold layer thickness of the electrode was . μ m. on the surface of electrodes, we grew a thermal isolator layer of silicon dioxide on the sensing zone by plasma enhanced chemical vapor deposition (pecvd) to lower the temperature rise of fluids due to microwave dielectric heating . we then used a network analyzer (anritsu, ms c) as the source to measure the absorption spectrum of h n viruses from ghz to ghz. the virus particle density in the solution was . × m − . the spectrum of the solution without viruses was first measured as a reference; solution with viruses was later measured. by removing the solution background, the microwave absorption spectrum of h n viruses in solution was thus obtained (fig. (b) ) following a procedure similar to ref. . microwave illumination measurement. in our experiments, we used two different microwave sources, depending on the utilized microwave power: a network analyzer (anritsu, ms c) or an yttrium iron garnet (yig) oscillator. then the microwave signal was amplified by a power amplifier (qpj- ) and radiated from the horn antenna (electro-metrics, em- ). the aperture size of the antenna was . cm × . cm. to avoid damage on oscillator and amplifier caused by back reflection, we added an isolator and a directional coupler. all the microwave systems were put in a p class flow hood. the antenna was directed toward the bottom (fig. (a) ) or the side (fig. (a) ) of flow hood and the microwave was normally incident on either microscope slides ( fig. (a) ) or acrylic cuvettes (fig. (a) ) at a distance of cm below the exit of horn antenna. to avoid large reflection from the metal hood surface, the microscope slide or cuvette was put in a plastic dish supported by a broad band pyramidal absorber. for each measurement, the viral solution ( . × m − particle density) under illumination was drop on the slide and covered with a cover glass or was contained inside the cuvettes. under such an experimental geometry, we illuminated viruses for minutes at various microwave frequencies or at various scientific reports | : | doi: . /srep microwave powers. after illumination, we used buffer to wash-down and collect the viruses, which introduced a -fold dilution of virus concentration. then the illuminated viral solutions, together with the control sets were sent for plaque assay. quantitative plaque assay analysis. to measure the activity of viruses, we employed a quantitative plaque assay. the mdck cells used for plaque assay were grown in wells plates by adding ml of cells ( × /ml) to individual well. after confluent growth of mdck cells in plates, the cells were successively washed with phosphate buffer saline (ph . ) and eagles' mem with μ g/ml tpck trypsin. after washed, μ l of the ten-fold serial dilution of viruses were added into each well. for better virus adsorption, the inoculated cells were incubated at °c in a % co incubator for one hour. after then, the virus inoculums were removed and the eagles' minimal essential medium with μ g/ml tpck trypsin and . % agarose was added. after the gel formation, the plates were put in a % co incubator for at least - hrs. after incubation, the plates were fixed with % formalin for hour. after pour off agarose, the fixed cells were stained with crystal violet for min and washed with tap water. below certain virus concentration, the plaques wouldn't overlap each other seriously and can be counted unambiguously. considering the corresponding dilution factor, the plaque forming unit per ml (pfu/ml) of the original virus can thus be quantified. the titer measurement will be performed three times on the same sample to reduce quantization error. quantified by the plaque assay, the concentration of active viruses in our prepared viral solutions was around /ml for h n and h n . real-time pcr analysis. the sample rna was extracted and amplified by the rt and quantitative real-time pcr (primerdesign precision onestep ™ qrt-pcr mastermix) with primer amf (sequence: ′ -gagtcttctaaccgaggtcgaaacgta- ′ ), primer flu-ar (sequence: ′ -caaagcgtctacgctgcagtcc- ′ ) and flu a ( ′ -fam-tttgtgtttacgctcaccgt-tamra- ′ ) probe. for control group, rnase was added for minutes digestion and stopped with rnase inhibitor. then viruses were fractured artificially by freeze-thaw treatment. we thus were able to observe that the rna signal of virus rose after cycles of the amplification. for pre-treated groups, the rnase treatment were done and stopped before ghz microwave illumination in order to make sure that rna outside the viral envelope was eliminated in the first place. for the post-treat groups, rnase was added right after the illumination. if the virus particles were fractured, the released rna would be degraded by rnase. ultrasonic absorption evidence for structural fluctuations in frog virus and its subparticles absolute measurement of enhanced fluctuations in assemblies of biomolecules by ultrasonic techniques resonant absorption of ultrasound energy as a method of hiv destruction low frequency mechanical modes of viral capsids: an atomistic approach vibrational modes of nano-template viruses atomistic modeling of the low-frequency mechanical modes and raman spectra of icosahedral virus capsids raman scattering studies of the low-frequency vibrational modes of bacteriophage m in water-observation of an axial torsion mode microwave resonant absorption of viruses through dipolar coupling with confined acoustic vibrations effects of hydration levels on the bandwidth of microwave resonant 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strains by electron microscopy architecture of ribonucleoprotein complexes in influenza a virus particles bending and puncturing the influenza lipid envelope water: a dielectric reference inactivation of bacteriophage by microwave irradiation inactivation of lactobacillus bacteriophage pl- by microwave irradiation influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling microwave dielectric heating of fluids in an integrated microfluidic device this project was sponsored by the ministry of science and technology of taiwan under most - -m- - -my . key: cord- -a whyds authors: hussein, hosni a. m.; akula, shaw m. title: mirna- inhibits kshv, ebv, hsv- infection of cells via stifling expression of interferon induced transmembrane protein (ifitm ) date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: a whyds kaposi’s sarcoma-associated herpesvirus (kshv) is etiologically associated with all forms of kaposi’s sarcoma worldwide. little is currently known about the role of micrornas (mirnas) in kshv entry. we recently demonstrated that kshv induces a plethora of host cell mirnas during the early stages of infection. in this study, we show the ability of host cell novel mir- to specifically inhibit kshv-induced expression of interferon induced transmembrane protein (ifitm ) to limit virus infection of cells. transfecting cells with mir- mimic specifically lowered ifitm expression and thereby significantly dampening kshv infection. in contrast, inhibition of mir- using mir- inhibitor had the direct opposite effect on kshv infection of cells, allowing enhanced viral infection of cells. the effect of mir- on kshv infection of cells was at a post-binding stage of virus entry. the highlight of this work was in deciphering a common theme in the ability of mir- to regulate infection of closely related dna viruses: kshv, epstein-barr virus (ebv), and herpes simplexvirus- (hsv- ). taken together, we report for the first time the ability of host cell mirna to regulate internalization of kshv, ebv, and hsv- in hematopoietic and endothelial cells. recently concluded study, we described a significant increase in the expression of host cell encoded mir- as early as min pi of cells . in the present study, we monitored expression of this mir- at early time points during kshv infection of human b and endothelial cells. we employed human b (bjab) and endothelial (hmvec-d) cells as they are physiologically relevant cells to kshv biology. expression of mir- gradually increased from min pi and peaked at min pi in kshv infected bjab (fig. a) and hmvec-d cells (fig. b) . uninfected bjab and hmvec-d cells did not express mir- (fig. a,b) . expression of known mirnas, hsa-let- c and hsa-mir- - p, were not significantly altered when compared to mir- during early stages of kshv infection of cells (fig. a,b) . treatment of cells with units/ml of heparinase i/iii for h at °c prior to kshv infection of cells resulted in a significant drop in the expression of mir- in bjab (fig. c) and hmvec-d cells (fig. d) . also, infection of both bjab and hmvec-d cells with uv.kshv could induce the expression of mir- to comparable levels as the wild-type kshv (fig. c,d) . these results demonstrate kshv to induce host cell mir- very early upon infection. key features of the novel mir- including the secondary structure are provided in table and supplemental fig. , respectively. to evaluate the biological effects of mir- in target cells, we analyzed the effects of mir- mimic and inhibitor on kshv infection. the range of doses tested in this study is comparable to those reported in the earlier studies [ ] [ ] [ ] [ ] [ ] . the doses of the mimic in panels 'c and d' , student t test was performed to study the effect of hs and compare infection of cells with kshv versus uv.kshv on mir- expression at , , , and min pi. two-tailed p value of . or less was considered statistically significant. *p < . ; **p, . ; ***p < . ; ns-not significant. and inhibitor used in the study did not significantly induce cell death in bjab and hmvec-d cells ( fig. a,b) . transfection of bjab (fig. c ) and hmvec-d (fig. d ) cells with mir- mimic significantly reduced kshv infection of cells as monitored by the expression of orf gene as early as min pi. kshv entry is a quick process and the ie gene, orf , is expressed immediately upon infection . kshv infection was not significantly altered in bjab (fig. c ) and hmvec-d ( fig. d ) cells that were either transfected with scrambled mirna control (mir-nc) or mock transfected. interestingly, the effect of mir- mimic on kshv infection of bjab and hmvec-d cells could be significantly reversed by co-transfecting cells with nm of mir- inhibitor (fig. e ). co-transfection of cells with mir-ns did not alter the effects of mir- mimic (fig. e ). to ascertain that the effect of mir- mimic was at a post-binding stage of infection, we performed a binding assay. the binding assay performed on bjab and hmvec-d cells demonstrated that mir- mimic and the mir- inhibitor did not block kshv from binding the target cells (fig. f ). incubating kshv with heparin but not csa significantly blocked kshv from binding cells (fig. f ). our results clearly implicate mir- to inhibit kshv infection of cells. to extend our understanding of the role of mir- on other related viruses, we tested the effect of mir- mimic and the mir- inhibitor on ebv and hsv- infection of bjab cells. interestingly, mir- mimic could significantly block ebv and hsv- infection of bjab cells and this inhibition could be specifically reversed by the mir-inhibitor (fig. ). mir- targets ifitm . by using the diana and mirmap tool algorithms, we identified a putative mir- binding site located in the ′-utr of ifitm mrna (supplemental fig. ). to confirm the ability of mir- to specifically inhibit ifitm expression, we monitored the expression of ifitm in target cells that were untransfected, transfected with mir- mimic, or mir-nc prior to infection. transfection of bjab and hmvec-d cells with mir- mimic significantly lowered the expression of ifitm at min pi compared to untransfected cells and cells transfected with mir-nc (fig. a) . transfection of cells with mir- mimic could specifically inhibit ifitm expression from min till h pi (data not shown). these results authenticate the fact that ifitm expression may well be regulated by mir- . in order to determine the bona fide target of mir- , a luciferase reporter assay was performed. in this assay, two quantifiable genes encoding luciferase proteins were put on a vector. the ifitm ′ utr with the target region was placed downstream gluc to regulate its translation, and seap was placed under no regulation for normalization. cells were co-transfected with the ifitm ′ utr vector plasmid and mir- mimic. mir- mimic significantly decreased the relative luciferase activity compared to the cells that were transfected with mir-nc (fig. b ). in contrast, transfection of cells with mir-inihibitor reversed the ability of mir- mimic from lowering the luciferase activity (fig. b ). there was an inverse correlation observed in the expression of mir- and ifitm during the course of kshv infection of bjab (fig. c ) and hmvec-d (fig. d ) cells. these results suggest that mir- directly targets ifitm and thereby downregulates its expression. was significantly elevated with kshv infection of bjab and hmvec-d cells (fig. a ). the expression of ifitm increased in kshv infected cells as early as min pi which was elevated by min and min pi in bjab and hmvec-d cells, respectively, but declined sharply by min pi (fig. a ). to confirm a role for ifitm in kshv infection of target cells, we transiently transfected bjab and hmvec-d cells with pqcxip/ifitm and the expression of ifitm was confirmed by flow cytometry (fig. b ). kshv infection of the above ifitm expressing bjab and hmvec-d cells was a measure of the expression of orf at min pi. the idea was to strictly understand the effects of ifitm expression on early stages of kshv infection. bjab (fig. c ) and hmvec-d ( fig. d ) cells expressing ifitm supported a significantly enhanced kshv infection compared to those cells that were left untransfected, mock transfected, or transfected with the empty vector. surprisingly, ifitm expression also enhanced hsv- and ebv infection of bjab and hmvec-d cells (fig. c,d) . interestingly, co-transfection of the above cells with mir- mimic could significantly drop kshv infection of cells compared to mir-nc (fig. c,d) . to further confirm the role of ifitm in kshv infection of cells, we first transfected cells with sirna specific for ifitm . northern blotting was performed at , , , and hours after transfection as per the standard protocols to monitor ifitm mrna expression (fig. a) . the levels of ifitm mrna was significantly suppressed in bjab and hmvec-d cells by sirna when compared with a (ns)sirna control (fig. a) inhibition of % ± %, % ± %, and % ± % was observed in bjab cells at , , and hours, respectively, after sirna transfection (fig. a) . a ifitm mrna inhibition of % ± %, % ± %, and % ± % was observed in hmvec-d cells at , , and hours, respectively, after sirna transfection. ifitm expression levels were not significantly altered by the (ns)sirna controls in both the cells tested, demonstrating the specificity of the sirna used (fig. a ). ifitm expression in target cells transfected with sirna specific to ifitm was significantly lowered at min post kshv infection (fig. b ). in contrast, kshv induced ifitm expression in untransfected or cells transfected with (ns)sirna were not altered (fig. b ). on the same lines, kshv infection in cells silenced for the expression of ifitm was significantly lower compared to cells that were untransfected or transfected with (ns)sirna (fig. c ). silencing the expression of ifitm also decreased ebv and hsv- infection of the above cells (fig. c) . the above viral infections were monitored by performing qrt-pcr. taken together, the results clearly implicate a role for ifitm in enhancing kshv, ebv, and hsv- infection of cells. since mirnas discovery over years ago, mirnas have been established as key players in the molecular mechanisms of mammalian biology including the maintenance of normal homeostasis and the regulation of disease pathogenesis. host mirnas also play a crucial role in mounting an immune response against microbial infections including those caused by viruses . several viruses belonging to herpesvirus, polyomavirus, hepadnavirus, and hmvec-d that were untransfected, mock transfected, transiently transfected with mir- mimic, cotransfected with mir- mimic and mir- inhibitor(mir-mimic + mir-inhib), co-transfected with mir- mimic and nonspecific inhibitor (mir-mimic + mir-ns), or untransfected and treated with heparin or csa. data was plotted to represent the percentage of kshv binding to target cells treated differently compared to the untransfected cells. bars (panels a-f) represent average ± s.d. of five individual experiments. student t test was performed to compare groups. two-tailed p value of . or less was considered statistically significant. *p < . ; **p, . ; ***p < . ; ns-not significant. scientific reports | ( ) : | doi: . /s - - -w adenovirus, and retrovirus encode mirnas , . virus-encoded mirnas (vmirna) identified in virus-infected cells significantly influence viral replication and disease progression by modulating viral as well as host cellular mrna. many of the cellular mirnas affect viral replication either directly by binding to the viral genome or indirectly by targeting host factors related to replication . there are several published works that describe virus and cellular encoded mirnas in kshv pathogenesis , . however, to date, there is no report on the role of cellular mirnas during early stages of kshv infection. recent studies from our laboratory using a combination of deep sequencing and qrt-pcr identified cellular mir- to activate as early as min pi . in the current studies, we employed physiologically relevant human b cells (bjab) and endothelial cells (hmvec-d) to make the study more meaningful to kshv biology. accordingly, using specific primers we analyzed the expression profile of cellular mir- during the first min of kshv infection of cells. a successful viral entry was a measure of kshv to enter cells and express immediate early gene, orf . the expression of orf was monitored by qrt-pcr , . kshv infection of bjab and hmvec-d cells rapidly triggered the expression of mir- as early as min pi that peaked at min pi (fig. ) . expression of mir- in kshv infected bjab is higher than those in hmvec cells. this could be attributed to the inherited variations observed between different cells in the manner by which they respond to virus infection . moreover, the expression level of mirnas has been shown to vary among tissues, cell types, and even between cells of the same lineage , . cellular mir- is triggered by uv.kshv infection of cells while treating target cells with heparinase i/iii prior to infecting cells with the wild-type kshv failed to induce expression of mir- . treatment of cells with heparinase i/iii cleave heparan sulfate (hs) at different sites and liberate them from the cell surface . kshv binds to a target cell via interacting with hs expressed on the cell surface . taken together, our results indicate the following: (i) it is the interactions between the virus envelope proteins and the host cells that trigger mir- response; and (ii) binding of kshv to cells is critical to mir- expression in cells. mir- mimic specifically inhibited kshv infection of bjab and hmvec-d cells (fig. c,d) . transfection of cells with mir- inhibitor reversed the effects of mir- mimic on kshv infection of cells (fig. e) . the effects of mir- mimic and inhibitor was specific as the scramble negative control (mir-nc) and non-specific inhibitor (mir-ns) did not significantly alter kshv infection of cells (fig. c,d,e) . it was concluded that the effects of mir- mimic and inhibitor on kshv infection was at a post attachment stage of internalization as they did not adversely affect virus binding to cells (fig. f) . in this study, we originally wanted to use two relevant viruses as negative controls to better understand the specificity of mir- on kshv infection of cells. ebv and hsv- were selected as controls: ebv, like kshv, belongs to γ-herpesvirinae while hsv- belongs to α-herpesvirinae. interestingly, we observed a similar effect of mir- mimic and inhibitor on ebv and hsv- infection of cells (fig. f ). this could be due to the fact that α, β, and γ-herpesviruses exhibit and share a common three-dimensional capsid structure along with the fact that there is quite a bit of homology in the glycoproteins being expressed on the viral envelope . one mirna may regulate many genes as its targets, while one gene may also be targeted by many mirnas . accordingly, mir- can possibly target several genes (supplemental fig. ). using bioinformatics tools, we identified ifitm to be the most promising targets to mir- . ifitm is a member of the interferon-induced - amino acid protein family including ifitm , ifitm , ifitm , ifitm and ifitm . this family of proteins is located on chromosome of the human genome and originally described as highly inducible genes by αand γ-interferons (ifns) , . the three members of the ifitm proteins (ifitm , ifitm , and ifitm ) have gained prominence as novel antiviral ifn-stimulated genes (isgs) . hence, we set out to test the effect of transfecting target cells with ifitm - genes on kshv infection of target cells. over-expressing ifitm significantly enhanced kshv infection of cells; ifitm moderately enhanced kshv infection while ifitm did not alter the viral infection (supplemental fig. ) . based on these results, we focused our further studies on ifitm in terms of mir- and early stages of kshv infection. using bioinformatic tools it was determined that the mir- target ifitm expression. luciferase assay demonstrated the ability of mir- to physically interact with ifitm (fig. b) . mir- mimic specifically inhibited ifitm expression that could be reverted by transfecting cells with mir- inhibitor (fig. b) . there was an apparent inverse correlation observed between the kshv-induced ifitm expression and mir- response (fig. c,d) . we propose the sharp decline in the expression of ifitm min pi is because of an increase in the expression of mir- (fig. c,d) . taken together, our study established a direct association between virus-induced ifitm and endogenous mir- expression in the biology of kshv. ifitm is expressed in many cell types including leukocytes and endothelial cells , . ifitm modulates cell functions including immunological responses, cell proliferation, cell adhesion, and germ cell maturation . as other ifitm proteins, ifitm is significantly upregulated by interferons type i and ii and is critical for anti-virus . in general, the ifitm family of proteins affects virus entry of cells. over-expression of ifitm significantly enhanced kshv infection of target cells (fig. c,d) while silencing expression of ifitm had the opposite effect (fig. ). more interestingly, we observed identical effects of ifitm in enhancing ebv and hsv- infection of cells (fig. c) . ifns are generally considered to be antiviral cytokines that inhibit virus infection of cells by stimulating isgs . in fact, we observed a significant increase in the expression of ifn-α and -γ during the early stages of kshv infection of bjab and hmvec-d cells (supplemental fig. ). if that is the case, how can ifitm enhance infection of not just kshv; but also of ebv and hsv- ? such a scenario can be possible only if the virus infection is not altered by ifns and associated proteins . interestingly enough, herpesviruses as a group (including kshv) is considered to be relatively insensitive to the antiviral effects of ifns in a variety of different cell systems , . in a way, our results demonstrate for the first time that herpesviruses, kshv, ebv, and hsv- , not only hijack ifitm to their benefit in facilitating virus entry into cells but also evade the ifn-induced antiviral effects. this study provides a new insight to virus infection. kshv (including ebv and hsv- ) interactions with target cells induce ifitm within minutes, which facilitate virus entry into cells. kshv-induced ifitm is part of the innate immune activation system that occurs in an antigen-independent fashion and relies on the ability of the host to recognize virus via specific pattern recognition receptors . to counter the effects of kshv-induced ifitm , infected cells respond within a short period of time by inducing expression of mir- . cellular mir- in turn inhibits expression of ifitm and thus limit virus infection of cells. perhaps, this could be a mechanism of superinfection resistance (sir) developed by cells towards kshv and other related viral pathogens. ifitm suppression by mir- may have direct and indirect effects of kshv pathobiology: (i) directly limit kshv infection of cells; (ii) block cell proliferation/division and thereby promote kshv latency ; and (iii) reduce virus-induced inflammation. such effects of ifitm on the biology of kshv will be better understood by employing the three-dimensional ( -d) cell culture models as they mimic certain aspects of the tissue environment , . for all this time, studies on virus entry have always focused primarily on the roles of virus encoded glycoproteins and their cognate host cell receptors. to our knowledge, this is the first report of a mirna influencing kshv infection of cells and this, we hope, will open doors to a further understanding of virus entry; after all, it is the mirnas that regulate the gene function. of late, mirna-based therapeutics have been used to effectively treat autoimmune diseases , and cancers including prostate cancer, and leukemia in animal models. the fact that mirnas can influence virus entry is fascinating as mirna-based therapeutics like the use of mir-mimics can effectively be used to treat virus entry including pathogenesis. there are several questions that need to be answered and they are as follows: (i) how does ifitm enhance kshv infection of cells? does it physically bind kshv envelope-associated protein and facilitate endocytosis? (ii) what is the role of host cell receptors in the ifitm -facilitated virus entry? (iii) what is the dynamics between the expression pattern of ifitm and in regulating kshv infection of cells? and (iv) can induction of mir- by kshv infection prevent infected cells from being superinfected with kshv and other related herpesviruses, in vivo? current studies in our lab are dedicated to answer these questions. fetal bovine serum (fbs; atlanta biologicals, lawrenceville, ga), l-glutamine, and antibiotics . hmvec-d cells were propagated in egm mv-microvascular endothelial cell medium (clonetics) as per standard protocols. the passage numbers for hffs and hmvec-ds used in this study ranged between - , and - , respectively. all the cells used in this study were negative for mycoplasma as tested by mycoplasma pcr elisa, roche life science. for culture conditions, refer to supplemental data. virus. the viruses used in this study were wild-type kshv , herpes simplexvirus- (hsv- ) , and epstein-barr virus (ebv) . we generated ultraviolet (uv) inactivated kshv (uv.kshv) as per early studies . cytotoxicity assay. the ldh assay was performed using the cytotox non-radioactive kit (promega) as per earlier studies . target cells were treated with different concentrations of mir- mimic and inhibitor at °c in a v-bottom -well plate. after a h incubation, the cells were analyzed for the expression of ldh, as an indicator of cell death. the ldh assay was performed using the cytotox non-radioactive kit (promega) as per earlier studies extracted rna was used to synthesize cdna and the expression of orf was monitored by qrt-pcr using specific primers as per earlier studies expression of orf was used as a scale to measure kshv infection of cells. as reported earlier , the lowest limit of detection in the standard samples was - copies for the orf gene. the results from the use of orf primers were consistently confirmed by monitoring the expression of another viral immediate early (ie) gene, vgpcr (data not shown). ebv and hsv- infection was monitored using specific primers to brlf (homolog of kshv orf ) binding assay. the effect of mir-mimic and inhibitor on kshv binding to target cells was assessed by pcr detecting the cell-bound kshv dna. briefly, untransfected cells or cells transfected with mir-mimic or inhibitor were infected with moi of kshv at + °c. after min of incubation with virus, cells were washed three times with pbs to remove the unbound virus. cells were pelleted, and total dna including those representing the cell bound kshv was isolated using dneasy kit (qiagen, valencia, ca) and subjected to qpcr analysis monitoring orf according to recently published work . incubating kshv with µg/ml of heparin and chondroitin sulfate a (csa; sigma-aldridge) for h at °c were used as known positive and negative controls. western blotting. all the buffers used in this project were made with water that was endotoxin and pyrogen free. western blotting was conducted as per earlier studies using the following primary antibodies: rabbit anti-ifitm polyclonal antibody (emd millipore) and mouse anti-actin antibodies (clone ac- ; sigma-aldridge). the quality of rna was tested using a spectrophotometer. only the rna samples with / ratios of . to . were used in the study. approximately ng of rna was reverse transcribed in a µl reaction volume using the all-in-one tm mirna qrt-pcr detection kit (genecopoeia, rockville, md). briefly, the cdna was synthesized in a μl reaction mix containing μl of x reaction buffer, . u/μl poly a polymerase, ng/μl ms rna, and µl rtase mix. the reaction was performed at °c for min and terminated at °c for min. cdna that was produced in the rt reaction was diluted ten-fold and was used as the template for the pcr reaction in an applied biosystems viia real-time pcr system (life technologies, usa). in this system, ms rna was used as an external reference for the quality of the extracted mirnas, and rnu b, rnu , rnu , and rnu were used for normalization. the expression levels of mirnas were measured employing qrt-pcr with the sybr green detection and specific forward primer for the mature mirna sequence and the universal adaptor reverse primer (genecopoeia, usa). expression of ifn-α, -β, and -γ by qrt-pcr was conducted as per earlier protocols using appropriate primers [ ] [ ] [ ] . scientific reports | ( ) : | doi: . /s - - -w dual-luciferase reporter assay. luciferase reporter plasmids with wild-type ifitm ′-utr were purchased from genecopoeia (rockville, md). cells were plated onto -well plates. at h post-plating, cells were co-transfected with ifitm ′-utr luciferase reporter plasmid and mir- mimic or mir-ncna scramble control (mir-nc) using fugene hd (promega). at , , h post transfection, supernatants were collected from each treatment and the luciferase activity measured using the secrete-pair dual luminescence assay kit (genecopoeia) as per the manufacturers' recommendations. northern blotting. northern blotting to monitor ifitm and β-actin expression was performed using a dig luminescent detection kit (roche, indianapolis, in) as per the manufacturer's recommendations . silencing ifitm using sirna. expression of ifitm was inhibited by the transfection of double-stranded (ds) rna oligos as per standard protocols . ifitm sirna was purchased from dharmacon rna technologies (lafayette, co). briefly, × cells were washed twice in rpmi and incubated in phenol red-free rpmi supplemented with % fbs at °c. after hours incubation (considered as h for experiments in fig. a) , the target cells were transfected with either ds short interfering rnas (sirnas) or the nonspecific (ns) controls using fugene hd as per manufacturer's recommendations (promega). at , , , and hours after transfection, total rna was isolated from the cells and subjected to northern blotting to monitor the expression of ifitm and β-actin mrna as per the protocol mentioned in the "materials and methods" section describing northern blotting. in another set of experiments, untransfected cells and cells transfected with sirna or (ns) sirna for h were infected with moi of kshv. at the end of min pi, kshv infection was assessed by monitoring orf expression by qrt-pcr. data availability statement. all data generated or analyzed during this study are included in this published article (and its supplementary information files). identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma kaposi's sarcoma-associated herpesvirus-like dna sequences in multicentric castleman's disease primary kaposi's sarcoma of the heart in nonimmunodeficient patient: case report and literature review cellular origin of kaposi's sarcoma and kaposi's sarcoma-associated herpesvirus-induced cell reprogramming serodiagnosis for tumor viruses chromatinization of the kshv genome during the kshv life cycle regulation of herpesvirus reactivation by host micrornas noncoding rnps of viral origin kinetics of kaposi's sarcoma-associated herpesvirus gene expression micrornas-based imaging 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fibrosis mir- -socs -jak axis regulates allergic inflammation and allergic inflammation-promoted cellular interactions microrna- - p regulates the chemotherapeutic resistance of hepatocellular carcinoma cells by targeting pten/ jnk/anxa pathway differential responses of pparalpha, ppardelta, and ppargamma reporter cell lines to selective ppar synthetic ligands cell membrane-bound kaposi's sarcoma-associated herpesvirus-encoded glycoprotein b promotes virus latency by regulating expression of cellular egr- the mammalian microrna response to bacterial infections role in hepatitis c virus pathogenesis disintegrin-like domain of glycoprotein b regulates kaposi's sarcoma-associated herpesvirus infection of cells upregulation of cd expression on endothelial cells infected with human cytomegalovirus identification of tissue-specific micrornas from mouse microrna expression differences in human hematopoietic cell lineages enable regulated transgene expression specific binding of the chemokine platelet factor to heparan sulfate human herpesvirus interaction with target cells involves heparan sulfate comparative virion structures of human herpesviruses multiple-to-multiple relationships between micrornas and target genes in gastric cancer evolution of vertebrate interferon inducible transmembrane proteins a single dna response element can confer inducibility by both alpha-and gamma-interferons interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms molecular analysis of a human interferon-inducible gene family ifitm-family proteins: the cell's first line of antiviral defense the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitm proteins restrict hiv- infection by antagonizing the envelope glycoprotein the interferon-induced transmembrane proteins, ifitm , ifitm , and ifitm inhibit hepatitis c virus entry ifitm proteins restrict viral membrane hemifusion the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus interferon induction of ifitm proteins promotes infection by human coronavirus oc antiviral actions of interferons sensitivity of the epstein-barr virus transformed human lymphoid cell lines to interferon kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha interferon-stimulated genes and their antiviral effector functions pattern recognition receptors and inflammation retroviral superinfection resistance silencing of interferon-induced transmembrane protein (ifitm ) inhibits proliferation, migration, and invasion in lung cancer cells kshv vcyclin counters the senescence/g arrest response triggered by nf-kappab hyperactivation -d microwell array system for culturing virus infected tumor cells kshv-initiated notch activation leads to membrane-type- matrix metalloproteinase-dependent lymphatic endothelial-to-mesenchymal transition inhibition of autoimmune diabetes in nod mice by mirna therapy & berindan-neagoe, i. micrornas and cancer therapy -from bystanders to major players b-raf-dependent expression of vascular endothelial growth factor-a in kaposi sarcoma-associated herpesvirusinfected human b cells kaposi's sarcoma-associated herpesvirus (human herpesvirus ) infection of human fibroblast cells occurs through endocytosis epstein-barr virus brlf inhibits transcription of irf and irf and suppresses induction of interferon-beta cellular transcription factor oct- interacts with the epstein-barr virus brlf protein to promote disruption of viral latency discrimination of herpes simplex virus type strains by nucleotide sequence variations ifn-alpha is constitutively expressed in the human thymus, but not in peripheral lymphoid organs rig-i and il- are negative-feedback regulators of sting induced by double-stranded dna a long noncoding rna signature for ulcerative colitis identifies ifng-as as an enhancer of inflammation we sincerely thank ikenna okafor and frank williams to have conducted the double-blinded qrt-pcr experiments. we thank dr. michael farzan (the scripps research institute, jupiter, usa) for kindly providing the plasmid, pqcxip encoding ifitm . technical help by dr. douglas weidner with the use of flow cytometer is highly appreciated. we thank dr. joseph pagano (unc-chapel hill, nc) to have provided us with the ebv stock virus. we thank dr. blossom damania (university of north carolina at chapel hill) to have kindly provided us with the bjab cells. we thank dr. adrian reber, centers for disease control and prevention, to have read and critiqued this report. supplementary information accompanies this paper at https://doi.org/ . /s - - -w. the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - ufjh o authors: liang, li-lin; tseng, ching-hung; ho, hsiu j.; wu, chun-ying title: covid- mortality is negatively associated with test number and government effectiveness date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: ufjh o a question central to the covid- pandemic is why the covid- mortality rate varies so greatly across countries. this study aims to investigate factors associated with cross-country variation in covid- mortality. covid- mortality rate was calculated as number of deaths per covid- cases. to identify factors associated with covid- mortality rate, linear regressions were applied to a cross-sectional dataset comprising countries. we retrieved data from the worldometer website, the worldwide governance indicators, world development indicators, and logistics performance indicators databases. covid- mortality rate was negatively associated with covid- test number per people (rr = . , p = . ), government effectiveness score (rr = . , p = . ), and number of hospital beds (rr = . , p < . ). covid- mortality rate was positively associated with proportion of population aged or older (rr = . , p < . ) and transport infrastructure quality score (rr = . , p = . ). furthermore, the negative association between covid- mortality and test number was stronger among low-income countries and countries with lower government effectiveness scores, younger populations and fewer hospital beds. predicted mortality rates were highly associated with observed mortality rates (r = . ; p < . ). increasing covid- testing, improving government effectiveness and increasing hospital beds may have the potential to attenuate covid- mortality. scientific reports | ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports/ the adequacy of resources to treat large numbers of patients . a recent study analyzed the association between covid- mortality and health care resource availability . in addition, increasing covid- testing has been advocated to attenuate its spreading . the resulting pieces of evidence have not been assembled or applied to explanations of country variations in covid- mortalities. countries vary widely in terms of capacities to prevent, detect and respond to disease outbreaks . we aim to explore factors associated with covid- mortalities at the country level. specifically, we examined whether a key strategy, covid- testing, can reduce covid- mortalities. we also examined whether the severity of covid- outbreak, as measured by the critical case rate and case number explains high numbers of covid- mortalities. furthermore, we investigated whether government effectiveness, or the government's capacity to formulate and implement sound policies to tackle the crisis, can reduce covid- mortality. finally, this study analyzed the associations of covid- mortality with proportions of aged persons, number of hospital beds, preexisting disease patterns and transport infrastructure, a proxy for human mobility. study design and data sources. for this worldwide cross-sectional study, we used data from open access databases. we retrieved covid- related data from the website "worldometer: coronavirus" variables. covid- mortality rate was defined as the number of deaths per covid- cases. since the distribution of covid- deaths was right skewed, we log-transformed the variable to make the data conform more closely to the normal distribution and to improve the model fit. the covid- related factors were the test number per people, case number per , people, and the critical case rate. the critical case rate was calculated by dividing the number of critical cases by the number of covid- infected cases. government effectiveness was measured by wgi government effectiveness scores. these scores captured perceptions of a diverse group regarding the quality of public and civil services (e.g. education and basic health services), the quality of policy formulation and implementation, and the government commitment to such policies . wgi applied a statistical method termed an unobserved component model to standardize data from various sources and to construct indicators. the scores for government effectiveness ranged from − . to . , with a lower value indicating a lower level of effectiveness . population age structure was measured by the percentage of the population aged or older. the number of beds was measured per , people. disease patterns were measured by the percentage of all-cause deaths attributable to communicable diseases. the range of communicable diseases was all diseases excluding non-communicable diseases such as cancer and diabetes mellitus. quality of transport infrastructure was measured by a lpi indicator, "quality of trade and transportrelated infrastructure". the indicator assessed the overall quality of ports, airports, rail, roads, and information technology. the quality score ranged from (worst) to (best), and was estimated to allow for cross-country comparisons . simple linear regressions were first applied to investigate the correlation between covid- mortality rate and test number, because the number of covid- testing is more controllable by government than other predictors in our model. we ranked countries on the basis of their per capita incomes, government effectiveness scores, proportions of population aged or older, and numbers of hospital beds. for each ranking, countries were divided into high, middle/moderate, and low. the goal was to examine whether the relationship between covid- mortality and testing varied with country characteristics. correlation coefficient and p-value of coefficient for test number were calculated for all subgroup analyses. in the multiple regression analysis, covid- mortality rate was regressed on covid- test number, case number, critical case rate, government effectiveness score, proportion of population aged or older, number of beds, deaths attributable to communicable diseases, and transport infrastructure quality score. country populations were used as weights to account for unequal variances in the potential distribution of the disturbance term. the use of weights did not change regression results substantially. all analyses were performed using stata descriptive statistics. relationships between covid- mortality rate and test number are illustrated in fig. . figure a , b, and c demonstrates that covid- mortality rate was negatively and significantly associated with test number for high-income (r = − . , p = . ), middle-income (r = − . , p = . ) and low-income (r = − . , p = . ) countries, respectively. figure e and f exhibits that the negative correlation between covid- mortality rate and test number was significant for countries with moderate (r = - . , p = . ) and low (r = − . , p = . ) government effectiveness scores, respectively. figure h and i displays that the negative correlation was significant for countries with moderate (r = − . , p = . ) and low (r = − . , p < . ) percentage of aged persons, respectively. finally, fig. l reveals that the negative correlation was significant in countries with fewest beds (r = − . , p = . ). results of multiple regression for predicting covid- mortality rates are shown in table . among the covid- related factors, one additional covid- screening test per people was associated with a % reduction in mortality risk (rr = . ; % ci . to . , p = . ). among the country related factors, a . increase in government effectiveness score was associated with a % reduction in mortality risk (rr = . ; % ci . to . , p = . ); a percentage point increase in the population aged or older is associated with a % increase in mortality risk (rr = . , % ci . to . , p < . ). one additional bed per , people was associated with a % reduction in mortality risk (rr = . ; % ci . to . , p < . ). a . increase in logistics infrastructure quality score was associated with a % increase in mortality risk (rr = . ; % ci . to . , p = . ). validation of the prediction model. to validate our regression model, we examined the association between the predicted and the observed mortality rates for each country (fig. ) . the predicted value was obtained from the multiple linear regression. the x axis was the observed morality rate and the y axis was the predicted mortality rate. we excluded singapore and qatar from fig. because they were outliers. the predicted mortality rates were significantly and positively correlated with the observed mortality rates (r = . , p < . ). as robustness checks, we included variables for gdp per capita, health expenditures and primary school enrolment rate in multiple regressions for covid- mortality rate. the variables are summarized in supplementary table s . none of the coefficients for these variables was statistically significant, and the main regression results did not change. therefore, these variables were excluded from the final model. in addition, we conducted analyses for the relationships of covid- mortality rate with gdp per capita and school enrolment rate for different income groups. the results are presented in supplementary s . to the best of our knowledge, this is the first country level study to systematically examine the factors related to covid- mortality. the multiple regression revealed that covid- mortality rate is negatively associated with test number. the effectiveness of population screening for covid- infection to reduce mortality risk is currently being debated. those supporting screening suggest the beneficial effect of identifying asymptomatic patients to attenuate covid- spread. opponents argue that reduced mortality risk is mainly due to increased detection of asymptomatic patients. in the present study, we found that one additional test per people was associated with a % reduction in mortality rate, even after adjusting for case number, critical case rate, and various country-related factors. www.nature.com/scientificreports/ notably, simple regression analyses indicated that the negative association of covid- mortality with test number varied with country characteristics. low-income countries and countries which had the lowest government effectiveness scores, lowest proportions of aged persons, and fewest beds (i.e. those at the bottom one-third of ranking) exhibited the most negative correlation (in terms of correlation coefficient) between covid- mortality and testing. we re-examined these results by including interactions terms between test number and country characteristics; similar conclusions were reached. these results suggest that scaling up testing might potentially serve as an effective approach to attenuate mortality when governments were less effective in controlling disease outbreaks or when hospital beds were less sufficient. greater government effectiveness was found in this study to be associated with lower covid- mortality rates. this indicator captures capacity of government to effectively formulate and implement sound policies, and is a key dimension of good governance. good governance is essential to long-term development outcomes, such as per capita incomes . the present study demonstrated that for short-term crises such as the covid- outbreak, government effectiveness remains critical. for example, an effective government would respond to covid- pandemic proactively by making policies to ensure sufficient supply of personal protective equipment . quick implementation of effective quarantine, lockdown and screening policies , , , as well as provision of good public health services in managing and treating covid- patients, also require an effective government . recent covid- clinical studies have reported associations for mortality with old age and multiple comorbidities , , . we confirmed these observations. countries with higher proportions of people aged or older had significantly higher mortality rates (p < . ). in the present study, bed number was negatively and significantly associated with covid- mortality rate (p < . ). this finding supports the argument that hospital bed is a critical input in treating covid- infected patients who need intensive care . in addition, countries with better trade and transport-related infrastructure appeared to have higher covid- mortality rates (p = . ). a possible explanation is that transport infrastructure facilitated human mobility and movement of goods, which might increase transmissions of covid- among high-risk populations. there are several limitations to the present study. first, this study is based on covid- cases reported by countries. inaccurate reporting and the rapid increases in cases may have influenced the predictive power of our model. however, the trends in the prognostic factors for predicting mortality rates may not have changed. second, the lack of completeness of the database limits our analyses in certain countries, for example test numbers in china and critical case numbers in new zealand and indonesia. third, the covid- related factors used in the present study are from country-level data, not patient-level data. if worldwide patient-level data is made available table . multiple regression for predicting covid- mortality rates. a total of countries were included in the regression analysis. the dependent variable was covid- mortality rate % (log). the r-squared value was . ; adjusted r-squared value was . . a rr: relative risk. b se: standard errors. c,d both government effectiveness and infrastructure quality scores were multiplied by . thus the corresponding relative risk should be interpreted on the basis of a . incremental increase in these indicators. www.nature.com/scientificreports/ for analyses, the prediction accuracy will further improve. fourth, we selected only a limited number of factors that potentially determine the covid- mortality in a country. future studies may explore other country-related factors to improve the prediction accuracy. finally, acquired community immunity after the worldwide spread of covid- may change the prediction accuracy. however, the results of this study can still contribute to future pandemic-related policymaking at the country level. in conclusion, we found that higher covid- mortality is associated with lower test number, lower government effectiveness, aging population, fewer beds, and better transport infrastructure. increasing covid- test number and improving government effectiveness have the potential to reduce covid- related mortality. received: april ; accepted: june a novel coronavirus from patients with pneumonia in china defining the epidemiology of covid- -studies needed covid- : uk lockdown is "crucial" to saving lives, say doctors and scientists covid- : india imposes lockdown for days and cases rise covid- and italy: what next clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical characteristics of deceased patients with coronavirus disease : retrospective study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china as covid- cases, deaths and fatality rates surge in italy, underlying causes require investigation clinical features of patients infected with novel coronavirus in wuhan the effectiveness of quarantine of wuhan city against the corona virus disease (covid- ): a well-mixed seir model analysis projecting hospital utilization during the covid- outbreaks in the united states potential association between covid- mortality and health-care resource availability covid- mass testing facilities could end the epidemic rapidly health security capacities in the context of covid- outbreak: an analysis of international health regulations annual report data from countries the worldwide governance indicators: methodology and analytical issues logistics performance index (lpi) connecting to compete: trade logistics in the global economy, the logistics performance index and its indicators report (the international bank for reconstruction and development/the world bank reflections on leadership in the time of covid- estimating the effects of non-pharmaceutical interventions on covid- in europe an investigation of transmission control measures during the first days of the covid- epidemic in china keeping governments accountable: the covid- assessment scorecard (covid-score) baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region this study is supported in part by yin yen-liang foundation development and construction plan (school of medicine, national yang-ming university) and ministry of science and technology (research center for epidemic prevention of national yang-ming university). the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - -x.correspondence and requests for materials should be addressed to c.-y.w.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - qoksn authors: shi, da; shi, hongyan; sun, dongbo; chen, jianfei; zhang, xin; wang, xiaobo; zhang, jialin; ji, zhaoyang; liu, jianbo; cao, liyan; zhu, xiangdong; yuan, jing; dong, hui; wang, xin; chang, tiecheng; liu, ye; feng, li title: nucleocapsid interacts with npm and protects it from proteolytic cleavage, enhancing cell survival, and is involved in pedv growth date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: qoksn porcine epidemic diarrhea virus (pedv) replicates in the cytoplasm of infected cells, but its nucleocapsid (n) protein localizes specifically to the nucleolus. the mechanism of nuclear translocation, and whether n protein associates with particular nucleolar components, is unknown. in this study, we confirm that a nucleolar phosphoprotein nucleophosmin (npm ) interacts and co-localizes with the n protein in the nucleolus. in vitro binding studies indicated that aa – of n and aa – of npm were required for binding. interestingly, n protein importation into the nucleolus is independent of the ability of npm to shuttle between the nucleus and the cytoplasm. furthermore, overexpression of npm promoted pedv growth, while knockdown of npm suppressed pedv growth. in addition, binding of n protein to npm protects it from proteolytic degradation by caspase- , leading to increased cell survival. taken together, our studies demonstrate a specific interaction of the n protein with the host cell protein npm in the nucleolus. the results suggest potential linkages among viral strategies for the regulation of cell survival activities, possibly through an interaction of n protein with npm which prevents its proteolytic cleavage and enhances cell survival, thus ultimately promoting the replication of pedv. how these interactions may lead to porcine disease. the coronavirus n protein is abundantly produced within infected cells. n protein has multiple functions, including as a structural protein that forms complexes with genomic rna, and plays an important role in enhancing the efficiency of virus transcription and assembly. the identification of host proteins targeted by viral proteins during the infection process provides important insights into the mechanisms of viral protein function. to date, interactions of n protein with numerous host cell proteins have been identified, including hcypa , proteasome subunit p , smad , hnrnp-a , the chemokine cxcl , translation elongation factor- alpha , cellular pyruvate kinase protein and - - . comparative studies among various coronavirus n proteins could aid the development of novel antiviral therapeutics that target interactions between host cell proteins and the n protein . manipulation of multiple host cell factors by a relatively small number of viral proteins is critical for virus replication and spread. given the limited coding capacity of the pedv genome, its protein products must be multifunctional in order to counter host cell antiviral defenses. although originally thought to serve purely structural roles, n proteins of coronavirus are emerging as important players at the virus-host interface. our research group has shown that the pedv n protein localizes not only in the cytoplasm, but also in the nucleolus in infected cells and cells expressing the n protein alone ; however, the factors that determine the nucleolar localization of pedv n protein and the effect of this localization on virus replication are not clearly understood. during infection, a number of viral proteins interact with the nucleolus and are able to reorganize nucleolar antigens , with examples from rna viruses, dna viruses and retroviruses. these include porcine reproductive and respiratory syndrome virus nucleocapsid protein , hepatitis d virus large-delta antigen , marek's disease virus meq protein , the adenovirus iva gene product and v protein , newcastle disease virus matrix protein , and human immunodeficiency virus type (hiv- ) rev and tat . the nucleolus is a highly structured and dynamic nuclear organelle that is involved in the transcription of rrna and in ribosome biogenesis . it contains many proteins, including nucleophosmin (npm /b ), fibrillarin, nucleolin, spectrin, and the ribosomal proteins s and l . in the nucleolus, npm plays a role in centrosome duplication, ribosome biogenesis, intracellular transport, apoptosis and mrna splicing . npm has been ascribed both growth promoting and tumor suppressive functions , . loss of npm results in genome instability, which is manifested by aneuploidy, increase in centrosome numbers, and dna damage checkpoint activation [ ] [ ] [ ] . several different types of cancer cell with elevated levels of npm are more resistant to uv-or hypoxia-induced apoptosis than those with low expression . the ability of npm to suppress apoptosis may play a significant pro-survival role during tumor development . to date, various studies have focused on the nuclear/nucleolar localization properties of the n protein of coronaviruses , , but information on the interactions of n protein with nucleolar proteins, and their impact on the outcome of pedv infection, is limited. here, we show that nucleolus protein npm interacts specifically with the pedv n protein and positively modulates pedv growth. shown that n protein is localized in the cytoplasm and nucleolus . in this study, to determine the intracellular distribution of n protein at the protein level, pedv-infected vero e cells were lysed, separated into nuclear and cytoplasmic fractions, and analyzed by western blotting. as shown in fig. a and b, pcna protein was detected only in the nuclear fraction, whereas gapdh was mostly present in the cytoplasmic fraction, confirming the successful separation of the nuclear and cytoplasmic fractions. the n protein was detected in the virus-infected nuclear and cytoplasmic fractions (fig. c , lanes , and ). in addition, multiple bands appeared on the western blots, because the n proteins of coronaviruses are phosphorylated in virus-infected cells (fig. d ). these results indicate that n is a protein that shuttles between the nucleus and the cytoplasm, which is consistent with the role of n protein in viral replication. n protein interacts and co-localizes with npm . the nucleolus is structurally divided into three major subcompartments: the fibrillar center, a dense fibrillar component, and a granular component . three distinct proteins that are mainly located in the nucleolus have been identified , : the major nucleolar protein fibrillarin, which is a component of a nucleolar small nuclear ribonucleoprotein that is involved in rrna processing; npm , which is a putative ribosome assembly factor; and nucleolin, which is involved in the processing of precursor rrna. to characterize the level of expression of n, npm , fibrillarin and nucleolin, encoding plasmids were constructed with myc or × flag at the n-terminus of each protein. individual expression plasmids were transiently transfected into hek t cells, and their expression was assessed using western blotting (fig. s ). as measured by western blotting using a monoclonal antibody (mab) against n (see supplementary fig. s a ), myc (see supplementary fig. s b ) or flag (see supplementary fig. s c ) tag, individual expression constructs encoding n, npm and fibrillarin proteins were expressed at different levels at the appropriate size, however, the construct encoding nucleolin failed to be expressed. to investigate the possible molecular target of n in the nucleolar, co-immunoprecipitation (co-ip) experiments were performed. the result indicated that n protein showed an interaction with npm in the co-ip assay ( fig. a,b ). to investigate whether n protein is able to interact with endogenous npm in the context of pedv infection, virus-infected vero e cell lysates were immunoprecipitated with an anti-npm mab and probed for the presence of n protein with anti-n mab. n protein was readily detected in pedv-infected vero e cells (fig. c) , indicating that n protein indeed interacts with endogenous npm protein in pedv-infected vero e cells. however, the interaction of n protein with fibrillarin or nucleolin was not observed in the co-ip assay (see supplementary fig. s a and b) . to verify and extend the binding data obtained in the co-ip assay, we performed glutathione s-transferase (gst)-pull down experiments. the gst or gst-npm protein was expressed in escherichia coli and immobilized on glutathione-conjugated sepharose beads. beads carrying gst or gst-npm were incubated with lysates from hek t cells transfected with pcmv-myc-n. after thorough rinsing, the protein complex captured on the beads was solubilized, subjected to electrophoresis in a denaturing gel, and immunoblotted with anti-myc or anti-gst antibody. as shown in fig. d , the gst-npm protein could pull down myc-n. in contrast, gst alone did not pull down myc-n. these data indicate that n protein can specifically interact with npm . to examine the co-localization of n protein with npm , vero e cells were co-transfected with plasmids expressing acgfp-n and dsred-npm proteins, and the subcellular localization of n protein and npm was examined by confocal microscopy (fig. e ). imaging indicated that, as previously shown, the acgfp-n protein localized to both the cytoplasm and the nucleolus, but not to the nucleus, in vero e cells; dsred-npm protein localized to the nucleolus; and co-localization result showed . ± . % of n protein positive cells were npm positive in the nucleolus. moreover, the interaction of n protein with porcine npm protein also was validated in our studies (fig. f ). in total, these data indicated that n protein is able to interact with npm protein. amino acids - of n protein are responsible for binding to npm . to define the specific region of n protein required for the interaction with npm , we used a series of gfp-tagged n protein deletions to map the npm binding site on n protein (fig. a) . the gst-pull down assay revealed that gst-npm bound to gfp-nr , gfp-nr + , gfp-nr + and gfp-n (fig. b ), but not to gfp-nr and gfp-nr . in contrast, gst alone did not pull down gfp-nr (fig. c) . furthermore, constructs lacking the nr domain (gfp-nΔ - ) failed to interact with npm , suggesting that the nr domain of n protein is critical in binding to npm (fig. d ). the c-terminal of npm mediates its interaction with n protein. various functional domains have been identified within npm , including an n-terminal oligomerization domain (oligod) bearing chaperone activity, the c-terminal nucleic acid binding domain (nbd), and two central acid domains for histone binding (histond). to characterize further the interaction of npm and n protein, we mapped the domains of npm necessary for its association with n protein, based on the well-known functional domain of npm , and using a series of gst-tagged npm deletion mutants ( - , - , - , - , - , and - ) fused to gst (fig. e) . the results indicated that the c-terminal (aa - ) is essential for the association of npm with n protein (fig. f) . after separation by sds-page, proteins were detected by immunoblotting with the indicated antibodies. a % aliquot of wcl was also probed to confirm protein expression. the identities of the protein bands are indicated on the right. (c) co-ip of pedv n protein with endogenous npm . pedv-infected (+ ) or mock-infected (− ) vero e cells were used for ip with anti-npm protein mab and immunoblotted with the indicated antibodies. the identities of the bands are shown on the right. (d) gst-pull down assay. glutathione beads conjugated to gst or the gst-npm fusion protein were incubated with recombinant myc-n. after washing, proteins were eluted from the beads and sds-page was performed. the presence of n protein was detected by immunoblotting with anti-myc mab. gst and gst-npm protein expression was confirmed by immunoblotting with mouse anti-gst mab. (e) co-localization of n protein with npm . vero e cells were co-transfected with pacgfp-n and pdsred-npm . the pedv n protein is colored green and the npm fusion protein colored red. merged images are also presented, and the position of the nucleus is indicated by dapi (blue) staining in the merged images. the nucleolus (no) is arrowed where appropriate. lower panels show boxed regions at high magnification. (f) co-ip of hek t cells co-transfected with recombinant constructs encoding myc-n and × flag-tagged porcine npm . scientific reports | : | doi: . /srep npm phosphorylation or sumoylation has no effect in mediating its binding to pedv n protein. it has been shown previously that cdk /cyclin e-mediated phosphorylation of npm on thr- promotes dissociation of npm from centrosomes, allowing the initiation of centrosome duplication . thus, when the t a unphosphorylatable mutant is ectopically expressed, t a binds continuously to centrosomes, resulting in suppression of centrosome duplication . a recent study showed that npm can be sumoylated on both lys- and lys- residues, although lys is the major sumoylation site. mutation of k alters its subcellular distribution, and k r mutation makes npm susceptible to caspase- cleavage and decreases cell proliferation. intriguingly, thr- , lys- and lys- are all located in the c-terminal domain of the npm protein, which is essential for the interaction of npm with n protein. to explore whether thr- , lys- and lys- have a role in the association between npm and n protein, we co-transfected various × flag-npm constructs into hek t cells with myc-n. co-ip assays demonstrated that the unphosphorylated t a, unsumoylated k r and k r were able to bind myc-n, and had either moderate effects or no effect on n protein binding (fig. ). regions of the nucleolus , is associated with preribosomal particles , and forms pentamers that may be important for the assembly of ribosomes . npm has the ability to shuttle between the nucleus and the cytoplasm ; it binds to nuclear/nucleolar localization signal containing peptides , and thus serves as a shuttle protein in nuclear/nucleolar import. interestingly, in this study we also found that the npm interacts with n protein, suggesting that npm may serve as a shuttle protein for transport of n protein into the nucleolus, although the interaction domain of npm -n (aa - ) is not within the fragment of n protein that contains the nucleolus localization signal (aa - ). it is hypothesized that transport of n protein into the nucleolus depends on the movement of npm between the nucleus and the cytoplasm. to examine this hypothesis, we co-transfected hek t cells with pacgfp-n and pdsred-npm , and time-lapse images were acquired at - hpt at min intervals. the transport into the nucleolus, interaction with npm and export of n protein in transfected cells were clearly observed at - hpt. as shown in fig. a , npm protein appeared first in the nucleolus (t = min) and then a small amount of n was observed in the nucleolus at hpt (t = min). n protein accumulated continuously in the nucleolus of transfected cells and interacted with npm until t = - min, and was exported from the nucleolus at t = - min. real-time visualization of the kinetics of nucleolar import, interaction with npm and export of n protein indicated that the process was rapid, taking only min in total, thereby ruling out the possibility that the nucleolar localization of n protein is npm independent. the time-lapse video showing the kinetics of nucleolar translocation of n protein and its interaction with npm is provided as supplementary material (video s in the supplementary materials). to corroborate our findings, we first tested whether depletion of npm by specific sirnas resulted in reduced nuclear import of n. for this purpose, three pairs of npm sirnas were synthesized. these sirnas were transfected into vero e cells, and it was found that npm sirna ( + ) reduced the level of expression of npm (fig. b ). in addition, we examined the viability of cells receiving sirna using the cck- assay. the results showed that there was no difference between npm rnai and rnai control in terms of the viability of transfected cells (see supplementary fig. s ). as shown in fig. c , transport of n protein was hardly affected in cells with reduced npm levels in comparison with that in the cells treated with a scramble sirna (siscr), mock-treated cells. as a control, we also co-transfected vero e cells with myc-n and × flag-npm , but did not observe a stimulation of nuclear import of myc-n with the increased expression of the npm protein (fig. d ). the observation that npm interacts with pedv n protein prompted investigation of the relevance of this interaction to the pedv life cycle. vero e cells were transfected transiently with × flag-npm and subsequently . npm phosphorylation or sumoylation has no effect in mediating its binding to pedv n protein. hek t cells were co-transfected with the indicated plasmids, and the wcl obtained at hpt were immunoprecipitated with anti-flag mab. after separation by sds-page, proteins were detected by immunoblotting with the indicated antibodies. a % aliquot of wcl was also probed to confirm protein expression. the identities of the protein bands are indicated on the right. densitometric data for myc-n/ × flag-npm and mutants from three independent experiments are expressed as mean ± sd. infected with pedv. in these cells, × flag-npm could be readily detected, and the expression of n protein was increased ( fig. a and b) . the results showed that overexpression of npm results in upregulation of n protein expression when compared with the empty vector. an increase in viral titer was also observed in the supernatants of these cells (fig. c) . furthermore, the expression of npm in vero e cells infected with pedv was assessed, and the results indicated that pedv infection increases the expression of endogenous npm (fig. d and e) . given that overexpression of npm significantly affected pedv replication in vero e cells, it was interesting to investigate whether knockdown of npm also affected pedv replication. to this end, sirna-mediated knockdown of npm in vero e cells infected with pedv was investigated. as shown in fig. a and b, knockdown of npm resulted in downregulation of n protein expression at or hpi, accompanied by a significant reduction of viral load in the cell culture supernatants in comparison with that in the cells treated with a scramble sirna (siscr), mock-treated cells, or normal vero e cells (no treatment) (fig. c ). this indicated that growth of pedv was arrested in cells with a reduced level of npm . together with the results of the overexpression experiments, these data highlight the synergistic action of cellular npm expression on pedv replication and n protein expression. programmed cell death, or apoptosis, is an essential event in animal development and is observed in many developing tissues in both invertebrates and vertebrates. the activation of the caspase family is a central event in apoptosis. downstream caspases include caspase- , the precursor form of which is predominantly synthesized in the cytosol . activated caspase- can be translocated from the cytoplasm into the nucleus . caspase- is activated by upstream caspases and then cleaves many intracellular target proteins to induce apoptotic cell death; for example, npm is a substrate of caspase- . to explore whether n protein binding has any role in mediating the apoptotic cleavage of npm , we transiently transfected myc-n and empty vector into vero e cells and then treated with or without m m of ac-devd-cho (caspase- inhibitor). the cells induced apoptotic cleavage of npm by treatment with an apoptosis inducer, staurosporine (sts), or not. apoptotic stimulus clearly demonstrated that npm protein cleavage and occurred in empty vector cells following sts treatment. in contrast, npm was almost intact when n was overexpressed in vero e cells with or no sts treatment, highlighting that nucleolus-targeted n prevents apoptotic degradation of npm (fig. a) . meanwhile, cleaved caspase- was found in sts treated cells (fig. a) . furthermore, ac-devd-cho, a caspase- inhibitor, was able to restore the sts-induced npm and caspase- protein cleavage (fig. b ). this suggests that interaction with n protein protects npm from apoptotic degradation. a number of studies have indicated that npm , one of the major nucleolar phosphoproteins, is involved in the regulation of nucleolar function during cellular differentiation and in antiapoptosis . to evaluate whether the n-npm complex is important in prevention of apoptosis, we studied vero e cells transiently expressing myc or myc-n and induced apoptosis by sts. compared with cells with no sts treatment, a dna fragmentation assay revealed that overexpression of pedv n protein slightly diminished dna degradation. by contrast, robust dna fragmentation was detected in cells transiently expressing myc tag (fig. c ). to evaluate further the antiapoptotic effect of n protein in vivo, we examined the sensitivity of vero e cells transfected with myc-n or empty vector and with induction of apoptosis by treatment with sts or not. dapi and tunel staining of the nucleus revealed that myc-n transfected cells displayed greater viability after stimulation by sts and were markedly less sensitive to sts-induced apoptosis than empty vector transfected cells (fig. d) . collectively, these data demonstrate that the n-npm interaction plays an essential role in protecting cells from apoptotic degradation, thus promoting cell survival. the interaction of viral proteins with nucleolar antigens may explain why viral proteins have been observed in the nucleolus and may also explain the viral exploitation of nucleolar function, leading to alterations in host cell transcription and translation, and disruption of the host cell cycle to facilitate viral replication. our previous and current studies (fig. ) indicate that the pedv n protein is actively transported to the nucleolus during the time course of pedv infection. the function of n protein during pedv infection is thought to require interaction with cellular proteins, therefore in this study we investigated whether the pedv n protein interacts with three major nucleolar antigens: npm , fibrillarin and nucleolin. interaction with one or all of these antigens may explain our previous observations that pedv n protein is localized to the nucleolus . proteins that localize to the nucleolus have been reported to be involved in cell growth, the cell cycle and cell survival , . in the current studies, we also wished to investigate whether interaction of the n protein with nucleolar proteins affects pedv replication. this study was based on different lines of evidence, reflecting both in vivo and in vitro situations, and demonstrated that pedv n protein is able to associate with the major nucleolar protein npm of vero e cells (fig. ) ; we failed to detect an interaction with the fibrillarin or nucleolin (see supplementary fig. s ). the n protein also interacted with porcine npm (fig. f ). in the immunoprecipitation ( fig. a,b ) and the gst-npm fusion protein pull down assay (fig. d) , both in vitro translated and cellular npm were shown to interact with n protein. the immunoprecipitation experiment in pedv-infected vero e cells provided further support for the in vivo binding of npm and n protein (fig. c) . the confocal microscopy analysis showed the co-localization of npm and n in the nucleolus (fig. e) , and the in vitro binding studies utilizing deletion mutants of n or npm defined the binding sites of these two proteins. as shown in fig. b, c and d , apart from the full-length protein, only the n variants containing the nr fragment, nr , nr + and nr + , but not the c-terminal or n-terminal fragment, were able to bind to npm , suggesting that the domain that interacts with npm is within amino acid residues - of n. interestingly, this region of n consists of an sr-domain, containing serine and arginine residues , and is involved in cell signaling and post-translational modifications such as phosphorylation . npm scientific reports | : | doi: . /srep has been reported to bind to the arginine-rich basic region of the human t-cell leukemia virus protein rex , and to the hiv proteins rev and tat . however, further studies will be required to determine more precisely the location of the interaction domain and the specific amino acid residues that participate in the interaction. the protein npm is multifunctional and exhibits nucleic acid binding, ribonuclease activity, and molecular chaperone activity , . these three activities reside in nearly independent but partially overlapping segments of the polypeptide chain . the n-terminal nonpolar region and the acidic region of the middle portion of npm are important for its chaperone activity, and the c-terminal is essential for nucleic acid binding . interestingly, analysis of the binding sites of targeting proteins on npm has revealed that most of them reside in the c-terminal portion of the molecule. for example, npm binds to the nucleolar proteins p , nucleolin , and tat through a fragment of npm containing amino acids - or - . this is in accordance with the interaction of n protein with npm , which occurs at the c-terminal portion of npm (fig. f) . the binding region of npm for viral proteins hdag and rex has been localized to these acidic regions. therefore, these results suggest that the interaction of npm with n protein is similar to the interactions with nucleolar protein p , nucleolin, and tat, but is different from the interaction of npm with the viral proteins hdag and rex. the localization of viral proteins to the nucleolus generally occurs through the interactions of basic regions on the viral protein with stretches of acidic residues on nucleolar proteins such as npm and nucleolin , . upon binding to viral protein in the cytoplasm or in the nucleus, npm and nucleolin function as shuttle proteins, directing the transport of viral proteins across the nuclear pore complex into the nucleoplasm and then to the nucleolus. however, the transport of n protein from the cytoplasm into the nucleolus was not dependent on the shuttle protein npm in this study (fig. and video s in the supplementary materials). one possible reason is that the n protein may bind to importin α and importin β ; both play essential roles in the nuclear transport of proteins through the nuclear pore complex. whether other viral proteins or host factors are involved in the nuclear transport of pedv n protein needs to be determined in the future. the role of n protein during its interaction with npm may represent a unique function of n protein in the nucleolus. npm is a multifunctional protein involved in many cellular and viral activities. in particular, npm interacts with viral proteins from several different viruses and promotes viral replication cycles. interaction between npm and adenoviral protein v promotes virus assembly during virion maturation . npm also forms a complex with hepatitis delta virus (hdv) antigens to enhance replication of hdv rna . in this study, we also demonstrated the synergistic action of cellular npm expression on pedv replication and n protein expression (figs and ) . these interactions link npm with the viral life cycle as an important protein for viral replication. apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. with few exceptions, most studies of virus-encoded antiapoptotic proteins have focused on dna viruses. the known exceptions are the picornavirus-encoded proteins leader and bc , , as well as the rubella virus capsid protein . although infection with these viruses induces apoptosis in many cell lines, this is generally observed late in the infection process. in this report, we have demonstrated that the major isoform of the pedv n protein in infected cells functions to block apoptosis. the protective capacity of n protein is dependent on interaction with npm ; it protects it from proteolytic cleavage, enhancing cell survival, and positively regulates pedv replication and growth. in summary, the key findings of this study are the identification of nucleolus protein npm as a novel interacting partner of the pedv n protein. that npm promotes pedv growth is due to n protein inhibition of caspase- -mediated cleavage of npm , which prevents proteolytic cleavage of npm and enhances host cell survival. the identification and characterization of the interaction of pedv n protein with npm with the resultant alteration in host cell survival may facilitate the development of vaccines and therapeutics for use in pigs. cells were purchased from atcc, grown in dulbeccos modified eagle's medium (dmem) supplemented with % heat-inactivated fetal bovine serum (fbs) and penicillin-streptomycin, and incubated at °c in % co . the pedv strain cv was propagated in pedv-infected vero e cells. virus titers in the culture supernatants of pedv-infected vero e cells were determined by the reed-muench method. plasmids. the plasmids expressing gfp-tagged n, nr , nr , nr , nr + and nr + have been described previously . the npm and fibrillarin genes were amplified from pdsred-npm and the genome of vero e cells, respectively. both were cloned into a p × flag-cmv- vector (e ; sigma) with the ecori and kpni restriction enzymes. the pedv n protein gene was cloned into the pcmv-myc vector ( ; clontech) with the sali and kpni restriction enzymes to generate the pmyc-n plasmid. for bacterial expression of the gst-tagged npm protein, the npm protein gene region was subcloned into the pgex- p- vector ( - - ; ge healthcare), creating pgex-npm . a series of mutant forms of npm was generated from pdsred-npm by conventional pcr with the mutagenesis primers listed in table . additionally, Δ nr , npm (t a), npm (k r) and npm (k r) were generated by overlapping pcr. all plasmids were verified by sequencing. plasmid dna transfection. cells in six-well plates (corning) cultured at °c in a humidified incubator with % co were transfected with the respective plasmids ( m g each) using the attractene transfection reagent ( ; qiagen) according to the manufacturer's instructions. at h post-transfection (hpt), the transfection mixture was replaced with complete growth medium and incubated for an additional h before being used for assays. after dna or small interfering rna (sirna) transfection, cells were infected with pedv strain cv at a multiplicity of infection (moi) of . . after h, the viral inoculum was removed and the infected cells were washed three times with phosphate-buffered saline (pbs; ph . ) and re-fed with dmem containing m g/ml trypsin. at various time points post-infection, cell-free culture supernatants and cell lysates were harvested and stored at − °c until use. preparation of nuclear and cytoplasmic fractions. nuclear and cytoplasmic fractions were prepared as described previously . briefly, treated vero e cells were scraped into ice-cold pbs, centrifuged at × g, and resuspended in ice-cold buffer a ( mm hepes [ph . ], mm kcl, . mm edta, . mm egta, mm dithiothreitol), and then nonidet p- (final concentration, . %) was added. the cells were lysed by five strokes of a dounce tissue homogenizer (bellco glass). the nuclear fraction was pelleted by centrifugation at , × g for s at °c. the supernatant was used as the cytoplasmic fraction. to ensure that the subcellular fractions were separated properly, subcellular lysates were verified by the antibodies against the corresponding fractions. these antibodies were anti-glyceraldehyde- -phosphate dehydrogenase (gapdh) for the cytoplasm and anti-proliferating cell nuclear antigen (pcna) for the nucleus. gst-pull down assays. for the gst-pull down assays, gst or gst-npm protein produced in escherichia coli bl (de ) cells was conjugated to glutathione beads ( ; ge biosciences) and blocked for h in % bovine serum albumin. the beads were then washed three times with tif buffer ( mm tris-hcl [ph . ], mm nacl, mm mgcl , . % nonidet p- , % glycerol, . mm dithiothreitol, mg/ml protease inhibitor) and incubated for h at °c with recombinant myc-tagged n harvested from transfected hek t cells. the beads were washed at least five times with tif buffer, followed by elution and detection of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and immunoblotting. hek t cells were transfected with the indicated constructs as described above. the transfected cells were harvested at hpt, washed three times with cold pbs (ph . ), and lysed with ip lysis buffer ( ; thermo) containing mm phenylmethylsulfonyl fluoride (pmsf) and mg/ml protease inhibitor cocktail ( ;roche) at °c for min. clarified extracts were precleared with protein a/g beads (sc- ; santa cruz) and then incubated with protein a/g beads plus anti-flag (f ; sigma), myc (c ; sigma) or npm (pla ; sigma) mab for - h. the beads were then washed with ip lysis buffer and boiled in sample buffer, and the proteins were subjected to sds-page, followed by immunoblotting analysis with anti-flag, anti-myc and anti-n mab. table . primers used in this study. objective. all images were acquired with -ms exposures under the same illumination conditions and analyzed using las af . . software (leica laser technik; germany). sirnas targeting npm were used at a final concentration of nm, unless otherwise stated. cells were transfected with sirnas with x-tremegene sirna transfection reagent ( ; roche) as described previously the sirna target sequences of npm were ggaagatgcagagtcagaatt (sinpm - ) and ggaagccaagttcatcaattt (sinpm - ). western blotting was used to analyze endogenous npm protein production with anti-npm mab. confocal imaging. vero e cells were seeded on microscope slide coverslips, which were set in -mm diameter dishes, and grown to a confluence of ~ %. at hpt, the cells were fixed with % paraformaldehyde for min. then the nucleus was stained with ′ , -diamidino- -phenylindole (dapi) ( . m g/ml) (d ; sigma) for min and analyzed by laser confocal scanning microscopy (leica laser technik; germany). western blotting analysis. total cellular proteins were extracted with the ripa lysis buffer (r ; sigma) and the concentrations were determined with a pierce ® bca protein assay kit ( ; thermo). the total proteins ( m g) were subjected to sds-page, and separated protein bands were electro-transferred onto a nitrocellulose membrane ( ; pall) using a semidry blotter (bio-rad). the membrane was soaked in blocking buffer (pbs containing % nonfat milk) for h and then reacted with the indicated antibodies: licor biosciences), and thereafter the blots were visualized using an odyssey infrared imaging system (licor biosciences). quantification of band intensities by densitometry was carried out using the image j software. dna fragmentation assay. oligonucleosomal fragmentation of genomic dna was investigated as described below. briefly, × cells in ml of medium were incubated with nm of staurosporine (sts) for h. after incubation, the cells were lysed on ice for min in m l lysis buffer ( . % sds/ % nonidet p- / . mg/ml proteinase k in pbs). genomic dna was extracted by the phenol/chloroform method. the pellet was dissolved in m l of te buffer ( mg/ml rnase) for h at °c. a total of m g of dna was loaded on a % agarose gel and visualized under uv light. terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labelling (tunel) assay. the tunel assay was performed using the in situ cell death detection kit, fluorescein ( ;roche), following the manufacturer's instructions. in brief, vero e cells treated or untreated with sts were fixed in % paraformaldehyde for min at room temperature, washed with pbs, and permeabilized with freshly prepared . % triton x- and . % sodium citrate for min on ice. after washing with pbs, the cells were overlaid with m l of tunel reaction mixture, according to the manufacturer's instruction, and incubated for h at °c. finally, the cells were washed with pbs, then the nucleus was stained with dapi for min and directly analyzed under a fluorescence microscope using an exciting wavelength in the range of - nm ( was used in this experiment) and detection in the range of - nm. quantification of the green fluorescence-positive cells was performed by taking the average of at least six fields of view. cell viability assay. the cell viability assay was performed using the cell counting kit- (cck- ) (ck ; dojindo) according to the manufacture's protocol. in brief, vero e cells were seeded in a -well plate at a density of , per well and incubated at °c for h. the cells were transfected with sirnas (or not), and the plates were incubated for h. subsequently, m l of cck- was added to each well, and the cells were further incubated for h. the optical density at nm was measured. the viability of the treated cells was 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cyclin-dependent kinase -cyclin e and its role in centrosome duplication sumoylation of nucleophosmin/b regulates its subcellular localization, mediating cell proliferation and survival localization of the nucleolar protein no in amphibian oocytes translocation of nucleolar phosphoprotein b ( kda/pi . ) induced by selective inhibitors of ribosome synthesis function of homo-and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins npm , npm and npm during sperm chromatin remodeling major nucleolar proteins shuttle between nucleus and cytoplasm caspases disrupt the nuclear-cytoplasmic barrier spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects increased stability of nucleophosmin/b in anti-apoptotic effect of ras during serum deprivation down-regulation of nucleophosmin/b during retinoic acid-induced differentiation of human promyelocytic leukemia hl- cells identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein intrinsically unstructured proteins and their functions nucleolar targeting signal of rex protein of human t-cell leukemia virus type i specifically binds to nucleolar shuttle protein b- protein b is an important human factor for the nucleolar localization of the human immunodeficiency virus protein tat preferential cleavage in pre-ribosomal rna byprotein b endoribonuclease nucleolar protein b has molecular chaperone activities mapping the functional domains of nucleolar protein b identification of the nuclear and nucleolar localization signals of the protein p . interaction with translocation protein b c interacts with b , a putative nucleolar-localization-signal-binding protein the nucleolar phosphoprotein b interacts with hepatitis delta antigens and modulates the hepatitis delta virus rna replication structure and functions of nucleolin molecular dissection of nucleolin's role in growth and cell proliferation: new insights antiapoptotic activity of the cardiovirus leader protein, a viral "security coxsackievirus protein bc blocks host cell apoptosis by inhibiting caspase- the rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of bax a high-efficiency hela cell nuclear transcription extract antagonistic effects of cellular poly(c) binding proteins on vesicular stomatitis virus gene expression key: cord- - ffuivil authors: cinelli, matteo; quattrociocchi, walter; galeazzi, alessandro; valensise, carlo michele; brugnoli, emanuele; schmidt, ana lucia; zola, paola; zollo, fabiana; scala, antonio title: the covid- social media infodemic date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ffuivil we address the diffusion of information about the covid- with a massive data analysis on twitter, instagram, youtube, reddit and gab. we analyze engagement and interest in the covid- topic and provide a differential assessment on the evolution of the discourse on a global scale for each platform and their users. we fit information spreading with epidemic models characterizing the basic reproduction number [formula: see text] for each social media platform. moreover, we identify information spreading from questionable sources, finding different volumes of misinformation in each platform. however, information from both reliable and questionable sources do not present different spreading patterns. finally, we provide platform-dependent numerical estimates of rumors’ amplification. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ dynamics of hatespeech and conspiracy theories , , the effect of bots and automated accounts , and the threats of misinformation in terms of diffusion and opinions formation , . in this work we provide an in-depth analysis of the social dynamics in a time window where narratives and moods in social media related to the covid- have emerged and spread. while most of the studies on misinformation diffusion focus on a single platform , , , the dynamics behind information consumption might be particular to the environment in which they spread on. consequently, in this paper we perform a comparative analysis on five social media platforms (twitter, instagram, youtube, reddit and gab) during the covid- outbreak. the dataset includes more than million comments and posts over a time span of days. we analyze user engagement and interest about the covid- topic, providing an assessment of the discourse evolution over time on a global scale for each platform. furthermore, we model the spread of information with epidemic models, characterizing for each platform its basic reproduction number ( r ), i.e. the average number of secondary cases (users that start posting about covid- ) an "infectious" individual (an individual already posting on covid- ) will create. in epidemiology, r = is a threshold parameter. when r < the disease will die out in a finite period of time, while the disease will spread for r > . in social media, r > will indicate the possibility of an infodemic. finally, coherently with the classification provided by the fact-checking organization media bias/fact check that classifies news sources based on the truthfulness and bias of the information published, we split news outlets into two groups. these groups are either associated to the diffusion of (mostly) reliable or (mostly) questionable contents and we characterize the spreading of information regarding covid- relying on this classification. we find that users in mainstream platforms are less susceptible to the diffusion of information from questionable sources and that information deriving from news outlets marked either as reliable or questionable do not present significant difference in the way it spreads. our findings suggest that the interaction patterns of each social media combined with the peculiarity of the audience of each platform play a pivotal role in information and misinformation spreading. we conclude the paper by measuring rumor's amplification parameters for covid- on each social media platform. we analyze mainstream platforms such as twitter, instagram and youtube as well as less regulated social media platforms such as gab and reddit. gab is a crowdfunded social media whose structure and features are twitterinspired. it performs very little control on content posted; in the political spectrum, its user base is considered to be far-right. reddit is an american social news aggregation, web content rating, and discussion website based on collective filtering of information. we perform a comparative analysis of information spreading dynamics around the same argument in different environments having different interaction settings and audiences. we collect all pieces of content related to covid- from the st of january to the th of february. data have been collected filtering contents accordingly to a selected sample of google trends' covid- related queries such as: coronavirus, coronavirusoutbreak, imnotavirus, ncov, ncov- , pandemic, wuhan. the deriving dataset is then composed of , , posts and , , comments produced by , , users. for more details regarding the data collection refer to methods. interaction patterns. first, we analyze the interactions (i.e., the engagement) that users have with covid- topics on each platform. the upper panel of fig. shows users' engagement around the covid- topic. despite the differences among platforms, we observe that they all display a rather similar distribution of the users' activity characterized by a long tail. this entails that users behave similarly for what concern the dynamics of reactions and content consumption. indeed, users' interactions with the covid- content present attention patterns similar to any other topic . the highest volume of interactions in terms of posting and commenting can be observed on mainstream platforms such as youtube and twitter. then, to provide an overview of the debate concerning the disease outbreak, we extract and analyze the topics related to the covid- content by means of natural language processing techniques. we build word embedding for the text corpus of each platform, i.e. a word vector representation in which words sharing common contexts are in close proximity. moreover, by running clustering procedures on these vector representations, we separate groups of words and topics that are perceived as more relevant for the covid- debate. for further details refer to methods. the results (fig. , middle panel) show that topics are quite similar across each social media platform. debates range from comparisons to other viruses, requests for god blessing, up to racism, while the largest volume of interaction is related to the lock-down of flights. finally, to characterize user engagement with the covid- on the five platforms, we compute the cumulative number of new posts each day (fig. , lower panel) . for all platforms, we find a change of behavior around the th of january, that is the day that the world health organization (who) issued its first situation report on the covid- . the largest increase in the number of posts is on the st of january for gab, the th january for reddit, the th january for twitter, the th january for youtube and the th of february for instagram. thus, social media platforms seem to have specific timings for content consumption; such patterns may depend upon the difference in terms of audience and interaction mechanisms (both social and algorithmic) among platforms. information spreading. efforts to simulate the spreading of information on social media by reproducing real data have mostly applied variants of standard epidemic models [ ] [ ] [ ] [ ] . coherently, we analyze the observed monotonic increasing trend in the way new users interact with information related to the covid- by using epidemic models. unlike previous works, we do not only focus on models that imply specific growth mechanisms, but also on phenomenological models that emphasize the reproducibility of empirical data www.nature.com/scientificreports/ most of the epidemiological models focus on the basic reproduction number r , representing the expected number of new infectors directly generated by an infected individual for a given time period . an epidemic occurs if r > ,-i.e., if an exponential growth in the number of infections is expected at least in the initial phase. in our case, we try to model the growth in number of people publishing a post on a subject as an infective process, where people can start publishing after being exposed to the topic. while in real epidemics r > highlights the possibility of a pandemic, in our approach r > indicates the emergence of an infodemic. we model the dynamics both with the phenomenological model of (from now on referred to as the exp model) and with the standard sir (susceptible, infected, recovered) compartmental model . further details on the modeling approach can be found in methods. as shown in fig. , each platform has its own basic reproduction number r . as expected, all the values of r are supercritical-even considering confidence intervals (table )-signaling the possibility of an infodemic. this observation may facilitate the prediction task of information spreading during critical events. indeed, according to this result we can consider information spreading patterns on each social media to predict social response when implementing crisis management plans. while r is a good proxy for the engagement rate and a good predictor for epidemic-like information spreading, social contagion phenomena might be in general more complex [ ] [ ] [ ] . for instance, in the case of instagram, we observe an abrupt jump in the number of new users that cannot be explained with continuous models like the standard epidemic ones; accordingly, the sir model estimates a value of r ∼ that is way beyond what has been observed in any real-world epidemic. www.nature.com/scientificreports/ questionable vs reliable information sources. we conclude our analysis by comparing the diffusion of information from questionable and reliable sources on each platform. we tag links as reliable or questionable according to the data reported by the independent fact-checking organization media bias/fact check . in order to clarify the limits of an approach that is based on labelling news outlets rather than single articles, as for instance performed in , , we report the definitions used in this paper for questionable and reliable information sources. in accordance with the criteria established by mbfc, by questionable information source we mean a news outlet systematically showing one or more of the following characteristics: extreme bias, consistent promotion of propaganda/conspiracies, poor or no sourcing to credible information, information not supported by evidence or unverifiable, a complete lack of transparency and/or fake news. by reliable information sources we mean news outlets that do not show any of the aforementioned characteristics. such outlets can anyway produce contents potentially displaying a bias towards liberal/conservative opinion, but this does not compromise the overall reliability of the source. figure shows, for each platform, the plots of the cumulative number of posts and reactions related to reliable sources versus the cumulative number of posts and interactions referring to questionable sources. by interactions we mean the overall reactions, e.g. likes or other form or endorsement and comments, that can be performed with respect to a post on a social platform. surprisingly, all the posts show a strong linear correlation, i.e., the number of posts/reactions relying on questionable and reliable sources grows with the same pace inside the same social media platform. we observe the same phenomenon also for the engagement with reliable and questionable sources. hence, the growth dynamics of posts/interactions related to questionable news outlets is just a re-scaled version of the growth dynamics of posts/reactions related to reliable news outlets; however, the re-scaling factor ρ (i.e., the fraction of questionable over reliable) is strongly dependent on the platform. in particular, we observe that in mainstream social media the number of posts produced by questionable sources represents a small fraction of posts produced by reliable ones; the same thing happens in reddit. among less regulated social media, a peculiar effect is observed in gab: while the volume of posts from questionable sources is just the ∼ % of the volume of posts from reliable ones, the volume of reactions for the former ones is ∼ times bigger than the volume for the latter ones. such results hint the possibility that different platform react differently to information produced by reliable and questionable news outlets. to further investigate this issue, we define the amplification factor e as the average number of reactions to a post; hence, e is a measure that quantifies the extent to which a post is amplified in a social media. we observe that the amplification e u (for unreliable posts posts produced by questionable outlets) and e r (for reliable posts www.nature.com/scientificreports/ posts produced by reliable outlets) vary from social media platform to social media platform and that assumes the largest values in youtube and the lowest in gab. to measure the permeability of a platform to posts from questionable/reliable news outlets, we then define the coefficient of relative amplification α = e u /e r . it is a measure of whether a social media amplifies questionable ( α > ) or reliable ( α < ) posts. results are shown in table . among mainstream social media, we notice that twitter is the most neutral ( α ∼ i.e. e u ∼ e r ), while youtube amplifies questionable sources less ( α ∼ / ). among less popular social media, reddit reduces the impact of questionable sources ( α ∼ / ), while gab strongly amplifies them ( α ∼ ). therefore, we conclude that the main drivers of information spreading are related to specific peculiarities of each platform and depends upon the group dynamics of individuals engaged with the topic. and engagements ( ρ eng ). in more popular social media, the number of questionable posts represents a small fraction of the reliable ones; same thing happens in reddit. among less popular social media, a peculiar effect is observed in gab: while the volume of questionable posts is just the ∼ % of the volume of reliable ones, the volume of engagements for questionable posts is ∼ times bigger than the volume for reliable ones. further details concerning the regression coefficients are reported in methods. table . the average engagement of a post is the number of reactions expected for a post and is a measure of how much a post is amplified in each social media platform. the average engagement e u (for unreliable post) and e r (for reliable post) vary from platform to platform, and are the largest in twitter and the lowest in gab. the coefficient of relative amplification α = e u /e r measures whether a social media amplifies more unreliable ( α > ) or reliable ( α < ) posts. among more popular social media platforms, we notice that twitter is the most neutral ( α ∼ % i.e. e u ∼ e r ), while youtube amplifies unreliable sources less ( α ∼ / ). among less popular social media platforms, reddit reduces the impact of unreliable sources ( α ∼ / ) while gab strongly amplifies them ( α ∼ ). in this work we perform a comparative analysis of users' activity on five different social media platforms during the covid- health emergency. such a timeframe is a good benchmark for studying content consumption dynamics around critical events in a times when the accuracy of information is threatened. we assess user engagement and interest about the covid- topic and characterize the evolution of the discourse over time. furthermore, we model the spread of information using epidemic models and provide basic growth parameters for each social media platform. we then analyze the diffusion of questionable information for all channels, finding that gab is the environment more susceptible to misinformation dissemination. however, information deriving from sources marked either as reliable or questionable do not present significant differences in their its spreading patterns. our analysis suggests that information spreading is driven by the interaction paradigm imposed by the specific social media or/and by the specific interaction patterns of groups of users engaged with the topic. we conclude the paper by computing rumor's amplification parameters for social media platforms. we believe that the understanding of social dynamics between content consumption and social media platforms is an important research subject, since it may help to design more efficient epidemic models accounting for social behavior and to design more effective and tailored communication strategies in time of crisis. table reports the data breakdown of the five social media platforms. different data collection processes have been performed depending on the platform. in all cases we guided the data collection by a set of selected keywords based on google trends' covid- related queries such as: coronavirus, pandemic, coronaoutbreak, china, wuhan, ncov, iamnotavirus, coronavirus_update, coronavirus_transmission, coronavirusnews, coronavirusoutbreak. the reddit dataset was downloaded from the pushi ft.io archive, exploiting the related api. in order to filter contents linked to covid- , we used our set of keywords. in gab, although no official guides are available, there is an api service that given a certain keyword, returns a list of users, hashtags and groups related to it. we queried all the keywords we selected based on google trends and we downloaded all hashtags linked to them. we then manually browsed the results and selected a set of hashtags based on their meaning. for each hashtag in our list, we downloaded all the posts and comments linked to it. for youtube, we collected videos by using the youtube data api by searching for videos that matched our keywords. then an in depth search was done by crawling the network of videos by searching for more related videos as established by the youtube algorithm. from the gathered set, we filtered the videos that matched coronavirus, ncov, corona virus, corona-virus, corvid, covid or sars-cov in the title or description. we then collected all the comments received by those videos. for twitter, we collect tweets related to the topic coronavirus by using both the search and stream endpoint of the twitter api. the data derived from the stream api represent only % of the total volume of tweets, further filtered by the selected keywords. the data derived from the search api represent a random sample of the tweets containing the selected keywords up to a maximum rate limit of tweets every minutes. since no official api are available for instagram data, we built our own process to collect public contents related to our keywords. we manually took notes of posts, comments and populated the instagram dataset. we consider all the posts in our dataset that contain at least one url linking to a website outside the related social media platfrom (e.g., tweets pointing outside twitter). we separate urls in two main categories obtained using the classification provided by mediabias/factcheck (mbfc). mbfc provides a classification determined by ranking bias in four different categories, one of them being factual/sourcing. in that category, each news outlet is associated to a label that refers to its reliability as expressed in three labels, namely conspiracy-pseudoscience, pro-science or questionable. noticeably, also the questionable set include a wide range of political bias, from extreme left to extreme right. using such a classification, we assign to each of these outlets a binary label that partially stems from the labelling provided by mbfc. we divide the news outlets into questionable and reliable. all the outlets already classified as questionable or belonging to the category conspiracy-pseudoscience are labelled as questionable, the rest is labelled as reliable. thus, by questionable information source we mean a news outlet systematically showing one or more of the following characteristics: extreme bias, consistent promotion of propaganda/conspiracies, poor or no sourcing to credible information, information not supported by evidence or unverifiable, a table . data breakdown of the number of posts, comments and users for all platforms. www.nature.com/scientificreports/ complete lack of transparency and/or fake news. by reliable information sources we mean news outlets that do not show any of the aforementioned characteristics. such outlets can anyway produce contents potentially displaying a bias towards liberal/conservative opinion, but this does not compromise the overall reliability of the source. considering all the news outlets that we retrieve from the list provided by mbfc we end up with outlets classified as questionable outlets classified as reliable. using such a classification we quantify our overall ability to match and label domains of posts containing urls, as reported in table .the matching ability that is low doesn't refer to the ability of identifying known domain but to the ability of finding the news outlets that belong to the list provided by mbfc. indeed in all the social networks we find a tendency towards linking to other social media platforms, as shown in table . to provide an overview of the debate concerning the virus outbreak on the various platforms, we extract and analyze all topics related to covid- by applying natural language processing techniques to the written content of each social media platform. we first build word embedding for the text corpus of each platform, then, to assess the topics around which the perception of the covid- debate is concentrated, we cluster words by running the partitioning around medoids (pam) algorithm on their vector representations. word embeddings, i.e., distributed representations of words learned by neural networks, represent words as vectors in r n bringing similar words closer to each other. they perform significantly better than the well-known latent semantic analysis (lsa) and latent dirichlet allocation (lda) for preserving linear regularities among words and computational efficiency on large data sets . in this paper we use the skip-gram model to construct word embedding of each social media corpus. more formally, given a content represented by the sequence of words w , w , . . . , w t , we use stochastic gradient descent with gradient computed through backpropagation rule for maximizing the average log probability where k is the size of the training window. therefore, during training the vector representations of closely related words are pushed to be close to each other. in the skip-gram model, every word w is associated with its input and output vectors, u w and v w , respectively. the probability of correctly predicting the word w i given the word w j is defined as where v is the number of words in the corpus vocabulary. two major parameters affect the training quality: the dimensionality of word vectors, and the size of the surrounding words window. we choose as vector dimension-that is typical value for training large dataset-and words for the window. www.nature.com/scientificreports/ before applying the tool, we reduced the contents to those written in english as detected with cld . then we cleaned the corpora by removing html code, urls and email addresses, user mentions, hashtags, stop-words, and all the special characters including digits. finally, we dropped words composed by less than three characters, words occurring less than five times in all the corpus, and contents with less than three words. to analyze the topics related to covid- , we cluster words by pam and using as proximity metric the cosine distance matrix of words in their vector representations. in order to select the number of clusters, k, we calculate the average silhouette width for each value of k. moreover, for evaluating the cluster stability, we calculate the average pairwise jaccard similarity between clusters based on % sub-samples of the data. lastly, we produce word clouds to identify the topic of each cluster. to provide a view about the debate around the virus outbreak, we define the distribution over topics c for a given content c as the distribution of its words among the word clusters. thus, to quantify the relevance of each topic within a corpus, we restrict to contents c with max � c > . and consider them uniquely identified as a single topic each. table shows the results of the text cleaning and topic analysis for all the data. epidemiological models. several mathematical models can be used to analyse potential mechanisms that underline epidemiological data. generally, we can distinguish among phenomenological models that emphasize the reproducibility of empirical data without insights in the mechanisms of growth, and more insightful mechanistic models that try to incorporate such mechanisms . to fit our cumulative curves, we first use the adjusted exponential model of since it naturally provides an estimate of the basic reproduction number r . this phenomenological model (from now on indicated as exp) has been successfully employed in data-scarce settings and shown to be on-par with more traditional compartmental models for multiple emerging diseases like zika, ebola, and middle east respiratory syndrome . the model is defined by the following single equation: here, i is incidence, t is the number of days, r is the basic reproduction number and d is a damping factor accounting for the reduction in transmissibility over time. in our case, we interpret i as the number c auth of authors that have published a post on the subject. as a mechanistic model, we employ the classical sir model . in such a model, a susceptible population can be infected with a rate β by coming into contact with infected individuals; however, infected individuals can recover with a rate γ . the model is described by a set of differential equations: where s is the number of susceptible, i is the number of infected and r is the number of recovered. in our case, we interpret the number i + r as the number c auth of authors that have published a post on the subject. in the sir model, the basic reproduction number r = β/γ corresponds to the ration among the rate of infection by contact β and the rate of recovery γ . notice that for the sir model, vaccination strategies correspond to bringing the system in a situation where s < n/r ; in such a way, both the number of infected will decrease. to estimate the basic reproduction numbers r exp and r sir for the exp and the sir model, we use least square estimates of the models' parameters . the range of parameters is estimated via bootstrapping , . linear regression coefficients. table reports the regression coefficient ρ , the intercept and the r values for the linear fit of fig. . high values of r confirm the linear relationship between reliable and questionable sources in information diffusion. ∂ t s = −βs · i/n ∂ t i = βs · i/n − γ i ∂ t r = γ i table . results of text cleaning and analysis for all the corpora. vocabulary size topics contents with max � > . the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. received: april ; accepted: september table . coefficients and r of the linear regressions displayed in fig. . www.nature.com/scientificreports/ namin g-the-coron aviru s-disea se-(covid - )-and-the-virus -that-cause s-it part -social and political challenges: . western democracy in crisis? ?sfvrs n= f _ how to fight an infodemic director-general's remarks at the media briefing on novel coronavirus on twitter under crisis: can we trust what we rt false flags, and digital vigilantes: misinformation on twitter after the boston marathon bombing incorporating media data into a model of infectious disease transmission italy prohibits travel and cancels all public events in its northern region to contain coronavirus how people decide what they want to know real-time influenza forecasts during the - season the future of influenza forecasts quantifying search bias: investigating sources of bias for political searches in social media anatomy of news consumption on facebook emergence of metapopulations and echo chambers in mobile agents polarization of the vaccination debate on facebook the spreading of misinformation online science vs. conspiracy: collective narratives in the age of misinformation selective exposure shapes the facebook news diet debunking in a world of tribes the emergence of consensus: a primer echo chambers: emotional contagion and group polarization on exposure to opposing views on social media can increase political polarization polarization and fake news: early warning of potential misinformation targets information disorder: toward an interdisciplinary framework for research and policy making. council of the spread of true and false news online the misinformation machine go eat a bat, chang!": an early look on the emergence of sinophobic behavior on web communities in the face of covid- hate multiverse spreads malicious covid- content online beyond individual platform control what types of covid- conspiracies are populated by twitter bots? first monday fighting the covid- infodemic: modeling the perspective of journalists, fact-checkers, social media platforms, policy makers, and the society an exploratory study of covid- misinformation on twitter influence of fake news in twitter during the us presidential election media bias/fact check, the most comprehensive meida bias check resource differences in the mechanics of information diffusion across topics: idioms, political hashtags, and complex contagion on twitter novel coronavirus ( -ncov) situation report- eight challenges for network epidemic models characterizing super-spreading in microblog: an epidemic-based information propagation model modeling the infectiousness of twitter hashtags phase transitions in information spreading on structured populations fitting dynamic models to epidemic outbreaks with quantified uncertainty: a primer for parameter uncertainty, identifiability, and forecasts estimating epidemic exponential growth rate and basic reproduction number an idea for short term outbreak projection: nearcasting using the basic reproduction number the mathematical theory of infectious diseases and its applications (charles griffin & company ltd, a crendon street the spread of behavior in an online social network experiment modeling confirmation bias and polarization modeling echo chambers and polarization dynamics in social networks fake news on twitter during the us presidential election linguistic regularities in continuous space word representations distributed representations of words and phrases and their compositionality learning representations by back-propagating errors an introduction to the bootstrap the authors declare no competing interests. correspondence and requests for materials should be addressed to w.q.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -v de k authors: lim, chia chiu; woo, patrick c. y.; lim, theam soon title: development of a phage display panning strategy utilizing crude antigens: isolation of mers-cov nucleoprotein human antibodies date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: v de k antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. target antigen preparation is a main bottleneck associated with the panning process. this includes production complexity, downstream purification, quality and yield. in many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as yin-yang panning. a naïve human scfv library with kappa light chain repertoire with a library size of ( ) was developed. the improved yin-yang biopanning process was able to enrich monoclonal antibodies specific to the mers-cov nucleoprotein. three unique monoclonal antibodies were isolated in the process. the yin-yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display. the blocking effect of ptm buffer and e. coli lysate towards αubi and m ko phages against rubi. initially, a proof of concept was conducted to investigate the binding efficiency of the anti-ubiquitin (αubi) scfv phage towards rubi in the complex microenvironment during biopanning. a total of four different conditions (complex microenvironment) were tested stepwise. first, both αubi and m ko phages were assimilated into different blocking agents: pbs, % ptm buffer, e. coli lysate and combinations of % ptm buffer with e. coli lysate. phage titres were determined after binding between αubi to rubi and m ko phage to rubi. the immunotube blocking with e. coli lysate and skimmed milk overnight, wash before use; . adding the desired amount of antibody phage library into the immunotube; . phage library preincubation takes place for h in the immunotube; . bring over the preincubated phage library into antigen-coated well for binding; . stringent washing and elution of phage with trypsin enzymatic action. www.nature.com/scientificreports www.nature.com/scientificreports/ unbound phages were collected as 'blocked phages' and bound phages as 'eluted phages' (fig. ) . based on the phage titres, αubi phage showed good binding capacity towards rubi in the presence of % ptm and e. coli lysate. some amount of m ko phage was also rescued despite incubation in different sets of blocking agents. nevertheless, it should be highlighted that the combination of ptm buffer and lysate managed to block a large amount of αubi and m ko phage in separate sample sets. the extent of blocking was further determined by calculating the ratios of the difference between blocked αubi in a mixture of ptm buffer and lysate against other sets of blocking agents. there was approximately a -fold difference between αubi blocked with a combination of ptm buffer and lysate against the ptm buffer only and lysate only. a . -fold difference was recorded between the ptm buffer and lysate combination against pbs only, where the cut off was -fold. the similar ratios of difference were determined for blocked m ko sample sets at a cut off of -fold. the amount of blocked m ko in the ptm buffer and lysate combination was higher by -fold with respect to m ko in pbs only, . -fold for m ko in lysate only and . -fold for m ko in ptm only. it showed that the combination of ptm buffer and lysate was able to capture more unbound phages than other blocking agents. next, a single round biopanning simulation was conducted in a similar fashion to further investigate the binding capacity of αubi phage towards rubi. αubi and m ko phage were mixed at a ratio of : and was used for biopanning simulation. the phage titres across samples were determined as - cfu/ml, - www.nature.com/scientificreports www.nature.com/scientificreports/ cfu/ml, - cfu/ml and - cfu/ml for 'blocked αubi' , 'eluted αubi' , 'blocked m ko ' and 'eluted m ko ' , respectively ( supplementary fig. s a ). the binding between αubi phage towards rubi was relatively good despite a higher amount of m ko was present in the sample. the ratios of difference between unbound and eluted fractions for αubi phage across samples were -fold, -fold, -fold and -fold (fig. a) . the fold change of unbound and eluted m ko across samples were large ( , -fold, , -fold, , -fold and , -fold). the highest ratio of difference was presented by m ko blocked in ptm buffer, followed by a combination of ptm buffer and lysate and lysate only. this implied that the blocking agents were able to capture the background phages and reduce non-specific binding towards the antigen. lastly, crude rubi was used as the target antigen in a similar biopanning simulation as mentioned above. collectively, the phage titres for unbound αubi phage ( cfu/ml) and unbound m ko ( - cfu/ml) were slightly higher (about - -fold for unbound αubi phage and - -fold for unbound m ko ) than those compared to previous simulations using the purified rubi fraction as shown in supplementary fig. s b . on the other hand, the differences between the eluted αubi phage with eluted m ko were lower compared to the previous simulation. it was - -fold in differences between the eluted αubi phage with eluted m ko for previous simulation whereas the differences dropped to only - -fold, with the exception for sample ( -fold). this was due to the abrupt drop of eluted m ko . the binding capacity of αubi phage towards rubi in crude fraction was consistent with previous simulations, as the similar amount of eluted αubi phages was determined to be in the range of - cfu/ml. the highest ratio of difference between unbound and eluted αubi phage was recorded from sample (ptm buffer only) with a , -fold difference followed by a -fold difference recorded from sample (ptm buffer and lysate). as for the ratios of difference between unbound and bound m ko , the highest fold change was , -fold recorded from sample (lysate only) while the lowest fold change was determined from sample (ptm buffer and lysate) with only , -fold difference (fig. b ). there were different trends exhibited from the fold change between unbound and bound αubi phage with m ko but the blocking effects of ptm buffer and lysate were able to be identified. the phage elisa was conducted to measure the performances and effects of different blocking agents on the binding of αubi phages against crude rubi. out of the four samples, the combination of ptm buffer and lysate showed the highest signal ( . ) detected with anti-c-myc-hrp as shown in fig. c . this combination of blocking agents was able to control the interference of background phages without compromising the binding capacity of αubi phage. therefore, this combination of blocking agents was used for the next stage of biopanning optimization. lysate preblocking and phage preincubation effects towards αubi and m ko phages against rubi. to further optimize the biopanning conditions against crude antigens, the 'yin-yang' (capture and eliminate) approach was designed to reduce non-specific binding by background phages. the approach utilized e. coli lysate and ptm to block the microwells, followed by incubation of phages with ptm buffer in the prepared microwells. this procedure imposes a capture and elimination effect to the phage mixture, enabling non-specific phages to be preoccupied from interfering in the binding activity. initially, αubi and m ko phages were subjected to binding with purified rubi. the unbound phages were collected as 'blocked phages' and bound phages as 'eluted phages' (fig. ) . the overall phage titres for unbound m ko were higher compared to www.nature.com/scientificreports www.nature.com/scientificreports/ the previous optimization without lysate preblocking and phage preincubation by approximately to -fold. also, the differences of unbound and bound m ko were larger compared to the last optimization by more than -fold. therefore, lysate preblocking and phage preincubation was found to reduce the non-specific binding of background phage more effectively. next, a mixture of αubi and m ko ( : ratio) was subjected to incubation with ptm buffer in lysate/ ptm-preblocked microwell ('yin' step) prior to binding with purified rubi ('yang' step). a higher number of unbound m ko phage at cfu/ml was rescued or determined ( supplementary fig. s a ). this was a -fold increase in comparison to the biopanning process devoid of preblocking and preincubation, indicating that the yin-yang process was more efficient in removing non-specific phage particles. however, higher numbers of eluted m ko phage particles were also obtained indicating more background phages were recovered during the simulation. nevertheless, the bound αubi phage was slightly higher than the bound m ko by - -fold. the www.nature.com/scientificreports www.nature.com/scientificreports/ ratio of difference between unbound and bound αubi phage was determined (fig. a ). the highest difference was recorded from sample (pbs only) at , -fold whereas the lowest difference was sample (ptm buffer and lysate) at only -fold. the highest fold change between the unbound and bound m ko was determined from sample (ptm buffer) at -fold, followed by sample (lysate) and sample (ptm buffer and lysate), both were , -fold. this showed that ptm buffer and lysate were suitable for preblocking and phage preincubation. lastly, biopanning was conducted using crude rubi as the target antigen. based on the phage titres in supplementary fig. s b , the amount of unbound m ko ( cfu/ml) were similar to that of the biopanning simulation using purified rubi. this indicates the efficiency of e. coli lysate and ptm buffer blocking to remove non-specific interaction from crude fractions. as for bound phages, the number of bound m ko ( - cfu/ml) however was almost the same as the bound αubi phages ( - cfu/ml) with a difference of only . - -fold. this pattern was expected, as the difference of bound phages in previous simulation (with pure rubi) was also minimal. notably, the amount of bound m ko for sample (ptm buffer and lysate) was -fold higher than αubi phage. this showed a dominant population of empty phages in the eluted phages rather than antibody presenting phages. the highest fold change between unbound and bound αubi phage was recorded from sample (ptm only) at , -fold, followed by sample (pbs only) ( , -fold) and sample (ptm and lysate) ( , -fold) (fig. b) . on the other hand, the highest ratio of difference between unbound and bound m ko was determined from sample (pbs only) at , -fold, followed by the ptm buffer and lysate combination ( , -fold). this implied that the 'yin' step was sufficient to deplete the non-specific binding of m ko . moreover, the binding capacity of αubi phage towards crude rubi was not affected by the 'yin' effect, but in contrast helped to control the interference of background phages. the eluted phages were amplified and packaged for the subsequent phage elisa to assess the 'yin-yang' effect utilising different blocking agents. out of the four samples, the combination of ptm buffer and lysate showed the highest signal ( . ) detected with anti-c-myc hrp as shown in fig. c . despite a drop in the binding of αubi towards the crude rubi that was evident with a lower od nm readout, it could be concluded that the 'yin' effect is essential and the use of the ptm buffer and lysate combination was suitable to reduce the non-specific interaction as designed by the 'yin' effect. human naive kappa light chain scfv phagemid library construction. a naïve scfv library was constructed using healthy human samples focusing on the igm v h and v k repertoire. the diversity of the constructed naïve antibody phagemid library was estimated to be . colony pcr was performed and resolved by agarose electrophoresis as shown in supplementary fig. s . a total of colonies from randomly selected colonies presented a single band of the expected amplicon size at approximately , bp. this indicated that full-length scfv constructs were incorporated into the phagemid vector at a reasonable success rate of %. 'yin-yang' biopanning and polyclonal elisa. the in-house human naïve kappa light chain scfv library was subjected to 'yin-yang' biopanning against crude rmers-np. a higher percentage of blocking agent and an increase in washing stringency were introduced during the biopanning rounds based on the conditions from the earlier optimization steps. in just two rounds of affinity selection, there was an enrichment of specific anti-rmers-np antibodies. supplementary fig. s shows the polyclonal elisa result of crude rmers-np biopanning at absorbance nm. a nm were slightly low at . and . for the first and second round of biopanning respectively. this could be attributed to the low amount of rmers-np being presented in the total crude lysate. the phage titre determination indicates approximately . folds of enrichment was observed. the phage enrichment was tabulated in supplementary table s that complements the polyclonal elisa result. monoclonal elisa. a total of individual clones were selected from the second polyclonal pool for monoclonal elisa assay against crude rmers-np. the positive clones were identified with a cut-off od nm value above . after normalizing with their background values. the readouts of positive controls in the monoclonal elisa were . and . respectively (h and h , labelled as p), while the negative controls were below . (h and h , labelled as n) (fig. ) . overall, the monoclonal elisa assay resulted in positive binders. the generated human naïve library was able to provide satisfactory enrichment for anti-rmers-np. a preliminary screening by colony pcr was subjected to these positive clones to identify clones with full-length inserts before identification by sequencing. sequence analysis. sequencing results of the selected binders were analysed by imgt/v-quest to determine the identity of the scfv monoclonal antibody clones. only full length antibody clones were obtained that showed unique combinations of heavy chain and kappa light chain families. in addition, clones were found to have deletions at their gene segments but not at the complementarity-determining regions (cdrs) and were categorized as partial clones. then, clones presented with umber and ochre stop codons while truncated clones were also obtained from the anti-rmers-np pool. a compilation of the gene segment usage for the full clones is presented in table . preparation of antibody fragments. the clones a , c and g were successfully expressed as inclusion bodies and subjected to solubilization using m urea buffer (ph ). western blotting using streptavidin-hrp was performed to detect the biotinylated antibody fragments as shown in fig. a . the purified recombinant antibody fragments showed a distinct band at the expected size of ~ kda for ra scfv and rg scfv. despite the expected size of rc scfv is ~ kda, it ran slightly higher at ~ kda. www.nature.com/scientificreports www.nature.com/scientificreports/ antigen binding elisa. the preparation of purified rmers-np(his ) and rgfp(his ) for elisa were detailed in the supplementary method . the expression profiles of the antigens were included in supplementary result . the binding profiles of the recombinant antibody fragments were determined against a range of rmers-np(his ) from μg to μg. each sample set was incubated with μg of purified antibody. in order to assess the cross-reactivity of the antibody towards other his -tagged proteins, rgfp(his ) was used. a total of μg of purified rgfp(his ) was coated and allowed to bind with μg of each antibody clone respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ after normalization. the cross-reactivity profiles of the antibody fragments towards rgfp(his ) at absorbance nm after normalizing with their background values are shown in fig. f . the data also takes into consideration cross reaction against streptavidin-hrp. overall, the antibody fragments had very little binding towards the control antigen as the readouts were lower than . . therefore, the antibody fragments could be identified as specific towards the rmers-np. to date, antibody phage display still persist as the method commonly used for the generation of recombinant antibodies against various targets for biomedical applications and research , . the robustness of bacteriophage to display antibodies has aided to expand the portfolio of target antigens which includes toxins, transmembrane proteins, peptides, haptens and even epitopes . unfortunately, the problems of expression and purification of e. coli produced recombinant proteins are atypical. some recombinant antigens have solubility and purity problems , stability issues, difficult to be immobilized and exist in multiple morphologies . due to these reasons, there is a need to develop an alternative procedure for selection towards relevant antigens presented in a complex mixture especially in situations when rapid antibody development is required or if the purified target is unavailable. therefore, we demonstrated a capture and elimination step prior to affinity selection in order to provide a 'subtractive' effect to the phage population by blocking the undesired binders via binding to other components in the blocking mixture while freeing the positive binders to favour binding towards the relevant antigen. the'yin-yang' panning method is somewhat akin to the negative/positive selection panning strategies whereby negative selection aims to remove unwanted binders and allowing positive selection of targeted binders , , . the main difference between this study and the previous methods is the approach and target of negative selection. the target for negative selection is the unspecific antibody phage binders that binds to proteins other than the target antigen. in light of this, the blocking mixture is mainly used to deplete the negative binders. in the previous approaches, polyclonal and mabs are used to target the unrelated epitopes presented on phages in order to select and remove them, allowing the remaining phage clones to select for antibodies that have affinity towards specific epitopes , , . moreover, the 'yin-yang' method offers two rounds of negative selection during a single round of biopanning, one before positive selection and one simultaneously carried out with the positive selection. this creates a competitive situation where a small number of unbound negative phage will compete with a large number of unbound positive phage for the target antigen in the presence of target-unrelated proteins. we demonstrated an inexpensive immobilisation strategy of target-unrelated proteins onto a solid phase for negative selection, whereby unrelated proteins preoccupy non-specific binders, either bound to surface or in phage mixture. the limitation of this method is the inability to remove the non-specific binders prior to binding. however, imposing www.nature.com/scientificreports www.nature.com/scientificreports/ a more stringent washing process during phage elution can compensate for this. therefore, the phages binding to unspecific proteins can be washed away under stringent conditions, subsequently rescuing bound phages that is immobilised on the plate. additionally, this method can also be adapted to membrane-bound proteins by using the western blotting system reported by ding, et al. and liu, et al. . a comparison of other published enrichment strategies and the 'yin-yang' method is provided in table . preliminary optimization was performed in a stepwise manner to investigate the strength of blocking agents towards phage binding capabilities for both antibody phage and phage without antibody using αubi and m ko . the use of αubi as a model phage for optimization represents the specific rubi positive binders while m ko represents the population of non-specific binders. the mixture of ptm and e. coli lysate exerts the highest blocking effect for both αubi and m ko . nonetheless, the bulk m ko population (about cfu/ ml) is expected to saturate the sample thus allowing it to adsorb and bind non-specifically to rubi which can be recovered later. m ko binds at low affinity towards other antigens as reported by lamboy et al. and modifications have been performed to reduce unspecific binding by m ko . to simulate the similar conditions during affinity selection, αubi and m ko were mixed to represent a cocktail of library phage for panning against rubi in purified and crude forms . competition is expected to occur between positive binders and non-specific binders against the target antigen. thus, a competitive selection could be imposed by saturating the non-specific binders, m ko with the competitive proteins (lysate and ptm) thereby promoting binding of αubi towards purified rubi . despite the presence of other unknown e. coli proteins in the crude fraction of rubi with the actual concentration of rubi being expected to be lower than the purified rubi, the binding capability of αubi is still unaffected as shown in phage elisa assay upon affinity selection against crude rubi. the blocking effect of ptm and e. coli lysate was the highest among the samples and was able to block m ko from competing with αubi. there was no bias in the titres of packaged phages for elisa assay as they were normalized to before assayed. during the 'yin-yang' (capture and elimination) step, preincubation of phage was performed in wells coated with e. coli lysate and subsequently with ptm. proper blocking of the surface with blocking agent such as skimmed milk helps to inhibit unspecific binding [ ] [ ] [ ] and phage preincubation can enhance the reduction of unspecific binders even to the treated surface . the idea of using e. coli lysate proteins as one of the competitive proteins in biopanning originates from the whole cell biopanning approach, where target antigen and un-relevant proteins are presented in the cell lysate mixture . theoretically, it could be expected that non-binders were depleted during the 'yin-yang' step, subsequently allowing free positive binders to initiate binding against the target during affinity selection. the 'yin-yang' step complements the blocking effect by the lysate and ptm, www.nature.com/scientificreports www.nature.com/scientificreports/ thereby allowing more non-binders to be trapped. this was evident as a higher number of unbound m ko was recorded. however, throughout the biopanning simulation against rubi in purified and crude forms, there were increasing numbers of non-binders being rescued, close to the amount of positive binders rescued. this could be attributed to insufficient washing that resulted in the rescue of non-binders together with positive binders during elution . despite this, the rescued phages from the crude rubi were amplified and subjected to phage elisa assay to determine the best performing sample. the best performing sample was the lysate and ptm combination that gave the highest readout after background subtraction. hence, we demonstrated that the 'yin-yang' step is crucial during biopanning against crude antigen while the combination of ptm and lysate can help to control and reduce the disturbance of non-binders during antigen binding. nevertheless, due to the rise of background phage elution, washing stringency should be revised accordingly. the rationale of coating the phage pre-incubation well with lysate and then with ptm is to inhibit the plastic binders from interfering in the optimization steps and for further negative selection. in this work, a naïve human antibody phage library was generated for panning against the mers-cov nucleoprotein. the naïve library was constructed from healthy donors comprising of three ethnic groups. this is aimed to expand the diversity of antibody repertoire for a highly diverse antibody library . the 'single pot' antibody library repertoire has a universal characteristic, which makes it suitable to be employed for antibody selection against a wide range of target antigens , . to generate a library of highest possible diversity, size and quality, combinatorial mixing of heavy and light chains was performed to create all possible gene combinations. the v-gene repertoire was amplified using a gene specific primer set with a high coverage of antibody genes . in addition to that, the genetic diversity from donor samples of three ethnic groups may offer additional sequence diversity. cloning efficiency was improved by performing an intermediary cloning to ensure highest incorporation of full antibody gene sequences into the destination phagemid vector. the library generated has a modest diversity of that is sufficient for antibody selection. despite naïve libraries have been reported to yield low affinity binders, it is still an important source for antibodies. a correlation between library diversity with antibody affinity was proposed to be directly proportional . even so, the low affinity binders can undergo in vitro affinity maturation to improve their binding affinity and specificity , , . kappa light chain isotype was solely used with the heavy chain igm isotype repertoire for this naïve library as more kappa antibodies are reported in the human peripheral blood than lambda antibodies. this is because rearrangement of kappa isotypes occurs earlier but when both kappa alleles failed to rearrange productively, this is followed by lambda rearrangement . however, this ratio changes upon encountering antigens, suggesting that light chain rearrangement occasionally takes place in b cell after the onset of somatic hypermutation . this is in line with the structural and physiochemical studies of kappa and lambda antibodies, with both exhibiting differential characteristics during adaptive immune response , . however, in this context, we sought to generate a naïve library for kappa antibodies while further studies on differential properties of lambda and kappa antibodies will be carried out later. in this study, the naïve library was subjected to the revised affinity selection to isolate antibodies against rmers-np from a crude preparation. 'yin-yang' biopanning was able to enrich antibodies against crude rmers-np after rounds of selections by . -fold. the low enrichment ratio was expected due to the loss of binders to interfering proteins from the crude fraction. the method is able to impose both the competitive and subtractive effect onto non-specific binders in order to increase the possibility of positive binders to bind the target in extremely low amounts. reproducibility of this method is fairly good as good enrichment was achieved in several attempts using other in-house antibody libraries against different targets (data not shown). mab selection was done and full antibody clones were obtained for this work. based on the sequence analysis, the v-gene combination of clone rmers-np-a is ighv -igkv , ighv -igkv for rmers-np-c and ighv -igkv for rmers-np-g . the antibody fragments were expressed, purified and their binding specificity determined towards rmers-np. rmers-np-a binds specifically to rmers-np with very minimal background signal while rmers-np-c and rmers-np-g presented some degree of background signal indicating the clones as low performing clones. we noticed a slight difference in the antibody band migration which may likely be due to the introduction of chemical modifications , which in this case is the biotinylation that contributed to the retardation of gel migration. in addition, previous reports examined the efficiency of biotinylation by comparing the gel migration of biotinylated products against non-biotinylated products in the presence of streptavidin and reveal a slower migrating of biotinylated products [ ] [ ] [ ] [ ] [ ] . despite all three antibody fragments underwent biotinylation, the gel shift effect was more drastic for rc scfv at approximately - kda difference in comparison to ra scfv and rg scfv. previously reported neutralizing mabs against mers-cov were specific to the receptor-binding domain (rbd) of the spike (s) glycoprotein of mers-cov . the rbd of mers-cov binds to human dipeptidyl peptidase (hdpp ) (cellular receptor) and gains entry into the target cell for viral infection. most of the reported mabs has been focused on the neutralization of rbd to block the interaction of rbd to hdpp and extreme potency has been exhibited by mabs targeting rbd. despite hdpp is another potential target for neutralization, it is also a particularly important molecule in the immune regulation of t cell activation and chemokine function. therefore, clinical use of it is unadvisable as adverse side effects may arise , . a recent report combined mabs specific to rbd and non-rbd (other domains on s glycoprotein) to initiate neutralizing enhancement as different antigenic sites on s proteins are targeted . nevertheless, immune escape is always endeavoured by coronaviruses by expression of mutant s proteins while nucleocapsid proteins have less mutations. the abundant np of mers-cov makes it another potential candidate target other than the s proteins. nps are responsible for viral genomic rna packaging, transcription and assembly . in a previous report, three mabs against mers-cov rbd with exceptional potency was found to originate from the ighv - heavy chain family . interestingly, in this study, we found that rmers-np-c clone targeting the np also belongs to ighv - heavy chain family. of note is that the ighv - gene was also found to be utilized by other antiviral antibodies targeting hiv- , influenza virus and hepatitis c virus . www.nature.com/scientificreports www.nature.com/scientificreports/ this 'yin-yang' method together with the naïve scfv library was able to select antibodies against a complex antigen by providing competitive and subtractive effects to favour binding of positive binders. this permits antigens in different states (complex mixture, low purity, protein cocktails) to gain excess for affinity binding without the limitations of protein purity and yield. it is likely that optimization of this approach is required for different antigens produced in different host cells due to the differences in cell contents. the generated kappa light chain scfv library was able to generate mabs against mers-cov np using the proposed approach with crude rmers-np. therefore, this kappa exclusive naïve library is potentially useful for isolation of antibodies against other interesting antigens in the future. in conclusion, the 'yin-yang' panning together with the kappa naïve scfv library can be used as an alternative selection process for mab generation against challenging target antigens. to prepare rubi and its complementary e. coli lysate, the recombinant plasmid prset-bh bearing the ubi gene and the empty plasmid with helper plasmid prare were introduced into e. coli bl (de ) strain. the transformed cells were grown in yt media supplemented with . % (v/v) glucose, µg/ ml ampicillin, µg/ml chloramphenicol and mm isopropyl β-d- -thiogalactopyranoside (iptg) at °c for h. the bacterial cell pellets were collected and subjected to cytoplasmic extraction with mg/ml lysozyme followed by min sonication on ice. imac purification was performed for rubi using a ml ni-nta agarose fast flow column (ge healthcare). the recombinant plasmid prset-bh bearing mers-np gene was expressed in a similar fashion using e. coli c (de ) strain and followed by cytoplasmic extraction. the crude proteins and lysates were subjected to sds-page analysis. 'yin-yang' biopanning optimization: blocking effects of ptm buffer and e. coli lysate towards anti-ubiquitin and m ko and the effect of phage preincubation. blocking effects of ptm buffer and e. coli lysate. optimizations were done step-wise to investigate the binding capacity of the antibody towards the antigen in various blocking agents and target-unrelated proteins. initially, a one-step affinity selection was performed separately with αubi and m ko against µg of purified rubi in the presence of various blocking agents. eight microwells were coated with μl of purified rubi in pbs buffer overnight at °c. the wells were washed three times with . % (v/v) tween- (pbs-t) and blocked with μl of ptm blocking buffer ( % (w/v) skimmed milk in pbs-t) for h with rpm agitation at room temperature (rt). simultaneously, the αubi phage ( cfu/ml) and m ko ( cfu/ml) were blocked separately with sets of blocking agents in a total volume of μl: i. pbs buffer, ii. % ptm, iii. e. coli lysate and iv. % ptm and e. coli lysate. the incubation took place for h at rt, with gentle agitation at rpm. then, the antigen-coated wells were washed three times with pbs-t and the blocked phages were transferred into the wells accordingly. the binding took place at rt for h at rpm. the phage mixtures were collected at the end of incubation as 'unbound phages' and the wells were washed three times with pbs-t. the bound phages were eluted by enzymatic digestion with μl of trypsin ( μg/ml in pbs buffer) for min at °c, static. serial dilutions were performed for both unbound and eluted phages. the serially diluted phages were allowed to infect μl of exponentially growing tg culture (od nm = . ) at °c for min, static. the phage titres were determined by μl spots on ampicillin-glucose- yt agar and kanamycin- yt agar plates. the titre data was collected as 'blocked phage' and 'eluted phage' . next, biopanning simulations were carried out using a mixture of αubi ( cfu/ml) and m ko ( cfu/ml) against purified and crude rubi in similar conditions. preparations for antigen-coated wells and ptm-preblocked wells were similar as above. the mixture of αubi and m ko phages was blocked in sets of blocking agents in a total volume of μl: i. pbs buffer, ii. % ptm, iii. e. coli lysate and iv. % ptm and e. coli lysate. the blocking of phages took place for h with rpm agitation at rt and subsequently transferred into antigen-coated wells for binding. phage titre data for unbound phage and eluted phage was determined. for biopanning simulation against crude rubi, μg of crude rubi was coated onto microwells in pbs buffer. the total protein concentration of crude rubi was measured with nanodrop (thermo fischer scientific, ma, usa). the procedures were similar as above. www.nature.com/scientificreports www.nature.com/scientificreports/ effect of phage preincubation. the second stage of optimization was performed with preincubation of phage prior to affinity selection against target antigen. then, μl of purified rubi ( μg) in pbs buffer was coated onto microwells overnight at °c for both αubi and m ko sample sets. concurrently, the same number of wells were blocked with μl e. coli lysate at °c overnight. the antigen-coated wells and lysate preblocked wells were subjected to three times pbs-t washing. the wells were then blocked with μl ptm blocking buffer for h at rt, rpm. the lysate/ptm-coated wells were washed three times with pbs-t and used for phage blocking. αubi and m ko were incubated with various blocking agents in lysate/ptm-coated wells, separately. then, the phages were transferred into antigen-coated wells for binding to occur. the phage titres for both 'blocked phage' and "eluted phage' were determined. for biopanning simulations, a mixture of αubi and m ko was preincubated prior to affinity selection against purified and crude rubi. the rescued phages from biopanning simulations against crude rubi for both optimizations were packaged and amplified. the phages were subjected to phage elisa assay to assess the binding performance in the presence of various blocking agents. the detailed workflow is included in the supplementary methods - . construction of human naïve kappa light chain scfv phagemid library. a total of cdna of healthy human donors from three local ethnic groups (malay, chinese and indian) was used as the starting material for library generation. collection of human blood samples, total lymphocytes isolation, preparation of cdna and library cloning were performed according to lim, et al. with slight modifications. sample collection was performed in accordance with the human ethical approval from the human ethics committee of universiti sains malaysia. all donors were informed about the project and gave their informed consent. all donors are healthy individuals with no known infections and have been physically healthy months before collection. detailed protocol for the library generation is described in supplementary method . 'yin-yang' biopanning of human antibody scfv phage library against crude rmers-np. the affinity selection process was separated into steps. the first step was carried out to capture the non-specific phages that are present in the phage library preparation. the second step was biopanning with 'filtered' phages from the previous steps against the target antigen. a total of . mg of crude rmers-np in µl of pbs buffer was coated to the surface of the microplate wells at °c for h. simultaneously, an immunotube with high binding surface was blocked with equal volumes of e. coli lysate (generated from e. coli c (de ) strain with empty prset-bh ) and % ptm at °c, rotating for h. all interval washes were performed times with pbs-t. the antigen-coated well was blocked with µl of % ptm buffer for h, rpm at rt. 'ying' affinity selection was done in the immunotube with preincubation of library phage ( µl of library phage at cfu/ml, µl of % ptm and µl e. coli lysate) for h, rotating at rt. then, the content in the immunotube was transferred into the well and incubated for h, rpm at °c for 'yang' affinity selection. at the end of binding, the wells were washed with % pbs-t (pbs buffer with % (v/v) tween ) for times and times for the subsequent rounds of biopanning. the bound phages were eluted with µl of trypsin for min at °c. e. coli tg culture was infected with trypsin-eluted phage particles after first round of biopanning. the phage-infected tg cells were propagated and the phage particles were packaged and used for the second round of biopanning. polyclonal antibody phage elisa. the trypsin-eluted phages from two biopanning rounds were amplified and packaged. two wells were coated with . mg of crude rmers-np while concurrently, another two wells were blocked with % ptm as background control. the enriched phages were blocked with equal volumes of % ptm and lysate in lysate/ptm-blocked immunotubes as described. then, the phage mixtures were incubated in antigen-coated and background wells for h at similar biopanning conditions. anti-m -horseradish peroxidase (hrp) was used to detect the bound phages for h at rt, rpm and , ′-azino-bis( -ethylbenzothiozoline- -sulfonic acid) diammonium salt (abts) was used to develop a colorimetric signal. the enrichment pattern was determined at absorbance nm. elisa was carried out using the protocol according to lim, et al. with slight modifications. briefly, phage particles with the highest enrichment was subjected to tg infection and single colonies were picked and cultured. the performance of the monoclones was assayed against . mg of crude rmers-np. % ptm was used as the sole blocking agent and three times washing with pbs-t was used at all the interval washes. the monoclonal phage particles were transferred into antigen-coated elisa microplate and incubated for h at °c, rpm. then, anti-m hrp was introduced to detect phage binding followed by abts developing solution. the absorbance was measured at od nm within min. the detailed protocol is outlined in supplementary method . dna sequencing. the positive clones from the monoclonal elisa were cultured at °c and rpm for h. the cell pellets were collected and plasmid extraction was performed using qiaprep spin miniprep kit (qiagen, germany). the purified plasmids were sent for sequencing (first base laboratories sdn bhd, malaysia). the sequencing results were then analysed by imgt/v-quest bioinformatics tool available at imgt ® , the international immunogenetics information system ® , . a scfv, c scfv and g scfv dna were amplified from their respective plabel vectors and cloned into e. coli expression vector prset-bh at restriction sites ncoi and noti (neb, ma, usa). the resulting vectors were named prset-bh a scfv, prset-bh c scfv and prset-bh g scfv. expression of a , c and g was performed with shuffle ® t with prare for in vivo biotinylation. the expression condition for biotinylated a , www.nature.com/scientificreports www.nature.com/scientificreports/ c and g was optimized to °c, rpm for h with iptg induction at od nm . at a final concentration of . mm and μm d-biotin. the inclusion bodies of biotinylated a , c and g were solubilized using m urea buffer, ph ( m urea, . m nah po , . m tris). the urea crude fractions were subjected to protein purification using ni-nta agarose gravity column (qiagen, germany) at denaturing conditions. initially, the column was equilibrated with m urea buffer at ph followed by sample loading. then, the column was subjected to washing with ml of m urea buffer (ph ). step elutions were performed by m urea buffer at ph . , . and lastly . , ml elution fractions were collected stepwise. the protein fractions were analysed on sds-page. the last fractions from a , c and g were combined and subjected to buffer exchange using amicon ® ultra . ml centrifugal filters (merck, darmstadt, germany). the antibody fragments were dissolved in x pbs buffer (ph . ). western blotting was then performed using streptavidin-hrp ( : ) (thermo scientific, ma, usa) to detect and confirm the respective antibody fragments. antigen binding elisa. the purified rmers-np (fused with his -tag only) fractions, purified rgfp (fused with his -tag only) fractions and purified antibody fragments were quantified (detailed in supplementary method ) . a serial dilution of purified rmers-np(his ) (μg: , , , , , ) was coated onto microwells in pbs buffer with a total volume of μl at °c overnight. all interval washes were performed times with pbs-t. the antigen-coated wells were blocked with % ptm blocking buffer for h at rt, rpm. the respective antibody fragments (a , c and g ) were resuspended in pbs buffer, with each μl aliquots representing μg. post-blocking, the wells were washed and each well was added with μl of antibody fragments ( μg). additionally, empty wells were also added μl of antibody fragments as negative control wells. to check the cross-reactivity of antibodies towards his -tagged proteins, wells were coated μg of rgfp(his ) and subsequently allowed to incubate with μl of antibody fragments ( μg). the antigen-antibody binding took place at rpm, °c for h. then, μl of streptavidin-hrp (diluted at : in ptm blocking buffer) was added into each well and incubated for h at rt, rpm to detect antigen-antibody binding. lastly, μl of abts developing solution was added after washing. the absorbance was measured at od nm within h. filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface designing human antibodies by phage display by-passing immunization: human antibodies from v-gene libraries displayed on phage developability and functional size: the holy grail of combinatorial antibody library generation molecular considerations for development of phage 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syndrome coronavirus spike glycoprotein to avoid neutralization escape development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germlinelike antibody generation and analysis of the improved human hal / antibody phage display libraries site-specific scfv labelling with invertase via sortase a mechanism as a platform for antibody-antigen detection using the personal glucose meter generation of a naïve human single chain variable fragment (scfv) library for the identification of monoclonal scfv against salmonella typhi hemolysin e antigen imgt/v-quest: the highly customized and integrated system for ig and tr standardized vj and vdj sequence analysis imgt standardized analysis of the immunoglobulin (ig) and t cell receptor (tr) nucleotide sequences rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library biopanning and rapid analysis of selective interactive ligands subtractive single-chain antibody (scfv) phage-display: tailoring phagedisplay for high specificity against function-specific conformations of cell membrane molecules microselection-affinity selecting antibodies against a single rare cell in a heterogeneous population selection of antibodies against a single rare cell present in a heterogeneous population using phage display masked selection: a straightforward and flexible approach for the selection of binders against specific epitopes and differentially expressed proteins by phage display optimized selection of anti-tumor recombinant antibodies from phage libraries on intact cells targeting membrane proteins for antibody discovery using phage display t.s.l. contributed to the conception of the study, analyzed the data and supervised the study. c.c.l. planned and performed the experiments, collected data and performed data analysis. p.c.y.w. provided important dna material for the study and assisted in data analysis. all authors provided critical feedback to this study and contributed to manuscript writing. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -zj ro j authors: dipaola, joshua d.; yindee, marnoch; plotnik, joshua m. title: investigating the use of sensory information to detect and track prey by the sunda pangolin (manis javanica) with conservation in mind date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: zj ro j pangolins are of conservation concern as one of the most heavily poached, yet least understood mammals. the sunda pangolin (manis javanica) in particular is a critically endangered species. here, we investigate the behaviour of these pangolins, for the first time, using a battery of cognitive tasks based on a manipulation of available sensory information. in an object-choice task in which only one of two containers was baited with food, the pangolins were able to find the food with olfactory information alone (n = ), but not with visual or acoustic information alone (n = ). the single subject tested on all three domains was further tested on how he used smell to find food by providing him with an opportunity to find it from a controlled distance or by using scent trails as a guide. the results suggest that our subject may have the capacity to exploit scent trails left by prey which can be tracked to a final source, though we found no evidence to suggest that he had the ability to initiate hunts based on distant prey odors. despite the small sample size, this is the first controlled experiment to investigate pangolin foraging behaviour and cognition, which may have implications for the future protection of pangolin habitat based on the location of prey species. pangolins are of conservation concern as one of the most heavily poached, yet least understood mammals. the sunda pangolin (manis javanica) in particular is a critically endangered species. here, we investigate the behaviour of these pangolins, for the first time, using a battery of cognitive tasks based on a manipulation of available sensory information. in an object-choice task in which only one of two containers was baited with food, the pangolins were able to find the food with olfactory information alone (n = ), but not with visual or acoustic information alone (n = ). the single subject tested on all three domains was further tested on how he used smell to find food by providing him with an opportunity to find it from a controlled distance or by using scent trails as a guide. the results suggest that our subject may have the capacity to exploit scent trails left by prey which can be tracked to a final source, though we found no evidence to suggest that he had the ability to initiate hunts based on distant prey odors. despite the small sample size, this is the first controlled experiment to investigate pangolin foraging behaviour and cognition, which may have implications for the future protection of pangolin habitat based on the location of prey species. one of the most important confounds in the design of comparative cognition research centers on the question of ecological validity. drawing cognitive comparisons across evolutionarily distant taxa requires a basic understanding of an animal's umwelt, with particular attention to how behaviours such as foraging and problem-solving are affected by sensation. for instance, while developing puzzles that require visual access and manipulation with tools makes ecological sense for animals like chimpanzees - and corvids , , apparatuses that provide olfactory information and can be manipulated using an animal's own body parts are more relevant for elephants [ ] [ ] [ ] and dogs , . understanding the sensory perspective of a particular species is even more critical when designing behavioural or cognitive tests for animals that have rarely, if ever been studied previously. investigations of the proximate, cognitive mechanisms that underlie an animal's decision-making processes can have important implications both from a theoretical (i.e., for understanding how cognition evolves across species to help an animal forage, interact with conspecifics, and avoid predation, for example) and an applied conservation perspective. for the latter, there is a growing need for conservationists developing endangered species strategies to collaborate with ecologists, biologists and psychologists in order to gain a greater understanding of animal behaviour and cognition. to this end, the experimental object-choice task can be used to better understand the importance of particular sensory modalities in an animal's foraging behaviour. this task typically involves the presentation of two or more options where only one is baited with food. the experimenter can control how much or how little sensory information is available to the animal when it investigates and subsequently makes a choice between the provided options. this task has been used to investigate different capacities, including cue-following in the visual (e.g. [ ] [ ] [ ] ), subjects. this study was conducted between january-march and june-july, with two wild-born, sexually mature sunda pangolins (m = , f = ) housed at the livestock and wildlife hospital of the faculty of veterinary science, mahidol university, kanchanaburi, thailand. permission to conduct this research was granted by the director of the hospital (co-author, m.y.), in whose care these pangolins were under. our initial sample included three subjects (m = , f = ), however, one pangolin showed no interest in the testing apparatus while being habituated to the research materials and thus, no data were collected on this subject. the two subjects that participated in the study were tested directly within their enclosures to minimize stress associated with handling. the pangolins' housing consisted of semi-enclosed rooms in an outdoor barn at the veterinary hospital, and the animals had access to water, shelter and climbing structures. the pangolins had strict diets developed by the veterinary staff to maintain their health. they were each given a predetermined quantity of dead weaver ants (oecophylla smaragdina) once per day (in the evening), with occasional supplements of live ants. due to the predetermined, singular feeding period as well as the relatively fragile nature of the pangolins' health in captivity, we did not withhold or reduce their diet to increase their motivation levels for testing. the hunter college institutional animal care and use committee (protocol # jp-pangolin- / ) reviewed and approved this research, and the methods were carried out in accordance with the relevant guidelines and regulations. procedure. these animals were first tested in phase i of this study on their ability to locate food, a sample of ants, across foraging tasks within three sensory domains: visual, acoustic, and olfactory (see plotnik et al. for similar procedures on which the current research is based). because sunda pangolins are nocturnal , , , , , all testing was conducted between - hrs. for initial sensory testing, the pangolins were presented with a basic object-choice task in which two hard plastic cm × cm × cm containers, one containing food the other not, were presented inside a wooden testing chamber. each container rested flat on top of its own sliding wooden platform, which had a cm vertical wooden handle attached so that the experimenter could manipulate the position of the containers throughout a trial without physically entering the testing chamber (fig. ) . because the pangolins were wild-caught and had limited experience with novel objects prior to testing, we first habituated the animals to the paradigm by allowing them to retrieve food from a single baited container without a lid. we then gave them an opportunity to search two open containers, with only one baited, and then repeated this inside the testing chamber. once they voluntarily entered the chamber and searched the two containers consistently, testing www.nature.com/scientificreports www.nature.com/scientificreports/ began on the two-object choice task. for each trial of testing in phase i, the pangolin could first approach two locked containers (with lids) placed adjacent to each other on their respective wooden platforms. during this time, the pangolin could directly touch and investigate both containers. after s, the experimenter used the vertical handles to pull the two containers to the back of the chamber where a short, cm partition separated the two containers. the purpose of this partition was to isolate each container to make the decision-making process as clear as possible to both the subject and experimenter. the pangolin was then given s to make a choice. the criterion for 'choice' required that the pangolin pass the partition and touch the container with its snout or claws. this criterion for 'choice' was used for all conditions except phase ii 'olfactory distance' testing (see below for details on this condition). the experimenter then manually unlocked the lid of the chosen container and withdrew the alternative by lifting it out of the apparatus using the vertical handle. if a pangolin failed to make a choice . the view is from behind the testing apparatus. once the pangolin enters the chamber (from the top of the image), the metal mesh screen is removed and the pangolin can investigate the two containers. the containers are then pulled to the back of the chamber, at which point the pangolin can make a choice and the lid of the chosen container is removed. (c) experimental setup for 'olfactory distance' testing in phase ii. similar to phase i, the pangolin entered the chamber (from the top of the image), the screen was removed, and the pangolin could investigate the two containers, which were positioned on top of pvc pipes above the pangolin's head. this photograph represents a -cm training trial. (d) experimental setup for 'scent trail' testing in phase ii. the pangolin could again enter the chamber but there was no screen to remove; the pangolin could follow the scent on either of two wooden planks that led to a baited ('food' plank) or unbaited ('water' or 'mint' plank) container. this image does not include the cloth tape applied to the planks prior to testing. all photos by j.d.d. in the time allotted, the trial was reset. all experimental testing was voluntary, with the pangolins being allowed to enter and exit the testing area at will as soon as the testing apparatus was rebaited after each trial. rebaiting of containers happened out of view of the pangolins between trials, and metal mesh screens were used to temporarily block off the apparatus while it was rebaited. because we did not want to handle the pangolins or restrict their movement within their enclosures, there was no specific inter-trial interval (iti) established for testing. in phase i of testing, the pangolins were presented with three conditions that each provided sensory information about the presented food from a single modality (see supplementary video s ). the containers were manipulated so that they were either: a) opaque, held g of dead ants and were locked with perforated lids (so that the pangolins could smell but not hear or see the ants -the 'olfactory' condition), b) transparent with solid lids and g of dead ants (so that the pangolin could see but not hear or smell the ants -the 'visual' condition), or c) opaque with solid lids and live ants (so the pangolin could hear but not see or smell the ants -the 'acoustic' condition). containers and lids were made opaque using black duct tape applied on the inside. in this last condition, the average weight of the live ant nests (the ants plus the leafy substrate from their environment) in the closed baited container was g. in addition, even though the pangolins are nocturnal, the 'visual' condition was conducted either in the presence or absence of an artificial light to investigate whether visual light made any difference in the pangolin's ability to locate the food. control trials utilized opaque containers, solid lids and g of dead ants (so that pangolins had no access to sensory information about the ants), and allowed us to investigate whether the pangolins were using cues independent of the food present -e.g., inadvertent experimenter cuing -to make a choice. the -g amount of dead ants for each trial within a non-acoustic condition was determined based on the average weight of a live ants' nest minus the substrate, as well as a relatively equal distribution of the pangolin's daily diet across an evening's test trials as determined by the hospital's veterinary staff. subjects were given the opportunity to participate in one testing session per day across both phase i and phase ii of this study. testing was conducted on consecutive days unless the subject failed to voluntarily participate, in which case testing was delayed until the next day. phase i conditions were presented in the following order: olfactory, visual no-light, visual light, acoustic untreated, and acoustic treated. the 'olfactory' condition consisted of four sessions of trials (eight test and four control). the 'visual' condition consisted of two parts: four sessions of trials (eight test and four control) presented in the absence of an artificial light source (no-light), followed by an equal number of trials in the presence of light. in the first three sessions of the 'acoustic' condition, live weaver ants were collected from the environment and immediately presented to the pangolin in the same containers for testing ('acoustic untreated' -three sessions of trials (eight test and four control)). it was very difficult to move the ants between containers once they had been collected due to their high level of aggression and fast movement. because we realized later that the ants may have left olfactory cues on the exteriors of the containers during collection, we ran an additional three sessions of trials in which we meticulously cleaned the outside of the containers between ant collection and presentation to the pangolin using a mild soap and water solution ('acoustic treated'). in all types of condition, containers were cleaned between sessions. in the 'acoustic' condition, the 'baited' container remained unchanged throughout a session (due to the impracticality of moving a live ant colony easily between trials), however, the 'baited' container was placed on each side of the apparatus an equal number of times. in this 'acoustic' condition, the pangolin was rewarded with a separate quantity of dead ants if it correctly chose the container with live ants, to avoid having to open and rebait the live ant container. because the 'olfactory' and 'visual' conditions utilized dead ants throughout, both containers were 'baited' or 'unbaited' an equal number of times within each session to avoid any confound of residual odor (e.g., from previously baited food or the pangolin's own scent). all trials across the 'olfactory' , 'visual' , and 'acoustic' conditions of phase i were pseudo-randomised within a session so that each session consisted of an equal number of trials of left and right correct choices (based on the side of the chamber in which a container was placed), no one side was baited with food more than three consecutive times in a row, and the first three trials for each session across all conditions were always tests rather than controls to avoid a frustration effect. the female pangolin, 'betty, ' was only tested on a portion of her first condition, the initial 'olfactory' condition (table ) , because we noticed a significant decrease in both her nightly food intake and her interest in entering the experimental apparatus as testing progressed. because we did not want to negatively impact her welfare state, she was not subjected to any further testing. the male pangolin, 'pluto' , was tested on all of the conditions as he remained highly motivated throughout testing. in phase ii of the study, the male pangolin was tested on two different olfactory conditions. the 'olfactory distance' condition specifically aimed to investigate whether pangolins may detect their prey or initiate a hunt based on distant olfactory cues (see supplementary video s ). this condition was similar to the phase i olfactory condition with the following exceptions. first, in 'olfactory distance' trials, the pangolin could not physically touch either container while he first investigated them. instead, he could only smell, but not touch the containers from a distance of , or cm. second, the pangolin was given s to investigate both containers in 'olfactory distance' testing compared to the s used for the olfactory condition in phase i. the investigation time was increased for 'olfactory distance' trials to ensure that the pangolin had sufficient time to navigate the additional spatial components of this condition (details below). third, the containers were no longer presented to the pangolin for investigation on the floor of the apparatus in 'olfactory distance' . they were now positioned overhead on a vertical plane to best mimic what the pangolin might spatially encounter in an arboreal hunt; he would now need to smell upwards in order to gain access to the food's olfactory information. the containers were placed porous lid-side down on top of a solid, pvc tube cut to the predetermined length/distance and set on top of a wooden plank overhanging the testing chamber (fig. ) . holes were cut into this plank so that the pangolin could smell into the tubes (and thus the distant containers) above it. in any given training or test trial of the 'olfactory distance' condition, after the pangolin entered the testing chamber, he had s to investigate the two sides. the containers were then placed on the floor of the chamber so the pangolin could choose one. the pangolin then had s to pass the -cm partition separating the two scientific reports | ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ containers, at which point the experimenter unlocked and removed the chosen container's lid, while the other container was removed (see supplementary video s ). this early designation of the pangolin's choice (i.e., before he made contact with a container) was used to minimize his opportunity to make a decision using olfactory information from within the containers once they were placed on the floor. the food reward used in this condition consisted of g of dead ants. since this was the pangolin's first experience with testing on a vertical plane, he was first trained with the containers placed directly overhead on the wooden plank without the added distance created by the pvc tubes. the pangolin reached the criterion of % correct in a single session of eight trials (i.e., seven out of eight trials correct) within his first training session. subsequently, he was trained on a distance of cm to ensure he understood the task with all of the apparatus components in place, reaching the same criterion in his third session. following completion of training, the pangolin participated in the test condition consisting of ten total sessions. each testing session included six test trials (two trials of each distance, randomised) and two control trials, for a total of eight trials per session. control trials across all 'olfactory distance' testing were conducted at the shortest distance ( cm) using the same control containers from phase i. as in phase i, in the 'olfactory distance' condition, once the containers were placed on the pvc by the experimenter, the metal mesh screen was removed so the pangolin could enter the apparatus. again, no inter-trial interval (iti) was set to avoid having to handle the pangolin or restrict his movements, however, he never entered the apparatus sooner than s after the containers were placed on the pvc (it usually took considerably longer), and he always had s to investigate them before they were removed and placed on the ground. thus, at least s passed between setting a trial and the pangolin having to make a choice. the 'scent trail' condition aimed to investigate whether pangolins could use scent trails to track their prey (see supplementary video s ). due to the pangolin's natural tendency to smell as it moved through the wooden chamber, no training was needed for this condition. test trials made use of two wooden planks on each of which a strip of cloth tape was applied to prevent the smeared liquid from running. a different scent was smeared on each tape strip using a sponge (approximately ml of scent was applied per strip, per trial). one of two stimulus containers (one baited and one unbaited) was placed at the end of each plank, on its side and with the locked lid facing the pangolin. once the animal voluntarily walked into the testing chamber, he had s in which to make a choice. as soon as he reached one of the two containers, the lid was unlocked and removed. for all test trials, the food (f) scented trail always lead to the baited container. this scent was extracted by taking dead weaver ants and pressing them within the barrel of a syringe, producing a concentrated liquid extract. in one type of test trial, the scent of ants (f) was applied to one trail while plain tap water (w) was applied to the non-food bearing trail (f vs. w). the w trail acted as a control for a scent 'smear' by providing comparable characteristics of the ant 'smear' without the odor of prey. for the other type of test trial, the scent of ants (f) was applied to one trail while the non-prey odor of mint (m) was applied to the alternative trail (f vs. m). the mint odor was produced by gathering wild mint leaves, dicing them and soaking them in warm water min prior to testing. the resulting extract (devoid of the leaves) was smeared on its own plank. this condition investigated whether the smell of a non-prey, naturally occurring odor was distracting or potentially more interesting than prey-derived odors. phase i control containers (i.e., opaque with solid lids) were used as the stimulus containers in the test trials for this condition as we wanted the subject to make olfactory decisions based on the trail scent without being influenced by the odor of the food inside the container. 'scent trail' control trials followed the same procedure as all prior testing in this condition except that the trail strips used were devoid of any smear. these trials again ensured the pangolins were not using any cues besides those which we intentionally provided for them to find the food. this condition consisted of ten sessions, with each session consisting of two control trials and six test trials (three f vs. m and three f vs. w trials). prior to each trial, scents were reapplied to their respective planks. phase ii pseudo-randomisation procedures were identical to that of phase i except that only the first two trials in visual -light visual -no light acoustic -untreated acoustic -treated betty ♀ / * / table . raw data for both subjects ('pluto' , the male, and 'betty' , the female) across all test and control conditions in phase i and ii. the ratios provided represent the number of trials in which the pangolin selected the correct, baited container (numerator) over the total number of trials for each respective condition (denominator). the top of each column provides an abbreviation of the condition tested with the type of trial listed below. control trials are represented by "c". test trials are represented by "t". in phase i, the olfactory condition was comprised of only one type of test trial, while visual and acoustic conditions included two separate types each. in phase ii, the 'olfactory distance' condition was comprised of three types of test trials based on the distance at which the stimuli were presented, and are delineated in the table by this distance in centimeters (cm). there were two types of test trials in the 'scent trail' condition. 'scent trail' test trials which provided a food vs. mint scent pair are represented by "f vs m". test trials which provided a food vs. water scent pair are represented by "f vs w". ratios that are bolded and marked with an asterisk indicate that the pangolin performed significantly better than chance (p < . ) (*binomial test, two-tailed). see the methods section for details on each condition across the two phases. a phase ii session were always test trials; the remaining trials within a session contained a randomised order of test and control trials. all experimental conditions were recorded using the built-in infrared nightshot capabilities on a sony fdr-ax (see supplementary videos s -s ). trials were coded for the pangolin's container choice in real-time by j.d.d. both subjects selected the baited container significantly more often than chance in the olfactory condition of phase i (p < . for both pangolins, two-tailed binomial test). the olfaction results for the female, however, reflect only partial completion for this condition. she did not participate in any other testing. the male did not perform significantly better than chance in any other condition in phase i or in the 'olfactory distance' condition in phase ii (table ). in the 'scent trail' condition, the male chose the food-scented trail significantly more often than chance across all trials ( / trials, p = . , two-tailed binomial test). although he chose the food-scented trail more often in both types of 'scent trail' test trial (the food vs. mint and food vs. water conditions) - . % in both -the results were not statistically significant when analysed separately ( / trials each, see table ). in this study, two pangolins (m = , f = ) demonstrated a capacity for finding food using olfactory information alone. one pangolin's performance (m = ) on the additional olfactory foraging tasks suggests he may have been using scent trails to track prey rather than olfactory information at a distance directly from the source. in addition, this pangolin was unable to use visual or acoustic information to find food when this was the only sensory information about the food's location available to him. the two pangolins' poor performance on control trials confirmed that they did not use any other inadvertent cuing from the experimenter to find the food. in order to discuss and interpret these results, we recognise the small sample size is a significant limitation. in particular, most of our results are based on the performance of a single pangolin. further testing with a larger sample size that focuses on different types of visual and acoustic stimuli in a foraging context would be illuminating, and would allow for extrapolation of results to a larger population within and potentially across pangolin species. without access to a larger sample size and the opportunity to test multiple subjects with a randomised order of conditions, we also cannot exclude the possibility that the data from our one subject were influenced by a possible order effect. though we did not find any evidence to suggest this, it is also possible that our subject had sensory impairments which could have impacted our results. however, due to the extreme difficulty of testing pangolins in controlled settings and the sunda pangolin's status as a critically endangered mammal on the verge of extinction, we present these results as a case study. we feel it is important to hypothesise about how a single pangolin's performance may help inform our understanding of pangolin behavioural ecology and cognition. the pangolin's performance on the visual condition, for example, makes sense considering the pangolin's morphology and known ecology. the sunda pangolin is a predator with relatively small eyes that forages on small insects at night , , , and thus vision may not be as relevant to its foraging strategy as olfaction. it is also possible that our use of dead ants as the rewarding stimulus was not ecologically salient enough for the pangolin to locate the food visually. in addition, the pangolin in this study did not demonstrate an ability to locate its food based on the acoustic cues provided by live ants. although we included leaves as a substrate in order to increase the sound produced by the ants inside the container, the plastic may have muted the acoustics enough that the pangolin could not differentiate between the baited and unbaited containers. however, from an ecological perspective, if pangolins do use their hearing to hunt insects in the wild, they would have to detect their acoustic signatures from varying distances and amid ambient noises within their natural habitats. they would also need to be able to hear their prey through tunnels in the ground, dense dirt mounds, or high within tree nests. thus, at close proximity, if the pangolin in our study used acoustic information to locate prey, he should have been able to detect it even behind the artificial plastic barrier. our own experience with the three pangolins suggests they do react to loud noises, and may adjust their tail posture based on the substrate on which they are walking. this behaviour may indicate that pangolins adjust their locomotion and their tail's position to minimize the noise they make in the wild. thus, although the pangolins may not use acoustic information when locating prey, it is likely they use it in predator detection or avoidance. plotnik et al. , for instance, found that asian elephants did not use auditory cues to find food in a similar object-choice task, even though elephants are well-known for their complex acoustic repertoires (e.g. [ ] [ ] [ ] . like for elephants, the "sound food makes" may not be important for the pangolins to find it. considering the coevolution that can take place between predatory and prey (e.g. [ ] [ ] [ ] , it might make more sense that the predatory habits of the pangolin would have evolved to exploit the more obvious chemosensory signatures generated by their prey. ants and termites rely heavily on the transmission of odors and pheromones to communicate amongst themselves [ ] [ ] [ ] [ ] . thus, a keen sense of smell would allow the pangolin to efficiently exploit the abundant availability of these olfactory signatures. from a foraging perspective, it seems likely that pangolins would prioritize olfactory information when searching for prey. while it is unknown if it is pheromones or other prey chemicals (such as odor from larvae) that influence the pangolin's navigation in a hunt, we tried to understand how olfaction was functionally employed by pangolins across different ecological scenarios. since the sunda pangolin has been noted for its semi-arboreal foraging behaviour , , 'olfactory distance' testing was conducted on a vertical plane to best replicate a foraging situation that a wild pangolin might encounter as it walks through a forest in search of arboreal prey. in the absence of nearby environmental cues, we found that our subject was either not sufficiently motivated by the distant prey odor offered, or was unable to locate it based on the olfactory information available. another possible explanation for our results in 'olfactory distance' testing was our inability to use a specific, longer inter-trial interval (iti) in this condition to ensure that the odor molecules had sufficient time to make their way from the container at the ( ) : | https://doi.org/ . /s - - -x www.nature.com/scientificreports www.nature.com/scientificreports/ top of the pvc down to the ground, thus creating an odor concentration gradient sufficient enough to be detected by the pangolin. due to the danger of the experimenter negatively impacting the pangolin's health by excessive handling or spending too much time inside his enclosure, we could not enact a longer iti. thus, the minimum time between trials plus the time the pangolin had to investigate was at least seconds. the food amount used in each trial, g of ants, was determined based on the average weight of an ant nest minus substrate, and thus this should also have enhanced the pangolin's capacity for finding them in a short period when the smell was confined within a tube and that smell was of a sufficient, ecologically relevant concentration. another potential confound in all olfactory conditions was the fact that we could not completely remove residual odor between trials, and thus the pangolin's choices may have been confused by olfactory information from prior trials. however, the pangolins' success on phase i olfaction trials and phase ii 'scent trail' trials suggests residual odor was likely not enough to overcome the pangolins' interest in odors emanating from a present food source, and thus the one pangolin's poor performance on 'olfactory distance' was likely not due to residual odor. in addition, the pseudo-randomisation of distance trials and the placement of different pvc tube lengths also meant that residual odor was regularly dispersed when tubes were removed and replaced, making it an unreliable source of information about food location. finally, if residual odor was a confound, we would expect the pangolin's performance on control trials to be influenced by the location of food in the test trials immediately preceding them. in a post-hoc review of these trials in 'olfactory distance' testing, we found that of the control trials that immediately followed a test trial, pluto the pangolin chose the same location in the control trial that had the baited container in the immediately preceding test trial only six of times (two-tailed binomial, p = . ). this suggests he was not using residual odor to find his food. in studies of elephants, another highly olfactory animal, they too did not seem to use residual odor to guide their choices about food , . despite the pangolin's poor performance on the 'olfactory distance' condition, it seems unlikely that a predator with a developed olfactory system , , , , , would be unable to detect its prey from the distances we investigated. scent trails would be most useful once they are found, but detection of distant odors may offer valuable information about directionality when there are no immediate environmental cues available. overall, the performance of our subject in the 'scent trail' condition suggests that the proximate environmental cues offered by scent trails may offer a more efficient method to localize live, mobile prey. our results were only significant in this condition when viewed in aggregate (i.e., across all 'scent trail' trials but not within food vs. mint or food vs. water trials). however, the similar percentage correct on each of these trial types ( . %) suggests that the pangolin likely could differentiate between the food and the non-food odor trails and followed the former. in fact, the anatomy of the pangolin may be conducive to following scent trails, as it has a low-hanging head and elongated nose that slopes towards the ground during terrestrial locomotion. the reliance on terrestrial scent trails, however, is somewhat confounding considering the semi-arboreal ecology of sunda pangolins , , , and we believe further testing is needed to understand whether the value of foraging cues changes as pangolins move from the forest floor into the trees. for instance, perhaps pangolins prioritize different sensory information as they pursue prey across different spatial planes, or they may follow olfactory information continuously as they track their food's movements. it is not always clear how the study of animal cognition fits into improving welfare and conservation in practice , . the goal of this research was to identify the pangolin's primary sensory modality in foraging behaviour, and then how that modality (olfaction) may be used to track prey. although this is only one study with a small sample size, research on the behaviour and cognition of animals like pangolins may have important applications for ex-situ captive management , , , . considering the high mortality rates of pangolins in captivity due to artificial diets and a lack of knowledge about their ecology , - , a greater understanding of the pangolin's natural behaviour (including foraging) is crucial for improving the welfare of these animals when reliant on human care. investigating pangolin behavioural ecology and cognition is also relevant to their conservation in the wild , , , . it is possible, for instance, that pangolins not only detect and value the scent of their prey, but also associated odors linked to it, such as the aroma of specific flora typically inhabited by their prey. in addition, although our study did not investigate ecologically relevant non-food odors in detail, it is possible that pangolins may prefer areas of their habitat which are devoid of predatory odors, while the presence of conspecific odors may influence their movement patterns based on whether the olfactory information comes from competitors or mates. our study, while limited, could complement future research on pangolin foraging behaviour to help create more efficient conservation protocols that focus on protecting pangolin food resources and habitat rather than on locating elusive, individual animals. a broader perspective on how pangolins use sensory information to forage and navigate through their environment could thus highlight key conservation points to protect not only pangolins, but the more discrete resources which they value as well. finally, the covid- pandemic highlights a crucial need to look beyond the charisma of endangered species when deciding which animals deserve the field of conservation's attention. it is vital that we work to better understand wildlife behaviour and ecology through research across scientific disciplines, even when focal species are difficult to study or lack popular or political attention. although it is still unknown whether the pangolin acted as a vector for the coronavirus between wildlife and humans , , what is clear is that the illegal wildlife trade and the increasing physical contact between wildlife and humans pose an existential threat not only to biodiversity in general, but also to our own existence as a species. all of the raw data that support the findings of this study are included in table . differences in the cognitive skills of bonobos and chimpanzees conformity to cultural norms of tool use in chimpanzees problem solving in great apes (pan paniscus, pan troglodytes, gorilla gorilla, and pongo abelii): the effect of visual feedback the mentality of crows: convergent evolution of intelligence in corvids and apes thinking with their trunks: elephants use smell but not sound to locate food and exclude nonrewarding alternatives elephants have a nose for quantity african elephants use plant odours to make foraging decisions across multiple spatial scales smelling more or less: investigating the olfactory experience of the domestic dog the world from a dog's point of view: a review and synthesis of dog cognition research a comparative analysis of animals' understanding of the human pointing gesture domestic dogs (canis familiaris) use human and conspecific social cues to locate hidden food cues that chimpanzees do and do not use to find hidden objects inferences about the location of food in the great apes (pan paniscus, pan troglodytes, gorilla gorilla, and pongo pygmaeus) domestic pigs' (sus scrofa domestica) use of direct and indirect visual and auditory cues in an object choice task asian pangolins: how behavioural research can contribute to their conservation in proceedings of the workshop on trade and conservation of pangolins native to south and southeast asia the global trafficking of pangolins: a comprehensive summary of seizures and trafficking routes from - in traffic report: southeast asia regional office overview of pangolin trade in southeast asia in proceedings of the workshop on trade and 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sources and secretions in pheromone communication in social insects: ants wasps, bees, and termites organization of the olfactory pathway and odor processing in the antennal lobe of the ant camponotus floridanus taking the elephants' perspective: remembering elephant behavior, cognition and ecology in humanelephant conflict mitigation comparative cognition for conservationists current status and future directions of applied animal behavioral research for animal welfare and conservation identifying sars-cov- related coronaviruses in malayan pangolins probable pangolin origin of sars-cov- associated with the covid- outbreak we thank the faculty and staff of the livestock and wildlife hospital of mahidol university in kanchanaburi, thailand for their support of this research and their care of the pangolins. we also thank parntep ratanakorn for his continued support of collaborative research on endangered species behaviour in thailand. we greatly appreciate andrew michalski's help with apparatus construction and design, and thank martin chodorow for his analysis advice. we also thank melissa schmitt, peter moller and reviewers for comments on an earlier version of this manuscript, and matthew rudolph for assistance with the figure. this paper is dedicated to the memory of pluto, our primary research subject, who passed away from natural causes in january, . his voluntary participation in this research was crucial to this study's success, and his curiosity and good nature made it a pleasure to work with him. this work formed the basis of the first author's master's degree thesis in the animal the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -x.correspondence and requests for materials should be addressed to j.m.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -gvzp h g authors: ling, shenglong; zhang, chengwei; wang, wei; cai, xiaoying; yu, lu; wu, fangming; zhang, longhua; tian, changlin title: combined approaches of epr and nmr illustrate only one transmembrane helix in the human ifitm date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: gvzp h g interferon-inducible transmembrane protein ifitm was known to restrict the entry of a wide spectrum of viruses to the cytosol of the host. the mechanism used by the protein to restrict viral entry is unclear given the unavailability of the membrane topology and structures of the ifitm family proteins. systematic site-directed spin labeling (sdsl) and electron paramagnetic resonance (epr) studies of ifitm in detergent micelles identified a single, long transmembrane helix in the c-terminus and an intramembrane segment in the n-terminal hydrophobic region. solution nmr studies of the same sample verified the secondary structure distribution and demonstrated two rigid regions interacting with the micellar surface. the resulting membrane topology of ifitm supports the mechanism of an enhanced restricted membrane hemi-fusion. scientific reports | : | doi: . /srep biophysical studies are urgently required to illustrate the three-dimensional structures, or at least the membrane topologies of ifitms. in this report, a combination of electron paramagnetic resonance (epr) and nuclear magnetic resonance (nmr) was applied to investigate the structure and membrane topology of the ifitm protein in detergent micelles. systematic site scanning of spin labeling, epr dynamic and accessibility analysis identified a c-terminal transmembrane α -helix and an n-terminal ifitm segment (composed of two short α -helices) lying on the surface of micelles. further triple resonance solution nmr studies verified the secondary structures of ifitm and also illustrated the backbone flexibility through nmr relaxation analysis. collectively, a tentative ifitm model was proposed. this model adopts a topology similar to model i (fig. a) , which is consistent with recent antiviral mechanism studies. epr analysis revealed the single transmembrane topology of ifitm . with site-directed spin labeling (sdsl), epr spectroscopy is a powerful tool to analyze the mobility and secondary structure of a membrane protein [ ] [ ] [ ] [ ] . before implementing sdsl-epr studies, three endogenous cysteine residues (c , c and c ) on ifitm were first substituted with serine to construct the cys-less "wt". then, a total of sequential residues (from w to y , except t , which was not expressed after cysteine mutation) covering the two predicted hydrophobic regions were mutated to cysteine residues (supplementary figure ). all purified ifitm mutants in dodecyl-phosphocholine(dpc) micelles were identified in a monomeric state using sds-page, same as the wild-type (wt) ifitm protein (fig. c) . the spin labeling reaction with methanethiosulfonate (mtsl) was subsequently performed to attach the side chain r to the ifitm mutants, showed as fig. b . then, the systematic site-specific continuous wave (cw)-epr spectra of spin-labeled ifitm were acquired in dpc micelles at ambient temperature( k). the epr spectra line shape could reflect the mobility of the nitroxide probe r , which also contains structural information of proteins . total of cw-epr spectra are presented in fig. , and the typical nitroxide three-line cw-epr spectra of spin-labeled ifitm variants were observed. although most of the spectra exhibited only one motional state, several residue sites (s r , l r , g r , a r , s r and d r ) had multiple components. for a clear presentation, spectra of spin labels at these residue sites were plotted in fig. a , indicating the presence of both the immobilized (i) and mobilized (m) components. interestingly, all these residues with multiple motional states were identified in the first half of the putative hydrophobic region(from w to g ), which strongly suggests that this segment adopts two different motional or conformational states. (a) three different topology models proposed recently for ifitm . the hydrophobic region of ifitm from w to y was analyzed using epr methods. (b) the spin labeling reaction for cysteine substituted ifitm mutants to introduce the nitroxide side chain, which is denoted r . (c) sds-page analysis of ifitm in detergent micelles. both wt-ifitm and ifitm -w c were purified as monomers in detergent micelles. figure . cw-epr spectra of spin-labeled cysteine mutants, covering the hydrophobic region of human ifitm (form w to y , except t ). spectra composed of multiple components were highlighted with . all spectra were normalized by the height of the central peak. the cw-epr spectra exhibit multi-components in detergent micelles. "i" and "m" represent the "immobile" and "mobile" components, respectively. (b,c) represent the oxygen accessibility (Π o ) and niedda accessibility (Π niedda ) of the hydrophobic region of ifitm in detergent micelles. sine waves with a periodicity of . were drawn to indicate the α -helix secondary structure. (d) the membrane immersion depth (Φ ) of sequential residues span the hydrophobic region of ifitm in detergent micelles. the grey region represents the putative transmembrane region and the dashed line indicates the suggested membrane interface. then, epr power saturation studies were conducted as shown in supplementary figure . these experiments illustrate the accessibility of spin radical side chains, which can be used to derive secondary structure and the membrane topology information of proteins , , . the systematic accessibility parameters (Π ) were investigated for the sequential residues (form w to y , except t ), covering the two putative hydrophobic regions of ifitm . oxygen in the air was used as the hydrophobic paramagnetic reagent to evaluate membrane exposure, while niedda served as the hydrophilic paramagnetic reagent to study the extent of aqueous exposure. Π o and Π niedda were plotted against the residue numbers in fig. b ,c. as we can see, the Π niedda values progressively decreased from a to g , followed by a lower flat region (from i to i ). then, the Π niedda values increased from residue i to a . on the contrary, the value of Π o first increased starting from residue a and then slowly decreased. these two typical changing patterns of Π o and Π niedda demonstrated that a highly hydrophobic transmembrane region exists in ifitm starting from residue a to a in detergent micelles. however, no similar patterns in the accessibility parameters (both Π o and Π niedda ) were observed in the first half of the putative hydrophobic region (from w to g ), indicating that no transmembrane property exist in this segment of ifitm . moreover, a close examination of the accessibility parameter values (Π o and Π niedda ) of the spin labeled residues revealed some periodic behaviors. the periodicity covered the residues from a to a in fig. b ,c, indicating that this segment adopts an α -helical secondary structure in detergent micelles. in addition, the accessibility parameter values of two short segments (l -f and i -r ) in the n-terminal region were also observed with same periodicity, suggesting that two short α -helices potentially exist in this segments. moreover, the immersion depth parameter (Φ ) was derived from the conjugated accessibility parameters of Π o and Π niedda . as shown in fig. d , the Φ values of the residues covering the entire putative hydrophobic region of ifitm can be separated into two apparently different patterns. in the first pattern starting from w to g , the Φ value fluctuated around a relatively low value, indicating that the residues in this segment were partially buried in the micelles. however, the second pattern highlighted in gray ( fig. d ) was totally different ( the Φ values progressively increased from residue a to t , followed by a decrease until residue y ), indicating that a transmembrane segment was embedded in this region of ifitm . collectively, the analysis of the accessibility parameter (Π o and Π niedda ) and the immersion depth parameter (Φ ) derived from the power saturation experiments demonstrated that the ifitm protein contains a long transmembrane α -helix covering the residue sequence from a to a in the putative hydrophobic region. this coincides with the previously predicted topology i and topology iii model, but totally different from the topology ii model (fig. a) . at the same time, two short α -helices without transmembrane properties were observed in the first half of the putative hydrophobic region, which was similar to the predicted topology model i or model ii (fig. a) . moreover, as shown in fig. a , several residues in the first half of the putative hydrophobic region were revealed to contain multi-motional states by the cw-epr spectra. thus, our epr analysis of ifitm in detergent micelles suggested that ifitm might adopt a membrane topology structure similar to the topology model i in fig. a . solution nmr structure determination and backbone relaxation analysis of ifitm . with information on the secondary structure and membrane topology of ifitm as determined by the epr studies, solution nmr studies were also conducted to determine the atomic resolution structure of ifitm in dpc micelles. the backbone chemical shift assignments ( h, n, co, c α , and c β ) of full-length ifitm protein in dpc micelles were achieved using series of two-and three-dimensional solution nmr spectra (fig. s ). heteronuclear single quantum coherence (hsqc) spectra of the specifically n labeled amino acid ( n-leucine, n-isoleucine, n-valine, n-methionine or n-phenylalanine) in the ifitm samples were acquired to assist the residue assignments (fig. s ). based on the backbone chemical shift values of assigned residues, the program talos + was used to estimate the secondary structure of ifitm . as shown in fig. a , three segments with relatively large negative talos + index values were obtained, representing three α -helix segment. the remainder of the ifitm protein exhibited talos + index values close to zero, indicating a random coil. compared with the previously predicted topology model that contains two long hydrophobic regions, the first two short α -helixes of ifitm resided in the first putative hydrophobic region while the third long α -helix correspond to the second putative hydrophobic region. to further verify the secondary structure, backbone relaxation analysis of ifitm was performed. as shown in fig. c , residues with small t values were mainly observed in three regions, suggesting high backbone rigidity. this finding is consistent with the α -helix structure predicted by program talos + in the same region. while most residues in the n-terminus have large t values, indicating high flexibility property of the backbone. thus, the overall results were consistent with the derived secondary structure of ifitm as determined by talos + analysis (fig. ) . however, the final atomic resolution structure of full-length ifitm could not be obtained, mainly due to the lack of an adequate number of noe restraints and the intrinsic random coil structure in the n-terminal region. instead, low resolution structural topology of full-length ifitm , especially the putative hydrophobic region, can still be derived from the limited nmr data. as shown in supplementary figure , an ensembles of conformers with the lowest energy and best convergence were selected from calculated structures of the n-terminally truncated ifitm (from w to a ). a kink or unusual dihedral angles could be found in the c-terminal of the transmembrane helix, which were probably derived from the unassigned residues nearby and the existence of pro ( figure s ). when superimposing the relatively long c-terminal α -helix (from a to a ), the root-mean-square deviation (rmsd) values of the backbone atoms and heavy atoms were . Å and . Å, respectively. for a clear presentation of the structure and predicted membrane topology of the putative hydrophobic region of ifitm , one conformer was selected out (fig. a ). the structural model of ifitm shows that a long c-terminal transmembrane helix and two short intramembrane α -helices in the n-terminal of the hydrophobic region was connected by a small flexible loop between them, which is perfectly consistent with the structural topology derived from the epr analysis. taken together with the epr and solution nmr results, we conclude that the hydrophobic region of ifitm adopts a topology containing two short intramembrane α -helices followed by a long transmembrane α -helix (fig. b) . scientific reports | : | doi: . /srep three different membrane topology models for ifitm proteins have been previously proposed . the first model suggested that ifitm adopted an intramembrane topology with both the n-and c-termini facing cytoplasm, based on the observation that the inserted n-linked glycosylation site within the n-and c-terminal region cannot be modified (model ii of fig. a) . the results from flow cytometry experiments and cell surface immune-staining experiments argued that ifitm is a dual-transmembrane protein with both its n-and c-termini exposed to the endoplasmic reticulum (er) lumen or extracellular space (model iii of fig. a) , . the third topology model was recently proposed demonstrating that ifitm is a type ii transmembrane protein (model i of fig. a) , . through the combined approaches of epr and solution nmr, the structural model and the membrane topology of human ifitm protein were determined. as illustrated in fig. b , ifitm adopts a transmembrane topology, supporting the proposed type ii transmembrane protein topology (model i in fig. a) . at the c-terminus of human ifitm , a long α -helix covering residues from a to a spans through the micelles. while two short discrete intramembrane α -helices were discovered in the first predicted hydrophobic segment of ifitm . as shown in fig. d , the epr immersion depth (Φ ) data demonstrated that these two short α -helices possessed no transmembrane properties, but only buried partially in the micelles. and the relatively larger Φ values of the residues in the second short helix indicated that it might be more deeply buried in micelles than the first short helix. moreover, the random coil property of the n-terminus of ifitm was revealed by nmr analysis. recently, functional studies by li et al. have suggested that ifitms could hamper viral membrane hemi-fusion through reducing membrane fluidity and conferring a spontaneous curvature , , . here in our topology structure of ifitm in micelles, two short intramembrane α -helices were discovered in the first predicted hydrophobic region. these two short helices were likely to induce membrane curvature when they inserted into only one leaflet of the lipid bilayers (fig. b) , although lipid bilayers adopt different curvature and membrane thickness from micelles. hence, our membrane topology of ifitm could provide direct structural evidence to support the antiviral mechanism proposed by li et al. moreover, three types of post-translational modifications have been reported to regulate the antiviral activity through influencing the subcellular localization, trafficking, clustering or interaction with potential receptors. the s-palmitoylation occurs at cysteines , and , and ubiquitination takes place at lysines , , and , whereas tyrosine is phosphorylated , . these post-translational modifications can only occur when these residues are exposed to cytoplasmic side of the membrane, which is consistent with our ifitm structure and proposed membrane topology model (fig. b) , with an intracellular n-terminal domain and an intramembrane segment facing the cytoplasm. moreover, cw-epr spectra analysis indicated that residues with multi-motional or multi-conformational states all resided in the intramembrane hydrophobic segments (w -g ). we suggested that the multi-conformational property of the intramembrane hydrophobic segments might closely relate to its multiple roles in post-translational modifications, such as different structural states might interact with different enzymes. therefore, the combined epr and solution nmr studies clearly supported the type ii transmembrane protein topology model for ifitm , which is consistent with the previously proposed mechanism of its antiviral function. it remains unclear why the other contradictory membrane topology models for ifitm are derived from functional studies. we cannot exclude the possibility that ifitm may be able to adopt multiple topologies in different stages of the viral infection period, or in different host cell types. further structural and functional correlation studies of ifitm in different cell types and studies focusing on other ifitm proteins are still required to completely unveil the antiviral mechanism of ifitm proteins. three endogenous cysteines (c , c and c ) were substituted by serine through site-directed mutagenesis to construct cysless "wt" ifitm . then, sequential residues spanning from w to y (except t ) on full-length ifitm were mutated to cysteine, respectively. the target ifitm gene sequences were subsequently inserted into the vector pet b (novagen) using the restriction sites nde i and xho i. all constructs were verified through gene sequencing. protein expression and purification. ifitm protein was over-expressed in bl -gold (de ) competent cells (novagen) transformed with vector pet b containing ifitm coding sequence in m media at °c. a final concentration of . mm iptg (isopropyl-d-thio-galactoside) was added at cell density around od = . to induce protein over-expression in the following hours. the cell pellets harvested by high-speed centrifugation (allegrax- r, beckman) were resuspended in lysis buffer( mm tris, mm nacl, ph . ). then, cell membrane was broken using ultrasonication, followed by separation procedure using high-speed centrifugation. after that, the pallets fraction containing ifitm protein was dissolved using the binding buffer ( mm tris, mm nacl, ph . ) containing % (w/v)sds. finally, iifitm protein was purified using ni-nta affinity chromatography (qiagen, germany) and eluted in binding buffer containing . % dpc. sds-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was applied to analyze the purified ifitm protein in dpc micelles. mtsl ( -oxyl- , , , -tetramethyl-Δ -pyrr-oline- -methyl methanethiosulfonate) spin labeling. spin labeling reaction was carried out through incubating purified ifitm mutants with fold molar ratio mtsl (toronto research chemicals, ontario, canada) at °c overnight. excessive free mtsl was removed using a pd- gravity flow desalting column (ge biosciences). then, spin labeled ifitm protein in binding buffer with . % dpc (w/v)were concentrated to approximately μm using amicon ultra- centrifugal filter units (millipore) for further epr experiments. continuous wave epr (cw-epr) spectroscopy. cw-epr spectra were acquired using a bruker a spectrometer (bruker biospin gmbh, rheinstetten, germany) at x-band ( . ghz) equipped with a high-sensitivity cavity (er hs, bruker biospin gmbh, rheinstetten, germany). samples were placed into a quartz capillary tube(about . mm, kimble micro-capillary pipets) with a volume of approximately μl. experimental parameters were optimized with khz modulation frequency, mw incident microwave power, gauss modulation amplitude, . ms time constant, . ms conversion time and gauss scan width. for power saturation studies and accessibility analysis, the experiments were performed using the same spectrometer equipped with an er d cw resonator (bruker biospin, germany). samples with a total volume of ~ μl were loaded into gas permeable tpx capillary tubes. during the power saturation analysis, the range of incident microwave power was from . mw ( db attenuation) to mw( db attenuation) with the step of db.to obtain the epr spectra of ifitm sample in the n atmosphere, o atmosphere of the sample was purged through n blowing or equilibration. at the same time, epr spectra were acquired in air (o as the hydrophobic paramagnetic reagent), or in n with the presence of mm ni + -edda complex (niedda) as the hydrophilic paramagnetic reagent. power saturation curves were measured as the vertical peak-to-peak amplitude(a) of the first derivative m i = line as a function of incident microwave power (p). to determine the value of p / , the data were fitted using an r software script according to the equation ( ): / / i is a scaling factor, p / is the incident power where the first derivative amplitude is reduced to half of its unsaturated value, and ε is a measure of the homogeneity of saturation of the resonance line. with the obtained p / , the accessibility parameter Π o and Π niedda were calculated according to the equation ( ): where Δp / is the value difference between two p / values in the presence and absence of relaxing agent, Δhpp is the peak-to-peak line-width of the first derivative spectrum, p / (dpph) and Δhpp (dpph) were the p / and peak-to-peak line-width values of a standard sample of crystalline , -diphenyl- -picrylhydrazyl (dpph) in kcl. then, the immersion depth parameter Φ could be calculated using the power saturation parameter: n-isoleucine, n-valine, n-methionine and n-phenylalanine selectively labeled ifitm samples. details of the experimental parameters were described previously. all the spectra were analyzed using nmrpipe and nmrview. backbone resonance assignment was then performed. dihedral angle constraints were predicted using talos + program, based on the obtained chemical shifts of cα , cβ , transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells molecular analysis of a human interferon-inducible gene family the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus a diverse range of gene products are effectors of the type i interferon antiviral response the n-terminal region of ifitm modulates its antiviral activity by regulating ifitm cellular localization characteristics of ifitm, the newly identified ifn-inducible anti-hiv- family proteins the small interferon-induced transmembrane genes and proteins bril: a novel bone-specific modulator of mineralization evolution of vertebrate interferon inducible transmembrane proteins the broad-spectrum antiviral functions of ifit and ifitm proteins ifitm proteins-cellular inhibitors of viral entry ifitm inhibits influenza a virus infection by preventing cytosolic entry ifitm proteins restrict viral membrane hemifusion palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein (ifitm )-mediated resistance to influenza virus tapa- , the target of an antiproliferative antibody, is associated on the cell-surface with the leu- antigen monoclonal antibody to the interferon-inducible protein leu- triggers aggregation and inhibits proliferation of leukemic b cells ifitm-family proteins: the cell's first line of antiviral defense a membrane topology model for human interferon inducible transmembrane protein interferon-induced transmembrane protein is a type ii transmembrane protein cw-epr studies revealed different motional properties and oligomeric states of the integrin beta a transmembrane domain in detergent micelles or liposomes ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter: sodium symporters three-dimensional architecture and gating mechanism of a k+ channel studied by epr spectroscopy watching proteins move using site-directed spin labeling motion of spin-labeled side chains in t lysozyme. correlation with protein structure and dynamics a collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin structure of the cytoplasmic domain of erythrocyte band hereditary spherocytosis variant p r: band tuscaloosa talos+ : a hybrid method for predicting protein backbone torsion angles from nmr chemical shifts membrane curvature and mechanisms of dynamic cell membrane remodelling mechanics of membrane fusion the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication we gratefully thank the national key science research grant of ministry of science and technology, china with grant number cb , national natural science foundation of china with the grant funding support no. u , and . t.c., z.l. and l.s. designed the experiments and t.c. oversaw the whole experimental processes. l.s., w.w., c.x. and y.l. expressed the protein and did the spin labeling; l.s. conducted the epr experiments and the analysis. z.c., l.s. and w.f. conducted the solution nmr studies. t.c. and l.s. wrote the manuscript under helps from all authors. key: cord- -f wccj authors: kim, hyun cheol; kim, soontae; kim, byeong-uk; jin, chun-sil; hong, songyou; park, rokjin; son, seok-woo; bae, changhan; bae, minah; song, chang-keun; stein, ariel title: recent increase of surface particulate matter concentrations in the seoul metropolitan area, korea date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: f wccj recent changes of surface particulate matter (pm) concentration in the seoul metropolitan area (sma), south korea, are puzzling. the long-term trend of surface pm concentration in the sma declined in the s, but since its concentrations have tended to incline, which is coincident with frequent severe hazes in south korea. this increase puts the korean government’s emission reduction efforts in jeopardy. this study reports that interannual variation of surface pm concentration in south korea is closely linked with the interannual variations of wind speed. a -year ( – ) regional air quality simulation was conducted over east asia ( -km) and over south korea ( -km) to assess the impact of meteorology under constant anthropogenic emissions. simulated pm concentrations show a strong negative correlation (i.e. r = − . ) with regional wind speed, implying that reduced regional ventilation is likely associated with more stagnant conditions that cause severe pollutant episodes in south korea. we conclude that the current pm concentration trend in south korea is a combination of long-term decline by emission control efforts and short-term fluctuation of regional wind speed interannual variability. when the meteorology-driven variations are removed, pm concentrations in south korea have declined continuously even after . particulate matter (pm; pm , particles with a diameter of µm or less, is used in the study unless specified) concentration is its interannual trend, which recently showed a signal of increase after a long downward trend. since the early s, annual mean surface pm concentrations have decreased continuously over the sma and south korea (fig. ). the annual mean concentration was . μg/m in , and continued to decrease until it reached its minimum ( . μg/m ) in , but since then it has begun to rise ( . μg/m , . μg/m , . μg/ m in , , and , respectively). although several reasons (e.g., regional transport changes in china , or increased use of diesel-engine passenger cars) have been suggested for the recent changes, the potential of meteorological variance has not been actively discussed until now. in addition to the changes in the amount of released emissions of pollutants and their precursors, meteorological conditions are also primary contributors to regional haze and air pollutions , . wind, in its velocity and transport pattern, is one of the major meteorological components that affects surface air pollution , . winds can control vertical mixing and regional ventilation . persistent stagnant conditions (i.e. calm wind) provide critical conditions that lead to the development of local pollution episodes. wind direction controls the source-receptor relationship , and it also directly initiates local emissions such as dust storms or sea salt emissions , . large scale transport, both regional or inter-continental, can affect the atmospheric composition by altering their lifetime (e.g. longer lifetime at higher altitudes), and influences global meteorology and climate , . identifying the major drivers in the long-term trend of surface pm concentration can be limited due to the intricacy of chemical and meteorological processes. a -year simulation with a constant anthropogenic emission inventory was designed to isolate the interannual variation of surface pm concentrations solely due to variations of meteorology (see method). after testing multiple meteorological variables, we found a significant association of wind speed and surface pm concentrations. normalized anomalies (annual averages subtracted and then divided by the - average) of surface pm concentration and -m wind speed averaged over three geographical coverages (i.e. -km domain-wide, korea, and the sma) are compared (fig. ) . noticeably, interannual variations of modeled surface pm concentrations show very similar patterns in all regions. the observed pm, averaged over sites in south korea, shows similar but slightly different interannual variability. its year-to-year variation pattern is very similar to that of modeled variation; a small drop in , strong positives in - , strong negative in , and an increase in - . its general pattern, however, is mostly positive in early years and negative in later years. it thus seems reasonable to say that the observations show mixed signals; likely a combination of a short-term year-to-year fluctuations (caused by meteorological changes) and a long-term decline (caused by changes in anthropogenic emissions) . normalized anomalies of -m wind speed, on the other hand, show good agreement between observations (averaged over sites in south korea) and modeled wind speed over three regions, demonstrating that the model reasonably simulated interannual variations of the wind field. the key feature from the interannual variations of surface pm concentrations and surface wind is that they show totally opposite phases in their interannual variabilities. it should be noted that the interannual variations of pm concentrations, in all three regions, are best explained by the changes of domain-wide averaged wind speed compared with smaller scale averages. this might imply that the controlling mechanism is not on a local scale (e.g. sea breeze or local circulation) but rather synoptic scale activities (e.g. frontal passage). considering the efficiency of the wipe-out mechanism by cold frontal activities, the annual wind speed variability seems to represent the efficiency of regional ventilation . northeastern asia is a routine passage of mid-latitude cyclogenesis, initiated on the lee side of the altai-sayan mountains . the role of altai-sayan cyclogenesis, as an efficient cleaning mechanism of pollutants over northern china and korea during cool season, is demonstrated and discussed in kim et al. . the change of summertime circulation (i.e. east asia summer monsoon) on regional pollution is also discussed in zhu et al. . scatter plots of normalized anomalies between wind speed and surface pm concentration from the model ( − . % and . %, while the wind speed anomaly varied between − . % and . %. the range of variability in pm concentration is impressive since it is almost compatible to total long-term changes; that is ~− % per year or ~− % during - . remarkably, even with excellent agreement between modeled and observed interannual anomalies in surface pm concentration and wind speed, a direct comparison between observed pm concentration and observed wind speed variability shows poor correlation (i.e. r = . ). it clearly shows the advantage of the emission-isolating modeling approach, otherwise the meteorology-driven interannual variability of pm concentration seems to be overshadowed by emission reduction in the long-term trends. finally, we tried to remove the meteorology-driven variances from the original pm concentrations. fractional interannual anomalies from fig. a are multiplied to an overall mean of pm concentrations from each region, and then subtracted from the original annual mean concentrations. considering the uncertainties from the original model bias, using the relative variances instead of absolute concentrations from the model is a more robust approach . adjusted annual pm concentrations, with meteorology-driven variances removed, are shown in fig. in relation to seoul, the sma, and south korea. the most surprising change of the annual pm concentrations, compared with those of fig. , is their trend in recent years. the increase since , which had frustrated apparently, the decreasing rate in seoul is faster than the national average, implying the effectiveness of special emission control efforts in the locale, including replacing diesel buses and diesel garbage trucks with natural gas vehicles , . the local minimum of in the adjusted pm concentrations is noteworthy. the most likely explanation for this decrease is the impact of the global recession during - , as indicated by several previous researches , . at this point, we are not able to distinguish if the impact is due to changes caused by the south korean economy's experience of the global recession, or due to changes of transported pollutants and precursors by chinese economic changes. the case of is also interesting. pm concentration was higher in early ( . μg/m in jan-mar and . μg/m in jan-mar), but the annual mean is lower than that of because its spring-to-fall concentration is very low, even with the weakest wind speed observed. this is likely an extraordinary case by the impact of the middle east respiratory syndrome (mers), which has seriously impacted the south korean economy and society . although further investigations are necessary, these cases in and may provide interesting examples of the socio-economic impact on the environment, in addition to emission control policy-driven impact. three factors, long-term trend by emission control, short-term variation by meteorology, and sporadic offsets by unexpected social or economic episodes, seem to be affecting pm concentrations in south korea. to conclude, we found that there is a strong correlation between variations of surface pm concentrations in south korea and variations of wind speed. this study addresses several important implications. first, interannual variation of meteorological conditions has the strong potential to affect long-term trends in surface pm pollution events. at this point, we do not have any evidence that the reduced wind speed is part of long-term change or just short-term fluctuation in general circulations or ventilation patterns. however, it is worth further investigation since the frequency of mid-latitude cyclones can be reduced in warmer weather conditions, as suggested through observations [ ] [ ] [ ] [ ] and models studies , - . mickley et al. used the goddard institute of space studies model to demonstrate that the severity and duration of summertime regional pollution can increase due to a decline in the frequency of mid-latitude cyclones under future warmer climates. this may provide a key feature to understand the frequent occurrence of severe haze episodes not only in south korea but also in east asia, especially in northern china. yang et al. also demonstrated haze days over eastern china has increased between and , and argued that the weakening of winds is the dominant factor leading the decadal increase. second, accurate simulation of surface wind field, especially in terms of wind speed, seems to be critical for an accurate regional air quality model and forecast. analysis suggests a % change in annual wind speed is associated with % annual surface pm concentration. in many air forecast simulations in south korea, including the current long-term simulation used in this study, modeled surface winds tend to be overestimated ( . third, most importantly, emission control efforts from the south korean government and community, as well as neighboring countries, seem to be effective. however, changes in meteorological conditions seem to offset those efforts. recent space-borne observations also confirm a considerable decrease of no and so vertical column densities over the sma, south korea, and northeastern china (i.e. the beijing-tianjin-hebei region) , . in order to investigate the interannual variation of surface pm concentration in the sma, we conducted a -year simulation using a regional air quality modeling system. the weather research and forecasting model (wrf), the sparse matrix operator kernel emission (smoke), and the community multiscale air quality (cmaq) models were utilized to simulate meteorology and chemistry over east asia ( - was used to prepare biogenic emissions. mcip-processed ground reaching solar radiation and -m temperature were used to adjust hourly biogenic emissions such as isoprene and monoterpenes. other vegetation data like leaf area index, plant function type, and emission factors were also used as released with megan v . . biomass burning and dust emissions were not included. the modeling configuration was tested for multiple years as part of the integrated multi-scale air quality study for korea (imaqs-k) system, which was initially developed as a prototype of the official national air quality forecast system in south korea. the imaqs-k system has been operational since . descriptions on physical options are provided in kim et al. , and basic model performance evaluations for -year period are provided in the supplementary materials. model performed well in reproducing spatial and temporal variations of meteorology and chemical components. while total particulate matter concentration generally underestimates in the model, current analysis, using percent change of inter-annual variation, is designed to be less affected by the model bias. surface monitoring data. hourly observations of surface pm concentrations and wind speed were obtained from nier and kma, respectively. pm measurements, based on the beta-ray absorption method , were collected from urban air monitoring network sites, and wind data were collected from meteorological monitoring sites. locations of monitors are shown in the supplementary material. note on other controlling factors. ( ) a recent study on the regional emission attribution to the south korea suggests that the relative attribution of foreign emission sources is not sensitive to inter-annual variation of meteorology , contributing around % of the sma pm concentration. combined with the current declining trend of anthropogenic emissions from china, we can rule out the possibility of the increased efficiency of chinese emissions transport. ( ) the number of diesel vehicles in south korea has increased % from to . since non-truck diesel vehicles emit . % of total no x emission in south korea , resultant impact is small (< % of total pm). however, careful and continuous monitoring of diesel vehicle emissions is necessary due to their high uncertainty in emissions factors as we have learned from the volkswagen emission scandal . exploring the severe winter haze in beijing: the impact of synoptic weather, regional transport and heterogeneous reactions characterization of submicron aerosols during a month of serious pollution in beijing possible influence of atmospheric circulations on winter haze pollution in the beijing-tianjin-hebei region, northern china air quality modeling in east asia: present issues and future directions korean national emissions inventory system and effects of the asian dust events on daily mortality in seoul a review on east asian dust storm climate, modelling and monitoring the chemical composition 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the east asian summer monsoon verification, validation, and confirmation of numerical models in the earth sciences co-benefit analysis of an air quality management plan and greenhouse gas reduction strategies in the seoul metropolitan area natural gas vehicles promotion program in urban areas trends in omi no observations over the us: effects of emission control technology and the economic recession reductions in nitrogen oxides over europe driven by environmental policy and economic recession middle east respiratory syndrome-advancing the public health and research agenda on mers-lessons from the south korea outbreak long-term trend and spatiotemporal variations of haze over china by satellite observations from to trends in cyclone and anticyclone frequency and comparison with periods of warming and cooling over the northern hemisphere multidecadal global and regional trends in mb and mb cyclone frequencies trends in northern hemisphere surface cyclone frequency and intensity changes in mid-latitude variability due to increasing greenhouse gases and sulphate aerosols possible change of extratropical cyclone activity due to enhanced greenhouse gases and sulfate aerosols-study with a high-resolution agcm model-simulated northern winter cyclone and anticyclone activity under a greenhouse warming scenario increase in winter haze over eastern china in recent decades: roles of variations in meteorological parameters and anthropogenic emissions aura omi observations of regional so and no pollution changes from a space-based, high-resolution view of notable changes in urban no x pollution around the world a time-split nonhydrostatic atmospheric model for weather research and forecasting applications research data archive at the national center for atmospheric research review of the governing equations, computational algorithms, and other components of the models- community multiscale air quality (cmaq) modeling system model representation of secondary organic aerosol in cmaqv documentation of the saprc- chemical mechanism for asian emissions in for the nasa intex-b mission estimates of global terrestrial isoprene emissions using megan (model of emissions of gases and aerosols from nature) influence of fossil-fuel power plant emissions on the surface fine particulate matter in the seoul capital area measurement methods to determine compliance with ambient air quality standards for suspended particles regional contributions to particulate matter concentration in the seoul metropolitan area, korea: seasonal variation and sensitivity to meteorology and emissions inventory impact of the volkswagen emissions control defeat device on us public health h.c.k. and s.k. planned the research, s.k., c.b., m.b. performed simulations. h.c.k. wrote the paper. b.u.k., r.p. and a.s. provided discussion and comments on chemistry. c.s.k., s.h., s.w.s., and c.k.s. provided discussions and comments on meteorological aspect. supplementary information accompanies this paper at doi: . /s - - - competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -wshvvgxg authors: he, shengyang; zhou, kefu; hu, mengyun; liu, chun; xie, lihua; sun, shenghua; sun, wenwu; chen, liangkai title: clinical characteristics of “re-positive” discharged covid- pneumonia patients in wuhan, china date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: wshvvgxg to analyze the clinical characteristics of re-positive discharged covid- patients and find distinguishing markers. the demographic features, clinical symptoms, laboratory results, comorbidities, co-infections, treatments, illness severities and chest ct scan results of patients were collected from st january to th february . covid- was diagnosed by rt-pcr. clinical symptoms and nucleic acid test results were collected during the days post-hospitalization quarantine. out of covid- patients were detected re-positive during the post-hospitalization quarantine. re-positive patients could not be distinguished by demographic features, clinical symptoms, laboratory results, comorbidities, co-infections, treatments, chest ct scan results or subsequent clinical symptoms. however, re-positive rate was found to be correlated to illness severity, according the acute physiology and chronic health evaluation ii (apache ii) severity-of-disease classification system, and the confusion, urea, respiratory rate and blood pressure (curb- ) score. common clinical characteristics were not able to distinguish re-positive patients. however, severe and critical cases classified high according apache ii and curb- scores, were more likely to become re-positive after discharge. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ disease progression, no differences were found, suggesting this group of covid- patients could be difficult to detect by using standard clinical data. to formulate a more effective covid- patients management strategy, in this investigation a novel method to asses and discriminate 're-positive patients' infectivity from common covid- patients is presented. study design and participants. patients' admission time ranged from january st to february th. covid- diagnoses were made according to criteria from the th version of the guidelines on the diagnosis and treatment of covid- issued by the national health commission of china. all raw clinical and laboratory results were collected from electronic medical records system of the central hospital of wuhan, followed by a follow up visit up to days (also known as the discharge quarantine) to test for a re-positive nucleic acid assay. all participants signed informed consent. the study was approved by the ethics committee of the central hospital of wuhan and was performed in accordance with the principles of the helsinki declaration ii. data collection. all patients enrolled in the study were from different covid- units of the central hospital of wuhan. all covid- tests were performed by different departments of the central hospital of wuhan. computer tomography (ct) scan evaluations were made by at least specialists from the radiology department. the sars-cov- nucleic acid rt-pcr test quality control was performed by specialists from the clinical laboratory department. the clinical data included: the demographic descriptions, main symptoms, comorbidities, changes of laboratory results, main treatments, etc. for privacy reasons, the raw data of these patients are not presented. clinical definition. diagnosis was performed by detecting sars-cov- rna in nasopharyngeal swabs. the rna detection kits were provided by sansure biotech (changsha, china) and zj bio-tech (shanghai, china), and used according manufacturer's protocol by specialized laboratory personnel. the severity of covid- patients was defined according to the th version of the guidelines on the diagnosis and treatment of covid- . briefly, ( ) mild type: mild clinical symptoms without any radiology findings; ( ) general type: limited clinical symptoms: i.e. fever, cough and other common pneumonia related symptoms with radiological abnormality; ( ) severe type: patients have any of the following: (a) respiratory distress, respiratory rate ≥ per min; (b) oxygen saturation on room air at rest ≤ %; (c) partial pressure of oxygen in arterial blood/ fraction of inspired oxygen ≤ mmhg; ( ) critical type: patients have any of the following: (a) respiratory failure occurs and mechanical ventilation is required; (b) shock occurs; (c) patients with other organ dysfunction needing intensive care unit monitoring treatment. covid- patients were considered discharged when they meet all the criteria from the th version of the guidelines on the diagnosis and treatment of covid- . briefly, ( ) normal body temperature for more than days; ( ) significantly recovered respiratory symptoms; ( ) lung imaging shows obvious absorption and recovery of acute exudative lesion; ( ) negative results of the nucleic acid tests of respiratory pathogens for consecutive two times (sampling interval at least day). definition of "re-positive": when a confirmed covid- patient is detected sars-cov- rna positive during the days post-discharge quarantine (random test timing). laboratory confirmation and treatment. all laboratory results were double checked by at least specialists from the clinical laboratory medicine department. hospitalized patients were tested for sars-cov- rna every - h before discharge. the brief indications for corticosteroid utility (intravenous injection) are described as follow: ( ) respiratory distress, respiratory rate ≥ per min; ( ) deteriorations on radiology results after initial treatments; ( ) oxygen saturation on room air at rest ≤ %. continuous variables are presented as median (interquartile range, iqr) and categorical variables as n (%). differences in clinical characteristics and laboratory findings between groups were compared using mann-whitney u test (continuous variables) and chi-squared test or fisher's exact test (categorical variables). the multivariate logistic regression has been made for data valid test. all analyses were performed using r software (the r foundation, https ://www.r-proje ct.org, version . . ). a two-sided significance level of . was used to evaluate statistical significance. demographic features and clinical symptoms. covid- patients admitted to the central hospital of wuhan from january to february , were enrolled in the present study. out of covid- patients (table ) were detected 're-positive' during the post-discharge quarantine. the demographic characteristics were found not to be associated with 're-positive' rate. common symptoms of hospitalized covid- patients, including fever, muscle ache, fatigue, headache, cough, chest tightness, chest pain, and diarrhea were taken into consideration, and none of these clinical symptoms could account for the 're-positive' outcome. comorbidities and co-infections. a large majority of patients (table ) had comorbidities such as hypertension ( %), diabetes ( %), chronic kidney disease ( %), lung diseases ( %) and tumor-related diseases ( %). still, none of these co-morbidities were found to correlate with 're-positive' patients. furthermore, common co-infections (i.e. mycoplasma, chlamydia and other respiratory virus- table ) detected at admission or during hospitalization, were also found to have no correlation with 're-positive' outcomes ( www.nature.com/scientificreports/ treatments and severities. according disease severity, different treatment plans were adopted, such as antibiotics including quinolone and cephalosporins and antivirus including ribavirin, oseltamivir, abidor and lopinavir/ritonavir. methylprednisolone, intravenous gamma globulin (ivig) and ventilation was also selectively utilized. no significance differences in treatments modalities, comparing with the re-positive patients (table ) were observed. however, severity of illness (as per classification described above) showed that the 're-positive' patients tend to be severe, along with apache ii and curb- score, which are both indicators of severities. similarly, the hospitalization length of stay and whole medical care costs results were consistent with apache ii and curb- (table ) . to simplify the ct scan evaluation, all the enrolled patients were divided into three group. group : lesions present in - % of the bilateral lung field; and group : - %; : more than %, respectively. ct scan outcomes were found statistically insignificant ( table ). the representative ct scan developments of both re-positive and non-re-positive patients are shown in fig. . laboratory results. routine blood tests, other blood biochemistry and blood gas analysis results at day , , and day , are presented in table . no statistically differences were identified in 're-positive' patients. to further evaluate the recovery of covid- patients, a phone follow-up visit was set up with each patient, focusing on the incidence of clinical symptoms (table ). results showed that many patients still had symptoms, including coughing ( %), phlegm ( %), palpitate ( %), chest tightness ( %), paracenesthesia ( %) and fatigue ( %). additionally, out of 're-positive' patients spent their quarantine at home with family, and no other family member have been reported to have been infected so far. a following study is underway to assess further outcomes of these "re-positive" patients. multivariate logistic regression of potential predictors for "re-positive". to further testify the predictors for "re-positive" cases, a multivariate logistic regression was conducted (table s ). the or were found www.nature.com/scientificreports/ greater than for all predictors when using univariate test. moreover, when adjusted by age (> = yo), cardiovascular comorbidities, severities and the utility of glucocorticoids, or values were decreased but still greater than , indicating potential predicting abilities. since the outbreak of the covid- crisis in late , millions of people have been diagnosed all over the world. fortunately, the majority of hospitalized covid- patients have been successfully discharged. however, many studies have reported that discharged patients could again be tested viral nucleic acid positive - , arising the possibility of a potential re-infection. results in this study showed that 're-positive' patients do not display any distinguishing clinical markers, except illness severity, making the existence of such group of patients questionable. researchers questioned the low sensitivity of many viral rna detection kit currently in use, that could also be affected by many other factors (e.g. quality control of the kits, quality and sample delivery method, etc.) . xiao and colleagues found this re-positive phenomenon could be due to false-negative of rt-pcr, since they observe that a certain number of covid- patients had a prolonged viral rna conversion time . yuan and colleagues retrospectively studied re-positive covid- patients in shenzhen, china and suggested the results of viral nucleic acid by rt-pcr were variable, even if patients showed two negative results for respiratory pathogens nucleic acid tests before discharge . this study, like ours, suggests that the re-positive phenomenon could be a technical bias rather than actual patient group. besides the possibility of false-negative results from rt-pcr, sample selection and collection could also lead to the 're-positive' detection and efficient virus load could be key to have positive rt-pcr results. it has been shown that sars-cov- binds to ace receptor which are mainly located in lower respiratory tract rather than www.nature.com/scientificreports/ upper . consequently, for instance, collected samples from a nasopharyngeal swab might have less virus load compared to other sample collected from the lower respiratory tract samples (e.g. alveolar lavage fluid), leading to an unreliable rt-pcr result. furthermore, even though samples are properly collected and analysed resulting in positive results, it could potentially not prove that patients are infective, as only those who can transmit live virus are defined as infective patients . some researchers argued that the use of corticosteroid may have potential risks, as it could suppress our immune functions, decreasing the ability of viral clearance . theoretically, this could be a reasonable hypothesis, accounting for the occurrence of re-positive cases, however, in the present study, the use of corticosteroid did not increase the number of 're-positive' patients, consistent with previous work by lan . lan and colleagues found those 're-positive' patients to be younger, with shorter hospitalization time and shorter seroconversion. however, in the present study, the opposite was found. specifically, 're-positive' patients showed to be severe cases, with higher apache ii and curb- score, and longer hospitalization time. another issue to consider is the many differences between covid- patients from china, europe and america, especially with sequalae. for instance, researchers reported patients from europe and america with more olfactory and gustatory complaints , , while chinese patients had less . one hypothesis could be related to the different virus strains present in different countries , leading to different clinical characteristics and even sequalae. however, there are currently no study comparing "re-positive" rate in different regions worldwide. some limitations of the present study merit consideration. firstly, no covid- mild cases patients were enrolled in this study due to different local medical care policies. specifically, wuhan was the first city with covid- outbreak, with the largest cases patients in the country. to increase medical care efficiency, many fangcang shelter hospitals were created for mild covid- cases . therefore, the central hospital of wuhan, as a large general hospital, mainly dealt with patients ranging from moderate to critical. additionally, at the beginning of covid- outbreak in wuhan, every large general hospital was overloaded, which may have resulted in an imperfect quality control of sample collection and delivery. consequently, patients enrolled in this study are different to previous published result by lan, which may lead to bias. moreover, in lan's study, the severity of their enrolled patients were different from ours, as most of the covid- patient in their study are general cases which may also cause bias. additionally, a novel pathogen as sars-cov- is, it is currently not certain that what, the virus itself or the excessive immune reaction, account for the severity of patients. therefore, it remains possible that severe and critical patients may have higher viral loads and longer clearance time. more research is necessary. furthermore, our is a single-center, retrospective study with limited number of participants, therefore, more prospective clinical research is needed. www.nature.com/scientificreports/ another important issues in our study is that viral load quantification was not conducted due to lack of skilled laboratory personnel at the beginning of this pandemic. yu and colleagues reported that the quantitative virus load detection would give rise to the diagnosis sensitivity and accuracy, especially in cases with low virus load . some researchers hypothesized that the re-positive covid- cases could be the virus re-infection . immunologically speaking, after the acute infection of the sars-cov- , the human body should generate specific neutralizing antibodies against the virus for at least days ; furthermore a recent animal experiment in rhesus macaque indicated re-infection phenomenon did not happen . these result are in line with other studies on severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) . in conclusion, in the present study, 're-positive' covid- patients were compared to non-'re-positive' patients, showing no significant differences between these two groups based on clinical characteristics, but correlated to illness severity. no evidence indicates 're-positive' patients were still infective, and those who have had close contacts with 're-positive' patients were currently safe, but follow up studies are in progress. since understanding of the mechanisms of sars-cov- infection is still lacking, a careful discharge protocol should be applied (e.g. negative results of the nucleic acid tests of respiratory pathogens for consecutive times), and post-discharge quarantine should be strictly observed, especially for severe and critical covid- patients. genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding positive rt-pcr test results in patients recovered from covid- persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients positive result of sars-cov- in sputum from a cured patient with covid- prolonged presence of sars-cov- in feces of pediatric patients during the convalescent phase detection of novel coronavirus ( -ncov) by real-time rt-pcr false-negative of rt-pcr and prolonged nucleic acid conversion in covid- : rather than recurrence pcr assays turned positive in discharged covid- patients potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody covid- disease with positive fecal and negative pharyngeal and sputum viral tests effect of corticosteroids on treatment failure among hospitalized patients with severe community-acquired pneumonia and high inflammatory response: a randomized clinical trial upper airway symptoms in coronavirus disease (covid- ) clinical characteristics associated with persistent olfactory and taste alterations in covid- : a preliminary report on patients clinical presentation of covid- : a systematic review focusing on upper airway symptoms introductions and early spread of sars-cov- in the new york city area fangcang shelter hospitals: a novel concept for responding to public health emergencies quantitative detection and viral load analysis of sars-cov- in infected patients will we see protection or reinfection in covid- ? breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid- reinfection could not occur in sars-cov- infected rhesus macaques t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus spike protein elicit a specific t-cell immune response in patients who recover from sars antibody response and disease severity in healthcare worker mers survivors with the present study, firstly we would like to send our gratitude to all the health worker and covid- patients who have made their valuable contributions. moreover, we would like to thank prof. daniela traini (woolcock institute of medical research, respitech group), prof. georges e.r. grau (university of sydney, vascular immunology unit) and miss wenzhu li (a warm-hearted and responsible citizen from wuhan, china) for their generous help with the proofreading of the manuscript. this project was funded by grants from the national natural the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to w.s. or l.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -zdf ji k authors: gu, hongjing; xie, zhengde; li, tieling; zhang, shaogeng; lai, chengcai; zhu, ping; wang, keyu; han, lina; duan, yueqiang; zhao, zhongpeng; yang, xiaolan; xing, li; zhang, peirui; wang, zhouhai; li, ruisheng; yu, jane j.; wang, xiliang; yang, penghui title: angiotensin-converting enzyme inhibits lung injury induced by respiratory syncytial virus date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: zdf ji k respiratory syncytial virus (rsv) infection is a major cause of severe lower respiratory illness in infants and young children, but the underlying mechanisms responsible for viral pathogenesis have not been fully elucidated. to date, no drugs or vaccines have been employed to improve clinical outcomes for rsv-infected patients. in this paper, we report that angiotensin-converting enzyme- (ace ) protected against severe lung injury induced by rsv infection in an experimental mouse model and in pediatric patients. moreover, ace deficiency aggravated rsv-associated disease pathogenesis, mainly by its action on the angiotensin ii type receptor (at r). furthermore, administration of a recombinant ace protein alleviated the severity of rsv-induced lung injury. these findings demonstrate that ace plays a critical role in preventing rsv-induced lung injury, and suggest that ace is a promising potential therapeutic target in the management of rsv-induced lung disease. the mechanisms underlying rsv-induced lung disease and the associated long-term consequences remain incompletely understood, although lung inflammatory responses likely play a central role in promoting pathogenesis , . rsv infection can progress to ards, the most severe form of ali that further contributes to morbidity in infants [ ] [ ] [ ] . notably, our recent study showed that ace is involved in influenza h n -induced lung injury and that use of a recombinant ace protein alleviates the severity of ali . in this study, we hypothesized that the ras system mediates the severity of rsv-induced lung injury. our findings demonstrate that ace plays an important role in rsv-induced lung injury, and that administration of a recombinant ace protein attenuates the severity of lung injury in a preclinical model of rsv infection. animals. three-week-old wild-type (wt) c bl/ (abbreviated to b ) mice (experimental animal center, beijing, china), and three-week-old ace knockout (abbreviated to ko) mice (b background) were housed in the animal facility at beijing institute of microbiology and epidemiology in accordance with institutional guidelines. all experiments involving human subjects were performed in accordance with the guidelines and regulations of beijing institute of microbiology and epidemiology. all experimental protocols were approved by the institutional animal care and use committee of beijing institute of microbiology and epidemiology (id: syxk - ) and carried out in accordance with the approved guidelines. experimental mouse models. the wild-type rsv virus (abbreviated to bj ) used in this study was isolated from a confirmed rsv-infected patient in beijing children's hospital. the genomic sequence of bj is available in the genbank database (accession number kc ). bj and a virus were amplified in hep- cells. after freeze-thaw, the infected cells were harvested by centrifugation at , × g for min at °c; the supernatants were concentrated by centrifugation at , × g (beckman rotor sw ) and the concentrate was re-suspended in x pbs. viral suspensions were overlaid onto a discontinuous sucrose gradient by centrifugation at , × g; the purified bj or a virus bands were collected, diluted with pbs, and concentrated by centrifugation at , rpm for h. purified viruses were re-suspended in x pbs and stored at − °c. for rsv-induced lung injury, -week-old wt b mice were anesthetized with μ l % (w/v) pentobarbital sodium, and then inoculated intranasally (in) with × . pfu of bj or μ l x pbs (mock-infection). for all mice, body weight was recorded daily before and after viral infection. progression-free survival was evaluated at the time of viral infection. for uv-inactivation, bj viral suspensions at . ml/well in a six-well plate were exposed to ultraviolet (uv) (approximately inches from uv light source) for min in a biosafety cabinet. ang ii levels were measured as described elsewhere . western blotting. lung tissues were lysed in ripa buffer. the following antibodies for western blotting were used: rat polyclonal anti-ace antibodies and rabbit monoclonal anti-ace antibodies (all r&d systems). histological examination. following anesthetization with pentobarbital sodium, -week-old mice were infected in with × . pfu bj , and sacrificed on day (days post infection; dpi). for hematoxylin and eosin (h&e) staining, the lungs from each mouse were fixed in % formalin, embedded in paraffin, cut into -μ m sections, and stained with h&e to analyze pathological changes in lung tissue. the number of inflammatory cells was counted, and data were analyzed statistically and presented as the number of cells per × field. lung wet-to-dry weight ratio. mice were sacrificed at dpi. the lungs were weighed and then dehydrated at °c for h. the severity of pulmonary edema was measured using the lung wet-to-dry weight ratio. viral titration. virus titers were measured in the supernatants of lung homogenates from mice at dpi as described previously . briefly, hep- cells were cultured as monolayers in -well plates, and infected with serial -fold dilutions of lung suspension in dmed/f ( : ). the overlay was prepared with agar (sigma-aldrich, st louis, mo, usa) at a final concentration of % dmem/f = : (hyclone laboratories, south logan, ut, usa) supplemented with % fetal calf serum (gibco, waltham, ma, usa). hep- cells were cultured for days at °c and % co . cells were stained with crystal violet, and viral plaques were counted. viral titers were calculated using the reed and muench method , and expressed as log pfu/g of lung tissue. were performed using graphpad prism software (ver. . ; san diego, ca, usa). a value of p < . was considered statistically significant. all experiments were performed in triplicate at least. to determine the role of ace in rsv-induced lung injury, we first measured the levels of ang ii in the plasma of rsv-infected patients. in total, pediatric patients and healthy children were recruited from beijing children's hospital, the china pla general hospital, and beijing hospital in and . the clinical characteristics underlying the conditions and outcomes of these individuals are described in tables s and s . all patients were confirmed to carry solely the rsv viral genomic fragment using pcr analysis. concomitantly, plasma levels of ang ii were significantly elevated in rsv-infected patients relative to healthy subjects (p < . ; fig. a ). to determine any correlation between plasma levels of ang ii and the onset of rsv infection, we collected plasma samples from these individuals at early-and late-stages of infection. plasma levels of ang ii were higher in the early-stage (up until days from onset) than in the late-stage (after days from onset), indicating that plasma levels of ang ii decreased rapidly from the infection to the recovery phase of rsv infection ( figure s a ). taken together, our data indicate that the renin-angiotensin system plays an important role in the host response against rsv infection. to evaluate the direct effect of rsv infection on ras further, mice were infected in with live rsv bj or a virus. three days after viral infection, plasma levels of ang ii were measured. rsv a viral infection resulted in a significant increase in ang ii levels relative to mock infection (p < . ; fig. b ). interestingly, rsv bj infection led to a greater increase in ang ii levels than did infection with a virus (p < . ; fig. b ). concomitantly, ang ii levels in lung homogenates were moderately elevated in mice infected with rsv bj (p < . ) or a virus relative to mock infection (fig. c) . to determine the mechanism responsible for the increased levels of ang ii, we examined the protein levels of ace , which converts ang ii to ang - . accumulation of ace protein was reduced in mice infected with rsv bj or a virus, relative to mock infection (fig. d) . importantly, ace protein levels were dramatically decreased in lung homogenates from mice infected with rsv bj virus at days post-infection (p < . ; fig. d ). interestingly, levels of ace protein were comparable in the lungs of mice infected with rsv or mock virus (fig. d) . these data demonstrate that rsv infection leads to a decrease in ace expression and an increase in ang ii levels in preclinical models, further supporting the critical role of ras in rsv infection. to investigate the impact of ace on live rsv-induced clinical signs associated with lung injury, we infected ace knockout (ace ko) or wild-type (wt) mice with rsv bj virus, and measured overall survival. fourteen days post infection, % of wt mice survived, whereas only % of ace ko mice survived (fig. a) . seven days post rsv infection, the average body weight of ace ko mice decreased by % relative to that of wt mice (p < . ; fig. a) . meanwhile, the lung histopathology scores, as defined by leukocyte infiltration cell counts, were significantly reduced in ace ko mice compared with wt mice (fig. c) . moreover, lung edema, defined by the lung wet-to-dry weight ratio, was significantly greater in rsv-infected ace ko mice compared with wt mice (p < . ; fig. d ), indicating a more severe lung injury phenotype. consistent with these findings, viral titers in lung tissue of rsv-infected ace ko mice were significantly higher by approximately five-fold relative to those of rsv-infected wt mice (p < . ; fig. e ). furthermore, plasma levels of ang ii were significantly elevated in ace ko mice compared with wt mice (p < . ) at days post bj infection (fig. f) . similar findings were observed in ace ko and wt mice infected with rsv a virus. in an independent experiment, % of wt mice were alive at dpi, whereas only % of ace ko mice survived ( figure s a) . concomitantly, body weight decreased in ace ko mice compared with wt mice at dpi ( figure s b ). the lung wet-to-dry weight ratio was significantly higher in rsv a -infected ace ko mice than in rsv a -infected wt mice (p < . ; figure s c ). viral titers of rsv a virus-infected ko mice were also significantly higher by nearly five-fold compared with wt mice (p < . ; figure s d ). collectively, these results suggest that ace plays a critical role in rsv-induced lung injury. thus, interfering with ace expression may attenuate disease severity following respiratory rsv infection. proteins efficiently protect against rsv infection in preclinical models, rsv bj virus-infected wt mice were treated with recombinant hace proteins ( . mg/kg) day before infection, as well as at and dpi. at dpi, a significant increase in body weight was observed -n hace -treated mice and vehicle-treated mice (p < . ; fig. a ). importantly, hace -treated mice exhibited only mild inflammatory reactions, whereas severe histopathological damage, including fragmentation of alveolar walls and infiltration of lymphocytes, was observed in vehicle-treated mice. concomitantly, leukocyte cell counts were significantly decreased in hace -treated mice compared with vehicle-treated mice (p < . ; fig. b ). moreover, the lung wet-to-dry weight ratio in hace -treated mice was significantly reduced (p < . ; fig. c ), indicating an attenuation of lung edema mediated by recombinant ace proteins. additionally, the lung viral titers of hace -treated mice were significantly decreased by nearly % compared with vehicle-treated mice (p < . ; fig. d ). furthermore, plasma levels of ang ii were significantly decreased in hace -treated mice (p < . ) at dpi (fig. e) . notably, hace -treated mice infected with rsv a exhibited mild pathological lung alterations ( figure s a ). the lung wet-to-dry weight ratio in hace -treated mice was decreased by % compared with vehicle-treated mice (p < . ; figure s b ). viral titers of hace -treated mice infected with rsv a were also significantly decreased by % compared with vehicle-treated mice (p < . ; figure s c ). these data therefore demonstrate the therapeutic effects of recombinant hace protein in improving lung physiology and histopathology in vivo, and support the potential use of hace protein to alleviate the symptoms of acute lung injury during rsv infection. tors mediate the action of ang ii, we next assessed the efficacy of inhibitors specific for the at or at receptors (at r/at r) by examination of in vivo clinical signs in the lungs. first, live rsv-infected wt mice that received the vehicle treatment exhibited lung edema and infiltration of inflammatory cells (fig. a) . the at r inhibitor (losartan) treatment markedly attenuated the severity of rsv-induced histopathologic alterations in rsv-infected mice (fig. a) . next, the lung wet-to-dry weight ratio was significantly reduced, by % (p < . ), in at r inhibitor-treated mice compared with vehicle-treated mice (fig. b) . moreover, lung viral titers decreased markedly in the rsv bj virus-infected wt mice treated with at r inhibitor (fig. c) . furthermore, plasma levels of ang ii were also reduced by treatment with the at r inhibitor (fig. d) . on the contrary, the at r inhibitor had no significant effects on the lung wet-to-dry weight ratio ( figure s a ) or viral titers (figure s b) , suggesting a lack of efficacy of this inhibitor in rescuing the lung injury induced by bj virus-infected wt mice. therefore, these data support the assertion that both ang ii and at r play critical roles in regulating rsv-induced ali in preclinical models. our previous demonstration that ace mediates rsv-induced lung injury (fig. ) , led us to hypothesize that suppression of at r attenuates rsv-induced lung injury in ace ko mice. to test this, rsv bj virus-infected ace ko mice were treated with at r ( mg/kg) at day before infection, dpi and dpi. at dpi, at r-treated ace ko mice exhibited mild inflammatory changes, whereas severe histopathological damage was observed in vehicle-treated ace ko mice. concomitantly, leukocyte cell counts were reduced by % in ace ko mice treated with at r (p < . ; fig. a ). the lung wet-to-dry weight ratio was decreased by % in at r-treated ace ko mice compared with vehicle-treated ace ko mice (p < . ; fig. b ). moreover, lung viral titers were decreased significantly in at r-treated ace ko mice compared with vehicle-treated ace ko mice (fig. c ). of note, there was no apparent effect on lung edema ( figure s a ) or viral titers ( figure s b ) in bj virus-infected ace ko mice treated with the at r inhibitor, indicating that at r was likely not to be involved in the process of rsv-induced lung injury. based on the above data, we propose that ace may play a crucial role in the process of rsv viral infection, mainly by affecting at r (fig. d) . our data suggest that rsv infection causes severe lung injury in an experimental mouse model and in patients, at least in part by modulating the ras system via down-regulation of the ace -at r axis. previous studies have reported on the use of rsv-induced lung injury models for testing drug efficacy . the selection of a reliable mouse model of rsv infection for investigation of associated clinical abnormalities has been a major challenge in rsv research. the a strain was originally isolated from clinical samples in , and has long been the standard laboratory rsv strain used in research , . however, this historical strain induces mild lung pathology in mice compared to other recently isolated strains . therefore, we attempted to isolate pathogenic viruses from clinical nasopharyngeal swabs collected from pediatric patients, and subsequently identified bj . bj virus is an isolated rsv belonging to subgroup a, and causes severe respiratory disease in preclinical models. the hospital-isolated bj strain was used in this study because of its direct clinical relevance. we anticipate that the bj strain will be widely used for the development of animal models for rsv research. previous studies have shown that ace , expressed in the lungs of patients with pulmonary diseases and also in healthy subjects, is involved in ali induced by sars and influenza (lethal h n and h n virus) , . importantly, yumiko imai et al. revealed that ace is an essential receptor for sars infection in vitro and in vivo , , and that it protects individuals from developing severe acute lung failure . moreover, as ace is a non-specific protease, it would also be interesting to investigate its role in the regulation of cellular metabolites . however, ace is non-functional during lung injury processes induced by various influenza virus subtypes such as h n and h n (data not shown). there are associations between influenza virus infection and ace levels, even with different influenza strains (avian high-path h n versus h n ) , . despite the reports that focused on the impact of these viruses on lung pathology, the underlying molecular mechanisms responsible for ras-and ace -mediated-lung injury have not been fully elucidated. we report here that ace plays an important role in rsv-induced lung injury. this was associated with increased plasma levels of ang ii in rsv-infected patients in china. nevertheless, a considerably larger cohort of rsv-infected individuals from various regions of china is required to confirm these findings. live rsv-induced lung injury resulted in significant down-regulation of ace at the early stage after the onset of infection, which controlled the function of ras, but not in the uv-bj group ( figure s ). we hypothesize that rsv growth is directly or indirectly related to rsv-induced pathology. moreover, our studies provide a molecular basis for the mechanism of lung injury in patients infected with rsv. importantly, the levels of ang ii were elevated following down-regulation of ace , causing severe lung injury via at r during the process of rsv infection (fig. d) . our discovery reveals that common molecular and cellular mechanisms are likely shared by animal models and patients with acid-aspiration , sepsis , sars-cov , influenza virus , or rsv infection. moreover, our data demonstrate that ace deficiency aggravates rsv-induced ali and that administration of a soluble ace recombinant protein ameliorates lung injury in vivo. nevertheless, further studies assessing the potential therapeutic efficacy of recombinant human ace protein are required using different animal models to verify the protective effects of ace against rsv-induced lung injury. other studies have used cotton rats and monkeys to study rsv pathogenesis and rsv vaccine or drug investigations . the combination of clinical findings and preclinical studies has revealed a critical role of ras in the pathogenesis of rsv-induced lung injury, and demonstrated that ace plays a key role in the development and progression of rsv infection. collectively, our findings suggest the intriguing possibility that targeting ace and ras represents a valuable strategy for treating rsv infections. modulation of ace represents a potential novel pharmacological approach to ameliorate rsv-induced lung inflammation. mechanisms leading to decreased expression/activity of ace have not been fully investigated. in our initial clinical study, we found that elevated plasma concentrations of ang ii correlated with the severity of lung injury. we therefore hypothesize that levels of ang ii in plasma samples of patients with rsv infection may reflect events in the lower respiratory tract. although our study was performed in a relatively small cohort of rsv-infected subjects, it included a balanced representation of the spectrum of disease that is caused by this pathogen in infancy. importantly, our preclinical studies suggest that infections caused by other viral respiratory pathogens that are known to cause lung disease in infants may also result in similar inhibition of ace expression. further clinical studies will be required to clarify this issue. in this regard, we are currently enrolling infants in a prospective study to test patients with a broad spectrum of disease 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peripheral blood neutrophils ace and ace in inflammation: a tale of two enzymes animal models of human respiratory syncytial virus disease this work was supported in part by the funding from national programs for high technology research and development of china (ss aa ), the ministry of science and technology of china ( cb , zx and ss aa ). p.h.y was supported by beijing nova program (no.z ).we will thank professor josef m. penninger and chengyu jiang for providing ace knockout mice. h.g., s.z., c.l., p.z., k.w., l.h., y.d., z.z., x.y and l.x contributed to experiment. p.y., h.g. and b.c. analyzed the data. z.x., t.l., p.z., z.w., r.l. and p.y. collected the samples. p.y., j.y. and x.w. designed the experiments and reviewed/edited the manuscript extensively. p.y., j.y. and x.w. wrote the manuscript. key: cord- -tid a authors: basso, luis g. m.; vicente, eduardo f.; crusca jr., edson; cilli, eduardo m.; costa-filho, antonio j. title: sars-cov fusion peptides induce membrane surface ordering and curvature date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: tid a viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. the s subunit of the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus (cov) contains internal domains called fusion peptides (fp) that play essential roles in virus entry. although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. here we employed differential scanning calorimetry (dsc) and electron spin resonance (esr) to gather information on the membrane fusion mechanism promoted by two putative sars fps. dsc data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. esr showed that both fps increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. therefore, bending moment in the bilayer could be generated, promoting negative curvature. the significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the sars-cov-mediated membrane fusion are discussed. corresponding to residues - (immediately positioned n-terminally to hr ) and to residues - (immediately positioned c-terminally to a second, internal cleavage site s ' at r ) , and another less conserved region corresponding to a hydrophobic stretch located between residues and (near the s /s boundary region at site r ) . these putative fps are thought to destabilize host cell membranes, driving the refolding of the s subunit into the post-fusion -hb configuration, one of the late steps in the viral membrane fusion process . although membrane fusion promoted by class i viral glycoproteins, such as sars-cov spike, human immunodeficiency virus (hiv) gp or influenza virus hemagglutinin (ha), has been broadly studied in recent years [ ] [ ] [ ] [ ] , many aspects of the molecular mechanism behind the virus-host cell membrane fusion remain unknown, including conformational changes of the lipid bilayers during peptide-membrane interactions. elucidating the nature of protein-lipid interactions as well as the conformational properties of both the membranotropic segments of the viral fusion proteins and the lipids in cell membranes can help to dissect the major steps of the orchestrated membrane fusion mechanism promoted by those biological machines. however, structural and dynamics information at the molecular level of peptide-induced membrane fusion in the context of the whole spike protein is difficult to obtain. thus, synthetic peptides corresponding to the putative fusion peptides might be very useful in providing detailed information on the interaction of those segments with lipid model membranes not only because the peptides themselves support membrane fusion, but also because there is a direct correlation between the effects of mutations in the intact protein and in the peptide analogues for membrane fusion [ ] [ ] [ ] . in the present study, we investigated the effects of two putative fusion peptides from sars-cov s glycoprotein, corresponding to residues - (sars fp ) and - (sars ifp ) , , , , on the structural dynamics, physicochemical properties, and thermotropic phase behavior of lipid model membranes by differential scanning calorimetry (dsc), continuous wave (cw) and pulsed electron spin resonance (esr) along with nonlinear least-squares (nlls) spectral fitting . we found that both peptides increase the lipid packing and decrease the water content inside the lipid bilayer only for membranes containing negatively charged lipids, as well as generating opposing curvature stresses on highly curved membranes containing non-bilayer-forming phospholipids. the significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids for the fusion mechanism mediated by the sars-cov s glycoprotein are discussed. we are interested in investigating the extent of perturbation of model membranes caused by a functional concentration of the peptides, i.e, peptide concentration that is already known to promote fusion of model membranes. at this concentration, what are the changes in the structural dynamics, curvature and hydration of the lipids and in the thermodynamic parameters of the membranes that lead to membrane fusion? it has been shown that sars fp and sars ifp are able to induce membrane fusion only at high peptide-to-lipid molar ratio , . thus, we chose a : lipid/peptide molar ratio for most of the experiments and compared the effects of the peptides on model membranes that present very different physicochemical properties. to examine the effects of the putative sars fusion peptides on the structural integrity of lipid model membranes, the thermotropic phase behavior of different multilamellar vesicles (mlv) in the absence and in the presence of mol% of peptides were determined (fig. ) . the thermodynamic parameters obtained from the analysis of the dsc curves are shown in table . it is worth mentioning that the membrane-associated peptides remain folded in the liquid crystalline phase of the phospholipids , . multilamellar vesicles of dppc and dppg exhibited two endothermic events in the temperature range studied. the low-enthalpic, broad pretransition arises from the conversion of the lamellar gel phase, l β' , to the ripple gel phase, p β' , and is observed at about . °c for dppc and at . °c for dppg (table ) . on the other hand, the more energetic and more cooperative (narrow) main phase transition arises from the conversion of p β' to the liquid-crystalline phase, l α , and is centered at . °c for dppc and at . °c for dppg. membranes composed of unsaturated lipids presented only a very asymmetric, low-enthalpic, and broad main phase transition at . °c for popa, at . °c for pope, and at ~ . °c for pops. these results reasonably agree with the literature [ ] [ ] [ ] [ ] . the slight discrepancies observed in comparison with literature data are likely due to differences in lipid preparations, buffers, and ionic strength [ ] [ ] [ ] . by contrast, the main phase transition of dpps vesicles is split into two endothermic events, one centered at . °c and the other centered at . °c (table ). this two-peak feature has been attributed to different protonation states of the head group polar moiety . binding of drugs, peptides, and proteins to membranes can promote structural and dynamic changes of lipid bilayers that significantly affect their thermotropic phase behavior , . shifts of the melting transition temperature of the lipids, for instance, can be related to alterations of the entropy change between the gel and fluid states, whereas a broadening of the dsc thermogram may be the result of a decreased transition cooperativity (~ /Δ t / ). as can be observed in fig. , incorporation of mol% of fusion peptides into the lipid bilayers did not strongly affect the main phase transition temperature of the liposomes. however, the enthalpy and entropy changes of the main phase transition were significantly altered, especially for membranes containing negatively charged lipids ( fig. and table ) . sars fp and sars ifp only slightly perturbed zwitterionic dppc liposomes. it was observed a small increase of both the melting temperature t m (less than %) and the calorimetric enthalpy change of the transition Δ h (less than . %), indicating a slight stabilization of the peptide-bound dppc gel phase. the increased pretransition temperature of the peptide-containing dppc vesicles also indicates structural changes in the dppc polar head group , . on the other hand, more prominent effects on the enthalpy change and on the cooperativity of the transition of zwitterionic pope vesicles were observed. sars fp decreased the transition Δ h by about %, whereas sars ifp decreased it by % (table ) . pope-sars fp interaction also markedly broadened the dsc endotherm (increase of Δ t / by ~ %). this result indicates that sars fp intercalates within pope bilayer and thus decreases the intermolecular cooperativity of the transition. overall, more significant effects on the thermodynamic (or dsc) parameters of the bilayer phase transitions were observed for membranes containing negatively charged lipids ( table ) : reduction of the calorimetric Δ h by about % ( %) for dppg and by about % ( %) for pops, for instance, after incorporation of sars fp (sars ifp ). unlike the previous cases, endotherms containing multiple peaks appeared when the peptides were mixed with popa vesicles (fig. ) . in this case, the lower-temperature endothermic peak certainly arises from peptide-bound membranes, whereas the higher-temperature peak can be due to peptide-free popa vesicles in the popa/sars fp samples and due to a mixture of peptide-free and peptide-bound popa vesicles in the popa/sars ifp samples. the two-component feature of dpps main phase transition remained in the peptide-containing dpps samples. although the calorimetric enthalpy change of the whole transition decreased in the presence of the peptides, the area under the higher-temperature endotherm increased. the peptides also promoted opposing effects on the cooperativity of the two endotherms: while sars fp narrowed both transitions (thus increasing lipid cooperativity), sars ifp broadened the endotherms. this may be related to different locations and topology of the peptides in the membranes. another interesting feature is the appearance of a third, low-enthalpic peak at around °c in dpps/peptide vesicles. this result indicates a different mechanism of ps-peptide interaction compared to the other negatively charged lipids. the low-enthalpy, higher-temperature peak ( °c) was also observed in dmso-treated peptide-containing dpps liposomes, but was not observed in the acetonitrile-treated or dmso-treated peptide-free dpps vesicles. this result indicates that the new peak is not due to the binding of acetonitrile or dmso from the peptide stock solutions to the dpps mlvs (see fig. s and table s in supplementary information). interestingly, a low-enthalpy peak also appeared in the peptide-bound pops vesicles at a temperature slightly higher than the t m of the peptide-free pops vesicles (fig. ) . thus, this more stable and much less energetic endothermic peak may be due to specific ps-peptide interaction. taken together, these results suggest different mechanisms for the interaction of the peptides with zwitterionic and negatively charged phospholipids. electrostatic interactions seem to play an important role for the sars-cov fusion peptide-membrane interactions, since both peptides are positively charged at ph . : sars fp , + e sars ifp , + . moreover, since both peptides also significantly changed the thermotropic phase behavior of dppg mlvs at high ionic strength ( mm nacl; reduction of Δ h by % for sars fp and by % for sars ifp - table ), hydrophobic interactions may also play an important role in peptide binding and penetration into membranes. interestingly, the peptides promoted different effects on the thermograms of dppg, dpps, pops, and popa, suggesting that not only the charge but also the lipid packing (dpps vs. pops) and the lipid polar head group (pg, ps, or pa) may contribute to the energetics of peptide-membrane interactions. membrane curvature-promoting properties. since changes in membrane curvature have been associated with the potential mechanistic role of fusion peptides in inducing membrane fusion , , we tested the ability of the sars-cov fusion peptides to promote curvature strain on dipope vesicles. below the lα -h ii phase transition temperature, t h , phosphatidylethanolamines, such as dipope, spontaneously form lipid bilayers in the liquid-crystalline state, whereas above t h , they usually pack together in a highly curved hexagonally inverted structure with negative membrane curvature , . stabilization of this concave curvature due to peptide binding, for instance, will favor the nonbilayer h ii phase, which will translate into a t h reduction . conversely, peptides that induce positive membrane curvature will increase t h of the dipope/peptide samples and thus will stabilize the liquid-crystalline bilayer phase . figure shows dsc traces of dipope without and with . or . mol% of sars fp and sars ifp at both low ( fig. a) and high (fig. b) effects of the peptides on the structural dynamics of lipid vesicles. the local ordering and rotational dynamics of the lipid head group and acyl chains were investigated by cw esr using the nitroxide-labeled lipids dpptc, -pcsl, and -pcsl (see structure of the spin probes in supplementary fig. s ), which monitor different regions of the lipid bilayers. dpptc reports on the lipid/water interface, whereas -pcsl and -pcsl monitor the hydrophobic core of the membranes at different depths of penetration , [ ] [ ] [ ] . despite the perturbation of the dppc thermotropic parameters caused by the peptides, esr spectra of the spin labels embedded into dppc and dppc/cholesterol / (mol/mol) mlvs at different temperatures did not show noticeable changes in the lipid structural dynamics of the peptide-containing vesicles as compared to the peptide-free lipid bilayers (see supplementary fig. s ). this means that the membrane-bound peptides do not significantly perturb the local structural dynamics of the head group or the hydrophobic core of the zwitterionic mlvs as investigated by cw esr. on the other hand, remarkable changes in the esr spectra of the spin labels in liposomes containing negatively charged lipids, such as dppg, dpps, and popa, were observed. the experimental and best-fit nlls simulations are presented in supplementary figs s and s and the best-fit magnetic tensor components, rotational diffusion rates, and order parameters are summarized in supplementary tables s -s . before analyzing the changes in the lipid ordering and mobility induced by the peptides, it is worth mentioning a general feature observed for the order parameter s of dpptc in the gel and the fluid phases of all lipid model membranes. we generally and consistently found a positive value for s in the gel phase of the lipids and a negative value in their fluid phase. for instance, we found s = − . for dpptc in dppg at °c (table s ) , s = − . in dpps at °c (table s ) , and s ~ − . in popa at °c and °c (table s ). the meaning of this negative value for s of dpptc was extensively discussed by ge and freed for this same spin label in the fluid phase of dppc dispersions (s = − . at °c) in terms of the analysis of the cartesian components of the restoring potential u(Ω) . in the gel phase, there is a molecular force field in the head group that tends to align the trimethyl ammonium (tma) group of the dpptc (supplementary fig. s ) perpendicular to the bilayer surface, i.e. parallel to the local director of the bilayer, giving rise to s > . however, in the fluid phase, that surface-orienting potential tends to align the tma group parallel to the surface, giving rise to a negative order parameter . by definition, s ≡ < ( cos θ − )/ > , hence s tends to − . when θ tends to ° and it is negative for ° < θ < ~ . °. thus, if we compare the s values of dpptc from our studies with that obtained by ge and freed, we might infer that the orientation of the tma group in dppc (s = − . ; ge and freed work) and in dpps (s = − . ; our work) lies in between the one in dppg (s = − . ) and the other in popa (s = − . ). those differences in the tma orientation are likely due to distinct orienting forces that arise from the particular hydrogen-bonding network provided by the pg, ps, and pa bilayers and the dipolar interactions between the zwitterionic phosphoryl-tempo-choline group of dpptc and the surrounding negatively charged head groups. additionally, irrespective of its sign, an increase of s (either by becoming more positive for s > or less negative for s < ) would lead to the same interpretation: a greater tendency of the preferential orienting axis of tma to orient along the local director of the membrane and an increased restriction of the amplitude of its rotational motion. figure shows the plots of the rotational diffusion rates r ⊥ and the changes in the order parameter s fig. ). thus, the dpptc esr spectra at °c may be regarded as a single averaged spectrum from which both gel-like and fluid-like populations are not resolved. therefore, the effect of the peptides on r ⊥ and s should be analyzed with caution in this case. the ordering effect of the peptides on the dppg head group region in the gel and fluid phases was also observed in the hydrophobic core of the bilayer at all temperatures, but with the most prominent changes in the ripple gel phase (fig. b ,c). the more fusogenic sars fp consistently increased the ordering of -pcsl and -pcsl more than sars ifp . particularly, since the -pcsl probe is more sensitive to molecular motions than dpptc and -pcsl , , the structural dynamics of the gel-like and fluid-like lipid states could be resolved and characterized (table s ) . although the peptides shifted the equilibrium between the two states towards the fluid-like one (the population of the fluid-like component increased from to ~ %), the packing (s ) of the center of the bilayer was also increased for both lipid states. as for the mobility, the peptides reduced the rotational diffusion of -and -pcsl mainly in the fluid phase. only sars fp was able to decrease r ⊥ below t m , with the most significant change observed for -pcsl at °c (r ⊥ decreased from . × s − to . × s − ; table s ). figure shows the r ⊥ and Δ s values for dpptc and -pcsl embedded in dpps at temperatures corresponding to the gel ( and °c) and fluid ( °c) phases of the membrane. the rotational mobility and order parameter of dpptc were both affected by the peptides at all temperatures, but the most striking changes were observed in the very ordered dpps gel phase (fig. a ). in the fluid phase, r ⊥ decreased by % and % for sars ifp and sars fp , respectively. at °c, r ⊥ decreased by - %: from . × s − to . × s − for sars ifp , and to . × s − for sars fp (table s ). the most prominent change in the dpps head group region induced by the peptides, based on our nlls simulations, took place on the molecular alignment of the dpptc tma group. firstly, the s at °c found in our simulations was . , much lower than that found in pure dppg at the same temperature (s = . ; table s ). ge and freed reported a value of . for dpptc in dppc dispersions in the gel phase ( °c) and a similar value was found by barroso et al. for a similar pc spin probe in an equimolar mixture of dppc/dpps (s = . ). this result indicates that the phosphoryl-tempo-choline group of dpptc is more loosely packed in the gel phase of the pure dpps bilayer than in the other model membranes reported. interestingly, peptide addition to pure dpps remarkably increased the ordering of the head group in the gel phase: s jumped from . to about . for both peptides at °c and similar changes were observed at °c. in the fluid phase, though, just a slight variation was obtained (Δ s = . - . ) (fig. a ). peptide binding to dpps head group caused a large ordering effect, thus restricting the mobility of the lipids. the spectra of -pcsl in the peptide-free dpps obtained at temperatures below t m presented very broad resonance lines (supplementary fig. s b ; °c). even in high ionic strength condition, a very broad lineshape in the ordered dpps gel phase persisted (not shown). this line broadening effect is due to strong dipolar interactions that arise from possible cluster formation of the pc spin labels in the dpps milieu. to test this hypothesis, we prepared dpps/ -pcsl samples with either . mol% or . mol% of the spin label. at the higher probe concentration, even broader lines were obtained as compared with the esr lineshape of . mol%, whereas narrower, but still broad, lines were found with . mol%. this result shows that -pcsl does not partition very well in the dpps gel phase but does so in the fluid phase, as illustrated in supplementary fig. s b (spectrum acquired at °c). after unsuccessful attempts to fit the -pcsl spectra with the addition of a heisenberg exchange coupling, we presented only the best-fit parameters from nlls simulations of the spectra acquired at °c (supplementary table s ). both r ⊥ and s were only slightly affected by the membrane-bound peptides at that temperature: r ⊥ slightly decreased from . × s − to . × s − for sars fp and s increased from ~ . to ~ . or . for sars ifp and sars fp , respectively (table s ) . interestingly, binding of the peptides to dpps promoted a better partition of -pcsl in the membrane. the structural organization of the dpps/peptide membranes altered in such a way that the pc spin probe became miscible in the binary system. as for the -pcsl in dpps, no change in the esr lineshape at both °c and °c upon peptide addition was observed (not shown), indicating no long range effect of the peptides on the center of the bilayer, despite the fact that they do bind and perturb the head group and the region around c of the lipid acyl chain. however, at °c, sars fp and sars ifp promote the appearance of a second, disordered component in the esr spectra table s ). in the fluid phase, the only parameter affected by peptide binding was the rotational mobility, which was diminished from . × s − to . or to . × s − for sars fp or sars fp , respectively. our results indicate the peptides bind to both gel and fluid phases of dpps, but peptide insertion into the membrane seems to be activated either by temperature or ultimately by loosening of the hydrophobic packing of the highly ordered dpps gel phase. as the temperature is increased, long range effects on r ⊥ of the acyl chain were observed in the bilayer center. we also studied the effect of the peptide binding on the structural dynamics of popa mlvs in the fluid phase ( °c and °c). as shown in table s , sars fp and sars ifp only changed the ordering and r ⊥ of the spin labels at °c (fig. ) . at °c, the esr spectra from peptide-free and peptide-bound vesicles are almost the same. differently from the other model membranes, peptide binding to popa head group causes an increase of the mobility: r ⊥ changed from . to . × s − for sars ifp and to . × s − for sars fp (fig. a ). this result is probably related to the lack of a bulky head group of popa (the surface area per lipid of pa head group is smaller than that of pg or ps), which may facilitate the rotational diffusion of the phosphoryl-tempo-choline group of dpptc, despite the small decrease in the amplitude of the tma molecular motion (s increased by about . to . ; fig. b ). on the other hand, the best-fit nlls simulations of -pcsl spectra showed a prominent effect on r ⊥ rather than s : while the rotational mobility diminished % for sars ifp , it decreased % for sars fp , whereas Δ s was only about . (supplementary table s ). as for the -pcsl, we found a very high value for s (− . ) in the peptide-free popa liposomes, indicating a large deviation from cylindrical symmetry of the molecular alignment of the end-chain label to the local director of the membrane. to gain further insights on the molecular orientation of the end-chain label we derived the orienting potentials along the cartesian directions from the potential coefficients obtained by the best fits of the nlls simulations (section si ): u x ≡ u( °, °)/kt = . , u y ≡ u( °, °)/kt = − . , and u z ≡ u( °, °)/kt = − . . this result indicates a strong preference for the rotational diffusion axes z r (parallel to the pz orbital of the nitrogen) and y r of the nitroxide moiety to align along the normal to the bilayer, while x r , which is parallel to the n-o bond, is prevented to align along the membrane local director. therefore, the unsaturation of the oleyl acyl chain of popa would allow for a dynamic bending of the end chain of the spin label that favors much more the alignment of both z r and y r axes to table s ). since the fluid-state lipids ( °c) present lower order (s = . ) and high degree of molecular misalignment (s = − . ) with r ⊥ ranging from . to . × s − (supplementary table s ), these results allow us to infer that this second component does not present the same properties of fluid-state lipids but rather peptide-bound lipids. that is, peptide binding to popa bilayers gives rise to peptide-enriched and peptide-free domains, where the latter is barely affected by the peptides. affected by the peptides. these results are in good agreement with our dsc experiments, in which, as discussed earlier, thermograms with multiple peaks arise from peptide-associated and peptide-free popa vesicles. additionally, it is important to investigate the effects of membrane fusion promoters and inhibitors on the bilayer properties in an attempt to identify the changes in the physicochemical parameters of the membrane that might be relevant for membrane fusion. generally speaking, insertion of inverted cone-shaped molecules such as the lysophosphatidylcholine -palmitoyl- -hydroxyl-pc (lpc) into the outer monolayer would prevent membrane fusion supposedly by promoting positive membrane curvature , . conversely, insertion of cone-shaped molecules such as phosphatidylethanolamine, arachidonic acid (aa), and linoleic acid (la) into the outer monolayer would facilitate membrane fusion presumably by inducing negative curvature . using esr, ge and freed have found opposing effects of lpc and aa on the order parameter of a headgroup spin label embedded in dmpc model membranes . in their work, an ordering (disordering) effect was observed for the fusion promoter (inhibitor) aa (lpc), which may help to induce (prevent) membrane fusion. since the putative fusion peptides from sars-cov s protein promote substantial membrane fusion only in the presence of negatively charged lipids, it is important to verify whether that correlation is also valid for negatively charged lipid-containing vesicles. to do so, we prepared equimolar mixtures of dppc/dppg and dppc/popa membranes and studied the effects of sars fp , sars ifp , la, and lpc on the order parameter s of dpptc at °c. the best-fit parameters of the esr spectra shown in fig. s are summarized in table s in supplementary information. figure shows that both fusion peptides and la caused an ordering effect on the head group of both model membranes. in contrast, lpc promoted a disordering effect of dpptc. these results are thus in accordance with those found by ge and freed for the zwitterionic dmpc and indicate that sars fp and sars ifp might facilitate membrane fusion similarly to how the fusion peptide from influenza hemagglutinin does. since the ordering effect of the lipid head group has been attributed to bilayer dehydration , we used eseem spectroscopy to find out whether the peptide-induced ordering of anionic vesicles is possibly related to membrane dehydration, as suggested as a general fusion mechanism of other class i fusion peptides , . eseem has been successfully applied to examine the penetration depth profile of deuterium-substituted molecules, such as water, glycerol, and sugar inside lipid bilayers [ ] [ ] [ ] [ ] as well as to investigate the water permeation at protein/peptide-lipid interface of membrane-interacting peptides and membrane proteins [ ] [ ] [ ] . here we used a d o-containing buffer to probe the deuterium environment surrounding the spin labels doptc, -pcsl, and -pcsl ( supplementary fig. s ) embedded in popc/popg / (mol/mol) mlvs. the different spin labels allowed investigating the changes promoted by the peptides in the water content from the lipid/water interface down to the hydrophobic core of the bilayers. changes in the modulation depth of the time-domain eseem spectra or in the deuterium spectral density yields information about d o molecules situated near the nitroxide within a distance range of up to . nm, which is the known spatial reach of the method . figure a shows the spectral density of the spin-labeled lipids in the peptide-free and peptide-containing lipid vesicles (the normalized time-domain stimulated echo signals are given in the supplementary fig. s a ). all spectra are dominated by two signals, one centered at . mhz, arising from the hyperfine interaction of the nitroxide with surrounding deuterium nuclei ( h-larmor frequency at mt), and the other centered at . mhz, which arises from matrix protons near the spin label. since lipids, peptides and possibly residual h o contribute to the spectral density at . mhz, the h signal was not further analyzed. two additional weak peaks were observed in the ft-spectra of doptc and -pcsl: one at ~ . mhz, which is assigned to n , and the other at ~ . mhz, which corresponds to the larmor frequency of p (fig. a) . those nuclei belong to the choline group of popc ( n) and to the phosphate group ( p) of the lipids. both peaks are absent in the -pcsl eseem spectra because the distance between the spin probe moiety in the bilayer midplane and the head group is beyond the limit of the technique. the deuterium peak is composed of two spectral components: a low-intensity broad signal that arises from a direct deuterium bond between d o and the nitroxide radical; and a narrow doublet of intensity i( h) and amplitude Δ (fig. a) , which arises from the quadrupole interaction between the electron spin and non- h-bonded water molecules inside the bilayer . both Δ and i( h) have been found to depend linearly and nonlinearly, respectively, on the concentration of free water molecules at a distance of . to . nm . the parameters i( h), measured at . mhz, and Δ for all lipid spin labels are shown in fig. b . as expected, the deuterium spectral density decreases from the bilayer/water interface toward the membrane center. particularly, i( h) is reduced from ( ± ) ns in the water/lipid interface to ( ± ) ns around th carbon position and to ( . ± . ) ns near the bilayer midplane in the peptide-free vesicles, indicating reduced water content in the hydrophobic core as compared to the head group region. those i( h) values are somewhat higher than previously reported spectral densities for popc/popg / (mol/mol) bilayers but are in the same order of magnitude of those for dppc , . the discrepancies are most likely due to different freezing protocols, sample preparation, and experimental conditions such as the choice of τ , the first interpulse delay, and the frequency chosen to measure the intensity of the spectral density . sars fp and sars ifp decreased both i( h) and Δ for the spin labels that monitor the hydrophobic region of the bilayer (fig. b) . this finding implies that both peptides interact with popc/popg membranes and displace free water molecules from their hydrophobic core. theoretical calculations from milov et al. indicated that nitroxide-deuterons interactions spread to around nm . therefore, the spin label at c position is able to monitor water density around c up to the level of the carbonyl-glycerol and phosphocholine regions (distant about . nm from c ) , . thus, the contribution from bulk water molecules to the spectral density of -pcsl is negligible , . the significant % decrease of Δ for -pcsl in the peptide-containing membranes, relative to the peptide-free vesicles (supplementary table s , fig. b ), allows us to infer that the intramembrane water in the outer membrane region is dramatically reduced upon peptide incorporation. the same reasoning holds true for the -pcsl. as for the head group spin label doptc, we found that the peptides surprisingly promoted opposing effects on i( h) and Δ : while they slightly raised the magnitude of the d o signal, Δ values were reduced (fig. b) . the source of this opposing effect remains unclear to us, but it may be of some interest to speculate on that. considering the electron density profile of pc lipids , , we may infer that the nitroxide radical of the doptc phosphoryl-tempo-choline group is in direct contact with both membrane surface and bulk water molecules. changes in the orientation of the phosphoryl-tempo-choline must therefore locally disturb the water density around the spin label. incorporation of cholesterol into pc membranes, for instance, decreases the ordering of the lipid head group, i.e., makes it less aligned along the bilayer director . this effect allows for water molecules to move from the bulk into the membrane (down to ~c ), thus increasing both i( h) and Δ (fig. s b and table s in supplementary information) . on the other hand, if the phosphoryl-tempo-choline group becomes more aligned along the bilayer normal, i.e. the s of doptc increases, membrane-surface dehydration takes place . in this situation, the concentration of membrane-surface water molecules is reduced in the 'membrane side' . in contrast, realignment of the head group dipole makes the nitroxide radical become more exposed to interact with bulk water molecules in the 'solvent side' . milov et al. have theoretically shown that the nonlinear dependence of the i( h) on water concentration is due to the formation of nitroxide-water complexes . therefore, if the water distribution around the nitroxide is locally perturbed in such a way that it allows for formation of nitroxide-water complexes (i.e., disturbances take place closer than . nm), it must affect mainly the i( h), but not Δ . that is, reorientation of phosphoryl-tempo-choline due to peptide-lipid interactions may be such that it allows formation of nitroxide-water complexes, affecting mainly i( h). this may correspond to the source of the increased i( h). the contribution to Δ stems mainly from water molecules belonging to the second hydration shell (> . nm) up to ~ nm. from the 'solvent side' , the second water coordination sphere for the water-exposed nitroxide radical should not change, thus the major contribution to Δ of doptc may arise from free water molecules located in the 'membrane side' . the reduced level of the head group hydration accounts therefore for the decrease of Δ values. however, further detailed studies are needed to investigate this hypothesis. viral membrane fusion is a concerted mechanism that involves remarkable protein and lipid conformational changes. the molecular mechanism by which viral fusion proteins catalyze the membrane fusion reaction and the molecular details of lipid rearrangements in the lipid bilayer are not fully understood yet, although the current model has been constantly revisited and refined [ ] [ ] [ ] [ ] [ ] [ ] . crystal structures of pre-and post-fusion states of class i viral fusion proteins along with nmr structures of fusion peptides in membrane mimetics have provided a mechanistic view of the membrane fusion process. in this process, different protein segments act in an orchestrated way to achieve the complex and kinetically unfavorable task of bringing together and fusing two lipid bilayers , . however, there are still major gaps in the molecular details involved in the fusion process. they include: the sequential order and kinetics of the events, changes in the structure, dynamics, and physicochemical properties of different protein domains and lipid bilayers during the whole membrane fusion mechanism, free energies of the corresponding pre-, post-(fusion pore) and intermediate (hemifusion) fusion states as well as the modulation of all those parameters by lipid composition. sars-cov s subunit possesses various membranotropic segments, i.e., relatively short hydrophobic domains that are able to bind to and perturb membranes. these segments can act independently from each other and may help to stabilize the contact between viral and cell membranes , [ ] [ ] [ ] [ ] . however, monitoring the structural rearrangements of different segments of the intact s subunit as well as the dynamics of their interaction with viral and cell membranes on a molecular level constitute a challenging task. the use of synthetic peptides and phospholipids has provided important thermodynamic, structural, biochemical, and functional details of peptide-membrane interactions from both peptide and lipid perspectives, which makes them a good platform to study membrane fusion , , . our results indicate that both sars fp and sars ifp significantly perturb the thermotropic phase behavior as well as the molecular ordering and phospholipid rotational mobility of model membranes containing negatively charged lipids, but only cause moderate effects on zwitterionic membranes. these findings are in agreement with previously reported studies that indicated greater disturbance and higher affinity of both peptides for model membranes containing anionic rather than zwitterionic phospholipids , . even though the peptides do not partition well into zwitterionic membranes, they are able to perturb the thermodynamic parameters of the zwitterionic dppc and pope phase transitions as well as to change the membrane curvature properties of dipope. on the other hand, our cw esr experiments showed that the peptides do not affect the structural dynamics of zwitterionic lipid bilayers, but they do promote an ordering effect on the lipid head group and on the acyl chains of negatively charged membranes. this latter effect seems to be correlated to membrane dehydration, as shown by our eseem experiments. ordering and dehydration effects have also been observed for other class i viral fusion peptides such as those from hiv-i gp and influenza ha glycoproteins , . the relevance of membrane-curvature induction and lipid dehydration for the viral membrane fusion of the sars-cov fusion peptides will be discussed below. peptides induce bending moment and membrane dehydration of anionic membranes. lipid molecules in bilayers experience a restoring torque from neighboring lipids that tends to reorient the lipid chain and/or the head group along the normal to the bilayer. the order parameter s , which is associated with the restoring torque through the orienting potential u(Ω), is a measure of the extent of that molecular alignment. generally, the higher the s the lower is the angular amplitude of the wobbling motion of the spin probe, i.e. the more aligned along the local director is the lipid segment to which the probe is attached. a better alignment can enhance the molecular interactions between lipid molecules in the bilayer. therefore, s directly reports on the lipid packing density. in particular, slight changes in s of the lipid head group would have a greater impact on the structural organization of the membrane than changes in the ordering of the acyl chain . this is primarily due to the strong hydrogen-bonding network in the head group region (~ . to . kcal/mol) . thus, although the molecular structure of dpptc head group is different from those of the pg, ps, and pa head groups, reorientation of dpptc due to conformational changes in the polar region might be associated with changes in the hydrogen-bonding network . that is, the more ordered the dpptc, the more condensed is the bilayer, which leads to changes in the ionic interactions between the head groups and between the head groups and surface-bound water molecules. by contrast, the van der waals interactions between the lipid acyl chains in the hydrophobic core of the membrane are much weaker, i.e. the strength of the interaction is about . kcal/mol, which is even smaller than the thermal energy at k (k b t ~ . kcal/mol) . nevertheless, enhancement of lipid-lipid interactions due to condensation of the hydrophobic core leads to an increased chain-packing energy. in our studies, the peptides were added into preformed vesicle solutions. therefore, the changes observed here are primarily due to the interaction of the peptides with the outer leaflet of the bilayers. sars fp and sars ifp increase the s of -pcsl and -pcsl in dppg and popa membranes, making the nonpolar core of the outer leaflet more solid-like. furthermore, both peptides increase the ordering of head group spin label dpptc in pure dppg, dpps, and popa as well as in dppg-and popa-containing membranes, but not in the zwitterionic dppc and dppc/chol. therefore, an increase of both the head group and acyl chain packing densities of the outer leaflet is the major effect of the peptides on the negatively charged phospholipid membranes investigated in this work, which is in agreement with previous studies , . interestingly, sars fp shows higher membrane fusion activity in pure pg or in ps-or phosphatidylinositol (pi)-containing membranes, but not in zwitterionic ones . thus, ordering of the lipid head group seems to be an important structural change in the bilayer necessary to induce membrane fusion , . indeed, we found the same ordering effect on the head group of membranes containing negatively charged lipids for the membrane fusion promoter la and an opposite effect for lpc, a known fusion inhibitor. this ordering or condensation effect on the outer monolayer leads to a shrinkage of its surface area that compresses the inner leaflet. because of the mechanical coupling between the two leaflets in a lipid bilayer vesicle, the inner layer counteracts the compressive force exerted by the outer monolayer and therefore creates a nonuniform tension in the two leaflets, which redistributes the stress profile across the bilayer. as a result, a uniform membrane bending moment toward the condensed leaflet is induced, which ultimately generates negative (positive) membrane curvature if the outer (inner) monolayer is condensed . due to the properties of the stress profile across the bilayer and to the nature of the molecular interactions in the head group and acyl chain regions, the largest contribution to the bending moment arises from the ordering of the head group region . thus, the increase of s of dpptc in the outer leaflet of negatively charged lipid membranes promoted by the sars-cov fusion peptides could potentially induce negative membrane curvature (please see section si of supplementary information for further details). this negative curvature effect induced by bending moment has also been proposed as the putative membrane fusion mechanism of other class i fusion peptides such as those from influenza ha or hiv- gp , . another important aspect to consider is the inverse correlation between head group ordering and hydration of lipid bilayers . if an increased head group packing density leads to membrane dehydration, this effect may help to overcome the high hydration repulsive energy that arises from membrane surface-bound water molecules . prior to fusion, two apposed lipid bilayers must approach. when the distance between the approaching bilayers is within to nm, a high hydration repulsion arises and dominates the interactions between them , thus preventing the membranes to make contact and to proceed to fusion. therefore, molecules that have the ability to overcome this hydration barrier could succeed in promoting membrane fusion. bilayer dehydration can be accomplished by different mechanisms depending on the molecular nature of the fusogenic molecule [ ] [ ] [ ] . particularly, hiv- gp and influenza ha fusion peptides promote membrane dehydration by increasing the ordering of the lipid bilayers , . that conclusion was inferred, though, from the aforementioned s /dehydration correlation and not by actually measuring the water content inside the bilayer. our eseem experiments, on the other hand, have undoubtedly shown that partial membrane dehydration actually takes place upon binding of the sars-cov fusion peptides to popc/popg membranes. this finding also provided a direct evidence of the correlation between head group ordering and membrane dehydration. however, in contrast to our eseem data, guillén et al have suggested that sars fp increases the water penetration depth into zwitterionic and anionic large unilamellar vesicles . this conclusion was based on the analysis of the fluorescence decay of diphenylhexatriene (dph) embedded in membranes, whose multi-component lifetimes were shortened in the presence of the peptide. although the increase of water penetration depth is a possible interpretation for their results , since the quantum yield of dph decreases in water, probe location and orientation might also influence the fluorescence lifetime of dph in lipid bilayers. conflicting results in the literature have indicated there is no consensus yet regarding the exact location of dph in pure model membranes [ ] [ ] [ ] [ ] . particularly, konopásek and coworkers , have shown that the short-lived component of dph embedded in lipid bilayers originates from a probe population located at the membrane-water interface. therefore, the subnanosecond short-lived component of dph in the very ordered gel phase of dimyristoyl phosphatidylglycerol (dmpg) found by guillen et al. in the absence of sars fp could thus potentially be due to a shallower interfacial location of dph in the dmpg bilayer. thus, the decrease of the dph lifetime in the presence of sars fp found by guillen and coworkers could potentially stem from the contact of the fluorophore with water molecules at the membrane-water interface due to a reorientational distribution of dph in the bilayer upon the condensing effect induced by the peptide. lastly, it is important to emphasize that the alterations of the bilayer structure observed for sars fp and sars ifp take place at high peptide concentrations and thus are most likely due to a cooperative behavior of membrane-bound self-associated β -sheet peptides. in fact, both peptides present membrane fusion activity only at high peptide-to-lipid molar ratio and regular extended β -sheet aggregates are the most populated membrane-bound peptide conformation , , . peptide oligomerization in membranes as extended β -like aggregates seems to play an important role in peptide-induced membrane fusion . peptide self-association in small areas of the membrane surface might actually be important in the very first stages of the membrane fusion process . indeed, since a high amount of work is required to merge large surface areas of two lipid bilayers, lipid merging most likely proceeds through local points of contact , , . thus, bending moments promoting negative curvature and membrane dehydration may not only help to decrease the hydration barrier between the proximal leaflets of the approaching bilayers but also minimize the work necessary for merging the monolayers. the latter is a consequence of the possible formation of point-like membrane protrusions, also referred to as local 'nipples' , a critical step that theoretically precedes the formation of the intermediate hemifusion stalk , , . peptides change membrane curvature of non-bilayer lipids in opposing ways. accumulating evidence suggests that membrane fusion involves the formation of strongly curved lipid bilayers in the pre-fusion, intermediate, and post-fusion states , . it is therefore tempting to investigate the potential ability of fusion peptides to generate membrane curvature, since that information can help to elucidate the role played by those molecules in the viral membrane fusion process. dsc experiments with non-bilayer-forming lipids such as phosphatidylethanolamines (pe) have been successfully used to indirectly probe changes in the intrinsic spontaneous curvature properties of pe vesicles by a wide variety of peptides , , . shift of the l α -to-h ii transition temperature is an excellent indicator of lipid phase changes and curvature alterations . we found that both sars fp and sars ifp peptides have the ability to bend dipope lipid bilayers. while sars fp induces positive curvature, sars ifp causes opposing stresses on the membrane depending on the ionic strength: it promotes positive (negative) curvature strain at low (high) ionic strength. the capacity of the sars fusion peptides to generate different curvature stresses on pe vesicles might actually be important in the context of the whole sars-cov s -mediated membrane fusion. the small stressed protrusions formed in the pre-fusion state are characterized by lipid domains possessing positive curvature flanked by negatively-curved lipid patches that stabilize the local 'nipple' and allow for the establishment of close intermembrane contact , , . therefore, both peptides can hypothetically act in this early stage by stabilizing the two curved domains depending on the ionic strength of the environment and on the exposure to pe or to negatively charged lipids . membrane fusion proceeds with the formation of the so-called hemifusion intermediate or lipidic stalk, which is characterized by lipid mixing between the outer leaflets of the two apposed bilayers with the distal monolayers remaining unfused. the resultant mixed outer leaflet presents a high degree of negative curvature , . stabilization of this intrinsic negative curvature has been traditionally interpreted as the common role played by various fusion peptides in the viral membrane fusion process , . the hemifusion intermediate would then subsequently progress to the formation of the fusion pore state , , which is characterized by lipid mixing of both the outer and inner leaflets of the merged bilayers, thus establishing the opening of a pore that allows content mixing between the two apposed membranes. in this pore state, both monolayers present strong and opposite curvature strains , . based on our findings, the outer, negatively-curved monolayer of both the lipidic stalk and the fusion pore states may be stabilized by the sars fusion peptides depending on the lipid composition: sars fp and sars ifp could act in membranes containing anionic lipids, and sars ifp could play a major role in pe-rich membranes at high ionic strength. on the other hand, the intrinsic positive curvature of the inner leaflet of the pore state can be stabilized by sars fp in the presence of pe lipids. that is, the more fusogenic sars fp peptide can also support membrane fusion by stabilization of porous structures. experimental and computational studies have also indicated stabilization of the pore state by fusion peptides from influenza ha , and parainfluenza virus (piv ) f protein . thus, our findings imply that both sars fusion peptides can act at different stages of the fusion process to facilitate membrane fusion. putative sars-cov membrane fusion model. taken together, our results reveal a functional plasticity of sars fp and sars ifp in helping to promote membrane fusion. both peptides can act in the early and late stages of the membrane fusion reaction by changing three properties of lipid bilayers, namely spontaneous curvature, hydration, and lipid packing density. incorporation of our findings into the currently proposed membrane fusion model induced by class i viral fusion proteins yields the following. upon receptor binding, s domain of sars-cov spike glycoprotein undergoes a large conformational change that releases and exposes sars fp and sars ifp to interact with target membranes . both peptides insert into but not significantly disturb (our data and data in references , and ) the highly-ordered, zwitterionic outer leaflet of the plasma membrane bilayer . this binding process bridges viral and cell membranes and thus facilitates trimerization of other s subunits. trimers are, in general, the fusion-active oligomeric state of class i fusion proteins . as a result, a trimeric extended prehairpin conformation is formed. at this point, it is important to emphasize that the actual conformational state of the sars-cov fusion peptides remains elusive. sars fp and sars ifp adopt, respectively, a v-shaped and a linear helical conformation in dodecylphosphatidylcholine micelles , but have a high tendency to aggregate and to form intramolecular β -sheets and extended β -strands stabilized by intermolecular interactions in models of lipid bilayers as well as to adopt, in small fractions, α -helical and unordered structures , . in the context of the intact protein, however, the structure and oligomerization state of those peptide segments still need to be addressed, although it has been proposed that membrane-bound self-associated peptides may provide the major driving force for trimerization of the whole protein , . peptide binding, conformational change and possibly aggregation into the membrane may trigger s refolding into a trimeric hairpin conformation. as a result, a six-helix bundle would form, bringing not only viral and target membranes into close proximity, but also the internal fusion peptide (sars ifp ) and the pretransmembrane (sars ptm ) domain of the s subunit . due to the high hydration repulsion of the closely apposed lipid bilayers and the requirement for bending membranes to minimize areas of strong interbilayer repulsion , , displacement of water molecules from the membrane surface and changes in membrane curvature seem to be the prerequisites for allowing close intermembrane contact and subsequent formation of the high-energy hemifusion intermediate state. interaction of sars fp and sars ifp with pe or with negatively charged lipids contained either in the plasma (via nonendocytic pathway) or in the endosome (via endocytic pathway) membranes may be important for the formation of point-like protrusions or for stabilization of the hemifusion stalk . since anionic phospholipids are mostly located in the inner leaflet of the membrane, it would be possible that the action of lipid flippases and scramblases could be endorsed by the peptide perturbation on the outer leaflet of the plasma membrane . the major effects of the peptides at the pre-fusion state could be the following: induction of positive curvature on pe-rich membranes, as indicated by our dsc data; and membrane dehydration and induction of bending moment on the outer leaflet of bilayers comprised of anionic lipids, as suggested by our esr data. the latter effects may also be responsible for triggering stalk formation, which is further stabilized by exposure of pe on the outer leaflet to sar ifp . hemifusion could be further facilitated by membrane interaction of a loop peptide segment located in between hr and hr domains and by a possible heteroligomerization of sars ifp with sars ptm . juxtaposition of sars ifp and sars ptm leads to a synergistic and cooperative action of both peptides that causes membrane destabilization and further peptide insertion . exposure of sars fp to the inner leaflet of the merged viral and cell membranes could have a great impact in the post-fusion state. indeed, sars fp could act by promoting positive curvature and stabilizing the high positively-curved inner leaflet that characterizes the porous state, thus facilitating pore formation (our dsc data). however, the molecular details of the above processes still need to be investigated. overall, the two putative fusion peptides from sars-cov s protein may help to regulate membrane fusion by acting in the early and late stages of the membrane fusion process. our main findings were: ( ) sars fusion peptides increase the ordering of the headgroup and acyl chain regions of mlvs containing negatively-charged, but not zwitterionic phospholipids; ( ) membrane fusion promoters induce similar effects on the head group ordering than do the fusion peptides, whereas membrane fusion inhibitors cause opposing effects; ( ) changes in the order parameters of the lipids are generally greater for the more fusogenic sars fp peptide than for sars ifp ; ( ) both peptides promote dehydration of pg-containing membranes and this effect is well correlated with the increased head group ordering; and ( ) dsc data support a hypothesis that sars fp induces positive curvature on dipope vesicles, whereas sars ifp promotes opposing stresses on the intrinsic negative curvature of dipope depending on the ionic strength. peptide-induced chain-packing energy and membrane surface ordering of the outer leaflet of negatively charged lipid bilayers promote partial membrane dehydration and could generate bending moment, as suggested by our esr studies. both effects may induce negative curvature and decrease the hydration repulsion of apposed bilayers. possible peptide involvement on the formation of the pre-fusion point-like protrusions and intermediate hemifusion stalk as well as on the stabilization of the fusion pore state suggest that the sars fusion peptides might play important roles in the whole membrane fusion process. taken together, our findings suggest that the sars fusion peptides have the ability to change the physicochemical properties of model membranes depending on the lipid composition and on the ionic strength. therefore, they can act in the early and late stages of the membrane fusion process, conferring them a functional plasticity that might be important to help overcome the high kinetic barrier involved in the sars-cov-induced membrane fusion. materials. n-terminally acetylated and c-terminally amidated sars fp ( mwktptlkyfggfnfsqil ) and sars ifp ( gaalqipfamqmayrf ) peptides were either purchased from genscript (piscataway township, nj) or manually synthesized according to the standard fmoc solid-phase peptide synthesis method on a rink-amide resin . the details of peptide synthesis are described in vicente et al. . purification was performed as described in supplementary section si . the phospholipids -palmitoyl- -hydroxy-sn-glycero- -phosphocholine (lpc), , -dipalmitoyl-snglycero-phosphatidylcholine (dppc), , -dipalmitoyl-sn-glycero- -phospho-( '-rac-glycerol) (dppg), , -dipalmitoyl-sn-glycero- -phospho-l-serine (dpps), , -dipalmitoleoyl-sn-glycero- -phosphoethanolamine (dipope), -palmitoyl- -oleoyl-sn-glycero- -phosphocholine (popc), -palmitoyl- -oleoyl-sn-glycero- -phospho-( '-rac-glycerol) (popg), -palmitoyl- -oleoyl-sn-glycero- -phospho-l-serine (pops), -palmitoyl- -oleoyl-sn-glycero- -phosphate (popa), and the spin labels -palmitoyl- -stearoyl(n-doxyl)-snglycero- -phosphocholine (n-pcsl, where n = and ), , -dioleoyl-sn-glycero- -phospho(tempo)choline (doptc), and , -dipalmitoyl-sn-glycero- -phospho(tempo)choline (dpptc) were purchased from avanti polar lipids, inc. (alabaster, al). cholesterol (chol) and linoleic acid (la) were obtained from sigma-aldrich (st. louis, mo). all reagents were used without further purification. sample preparation. phospholipids ( . mg for dsc and . mg for esr) and spin labels ( . mol% for cw esr and mol% for pulsed esr) either in chloroform or chloroform/methanol : (v/v) stock solutions were mixed in a glass tube. after dried under a n flow, the lipid film was ultracentrifuged under vacuum overnight to remove traces of solvent. for cw esr experiments, the sample was hydrated in mm potassium phosphate buffer, ph . , sonicated in a bath type sonicator for a few seconds and maintained at a temperature above the main phase transition of the lipid for at least two hours for complete hydration. samples were then subjected to at least six freeze-thaw cycles. a measured volume of sars fp or sars ifp stock solutions in dimethyl sulfoxide (dmso) was added to the preformed multilamellar lipid dispersions. for dsc experiments, peptides dissolved in either acetonitrile/water : (v/v) or in dmso solutions were diluted into buffer and added to the lipid film for hydration. samples were vortexed for few seconds, maintained at a temperature above the phase transition for each lipid during at least min, and subjected to six freeze-thaw cycles. the amount of phospholipid (final lipid concentration of mg/ml for esr and mg/ml for dsc) and peptides used provided a : lipid/peptide molar ratio for most of the experiments. it is worth mentioning that the same amount of dmso or acetonitrile/water : added in the peptide-containing samples was also used in the peptide-free samples as controls for the esr and dsc experiments. the control samples were prepared using the same protocol as those of the peptide-containing samples. for dipope/peptide samples, peptides and lipids dissolved in chloroform/methanol : (v/v) stock solutions were mixed in a glass tube, dried to a lipid film under n gas and lyophilized overnight. samples were hydrated in mm sodium phosphate buffer, ph . , with or without mm sodium chloride, and freeze-thaw cycled six times below the liquid crystalline-to-inverted hexagonal (lα -h ii ) phase transition temperature (t h ) of the lipid. dipope concentration was mg/ml and peptide concentration varied from . to . mol% ( : and : lipid/peptide molar ratio, respectively). for eseem experiments, popc/popg : mol/mol and peptides ( : lipid/peptide molar ratio) were prepared as above, but hydrated in mm sodium phosphate, mm nacl d o buffer, pd = . (actual ph measurement). peptide concentration was confirmed spectrophotometrically by using the theoretical molar extinction coefficients of , m − cm − for sars fp esr experiments. cw-esr experiments were carried out on a varian e- spectrometer operating at . ghz. temperature was controlled by a homemade temperature control unit coupled to the spectrometer, whose accuracy is about . °c. samples were transferred to glass capillaries ( . mm i.d.), which were set into a quartz tube containing a mineral oil bath to help stabilize the sample temperature. the following acquisition parameters were used: center field, , g; scan width, to g; modulation amplitude, . or . g; modulation frequency, khz; microwave power, or mw; time constant, ms, and acquisition time, s. nonlinear least-squares simulations (nlls) of the cw-esr spectra were performed using the multicomponent labview (national instruments) software developed by dr. christian altenbach (university of california, los angeles, california) , . the rotational diffusion rates (r ⊥ , r ∥ ) and order parameters (s , s ) were obtained as described in ref. with further details in the section si of supplementary information. seed values for the magnetic parameters of both -pcsl and -pcsl were obtained from earle et al. and those of dpptc were taken from ge and freed . the strategy of the nlls simulation was performed as described elsewhere . pulsed esr experiments were performed on a bruker elexsys x-band pulsed esr spectrometer equipped with the bruker flexline er x-ms split-ring resonator and the itc oxford cryogenic system for temperature control. samples were immersed into liquid nitrogen prior to the measurements at k. three pulse electron spin echo envelope modulation (eseem) experiments were carried out with the π / -τπ / -t -π / -τecho pulse sequence and using a four-step phase cycling to suppress unwanted echoes . the microwave power was adjusted to give ns π / pulses and an interpulse delay τ of ns, kept constant in all experiments, was chosen to maximize deuterium modulations at the magnetic field where the echo intensity is maximum. starting at time delay t = ns, points were recorded with Δ t = ns steps to obtain the three-pulse stimulated echo decays. the integration gate length was ns and the shot repetition time was , μ s. the number of accumulations varied from to depending on the signal-to-noise ratio and on the modulation depth. data analysis was performed as described in bartucci et al. . briefly, the contribution of the spin relaxation to the eseem signal was eliminated by dividing the time-dependent echo amplitudes, v(τ , t), by a bi-exponential decay, 〈 v(τ , t)〉 , followed by subtraction of unity, as v norm (τ , t) = v(τ , t)/〈 v(τ , t)〉 − . the remained oscillations about zero were apodized with a hamming window and zero-filled to increase the total number of points to about k. numerical fourier transformation was performed and the resultant magnitude spectrum was 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cecropin-a( - ) n-terminal microdomain peptide from human dihydroorotate dehydrogenase: structure and model membrane interactions labview programs for the analysis of epr data polarity profiles in oriented and dispersed phosphatidylcholine bilayers are different -an electron spin resonance study elimination of unwanted echoes and reduction of dead time in three-pulse electron spin-echo spectroscopy key: cord- -jmpterrj authors: eilersen, andreas; sneppen, kim title: cost–benefit of limited isolation and testing in covid- mitigation date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: jmpterrj the international community has been put in an unprecedented situation by the covid- pandemic. creating models to describe and quantify alternative mitigation strategies becomes increasingly urgent. in this study, we propose an agent-based model of disease transmission in a society divided into closely connected families, workplaces, and social groups. this allows us to discuss mitigation strategies, including targeted quarantine measures. we find that workplace and more diffuse social contacts are roughly equally important to disease spread, and that an effective lockdown must target both. we examine the cost–benefit of replacing a lockdown with tracing and quarantining contacts of the infected. quarantine can contribute substantially to mitigation, even if it has short duration and is done within households. when reopening society, testing and quarantining is a strategy that is much cheaper in terms of lost workdays than a long lockdown. a targeted quarantine strategy is quite efficient with only days of quarantine, and its effect increases when testing is more widespread. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ within families, workplaces, and friend groups, everyone is assumed to know everyone. each agent is assigned one family and workplace, as well as two groups of friends. workplaces on average contain ten people, whereas each friend group on average contains five. in the simulation runs presented here, we use a population of n = agents. increasing the number of agents changes the outcome very little, except for minimising stochastic noise. we also do not allow migration in or out of the system. we use a discrete-time stochastic algorithm. at each time-step ( . days), each person has one interaction with some other person. a "die roll" decides whether the person will interact with family, friends, work, or the public. the respective odds are the above-mentioned percentages : : : . if the public is chosen, an entirely random person is selected, otherwise a person is drawn from a predefined group (family etc.). for each interaction, an infectious person has a fixed probability of passing on the disease to the person they interact with. the family size distribution of is based on the distribution of danish households . the average number of people per household is approximately , and large households of more than people have been ignored, as they account for less than % of the population. we believe that in a country where family sizes are larger and there are fewer singles, the family would be more important to the spread of disease. we test the effect of larger families in the supplement (figs. s -s ) and find that it does not change our overall conclusions. we simulate the progression of disease using an seir model with four exposed states, e = e + e + e + e , each lasting on average . days, corresponding to a mean incubation period of days. the exposed states are presymptomatic, meaning that people will not get tested in the incubation period. we let stages e ; be as infectious as the i-stage, as data suggest that a substantial fraction of covid- transmission happens before the onset of symptoms . multiple exposed states are included in order to get a naturalistic distribution of incubation periods. li et al. report that the mean incubation period is approximately five days and the reported distribution is fitted well by the gamma distribution we obtain from our four e-stages. a further problem is the duration of the infectious period (i). viral shedding has been observed to last up to eight days in moderate illness . on the other hand, according to linton et al. , the median time from onset to hospitalisation is three days. a bedridden patient (even if not hospitalised) is likely to transmit the disease less. to fit the observed mean serial intervals of . days of nishiura et al. we model the infectious period as a single state with an average duration of three days. in addition, the infectious presymptomatic period lasts on average . days. in comparison ref. uses a serial interval distribution with mean of . days. other authors have suggested a longer serial interval with presymptomatic infections. finally, the transmission rate of the disease is estimated from an observed rate of increase of % per day in fatalities in the usa. this also fits the observation of a growth rate of icu admissions of about . % per day in italy . with our parameters this is reproduced by a basic reproduction number r ~ (as we allow transmission figure . a diagram of the model structure. each agent has a network consisting of a family, a workplace and two groups of friends. the family accounts for % of interactions. work accounts for % and socialisation with friends accounts for a further %. the members of each of these groups are fixed throughout the simulation. finally, % of interactions happen "in public", which we implement as an interaction with a randomly chosen other agent. everyone in the work and friend sub-graphs are assumed to be connected to each other. below the graph, the underlying mechanisms of the disease are shown. we divide the exposed state into four in order to get a more naturalistic gamma distribution of incubation periods. the two last exposed states are infectious, but asymptomatic, meaning that individuals will not get tested. this is to include presymptomatic infection. in our simulation we set the family groups to an average of people, and the work network to completely interconnected people. the friend network consists of two groups with five in each. having calibrated the model in this way, we want to explore mitigation strategies for the corona epidemic. specifically, we will investigate the relative importance of the areas of social life, and the extent that reducing workplace size reduces disease spread. moreover, we will examine the possible gain and cost by simple contact tracing and light quarantine practices. to illustrate the relative importance of the workplace and public life, we consider the scenarios in fig. a . in the first scenario, nothing is done. in the second, contacts within the workplace are reduced by %, while in the third, contacts with friends and the public are reduced. finally, we compare these with similar scenarios, but where good hygiene or keeping a distance reduces the probability of infection from all types of encounters by half. in the figure, we see that the effects of reducing workplace and social contacts are roughly of the same magnitude. this reflects the assignment of % weight to each of these contact types. the slightly larger effect of social contacts reflects our assumption that these connections are less clustered than the workplace network. the two latter graphs show the scenarios where we both reduce infection probability within one group by % and overall infection probability by %. they show that an effective lockdown requires both restrictions of the time spent in the workplace and in the public sphere, and measures that reduce infection probability by increased hygiene and physical distancing. the above results provide one useful piece of information. if the effect of workplace and social contacts are of the same order, it is of little importance which one is restricted. ideally, both will be restricted for a period. however, when restrictions need to be lifted, authorities will primarily be able to control the workplace, whereas the social sphere relies on local social behavior. obviously, it is economically more sustainable to lift the one with the largest social consequences first, by allowing people to return to work while encouraging keeping social gatherings at a minimum. if restrictions are lifted before a substantial level of immunity is achieved, the epidemic will re-ignite. therefore, we now examine what can be done to minimise spread in the reopened workplaces. one possible strategy is to reduce the number of people allowed at any one time in each workplace. in fig. b , we compare an epidemic scenario where the average number of employees per workplace is with an epidemic where this number is reduced to . we further assume that the number of contacts per coworker remains the same, meaning that the number of contacts per person drops when workplace size is reduced. it can be seen that fragmentation of physical spaces at workplaces could have a significant effect on the peak number of infected. in a situation with a risk of straining the healthcare system, this could be part of a mitigation strategy. once again, the strategy becomes relatively more effective if the infection probability per encounter is also reduced. compared to the cases with no workplace size reduction, making workplaces smaller leads to a greater relative reduction in peak size if infection probability is lower, completely eliminating the epidemic at an infection probability reduction of %. a more local strategy that can be employed when reopening society is widespread testing and contact tracing. as mentioned above, hellewell et al. have suggested that this can be effective in containing covid- www.nature.com/scientificreports/ outbreaks provided high efficiency in detecting infected individuals. contact tracing has previously been modeled in relation to other epidemics , and used successfully against smallpox and sars . one obstacle to the widespread implementation of this strategy is the difficulty of tracing contacts. therefore, we will here implement a crude form of contact tracing where we ( ) close the workplaces of people who are tested positive for the disease, ( ) isolate their regular social contacts for a limited period, and ( ) keep symptomatic individuals in quarantine until they recover. we will see that such a step tracing and quarantine strategy ( stq) can give a sizeable reduction in disease spread while costing fewer lost workdays than overall lockdown. our simulations include the limitations imposed by not being able to trace the estimated % of infections from random public transmissions. thus, the strategy does not require sophisticated contact tracing but could be implemented based on infected people being able to recollect their recent face-to-face encounters with friends. it should be noted that we here quarantine persons in their own households, thereby making our contact tracing strategy easier to implement in practice. in particular, family members of a quarantined person are still free to interact outside their home if they are not themselves tested positive. the drawback of such light quarantine practices is that infected persons in quarantine may still transmit the infection to their families. figure examines how increased testing efficiency systematically improves our ability to reduce the peak disease burden. this would then be a more cost efficient way to mitigate the pandemic than a complete lockdown where each person would lose several man-months. even detecting as little as % of covid- infected per day (which with an average symptomatic disease duration of days corresponds to finding approximately % of the infected) can potentially reduce the peak number of cases by %. if % efficiency is possible, corresponding to detecting about a third of infectious cases, then peak height could be reduced by a factor of almost three with to a % drop, if the probability of infected people being tested is only % per day of illness. however, the price of this is that each person is on average quarantined once during the epidemic. if testing is more widespread, the epidemic peak can be further reduced, until it finally becomes unstable at a testing probability of around % per day. (d) epidemic peak and time spent in quarantine as a function of quarantine length for a testing probability of % per day. the average time spent in quarantine increases linearly with the length of quarantine. on the contrary, the effect of quarantine on the peak height appears to stagnate at approximately days. www.nature.com/scientificreports/ less than two weeks in quarantine per person during the entire epidemic. this is illustrated in fig. a where peak height is reduced from . to . at % testing efficiency. the main cost of the quarantine option is the quarantine time. figure d examines the efficiency versus cost of as a function of quarantine length. it can be seen that there is little gain in extending the quarantine period beyond the -day duration of the incubation period. for this reason we opted for days in quarantine in panel (a, b). as a consequence, an average person will stay around days in quarantine during the course of the epidemic with a testing probability of % per day. this time can be reduced if people can be convinced of smaller work environments and fewer face-to-face contacts per week. fragmentation of our networks into smaller groups will reduce both quarantine overhead and the direct transmission of the disease (fig. b, orange curve) . a prolonged lockdown will hugely disrupt society, and it is questionable whether a complete eradication of the virus is possible anyway. therefore, most governments have aimed at softening the epidemic curve, with varying degrees of success. the one step contact tracing with testing and quarantine is a means to this end and would work most effectively in combination with other efforts to reduce r . finally, we investigate whether an aggressive testing and contact tracing strategy could work if implemented at a late stage in an epidemic. this could be relevant if for example the strategy is part of an effort to reopen society after a period of lockdown. in fig. , we show two possible scenarios where testing and contact tracing is implemented after a -day lockdown with a % reduction of the work and social spheres. the lockdown is initiated when % of the population is infected. in (a) we subsequently test and quarantine the infected and their contacts for days, while in (b) the required quarantine is set to days. we assume a testing efficiency of % chance of detection for each day a person is symptomatic. the progression of the epidemic without testing is marked by a black graph for comparison. from the figure one sees that the strategy of even relatively short quarantines also works with a late onset. at a realistic detection probability, it prevents a resurgence of the epidemic. nonetheless, it is quite costly initially, with a very high peak in number of quarantined people. importantly, the effect does not increase with a longer quarantine period, but the cost is substantially larger. pandemics such as the one caused by covid- can pose an existential threat to our social and economic life. the disease itself is serious and leaves specific epidemic signatures and characteristics that make traditional contact tracing difficult. in particular it is highly infectious, can sometimes be transmitted already two days after exposure, and a large fraction of transmission happens before the onset of symptoms. as such it is difficult to contain without a system-wide lockdown of society. nonetheless, a successful containment in south korea used contact tracing. this motivated us to explore a one-step contact tracing/quarantine strategy ( stq). using reasonable covid- infection parameters we find that the stq strategy can contribute to epidemic mitigation, in the sense that it can reduce the peak number of infected individuals by about a factor of two even with a realistic testing rate of % per day of illness. this was illustrated systematically in fig. . the main cost was people in self-quarantine and not contributing to the workforce. in comparison one has to consider that a society-wide lockdown with similar reduction in peak height would have to last for about days (see fig. ). thus, the lockdown would require of order days of quarantine (or at least extensive social distancing) per person, whereas testing and isolation only requires on average around days per person with a -day quarantine www.nature.com/scientificreports/ even at high testing probabilities. importantly these numbers can be reduced if people are able to lower their number of contacts. a noticeable objection to the stq strategy is the fraction of cases with so weak symptoms that people do not contact health authorities. the effect of such limitations is in our model parameterized through the detection probability. from fig. c one sees that when the detection probability goes below % (a rate of % per day) the peak reduction of the stq strategy becomes only of the order percentage point. it should also be noted that, since we rely on symptoms to determine who stays in quarantine, and people in the infectious/symptomatic stage are assumed to always stay in quarantine, we implicitly assume that all infected persons develop at least some symptoms at some point. this may be a break from reality. the increasing availability of tests may also change the perspectives of the stq strategy. with widely available rapid tests, it will be possible to test everyone regularly, and to test all quarantined persons before they leave quarantine. supplementary figure s deals with the results of such a testing strategy and finds that it makes it possible to totally control the epidemic, or to mitigate it without quarantining any healthy individuals. to put this into perspective, the drawbacks of widespread, but slow testing is examined in the supplementary fig. s . here, we find that the stq strategy is most efficient with no test delay, and that delayed contact tracing is comparable to a primitive lockdown. one interesting point which we have not examined here, is that real-world social networks are heterogeneous, with a large variance in number of contacts. it may be expected, for example, that workers in customer-facing positions in shops will have a high risk of catching the disease and passing it on. the effects of this heterogeneity is examined more closely in ref. here, it is concluded that heterogeneity in the number of contacts enhances the effect of contact tracing, since persons with many contacts are both more likely to pass on the disease and more likely to be quarantined. in ref. , the authors suggest a stq strategy similar to the one we here model. the main points of the present analysis is the focus on mitigating instead of eradicating the epidemic, our suggestion of a shorter quarantine length, and the implementation of quarantine together with other members of the household instead of total isolation. our stochastic, agent-based approach also allows for local failures due to the limited duration of quarantine (people may not yet be symptomatic when exiting quarantine) and the non-traceable public contacts (set to %). finally, one noticeable finding is that contact tracing and reduction of contacts per person is still feasible even at a later stage of the epidemic. as can be seen in fig. , a lockdown and subsequent reopening with testing and contact tracing is highly effective in controlling the epidemic. our study that lockdowns have an important role to play in epidemic mitigation, but that they can be replaced by a stq strategy once the epidemic is under control. the covid- pandemic has set both governments, health professionals, and epidemiologists in a situation that is more stressful and more rapidly evolving than anything in recent years. due to the uncertainties caused by a situation in flux, it is difficult to predict anything definite about what works and what does not. the empirical observation that lockdowns worked in both china, and in a milder form in denmark shows that our assumption of a % reduction in specific infection rates under lockdown is realistic. our main result is that some of these restrictions can be replaced by testing, one-step contact tracing and short periods of quarantine. this is far cheaper than total lockdowns. perhaps most importantly, these measures work best in combination. as is highly relevant to the current epidemic stage of covid- , we pinpoint that stq can be successfully implemented also at a late stage of the epidemic where testing may become massively available. plots of alternative variants of our model (including alternative testing strategies and larger family sizes) can be found in the supplementary material. the code used to produce the plots shown in this article is available on figshare under the url https ://doi.org/ . /m .figsh are. .v . how will country-based mitigation measures influence the course of the covid- epidemic report : impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand social contacts and mixing patterns relevant to the spread of infectious diseases epidemic analysis of covid- in china by dynamical modeling the effect of control strategies to reduce social mixing on outcomes of the covid- epidemic in wuhan, china: a modelling study modelling transmission and control of the covid- pandemic in australia substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) contagion! the bbc four pandemic: the model behind the documentary contacts in context: large-scale setting-specific social mixing matrices from the bbc pandemic project early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia feasibility of controlling covid- outbreaks by isolation of cases and contacts fam n: families . january by municipality, type of family, size of family and number of children serial interval of novel coronavirus (covid- ) infections ) pandemic: increased transmission in the eu/eea and the uk-seventh update epidemiological characteristics of novel coronavirus infection: a statistical analysis of publicly available case data report : estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european countries covid- and italy: what next? the effectiveness of contact tracing in emerging epidemics smallpox and its eradication epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong heterogeneity is essential for contact tracing we thank gorm gruner, bjarke frost nielsen, andreas roepstorff, and lone simonsen for enlightening discussions. this project has received funding from the european research council (erc) under the european union's horizon research and innovation program under grant agreement no. . a.e. and k.s. both participated in devising the model. code was written and plots produced by a.e.. the functionality of the code was checked by comparison with an alternative algorithm written by k.s. a.e. and k.s. wrote and edited the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to a.e.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - b rvr authors: abrantes, joana; droillard, clément; lopes, ana m.; lemaitre, evelyne; lucas, pierrick; blanchard, yannick; marchandeau, stéphane; esteves, pedro j.; le gall-reculé, ghislaine title: recombination at the emergence of the pathogenic rabbit haemorrhagic disease virus lagovirus europaeus/gi. date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: b rvr rabbit haemorrhagic disease is a viral disease that emerged in the s and causes high mortality and morbidity in the european rabbit (oryctolagus cuniculus). in , a new genotype of the rabbit haemorrhagic disease virus emerged and replaced the former circulating lagovirus europaeus/gi. strains. several recombination events have been reported for the new genotype lagovirus europaeus/gi. , with pathogenic (variants gi. a and gi. b) and benign (genotype gi. ) strains that served as donors for the non-structural part while gi. composed the structural part; another recombination event has also been described at the p /p junction involving gi. strains. in this study, we analysed new complete coding sequences of four benign gi. strains and four gi. strains. phylogenetic and recombination detection analyses revealed that the first gi. strains, considered as non-recombinant, resulted from a recombination event between gi. and gi. , with gi. as the major donor for the non-structural part and gi. for the structural part. our results indicate that recombination contributed to the emergence, persistence and dissemination of gi. as a pathogenic form and that all described gi. strains so far are the product of recombination. this highlights the need to study full-genomic sequences of lagoviruses to understand their emergence and evolution. in this study, we obtained eight new genome sequences: four for gi. and four for gi. . these included four strains previously identified based on their capsid sequences as gi. (ja / - , bo / - , ja / - and cha / - ; genbank accession numbers: lt , lt , lt and lt , respectively) and two strains as gi. ( - and - ; genbank accession numbers: he and he , respectively) , . we obtained the full-length genomic sequences of strains - and - , the complete coding sequences (cds) of strains - and - , and the cds along with the ′ untranslated region (utr) of the strains ja / - , bo / - , ja / - and cha / - . the genomes of the earliest gi. strains - and - were , nucleotides (nt) long and shared . % nucleotide identity. the length of the ′ utr sequences is nt and that of the ′ utr is nt. the closest strains were gi. strains with an average nucleotide identity of . % ( . % for the non-structural part of the genomes), while the average identity with gi. strains was % ( . % for the non-structural part of the genomes). the genomes of the gi. strains were , nt long (excluding the ′ utr), except the cha / - strain that was , nt long. the ′ utr sequences had the same length ( nt) . when including the gi. - sequence (genbank accession number mn ), the average nucleotide identity between the five gi. strains was . %. the cds of the four gi. showed between . % and . % of nucleotide identity ( . % between - and - strains) , and when compared to gi. cds presented no deletions or insertions ( , nt long), while the gi. cds presented a deletion of six consecutive nucleotides within the capsid gene ( , nt long instead of , nt). this deletion is present in the benign strains - , ashington and the italian rcv (genbank accession numbers: ef and x ) , , and comprises a codon putatively under positive selection in gi. strains . the strain cha / - had a further three nucleotide deletion in the minor structural protein vp (positions - , amino acid ). the complete dataset ( sequences; , nucleotides) was screened for recombination with rdp (recombination detection program) . the different methods available in rdp detected as recombinants, with strong statistical support (p-values < . ; table ), all the strains from the early gi. outbreaks (strains - , - and n ), all the gi. strains previously identified as non-recombinants (strains cbval , zar - , rij - , tar - , zar - and seg - ) and gi. strains more recently detected ( plm , nl , canada , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a single recombination breakpoint was determined at the non-structural/structural parts boundary (table ) . for most of these strains, the gi. strain - was identified as the most likely donor for the non-structural part, with the exception of strain - for which the most likely donor is cha / - . a gi. strain ( - _barrancos_portugal_ or sos _portugal_ , genbank accession numbers kf and mg , respectively) was the most likely donor for the structural part. thus, according to rdp all these sequences (gi. strains from the early outbreaks and gi. non-recombinant strains) are gi. /gi. recombinants. the tanglegram based on the alignments for the non-structural and the structural genome partitions also evidenced as recombinants the strains identified by rdp (fig. ) . the phylogenetic analysis was performed taking into account the location of the recombination breakpoint detected by rdp at the junction of the non-structural and structural parts. in the ml tree of the structural part, which includes vp and vp (fig. i) , the gi. strains formed a strongly supported group (bootstrap value ), that was a sister group to gi. (bootstrap value ). the mrcv strain, a gi. -like/gi. -like recombinant , also grouped with the gi. strains (bootstrap value ). according to the nomenclature recently proposed for lagoviruses, mrcv does not meet the criteria to be considered as a member of the gi. group and is currently unclassified . indeed, the genetic distance between mrcv and gi. vp gene sequences is > % (data not shown) and no other similar strains (from independent outbreaks) had been identified. as for the four gi. sequences obtained in this study, they clustered within a highly supported group (bootstrap value ) formed , gi. strains previously considered as non-recombinants (marked with an *), and the strains from canada (canada ) and the netherlands (nl ). the ml tree for the non-structural protein p (see supplementary information), recapitulated most of the results observed in the ml tree for the non-structural part with the exception of the positioning of the portuguese strains sos , sos , sos , sos , sos , sos , sos and sos , that appear clustered with gi. . strains sos and sos were previously shown to be triple recombinants between gi. , gi. b and gi. (gi. for p , gi. b for the remaining non-structural proteins and gi. for the structural proteins) which is in agreement with the results obtained in this study. as for strains sos , sos , sos , sos , sos and sos , the tree suggests that their genome also originated from three different strains: gi. for p , gi. for the remaining non-structural proteins and gi. for the structural proteins. in this case, it most likely involved recombination between a gi. strain and a gi. /gi. recombinant strain. a tanglegram analysis and rdp further confirmed these results (see supplementary information). gi. was identified as a novel pathogenic form of lagovirus in france in . field observations and further characterisation of the strains revealed unique characteristics in comparison with former strains such as the ability to cross the species boundaries [ ] [ ] [ ] [ ] [ ] and to kill young rabbits , . previous studies also revealed the occurrence of recombination of gi. strains with non-pathogenic (gi. ) and pathogenic (gi. a and gi. b) strains - , showing an important role of recombination in generating diversity in gi. and confirming the high capacity of recombination within lagoviruses. our results show that the strains circulating at the time of the first noticed gi. outbreaks were already recombinants between a european non-pathogenic strain (gi. ), that was donor for the non-structural part, and the new lagovirus genotype, gi. , that formed the structural part. indeed, in our ml tree for the non-structural part, the strains from the first gi. outbreaks ( - and - ) grouped with the non-pathogenic gi. group, while for the structural part these strains clustered with all the strains of the newly emerged gi. genotype. strains from the earliest outbreaks in spain and portugal in - , and strains collected in the canary islands (spain) , france, the netherlands and canada in are also gi. /gi. recombinants. these results, that were confirmed by the rdp, the ml trees and the tanglegram analyses, demonstrate that all the gi. strains described so far, including those that were considered non-recombinants and that in some instances co-circulated with other recombinant gi. strains , resulted from recombination. several recombination events are thus associated with the evolution of this new genotype: with gi. , gi. , gi. b and gi. a, with a recombination breakpoint at the non-structural/structural boundary , . an additional recombination event was further previously reported with a breakpoint at the p /p boundary and involved gi. as the donor of p , while gi. /gi. (see also supplementary information) or gi. b/gi. recombinants were the donors for the remaining viral genome . notably, the pattern described here for lagoviruses, with a recombination hotspot at the start of the major structural gene that encodes the capsid, is typical of caliciviruses [ ] [ ] [ ] . indeed, the combination of low sequence divergence , presence of complex rna secondary structures that may promote template switching by the virus polymerase, and the existence of a subgenomic rna that may act as a secondary template when rna replication cbcoruche - _portugal_ www.nature.com/scientificreports/ is resumed upon template switching, seem to contribute for the high rates of recombination observed in this region in the genome of caliciviruses and that likely constitutes an important survival strategy in the evolution of this family . this strategy, that resembles antigenic shift in influenza virus , allows caliciviruses to rapidly generate diversity by producing new genomic combinations, which might be beneficial for the adaptation to new hosts and environments, and to overcome selective pressures. recombination is important in shaping the evolution of rna viruses, including the closely related picornaviruses and coronaviruses and, more importantly, is often associated with the emergence of new viruses and even families, e.g. the hepeviridae family . as for the recombination involving the breakpoint at the p /p boundary, it may have originated from a non-replicative recombination mechanism where rna strands are randomly self-ligated or joined by cellular ligases . atypical recombination breakpoints such as this have been also observed for other caliciviruses and a similar mechanism proposed . estimation of the time to the most recent common ancestor (tmrca) of gi. by using capsid sequences pointed to an emergence in july . although the estimated substitution rates may be inaccurate due to limited sampling, reduced sequence variation and low temporal spread, they tend to underestimate rather than overestimate the real tmrca , . thus, it is possible that despite surveillance efforts, gi. circulated unnoticed in wildlife prior to its detection . the virus that recombined with gi. to produce this new genotype is currently unknown and undetected, even with the molecular surveys of lagoviruses performed in european leporids and our improved knowledge of the complete coding sequences of both pathogenic and benign lagoviruses. the same occurs for some older recombinant iberian strains where the virus that originated the non-structural part has never been detected . both viruses, the one originating gi. and the one of these iberian strains, could have either circulated harmlessly prior to their detection, thus making difficult to detect them, or have become extinct due to their lower fitness as a non-recombinant form, as suggested for noroviruses that recombined and whose partial sequences were never found . the evidence for recombination with gi. a , gi. b , gi. and gi. , (see also supplementary information) reveals that gi. successfully recombines with a great diversity of pathogenic and non-pathogenic lagoviruses. this, together with the tmrca estimates and the hypothesis of circulating harmlessly before its emergence, supports an evolution from a non-pathogenic form that acquired pathogenicity . we suggest that evolution of www.nature.com/scientificreports/ pathogenicity was not driven solely by point mutations, but was aided by recombination events. similarly to other rna viruses, lagoviruses genomes may function as interchangeable modules rather than as strict genomes, which leads to the appearance of mosaic-like genomes through recombination, resulting in a semi-independent evolution of structural and non-structural genes , and with low-fitness regions being eliminated by recombination . this might help to explain how a non-pathogenic strain (gi. ), combined with a potentially benign strain (gi. ), gave rise to a highly lethal, pathogenic strain. non-pathogenic forms are thus likely to be involved in the evolution of pathogenic forms and recombination might have precipitated the emergence of lagovirus europaeus/ gi. . the hypothesis of a species jump may not be discarded. indeed, reservoir species could have acted as source for the original virus that jumped to the final host, the european rabbit . whether recombination occurred in the reservoir species or in the final host is unknown. nonetheless, reservoir species have yet to be identified in which lagoviruses could have evolved and replicated without being detected before "jumping" into the european rabbit (le gall-reculé, personal communication) [ ] [ ] [ ] [ ] . genetic mechanisms such as recombination require coinfection of a host cell by two (or more) viruses. the high frequency of recombination detected in lagoviruses also implies a high frequency of co-infection, either in the host population or in the reservoir species. leeks and colleagues established a link between co-infection and viral diversity, as more diverse populations can produce more virulent infections and better adapt to new hosts . this might explain the array of hare species infected by gi. [ ] [ ] [ ] [ ] [ ] , which constitutes a novelty in relation to gi. . our results add complexity to the diversity of lagoviruses, but help to elucidate our current understanding on the emergence of pathogenic forms. in addition, they show that full genomic sequences of lagoviruses are crucial to understand their evolutionary history and genetic relationships. future studies should focus on unravelling the role of the structural and the non-structural parts in the emergence of pathogenicity in lagoviruses. virus samples and full-length genome sequencing. no animals were captured, handled or killed specifically for the purpose of this study. duodenum samples were collected from four hunted french rabbits by the national hunting and wildlife agency (oncfs) during the hunting seasons in - and - . presence of lagoviruses had previously been detected in these samples and phylogenetic relationships of the obtained capsid vp gene sequences had been determined ; these were identified as gi. strains. four liver samples were further collected in france from dead rabbits collected in three rabbitries affected by rhd, two on november ( - and - gi. strains ) and one in june (strain [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and in the field on june (strain . the presence of gi. in the - sample was diagnosed by labovet conseil (les herbiers, france). we characterised this strain as well as the - one based on their complete vp gene sequences, confirming that they belong to the gi. genotype. for the sequencing of the complete coding genome of gi. strains, ~ mg of duodenal tissue were homogenized in a mixer-mill disruptor (tissuelysed, qiagen, hidden, germany). rnas were first extracted with trizol ® (trizol ls reagent, ambion, thermo fisher scientific, villebon-sur-yvette, france) before using the rneasy mini kit (qiagen, hilden, germany) to increase sensitivity . for gi. strains, rna was extracted from µl of liver exudate using the rneasy mini kit. except for two gi. strains ( - and - ) for which the coding sequence was obtained using ngs (see below) from purified and quantified rna using the rneasy minelute cleanup kit (qiagen, hilden, germany) and the qubit rna hs assay kit with the qubit . fluorometer (thermo fisher scientific, villebon-sur-yvette, france), respectively, rnas were reverse-transcribed using oligo-dt as a primer and maxima reverse transcriptase (thermo fisher scientific, villebon-sur-yvette, france). protocols were performed as recommended by the manufacturers. according to the strains, different pcr strategies were attempted for genome sequencing either by using combinations of newly designed primers (primer sequences available upon request) or primers previously published , , . thus, cdnas of ja / - , bo / - and cha / - gi. strains were amplified using several overlapping pcrs, including one that covered the recombinant breakpoint between the non-structural and structural encoding genes, using expand high-fidelity pcr system (roche, sigma-aldrich, saint-quentin-fallavier, france). for ja / - gi. strain, due to lower viral loads, after two first pcrs that amplified two overlapping fragments of about , and , bp, respectively, several nested or semi-nested overlapping pcrs were performed. the complete coding sequences of the four gi. genomes were obtained following either one pcr amplification of about , bp using the primers u and l followed by smaller overlapping pcrs for sequence confirmation ( - and - ; primer sequences available upon request), or ngs ( - and - , see below). for these two last strains, the ′ end sequence of the cds ( bp) was confirmed using the pcr primers u and l as described in le gall-reculé et al. ( ) . pcrs were performed using expand high fidelity enzyme (roche-applied-science). the different pcr products were analysed by electrophoresis, purified using the minelute pcr purification kit (qiagen, hilden, germany) and quantified using the qubit dsdna hs assay kit with the qubit . fluorometer (thermo fisher scientific, villebon-sur-yvette, france). dna sequences were determined with an abi prism genetic or a series genetic analysers in both directions (applied biosystems, foster city, ca, usa), using the pcr and several inner primers and big dye terminator v . (life technologies) as recommended by the manufacturer. the ′ and ′ ends of the - and - strains were obtained using the rapid amplification of cdna ends (race) method following the protocol developed in lemaitre et al. ( ) . pcr products were purified and sequenced as described above. consensus sequences were compiled using vector nti advance software (life technologies, thermo fisher scientific, villebon-sur-yvette, france). for ngs, cdna libraries were prepared using the ion total rna-seq kit (life technologies, carlsbad, ca, usa) according to a protocol adapted from the supplier's instructions (available upon request from the authors). sequencing was performed using ion proton sequencer (life technologies). the reads were cleaned with the the aligned reads of this third alignment were collected and then down-sampled to fit a global coverage depth estimation of x. these cleaned reads were submitted to the spades (version . . ) de novo assembler and their raw related reads to the mira (version . . ) de novo assembler. the de novo contigs were then submitted to megablast on a local copy of nucleotide databank. the best lagovirus reference identified with megablast was used in a last bwa alignment of cleaned reads. the genomic sequences produced in this study have been deposited in genbank under the accession numbers: mn , mn , mn , mn (gi. strains ja / - , bo / - , ja / - and cha / - , respectively); mn , mn , mn , mn (gi. strains - , - , - and - , respectively). complete coding sequences obtained in this study were aligned with publicly available complete coding sequences of lagovirus europaeus representing genotypes gi. , gi. , gi. and gi. in bioedit software version . . . the final dataset included sequences, , nucleotides in length (see supplementary information for the list of the sequences used). the dataset was screened for recombination by seven methods available in the rdp software version . (rdp, geneconv, bootscan, maxchi, chimaera, siscan and seq) with the following parameters: sequences were set to linear, bonferroni correction, highest acceptable p-value of . and permutations. only recombination events detected by three or more methods were considered. recombinant strains were visualized by plotting a tanglegram using the "dendextend package" in the rstudio software (version . . ) . phylogenetic analysis. pairwise nucleotide distance comparison (p-distance model) was computed using mega . following the results obtained with rdp, phylogenetic analyses were carried out separately for the following genome partitions: nucleotide positions (i) - (p ; supplementary information), (ii) - , (p , p , p , vpg, protease and rdrp), and (iii) , - , (vp and vp ). maximum-likelihood phylogenetic trees were inferred in mega using the best model of nucleotide substitution determined in the same software for the different genome partitions, according to the lowest aicc value (akaike information criterion, corrected). support for each cluster was obtained from , bootstrap replicates. rabbit haemorrhagic disease (rhd) and rabbit haemorrhagic disease virus (rhdv): a review characterisation of a non-pathogenic and non-protective infectious rabbit lagovirus related to rhdv identification and partial characterisation of a new lagovirus in australian wild rabbits detection and preliminary characterization of a new rabbit calicivirus related to rabbit hemorrhagic disease virus but nonpathogenic benign circulation of rabbit haemorrhagic disease virus on lambay island novel calicivirus identified in rabbits molecular epidemiology of rabbit haemorrhagic disease virus detection of a new variant of rabbit haemorrhagic disease virus in france proposal for a unified classification system and nomenclature of lagoviruses spread of new variant rhdv in domestic rabbits on the iberian peninsula emergence of a new lagovirus related to rabbit haemorrhagic disease virus variant rabbit 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and g.l.g.r. performed the analyses, discussed the data, wrote and revised the first draft of the manuscript. c.d. obtained the molecular data, performed the analyses, discussed the data and revised the first draft. e.l. obtained the molecular data, discussed the data and revised the first draft. p.l. and y.b. analysed the ngs data. s.m. was responsible for the gi. strains sample collection, discussed the data and revised the first draft. p.j.e. discussed the data and revised the first draft. all authors contributed to the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.a.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -i odcsx authors: sahlol, ahmed t.; yousri, dalia; ewees, ahmed a.; al-qaness, mohammed a. a.; damasevicius, robertas; elaziz, mohamed abd title: covid- image classification using deep features and fractional-order marine predators algorithm date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: i odcsx currently, we witness the severe spread of the pandemic of the new corona virus, covid- , which causes dangerous symptoms to humans and animals, its complications may lead to death. although convolutional neural networks (cnns) is considered the current state-of-the-art image classification technique, it needs massive computational cost for deployment and training. in this paper, we propose an improved hybrid classification approach for covid- images by combining the strengths of cnns (using a powerful architecture called inception) to extract features and a swarm-based feature selection algorithm (marine predators algorithm) to select the most relevant features. a combination of fractional-order and marine predators algorithm (fo-mpa) is considered an integration among a robust tool in mathematics named fractional-order calculus (fo). the proposed approach was evaluated on two public covid- x-ray datasets which achieves both high performance and reduction of computational complexity. the two datasets consist of x-ray covid- images by international cardiothoracic radiologist, researchers and others published on kaggle. the proposed approach selected successfully and out of k features extracted by inception from dataset and dataset , while improving classification accuracy at the same time. the results are the best achieved on these datasets when compared to a set of recent feature selection algorithms. by achieving . %, . % and . %, % of classification accuracy and f-score for dataset and dataset , respectively, the proposed approach outperforms several cnns and all recent works on covid- images. medical imaging techniques are very important for diagnosing diseases. image segmentation is a necessary image processing task that applied to discriminate region of interests (rois) from the area of outsides. also, image segmentation can extract critical features, including the shape of tissues, and texture , . in general, feature selection (fs) methods are widely employed in various applications of medical imaging applications. for example, lambin et al. proposed an efficient approach called radiomics to extract medical image features. they showed that analyzing image features resulted in more information that improved medical imaging. chong et al. proposed an fs model, called robustness-driven fs (rdfs) to select futures from lung ct images to classify the patterns of fibrotic interstitial lung diseases. they applied the svm classifier with and without rdfs. the evaluation showed that the rdfs improved svm robustness against reconstruction kernel and slice thickness. in , to classify ultrasound medical images, the authors used distance-based fs methods and a fuzzy support vector machine (fsvm). moreover, a multi-objective genetic algorithm was applied to search for the optimal features subset. more so, a combination of partial differential equations and deep learning was applied for medical image classification by . they employed partial differential equations for extracting texture features of medical images. acharya et al. applied different fs methods to classify alzheimer's disease using mri images. the shearlet transform fs method showed better performances compared to several fs methods. also, in , an fs method based on svm was proposed to detect alzheimer's disease from spect images. duan et al. applied the gaussian mixture model (gmm) to extract features from pulmonary nodules from ct images. the optimum path forest (opf) classifier was applied to classify pulmonary nodules based on ct images. in , the authors proposed an fs method based on a convolutional neural network (cnn) to detect pneumonia from lung x-ray images. afzali et al. proposed an fs method based on principal component analysis and contour-based shape descriptors to detect tuberculosis from lung x-ray images. they used k-nearest neighbor (knn) to classify x-ray images collected from montgomery dataset, and it showed good performances. zhang et al. proposed a kernel feature selection method to segment brain tumors from mri images. they applied the svm classifier for new mri images to segment brain tumors, automatically. to segment brain tissues from mri images, kong et al. proposed an fs method using two methods, called a discriminative clustering method and the information theoretic discriminative segmentation. harikumar et al. proposed an fs method based on wavelets to classify normality or abnormality of different types of medical images, such as ct, mri, ultrasound, and mammographic images. it can be concluded that fs methods have proven their advantages in different medical imaging applications . furthermore, deep learning using cnn is considered one of the best choices in medical imaging applications , especially classification. cnns are more appropriate for large datasets. also, they require a lot of computational resources (memory & storage) for building & training. in some cases (as exists in this work), the dataset is limited, so it is not sufficient for building & training a cnn. in such a case, in order to get the advantage of the power of cnn and also, transfer learning can be applied to minimize the computational costs , . in transfer learning, a cnn which was previously trained on a large & diverse image dataset can be applied to perform a specific classification task by . therefore, several pre-trained models have won many international image classification competitions such as vggnet , resnet , nasnet , mobilenet , inception and xception . however, some of the extracted features by cnn might not be sufficient, which may affect negatively the quality of the classification images. therefore, a feature selection technique can be applied to perform this task by removing those irrelevant features. among the fs methods, the metaheuristic techniques have been established their performance overall other fs methods when applied to classify medical images. for example, da silva et al. used the genetic algorithm (ga) to develop feature selection methods for ranking the quality of medical images. they used different images of lung nodules and breast to evaluate their fs methods. evaluation outcomes showed that ga based fs methods outperformed traditional approaches, such as filter based fs and traditional wrapper methods. johnson et al. applied the flower pollination algorithm (fpa) to select features from ct images of the lung, to detect lung cancers. they also used the svm to classify lung ct images. the evaluation confirmed that fpa based fs enhanced classification accuracy. kharrat and mahmoud proposed an fs method based on a hybrid of simulated annealing (sa) and ga to classify brain tumors using mri. the combination of sa and ga showed better performances than the original sa and ga. narayanan et al. proposed a fuzzy particle swarm optimization (pso) as an fs method to enhance the classification of ct images of emphysema. they applied a fuzzy decision tree classifier, and they found that fuzzy pso improved the classification accuracy. li et al. proposed a self-adaptive bat algorithm (ba) to address two problems in lung x-ray images, rebalancing, and feature selection. they compared the ba to pso, and the comparison outcomes showed that ba had better performance. dhanachandra and chanu proposed a hybrid method of dynamic pso and fuzzy c-means to segment two types of medical images, mri and synthetic images. they concluded that the hybrid method outperformed original fuzzy c-means, and it had less sensitive to noises. li et al. proposed an fs method using a discrete artificial bee colony (abc) to improve the classification of parkinson's disease. the evaluation outcomes demonstrate that abc enhanced precision, and also it reduced the size of the features. in this paper, we proposed a novel covid- x-ray classification approach, which combines a cnn as a sufficient tool to extract features from covid- x-ray images. then, using an enhanced version of marine predators algorithm to select only relevant features. in general, mpa is a meta-heuristic technique that simulates the behavior of the prey and predator in nature . this algorithm is tested over a global optimization problem. however, it has some limitations that affect its quality. in addition, up to our knowledge, mpa has not applied to any real applications yet. so, based on this motivation, we apply mpa as a feature selector from deep features that produced from cnn (largely redundant), which, accordingly minimize capacity and resources consumption and can improve the classification of covid- x-ray images. the proposed covid- x-ray classification approach starts by applying a cnn (especially, a powerful architecture called inception which pre-trained on imagnet dataset) to extract the discriminant features from raw images (with no pre-processing or segmentation) from the dataset that contains positive and negative covid- images. then, applying the fo-mpa to select the relevant features from the images. this task is achieved by fo-mpa which randomly generates a set of solutions, each of them represents a subset of potential features. the next process is to compute the performance of each solution using fitness value and determine which one is the best solution. thereafter, the fo-mpa parameters are applied to update the solutions of the current population. the updating operation repeated until reaching the stop condition. then the best solutions are reached which determine the optimal/relevant features that should be used to address the desired output via several performance measures. inspired by our recent work , where vgg- besides statistically enhanced salp swarm algorithm was applied to select the best features for white blood cell leukaemia classification. also, other recent published works , who combined a cnn architecture with weighted symmetric uncertainty (wsu) to select optimal features for traffic classification. it is obvious that such a combination between deep features and a feature selection algorithm can be efficient in several image classification tasks. the main contributions of this study are elaborated as follows: . propose an efficient hybrid classification approach for covid- using a combination of cnn and an improved swarm-based feature selection algorithm. this combination should achieve two main targets; high performance and resource consumption, storage capacity which consequently minimize processing time. . propose a novel robust optimizer called fractional-order marine predators algorithm (fo-mpa) to select efficiently the huge feature vector produced from the cnn. . test the proposed inception fractional-order marine predators algorithm (ifm) approach on two publicity available datasets contain a number of positive negative chest x-ray scan images of covid- . . evaluate the proposed approach by performing extensive comparisons to several state-of-art feature selection algorithms, most recent cnn architectures and most recent relevant works and existing classification methods of covid- images. we do not present a usable clinical tool for covid- diagnosis, but offer a new, efficient approach to optimize deep learning-based architectures for medical image classification purposes. such methods might play a significant role as a computer-aided tool for image-based clinical diagnosis soon. remainder sections are organized as follows: "material and methods" section presents the methodology and the techniques used in this work including model structure and description. the experimental results and comparisons with other works are presented in "results and discussion" section, while they are discussed in "discussion" section finally, the conclusion is described in "conclusion" section. features extraction using convolutional neural networks. in this paper, we apply a convolutional neural network (cnn) to extract features from covid- x-ray images. we adopt a special type of cnn called a pre-trained model where the network is previously trained on the imagenet dataset, which contains millions of variety of images (animal, plants, transports, objects,..) on classe categories. so, transfer learning is applied by transferring weights that were already learned and reserved into the structure of the pre-trained model, such as inception, in this paper. in inception, there are different sizes scales convolutions (conv.), such as × , × , × . for instance, × conv. is applied before larger sized kernels are applied to reduce the dimension of the channels, which accordingly, reduces the computation cost. pool layers are used mainly to reduce the input's size, which accelerates the computation as well. so, for a × matrix, will result in × matrix after applying max pooling. there are three www.nature.com/scientificreports/ main parameters for pooling, filter size, stride, and max pool. in this paper, filters of size , besides a stride of and × as max pool, were adopted. inception architecture is described in fig. . the main purpose of conv. layers is to extract features from input images. in this paper, different conv. layers are applied to extract different types of features such as edges, texture, colors, and high-lighted patterns from the images. the combination of conv. and pool layers, three fully connected layers, the last one performs classification. the softmax activation function is used for this purpose because the output should be binary (positive covid- negative covid- ). inception's layer details and layer parameters of are given in table . as seen in table , we keep the last concatenation layer which contains the extracted features, so we removed the top layers such as the flatten, drop out and the dense layers which the later performs classification (named as fc layer). we have used rmsprop optimizer for weight updates, cross entropy loss function and selected learning rate as . . in this paper, inception is applied as a feature extractor, where the input image shape is ( , , ). since its structure consists of some parallel paths, all the paths use padding of pixel to preserve the same height & width for the inputs and the outputs. one of the drawbacks of pre-trained models, such as inception, is that its architecture required large memory requirements as well as storage capacity ( m.b), which makes deployment exhausting and a tiresome task. the shape of the output from the inception is ( , , ), which represents a feature vector of size . so some statistical operations have been added to exclude irrelevant and noisy features, and by making it more computationally efficient and stable, they are summarized as follows: • chi-square is applied to remove the features which have a high correlation values by computing the dependence between them. it is calculated between each feature for all classes, as in eq. ( ): where o k and e k refer to the actual and the expected feature value, respectively. in this paper, after applying chi-square, the feature vector is minimized for both datasets from to . • tree based classifier are the most popular method to calculate feature importance to improve the classification since they have high accuracy, robustness, and simple . for each decision tree, node importance is calculated using gini importance, eq. ( ) calculated two child nodes. where ni j is the importance of node j, while w j refers to the weighted number of samples reaches the node j, also c j determines the impurity value of node j. left(j) and right(j) are the child nodes from the left split and the right split on node j, respectively. in eq. ( ), the importance of each feature is then calculated. ( ) www.nature.com/scientificreports/ where fi i represents the importance of feature i, while ni j refers to the importance of node j. in order to normalize the values between and by dividing by the sum of all feature importance values, as in eq. ( ). finally, the sum of the feature's importance value on each tree is calculated then divided by the total number of trees as in eq. ( ). where refi i represents the importance of feature i that were calculated from all trees, where normfi ij is the normalized feature importance for feature i in tree j, also t is the total number of trees. after applying this technique, the feature vector is minimized from to and from to for dataset and dataset , respectively. fractional-order calculus (fc) gains the interest of many researchers in different fields not only in the modeling sectors but also in developing the optimization algorithms. the memory properties of fc calculus makes it applicable to the fields that required non-locality and memory effect. fc provides a clear interpretation of the memory and hereditary features of the process. accordingly, the fc is an efficient tool for enhancing the performance of the meta-heuristic algorithms by considering the memory perspective during updating the solutions. one from the well-know definitions of fc is the grunwald-letnikov (gl), which can be mathematically formulated as below : where where d δ (u(t)) refers to the gl fractional derivative of order δ . Ŵ(t) indicates gamma function. the gl in the discrete-time form can be modeled as below: where t is the sampling period, and m is the length of the memory terms (memory window). the δ symbol refers to the derivative order coefficient. for the special case of δ = , the definition of eq. ( ) can be remodeled as below: where d [x(t)] represents the difference between the two followed events. marine predators algorithm. the marine predators algorithm (mpa)is a recently developed meta-heuristic algorithm that emulates the relation among the prey and predator in nature . mpa simulates the main aim for most creatures that is searching for their foods, where a predator contiguously searches for food as well as the prey. inspired by this concept, faramarzi et al. developed the mpa algorithm by considering both of a predator a prey as solutions. the mpa starts with the initialization phase and then passing by other three phases with respect to the rational velocity among the prey and the predator. • initialization phase: this phase devotes for providing a random set of solutions for both the prey and predator via the following formulas: where the lower and upper are the lower and upper boundaries in the search space, rand is a random vector ∈ the interval of ( , ). according to the formula , the initial locations of the prey and predator can be defined as below: ( ) fi i = j:node j splits on feature i ni j k∈all nodes ni k www.nature.com/scientificreports/ where the elite matrix refers to the fittest predators. • stage : after the initialization, the exploration phase is implemented to discover the search space. therefore in mpa, for the first third of the total iterations, i.e., t max ). accordingly, the prey position is upgraded based the following equations. where r ∈ [ , ] is a random vector drawn from a uniform distribution and p = . is a constant number. the symbol r b refers to brownian motion. indicates the process of element-wise multiplications. • stage : the prey/predator in this stage begin exploiting the best location that detects for their foods. stage has been executed in the second third of the total number of iterations when t max < t < t max . faramarzi et al. divided the agents for two halves and formulated eqs. ( )- ( ) to emulate the motion of the first half of the population (prey) and eqs. ( )- ( ) for the second half (predator) as represented below. where r l has random numbers that follow lévy distribution. eq. ( )- ( ) are implemented in the first half of the agents that represent the exploitation. while the second half of the agents perform the following equations. where cf is the parameter that controls the step size of movement for the predator. • stage : this stage executed on the last third of the iteration numbers ( t > t max ) where based on the following formula: • eddy formation and fish aggregating devices' effect: faramarzi et al. considered the external impacts from the environment, such as the eddy formation or fish aggregating devices (fads) effects to avoid the local optimum solutions. this stage can be mathematically implemented as below: in eq. ( ), fad = . , and w is a binary solution ( or ) that corresponded to random solutions. if the random solution is less than . , it converted to while the random solution becomes when the solutions are greater than . . the symbol r ∈ [ , ] represents a random number. r and r are the random index of the prey. • marine memory: this is the main feature of the marine predators and it helps in catching the optimal solution very fast and avoid local solutions. faramarzi et al. implement this feature via saving the previous best solutions of a prior iteration, and compared with the current ones; the solutions are modified based on the best one during the comparison stage. recently, a combination between the fractional calculus tool and the meta-heuristics opens new doors in providing robust and reliable variants . for this motivation, we utilize the fc concept with the mpa algorithm to boost the second step of the standard version of the algorithm. hence, the fc memory is applied during updating the prey locating in the second step of the algorithm to enhance the exploitation stage. moreover, the r b parameter has been changed to depend on weibull distribution as described below. by taking into account the early mentioned relation in eq. ( ), the general formulation for the solutions of fo-mpa based on fc memory perspective can be written as follows: after checking the previous formula, it can be detected that the motion of the prey becomes based on some terms from the previous solutions with a length of (m), as depicted in fig. (left) . with accounting the first four previous events ( m = ) from the memory data with derivative order δ , the position of prey can be modified as follow; • second: adjusting r b random parameter based on weibull distribution. for the exploration stage, the weibull distribution has been applied rather than brownian to bost the performance of the predator in stage and the prey velocity in stage based on the following formula: where k, and ζ are the scale and shape parameters. the weibull distribution is a heavy-tied distribution which presented as in fig. (right) . in the current work, the values of k, and ζ are set to , and , respectively. our proposed approach is called inception fractional-order marine predators algorithm (ifm), where we combine inception (i) with fractional-order marine predators algorithm (fo-mpa). the proposed ifm approach is summarized as follows: . extracting deep features from inception, where about k features were extracted. . initialize solutions for the prey and predator. the prey follows weibull distribution during discovering the search space to detect potential locations of its food. . the predator tries to catch the prey while the prey exploits the locations of its food. the predator uses the weibull distribution to improve the exploration capability. meanwhile, the prey moves effectively based on its memory for the previous events to catch its food, as presented in eq. ( ). . finally, the predator follows the levy flight distribution to exploit its prey location. all above stages are repeated until the termination criteria is satisfied. these datasets contain hundreds of frontal view x-rays and considered the largest public resource for covid- image data. they were manually aggregated from various web based repositories into a machine learning (ml) friendly format with accompanying data loader code. they were also collected frontal and lateral view imagery and metadata such as the time since first symptoms, intensive care unit (icu) status, survival status, intubation status, or hospital location. both datasets shared some characteristics regarding the collecting sources. for both datasets, the covid images were collected from patients with ages ranging from - from both genders. it is also noted that both datasets contain a small number of positive covid- images, and up to our knowledge, there is no other sufficient available published dataset for covid- . table shows some samples from two datasets. table depicts the variation in morphology of the image, lighting, structure, black spaces, shape, and zoom level among the same dataset, as well as with the other dataset. • best accuracy: • best fitness value: • worst fitness value: • average of fitness value: • standard deviation of fitness value where r is the run numbers. fit i denotes a fitness function value. google colaboratory , commonly referred to as "google colab, " which is a research project for prototyping machine learning models on powerful hardware options such as gpus and tpus. in this paper, we used tpus for powerful computation, which is more appropriate for cnn. the model was developed using keras library with tensorflow backend . performance of the proposed approach. as inception examines all x-ray images over and over again in each epoch during the training, these rapid ups and downs are slowly minimized in the later part of the training. after feature extraction, we applied fo-mpa to select the most significant features. in this subsection, the results of fo-mpa are compared against most popular and recent feature selection algorithms, such as whale optimization algorithm (woa) , henry gas solubility optimization (hgso) , sine cosine algorithm (sca), slime mould algorithm (sma) , particle swarm optimization (pso), grey wolf optimization (gwo) , harris hawks optimization (hho) , genetic algorithm (ga), and basic mpa. in this paper, each feature selection algorithm were exposed to select the produced feature vector from inception aiming ( ) f score = × specificity × sensitivity specificity + sensitivity tables and . table shows the numerical results of the feature selection phase for both datasets. four measures for the proposed method and the compared algorithms are listed. as seen in table , on dataset , the fo-mpa outperformed the other algorithms in the mean of fitness value as it achieved the smallest average fitness function value followed by sma, hho, hgso, sca, bgwo, mpa, and bpso, respectively whereas, the sga and woa showed the worst results. the results of max measure (as table . results of the feature selection phase based on fitness function. highest results are in bold. www.nature.com/scientificreports/ in eq. ( )), showed that fo-mpa also achieved the best value of the fitness function compared to others. sma is on the second place, while hgso, sca, and hho came in the third to fifth place, respectively. according to the best measure, the fo-mpa performed similarly to the hho algorithm, followed by sma, hgso, and sca, respectively. although the performance of the mpa and bgwo was slightly similar, the performance of sga and woa were the worst in both max and min measures. generally, the most stable algorithms on dataset are woa, sca, hgso, fo-mpa, and sga, respectively. however, woa showed the worst performances in these measures; which means that if it is run in the same conditions several times, the same results will be obtained. for dataset , fo-mpa showed acceptable (not the best) performance, as it achieved slightly similar results to the first and second ranked algorithm (i.e., mpa and sma) on mean, best, max, and std measures. also, woa algorithm showed good results in all measures, unlike dataset , which can conclude that no algorithm can solve all kinds of problems. whereas, the worst algorithm was bpso. for more analysis of feature selection algorithms based on the number of selected features (s.f) and consuming time, fig. and table list these results for all algorithms. regarding the consuming time as in fig. a , the sma was considered as the fastest algorithm among all algorithms followed by bpso, fo-mpa, and hho, respectively, while mpa was the slowest algorithm. also, as seen in fig. b , fo-mpa algorithm selected successfully fewer features than other algorithms, as it selected and features from dataset and dataset , respectively. hgso was ranked second with and selected features from dataset and dataset , respectively. the largest features were selected by sma and sga, respectively. the convergence behaviour of fo-mpa was evaluated over independent runs and compared to other algorithms, where the x-axis and the y-axis represent the iterations and the fitness value, respectively. figure illustrates the convergence curves for fo-mpa and other algorithms in both datasets. figure , shows that fo-mpa shows an efficient and faster convergence than the other optimization algorithms on both datasets. whereas, the slowest and the insufficient convergences were reported by both sga and woa in dataset and by sga in dataset . to further analyze the proposed algorithm, we evaluate the selected features by fo-mpa by performing classification. in this experiment, the selected features by fo-mpa were classified using knn. table show classification accuracy of fo-mpa compared to other feature selection algorithms, where the best, mean, and std for classification accuracy were calculated for each one, besides time consumption and the number of selected features (sf). in table , for dataset , the proposed fo-mpa approach achieved the highest accuracy in the best and mean measures, as it reached . %, and . % of correctly classified samples, respectively. while, mpa, bpso, sca, and sga obtained almost the same accuracy, followed by both bgwo, woa, and sma. the lowest accuracy was obtained by hgso in both measures. based on standard deviation measure (std), the most stable algorithms were sca, sga, bpso, and bgwo, respectively. whereas, fo-mpa, mpa, hgso, and woa showed similar std results. the hgso also was ranked last. in dataset , fo-mpa also is reported as the highest classification accuracy with the best and mean measures followed by the bpso. the classification accuracy of mpa, woa, sca, and sga are almost the same. whereas the worst one was sma algorithm. besides, all algorithms showed the same statistical stability in std measure, except for hho and hgso. generally, the proposed fo-mpa approach showed satisfying performance in both the feature selection ratio and the classification rate. www.nature.com/scientificreports/ moreover, from table , it can be seen that the proposed fo-mpa provides better results in terms of f-score, as it has the highest value in datatset and datatset which are . and . , respectively. comparison with other cnn architectures. in this subsection, the performance of the proposed covid- classification approach is compared to other cnn architectures. it noted that all produced feature vectors by cnns used in this paper are at least bigger by more than times compared to that produced by fo-mpa in terms of the size of the featureset. for example, as our input image has the shape × × , nasnet produces k features, resnet and xception produce about k features and mobilenet produces k features, while fo-mpa produces and features for both dataset and dataset , respectively. figure shows a comparison between our fo-mpa approach and other cnn architectures. from fig. (left), for dataset , it can be seen that our proposed fo-mpa approach outperforms other cnn models like vggnet, xception, inception, mobilenet, nasnet, and resnet. it also shows that fo-mpa can select the smallest subset of features, which reflects positively on performance. accordingly, that reflects on efficient usage of memory, and less resource consumption. on the second dataset, dataset (fig. , right) , our approach still provides an overall accuracy of . %, putting it first with a slight advantage over mobilenet ( . %). comparison with related works. in this subsection, a comparison with relevant works is discussed. figure shows the most recent published works as in - and on both dataset and dataset . in , alexnet pre-trained network was used to extract deep features then applied pca to select the best features by eliminating highly correlated features. based on , the later step reduces the memory requirements, and improve the efficiency of the framework. while used different cnn structures. however, it was clear that vgg and mobilenet achieved the best performance over other cnns. also, in a new cnn architecture called efficientnet was proposed, where more blocks were added on top of the model after applying normalization of images pixels intensity to the range ( to ). also, some image transformations were applied, such as rotation, horizontal flip, and scaling. in , resnet- cnn has been applied after applying horizontal flipping, random rotation, random zooming, random lighting, and random wrapping on raw images. as seen in fig. , most works are pre-prints for two main reasons; covid- is the most recent and trend topic; also, there are no sufficient datasets that can be used for reliable results. however, the proposed fo-mpa approach has an advantage in performance compared to other works. also, all other works do not give further statistics about their model's complexity and the number of www.nature.com/scientificreports/ featurset produced, unlike, our approach which extracts the most informative features ( and features for dataset and dataset ) that imply faster computation time and, accordingly, lower resource consumption. compared to which is one of the most recent published works on x-ray covid- , a combination between you only look once (yolo) which is basically a real time object detection system and darknet as a classifier was proposed. they achieved . % and . % of accuracy and f-score, respectively compared to our approach with . % and . % for accuracy and f-score, respectively. while no feature selection was applied to select best features or to reduce model complexity. the proposed imf approach successfully achieves two important targets, selecting small feature numbers with high accuracy. therefore, reducing the size of the feature from about k as extracted by deep neural networks (inception) to be . and in dataset and dataset , respectively, after applying fo-mpa algorithm while increasing the general performance can be considered as a good achievement as a machine learning goal. besides, the used statistical operations improve the performance of the fo-mpa algorithm because it supports the algorithm in selecting only the most important and relevant features. it also contributes to minimizing resource consumption which consequently, reduces the processing time. in addition, the good results achieved by the fo-mpa against other algorithms can be seen as an advantage of fo-mpa, where a balancing between exploration and exploitation stages and escaping from local optima were achieved. as a result, the obtained outcomes outperformed previous works in terms of the model's general performance measure. furthermore, using few hundreds of images to build then train inception is considered challenging because deep neural networks need large images numbers to work efficiently and produce efficient features. however, the proposed imf approach achieved the best results among the compared algorithms in least time. one of the main disadvantages of our approach is that it's built basically within two different environments. the first one is based on python, where the deep neural network architecture (inception) was built and the feature extraction part was performed. the second one is based on matlab, where the feature selection part (fo-mpa algorithm) was performed. so, there might be sometimes some conflict issues regarding the features vector file types or issues related to storage capacity and file transferring. computational image analysis techniques play a vital role in disease treatment and diagnosis. taking into consideration the current spread of covid- , we believe that these techniques can be applied as a computer-aided tool for diagnosing this virus. therefore, in this paper, we propose a hybrid classification approach of covid- . it based on using a deep convolutional neural network (inception) for extracting features from covid- images, then filtering the resulting features using marine predators algorithm (mpa), enhanced by fractionalorder calculus(fo). the proposed imf approach is employed to select only relevant and eliminate unnecessary features. extensive evaluation experiments had been carried out with a collection of two public x-ray images datasets. extensive comparisons had been implemented to compare the fo-mpa with several feature selection algorithms, including sma, hho, hgso, woa, sca, bgwo, sga, bpso, besides the classic mpa. the results showed that the proposed approach showed better performances in both classification accuracy and the number of extracted features that positively affect resource consumption and storage efficiency. the results are the best achieved compared to other cnn architectures and all published works in the same datasets. according to the promising results of the proposed model, that combines cnn as a feature extractor and fo-mpa as a feature selector could be useful and might be successful in being applied in other image classification tasks. all data used in this paper is available online in the repository, [https ://githu b.com/ieee /covid -chest xraydatas et], [https ://stanf ordml group .githu b.io/proje cts/chexn et], [https ://www.kaggl e.com/pault imoth ymoon ey/ chest -xray-pneum onia] and [https ://www.sirm.org/en/categ ory/artic les/covid - -datab ase/]. the code of the proposed approach is also available via the following link [https ://drive .googl e.com/file/d/ -ok-eeegd cmcny kh ikak opmqa rvas x/view?usp=shari ng]. isolation and characterization of a bat sars-like coronavirus that uses the ace receptor optimization method for forecasting confirmed cases of covid- in china transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart use of chest ct in combination with negative rt-pcr assay for the novel coronavirus but high clinical suspicion medical image segmentation using fruit fly optimization and density peaks clustering brain tumor 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applications classification of covid- in chest x-ray images using detrac deep convolutional neural network covid- : automatic detection from x-ray images utilizing transfer learning with convolutional neural networks towards an efficient deep learning model for covid- patterns detection in x-ray images the diagnostic evaluation of convolutional neural network (cnn) for the assessment of chest x-ray of patients jcs: an explainable covid- diagnosis system by joint classification and segmentation automated detection of covid- cases using deep neural networks with x-ray images the authors declare no competing interests. correspondence and requests for materials should be addressed to r.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -ncdy qgb authors: wang, ji-gan; cui, hai-rong; tang, hua-bo; deng, xiu-li title: gastrointestinal symptoms and fecal nucleic acid testing of children with coronavirus disease: a systematic review and meta-analysis date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ncdy qgb in order to understand the clinical manifestations and incidence of gastrointestinal symptoms of coronavirus disease (covid- ) in children and discuss the importance of fecal nucleic acid testing.we retrospectively analyzed studies on gastrointestinal symptoms and fecal nucleic acid detection in pediatric covid- patients from january , to august , , including prospective clinical studies and case reports. the results of fecal nucleic acid detection were analyzed systematically. stata . software was used for meta-analysis.the results showed that the most common gastrointestinal symptoms in children with covid- were vomiting and diarrhea, with a total incidence of . % ( % cl . – . %). however, the prevalence of gastrointestinal symptoms in other countries ( . %, % ci . – . %) was higher compared to china ( . %, % ci – . %). in wuhan, the pooled prevalence was much higher ( . %, % ci . – . %) compared to areas outside wuhan in china ( . %, % ci . – . %). the positive rate of fecal nucleic acid testing in covid- children was relatively high at . % ( / ). additionally, . % ( / ) were still positive for fecal nucleic acid after respiratory tract specimens turned negative. one and two weeks after the respiratory tract specimens turned nucleic acid-negative, . % ( / ) and . % ( / ) patients, respectively, remained fecal nucleic acid-positive. the longest interval between the respiratory tract specimens turning negative and fecal specimens turning negative exceeded days. conclusions and relevance: gastrointestinal symptoms in pediatric covid- are relatively common. attention should be paid to the detection of fecal nucleic acids in children. fecal nucleic acid-negative status should be considered as one of the desegregation standards. literature screening and data extraction. two researchers independently searched and screened the literature and collected and cross-checked the relevant data. if there was any dispute, it was discussed or resolved with the help of a third researcher. inclusion criteria . research types: cohort study, case-control study, and case analysis; . subjects: children with covid- ; . observation index: clinical manifestations of covid- in children including gastrointestinal symptoms such as diarrhea and vomiting. exclusion criteria . repeated publication of the same research; . studies on adults; . incomplete or missing data or analysis, and inability to obtain the data literature. we included a case series study, using the national institute for clinical optimization. clinical excellence (nice) for quality evaluation . the evaluation items were as follows: the cases included in the case series should ( ) come from different levels of medical institutions that carry out multi-center research; ( ) clearly describe the research hypothesis or purpose; ( ) have clear exclusion criteria; ( ) have a clear definition of the measurement of results; ( ) present collected data that achieves the expected purpose; ( ) accurately describe that patients are continuously recruited; ( ) describe the main findings clearly; ( ) analyze and report the results in layers. one point is given for each item, and a total score ≥ out of points is considered as highquality research. two researchers independently evaluated the quality and cross-checked the results. statistical analysis. meta-analysis was performed using the stata . software. first, the original ratio (r) was transformed by double arcsine to make it conform to a normal distribution, and then the transform ratio (tr) was analyzed by meta-analysis. subsequently, the final rate (r) with the % confidence interval (ci) were obtained by converting the results with the formula r = (sin[tr/ ]) . meta-analysis was carried out by using the random effect model for all studies. the funnel chart was utilized to assess publication bias, and the metaanalysis significance level was designated as α = . . as this is a systematic review, ethical approval was not required. gastrointestinal symptoms of covid- . figure summarizes the article retrieval and abstraction method using the prisma guidelines. most research data are concentrated in articles published by china, the united states, and europe. a total of studies were included in this analysis - , of which described the gastrointestinal symptoms of patients (supplementary table s ). a total of patients were evaluated in the study, among whom had digestive tract symptoms, accounting for . % ( % ci . - . %) of the patients (fig. ) . vomiting and diarrhea were the most common gastrointestinal symptoms. when analyzing by country (studies from china versus studies from other countries), the prevalence of gastrointestinal symptoms in countries outside china was . % ( % ci . - . %), which was higher than that in china ( . %, % ci - . %). among patients in wuhan, the pooled prevalence was much higher at . % ( % ci . - . %) than in areas outside wuhan in china ( . %, % ci . %- . %) ( table ) . a majority of studies did not describe the stool characteristics or number of bowel movements. wang duan et al. reported that in the six northern provinces of china, patients had bowel movements - times per day, while wu huaping found that in the jiangxi province of china, the frequency of diarrhea in affected children was - times per day . subgroup analysis. the heterogeneity of this study was relatively large. to explore the source of heterogeneity, we analyzed according to the region (country or region) where the research object was located. it was discovered that the analysis results of each subgroup were basically consistent with the overall results, and there were no significant differences between the heterogeneity of each subgroup and the overall heterogeneity. therefore, was considered that the region of the research object was not the main source of heterogeneity (table ) . fecal testing for viral nucleic acid. thirteen reports included in the present study described fecal nucleic acid examination ( . however, in a study on three neonates , respiratory tract and fecal nucleic acid tests were positive and days after birth, respectively, and the fecal and respiratory tract specimens were negative on the th day after birth. publication bias. the funnel plot (fig. ) shows the presence of a possible publication bias. most of the research quality scores were not high. our confidence in the pooled estimates of prevalence was reduced because of concerns regarding risk of bias (selection bias, detection bias, and attrition bias), heterogeneity of the tested incidence of gastrointestinal symptoms. in the early stages of the pandemic, there was a shared misconception that children were not easily infected . however, with the spread of the pandemic, the number of infected children is increasing and several severe pediatric cases have been reported . it is sometimes difficult to distinguish the gastrointestinal symptoms of pediatric covid- from those caused by another viral illness, side effects of drugs, and digestive tract symptoms such as nausea and diarrhea caused by the disturbance of gastrointestinal flora by the fever itself. some studies have found that . % children use antibiotics that cause diarrhea, and the diarrhea is more severe in younger patients with lower respiratory tract infections treated with intravenous antibiotics. moreover, we discovered that the total incidence of gastrointestinal symptoms in children with covid- was . %; unfortunately, not all the studies described a control group when investigating the incidence of gastrointestinal symptoms in an antibiotic treatment group and non-antibiotic treatment group. in a meta-analysis of predominantly adult studies, studies (including patients with covid- ) were analyzed and the incidence of gastrointestinal symptoms was found to be . %, which is almost equal to . % found in this study. in addition, we discovered that the incidence of gastrointestinal symptoms in other countries ( . %) was significantly higher than that in china ( . %). one reason may be that the gastrointestinal symptoms were not paid attention to in the early stage of the epidemic. however, once the literature was published, gastrointestinal symptoms were described in detail. regarding the mechanism of infection of the severe acute respiratory syndrome coronavirus (sars-cov- ), it is currently believed that the major determinant of sars-cov- infection is the s protein, which binds to membrane receptors on host cells and mediates the fusion of the virus and cell membrane. angiotensin converting enzyme (ace ) is a homolog of ace and one of the important receptors on the cell membrane of host cells. the interaction between the s protein and ace promotes the invasion of host cells by sars-cov- . the structure of the sars-cov- s protein is highly similar to that of the sars coronavirus (sars-cov) s protein; however, sars-cov- s protein binds to ace with a higher affinity than the sars-cov s protein, indicating that sars-cov- possesses a stronger invasion ability . ace can control intestinal inflammation and diarrhea, and the interaction between sars-cov- and ace may lead to diarrhea . ace is highly expressed in the small intestine, especially in the proximal and distal intestinal epithelial cells; therefore, the small intestine is more vulnerable to sars-cov- infection. previous investigations may have underestimated the incidence of diarrhea among those infected with sars-cov- . further research is needed to determine whether diarrhea has diagnostic value for sars-cov- . in case of the middle east respiratory syndrome coronavirus (mers-cov), which is highly homologous to sars-cov- , it is believed that the intestinal tract is another route of infection and the incidence rate of diarrhea is - % . pathological examination. till date, there have been no endoscopic and pathologic studies of the digestive tract in pediatric covid- cases. however, a study in adults demonstrated that there was no obvious damage to the mucosal epithelium of the esophagus, stomach, duodenum, and rectum. in the inherent layers of the stomach, duodenum, and rectum, a large number of infiltrating plasma cells and lymphocytes were seen accompanied by interstitial edema. ace , the virus host receptor, is mainly found in the cytoplasm of gastro- positive rate and significance of fecal nucleic acids. in a recent study on hospitalized adult patients in china, the feces of . % of the patients were positive for the viral rna, the duration for which positive fecal results were obtained ranged from to days, and . % of the patients were still fecal nucleic acid-positive after being confirmed respiratory nucleic acid-negative. compared with adults, the present study found that the nucleic acid positivity rate of feces in children was higher ( . %). a study reported that among patients with covid- in hong kong, ( . %) had gastrointestinal symptoms and nine ( . %) had positive stool viral rna test results. the detection rates of fecal viral rna were . % and . % in people with and without diarrhea, respectively . at present, there is no relevant study on whether there is a difference in the positive rate of fecal nucleic acid testing in covid- children with and without diarrhea. in a recent study conducted from january , to february , , the chinese cdc reported pediatric covid- patients (including confirmed and suspected cases), of whom were asymptomatic ( . %) . however, a recent study from new york claimed that ( . %) out of pregnant women who tested positive for sars-cov- on admission did not have symptoms of covid- at the time of treatment. this is very worrying data, because it shows that there are more asymptomatic than symptomatic patients; therefore, controlling asymptomatic patients is the key to controlling the pandemic. in children with asymptomatic covid- , there is no relevant study on whether the nucleic acid sensitivity of respiratory specimens is higher than that of feces. furthermore, it remains unknown whether the children in whom the symptoms have resolved and respiratory tract specimens are negative while the stool samples remain positive for viral nucleic acids, are asymptomatic infectious sources. consequently, it is important to recommend that after recovery and discharge, pediatric patients be isolated at home for more than weeks. in terms of prognosis, a retrospective comparative study was carried out in patients over years old in the united states . the experimental group included patients with fever and cough due to covid- , and the control group included patients with fever and cough attributable to a common respiratory tract infection. the incidence of gastrointestinal symptoms in the two groups was . % and . %, respectively (p = . ). in the patients with covid- , the course of gastrointestinal symptoms was longer, but the mortality rate and rate of severe disease were lower in patients with gastrointestinal symptoms than in those without such symptoms. at present, there is no prognostic study on children with covid- . prevention and treatment. transmission through respiratory droplets and contact are currently considered to be the main routes of transmission of covid- . nevertheless, there is now increasing evidence of fecal-oral transmission . in clinical practice, doctors mostly pay attention to the manifestations of respiratory infection in children with covid- such as fever, cough, fatigue, etc. for patients in the gastroenterology department who have no respiratory symptoms, it is recommended to adopt the appointment system and timedivision diagnosis and treatment to reduce patient aggregation and avoid cross infections. the clinic should be well-ventilated and disinfection of the clinic should be performed daily at the beginning and end of the clinic. although gastrointestinal symptoms are often ignored, in children with diarrhea, abdominal pain, nausea, vomiting, and other gastrointestinal symptoms accompanied by a low fever, attention should be paid to their epidemiological history with screening of suspected patients. nucleic acid examination should be performed using throat swabs and anal tests. in daily life, the risk of transmission can be reduced by good hygiene practices, such as washing hands frequently and closing the toilet lid when flushing. at present, there is no specific drug for covid- . plasma therapy from convalescent patients is considered for those with severe disease ; however, this treatment is controversial . dexamethasone has now been proven to be a good treatment option for the covid- . diarrhea in covid- patients is mostly self-limiting, and symptomatic treatment such as montmorillonite powder can be used. for critically ill patients, intestinal microecological regulators may be used to maintain the balance of the intestinal flora and prevent secondary infection by intestinal bacterial translocation. the number of studies included in the meta-analysis was relatively small, with a relatively large proportion of case reports. most studies did not report on the duration of the gastrointestinal symptoms preceding the presentation. additionally, the number of patients was relatively small and the description of the gastrointestinal tract of children in the included studies was not sufficiently detailed. the heterogeneity is large and subgroup analysis can not find the source of heterogeneity, which will affect the accuracy of the results. therefore, it is necessary to conduct a large-scale double-blind randomized controlled study and include additional research factors such as stool frequency, stool characteristics, number of patients with gastrointestinal symptoms and positive fecal nucleic acid test results, length of hospitalization of fecal nucleic acid-positive patients, severity of illness, and the interrelation between respiratory tract sample nucleic acid and stool nucleic acid findings. gastrointestinal symptoms in pediatric covid- are relatively common. 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missing link in novel coronavirus human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus evidence for gastrointestinal infection of sars-cov- epidemiology of covid- among children in china universal screening for sars-cov- in women admitted for delivery gastrointestinal symptoms and coronavirus disease : a case-control study from the united states covid- : gastrointestinal manifestations and potential fecal-oral transmission effectiveness of convalescent plasma therapy in severe covid- patients the use of dexamethasone in the treatment of covid- j.w. and h.t. contributed to the study design, while h.c. and x.d. contributed to the data collection. statistical analyses and interpretation of results were performed by j.w. and h.c., whereas j.w. and h.t. drafted the manuscript and edited the language. all the authors participated in the critical revisions, and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.w.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - y yxcp authors: lim, hocheol; baek, ayoung; kim, jongwan; kim, min sung; liu, jiaxin; nam, ky-youb; yoon, jeonghyeok; no, kyoung tai title: hot spot profiles of sars-cov- and human ace receptor protein protein interaction obtained by density functional tight binding fragment molecular orbital method date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: y yxcp the prevalence of a novel β-coronavirus (sars-cov- ) was declared as a public health emergency of international concern on january and a global pandemic on march by who. the spike glycoprotein of sars-cov- is regarded as a key target for the development of vaccines and therapeutic antibodies. in order to develop anti-viral therapeutics for sars-cov- , it is crucial to find amino acid pairs that strongly attract each other at the interface of the spike glycoprotein and the human angiotensin-converting enzyme (hace ) complex. in order to find hot spot residues, the strongly attracting amino acid pairs at the protein–protein interaction (ppi) interface, we introduce a reliable inter-residue interaction energy calculation method, fmo-dftb /d/pcm/ d-spies. in addition to the sars-cov- spike glycoprotein/hace complex, the hot spot residues of sars-cov- spike glycoprotein/hace complex, sars-cov- spike glycoprotein/antibody complex, and hcov-nl spike glycoprotein/hace complex were obtained using the same fmo method. following this, a d-spies-based interaction map was constructed with hot spot residues for the hace /sars-cov- spike glycoprotein, hace /hcov-nl spike glycoprotein, and hace /sars-cov- spike glycoprotein complexes. finally, the three d-spies-based interaction maps were combined and analyzed to find the consensus hot spots among the three complexes. as a result of the analysis, two hot spots were identified between hace and the three spike proteins. in particular, e , k , g , and d of the hace receptor strongly interact with the spike proteins of coronaviruses. the d-spies-based map would provide valuable information to develop anti-viral therapeutics that inhibit ppis between the spike protein of sars-cov- and hace . domain (rbd) of s undergoes hinge-like conformational changes that transiently conceal or reveal the determinants of receptor binding . sars-cov- could possibly use angiotensin-converting enzyme (hace ), the same receptor as sars-cov and hcov-nl . since the essential function of the s protein is to penetrate host cells, it is considered as the optimal target for the prevention of cell infection. for this reason, s protein-targeted antibody-mediated neutralization has been considered as a suitable treatment for sars-cov diseases. therefore, the hot spot analysis on the interface between the rbd domain of the s subunit and the hace receptor would provide crucial information for antibody engineering and for small-molecular drug development. to investigate protein-protein interactions (ppis) between hace and the rbd domain of s subunit at the molecular level, an ab initio quantum mechanical (qm) method was introduced. this method was used to obtain the most accurate information on the ppis through analysis of the wave function obtained from the qm calculation, especially of the fragment molecular orbital (fmo) approximation method. even with the fmo method, the calculations in a biomolecular system need a huge amount of computer resources. in order to obtain results within a reasonable computation time while maintaining a certain degree of accuracy of ab initio mo, we introduced the density functional tight-binding (dftb) method, which is an efficient parameterized qm method and is expected to exhibit reasonable accuracy at a remarkably reduced computational cost . the fmo method is one of various linear-scaling methods to reduce the huge computational cost of qm calculations by the fragmentation of target molecules. the energies of fragment and their pairs are computed in the embedding electrostatic potential . recently, the fmo method has been combined with dftb, and the polarizable continuum model (pcm) was introduced to consider the effect of a solvent on a model system . pair interaction energies (pies) among the fragments of the model system from the fmo-dftb/pcm method correlate well with pies from ab initio dft fmo/pcm and with an ignition møller-plesset perturbation theory (mp ) fmo/pcm . in our earlier work, we investigated ppis between programmed cell death and its ligand pd-l using fmo-mp / pcm and the results efficiently explained the experimental site-directed mutagenesis data . in this work, to find common hot spot amino acids on the interfaces between the rbd domain and hace of the three complexes, rbd-sars-cov- /hace (twelve experimental structural data), rbd-sars-cov- / hace (four experimental structural data), and rbd-hcov-nl /hace (one experimental structural data), we performed fmo-dftb /d/pcm calculations. to visualize the interaction energy and the distance of the interacting amino acid pairs, the fmo/ d-spies analysis tool was introduced. to narrow down the hot spot region, we also performed the same calculation with rbd-sars-cov- /antibody complexes (five experimental structural data). based on the fmo/ d-spies results, we constructed d-spies-based interaction maps of the hace and rbd domains from sars-cov- , hcov-nl , and sars-cov- . in order to validate the ppi predictability of the fmo-dftb /d/pcm results, we compared them with the site-directed mutagenesis results. consequently, we summarized the fmo-dftb /d/pcm/ d-spies results as interaction maps and found the hot spot regions in rbd-sars-cov- and hace at a qm level. all experimental structures calculated in this work are summarized in table . all missing side chains were filled using prime implemented in maestro program . hydrogen atoms were added to the crystal structures at ph . and their positions were optimized with the propka function implemented in maestro program . water molecules in the crystal structures were included in the fmo calculations to explore their roles in ppis. in the fmo calculations, all n-acetylglucosamine (glcnac) residues in the rbd domains and hace were included, only rbd domains of three coronaviruses were included, and only fv domains of antibodies that bind to rbd domains were included. all fmo calculations were performed with the version feb , gamess . the two-body fmo method was applied to all calculations in this work for the fmo /dftb method; this is a recent extension of the selfconsistent-charge density functional tight-binding method and derived via a third-order expansion of the dft method . dftb calculations were performed using the ob parameter set , , and the uff-type dispersion correction (dftb /d) , . due to the exchange-repulsion term is not computed in dftb, the ex terms are all zero (see supplementary table s -s ) . the polarizable continuum model (pcm), an implicit solvation model, was employed with the explicitly expressed water molecules present in the x-ray crystal and cryo-em structures. all input files were prepared in compliance with the hybrid orbital projection (hop) scheme fragmentation . each residue and water molecule was defined as one fragment. two cysteine residues forming the disulfide bridge were defined as one fragment, and glcnac, with which the asparagine residue formed covalent bonds, was defined as one fragment. all d-spies results were generated with the reported protocol . in the protocol, we selected only pies within a specific distance ( . Å) between two fragments, which reflected the distance used for the approximate of electrostatic potential in fmo method . we considered the interaction with an pie more stable than − . kcal/mol to be significant on the basis of previous reports , , . to investigate ppis between hace and rbd domains of the three coronaviruses, we collected experimental structures summarized in table and performed fmo-dftb /d/pcm calculations for all the experimental structures. due to structural arrangements from mutations summarized in table s , we collected all available structures to consider them together. subsequently, we performed hot spot analysis using the fmo/ d-spies tool. hot spot region between hace and rbd-sars-cov- . in order to investigate the hot spot region in the rbd of the sar-cov- and hace receptor complex, we performed fmo calculations on rbd-sars-cov- /hace complexes (supplementary table s -s ). we summarized the fmo results in fig . when comparing the amino acid pairs of this study with the mutagenesis experimental results from two papers, it was confirmed that of the amino acid pairs correlated with the experimental results. the changes in the binding affinity between the proteins that form a complex by mutation can be explained by comparing the structural changes (i.e. changes in the amino acid pairs that contribute to the increase or decrease of the binding affinity) of the mutated proteins with those of the wild-type proteins. qu et al. reported that the n k/t s mutation on rbd-sars-cov- lowers the binding affinity . one complex (pdb id: d h) has the t s mutation in rbd-sars-cov- . t in wt rbd-sars-cov- attractively interacts with amino acids, y , g , n , g , f , and r , whereas s in the mutated complex attractively interacts with only amino acids, n , g , and r . wu et al. reported that the k t mutation on hace increases the binding affinity , because the k in wt hace (pdb id: ajf) interacts only with y of rbd-sars-cov- , whereas t in the mutated hace (pdb id: d g) interacts with two amino acids, y and y . the common hot spot region in rbd-sars-cov- against hace and sars-cov- antibodies. in order to narrow down the hot spot regions between hace and rbd-sars-cov- , we performed fmo calculations on four rbd-sars-cov- /antibody complexes (supplementary table s -s ). we summarized the fmo results in fig. . when comparing the amino acid pairs of this study with the previously reported results, it was confirmed that of the amino acid pairs are correlated: r /y , t /w , n /r , y /d , p /d , n /d , d /r , d /s , d /n , d /r , y /r , y /y , t /y , t /y , t /d , g /a , and y /d . in rbd-sars-cov- /m (pdb id: dd ) complex, the fmo results detected amino acid pairs, which are summarized in supplementary table s . the amino acid pairs that contributed to the stability of the complexes are well correlated with the published sitedirected mutagenesis study, in which the t mutation does not significantly affect the neutralizing activity of the antibody . the fmo results supported that t s mutation would change only minor van der waals interactions between t and hc y . in the rbd-sars-cov- /s (pdb id: nb , nb ) complex, the fmo results detected amino acid pairs, which are summarized in supplementary table s -s . the s binds to rbd-sars-cov- in different two states. the fmo results of state are detailed in supplementary table s , and those of the state are mentioned in supplementary table s -s . in the rbd-sars-cov- / the interactions between four antibodies ( r, m , s , and f g ) and rbd domain from sar-cov- are shown in the right-hand with color bars. the main hot spot region is colored in light red, and the secondary hot spot region in hace is colored in light blue, and all interactions shown in this map have attractive pie value more stable than − . kcal/mol, whose magnitudes are ignored. in order to find common hot spot amino acids in rbd-sars-cov- against hace and sars-cov- antibodies, we illustrated the fmo results with a d-spies-based map. (see fig. ). all four antibodies ( r, m , s , and f g ) and hace have two common amino acids, r and t , in rbd-sars-cov- . three of the four antibodies and hace have four common amino acids, t , g , i , and y , in rbd-sars-cov- . two of the four antibodies and hace have two common amino acids, f and q , in rbd-sars-cov- . only s and hace share four common amino acids, d , n , y , and y , in rbd-sars-cov- . only r and hace share two common amino acids, q and y , in rbd-sars-cov- . other interactions between antibodies and rbd-sars-cov- do not share interactions between hace and rbd-sars-cov- . considering the possibility of mutation prediction in viruses by the fmo methods , , the evolutionary process of sars-cov- can be performed to elude neutralization of antibody by switching the unshared interactions between the antibody and hace receptor. according to the map, there are two hot spot regions between hace and rbd-sars-cov- (see fig. ). the main hot spot region on hace consists of d , y , k , d , and several residues. the counter part of that on rbd-sars-cov- comprises r , t , t , i , y , and so on. the secondary hot spot region on hace receptor consists of d , k , and several residues. the counter part of that on rbd-sars-cov- comprises y , d , n , n , and so on. we found that sars-cov- antibodies focus on the main hot spot to block the formation of amino acid pairs between hace and rbd-sars-cov- . although the rbd of hcov-nl does not share structural homology with the rbds of sars-cov- and sars-cov- , the three viruses recognize the same hace receptor to invade host cells. in order to investigate the hot spot region between hcov-nl and hace , we performed fmo calculations on the hcov-nl /hace complex (pdb id: kbh). the fmo results in which amino acid pairs were detected are summarized in supplementary table s . the fmo results were in agreement with the six amino acid pairs (hace /rbd-hcov-nl ) previously reported by wu et al. : d /s , h /g , h /s , e /y , m /h , and g /g . in order to find amino acids in hot spot regions in the ppi interface between sars-cov- and hace , we performed fmo calculations on four sars-cov- /hace complexes (supplementary table s to investigate the common hot spot region on hace against rbds from the three viruses, and vice versa, we illustrated the fmo results in fig. . in the three viruses, all rbds have common interactions with d , k , e , k , g , and d in hace . sars-cov- and sars-cov- have common interactions with the s , q , f , e , a , d , y , q , y , e , n , and r in hace . only sars-cov- and hace share interactions with e , a , f , t , q , g , and f , whereas only nl -cov and hace share interactions with n , m , and f . the common interactions between sars-cov and nl -cov were h and r in hace . we created a d-spies based interaction map to find the hot spot regions from the ppi information between hace and rbd-sars-cov- (see figs. and ) . when comparing the interacting residues between hace and rbd of the three viruses, there are two hot spot regions consisting of shallow grooves on the hace receptor. the main hot spot is formed by e , k , g and d . the secondary hot spot consists of d and k . according to the map, the main hot spot is expected to be the most important hot spot between hace and rbd-sars-cov- . we observed that the secondary hot spot on hace has interactions with k , l , e , p , and q in rbd-sars-cov- , whereas the main hot spot has interactions with r , f , q , t , n , g , y , and q in rbd-sars-cov- . the results from the common hot spot region in sars-cov- antibodies supported the results that the main hot spot region was important for the ppi between rbd-sars-cov- and its antibodies. in the results of sars-cov- and its antibody (b ) summarized in supplementary table s , the antibody had interactions with r , q , n , g , and y of rbd-sars-cov- , which are the counterpart of the main hot spot. it can be used to develop antibodies and antiviral agents by using the information of the hot spot regions suggested in this work. even though the fmo method was successfully applied to evaluate ppis, analysis of biomolecular systems still requires huge computational costs. here, we combined parameterized quantum chemical approaches (fmo-dftb /d/pcm) and the d-scattered pair interaction energies ( d-spies) protocol to analyze ppis between sars-cov- and hace complex. the fmo-dftb /d/pcm/ d-spies results also showed a qualitative scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ www.nature.com/scientificreports/ correlation with site-directed mutagenesis results, such as the fmo-mp /pcm/ d-spies results in our earlier work . the reliable inter-residue interaction energy calculation method, fmo-dftb /d/pcm/ d-spies, would be a powerful tool for drug discovery and protein engineering in the future. furthermore, the quantum-mechanical-level hot spot analysis results will provide new directions for antibody engineering and small-molecule development. the d-spies-based map would provide valuable information for the discovery of anti-viral therapeutics that inhibit ppis between the spike protein of sars-cov- and hace . a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical features of patients infected with novel coronavirus in wuhan cryo-em structure of the -ncov spike in the prefusion conformation the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak-an update on the status structure, function, and evolution of coronavirus spike proteins the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex dftb : extension of the self-consistent-charge density-functional tight-binding method (scc-dftb) fragment molecular orbital method: an approximate computational method for large molecules the fragment molecular orbital method combined with density-functional tight-binding and the polarizable continuum model investigation of protein-protein interactions and hot spot region between pd- and pd-l by fragment molecular orbital method on the role of the crystal environment in determining protein side-chain conformations propka : consistent treatment of internal and surface residues in empirical p k a predictions gamess as a free quantum-mechanical platform for drug research parametrization and benchmark of dftb for organic molecules parameterization of dftb / ob for sulfur and phosphorus for chemical and biological applications an efficient a posteriori treatment for dispersion interaction in density-functional-based tight binding uff, a full periodic table force field for molecular mechanics and molecular dynamics simulations fragment molecular orbital method: application to polypeptides fragment molecular orbital method: use of approximate electrostatic potential the fragment molecular orbital method reveals new insight into the chemical nature of gpcr-ligand interactions exploring chemistry with the fragment molecular orbital method a virus-binding hot spot on human angiotensin-converting enzyme is critical for binding of two different coronaviruses receptor and viral determinants of sars-coronavirus adaptation to human ace identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, r structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody possibility of mutation prediction of influenza hemagglutinin by combination of hemadsorption experiment and quantum chemical calculation for antibody binding prediction of probable mutations in influenza virus hemagglutinin protein based on large-scale ab initio fragment molecular orbital calculations crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor h.l. a.b., and j.k. contributed equally to this work. m.k. and j.l. supported this work by collecting mutation data. all authors contributed to writing the manuscript and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to k.t.n.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -hbovh s authors: alsved, malin; widell, anders; dahlin, henrik; karlson, sara; medstrand, patrik; löndahl, jakob title: aerosolization and recovery of viable murine norovirus in an experimental setup date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: hbovh s noroviruses are the major cause for viral acute gastroenteritis in the world. despite the existing infection prevention strategies in hospitals, the disease continues to spread and causes extensive and numerous outbreaks. hence, there is a need to investigate the possibility of airborne transmission of norovirus. in this study, we developed an experimental setup for studies on the infectivity of aerosolized murine norovirus (mnv), a model for the human norovirus. two aerosol generation principles were evaluated: bubble bursting, a common natural aerosolization mechanism, and nebulization, a common aerosolization technique in laboratory studies. the aerosolization setup was characterized by physical and viral dilution factors, generated aerosol particle size distributions, and the viral infectivity after aerosolization. we found a lower physical dilution factor when using the nebulization generator than with the bubble bursting generator. the viral dilution factor of the system was higher than the physical dilution; however, when comparing the physical and viral dilution factors, bubble bursting generation was more efficient. the infectivity per virus was similar using either generation principle, suggesting that the generation itself had a minor impact on mnv infectivity and that instead, the effect of drying in air could be a major reason for infectivity losses. the hypothesis that the human norovirus (nov) to some extent can be spread through air is gaining more support as recent studies detected nov rna in air during outbreaks in hospitals , close to symptomatic patients , and after simulated floor cleaning procedures . possible sources of airborne nov in real-life settings are bubble bursting during vomiting and toilet flushing , or resuspension from contaminated surfaces . our recent findings showing that aerosol particles smaller than µm in diameter contain detectable amounts of nov rna indicate that viruses can travel longer distances than splashing droplets ( m) and also remain airborne for longer periods of time . today, there are only scarce estimates of the importance of airborne nov contribution to the overall transmission of the disease . a key knowledge gap concerns the ability of viruses to preserve their infectivity in air under different conditions. improved knowledge of this issue is necessary to establish effective measures for disease control. in order to address this issue, information on parameters that influence infectivity of airborne noroviruses is crucial. however, the infectivity of airborne human noroviruses has not been possible to assess in real-world environments, nor in laboratory experiments, since there is no well-established cell culturing system for these viruses working at the low concentrations obtained in air samples . to be able to study nov viability, the cultivable murine norovirus (mnv) is frequently used as a model , . moreover, mnv infections are a common problem in mice in laboratory animal facilities . evaluations of viral infectivity after aerosolization have been performed in previous studies , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . yet, many of these studies lack a detailed representation of the generated aerosol, such as size characterization from nanometer to micrometer sized aerosol particles, physical dilution factor (or spray factor) of the setup, and the effect of drying on particle size . the aerosol particle size is important since it influences the residence time in air, the amount of protective material surrounding the viruses, and in some cases also the severity of the infection in the host . several studies report detection of viruses in airborne sub-micrometer particles , [ ] [ ] [ ] . these smaller aerosol particles may be of special importance in disease transmission as they may remain airborne for long periods of time (hours) and easily deposit in the respiratory tract during inhalation. thus, there is a need for more research targeting such small aerosol particles. we developed an experimental setup for aerosolization and collection of mnv with subsequent infectivity assessment in cell cultures (fig. ) . mnv was aerosolized through either bubble bursting (by the slag) or twin fluid nebulization (by the atomizer). the generated liquid aerosol droplets were dried to droplet nuclei particles in the air, similar to aerosol droplets formed in real-world settings in air at regular relative humidity (rh < %). the mnv containing aerosol was collected in a liquid impinger and analyzed for infectivity by inoculation of permissive cell cultures. detection of intracellular nsrna by strand specific quantitative reverse transcription (qrt) pcr in these cell cultures was used as a measure of virus infectivity since the nsrna is specific for replication. the viral dilution factor was determined by qrt-pcr targeting the mnv genome -the positive sense rna (psrna) -in the starting liquid and the collection liquid. the physical dilution factor of the aerosolization setup was evaluated using a radioactive tracer ( m technetium). black arrows represent the aerosol flow, and the circled 'p's indicates where pressurized air was connected to the aerosol generators. the aerosol was dried in a diffusion dryer before entering the flow tube, where it was diluted with particle-free air. at the end of the flow tube, the aerosol particle concentration was monitored with a scanning mobility particle sizer (smps) and an aerodynamic particle sizer (aps), and collected into a biosampler impinger for analysis. all ports for excess air at the end of the flow tube were connected to hepa filters. the aerosol generation mechanisms for the slag and the atomizer are outlined in b). the collection liquid in the biosampler was analyzed by a viral infectivity assay and by viral rna quantification, outlined in c). slag: sparging liquid aerosol generator; hepa filter: high efficiency particulate air filter; nsrna: negative sense rna; psrna: positive sense rna; mnv: murine norovirus; qrt-pcr: quantitative reverse transcriptase polymerase chain reaction. physical and viral dilution factors of the system. the radioactive nuclei m tc was used as a tracer molecule in the aerosolized solution to allow for high-resolution determination of the dilution factor from starting liquid to collection liquid in the setup. the collection of aerosol generated by the slag resulted in a physical dilution factor of . log , three times higher than the dilution factor for the atomizer, which was . log (mean of triplicate runs). aerosolization of m tc was also used to evaluate the collection efficiency of the biosampler for the generated aerosols by comparison with collection on filters. relative to the particle filters, the biosampler collected % and % of the particle volume generated by the slag and the atomizer, respectively. the lower collection efficiency of the slag aerosol can be explained by the larger amount of particle mass below . µm (fig. b ) that is not efficiently collected by the biosampler . this difference in particle size can also explain the higher physical dilution factor observed for the slag aerosol than for the atomizer aerosol. the viral dilution factor was determined as the psmnv concentrations in the start solution compared to the collection solution. for both aerosol generators, the viral dilution factors were higher than the physical. however, the difference was threefold for the slag aerosol while eightfold for the atomizer aerosol. taken together, as compared to the atomizer, a smaller amount of slag aerosol is collected by the biosampler (i.e., higher physical dilution), but these particles contain more mnv psrna copies (lower viral dilution relative the physical dilution). negative sense rna detection in infected raw . cells. detection of nsrna by qrt-pcr in cell homogenates was performed with a limit of detection of rna copies per reaction (cycle threshold; ct ~ ). both the positive and negative strand specific qrt-pcr assays were run with their respective opposite sense rna standards to determine the concentrations needed to avoid false priming amplification. using the the corresponding ct-value for falsely detected nsrna using the positive sense primers was for an nsrna concentration of × copies. as noted by vashist et al. , unspecific priming could occur from the rt enzyme itself, which showed up in our analysis as a secondary melting temperature in the melting curve in samples with low template rna. thus, samples without the correct melting temperature were discarded and a cut-off at ct was used. optimization of incubation time for negative sense rna detection. optimal incubation time for nsrna detection inside the infected cells was evaluated by a time series analysis of the nsrna concentrations. the nsrna concentration increased with longer incubation time up to h (fig. a) . a maximum nsrna was reached at h for the wells inoculated with the highest viral load due to rapid cell death (verified by microscopy). the cells inoculated with the lowest viral concentration ( dilution factor) displayed nsrna quantities close to or below the limit of detection after and h incubation. therefore, the incubation times h and h were evaluated in a second time series experiment (fig. b) . however, h also resulted in undetectable nsrna values with the diluted inocula and h resulted in decreased nsrna concentrations for all dilutions compared to h. thus, h was chosen as the optimal incubation time for the low infectivity aerosol samples. regression lines were fitted to the same data as in fig. a , for evaluation of the relation between nsrna quantities and the viral inoculation concentration after the different incubation times (fig. ) . here, the samples that had already reached maximum nsrna quantities ( after h, and and after h) due to cell death were excluded from the fit. significant correlations between nsrna quantity and viral inoculation concentration infectivity of aerosolized murine norovirus. virus infectivity after aerosolization and collection were evaluated by cell inoculations with serially diluted aerosol samples. each inoculated cell culture was assessed qualitatively for infectivity by detection of intracellular nsrna, and was then used to calculate the % tissue culture infectious dose (tcid ) ml − (fig. ). the tcid ml − values were similar for the mnv from both generators, which agrees well because the genome concentrations (psrna) were also analogous. the . log viral dilution factor, together with the viral infectivity loss, resulted in a log factor reduction in tcid ml − . the viral infectivity loss per viral genome copy was, hence, reduced a factor of ~ log for both aerosol generators. understanding the mechanisms by which viral diseases can be transmitted through air is of importance for the development of accurate infection prevention guidelines and practices. noroviruses, which traditionally have not been considered to transmit via air, have nevertheless been detected in air samples , , . this gives reason to study the ability of noroviruses to maintain infectivity after aerosolization. in this study, we developed a methodology for studying infectivity of aerosolized mnv, a model virus for human norovirus. we observed that viable virus could be transferred but that the infectivity levels were reduced by two orders of magnitude after airborne transport. the aerosolization setup developed in this study has similarities with previously described systems , , , , [ ] [ ] [ ] [ ] , however our characterization of the setup is extensive and contributes detailed information on system performance. we also evaluated two aerosol generators for usage in aerosolization studies on viruses, since their aerosolization mechanisms are physically different: the slag simulates natural bubble bursting, while the collison type atomizer utilizes twin-fluid nebulization frequently used in laboratory studies. in addition, we used a radioactive tracer to obtain the physical dilution factor with higher precision than with common chemical tracers, and an alternative method for validation of infected cell cultures using negative strand specific qrt-pcr. previous studies have focused either on the physical dilution factor , or the viral genome dilution factor (together with infectivity reduction) , , . here we compared these two, and found that the physical dilution factors for both aerosol generators were lower than the viral dilution factors (fig. ) . both mnv and radioactive tracer were mixed in the same starting solution and thus, the observed disparity could be due to an uneven distribution of mnv in the droplets. as the atomizer is based on shear forces, supposedly both the radioactive molecule and the virus should be evenly distributed in the liquid, and hence, also in the generated aerosol droplets, as has been shown by pan et al. . another reason could be due to losses of mnv that stick to the biosampler glass container surfaces, while m tc molecules to a higher degree remain dissolved in the sampling liquid. compared to the atomizer, the slag aerosol was collected less efficiently by the biosampler due to the generation of fewer large particles (fig. a) . this was also demonstrated by a larger physical dilution of the radioactive tracer (fig. ) . nevertheless, the viral dilution factors for both generators were similar. thus, the bubble bursting aerosolization appears to be more efficient than the atomizer in generating virus-containing particles that are collected. presumably, a higher concentration of mnv in larger droplets could result in this. the physics and fluid mechanics behind bubble bursting in non-clean water have recently been investigated by poulain and bourouiba , showing that surfactants released from microorganisms may enhance aerosol droplet formation www.nature.com/scientificreports/ upon bubble bursting. if the same mechanisms can explain enhanced virus aerosolization from bubble bursting remains to be explored. the slag has only been used for aerosolization of influenza virus in one previous study , but then no infectious virus was detected in the collected samples. this may be due to a lower emission rate of the slag compared to most atomizers. in our study, the atomizer generated approximately the same amount of aerosol at an airflow rate of l min − as the slag at l min − . the mnv (about nm in diameter) is at least one order of magnitude smaller than the generated aerosol droplets and do seemingly not influence the droplet size distribution , . the majority of the droplets generated here were smaller than µm. we have shown in a previous study that human norovirus can be present in sub-micrometer particles in hospital wards during outbreaks . hence, there is a need to also include also these smaller particles in studies on viral aerosolization. considering that the majority of bioaerosol particles in the environment are - µm in diameter , most aerosol samplers have their highest collection efficiencies in that size range. however, viruses have been found in sub-micrometer particles both in the environment and in laboratory studies , , , , , , thus, a higher collection efficiency for small particles would greatly improve studies on aerosolized viruses. a recently developed technology for bioaerosol collection based on condensational growth has been shown to have > % collection efficiency for sub-micrometer aerosol particles, which would give a more representable result as viruses in all particle sizes are included in the downstream analysis . the condensational growth collector has a liquid collection volume that is ten times smaller than that of the biosampler, which would increase the virus concentration in the collected sample. this up-concentration would be advantageous for further studies on virus infectivity using the setup, because the infectivity detection assay presented here was close to the detection limit. the decreased mnv infectivity after aerosolization in this setup was reduced almost to the same amount (comparing infectivity/genome copy number) by the slag and the atomizer, which suggests that for this mnv, the aerosolization mechanism had little impact on infectivity. the atomizer recirculated the liquid, which is known to degrade biological cell membranes and cause water to evaporate . previous studies also did not find any loss of viability in mnv after min recirculation in a single-jet atomizer , and in coronavirus after recirculation in a collison nebulizer , even though coronaviruses are enveloped viruses. instead, it has been shown that drying can be detrimental for non-enveloped viruses . moreover, viruses aerosolized from liquids with low-solute concentrations showed reduced infectivity . these two parameters, drying and low-solute solution, could explain the difference in mnv infectivity observed here and in the previous study by bonifait et al. . bonifait et al. used an aerosol generator that produced droplets around . µm in diameter, which is about times larger in diameter (> times larger volume) than the droplets produced in this study. they also aerosolized mnv in virus growth medium, while in our study, mnv in growth media was diluted : in pbs (due to foaming in the slag). drying of mnv in laboratory settings has also been performed on surfaces , . these studies found that mnv dried in larger droplets ( - µl) or at higher relative humidity was more likely to be inactivated, and it was hypothesized that the drying time, rather than the fact that water was removed, was the likely reason for mnv inactivation . these results taken together, suggest that mnv is likely to remain infectious if dried fast (however, not completely) in a solution with protective molecules (proteins and sugars). samples collected from aerosols often exhibit low viral concentrations and therefore require a method with high sensitivity for infectivity detection. a previous study developed a method for molecular detection of an infection-specific molecule for mnv: the negative strand rna (nsrna), which is the complementary strand to the viral genome . the nsrna is produced inside cells exclusively during ongoing replication, thereby serving as a qualitative marker for infection. this method can result in fewer false negatives than the conventional observation of the cytopathic effect (cpe) in cell cultures, since the sensitivity of qrt-pcr is high. other advantages are that the molecular analysis is objective, and that it does not require personnel with long experience of cell work. however, using nsrna detection as a qualitative determiner still requires inoculation with serial dilutions to obtain a quantitative value of infectivity, such as the tcid . the risk for false priming of the opposite sense rna in the qpcr step was investigated by adding psrna to the nsrna assay (primers for nsrna), and vice versa. since we needed to add a high psrna concentration of × copies per reaction to reach a borderline positivity (ct value of ), the specificity of the key nsrna assay is sufficiently high. in addition, vashist et al. showed that including both negative and positive sense rna in the rt step for cdna synthesis did not alter the qpcr results for either set of primers. unspecific priming of the rt enzyme occurred only when there was no amount or too low amounts of rna template and could be identified by lower melt temperatures; hence, samples with aberrant melting temperatures were not included in the analysis. santiana et al. showed that mnv replication occur mainly within h of incubation, while % of the cells were still intact . most of the cell membrane damages took place after h and during this period mnv replication was limited. this pattern is in agreement with our results that show the highest nsrna concentrations around h, and then decreasing amounts due to cell death (fig. ). in addition, the time series analysis can be used for estimates of the infectivity of samples by simply harvesting cells at h, purify and amplify nsrna and plot the nsrna ct values against a standard curve. this would simplify comparisons in future studies of incremental changes in aerosol parameters (humidity, temperature, etc.). to observe a cpe in cell cultures, an incubation time of h was needed. however, after h, many cells were destroyed and the intracellular nsrna was degraded. as the infectivity was strongly reduced after aerosolization, the nsrna detection after h was chosen instead of cpe observation after h. the experimental setup described here highlights some difficulties in studies on aerosolized viruses: ( ) the lack of standards in how to generate bioaerosol that results in significant differences in aerosol particle size and concentration, ( ) the necessity to determine both physical and viral dilution factors, and ( ) www.nature.com/scientificreports/ during airborne transport due to the low-solute solution and dilution in the setup. by thorough characterization and description of the system performance and the aerosol characteristics, we provide new information on submicrometer aerosol particle generation and the effects on mnv infectivity. in addition, our results suggest that virus aerosolization by bubble bursting may be advantageous. the aerosolization setup developed in this study allows for further analysis of how the infectivity of airborne mnv is affected by factors in the environment, such as temperature, relative humidity and aerosol particle size. this may lead to a deeper understanding of seasonal and regional differences in virus transmission and offer better tools to minimize the spread of viral aerosols, and thus, prevent spreading of diseases. methods experimental aerosolization setup. an experimental setup for aerosolization and collection of viruses was developed (fig. ) , where either a collison type atomizer (constant output atomizer, model , tsi inc.) or a slag (sparging liquid aerosol generator, ch technologies) was used as the aerosol generator. the atomizer generated droplets by using a high perpendicular airflow to break up a thin liquid column (see schematic in fig. , lower left panel) . the largest droplets impacted on the wall inside the generator and were recirculated. in the slag, the solution to be aerosolized was dropped onto a porous stainless steel plate at a flow rate of . ml min − delivered by a syringe pump. at the same time, air was blown through the porous plate from below, bubbling through the liquid film of the mnv suspension, generating droplets though bubble bursting (see schematic in fig. , lower left panel) . the atomizer was operated at . bar and a l min − output airflow rate, and the slag at a l min − output flow rate. the generated aerosols were dried in a silica diffusion dryer (ddu , topas gmbh) and then mixed with a perpendicular dilution flow in a stainless steel flow tube to a total airflow rate of l min − . at this flow rate the particles were airborne about s before collection at the end of the flow tube. aerosol particles were collected by a biosampler impinger (skc inc.), with ml pbs as collection liquid, at an airflow rate of . l min − . aerosol particle size characterization. the characterization of the initial droplet sizes of the generated aerosol was performed using a % ammonium sulphate solution, which was chosen because it forms spherical particles when dried . using the measured dry salt aerosol particles d dry , the originally generated droplet diameters d wet were calculated with eqs. and : where m wet is the mass of the wet droplet, m dry is the mass of the dry particle, f salt is the mass fraction of salt in the solution, and ρ dry and ρ wet are the densities for dry particles and wet droplets. the wet droplet diameters range was . - µm, with a gap between . and . µm where the instrumental measurements did not overlap. physical dilution factor by radioactive tracer. the physical dilution factor of the aerosolization setup was determined by aerosolization of radioactive m tc in a solution of pbs and % growth media containing mnv (same solution properties as those used for mnv aerosolization). three runs of min aerosolization were performed with each aerosol generator and triplicate ( ml) aliquots were used for the analysis. the same start solution was used for both generators. gamma-ray spectroscopy was used to measure the radioactivity from m tc by means of a sodium iodine well count detector ( wizard, perkin elmer). the gamma radiation emitted from the start solution was then compared to that of the collection liquid. in addition, pbs was analyzed as a reference. the measuring time for each sample was set to s, and the results were adjusted for radioactive decay during the measurement time. the instrument automatically corrected for background activity. all samples were below the max count rate of million counts per minute, where the dead time error is < %. the collection efficiencies of the biosampler for the two generated aerosols were evaluated by parallel collection of radioactive aerosol particles on a mm ha filter with . µm pore size (millipore). the filter sampled close to % of all particles at a flow rate of l min − (data not shown). filters were placed in sample tubes (same as for liquid samples) with a pincer and analyzed together with the liquid samples by gamma-ray spectroscopy. for comparison, the radioactivity from the biosampler and the filter were normalized by their respective sampling airflow. aerosolization of murine noroviruses. the mnv stock solution in growth media was diluted : in pbs to decrease foaming due to high viscosity (primarily problematic in the slag) before aerosolization. the start solution was inserted into the atomizer flask or the syringe connected to the slag, and aerosolization with collection into the biosampler was run for min. aerosolization was performed in triplicates for each aerosol generator. samples were taken from the start solution before the experiment and from the collection liquid in the biosampler after the experiment. samples were stored in − °c freezer until analysis was performed, either by the mnv infectivity assay or by qrt-pcr for quantification (viral dilution factor). cells were grown in ml media at °c and % co . the cell culture was split three times per week by gently scraping cells off the flask bottom, and transferring / of the cells to fresh growth media in a new flask. the cell concentration before every split was determined by trypan blue staining ( : ) and cell counting (eve automated cell counter, nanoentek). infectivity analysis of mnv. prior to viral inoculations in -well plates, , freshly split cells were added per well (in µl media) and allowed to grow overnight to a monolayer. after the incubation, the supernatant from each well was removed and the cells were washed with pbs. viral tenfold dilution series in pbs were prepared and µl were added to each well and incubated for h, and were then replaced by fresh growth medium and incubated for the assigned incubation time ( h for aerosol samples). after incubation, the supernatant was removed and the cells were washed with pbs, and thereafter lysed with µl rlt lysis buffer (rneasy mini kit, qiagen, inc.) and all samples were stored at − °c. intracellular rna was extracted from the samples using the rneasy mini kit (qiagen, inc.) and then treated with dnase (thermo fisher scientific) for min at °c, according to the protocol of the manufacturer. extracted rna samples were stored at − °c. strand specific quantitative reverse transcription pcr. during replication of the genomic psrna, mnv produces an intermediate stage of nsrna that is used for generating multiple copies of psrna. the number of nsrna copies is usually orders of magnitudes lower than the number of psrna copies. hence, detection of the nsrna, unique for viral replication, calls for a specific reverse transcription step starting from a primer that is uniquely hybridizing to the negative strand. for mnv, such a strand-specific method has been described by vashist et al. and shown to be specific. we have used this methodology here (as well as in previous studies ). in short, the transcription primer targeting the nsrna has an added non-viral oligonucleotide sequence in its ′-end. in the subsequent qpcr reaction, a primer identical to the non-viral oligonucleotide tag is used in order to only amplify what was transcribed using the tagged primers. a similar reverse tagged transcription step was used to target the more abundant psrna. both negative and positive strand transcriptions were conducted with superscript iv in a thermal cycler (applied biosystems ) according to previously developed methodology , . these samples containing cdna transcripts were stored at − °c until quantification by qpcr. sybr select master mix (thermo fisher scientific) was used for qpcr in µl reactions using a steponeplus real-time pcr system (applied biosystems). for relative quantification, serially diluted positive sense and negative sense cdna respectively, with a concentration of . × copies µl − were included in the qpcr setup. the data was analyzed in stepone software v . . determining the tcid of the aerosolized samples. detection of nsrna by qrt-pcr was used as a qualitative measure of infection in each well sample and used to calculate the tcid ml − . the tcid was determined per aerosolization run (three per aerosol generator) based on triplicate wells per dilution, and the mean and standard deviation of these were used for comparisons. the tcid ml − was correspondingly determined for the start solution. time series analysis of intracellular negative sense rna. to find the optimal incubation time for viral samples with low concentrations (i.e., the collected aerosol samples) a time series analysis was performed to evaluate the nsrna concentration at various incubation times. the mnv stock solution was diluted in growth media in tenfold dilution series, ranging from zero to times dilution. three -well plates were inoculated with viral solution (duplicate wells) as described above. after the inoculation, the plates were incubated with fresh media for , and h, respectively. after the assigned incubation time, the supernatant was removed and the cells were lysed as above. the plates were stored at − °c until rna extraction and further analysis by qrt-pcr for detection and quantification of nsrna, as described above. a second time series analysis experiment was performed with the incubation times , , and h. statistics. aerosolization and collection were performed in triplicate runs for each aerosol generator, and the mean and standard deviation for each set of triplicate runs were calculated. the dilution factors were calculated by dividing the mean value of the start liquid concentration by the mean value of the collection liquid concentration. triplicate aliquots were extracted from the collection liquids for the m tc analysis, as well as for the psrna quantification, and the means of those were used to represent one sample. the qpcr analysis was performed using duplicate reactions and the mean of those was used. linear regression analysis using the anova test on the log-transformed data was used to compare the quantities of nsrna with inoculation dilutions in fig. . detection and quantification of airborne norovirus during outbreaks in healthcare facilities sources of airborne norovirus in hospital outbreaks norovirus recovery from floors and air after various decontamination protocols aerosol generation by modern flush toilets routes of transmission of influenza a h n , sars cov, and norovirus in air cabin: comparative analyses replication of human noroviruses in stem cell-derived human enteroids surrogates for the study of norovirus stability and inactivation in the environment: a 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particles consisting of ammonium sulfate, adipic and humic acid mixtures determination of % endpoint titer using a simple formula robert lange is recognized for his contribution to the aerosol particle size measurements and calculations using the experimental setup. great thanks is given to per wollmer, birgitta roos, lennart bergqvist and hanna holstein for their help and enthusiasm in enabling the radioactivity measurements. thanks also goes to tobias svensson for his contributions in the construction of the flow tube. the schematic illustration in the authors declare no competing interests. correspondence and requests for materials should be addressed to j.l.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -gb m fue authors: altschul, david j.; unda, santiago r.; benton, joshua; de la garza ramos, rafael; cezayirli, phillip; mehler, mark; eskandar, emad n. title: a novel severity score to predict inpatient mortality in covid- patients date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: gb m fue covid- is commonly mild and self-limiting, but in a considerable portion of patients the disease is severe and fatal. determining which patients are at high risk of severe illness or mortality is essential for appropriate clinical decision making. we propose a novel severity score specifically for covid- to help predict disease severity and mortality. patients with confirmed sars-cov- infection were included. we derived a risk model using the first half of the cohort (n = patients) by logistic regression and bootstrapping methods. the discriminative power of the risk model was assessed by calculating the area under the receiver operating characteristic curves (auc). the severity score was validated in a second half of patients. mortality incidence was . % in the derivation cohort and . % in the validation cohort. a covid- severity score ranging from to , consisting of age, oxygen saturation, mean arterial pressure, blood urea nitrogen, c-reactive protein, and the international normalized ratio was developed. a roc curve analysis was performed in the derivation cohort achieved an auc of . ( % ci . – . ) and an auc of . ( % ci . – . ) in the validation cohort. furthermore, based on the risk categorization the probability of mortality was . %, % and % for patient with low ( – ), moderate ( – ) and high ( – ) covid- severity score. this developed and validated novel covid- severity score will aid physicians in predicting mortality during surge periods. the first confirmed case of covid- in new york city was on march st, . within a few short weeks all of the hospitals in the area were overwhelmed hitting a peak on april th, of , confirmed positive cases that day. as of july rd, , there have been , deaths, , hospitalizations and a total of , cases in this city . new york city is an international travel hub with a high population density, and a heavy reliance on mass transportation that provided the permissive substrate for rapid viral spread . as such, this region was one of the earliest areas in the united states to encounter the full impact of the pandemic . over these first few months much has been learned about the disease, its deadliness, and those who are at higher risk for dying. in many people the disease is mild and self-limiting, but in a considerable portion of patients the disease is severe and fatal. determining which patients are at high risk of severe illness or mortality is an essential part of understanding this illness. prior reports from wuhan identified certain comorbidities as diabetes, hypertension and coronary artery disease as patients more likely to present to their hospital . they also discovered that patients with older age, higher sequential organ failure assessment (sofa) score, and elevated d-dimers were significantly associated with inpatient mortality . further reports have shown other predictors of poor outcome such as acute kidney injury, acute hepatic injury, the need for mechanical ventilation, elevated c-reactive protein (crp), interleukin- (il- ), lymphocyte count, and procalcitonin levels [ ] [ ] [ ] [ ] . covid- is unique in its ability to not only cause sepsis, and multi-system organ failure, but also to cause a severe inflammatory response that can lead to systemic multi-vascular thrombosis , . while the sofa score is also predictive of mortality for covid- , it does not address the additional thrombotic mitigators of severe illness www.nature.com/scientificreports/ development of disseminated intravascular coagulation (dic), and now being used to help guide the use of anticoagulation for patients with covid- [ ] [ ] [ ] . we propose a novel score specifically for covid- in-hospital mortality, combining elements of both of these scores to help predict disease severity and mortality. after approval of this study by the montefiore medical center/albert einstein college of medicine institutional review board, information on demographics, comorbidities, admission laboratory values, admission medications, admission supplemental oxygen orders, discharge and mortality was identified through a healthcare surveillance software package (clinical looking glass [clg] ; streamline health, atlanta, georgia) and review of the primary medical records. the montefiore medical center/albert einstein college of medicine institutional review board approved waiver of patient informed consent due to the retrospective design of the study. to our knowledge, a description of the entire cohort of patients, as in the current manuscript, has not been reported in other submissions. in the interest of transparency, anonymized data will be made available at https ://figsh are. com/s/ c a f df b d . all methods were carried out in accordance with relevant guidelines and regulations laboratory measures were extracted by identifying those obtained-on-admission. comorbidities were identified based on the international coding disease coding system (icd- ). the comorbidities chosen for this study are those used in the charlson comorbidity index. each patient's medical record was queried for any diagnosis occurring within years of his or her index admission. we included the laboratory markers that were made part of the routine tests on admission during the period of the study in our institution, among the available markers we selected the ones that have been reported to be commonly altered accordingly to recent studies (online appendix ). this study is an observational cohort study validating a novel, simple covid- in-hospital mortality score to predict inpatient mortality risk in patients with confirmed sars-cov- infection using a combination of presentation vital signs, and basic admission laboratory values. this model was created on patients presenting from march st to april th. we used the first numeric half of patients during this period (n = ) as the "derivation cohort" in which the severity score was developed and internally validated. the second numeric half of our cohort (n = ) was used to confirm the power of the prediction score; this part of the cohort was considered the "validation cohort". inclusion criteria was defined as all patients admitted to a hospital within a large healthcare network that were positive by detection of sars-cov- rna using real-time reverse transcriptase-polymerase chain reaction (rt-pcr) assay testing, performed within the hospital system or documented at an outside system prior to transfer. patients evaluated in the emergency room but not admitted, or those that died in the emergency room, were excluded from the analysis, given the relative paucity of data. most patients had only one admission, and we only considered the last hospitalization for those that had multiple admissions during this period. continuous values were represented using mean ± standard deviation (sd), or median and interquartile range (iqr). categorical variables were described using frequencies and proportions. comparisons were performed using student's t test, the nonparametric mann-whitney test or χ tests as appropriate. no imputation was made for missing data. the primary outcome of this study was in-hospital mortality. hence, all the following statistical steps were done with in-hospital mortality as the only dependent variable. for easier application to a risk score model, when performing multiple regression analysis, most continuous variables were converted to categories based on published data as follows: advanced age (≥ years, ≥ years, and ≥ years), body mass index (< . or > . kg/m ), oxygen saturation (< %), temperature (> °c), mean arterial pressure (map < mmhg, < mmhg, < mmhg), white blood cell count (< or > , per mm ), lymphocytes (< per mm ), platelet count (≤ , per mm ), alanine aminotransferase (alt > u/l), aspartate aminotransferase (ast > u/l), ferritin (> µg/l), inr (> . ), d-dimer (> mg/ml), creatinine (> µmol/l), blood urea nitrogen (bun) (> mg/dl), glucose (< or > mg/dl), sodium (< or > mmol/l), interleukin- (il- ) (> pg/ml), c-reactive protein (crp) (> mg/l), procalcitonin (> . ng/ml), and troponin (> . ng/ml). candidate predictors with p < . in univariate analyses were included a multiple logistic regression. in addition, a backward stepwise bootstrap regression model, in which random samples patients were generated with replacement, was also performed to investigate the relative importance of each variable included in our model . frequencies of occurrence of each covariate in the final model were noted; if predictors occurred in % or more of the bootstrap models, they were retained in the final multiple regression model. beta coefficients and odds ratios (or) were calculated with % confidence intervals (ci). the multiple regression coefficients of the predictive factors were used to assign integer points for the prediction score. however, for the simplicity of the score we allocated points in sequential order for variables with multiple categories (e.g., age in years < , ≥ , ≥ , and ≥ would equal to , , and points in the score, respectively). as described in previous validation methods , we assessed the discriminative power of the prediction score by calculating the area under the receiver operating characteristic (roc) curves (auc). a predictor with an auc above . was considered to be useful, while an auc between . and . indicated good diagnostic accuracy. risk categories were determined using the classification and regression tree (cart) analysis. the cart algorithm builds decision tree based on gini's impurity index as splitting criterion; the score was iteratively subdivided to find the cut-off point that produces the greatest reduction of impurity, meaning that it measures how often a random patient that died will be incorrectly labeled as low-risk and vice versa, a patient that survived will be labeled as high-risk . calibration of the risk score reflecting the link between predicted and observed risk, was evaluated by the hosmer-lemeshow goodness of fit test. a p value < . was considered statistically significant for all analyses. data were analyzed using the stata version and ibm spss version . www.nature.com/scientificreports/ distribution of socio-demographics, comorbidities, vital signs and laboratory values between the validation and derivation cohorts are shown in table the univariate analysis showed potential predictors with a p < . ( table ). out of the candidate predictors, variables remained as independent predictors in the multiple logistic regression analysis, including age (> , > and > years), female sex, oxygen saturation < %, mean arterial pressure (map) (< , < and < mmhg), international normalized ratio (inr) > . , creatinine > µmol/l, blood urea nitrogen (bun) > mg/dl, interleukin- (il- ) > pg/ml mol/dl, c-reactive protein (crp) > , and procalcitonin > . ( table ) . the bootstrap analysis revealed that, out of the independent predictors of mortality, age, oxygen saturation, map, bun, crp, inr and procalcitonin were reproducibly selected in more than %. due to the large number of missing data for procalcitonin ( %), this variable was excluded in order to avoid noise predictors. allocation of points for the covid- severity score was made based on beta coefficients and bca %ci, however for the simplicity of the score we allocated points to in subcategorized variables (age & map) ( table ). the total prediction score ranges between and with a high score indicating high risk of in-hospital mortality. a roc curve analysis was performed in the derivation cohort (fig. ) , the novel covid- severity score achieved an auc of . ( % ci . - . ) indicating a good discrimination for patients with higher risk www.nature.com/scientificreports/ www.nature.com/scientificreports/ of in-hospital mortality. furthermore, the hosmer-lemeshow goodness of fit test of tenfold cross-validation did not reach statistical significance (p = . ) indicating a good match of predicted risk over observed risk. finally, we applied the score to the patients in the validation cohort. the roc curve analysis showed an auc of . ( % ci . - . ) still indicating a useful discrimination for our model (fig. a) . then, we determined that low risk patients ( - points) had a . % risk of mortality, moderate risk patients ( - points) had a % risk of mortality and high-risk patients (> points) had a % risk of mortality (fig. b) . www.nature.com/scientificreports/ we propose a novel scoring system to aid in the prediction of inpatient mortality for patients presenting with sars-cov- infection to hospital emergency rooms. the score is based on simple pragmatic demographic data, and presenting biomarker values. this score incorporates the unique constellation of various presentations in which covid- can manifest in severe illness. we avoided incorporating mechanical ventilation use into the score as this was tied to a clinical decision, which over time with more knowledge an approach that changed. while il- also seems to predict mortality, we avoided incorporating this biomarker, as it is a non-routine test, and were not available in a large percentage of our patient population. as of yet there are no scoring systems created that are specific to the elements of covid- illness manifestations and that can predict mortality. the limitations of this study are its retrospective design, its cohort, which is primarily a minority urban population, and the epoch at which the data was required. since the data and outcomes were recorded during the highest surge of the pandemic this may bias the results towards higher mortality as this was a great strain on treating hospitals at the time. prior reports also have shown increased mortality in racial and ethnic minority patients . given the sociodemographic background of our patient population the score may again be biased towards higher mortality risk. while the design of the study may limit its generalizability to other populations, these findings are meaningful in that they are specifically applicable to minority urban centers that are suffering from large surge populations of infected patients, which in the first wave of the pandemic across the united states of america suffered the most. the encountered mortality rate is certainly high, but most likely the result of the high comorbidity burden in our population, the fact that all of these patients had enough symptom severity to warrant admission, and the fact that the study period was early in the pandemic when there was limited understanding regarding the disease. nonetheless, given the diverse patient population of the bronx, it is possible that this score can be generalized to other large inner-city populations. future research is needed to validate this score in other populations, as well as to compare this score to the sofa and isth dics score. the health network from which this data was captured is comprised of a network of major hospitals in the bronx in new york city, one of which is a large quaternary care facility accepting transfers for complex and severely ill patients in the region beyond the bronx into westchester county. the mortality rates reported here are for hospitalized patients who tended to be older and more severely affected than others infected with the virus. hence the mortality rate for hospitalized patients is higher than the more commonly reported case-fatality rate that reflects the number of deaths per documented infection. in any case, the rates reported here are broadly comparable to mortality rates for hospitalized patients in other countries at comparable time points in their respective pandemic outbreaks: china- % , italy- % , and new york- % . the mortality rates were slightly different between the training set ( . %) and the testing set ( . %). this is likely secondary to the temporal difference between the sets. during the first weeks of the pandemic surge, there was still little known about optimal management strategies for severely ill patients. as time went on, mortality rates decreased. in addition, there was more community awareness of the potential impact of the virus and it is possible patients were more likely to seek medical attention sooner and arrived in less severe states. despite this mortality rate differences, the severity score itself remained valid. there were also variances in racial distribution between the two cohorts. despite these differences in race, the severity score remained valid in predicting in-hospital mortality. www.nature.com/scientificreports/ in other metropolitan areas outside of new york city there have been reports of racial disparity and outcome, we found no difference in mortality rates between races , . there are a number of possibilities why. the bronx is uniquely diverse in its racial and ethnic populations however also one of the poorest regions in the united states of america with median income of $ , and . % of persons living in poverty . one reason could be that other social determinants of health, including poverty level are more powerful predictors of mortality rather than race alone. while mortality prediction is neither perfect nor absolute, having a simple score to predict how severe a patient's illness and hospital course will be, can aid admitting and emergency room physician's ability to triage severity and predict prognosis during surge periods. this can also be used to guide recommendations for palliative care consultation early in a patient's hospital course. www.nature.com/scientificreports/ open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. covid- data. department of health johns hopkins coronavirus resource center clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a singlecentered, retrospective, observational study presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area kidney disease is associated with in-hospital death of patients with covid- identification and validation of a novel clinical signature to predict the prognosis in confirmed covid- patients clinical features of patients infected with novel coronavirus in wuhan china coagulopathy in covid- the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine isth) ssodicdotisotah. towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation modified score for disseminated intravascular coagulation in the critically ill isth interim guidance on recognition and management of coagulopathy in covid- development and validation of a risk prediction score for severe acute pancreatitis using classification and regression trees (cart) and random forests to analyze attrition: results from two simulations hospitalization and mortality among black patients and white patients with covid- baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region epidemiology, clinical course, and outcomes of critically ill adults with covid- in new york city: a prospective cohort study covid- outcomes, risk factors and associations by race: a comprehensive analysis using electronic health records data in michigan medicine author contributions formal analysis, investigation, writing-original draft the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to d.j.a. or s.r.u.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -ez lif authors: wada, yoshiko; wada, kennosuke; iwasaki, yuki; kanaya, shigehiko; ikemura, toshimichi title: directional and reoccurring sequence change in zoonotic rna virus genomes visualized by time-series word count date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ez lif ebolavirus, mers coronavirus and influenza virus are zoonotic rna viruses, which mutate very rapidly. viral growth depends on many host factors, but human cells may not provide the ideal growth conditions for viruses invading from nonhuman hosts. the present time-series analyses of short and long oligonucleotide compositions in these genomes showed directional changes in their composition after invasion from a nonhuman host, which are thought to recur after future invasions. in the recent west africa ebola outbreak, directional time-series changes in a wide range of oligonucleotides were observed in common for three geographic areas, and the directional changes were observed also for the recent mers coronavirus epidemics starting in the middle east. in addition, common directional changes in human influenza a viruses were observed for three subtypes, whose epidemics started independently. long oligonucleotides that showed an evident directional change observed in common for the three subtypes corresponded to some of influenza a sirnas, whose activities have been experimentally proven. predicting directional and reoccurring changes in oligonucleotide composition should become important for designing diagnostic rt-pcr primers and therapeutic oligonucleotides with long effectiveness. viruses have always posed significant threats to public health, as highlighted by the recent ebolavirus outbreak in west africa [ ] [ ] [ ] [ ] and the emerging and re-emerging nature of influenza viruses . to face the world-wide threats caused by zoonotic rna viruses, which suddenly cause serious outbreaks by invasion from nonhuman hosts and mutate rapidly, we must understand the molecular evolutionary changes in their genome sequences extensively by innovating various technologies, including those used in big data analyses, e.g., large-scale word count. time-series word count of oligonucleotides in genome sequences can be conducted without specialized assumptions and prior knowledge and is useful for unsupervised data mining. importantly the obtained results are easily understandable, even for those unfamiliar with molecular evolutionary studies. oligonucleotide composition varies significantly among species even with the same genome g+ c%; therefore, the composition, especially of short oligonucleotides, has been used as a phylogenetic parameter "genome signature" to classify microbial genomes, including viral genomes. viruses are inevitably dependent on many host factors for their growth (e.g., nucleotide pools, proteins and rnas) and must escape from host antiviral mechanisms (e.g., interferon-induced systems) [ ] [ ] [ ] . therefore, after the invasion of zoonotic viruses from nonhumans, a certain level of directional change in genomic sequences and thus in mono-and oligonucleotide compositions should occur during human-to-human transmission. in fact, the g+ c% of influenza a virus strains isolated from humans is lower than that of strains isolated from natural hosts, which were avian , and the cg dinucleotide in an a/u context has been preferentially eliminated from classical h n influenza viruses during human-to-human transmission . by using blsom (batch learning self-organizing map) , , we previously found directional compositional changes in a wide range of oligonucleotides , that occurred after the onset of the influenza h n / pandemic of [ ] [ ] [ ] . in this study, we first conducted time-series word counts on changes in mono-to pentanucleotide compositions, occurring after invasion from nonhumans, for ebolavirus, mers coronavirus and influenza viruses. then, scientific reports | : | doi: . /srep as an example of long oligonucleotides, we analyzed -mers. because -mers are composed of (ca. . trillion) variables, the analysis becomes a big data analysis. long oligonucleotides are key elements of diagnostic pcr primers and potentially promising nucleic-acid therapeutics (e.g., antisense rna, sirna and mirna) , [ ] [ ] [ ] [ ] . rna viruses mutate very rapidly, and therefore, their sequence changes should be intensively characterized for designing diagnostic and therapeutic oligonucleotides with long effectiveness. the present time-series analysis on influenza a genomes showed that the -mers exhibiting the most evident directional changes observed in common for three different subtypes corresponded to several influenza a sirnas, which were experimentally validated. time-series analyses can provide information on changes possibly reoccurring after invasions from natural reservoir hosts. spatiotemporal analysis of mononucleotide composition in ebolavirus genomes. because of the recent major threat to public health in west africa, more than genomes of zaire ebolavirus (ebov) strains, isolated from humans from to , have been sequenced, and the sequences are available from the ncbi virus variation database (http://www.ncbi.nlm.nih.gov/genome/viruses/variation/ebola/). to study molecular evolutionary changes in ebov genomes in the current epidemic, we obtained strains, for which the isolated dates are given. although we focused on full-length genomes, a minor portion was relatively short in length, or many unidentified "n" bases were included, partly because of the stringent sterilization treatment obligated by the local government . because these will give odd values of mono-and oligonucleotide compositions, we focused on genomes longer than . kb after omitting n bases, which were derived from guinea, liberia and sierra leone strains. to conduct the present spatiotemporal analysis, strains isolated from several other geographic areas were omitted. we first analyzed time-series change in mononucleotide composition for strains isolated in the three areas separately. figure a plots four mononucleotide compositions (%) in each genome according to the isolated day, which started on march , . because mutations occur primarily as random processes, mononucleotide compositions for strains isolated even around the same day clearly differed from each other, which resulted in evident compositional diversity. despite this diversity, which should also be due to sequencing uncertainty, linear regression lines appear to show a time-dependent increasing or decreasing trend, a possible increase for c% and g% and a possible decrease for a% and u%. this is consistent with the excess u-to-c mutations found by two groups , . . data and regression lines separately analyzed for different geographic areas are differentially colored: guinea (blue), liberia (brown) and sierra leone (green). (b) averaged mononucleotide compositions for strains in each month are plotted according to the elapsed months from march . data and regression lines for the elapsed months are colored as described in (a). a separate and additional regression analysis for data without the averaging (a) that can examine time-series changes from the epidemic start for all three areas showed that the null hypothesis (i.e. no correlation) was rejected for all mononucleotides in the three areas ( cases) at the significance level of . , except for t% and g% for guinea; this was rejected for the t% for guinea at the significance level of . . for data after averaging (b), the null hypothesis was rejected at the significance level of . for mononucleotides in the three areas, except for g% and t% in guinea. scientific reports | : | doi: . /srep the present study focused on time-series changes, but not on data variations caused largely by random mutations and sequencing uncertainty, and therefore, average characteristics of strains isolated in a similar period were analyzed, summing up the sequences isolated in one month and calculating an averaged mononucleotide composition for each month (fig. b) . this could be done because the genome sequence data were distributed rather evenly by area and month; the average number of strains per month was . , . and . for guinea (blue), sierra leone (green) and liberia (brown), respectively. we omitted the data for a few months, for which no more than strains were available in the respective area. figure b shows a more conspicuous increasing or decreasing trend than fig. a , and the time-series increasing or decreasing trend revealed by the regression line for the elapsed months was the same among the three areas for all nucleotides, except for an ambiguous case of g% for guinea. in more detail, positive and negative directions of the regression line were primarily common among the three areas, although the slope angle appear to differ; table lists pearson's correlation coefficients calculated separately for the three areas. the highest negative (− . ) or positive ( . ) correlation among mononucleotides was observed for a% or c%, respectively, in liberia, which corresponded to a decrease of . as or an increase of . cs per genome at the final stage of the current outbreak. notably, the increasing or decreasing trend detected by these averaged data for one month (fig. b) is common to the result without averaging (fig. a) . in these regression analyses, the strains that were isolated in the three areas were independently analyzed. a separate regression analysis that could examine the statistical significance of directional changes from the epidemic start for all three areas, was conducted, by including the data of strains isolated at or near the beginning of the current epidemic (i.e., march strains in guinea) in liberia and sierra leone data. the results thus obtained by the latter analysis were consistent with those obtained by the former, and the null hypothesis (i.e. no correlation) was rejected at statistically significant levels in most cases, as described in figure legend short oligonucleotide composition in ebolavirus genomes. next, we analyzed each dinucleotide composition in the genomes grouped for each month. the results presented in fig. a , table and supplementary fig. a show that a common increasing or decreasing trend was observed among the three areas for more than half of the sixteen dinucleotides. correlation coefficients higher than . or less than − . are colored in table . for nine of the ten colored dinucleotides, the coefficient in the other two areas primarily shows the common directional trend. these high correlation coefficients should reflect the evolutionary dependence of the genome sequences. a decrease in uu and au and an increase in cc (fig. a) are predictable, even as a cumulative effect of their constituent mononucleotides (fig. b) , but an increase in ac, cu, gu and ug is not predictable as a simple cumulative effect and may indicate the characteristics of the contiguous sequence itself. we thus calculated the ratio of the observed dinucleotide occurrence to that expected from the mononucleotide composition (obs/exp) and found a decrease for uu and gc and an increase for gu, ug, ac and ua, which was common among the three areas ( fig. b and supplementary fig. a) . the slope direction of the regression line was common among the three areas, but the slope angle appeared to differ among the areas for a wide range of dinucleotides, as well as for tri-and tetranucleotides (data not shown). a spatiotemporal word count can be conducted without specific assumptions or prior knowledge and visualizes viral evolutionary changes in an easily understandable way. two distinct models can explain the common increasing or decreasing trend among the three areas, which was observed for a wide range of mono-and oligonucleotides. [model ] throughout the current ebov epidemic, virus movement between the three areas was extensive; therefore, the time-dependent trend of directional changes in oligonucleotide composition was common among the areas. [model ] even without the extensive mixing of viruses, common directional changes can occur. for example, viruses inevitably depend on many host factors for their growth, but human cells may not provide an ideal growth environment for invading viruses; therefore, common directional changes occur after invasions from nonhumans for supporting more efficient growth in human cells. in this connection, park et al. reported there was no clear evidence for import or export of ebov across national borders after its initial introduction. furthermore, although an increasing or decreasing trend for a wide range of mono-and oligonucleotides is common among the three areas, their actual composition (%) and the slope angle of the directional change often differ among the areas. this observation is consistent with model and the report by park et al. but not with model , as discussed below from various aspects. when considering the cellular environment differences between natural hosts (bats) and humans, not only the low-molecular substances (e.g., nucleotide pool) but also the macromolecules (e.g., proteins) involved in viral growth must be considered. additionally, antiviral mechanisms - should differ between humans and natural hosts. host macromolecules involved in viral growth (either supporting or inhibiting) should primarily recognize oligonucleotides, such as several nucleotides or longer, rather than mononucleotides. hence, we analyzed time-series changes in pentanucleotide ( -mer) occurrences. we first calculated correlation coefficients for all (= ) -mers and sorted them by the averaged correlation coefficients for the three areas. figure a and supplementary fig. b show time-series patterns of -mers with evidently high or low correlation coefficients; or (of types) -mers showed a common decreasing or increasing trend among the areas, respectively. examination of the ratio of the observed occurrences to those expected from the mononucleotide composition (obs/exp) showed that directional changes of many -mers cannot be explained by a cumulative effect of mononucleotide changes (data not shown), as found for various dinucleotides. when further examining the -mers showing the common directional change in more detail, their actual occurrence level and slope of supplementary fig. b) , and the difference often became clearer compared to that for mono-and dinucleotides. this cannot be explained by the aforementioned model . if the time-series directional change in mono-and oligonucleotide composition found for ebov is actually due to the necessity of supporting efficient growth in human cells (model ), such directional changes should be observed for other zoonotic rna viruses. directional change in mers virus genomes. we next analyzed mers coronaviruses isolated in the recent epidemic starting in april in the middle east , . the ebov outbreak analyzed above was initiated by a single virus strain being introduced from a nonhuman animal; this virus was then transmitted among humans during the outbreak. in contrast, mers viruses were repeatedly transmitted from nonhumans, mainly camels, in this epidemic , although its original natural host is thought to be bats . because direct nonhuman hosts for different human isolates differ among isolates, information on camel-derived strains in the current epidemic is important. from the ncbi virus variation database , we found and strains from humans and camels, respectively, with known isolated dates. the total number of mers virus genomes is approximately one tenth of ebov genomes, but their sequence length, even after omitting n bases, is mostly similar to its genome size (ca. kb); only one sequence that was evidently short ( . kb) was omitted from this analysis. we grouped sequences in each month and analyzed the averaged mono-and oligonucleotides (from to -mers) for each month and host. clear time-dependent increasing or decreasing trends were observed for a wide range of mono-and dinucleotides (fig. a,b) , but the increasing or decreasing trend found for actual mono-and oligonucleotides clearly differed from ebov; e.g., c% and g% decrease and u% increases in mers viruses. ebov is a negative-sense single-stranded rna virus, and the sequences registered in the database and analyzed here were complementary to viral genome sequences, but mers virus is a positive-sense single-stranded rna virus and, therefore, genome sequences were analyzed. this difference, however, cannot explain differences in directional changes in monoand oligonucleotide composition of ebov and mers viruses. figure b presents four -mers with a high increasing or decreasing trend; e.g., during this -month epidemic, approximately six uuuuus or four ccacus have been gained or lost per genome, respectively. the time-dependent gain of u (fig. b) clearly differs from its loss for ebov (fig. a) . although the regression lines (blue lines in figs b and ) were calculated only for human strains, occurrence data for camel strains (brown) were primarily positioned around the regression line, thus fitting the previous report that mers viruses were repeatedly transmitted between camels and humans in the present epidemic . because time-series directional changes in mono-and oligonucleotide compositions were observed for both ebov and mers viruses, the directional change should occur for other zoonotic rna viruses. mononucleotide composition in influenza virus genomes. we next analyzed influenza viruses, for which a large number of sequences are available from the influenza research database for different human a subtypes, epidemics of which have started independently at long intervals, such as several decades. importantly, the aforementioned model , in which the common directional change between different viral populations is expected without extensive virus mixing, is testable more rigorously than for the ebov outbreak, which started from a single invasion in guinea and expanded to other countries. furthermore, we may examine the following important issue. if a common directional change is observed, what types of changes likely reoccur in future epidemics caused by invasions from natural reservoir hosts? in addition to human a subtypes, genome sequences of human b type, which can currently infect humans but not birds, and of various avian a subtypes were available. the genomes of influenza a and b are composed of eight segments, and we first selected ca. , strains with a full set of eight sequences, categorized the full genome sequences according to host and serological type, and grouped them in each year. the sequence length cut-off was not included because the genome is composed of eight segments of various lengths and a simple, satisfactory criterion for length cut-off could not be set. alternatively, because times more sequences are available than for ebov, we selected years with at least ten strains of one category to reduce the artefactual effects derived from sequencing uncertainty. then, to analyze time-series changes, we selected human or avian a subtypes with more than five years fulfilling the above threshold (≥ten strains per year); the human b type also fitted these criteria. in the case of h n / [ ] [ ] [ ] (starting from and abbreviated ph n in the database), a sufficient number of sequences fulfilling the above threshold was available even per month and, therefore, ph n sequences in each month were analyzed; importantly, this allowed the study of changes occurring within one outbreak in detail. collectively, we focused on thirteen subtypes (three human and nine avian a subtypes and a human b type), and first analyzed time-series changes in mononucleotide compositions. three human subtypes of influenza a virus (human h n , h n and ph n ) changed their a%, c% and g% with common increasing or decreasing trends among the three subtypes (fig. a) , despite different epidemics having started by independent invasions from nonhumans; the g+ c% increase in human a subtypes was previously reported by rabadan et al. . figure a shows that the a%, c% and g% move time-dependently apart from those of avian a subtypes (sky blue) and toward those of the human b type (violet). because the b type can currently infect humans, but not birds, this type has possibly adapted better to growth in human cells than a subtypes. the directional change of human a subtypes "apart from avian a subtypes and toward the human b type" may visualize their evolutionary journey from each start of human-to-human transmission. in the case of u%, human h n (blue) and h n (brown) have already reached the b's level, and only ph n (green) shows an increasing trend toward b. averaged mono-and dinucleotide compositions (a,b), for strains in each month are plotted according to the elapsed months from april . camel-derived strains are specified by small brown symbols, and regression lines (blue) were calculated only for human-derived strains. the null hypothesis was rejected for all data ( cases) at the significance level of . , except for a% and g%, with or without camel-derived strains; this was rejected for the g% at the significance level of . . scientific reports | : | doi: . /srep a large number of ph n strains isolated from to could provide detailed information on changes occurring within and after one outbreak because the averaged mononucleotide compositions for each month were analyzed. in fact, a horizontally long panel examining three human a subtypes (fig. b) revealed that a% monotonically increased from to but it abruptly lost half of the increase in (arrowed), followed by a possible partial recursion increase in ; c% and g% ( supplementary fig. a ) also showed a monotonic decrease from to , followed by an abrupt backset. we next analyzed the di-to tetranucleotide compositions. concerning correlation coefficients for the elapsed years in the case of h n and h n and for the elapsed months in the case of ph n , three subtypes show a common increasing or decreasing trend for thirteen of sixteen dinucleotides. the top four cases for the absolute value of averaged correlation coefficients for the three subtypes were examined in fig. a . as observed for mononucleotides, all three subtypes appeared to move from avian a subtypes toward the human b type. a decrease in cg dinucleotides in a/u motifs during human-to-human transmission was previously reported . detailed inspection of ph n (fig. b) showed a monotonic increase of aa% from to was followed by an abrupt backset in (arrowed). here, we discuss the backset occurring after one outbreak may relate to the differential slopes of directional changes observed for different subtypes (figs and ). ph n strains, which are primarily derived from one outbreak, show an evidently steeper slope than h n and h n strains derived from multiple outbreaks, indicating a higher rate of ph n genome sequence changes. the first discussion was whether ph n had an evidently higher mutation rate than h n and h n . we propose here that without introducing the higher mutation rate of ph n , its seemingly higher rate of genome sequence changes can be explained by the model that conforms to the abrupt backset that occurred after an outbreak. when considering a viral infection spread, the ratio of persons with or without proper resistance to the viral infection (e.g., antibodies the same colored and smaller symbols than for human subtypes are used for avian a subtypes because differences among avian subtypes were not a concern in the present study. because the b type has moved gradually toward human a subtypes, this type appears not to have reached its hypothesized goal, which may possibly lie between human a and b types, but nearer the latter. (b) a horizontally long panel for a% of three human a subtypes is presented for clarifying detailed changes occurring within and after one outbreak of ph n . data of c% and g% are presented in supplementary fig. a . scientific reports | : | doi: . /srep that are highly effective against the respective strain) becomes important. therefore, the blooming strains (and their close relatives) in a certain outbreak (especially in a pandemic) may not be a very suitable strain for restarting the next outbreak because antibodies highly effective against the blooming strains are held by a large portion of humans. less blooming strains, that are less adapted to humans, may become good starting strains for the next outbreak, resulting in a backset after one outbreak, as observed in figs b and b and supplementary fig. . hence, the evolutionary change focusing on strains belonging to a single outbreak looks higher than those belonging to multiple outbreaks, giving a steeper slope. to illustrate this interpretation, we added hypothetical data of h n strains belonging to one outbreak as a series of small light-brown dots representing the dinucleotide compositions hypothesized for individual months, whose slope angle simply mimics that of ph n . although other various mechanisms must be included to explain the backset after one outbreak, we propose that the steep slope of ph n can be explained without introducing the higher mutation rate of ph n . a wide range of tri-and tetranucleotides also showed decreasing or increasing trends common among the three subtypes (data not shown). the common increase or decrease of mono-and oligonucleotides should reflect the convergent-type evolution occurring in independently evolving viruses, which have invaded independently from nonhumans. we predict that the time-dependent changes required for efficient growth in human cells will repeat with a significant probability after future invasions from natural reservoir hosts and that this view is most likely applicable to a wide range of zoonotic rna viruses. long oligonucleotides in human influenza a genomes. next, a time-series analysis was performed on long oligonucleotide occurrences. long oligonucleotides are key elements of diagnostic rt-pcr primers , and nucleic-acid therapeutics (e.g., sirnas) , [ ] [ ] [ ] . when designing diagnostic and therapeutic oligonucleotides with long effectiveness for highly mutable viruses, we have to understand fundamental features of time-series directional changes of long oligonucleotides and to predict changes that will reoccur with a high probability. the sizes of pcr primers and therapeutic oligonucleotides range primarily from to nucleotides , [ ] [ ] [ ] . thus, we analyzed -mer occurrences in genomes of three human influenza a subtypes, which independently invaded from nonhumans. of a total of (ca. . trillion) types, ca. . million types of -mers were found in these genomes. importantly, a time-series word count can analyze such big data without difficulty. after calculating correlation coefficients for each -mer for each subtype, we first selected all -mers having an evidently high or low correlation coefficient (> . or < − . ) in common for the three subtypes; all five -mers thus selected had negative correlation coefficients and were found to be mutually overlapped; i.e., these were components of one -mer. figure a presents time-series occurrences for one -mer, acagcagagugcuguggaug; other four -mers gave practically the same decreasing pattern. at the beginning stage of three independent epidemics, most strains had one copy of the -mer, but during the course of human-to-human transmission the copy number decreased in the viral population and were almost completely lost at the final stage in all three subtypes; i.e., the strains having this -mer sequence progressively reduced their share in viral populations, regardless of differences in subtypes. we think that this common decrease reflects a certain inconvenience for efficient growth in human cells and will reoccur with a high probability after a future invasion. we next discuss this common decrease, in connection with sirna designs. because of the social importance of influenza viruses, varieties of sirnas have been designed as promising nucleic-acid therapeutics. actually, virsirnadb , which have complied the experimentally validated sirna sequences, contains sirnas for influenza a viruses; and their length ranges from to . when we searched viral sirna sequences that were highly homologous to the -mers with an evident common increasing or decreasing trend with a blast search against virsirnadb, we found that four -mer influenza a sirnas (virsi , , and ), which had the same sequence (cagcagagugcuguggaug) but were experimentally validated in different cell lines, showed a complete match to the -mer sequence harbored by the aforementioned five -mers, which are components of one -mer (aggaacagcagagugcuguggaug). although these five -mers gave practically the same decreasing pattern to that listed in fig. a , the compensatorily increased -mer responding to their decrease differed slightly in changed positions among the subtypes. figure b ,c present patterns of the compensatorily increased -mers, which had nonsynonymous and synonymous changes from the original -mer, respectively (for details, see the legend). this indicates that the sequence ranging over a certain length is possibly inconvenient for efficient growth in human cells ranges and that changes within the range can resolve the possible inconvenience. since both synonymous and nonsynonymous changes were observed, the possible inconvenience should not relate to protein sequence. the information about the time-dependent directional change and the changed base should become important for designing sirnas with long effectiveness. future prospects. finding time-series directional changes in long oligonucleotide occurrences for a large number of influenza a genomes can provide a new refinement strategy for designing therapeutic oligonucleotides and diagnostic rt-pcr primers with long effectiveness. three influenza a subtypes may not be sufficient to judge the reoccurrence of the change, and therefore, our group has started to analyze the time-series changes in experimentally validated sirna sequences, by focusing on both human and avian strains. an important issue is clarification of biological mechanisms responsible for directional changes in oligonucleotide occurrences after host changes. if the mechanism is clarified, reoccurrence in the future should become much clearly defined. systematic analyses on a wide range of oligonucleotides with various lengths for the genomes of human and avian strains may give useful information about the possible mechanisms. judging from the directional changes observed in ebov and ph n outbreaks, genome sequences from strains isolated within several months after a new invasion appear to give reliable information about the directional change. if a large number of viral sequences becomes available, an averaging even per week for strains that are isolated in properly assigned geographic areas may clarify time-dependent directional changes. undoubtedly, past sequences from the same or closely related viral genomes become important for comparison. in the present study, we focused on zoonotic rna viruses whose reservoir hosts are vertebrates, but it is undoubtedly important to analyze other rna viruses spread by bugs (e.g., ticks and mosquitos), which have caused serious emerging infectious diseases, such as dengue fever and zika virus disease , . results obtained with time-series word count are easily understandable even for those unfamiliar with evolutionary biology. when social importance of time-series word count of oligonucleotides becomes clear, experts in various fields will participate actively. huge numbers of genome sequences, including those from disease-causing microbial strains, have accumulated rapidly because of revolutionary development in sequencing technologies and of social importance. in this era of big data accumulation, participation of experts in big data analysis becomes increasingly important for interdisciplinary efforts against big threats presented by infectious microorganisms. genome sequences of human zaire ebolavirus (ebov) and mers coronavirus strains were downloaded from the ncbi virus variation database (http://www.ncbi.nlm.nih.gov/genome/viruses/variation/) on dec. and dec. ( ) , respectively. because the number of mers virus sequences is small, compared with the other two viruses analyzed, a time-series analysis was conducted for the months having at least three strains, and when the number of strains for a certain month was less than , these were combined with those of the nearest neighboring month that had the lower number of strains than the other neighbor. from the ncbi influenza virus resource (http://www.ncbi.nlm.nih.gov/genomes/flu/), a total of ca. , segment sequences derived from ca. , influenza a and b virus strains were obtained on sep. ( ) . we calculated mono-and oligonucleotide occurrences in eight genome segments of influenza virus strains and summed up the occurrences for each strain. to prevent possible misassignment from a large number of ph n strains, relatively small numbers of human classical h n strains isolated from were omitted from the present analysis. influenza virus is a negative-sense single-stranded rna virus, and therefore, the sequences analyzed are complementary to viral genome sequences. computer codes are available from k.w. (k_wada@nagahama-i-bio.ac.jp). response team. ebola virus disease in west africa -the first months of the epidemic and forward projections genetic diversity and evolutionary dynamics of ebola virus in sierra leone ebola virus epidemiology, transmission, and evolution during seven months in sierra leone temporal and spatial analysis of the - ebola virus outbreak in west africa emerging viral diseases comparative dna analysis across diverse genomes inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses induction and suppression of rna silencing: insights from viral infections interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures comparison of avian and human influenza a viruses reveals a mutational bias on the viral genomes oligonucleotide motifs that disappear during the evolution of influenza in humans increase ifn-α secretion by plasmacytoid endritic cells analysis of codon usage diversity of bacterial genes with a self-organizing map (som) -characterization of horizontally transferred genes with emphasis on the e. coli o genome informatics for unveiling hidden genome signatures prediction of directional changes of influenza a virus genome sequences with emphasis on pandemic h n / as a model case novel bioinformatics strategies for prediction of directional sequence changes in influenza virus genomes and for surveillance of potentially hazardous strains novel swine-origin influenza a (h n ) virus investigation team. emergence of a novel swine-origin influenza a (h n ) virus in humans emergence and pandemic potential of swine-origin h n influenza virus origins and evolutionary genomics of the swine-origin h n influenza a epidemic progress toward oligonucleotide therapeutics: pharmacodynamic properties broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr nucleic-acid therapeutics: basic principles and recent applications mechanisms of gene silencing by double-stranded rna rna targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform virus variation resource -recent updates and future directions evidence for camel-to-human transmission of mers coronavirus close relative of human middle east respiratory syndrome coronavirus in bat influenza research database: an integrated bioinformatics resource for influenza research and surveillance. influenza and other respir a multiplex rt-pcr for detection of type a influenza virus and differentiation of avian h , h , and h hemagglutinin subtypes virsirnadb: a curated database of experimentally validated viral sirna/shrna the global distribution and burden of dengue history, epidemiology, and clinical manifestations of zika: a systematic review this work was supported by a grant-in-aid for scientific research (c: no. ) from the ministry of education, culture, sports, science and technology, japan. t.i. and s.k. conceived and designed this project. k.w., y.w. and y.i. developed computer programs. t.i. wrote the manuscript. all authors participate in the discussion through the project. key: cord- -pwlbrxn authors: zhang, xiao-ai; lu, qing-bin; wo, ying; zhao, jin; huang, dou-dou; guo, chen-tao; xu, hong-mei; liu, en-mei; liu, wei; cao, wu-chun title: prevalence and genetic characteristics of saffold cardiovirus in china from to date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: pwlbrxn the epidemiology and clinical features of the saffold cardiovirus (safv) remain ambiguous. the present study was designed to systematically and intensively investigate the epidemiological features of safv in pediatric patients in china. three cohorts of pediatric patients were recruited from to . cohort comprised patients with acute respiratory tract infections. cohort comprised patients with diarrhea. cohort comprised hand, foot, and mouth disease (hfmd) patients. a total of patients ( . %) among ( / , / , and / in cohorts , , and , respectively) were safv-positive. the samples from safv-positive patients were successfully sequenced, and four genotypes were identified: safv- , safv- , safv- , and safv- . a significantly higher detection rate was found in the hfmd patients than in other two cohorts (both p < . ). a higher frequency of severe clinical outcome and nervous system manifestation were also observed in the safv-positive hfmd patients. additionally, ( . %) cerebrospinal fluid and ( . %) serum samples from the hfmd-associated encephalitis patients were safv-positive. based on the vp sequences, all four genotypes displayed distinct geographical clustering. safv infection might be associated with a wide clinical spectrum and contribute to hfmd. myocardium of a previously healthy child who experienced sudden death, suggesting that the virus might cause serious invasive infection in children . all these aforementioned studies provided evidence that safv might have diverse pathogenicity that targets different tissues. however, given the insufficient systematic data, the epidemiological and genetic characteristics of safv infection remain ambiguous. the present study was designed to systematically and intensively investigate the epidemiological features of safv in pediatric patients in china. patient recruitment and sample collection. three cohorts of pediatric patients were recruited from march to december in chongqing children's hospital, chongqing, china. cohort comprised hospitalized patients with acute respiratory tract infection (arti), which was determined based on cough, rhinorrhea, dyspnea, and/or acute fever. . uc. for recruited patients, nasopharyngeal aspirates (npa) samples were collected upon hospital admission. cohort comprised outpatients with diarrhea, who had. loose stools in the previous hours. patients who had confirmed inflammatory bowel disease, celiac disease, cystic fibrosis, food intolerance, or patients who had any apparent clinical respiratory signs or symptoms were excluded. stool samples were collected from the recruited patients. cohort consisted of patients who were diagnosed with hand, foot, and mouth disease (hfmd), according to the guidelines released by the ministry of health of the people's republic of china (http://www.moh.gov.cn/publicfiles/business/htmlfiles/ mohyzs/s / / .htm). the outpatients were generally mild, with fever, and manifested at least one of the following features: maculopapular or vesicular rash on the palms, and/or soles, vesicles or ulcers in the mouth. hospitalized patients were diagnosed with severe hfmd if they manifested one of the following additional complications: encephalitis, meningitis, afp, or cardiorespiratory failure. for all hfmd patients, stool samples were collected, whereas for some cases, stool, throat swab, and serum samples were collected. in addition, csf was additionally collected for some cases diagnosed with encephalitis. healthy children who were recruited from the community during the same study period were used as controls, from whom stool, throat swab, and serum samples were also collected. none of the recruited healthy children exhibited arti, diarrhea, or hfmd-related symptoms during the recent one month of recruit. laboratory detection. all collected samples were tested for safv using real-time reverse-transcription polymerase chain reaction (rt-pcr) assay targeting the untranslated region (utr) as previously described . all samples with positive reactions were further amplified using nested rt-pcr targeting the -utr, which generated a -bp amplicon . a patient was considered to be infected with safv when positive detection was obtained in any type of the samples. the viral protein (vp) gene of all positive samples were amplified using nested rt-pcr approach as previously described . the detection of other viral pathogens varied for three cohorts. for cohort , npa samples were screened by rt-pcr or pcr for common respiratory viruses, which include influenza viruses a and b, respiratory syncytial viruses (rsv) a and b subtypes, parainfluenza viruses (piv) types , , , and , metapneumovirus (mpv), human rhinovirus (hrv), coronavirus (cov), human adenovirus (hadv), and human bocavirus (hbov) as previously described , . in cohort , stool specimens were tested for the following viruses using the corresponding tools: rotaviruses a, ideia rotavirus direct antigen detection kit (ideia, oxiod, uk); rotaviruses b and c, hadv, sapovirus, and astrovirus, pcr or rt-pcr ; and noroviruses gi and gii and hbov, real-time pcr , . in cohort , all collected stools, throat swabs, csfs, and sera were subjected to detection of enterovirus (ev), and further classification of ev and coxsackievirus a (cva ) was performed using real-time pcr using previously described primers . to further identify the ev serotypes other than ev and cva , semi-nested rt-pcr, which is specific for a partial region of vp , was performed for other ev-positive samples using previously reported primers . the amplicons were subjected to sequencing and blast analysis. this study was performed with the approval of the ethical committees of chongqing children's hospital and beijing institute of microbiology and epidemiology. the methods were carried out in accordance with the approved guidelines. at recruitment, written informed consent was obtained from all guardians of participants. safv strains accession numbers. the sequences obtained from the study were submitted to ncbi with the genbank accession numbers kj -kj . the genomic sequences were assembled using lasergene's dna seqman software (version . . , dna star inc. madison, wi, usa). the mega program (version . , sudhir kumar, arizona state university) was used for alignments and phylogenetic tree construction by maximum likelihood method using bootstrap pseudo replicates. nucleotide identities among strains were calculated using bioedit (version . , www.mbio.ncsu.edu/bioedit/bioedit.html). statistical analysis. descriptive statistics were performed, with continuous variables summarized as median and range, and categorical variables summarized as frequencies and proportions. the statistical significance between various groups was tested using the x -test or fisher exact test for categorical variables and independent t test or nonparametric test for continuous variables. a two-sided p value of less than . was considered to be statistically significant. analyses were performed using spss version . (spss). detection and genotyping of safv in three cohorts of patients. a total of patients ( , , and patients in cohorts , , and , respectively) were recruited into the study, ( . %) of which were boys. ages ranged from days to years (median, months). more boys were included in cohort (p . ), and older age was presented in cohort (p , . ) ( table ). among all the tested patients, ( . %) were safv-positive, [ ( . %), ( . %), and ( . %) from cohorts , , and , respectively (table ) ]. a significantly higher detection rate was found in hfmd patients among the three cohorts (p , . ). a total of specimens ( throat swab, sera, and stools) were collected from asymptomatic children. the age ranged from months to months (median, months), and ( . %) were boys. no positive detection was found in any type of samples from asymptomatic children. from the safv-positive patients, patients were successfully sequenced for vp nucleotide sequences, and four genotypes were identified: safv- ( , . %), safv- ( , . %), safv- ( , . %), and safv- ( , . %). for cohort , the safv determined from of the positive patients were successfully grouped into three genotypes: safv- ( , . %), safv- ( , . %), and safv- ( , . %). in cohort , nine of the detected safv were assigned to two genotypes: safv- ( , . %) and safv- ( , . %). in cohort , of the safv-positive patients were successfully sequenced, and four genotypes were identified: safv- ( , . %), safv- ( , . %), safv- ( , . %), and safv- ( , . %). the uncommonly seen safv- was only found in the hfmd patients (table ) . co-detections with other viruses were found in . % ( / ) of the safv-positive patients, including co-detections with one virus, three with two viruses, and one with three viruses. the patients with safv single infection had a median age of months (range, months- months), and ( . %) were boys, which were comparable with codetection patients (all p. . ). three cohorts had comparable codetection rates ( . %, . %, and . %, p . ) ( table ). in cohort , the most frequent co-detection occurred between safv and piv ( patients), followed by safv and rsv-b ( ), hadv ( ), hbov ( ), cov ( ), influenza virus a ( ), influenza virus b ( ), and hrv-a ( ). in cohort , nine patients were found to have co-detections, including co-detection with norovirus gii ( ), rotavirus group a ( ), and hadv ( ). in cohort , patients were found to have co-detection of safv with ev ( ), cva ( ), and other evs ( ) . demographic and clinical characteristics of the safv-positive patients. the median age of the safv-positive patients was months, and ranged from two months to months. of these patients, . % were males. in cohort , the safv-positive patients were significantly older than the safv-negative patients ( . months vs. . months, p , . ; table ). the infection rate increased significantly with age among children below years old (cochran-armitage trend . , p , . ). the highest positive rate ( . %) was found in -to -year-old patients (supplemental table ). in cohorts and , the age and gender profiles were highly comparable between the safv-positive and safv-negative patients (all p. . ). in cohort , seven ( . %) of the safv-positive patients, which is significantly higher than that of the safv-negative patients ( . % vs. . %, p . ), developed asthma that were related with this disease episode. by contrast, the opposite trend was found for pneumonia ( . % vs. . %, p . ). other clinical manifestations were highly comparable between safv-positive and safv-negative patients (all p. . ). in cohort , the diarrhea duration was comparable between safv-positive and safvnegative patients. no safv-positive patients exhibited symptoms of vomiting, but ( . %) safv-negative patients did (p . ). within cohort , the severe hfmd patients had significantly higher safv-positive rate than the mild hfmd patients ( . % vs. . %, p . ). furthermore, safv-positive patients presented with significantly higher frequency of nervous system manifestation than the safv-negative patients ( . % vs. . %, p . ). for all three cohorts, the clinical manifestations of patients with safv single detection were insignificantly different from those with safv co-detection ( table ). we additionally analyzed whether the co-detection of safv might aggravate the clinical outcome of ev infected hfmd patients ( table ). the results demonstrated that out of ( . %) safv/ev co-detected patients developed severe hfmd, significantly higher than that of patients with ev single detection ( . % vs. . %, p . ). by contrast, there was no significant difference between patients with cva single detection and patients with safv/cva co-detection (p. . ). similarly, no difference was attained for other evs co-detected patients either. genetic characterization of safvs. a phylogenetic tree was constructed based on the vp nucleotide sequences of saffold cardiovirus detected in clinical samples of patients in the current study, and sequences downloaded from genbank by maximum likelihood method using mega . (figure ) . a spatial pattern of the genetic clustering was identified for each genotype. for safv- , four lineages were formed, and all the chinese strains were grouped into one lineage (figure a ). for the chinese lineage, an obvious geographic-specific distribution was observed, with strains from three locations grouped separately into three sublineages. all the safv- cases from the current study were grouped into the chongqing sublineage, regardless of the cohort group. in safv- , five lineages were constructed, which included european, north american, middle-eastern, japanese, and east asian lineages ( figure b ). the strains from the present study were grouped into east asian lineage, which, however, diverged from those from beijing or thai strains. in safv- , six lineages were formed depending on the geographical origin. the chongqing strains in this study were classified into the asian lineages, forming a sublineage that was distinct from the strains of other regions ( figure c ). in safv- , the strains from three locations were classified into three clusters. the chongqing strains formed a separate cluster, which is different from those of japanese and pakistani strains ( figure d ). detection of safv in sera and csfs. additional csfs were collected from patients with hfmd-associated encephalitis, and six ( . %) were found to be safv-positive using real-time rt-pcr, and five were further confirmed by nested rt-pcr targeting the -utr. two of six safv-positive csfs were successfully sequenced for vp , and only safv- was identified. among serum samples collected from hfmd patients ( from mild hfmd patients and from severe hfmd patients), eight hfmd patients ( . %) were detected positive for safv, including seven patients with encephalitis and one patient with myocarditis. among sera from severe hfmd patients, were collected from patients with hfmd-associated encephalitis, and seven ( . %) were found to be safv-positive using real-time rt-pcr, and five were further confirmed by nested rt-pcr targeting the -utr. two out of eight safv-positive sera were successfully identified to be safv- and all from patients with hfmd-associated encephalitis. from three of the six safv-positive csfs and four of eight safvpositive sera, no hev was detected. information on the potential disease associations of safv has increased over the past six years, with data concentrated particularly on the involvement of safv in pediatric respiratory and gastrointestinal tract infections. in the current study, the prevalence of safv in pediatric patients in the recent four years in chongqing, china was determined by performing molecular epidemiological investigation on three cohorts of patients. in addition to arti and diarrhea patients, an unexpected significantly higher prevalence of safv was found in hfmd patients, especially in patients with hfmd-associated encephalitis. multiple genotypes of safv, which displayed geographic specific distribution pattern, were also found to be co-circulating in china. these data provided a reliable estimation based on a large sample size and long observation period, thereby expanding the understanding on the epidemiological features of safv. one might query the higher frequency of safv in the hfmd patients was due to the old age and more sampling types of this cohort, in comparison with the arti and diarrhea cohorts. an increasing prevalence of safv was found in arti patients, but not in the hfmd patients. thus, the age mismatch among the three cohorts might play minor roles in determining the detection rates. on the other hand, although the hfmd cohort was tested using stool, respiratory, blood, and csf samples, an over-estimation of infection rate was not generated, because stool samples offered the highest diagnostic sensitivity. when we took no account of the patients with stool negative while other sample types are positive, a prevalence rate of . % was still obtained. thus, this effect alone might not explain the higher prevalence observed in hfmd. several lines of evidence indicate that safv might act as the agent responsible for the clinical syndromes, at least for some of the subjects. first, although co-detection with other viruses existed, safvs were the only pathogens identified in ( . %) safv-positive specimens in this study. the co-detection of safv with other viruses ranged from % to % in previous studies, and the variation in results is probably due to different diagnostic assays applied [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the present study, common respiratory viruses in arti patients, common viruses in diarrhea patients, and all evs in hfmd patients were detected. the current results might represent a higher probability of single infection than the previous studies. still, single-infection by safv is only true relative to the list of viruses that are being tested. second, this study also revealed the negative detection of safv in healthy children, indicating that safv is less likely to be present in asymptomatic subjects. this result is consistent with a previous study that was conducted among asymptomatic patients in northern germany . finally, more severe clinical outcome and manifestation of nervous system were exhibited by safv-positive hfmd patients than in safv-negative hfmd patients. in our study, safvs were co-detected with other viruses in ( . %), ( . %), and ( . ) specimens in arti, diarrhea and hfmd patients respectively. for arti and diarrhea cohorts, no differential clinical manifestations were inferred from the patients with safv single detection or safv co-detection, probably due to small sample size for comparison. however, for hfmd patients, we found the co-detection of safv could aggravate the clinical outcome of ev infection. we are uncertain whether safv/ev co-detection can lead to synergies in the hfmd pathogenic mechanism, and therefore responsible for the pathogenesis of severe hfmd, which need further investigation in the future. previous studies have demonstrated the existence of safv in csf, but it was only limited to the case report , . in , safv- was isolated from the csf sample of a -year old boy with aseptic meningitis . safv- was subsequently detected in the csf and fecal samples of one child with cerebellitis as well as in the csf, blood, and myocardium of another child who died suddenly with no history of illness . in the current study, the existence of safv- was observed in the csf and safv- was observed in serum samples from the patients with hfmd-associated encephalitis. furthermore, among half of the safv-positive csfs and sera, safv was the solely detected pathogen. this information provided the first valid estimation of safv in pediatric encephalitis. pediatric encephalitis caused by viral infection has always been of great public health concern worldwide because of its high morbidity and mortality. the common agents of viral encephalitis include herpes simplex virus , varicella zoster virus, ev, epstein-barr virus, human herpesvirus , and measles virus , . however, an etiological diagnosis was reached in less than % of the cases despite the use of modern microbiological and radiological methods , . the list of uncommonly observed etiological agents for pediatric encephalitis remained far from complete. animal experiments have suggested showed that both safv- and safv- can infect the heart and the central nervous system of /sv mice . furthermore, they found that safv- was more neurotropic than safv- , and intracerebral inoculationof safv- into fvb/n mice caused acute encephalitis . in the current study, the existence of safv- and safv- was observed in the patients with hfmd-associated encephalitis. the present results, together with those of the earlier studies, indicate that safv- and safv- might play a role in the pathogenesis of viral encephalitis. however, these results warrant further confirmation by independent studies. to the best of the authors' knowledge, this study is the first to document safv in hfmd patients. the high frequency of safv in the stools, csfs, and sera of children with hfmd and the absence of safv in healthy children suggest that this new virus might contribute to hfmd, especially in hfmd-associated encephalitis. this finding has important public health implication for hfmd disease control, especially among severe hfmd patients. although with various patient types and large sample size, the major findings in the present study, especially the causal relationship between safv and the clinical disease, should be confirmed by serological study in the future. diverse patterns of immune and non-immune-mediated disease in emc m-variant-infected mice viruses in type diabetes: brief review the genetics of the persistent infection and demyelinating disease caused by theiler's virus theiler's virus infection: a model for multiple sclerosis characterization of vilyuisk virus as a picornavirus nucleotide sequence identifies vilyuisk virus as a divergent theiler's virus human vilyuisk encephalitis discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin new saffold cardioviruses in children identification of cardioviruses related to theiler's murine encephalomyelitis virus in human infections circulation of lineages of a novel saffold cardiovirus in humans saffold cardiovirus in children with acute gastroenteritis saffold cardioviruses of lineages in children with respiratory tract infections three clusters of saffold viruses circulating in children with diarrhea in japan saffold cardioviruses in children with diarrhea co-circulation and persistence of genetically distinct saffold viruses sequencing and phylogenetic analyses of saffold cardiovirus (safv) genotype isolates from children with upper respiratory infection in gunma saffold virus infection in children cardioviruses are genetically diverse and cause common enteric infections in south asian children saffold virus, a human theiler's-like cardiovirus, is ubiquitous and causes infection early in life seroepidemiology of saffold cardiovirus type seroepidemiology of saffold cardiovirus (safv) genotype in japan cultivation and serological characterization of a human theiler'slike cardiovirus associated with diarrheal disease serious invasive saffold virus infections in children sequence and phylogenetic analyses of saffold cardiovirus from children with exudative tonsillitis in yamagata development and implementation of a molecular diagnostic platform for daily rapid detection of respiratory viruses species-specific identification of human adenoviruses by a multiplex pcr assay a simple and rapid single-step multiplex rt-pcr to detect norovirus, astrovirus and adenovirus in clinical stool samples human bocavirus infection, people's republic of china multiplex real-time rt-pcr for simultaneous detection of gi/gii noroviruses and murine norovirus evaluation of a rapid real-time rt-pcrassay for detection of enterovirus rna in cerebrospinal fluid specimens sensitive, seminested pcr amplification of vp sequences for direct identification of all enterovirus serotypes from original clinical specimens the preparation of an infectious full-length cdna clone of saffold virus viral encephalitis: etiology, clinical features, diagnosis and management. the open infect principles and practice of infectious diseases, seventh edition viral encephalitis adaptation of saffold virus for high-titer growth in mammalian cells saffold virus infection of the mouse the authors would like thank all the subjects, their families, and collaborating clinicians for their participation. key: cord- -j gpww q authors: sun, wei; shi, zhencai; gao, fuqiang; wang, bailiang; li, zirong title: the pathogenesis of multifocal osteonecrosis date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: j gpww q our objective was to study the incidence, etiology, and diagnosis of multifocal osteonecrosis (mfon) and its treatment options to facilitate an earlier diagnosis and to optimize treatment. a radiological investigation was performed in osteonecrosis patients with a high risk of mfon for a more accurate diagnosis between january and june . for patients with osteonecrosis of both the hip and knee joints or for patients with a history of corticosteroid use or alcohol abuse who had osteonecrosis of one or more joints in the shoulder, ankle, wrist or elbow, magnetic resonance imaging (mri) was also performed on other joints, regardless of whether these joints were symptomatic. furthermore, we performed a radiological screening of patients who had a negative diagnosis of mfon but were at a high risk; among them, another mfon cases were successfully identified ( . %). thus, the incidence of mfon during the study period increased from . % to . %. patients diagnosed with osteonecrosis and who are at a high risk of mfon should have their other joints radiologically examined when necessary. this will reduce missed diagnosis of mfon and facilitate an earlier diagnosis and treatment to achieve an optimal outcome. between january and june , only of the osteonecrosis patients under our care were diagnosed with mfon ( . %) upon admission. after the comprehensive evaluation of the joints of patients who had a negative diagnosis of mfon but who were at a high risk, another cases of mfon were successfully identified ( . %). consequently, the incidence of mfon during the study period increased to . %. the mfon patients admitted to our center had a mean number of . osteonecrotic lesions, similar to the mean number of lesions ( . ) found in post-sars mfon patients. the associated factors, diseases, and comorbidities are listed in table . of the patients diagnosed with mfon on admission between and , were male and were female. forty-seven of the mfon patients had a history of corticosteroid use, and the remaining one patient had a history of alcohol use. of the patients with a history of corticosteroid use, had sle, nine had chronic nephropathy, five had hematological diseases (four had acute lymphoblastic leukemia and one had non-hodgkin's lymphoma), five had an organ transplantation (four had a renal transplantation and one had a cardiac transplantation), three had sjogren's syndrome, two had dermatomyositis, two had multiple sclerosis and three received steroid therapy for trauma emergency. of the newly diagnosed mfon patients, were male and were female. all of them had a history of corticosteroid use. fifteen patients had sle, eight had acute lymphoblastic leukemia, five had chronic nephropathy, and three had an organ transplantation (two had a renal transplantation and one had a hepatic transplantation). the mfon patients most commonly had osteonecrosis of the femoral head, followed by the knee, shoulder and ankle bones. the majority of patients had bilateral lesions (hips, knees and shoulders) (fig. , table ). there was a significant difference in the number of mfon patients identified after the radiological screening based on risk factors compared with that before the screening (p < . ). incidence of mfon. there have not been any journal articles in china concerning mfon, and such reports were also limited in other countries. mont et al. studied mfon cases diagnosed between and from centers in the united states. according to the available data from centers concerning their total number of patients with osteonecrosis, patients had mfon of the patients ( . %) diagnosed with osteonecrosis. in , we screened post-sars medical workers with corticosteroid use and found patients had mfon of the patients ( %) with a diagnosis of osteonecrosis. by contrast, between and , we found only patients with mfon of the patients ( . %) diagnosed with osteonecrosis who were admitted to our center. the incidence of mfon in post-sars osteonecrosis patients was obviously higher than that in daily clinical practice. this is because the lesions found in joints, such as the knees, shoulders and ankles, in the early stages of mfon cases may not have any signs or symptoms. in our screening of post-sars patients, all of them had a comprehensive mri evaluation of their bilateral hips, knees, shoulders and wrists or ankles, which revealed the asymptomatic osteonecrosis. however, in daily clinical practice, we often focus on the osteonecrosis diagnosis of the femoral head without performing an mri on the other joints. this implies that there are possible missed diagnoses of asymptomatic mfon patients. a recent study also showed a higher incidence of mfon than that typically reported in literature . dose of corticosteroids is the main risk factor for mfon. in france, hernigou reported on mfon cases diagnosed between and , all of which were associated with corticosteroid use. in the cases reported by mont et al. , % had a history of corticosteroid use, and the rest had a coagulation disorder. our study showed that of the ( %) mfon patients admitted to our center had a treatment history of high-dose corticosteroids, and the two remaining patients had a history of alcohol use. moreover, the dosage and route of administration of the corticosteroids were obviously related to the incidence of mfon. a study by hernigou demonstrated that the total dose and the daily dose of venous injection were closely related to the occurrence of mfon. this was also found in our study of post-sars osteonecrosis patients caused by the use of corticosteroids . there have been a limited number of mfon case reports and a high occurrence of mfon in asymptomatic patients. therefore, the exact incidence of mfon in patients with various diseases remains unclear. the incidence of mfon in patients with sickle cell disease was reported to be % ( of ) as a complication in the maintenance treatment of acute lymphocytic leukemia and non-hodgkin's disease, and its incidence in these diseases is also higher than that reported in the literature. solarino et al. performed mri screening in patients with acute lymphoblastic leukemia after chemotherapy and found that % of them had mfon . in the mfon cases presented in this study, most were sle, followed by hematological diseases, nephropathy, organ transplantation, dermatomyositis and multiple sclerosis. mfon was especially prevalent in leukemia patients; of the osteonecrosis patients with leukemia under our care were found to have mfon. three of the four patients who received pulse steroid therapy for trauma emergency had a spinal cord injury, for which steroid therapy was considered appropriate. however, one patient received pulse steroid therapy for only an eye injury and was found to have osteonecrosis in eight joints, including the hips, knees, shoulders and ankles. caution should be taken for such cases in the future. mfon patients most commonly had osteonecrosis of the femoral head, followed by the knee, shoulder and ankle bones. osteonecrosis of the shoulder, ankle and wrist never occurred aloneand was always accompanied by osteonecrosis of the hip and knee. among the three populations of mfon patients presented in this study, - % had femoral head involvement, - % had knee involvement, and - % had humeral head involvement. the average number of osteonecrotic lesions was . per patient. to date, mri is the most sensitive and specific tool to diagnose mfon. initial studies found that low field mri was as sensitive as a radionuclide scan in diagnosing osteonecrosis. nevertheless, recent studies have demonstrated that high field ( . - t) mri has a higher sensitivity, specificity and accuracy in diagnosing osteonecrosis. mont et al. compared the sensitivity of radionuclide scans with mri, and found that mri has a sensitivity of %. by contrast, the sensitivity of radionuclide scans is only %. radionuclide scans have a higher sensitivity in detecting advanced stage osteonecrosis of the hip and knee than of the shoulder and ankle. although the diagnosis of osteonecrosis by mri has its advantages, the screening for mfon with mri is faced with a long scanning time and high cost. there have been debates regarding the optimal method to screen symptomatic and asymptomatic mfon , . plain film x-ray is not sensitive enough to diagnose asymptomatic mfon, but there is still an advantage to carrying out an x-ray examination. even if the x-ray result of a joint with pain is negative for osteonecrosis, it provides a preliminary examination of the local bone structure, which serves as a reference for further investigation by mri. other methods include a short time inversion recovery (stir) mri test followed by mri or a radionuclide scan of specific joints. nevertheless, further studies are required to determine the optimal method for early detection of osteonecrosis. moreover, as the majority ( - %) of osteonecrosis cases reported in the literature are bilateral, patients diagnosed with osteonecrosis of a joint on one side should be radiologically examined on the other side. missed diagnoses of mfon have frequently occurred in clinical practice. to reduce this, we recommend the following measures. ) patients with associated diseases and long-term use of high-dose corticosteroids should have their hips and knees evaluated by mri within six to twelve months after the medication. ) patients diagnosed with osteonecrosis of the hip and knee should have their bilateral shoulders evaluated by mri. ) patients with corticosteroid use or alcohol abuse and who are diagnosed with osteonecrosis of one or more joints in the shoulder, ankle, wrist and elbow should have their hips and knees evaluated by mri. mfon patients usually start with silent lesions. as the disorder gradually progresses, patients will experience joint pain. at first, the pain is mild and patients may only experience increased pain during movement or weight bearing on the affected bone or joint. as significant damage develops in the joints, patients will experience severe pain. the period of time between the first symptoms and the loss of joint function varies for each patient and ranges from several months to several years. in the late stages of mfon, patients often have symptoms at rest and reduced joint activity, resulting in severe joint dysfunction. the treatment of mfon remains controversial. the general principle is to estimate the prognosis of each lesion. in addition, predicting the presence of collapse of the joint surfaces is the basis for determining the order of treatment. the progress of osteonecrosis is often different for each joint. osteonecrosis of the femoral head progresses the fastest and attention should be paid to the early appearance of symptoms. this is followed by the progression of osteonecrosis to the knees and shoulders. patients who have osteonecrosis of multiple weight-bearing joints and a large necrotic lesion area may develop joint destruction, and early intervention should be used. the progression of osteonecrosis to the elbow and wrist is relatively slow; however, we still lack experience regarding the appropriate treatment method for this area. bone infarction often leads to calcification and self-repair. hence, osteonecrotic elbows and wrists should mainly be managed by monitoring, and over treatment should be avoided if there are no clinical symptoms. in addition, an appropriate treatment strategy should be selected according to the stage of osteonecrosis in each joint involved . for symptomatic patients at an early stage (i.e., arco stages i, ii or china stages i-iii), joint-preservation treatment options are commonly used, such as extracorporeal shock wave, core decompression, and vascularized and nonvascularized bone grafting. when > mm of the articular surface has collapsed or the necrotic lesion area is > %, joint-preservation treatments may not provide good efficacy. in the advanced stages of the disease (i.e., arco stages iiic and iv, or china stages v-vi), arthroplasty is required when joint preservation is not possible. there are some limitations of this study. this study is limited by virtue of the retrospective analysis at only one center. and there was no randomized and blinded control group with conservative treatment in this study. the incidence of mfon was high when clinical risk factors were present, such has high-dose steroid use, alcohol abuse, sle, chronic nephropathy and leukemia. for a highly suspected case of mfon, a radiological screening of multiple joints is necessary, and mri is still the gold standard for diagnosing mfon. such screening can help to effectively reduce missed diagnoses. the goals of the treatment should be to take measures to delay the progress of the disease, to preserve the joint function and to avoid joint surface collapse and destruction by diagnosing the disease early. in addition, an appropriate treatment strategy should be selected according to the stage of mfon. conservative treatment and joint-preservation treatment options should be adopted during the early stages, while arthroplasty should be performed during the advanced stages. this study was performed in accordance with the principles expressed in the declaration of helsinki and approved by the ethics committee of the china-japan friendship hospital. informed consent was obtained from all patients. a standardized mri was taken of patients, including their bilateral joints. the apparatus was a ge sigma profile/gold, and t weighted, coronal stir, and transverse t wi sequences were used. the parameters of the scan included a layer thickness of mm and a distance between layers of mm. the t wi were obtained with a tr from ~ ms and a te of ms, and the stir images were obtained with a tr of ms and a te of ms. if an abnormal signal was found, then a t weighted scan was added, and for some of the patients, we also performed fat suppression and water-fat separation sequences. we only used t and t sequences for the other joints of the patients. osteonecrosis was defined as either a subchondral or an intramedullary area demarcated by a distinct marginal rim with low signal intensity that encompassed the medullary fat on the mri images. in addition to the mri, all patients were subjected to an x-ray that included the affected joints. our general survey on the bones and joints of post-sars medical workers who used corticosteroids revealed that the corticosteroid dose was approximately mg in unifocal osteonecrosis patients. by contrast, the dose was > mg in all mfon patients except for one patient who received mg, with the highest dose at , mg. the mfon patients also had prolonged corticosteroid treatment (≥ days) . of the mfon patients diagnosed between january and december , one had a -year history of alcohol abuse (average daily alcohol consumption of g). the remaining patients had a history of intensive steroid use for the following diseases: sle ( patients), acute lymphoblastic leukemia ( patients), chronic nephropathy ( patients), anaphylactoid purpura ( patient) and pulse steroid therapy for traumatic shock ( patient). therefore, we summarized the high risk factors for mfon to include: a medical history of sle, chronic nephropathy, hematological diseases or coagulation abnormalities (especially leukemia); total corticosteroid (methylprednisolone) dose > mg, especially for patients with a history of intravenous pulse therapy; and a total corticosteroid administration time of > days. between january and june , a total of osteonecrosis patients were admitted to our center, and only of them were diagnosed with mfon ( . %) upon admission. a radiological investigation was performed in patients with a negative diagnosis of mfon but who had a high risk of mfon and complaints of pain in other joints. for patients found to have osteonecrosis of both the hip and knee, mri was performed on their bilateral shoulders and ankles, and also their bilateral wrists and elbows when necessary, regardless of whether other joints were symptomatic. for patients with a history of corticosteroid use or alcohol abuse and who were found to have osteonecrosis of one or more joints in the shoulder, ankle, wrist and elbow, mri was performed on their hips and knees and their other joints when necessary. the results were analyzed by a chi-square test the using spss software. a p value < . was considered statistically significant. symptomatic multifocal osteonecrosis. a multicenter study. collaborative osteonecrosis group clinical research of correlation between osteonecrosis and steroid multifocal joint osteonecrosis in sickle cell disease thrombophilia, hypofibrinolysis, the enos t- c polymorphism,and multifocalosteonecrosis multifocal osteonecrosis in systemic lupus erythematosus: case report and review of the literature multiple site osteonecrosis in hiv infection osteonecrosis as a complication of treating acute lymphoblastic leukemia in children: a report from the children's cancer group on a case of multifocal osteonecrosis in a patient suffering from acute lymphoblastic leukemia multifocal avascular necrosis after liver transplantation: an unusual presentation of the antiphospholipid syndrome multifocal osteonecrosis associated with human immunodefi ciency virus infection symptomatic steroid-induced multifocal diaphyseal osteonecrosis in a patient with multiple sclerosis widespread osteonecrosis in children with leukemia revealed by whole-body mri vibration-induced multifocal carpal osteonecrosis in a -year-old man a rare cause of peripheral arthralgia in inflammatory bowel disease: multifocal osteonecrosis non-traumatic osteonecrosis of the femoral head: ten years later bone scanning of limited value for diagnosis of symptomatic oligofocal and multifocal osteonecrosis detection of multifocal osteonecrosis in an adolescent with dermatomyositis using whole-body mri fast sequences mr imaging at the investigation of painful skeletal sites in patients with hip osteonecrosis chinese specialist consensus on diagnosis and treatment of osteonecrosis of the femoral head this study was supported by the national natural science foundation of china ( ) and the china-japan friendship hospital youth science and technology excellence project ( -qnyc-a- ). the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the paper. w.s., z.c.s. and z.r.l. conceived the experiments. w.s., z.c.s., f.q.g. and w.b.l. performed the experiments. w.s., z.c.s., f.q.g. and w.b.l. analyzed the data. w.s., z.c.s. and z.r.l. wrote the manuscript. all authors reviewed the manuscript. competing financial interests: the authors declare no competing financial interests. this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- - fcc i i authors: hassani, abdelkader; azarian, mohammad mahdi sabaghpour; ibrahim, wisam nabeel; hussain, siti aslina title: preparation, characterization and therapeutic properties of gum arabic-stabilized gallic acid nanoparticles date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: fcc i i gallic acid (ga) is a natural phenolic compound with therapeutic effects that are often challenged by its rapid metabolism and clearance. therefore, ga was encapsulated using gum arabic into nanoparticles to increase its bioavailability. the formulated nanoparticles (ganps) were characterized for physicochemical properties and size and were then evaluated for antioxidant and antihypertensive effects using various established in vitro assays, including , -diphenyl- -picrylhydrazyl (dpph), nitric oxide scavenging (no), β-carotene bleaching and angiotensin-converting enzyme (ace) inhibitory assays. the ganps were further evaluated for the in vitro cytotoxicity, cell uptake and cell migration in four types of human cancer cell lines including (mcf- , mda-mb ) breast adenocarcinoma, hepg hepatocellular cancer, ht- colorectal adenocarcinoma, and mcf- a breast epithelial cell lines. the ganps demonstrated potent antioxidant effects and have shown promising anti-cancer properties in a dose-dependent manner with a predilection toward hepg and mcf cancer cells. the uptake of ganps was successful in the majority of cancer cells with a propensity to accumulate in the nuclear region of the cells. the hepg and mcf cancer cells also had a significantly higher percentage of apoptosis and were more sensitive to gallic acid nanoparticle treatment in the cell migration assay. this study is the first to confirm the synergistic effects of gum arabic in the encapsulation of gallic acid by increasing the selectivity towards cancer cells and enhancing the antioxidant properties. the formulated nanoparticles also had remarkably low toxicity in normal cells. based on these findings, ganps may have promising therapeutic applications towards the development of more effective treatments with a probable targeting precision in cancer cells. . powder x-ray diffraction patterns of xrd analysis of (a) the none capsulated gallic acid, (b) gum arabic, (c) physical mixture of ganps, (d) the nanoparticles of gallic acid prepared using the freeze-drying technique. evaluation of the antioxidant properties. the free radical scavenging activity of the free and nano encapsulated gallic acid was assessed in vitro by the dpph assay. dpph is a stable organic free radical used to estimate the antioxidant activity of various compounds. trolox served as a positive control due to its capacity to dissolve in the aqueous system. in this assay, the color of the dpph changed from deep violet to a pale or colorless solution in the presence of ganps nanoparticles. as illustrated in fig. , the percentage of dpph scavenging activity of ganps was . %, . %, and . % for the concentrations of , , and µg/ml respectively; whereas for free gallic acid the scavinging activity was . %, . %, and . %, respectively for the same concentrations (p < . ). this confirms the retention of the complete function of gallic acid after nanoencapsulation in gum arabic nanoparticles. trolox revealed strong scavenging activity of . % against dpph at similar concentrations. the antioxidant evaluation of free and nano encapsulated gallic acid was also determined in murine macrophage cell line (raw . ) . initially, the cell viability of raw . cells in response to treatments was evaluated using the mtt reduction assay in the presence or absence of lps. as shown in fig. , the lps induction had shown a relative toxic effect on the raw . cells upon treatment with ga and ganps. both treatment forms had negligible cytotoxicity activity against raw . cells in comparison to the untreated lps-stimulated cells. treatment of raw . cells with ga and ganps caused a considerable inhibition of nitric oxide (no) production as depicted in fig. . nonetheless, the nano encapsulated ga exhibited potent antioxidant activity, when compared to the free ga. the antihypertensive activity of ganps. the in vitro antihypertensive activity of free gallic acid and ganps was evaluated by measuring the ace inhibitory activity of the enzyme following the method outlined by cushman and cheung . this assay is based on the conversion of hippuryl-histidyl-leucine to hippuric acid. the in vitro antihypertensive capacity of free ga and ganps was investigated by measuring its absorbance fig. . according to the results, significant cytotoxicity was elicited among the hepg , mcf , mda-mb , and ht cell lines after treatment with ic concentrations of free and nano encapsulated gallic acid as shown in fig. . there was negligible cytotoxicity among the mcf- a cells especially at concentrations equivalent to the ic concentrations used in cancer cells (fig. ) and also in comparison with the untreated mcf- a cells. interestingly, the hepg and mcf cells were more sensitive to the treatments than the ht and mda-mb cells with a significantly higher selectivity with ic of . and . µg/ml for free ga and ganps respectively as shown in fig. . the ic value of ganps was approximately half that of free gallic acid in hepg cells as shown in fig. . as demonstrated, the ht and mda-mb cells were less sensitive to the treatments with an ic concentration of . and . µg/ml respectively. the ganps demonstrated variable patterns of cellular uptake in hepg , mda-mb , ht , and mcf cancer cell lines. as shown in fig. , the ga/c nps labeled with c green fluorescent dye were successfully uptake by the majority of the cancer cell types with condensation in the nuclear region as shown in fig. a . in section b, the empty nanoparticles labeled with c didn't have any of the cells stained with pi indicating non of . inhibition (%) of free ga and gallic acid nanoparticles by β-carotene bleaching assay with statistical analysis using a one-way anova test. data shown are mean value ± sd (n = , *p-value ≤ . , **p-value ≤ . ). figure . ace inhibition (%) for free gallic acid and ganps after , , and min. data shown are mean value ± sd (n = , t test, *p-value ≤ . , **p-value ≤ . ). . therefore, with more dead cells due to ga/c nps, it will be challenging to have the real background color of c nanoparticles. the cell uptake study findings were consistent with the cytotoxicity study of cancer cells using the mtt reduction assay. the ga/c nps showed higher cellular uptake and toxicity in hepg and mcf cells by internalizing in the nucleus and cytoplasm of cancer cells. the mda-mb and ht cells had a lower fluorescence intensity of pi attributed to the lower toxicity of ga/c nps. www.nature.com/scientificreports/ the migration assay was carried out to assess the effect of ganps and free ga with the ic value of concentrations on mcf , mda-mb- , hepg , and ht . the pictures were captured using an inverted light microscope at h and h and the cell migration was calculated as percentages as illustrated in fig. . the findings of this study had shown that the ganps treatment significantly retarded the migration of hepg and mcf cells compared with mda-mb and ht cancer cells (fig. ). ganps had also demonstrated potent anti-proliferative properties in the wound zone. the ganps were prepared by the freeze-drying method with slight modifications including the high-pressure of homogenization at the pressure of , bar for cycles to formulate smaller particles. deionized water was used in the preparation to improve the dispersion of nanoparticles during the sonication process. the x-ray diffraction technique was carried out to investigate the amalgamation between gallic acid and gum arabic as shown in fig. . the physical mixture pattern as shown in the figure demonstrated ga peaks with a reduction of intensity. however, the diffraction peaks of the ganps were completely diffused due to the amorphous state and the lack of crystallinity of the nanostructure indicating the formation of a new state in the ganps. these changes were significantly related to the interaction between ga and gum arabic. furthermore, the lack of a clear peak in the x-ray diffraction of ga might be the cause for the appearance of non-crystalline large peaks. hence, the absence of crystallinity of pure compounds indicates the formation of a new phase, specifically by the conversion to the amorphous state. in the tem, the prepared nanoparticles had spherical elliptical shapes with the size distribution of individual particles ranging from to nm as shown in fig. . according to the zeta sizer, the nanoparticles size measurement was in a higher range between to nm in aqueous solution probably due to the dry phase of tem analysis. as demonstrated, the produced droplets had a small size to increase the surface area and the bioavailability of ga incorporated into gum arabic nanoparticles. the use of ga is limited by its fast metabolism, low bioavailability, and poor absorption. these pharmacokinetic challenges reduce the concentration of drug reached (cmax) and therefore lead to its rapid elimination . therefore, the encapsulation of ga into gum arabic nanoparticles was justified to effectively enhance its bioavailability and improve the therapeutic effects in cancer tissue by enhancing the uptake via transcellular or paracellular mechanisms . increasing the dispersion of the small nanoparticles aimed to increase the reactive surface area of gallic acid in the formulated nanoparticles . the release profile of gallic acid is depicted in fig. where the ganps released a burst of gallic acid with a remarkably higher percentage of . % at ph . compared to . % at ph . after , min. this release pattern may be attributed to the weak bonds between gallic acid and gum arabic that are prone to break faster in acidic ph. this feature might offer a therapeutic privilege in cancer tissue by enhancing the release of gallic acid among cancer cells. these cells are continuously exposed to rapid shifts in the acid-base balance due to the limited blood supply exposing them more to acidic ph . there was also a slow release of ga from ganps within , min at ph . and ph . . the release of ga was relatively slow at this phase compared with the initial release pattern. this pattern of release is probably due to the diminution of ga content in gum arabic www.nature.com/scientificreports/ nanoparticles. hence, there was a plateau stage that lasted till , min at ph . and ph . , in which about of . % and . % of ga was released from ganps at ph level of . and ph . , respectively. the release behavior of ga from ganps was dependent on the negative charge of phosphate anion which gave a higher affinity for ion exchange with ga as an encapsulated agent. at ph . , solubility decreased with an increase in particle binding properties due to the ionization properties of gum arabic. these findings are consistent with other studies in which the gum arabic polymer tended to disturb nps at ph close to and higher than . . antioxidants play a crucial role in the protection against the attack of free radicals thus helping to prevent cardiovascular and cancer diseases which represent the most common causes of mortality. oxidative stress significantly contributes to the etiology of these diseases through multiple pathways. therefore, it is vital to keep the physiological balance between antioxidants and free radicals through the administration of potent antioxidants. therefore, several in vitro assays were used in this study to confirm the antioxidant properties of ganps. the dpph test was based on the reduction of the purple-colored stable free radical dpph to the yellow diphenylpicrylhydrazine in the presence of free gallic acid and ganps. due to the existence of hydrogen-donating www.nature.com/scientificreports/ properties of ganps, the phenolic compound has been shown to quench oxygen derived from free radicals by donating an electron or hydrogen atom to the free radicals in various systems from in vitro studies , . trolox was used as a positive control to calculate the percentage of dpph inhibition. interestingly, ganps nanoparticles and gallic acid exhibited an in vitro scavenging activity in a dose-dependent manner at the concentrations gradient of - µg/ml in dpph as depicted in fig. . this property of nanostructured gallic acid was reported in the literature in which the underlying mechanism of dpph radical scavenging activity was attributed to the single electron transfert (set) and hydrogen atom transfer (hat) . set refers to a rearrangement of structure with a loss of a proton. usually, this mechanism produces quinone derivatives, resulting in the formation of a double bond with a change of proton signals. the gallic acid is stabilized after the reaction as a radical. in contrast, hat is attributed to the loss of proton with the stabilization action of nearby groups on the charge of the molecule. www.nature.com/scientificreports/ the quick proton exchange is considered as a contraction of the proton signals and it doesn't indicate any change in the chemical structure of the molecules . although the shown antioxidant effect of ga was lower than that of trolox, the formulated nanoparticles form had significantly enhanced these effects compared with free gallic acid due to the dpph scavenging properties of gallic acid combined with the hydroxyl units of gum arabic. from the data in fig. , it is apparent that the dpph test has examined the impact of free radical on test compounds. therefore, the strong absorption at nm in visible spectroscopy was influenced by the odd electron of dpph. the odd electron was paired in the presence of hydrogen of a free radical scavenger, indicating the capacity of gallic acid nanoparticles to scavenge free radicals without prior enzymatic activity . the antioxidant properties of gum arabic may be attributed to its hydroxyl groups, polypeptide, and its highly branched structure which may improve the stability and antioxidant properties of gallic acid within the nanoparticles . gum arabic is, therefore, a suitable encapsulant capable of forming stable nanoparticles for safe delivery and can be used as a potent antioxidant in future formulations. the in vitro cytotoxicity effect of the free ga and ganps in raw . cells was evaluated using mtt assay as demonstrated in fig. . the mtt results which reflect the number of viable cells confirmed that both ganps and ga did not have a significant cytotoxic effect on the raw . cells. in these cells, another antioxidant test was performed known as the nitric oxide inhibition assay which is important for evaluating the antioxidant and anti-inflammatory properties of potential agents. the assay is based on the production of nitrite metabolite from nitric oxide and its quantification using griess reagent . nitric oxide is an important molecule in the regulation of numerous physiological mechanisms such as blood pressure and pathological conditions such as inflammation, shock and neurotoxicity . due to the scavenging capacities of antioxidants, these agents are used to treat those deleterious disorders. the cells were challenged with lps which induces the expression of nitric oxide synthase protein. this enzyme in turn will enhance the production of nitric oxide from sodium nitroprusside and oxygen that are also induced by the oxidative challenge of lps. the production of peroxynitrite anion, a strong oxidant is based on the interactions between superoxide radical and no , . the results of the nitric oxide inhibition assay confirmed significant antioxidant effects in raw . cells. the free and nano encapsulated gallic acid at various concentrations accordingly inhibited the production of nitrite radical in the culture medium in a dose-dependent manner. as depicted in fig. , the hydroxyl radical scavenging activity of ganps was times higher than that of ga at concentrations ranging between . and µg/ml with ic values of . µg/ml and . µg/ml, respectively (p < . ). this result also confirmed that gallic acid maintained its antioxidant properties that were consistent with other reports . gallic acid was reported to have cyclooxygenase (cox- ) and nitric oxide synthase (inos) inhibitory properties in lpsinduced raw . cells modulating the pro-inflammatory cytokines such as il- , ifn-γ, and tnf-α . the ganps nanoparticles exhibited strong inhibition on no production at concentrations ranging from . to µg/ml. gum arabic was also reported to enhance the free radical scavenging properties of therapeutic agents by neutralizing and absorbing free radicals . nitric oxide produced as a proinflammatory mediator during the immunopathological phenomenon may cause chronic inflammation that is circumvented by macrophages. these cells were enhanced by gum arabic to exhibit antioxidant and hepatoprotective properties . the β-carotene bleaching assay is a quick and simple test based on the competition between ganps and β-carotene to neutralize the hydroperoxide-derived free radicals produced by the oxidation of linoleic acid. as shown in fig. , a concentration-dependent inhibition of β-carotene bleaching was recorded due to the presence of phenolic groups in gallic acid. the highest reducing power was recorded at the concentration of µg/ml, which exhibited approximately . % of antioxidant activity (p < . ). although both ga and ganps showed potent antioxidant activity; however, the nano encapsulated gallic acid appeared to be significantly more effective than free gallic acid. these findings may be attributed to the phenolic groups in gallic acid and the functional groups of gum arabic used as the coating material . oxidative stress is largely implicated in the pathogenesis of hypertension disease; where it is responsible for the activation of several mechanisms leading to vasoconstriction . therefore, the in vitro antihypertensive activity of the free and nano encapsulated gallic acid was assessed by measuring the ace-inhibitory capacity using a uv-visible spectrophotometer at nm. the inhibition of ace function is also one of the widely used therapeutic options in the hypertension disease as this enzyme is involved in the renin-angiotensin axis of blood pressure regulation which is largely involved in the pathogenesis of hypertension . the ace that is renowned now being the target of the recent coronavirus infection is the catalyst converting angiotensin i to angiotensin ii in lung and kidneys . this study is the first evidence confirming the ace inhibitory activity of ganps in addition to its antioxidant activity providing a promising treatment for hypertension disease. as shown in fig. , the ace-inhibitory capacities of gallic acid and ganps were time-dependent in which the ace inhibitory activity of ganps and gallic acid at min was . % and . %, respectively using µl of µg/ml treatment solution. the use of gum arabic polymer as nanocarrier improved the antihypertensive capacity of ganps that explains the high percentage of ace inhibition compared to that of free gallic acid. such antihypertensive effects were reported in animal models of hypertension disease using free gallic acid and gum arabic treatments. several phenolic natural products including gallic acid are known to have ace inhibitory activity but with a poorly understood mechanism . the enhanced antihypertensive property ganps could be imputed to the high amount of gallic acid released from the nanoparticles. the evaluation of cell viability is a common assay to determine the in vitro cytotoxicity of various biomaterials. the mtt assay is largely known as a rapid quantitative and colorimetric assay to evaluate cell viability for different purposes. the assay depends on the reduction of mtt salt by mitochondrial dehydrogenases inside the viable cells. upon reduction, the yellow tetrazolium salt changes to the corresponding purple formazan. in this study hepatocellular, colorectal, and breast adenocarcinoma cell lines were used in addition to normal epithelial www.nature.com/scientificreports/ cells. figure demonstrates the cytotoxic effect of ga, ganps in these cell lines after h of exposure. it has been observed that both ga and ganps exerted potent anti-proliferative effects in cancer cells with minimal toxicity in normal epithelial cells as shown in section a of fig. . as depicted, the ic values were significantly low in all the cancer cells compared with epithelial cells. these results were consistent with previous reports confirming the antineoplastic effects of gallic acid where it significantly increased apoptosis and apoptotic markers in esophageal and melanoma cancer cells by interfering with akt/mtor pathway with no apparent toxicity in normal cells , . interestingly as shown in fig. , hepatocellular cancer (hepg ) and breast cancer (mcf- ) cells appeared to be more sensitive to gallic acid nanoparticles than the mda-mb breast, and ht colorectal cancer cells. ganps at the concentration of . µg/ml caused a reduction to . % of viable hepg cells n compared to untreated cells (p < . ). the nanoparticles appeared to be more potent in hepg cells where a lower percentage of cell viability ( . %) was recorded at ( µg/ml) concentration compared to . % of free gallic acid. these findings could be attributed to the effect of gum arabic. in one study, gum arabic was found to have a targeting specificity towards hepatic cells by its galactose groups that attach with asialoglycoprotein receptors (asgrp) of liver cells in which the nanocarriers stimulated receptor-mediated endocytosis of the therapeutic agents . thus, the chain in gum arabic reacted as natural targeting ligands for asialoglycoprotein receptors (asgrp) on hepatocytes, which have improved the cytotoxicity effect of gallic acid nanoparticles. consequently, the high cytotoxicity effect of ganps against hepg cells might be attributed to the galactose units of gum arabic polymer. ganps had also demonstrated enhanced cytotoxicity in mcf breast cancer cells compared to mda-mb breast cancer cells. this significant potency in mcf cancer cells may be attributed to the modulation of estrogen receptor function as reported in several studies [ ] [ ] [ ] . these receptors are present in mcf cells and are potent targets approached in the treatment of breast cancer; however, the mda-mb cells lack these receptors. therefore ganps have a better selective index toward hepatic cancer cells and mcf breast cancer cells probably due to the chemical structure of gum arabic polymer and the selectivity toward hepatic and breast cancer cell receptors as demonstrated in fig. . the aforementioned toxicity findings were consistent with the cellular uptake study. in this assay, coumarin- (c ) was selected for the observation of ganps penetration to the cells with propidium iodide (pi) counterstain that is characterized by its penetration into nonliving cells. as shown in fig. , there was a significant increase in the uptake of pi among hepg and mcf cells compared with other cancer cells that may be attributed to the higher uptake of ganps leading to more apoptosis. the high fluorescence of pi as shown in fig. masked the green color of c due to the förster resonance energy transfer phenomena. as mentioned earlier these cells were more sensitive to ganps due to the chemical structure of gum arabic as confirmed in another report where the use of gum arabic assisted the delivery of curcumin and c into hepatocellular carcinoma cells . the enhanced uptake of pi was consistent with several reports of the proapoptotic and cell cycle arrest properties of gallic acid in cancer cells , , , . in these studies, the mechanism of action was revealed in the form of enhancement of caspase, endonuclease dependant pathways, extrinsic apoptotic pathways, and disruption of p kip /skp complexes that are crucial in regulating cell cycle procession in addition to modulating the level of il- . these findings confirm the therapeutic properties and the efficient cell internalization of gallic acid nanoparticles. thus the use of gum arabic as a carrier permit facile passage of gallic acid through the cell membrane as well as increase its solubility. ganps treatment was further evaluated for its effect on the migration of hepg , ht , mcf , and mda-mb cancer cells as a measure of invasiveness and metastasis . the treatment of cells using ganps has revealed a significant reduction in the percentage of migration of hepg and mcf cells. these findings are consistent with the reported antimetastatic properties of gallic acid in gastric, cervical, melanoma, and oral cancer cells that are attributed to the interference of gallic acid with nf-kappab activity, matrix metalloproteinases, ras-erk and the pi k/akt signaling pathways [ ] [ ] [ ] [ ] . this study is the first to report the augmenting property of gum arabic in encapsulating gallic acid improving the therapeutic outcomes of several in vitro assays. the potent antioxidant properties of gallic acid were significantly enhanced by gum arabic coating attributed to the chemical properties of both compounds; this study also revealed several antineoplastic properties offered by the nanoformulation. the ganps enhanced the selective uptake in hepatic cells enhanced the cytotoxicity in breast cancer (+ estrogen receptor) and confirmed its proapoptotic effects. therefore ganps formulation is a potential candidate for the prevention and treatment of hepatic and breast cancers. extrapolation of the in vitro cytotoxicity effects of ganps to in vivo cytotoxicity effects demands further investigation in light of its application as a cancer chemotherapeutic agent. materials. gum arabic polysaccharide was bought from ennasr company (sudan). gallic acid (ga) used in this study was purchased from sinar scientific company (malaysia). dexamethasone, trolox, , -diphenyl- -picrylhydrazyl (dpph), hippuryl-histidyl-leucine sulfonamide, angiotensin-converting enzyme, n-( -naphtyl) ethylenediamine dihydrochloride, sodium nitrite, linoleic acid, dulbecco's modified eagle's medium (dmem), fetal bovine serum (fbs) and streptomycin were purchased from sigma-aldrich company (malaysia). tween , b-carotene, quercetin, and α-tocopherol were purchased from r&m company (china). hepg , mcf , mda-mb , ht , mcf- a, and raw . cells were obtained from atcc (american type culture collection). throughout all experiments, deionized water was used. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ preparation of gallic aid nanoparticles. the ganps were synthesized using the freeze-drying technique with few modifications. the aqueous solution containing gallic acid (ga) and gum arabic in : molar ratio was obtained by dissolving . g of ga in ml deionized water containing . g/ml of gum arabic. the mixture was kept under mild agitation at room temperature for h. the final suspension was subjected to a high-pressure homogenizer at a pressure of , bar for cycles and was then frozen at − °c. the final product was freeze-dried for h at − °c. in the confocal microscope study of cell uptake and apoptosis, the ganps labeled with c was prepared using a modified protocol of the same freeze-drying technique as described in some reports . an aqueous solution containing gallic acid (ga) and gum arabic in : molar ratio was obtained by dissolving . g of ga and . mg of coumarin- in ml of ethanol then mixed with . g of gum arabic dissolved in ml of dmso. the mixture was kept under mild agitation at room temperature for h. the final product was then freeze-dried for h at − °c. preparation of the physical mixture. the physical mixture of gallic acid (ga) and gum arabic was performed in a : ratio as lyophilized complex. ga and gum arabic were admixed into homogeneous powder using pestle and mortar. characterization of ganps nanoparticles. x-ray diffraction (xrd). the ganps, gum arabic, and ga powder samples were investigated using shimadzu refractometer, xrd (tokyo, japan). powder x-ray diffraction (xrd) patterns of ganps, gum arabic, and ga were recorded using cuk α incident beam, λ = . Å, and voltage of kv. analysis of samples was performed at θ = °- ° and a scan speed of ° per minute. size and zeta potential. the zeta potential and size of ganps were characterized using a zeta sizer (malvern nano-zs-zs, zeeman) with dynamic size. www.nature.com/scientificreports/ deionized water were added to the flask with vigorous shaking for min. blank emulsion, devoid of β-carotene was prepared. aliquots ( µl) of β-carotene emulsion were transferred into the -well plate containing µl of ga and ganps at various concentrations and incubated in the dark at °c. different concentrations of α-tocopherol were used as a positive control reflecting nearly complete inhibition of β-carotene bleaching. the absorbance measurements were recorded immediately at nm at min intervals for min. the antioxidant activity (aa) was assessed using the following equation: where a , a t , and a c , a tc are the absorbances of sample at t = , t = min and absorbance of control at t = , t = min. the anti-hypertensive assay using angiotensin-converting enzyme (ace) inhibition assay. the in vitro ace inhibition activity of gallic acid ga in gum arabic nanoparticles was assessed in vitro based on the conversion of hippuryl-histidyl-leucine to hippuric acid in the presence of ace enzyme . one hundred microliters of gallic acid ga and ganps were mixed with ace ( µl, ph . ) and incubated at °c for min. next, . mm of hippuryl-histidyl-leucine ( µl) was added to the mixture and incubated for , , and min. the enzymatic reaction was halted after the addition of µl of mol/l hci. hence, ml of ethyl acetate was added to remove the resultant hippuric acid. the solvent was evaporated at °c and re-dissolved in ml of n of nacl. the concentration of hippuric acid was evaluated by measuring the absorbance at nm. a blank devoid of ga and ganps was prepared at background subtraction. cell culture and cytotoxicity assay. a list of cancer cell lines selected to demonstrate the antineoplastic properties using the mtt assay which is based on the enzymatic reduction of tetrazolium salt -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) by active living cells. the cancer cell lines were all of an epithelial type representing a list of common cancer types including colorectal adenocarcinoma (ht ), hepatocellular cancer (hepg ), breast adenocarcinoma (mda-mb and mcf ), and mcf- a breast epithelial cell lines. all the cell lines were free of mycoplasma infection and the number of passages used in the experiments ranged between ( ) ( ) ( ) . the cells were maintained in dmem supplemented with % of penicillin-streptomycin and % of fetal bovine serum (fbs). the cells were harvested using trypsin-edta solution and examined using an inverted microscope (olympus ck ). a hemocytometer was used to determine cell density. standard solutions of ga and ganps were prepared and dissolved to concentrations ranging between . - µg/ml. this assay was performed as described in our previous reports to assess cytotoxicity activity of treatments in the selected cell lines . two hundred microliters of cell suspension with a density of × cells/ ml were placed into each well of -well plate and later incubated at % co and °c for h. after incubation time, the medium was replenished and the cells were treated with ga and ganps at concentrations ranging between . and µg/ml for cancer cells and . - , µg/ml for normal cell lines to determine the ic values. chemotherapeutic controls were also used with different concentration gradient from . to µg/ml ( -fluorouracil for ht , tamoxifen for hepg cells, and doxorubicin for mcf , mcf- a, and mda-mb . the last row of the -well plate was left for the control experiment under similar conditions. after h of postincubation, µl of mg/ml of mtt solution was added to each well, in which the plate was covered with aluminum foil and incubated for h. the medium was removed and replaced with µl dmso to dissolve the remaining purple formazan precipitate. the absorbance was recorded using an elisa reader at nm. this colorimetric assay focused on the reduction of mtt to purple formazan by the mitochondrial enzymes of the metabolically active cancer cells . the ic was calculated from the concentration of drug that inhibits % of cell growth. the experiment also included the mcf- a normal breast cell lines in the mtt assay to calculate the selective index for each of the cancer cell lines. all experiments were carried out in triplicate and the outcomes are presented in standard deviation and mean values. cellular uptake of ganps. the study also included a qualitative assessment of cell uptake in hepg , mcf , mda-mb , ht cancer cell lines based on the use of coumarin (c ) and propidium iodide (pi) fluorescent dyes. the use of both dyes in this experiment was adapted from win et al. . c is widely used for tracking of nanoencapsulation emitting green light at nm . while pi fluorescence dye was used as a counterstain selective to nonviable cells and cells with compromised cell membranes where it binds with base pairs of double-stranded dna giving the unique red fluorescence emission while sparing viable cells with intact cell membranes . the experiment started by culturing the cancer cells on glass coverslips with a cell suspension of cells/ cm in complete media inside the co incubator at °c for h allowing cell adherence to the coverslip. the cells were then subjected to the ic values of ga/c nps in serum-free medium for h. consequently, the cells were incubated with pbs buffer containing pi ( µg/ml) for min at room temperature after washing with pbs buffer; then washed again with pbs buffer followed by fixation with % ethanol in the freezer (− °c) for min. the coverslips were then examined using the confocal microscope with excitation wavelength nm under multichannel mode. scratch migration assay. the in vitro cell migration assay was performed by scratch assay to assess cell mobility following the protocol adapted from kovarikova et al. . the hepg , mcf , mda-mb , and ht cells were seeded in a -well plate with the cell density of ( × ). upon reaching % of confluence level, the scratch was carried out using a yellow pipette tip with an average diameter of mm. the cells were then washed www.nature.com/scientificreports/ times with pbs and treated with ganps at concentrations of . - µg/ml and were further incubated for h. based on the dino eye application connected to the inverted microscope, a marker line was used to select the position of the picture captured. various pictures were captured at h and h after the treatment at a magnification of ×. the experiment was performed in triplicate. hence, the percentage of migration of the aforementioned cell lines was calculated using the following formula: statistical analysis. t tests and one-way anova test followed by the holm-sidak multiple comparisons were performed using graphpad prism version . . 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migration and invasiveness the publication of this article was funded by the qatar national library. the authors would also like to acknowledge universiti putra malaysia for awarding the funds toconduct this research under project number gp-ips/ / . the authors declare no competing interests. correspondence and requests for materials should be addressed to w.n.i. or s.a.h.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -sqxlnk e authors: park, jiyeon; yoo, seung yeon; ko, jae-hoon; lee, sangmin m.; chung, yoon joo; lee, jong-hwan; peck, kyong ran; min, jeong jin title: infection prevention measures for surgical procedures during a middle east respiratory syndrome outbreak in a tertiary care hospital in south korea date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: sqxlnk e in , we experienced the largest in-hospital middle east respiratory syndrome (mers) outbreak outside the arabian peninsula. we share the infection prevention measures for surgical procedures during the unexpected outbreak at our hospital. we reviewed all forms of related documents and collected information through interviews with healthcare workers of our hospital. after the onset of outbreak, a multidisciplinary team devised institutional mers-control guidelines. two standard operating rooms were converted to temporary negative-pressure rooms by physically decreasing the inflow air volume (− . pa in the main room and − . pa in the anteroom). healthcare workers were equipped with standard or enhanced personal protective equipment according to the mers-related patient’s profile and symptoms. six mers-related patients underwent emergency surgery, including four mers-exposed and two mers-confirmed patients. negative conversion of mers-cov polymerase chain reaction tests was noticed for mers-confirmed patients before surgery. mers-exposed patients were also tested twice preoperatively, all of which were negative. all operative procedures in mers-related patients were performed without specific adverse events or perioperative mers transmission. our experience with setting up a temporary negative-pressure operation room and our conservative approach for managing mers-related patients can be referred in cases of future unexpected mers outbreaks in non-endemic countries. high-volume healthcare facility, which is how the previous south korea outbreak occurred . moreover, there may be very few hospitals that are prepared to provide perioperative care for mers patients. therefore, herein, we share our experience of providing infection prevention and control measures for surgeries for mers-related patients in our hospital. during the mers outbreak in our hospital, six mers-related patients underwent surgery including three possibly exposed patients, one directly exposed patient, and two mers-confirmed patients who recovered from the disease. all patients were negative during two preoperative mers screenings using real-time reverse transcription polymerase chain reaction (rrt-pcr) . figure shows the total number of surgeries performed during the outbreak period at our hospital and the distribution of mers-related patients undergoing surgery. temporary set-up of a negative-pressure operating room. the operating rooms in our hospital were generally positive-pressure environments, and we had no permanent negative-pressure operating rooms. because a negative-pressure operating room is the optimal environment to prevent airborne virus spreading to adjacent areas , two of our operating rooms in the main operating suite of the hospital were temporarily converted into negative-pressure operating rooms to perform surgical procedures on mers-related patients. operating rooms no. and were selected because they were connected to each other, but each room had separate atmospheric air inlets and exhaust systems. they also had separate air-conditioning and humidification systems. of the two connected rooms, one was used as the main operating room and the other was used as the anteroom where healthcare workers (hcws) applied and removed the personal protective equipment (ppe). in each room, fresh air was supplied from an inlet duct and discharged outside through the exhaust duct (fig. ) . because a constant exhausting air volume was maintained through the outlet duct, negative pressure in the operating room was achieved by decreasing the inflow air volume that entered through the inlet duct. first, the blades of the air volume control damper in the inlet duct were closed as much as possible (fig. ) . however, because the damper was not intended to be air-tight, the inflow volume to the operating room did not decrease sufficiently. second, as an additional measure to decrease the inflow volume, we opened the access hole in the inlet duct, which was originally used for duct inspection purposes (fig. ) . finally, a smoke test was carried out to ensure negative pressure. the room pressure was maintained at − . pa in the main operating room and at − . pa in the anteroom (fig. ) ; − . pa is below the negative pressure room standard of − . pa . www.nature.com/scientificreports www.nature.com/scientificreports/ airflow in both rooms reached - air exchanges per hour, according to airflow velocity measurements with an anemometer (ebt balometer; tsi alnor ® , minnesota, united states). in this environment, removing airborne contaminants requires minutes for % efficiency and minutes for . % efficiency . therefore, minutes of room ventilation was required after aerosol forming high-risk procedures, such as endotracheal intubation or extubation , . the cleanliness level of each room was also measured using a particle counter (tsi ; tsi, united states): main operating room = , and the anteroom = , , which were much lower than the institutional target level of < , for general surgery (fig. ) . cleanliness level was defined as the number of particles smaller than . µm in . m . equipment preparation and disinfection. all built-in instruments such as computers, telephones, and ventilators were covered with plastic paper. sufficient amounts of drugs, fluids, and other equipment were prepared in the operating room before surgery, and other unnecessary equipment was moved out. additionally, we used disposable equipment, when possible. high efficiency particulate air (hepa) filters were installed in the breathing circuits, both on the inspiratory and expiratory limbs of the ventilators and at the patient's site that connected to endotracheal tube. after operations with mers-exposed patients, minutes of room ventilation was followed by surface disinfection with diluted chlorine bleach ( ppm) , . cleaners wore standard ppe while disinfecting surfaces. for mers-confirmed patients, surface disinfection was performed twice. institutional guidelines for perioperative management of mers-related patients. during the mers outbreak, we set the following principles for perioperative management of mers-related patients: all elective surgeries for mers-confirmed patients were postponed to reduce the risk of potential in-hospital transmission. for mers-exposed patients, surgical procedures were delayed until after the potential incubation period of days . when a mers-related patient required an urgent or emergency operation, mers-cov pcr tests were performed twice with distinct specimens preoperatively, to account for asymptomatic mers patients or delayed positive conversion in symptomatic mers-exposed patients. for patients with ambiguous pcr results or without a pcr test, operations were performed according to the management guidelines for mers-confirmed patients. all the surgical procedures for mers-related patients were performed in the last order of the day as possible. perioperative protection level for hcws. when an operation for a mers-related patient was scheduled, the division of infectious diseases and infection control department confirmed the protection level of the hcws, according to institutional guidelines (table ). in principle, standard ppe was applied to hcws who cared for asymptomatic mers-exposed patients. standard ppe includes surgical gloves, surgical gowns, eye shields, and n respirators. while managing mers-confirmed or mers-exposed patients with mers-associated symptoms including fever, myalgia, respiratory symptoms, or diarrhea, hcws implemented enhanced ppe, which included coverall clothes with head cover, shoe covers, goggles, two pairs of surgical gloves, and powered air purifying respirator (papr) or n respirators. although we performed preoperative mers-cov pcr screening, enhanced ppe was still recommended when managing symptomatic mers-exposed patients regardless of their pcr results. anesthesiologists were recommended to apply enhanced ppe (including papr from the middle of the outbreak) when managing all mers-related patients because they were most directly exposed to the aerosol-producing high-risk procedures, such as endotracheal intubation and extubation. only minimal numbers of hcws were present in the operating room. institutional education regarding the precise use of ppe was provided to the all associated hcws and they were assisted by skilled nurses in the operating room during the ppe donning and doffing processes. patient transfer for operation. mers-related patients were transferred directly to the negative-pressure main operating room through an exclusive path and elevator by a physician wearing proper ppe. the walls and the floor of the passageways and the elevator were covered with plastic paper. mers-related patients wore a www.nature.com/scientificreports www.nature.com/scientificreports/ surgical mask during transfer. because only anesthesiologists wore enhanced ppe when in proximity to asymptomatic mers-exposed patients, minutes of room ventilation was performed after anesthetic induction, including endotracheal intubation. the surgical team then entered the main operating room through the anteroom. in the cases of symptomatic mers-exposed patients or mers-confirmed cases, all hcws wore enhanced ppe and the -minute ventilation time was not required. after completion of operation procedures, patients who were moved to the general ward recovered in the main operating room without going through the post-anesthesia care unit. thirty minutes of room ventilation was performed after tracheal extubation. a physician in the main operating room sent the patient into the corridor, while the other physician outside the main operating room wearing ppe took over and transferred the patient to the general ward directly through the exclusive pathway (fig. ) . patients moving to the intensive care unit (icu) were transferred while remaining intubated. before transfers, we injected patients with a sufficient amount of intravenous muscle relaxant and sedative drugs to prevent coughing or movement and we applied a portable ventilator or bag-valve mask with a hepa filter to the patient. operations for six mers-related patients. the details of the six mers-related patients undergoing surgery are presented in table . two patients had operations during phase and four patients during phase of the outbreak (fig. ) . the negative-pressure operating room was set up to be used from phase . regarding ppe levels for the hcws attending these six patients, standard ppe was applied during management of patient a (asymptomatic mers-exposed patient), while anesthesiologists wore enhanced ppe for high-risk procedures (tracheal intubation). enhanced ppe was applied to hcws for patient b because the patient was symptomatic and still within the two-week incubation period, even though both pcr results were negative. enhanced ppe, including papr to reduce risk of mers-transmission, was applied for patient c who underwent surgery in the middle of the outbreak (phase ). papr provides more perfect sealing and protection of the head surface. patients d and e had documented mers-cov infection and their recovery was confirmed with symptom resolution and two negative mers-cov pcr tests. however, enhanced ppe with papr was applied to hcws because the infection risk could not be eliminated during exposure to a large amount of body fluid, especially during cesarean section (patient d). after spinal anesthesia, she recovered in the main operating room and was transferred directly to the general ward. patient f had a history of exposure to a mers patient in the emergency room and was isolated due to a fever. enhanced ppe was applied to the hcws for patient f and she underwent surgery with only one set of negative pcr results because of her emergency condition. mers, as well as sars, is associated with coronaviruses, both of which have high affinity for the lower repiratory tract and easily produce severe pneumonia [ ] [ ] [ ] [ ] [ ] . although mers has lower human-to-human transmission potential and has resulted in fewer large outbreaks than sars, there may be occasional amplification of clusters in healthcare settings [ ] [ ] [ ] . moreover, mers case fatalities are reported to be much higher than sars ( - % for mers and - % for sars) , , , , . unlike sars, ongoing small and large mers outbreaks in the arabian peninsula foster potential future mers outbreaks in non-endemic countries. however, there is likely to be a very limited number of hospitals that are prepared with negative-pressure operating rooms, except for a few hospitals in hong kong that experienced the sars outbreak . almost all hospitals generally have positive-pressure operating rooms and they may experience an outbreak without facilities that are prepared for perioperative management of mers patients, as our hospital did in . one of the highlights of our experience during the outbreak was the temporary set-up of a negative-pressure operation room with an adequate pressure gradient (≥ . pa) by modifying two connected operating rooms according to us centers for disease control and prevention (cdc) guidelines . continuous negative pressure was maintained in the main operating room (− . pa) and the anteroom (− . pa). this temporary setting was possible because the two adjacent rooms had separate atmospheric air inlets and exhaust systems. although we could not measure the airflow pattern or dispersion of infectious particles directly , the cleanliness levels in both operating rooms were , particles, well below the institutional target cleanliness for general surgery (< , particles). although the precise route of mers-virus transmission is currently not clearly understood , mers, as well as sars, is known to spread by direct contact with infectious material, such as large respiratory droplets, and also by airborne routes , . touching contaminated objects may also be a source of transmission; this is different from tuberculosis, which is transmitted by airborne routes , . therefore, when performing procedures that generate aerosols, such as endotracheal intubation, in patients with mers or sars, hcws must wear enhanced ppe, including gloves, a gown, either a face-shield that fully covers the front and sides of the face or goggles, and respiratory protection at least as protective as an n filtering face piece respirator , , . when removing ppe, care should also be taken not to contact contaminated materials. considering potential aerosol generation in operating rooms and the transmission risk of mers-cov while changing ppe, the temporary modification of an operating room to a negative-pressure room with an anteroom should provide suitable protection for hcws participating in operations on mers-related patients . a second highlight of our experience is the highly conservative application of ppe to hcws. at the time of the outbreak, there were no specific guidelines for perioperative management . therefore, we used a conservative approach based on our experience and previous reports. first, although the previous guidelines recommended that asymptomatic mers-exposed patients be managed as general patients undergoing surgery, we applied standard ppe to hcws and we performed mers-cov pcr screening twice. although mers progressed gradually after symptom onset , we could not exclude the possibility that asymptomatic mers-exposed patients had the potential to develop symptomatic disease perioperatively. moreover, we observed development of mers after the known incubation period of days in an immunocompromised host , ; thus, the possibility of exceptional cases could be considered. furthermore, a certain proportion of asymptomatic mers-exposed patients could actually be asymptomatic mers-infected patients. approximately % of laboratory confirmed mers patients have been classified as asymptomatic or having nonspecific mild symptoms at the time of testing . the potential for transmission from asymptomatic mers-cov pcr-positive person is currently unknown, but there are reports about prolonged viral rna detection in the upper respiratory tract in asymptomatic pcr-positive person . considering these points, it would be reasonable to prepare more conservatively than the existing guidelines call for. www.nature.com/scientificreports www.nature.com/scientificreports/ another point on which our preparations differed from the guidelines was the application of enhanced ppe, which emphasizes full protection of the body surface with a hooded coverall. during the outbreak in our hospital, mers transmission events occurred among hcws who were equipped with standard ppe, including n masks. transmission may have occurred after possible contamination of uncovered head or face surfaces . therefore, if a patient with a mers contact history had mers-associated symptoms, applying enhanced ppe (either a n respirator or papr) during surgery would be appropriate for hcws because numerous droplets and aerosols may be produced during airway interventions. because the n may fit inadequately if worn for a long time or after movement during surgery, wearing papr will be more beneficial. however, unlike hcws dealing with ebola virus, impermeable and fluid-resistant gowns are not required because body fluids are not infectious as with ebola virus diseases , , , . our experience was limited in that, as a mers outbreak outside the endemic country, we did not have an opportunity to perform surgical procedures in actively virus-shedding mers-infected patients. additionally, our infection-prevention protocols would be too conservative to apply in mers-endemic situations. however, considering the potential risk of infected hcws, preventing mers transmission is extremely important in the management of a mers outbreak. importantly, our experience can be generalized to other non-endemic countries for managing potential outbreaks of emerging respiratory diseases. in the era of globalization, a mers outbreak can occur in any country outside the middle east. a very limited number of hospitals are equipped with negative-pressure operating rooms, and therefore, most hospitals are likely to experience a mers or other outbreak in an unprepared circumstance. we hope that this report will help other hospitals in preparing for future mers outbreaks and infection control in unexpected conditions. this study was based on all available data at the samsung medical center from the mers outbreak and on interviews with hcws associated with the outbreak. the study was approved by samsung medical center institutional review board. the documents for review included electronic medical records of mers-related patients who underwent operative procedures and institutional guidelines for perioperative management of mers-related surgical patients. the mers guidelines were prepared through multidisciplinary team discussions that were held by our hospital's infection control department during and after the mers outbreak. the records about the temporary set-up of a negative-pressure operating room were also reviewed. we also collected data through interviews with hcws who participated in surgery and anesthesia for mers-related patients. we defined mers-related patients as those who were possibly or directly exposed to mers or who had a previously confirmed mers diagnosis . in brief, patients who had a potential but unconfirmed close contact history with a mers patient were defined as possibly exposed patients, and they were allowed to continue their normal activities until mers-like symptoms developed. directly exposed patients included those who had close contact with a known mers patient and who did not wear proper ppe; these patients were isolated in their homes or in private negative-pressure rooms at our hospital. because the number of mers-infected patients continuously increased at our hospital, we partially closed the hospital on june , at which point outpatient-care clinics were closed and the emergency department was only available for life-threatening emergencies. all elective surgeries were postponed if possible . we defined the early phase of the outbreak (before june ) as phase and the middle phase of the outbreak (from june ) as phase . for mers-cov pcr tests, either sputum or nasopharyngeal swab samples were collected and sputum samples were preferred if available . sputum was collected directly into a sterile, leak-proof, screw-capped sputum collection sterile container and nasopharyngeal swab was collected with an eswab ( c, copan diagnostics inc., murrieta, ca, usa). clinical samples were screened by rrt-pcr testing with amplification targeting the upstream e region (upe) and confirmed by subsequent amplification of the open reading frame (orf) a using powerchek ™ mers real-time pcr kits (kogene biotech, seoul, korea). all rrt-pcr reactions were performed using the fast real-time pcr system (applied biosystems, foster city, ca, usa). the pcr reaction was performed in a total volume of μl ( μl pcr reaction mixture and μl template rna). thermocycling conditions included a step at °c for min, followed by °c for min and then cycles of s at °c and s at °c. positive viral template control and no-template control were included in each run. the glyceraldehyde- -phosphate dehydrogenase (gapdh) gene was amplified simultaneously as a heterologous internal control to monitor pcr inhibition. a positive test result was defined as a well-defined exponential fluorescence curve that crossed the cycle threshold (ct) < cycles for both upe and orf a. a sample was considered "equivocal" if the upe result was positive but the ct value for orf a was > and < . we interpreted the result as "indeterminate" if ( ) the upe result was positive but the ct value for orf a was undetected or if ( ) the ct value for upe was > and < . institutional review board statement. the study was performed in accordance with the declaration of helsinki and experimental protocols were revised and approved by irb at samsung medical center. (irb no. smc - - - ). informed consent statement. irb at samsung medical center has approved the waiver of patient consent form because of the retrospective nature of this study. patient confidentiality is maintained at all time in accordance with samsung medical center policies. middle east respiratory syndrome: new disease, old lessons isolation of a novel coronavirus from a man with pneumonia in saudi arabia contact investigation for imported case of middle east respiratory syndrome enhanced mers coronavirus surveillance of travelers from the middle east to england laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities family cluster of middle east respiratory 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hospital-associated outbreak surgical protocol for possible or confirmed ebola cases anesthetic implications of ebola patient management: a review of the literature and policies j.p. this author helped with data collection, data analysis, writing of the first draft and revision of the manuscript, and archiving of the study files. s.y.y. this author helped with data analysis, discussion of results, writing and revision of the manuscript, archiving of the study files and approval of final version. j. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.r.p. or j.j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -jidvkij authors: robitaille, alexa c.; caron, elise; zucchini, nicolas; mukawera, espérance; adam, damien; mariani, mélissa k.; gélinas, anaïs; fortin, audray; brochiero, emmanuelle; grandvaux, nathalie title: dusp regulates apoptosis and cell migration, but not the jip -protected cytokine response, during respiratory syncytial virus and sendai virus infection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: jidvkij the host antiviral response involves the induction of interferons and proinflammatory cytokines, but also the activation of cell death pathways, including apoptosis, to limit viral replication and spreading. this host defense is strictly regulated to eliminate the infection while limiting tissue damage that is associated with virus pathogenesis. post-translational modifications, most notably phosphorylation, are key regulators of the antiviral defense implying an important role of protein phosphatases. here, we investigated the role of the dual-specificity phosphatase (dusp ) in the host defense against human respiratory syncytial virus (rsv), a pathogenic virus of the pneumoviridae family, and sendai virus (sev), a model virus being developed as a vector for anti-rsv vaccine. we found that dusp is upregulated before being subjected to proteasomal degradation. dusp does not inhibit the antiviral response, but negatively regulates virus-induced jnk/p mapk phosphorylation. interaction with the jnk-interacting protein scaffold protein prevents dephosphorylation of jnk by dusp , likely explaining that ap- activation and downstream cytokine production are protected from dusp inhibition. importantly, dusp promotes sev-induced apoptosis and suppresses cell migration in rsv-infected cells. collectively, our data unveils a previously unrecognized selective role of dusp in the regulation of tissue damage and repair during infections by rsv and sev. induction of dusp during sev and rsv infection. to first assess a potential function of dusp , we analyzed dusp expression during the course of sev and rsv infection in a cells by immunoblot. dusp was induced by about fold at h of rsv infection and was returned to basal levels at h post-infection (fig. a) . dusp steady state levels remained unchanged during sev infection (fig. a) . because dusp turnover was previously reported to be affected by the ubiquitin-proteasome pathway , we further analyzed dusp levels during sev and rsv infection in the presence of the s proteasome inhibitor mg . in the presence of mg , induction of dusp is detectable in both infections and the overall dusp levels were markedly increased compared to control cells (fig. b) . these results imply that dusp protein levels are induced in response to sev and rsv infection and that dusp is subjected to proteasome-mediated degradation. dusp does not affect sev and rsv replication. next, we sought to determine whether dusp belongs to the numerous negative regulators of the antiviral response induced in response to virus infections , . first, because of their importance in the control of the ifn-mediated antiviral defense, we evaluated whether dusp has an impact on the signaling cascades leading to nf-κb and irf activation. considering previous characterization of nf-κb activation in the context of rsv infection , iκbα-s and p -s phosphorylation were analyzed. activation of irf was monitored through detection of irf -s phosphorylation . ectopic expression of dusp in a cells did not alter iκbα, p or irf phosphorylation profiles observed during the course of sev ( fig. a,b ) or rsv infection (fig. c) , showing that dusp does not play a role in the regulation of these defense pathways. additionally, ectopic expression of dusp in a cells neither significantly altered sev n mrna levels measured by qrt-pcr ( fig. d top panel) nor the de novo production of rsv virions quantified by plaque assay (fig. d bottom panel) . consistently, rnai-mediated silencing of dusp did not alter the capacity of rsv to replicate in a cells (fig. e ). together these data indicate that dusp does not negatively regulate the capacity of the cell to mount an antiviral response to restrict rsv or sev replication. because dusp preferentially dephosphorylates jnk and p in a variety of inflammatory contexts , , we next sought to evaluate the role of dusp in the negative regulation of rsv-and sev-induced jnk and p activation. first, we monitored the effect of ectopically expressed dusp on the sev-and rsv-induced jnk t /y and p t /y phosphorylation in a cells. as expected, immunoblot analysis revealed that sev (fig. a,b) , and to a lesser extent rsv (fig. c) , induce jnk and p phosphorylation. importantly, dusp expression abrogates jnk and p phosphorylation ( fig. a-c) . subsequently, we confirmed the role of dusp through analysis of the impact of dusp silencing on the profile of virus-induced jnk and p phosphorylation. immunoblot analysis confirmed the efficiency of dusp silencing in a cells throughout the course of sev and rsv infection (fig. ) . consistent with a role of dusp in the dephosphorylation of jnk and p , we observed a marked increase of jnk and p phosphorylation in the absence of dusp during sev (fig. a ) and rsv infection (fig. b) . altogether, these observations reveal a critical role of dusp in the negative regulation of jnk and p activation during rsv and sev infection. interaction with jip protects jnk from dusp dephosphorylation. scaffolding proteins of the jip family interact with distinct pools of jnk to specifically place them next to their upstream activators and downstream substrate mediating specific functional pathways , . therefore, we aimed to investigate the role of jip proteins in rsv-and sev-induced jnk activation and regulation by dusp . the jip family is composed of members, jip - , with distinct expression profiles. here, we assessed the role of jip and jip in a cells based on previous reports indicative of their expression in lung cells [ ] [ ] [ ] [ ] . in contrary, jip was shown to be restricted to human brain and human jip was found only in testis , . first, we tested the interaction between jnk and jip or jip during sev and rsv infection. ectopically expressed jip coimmunoprecipitated with endogenous jnk, both in uninfected and sev-and rsv-infected cells (fig. a ). by contrast, jip did not interact with jnk (fig. a ). since we demonstrated the existence of a jip /jnk complex, we examined the role of jip in the regulation of dusp dephosphorylation during sev and rsv infection. a cells were cotransfected with jip together with empty or dusp -expressing constructs before infection with sev (fig. b) or rsv (fig. c ). confirming our finding (fig. ) , ectopic expression of dusp resulted in inhibition of sev-and rsv-induced jnk phosphorylation (fig. b,c) . importantly, when jip was coexpressed with dusp , the levels of phosphorylated jnk was increased . and times compared to cells transfected with dusp only during sev and rsv infection, respectively (fig. d ). collectively, these data demonstrate that jip interacts with jnk and that recruitment of jnk by jip prevents dephosphorylation by dusp during sev and rsv infection. in a-c, immunoblot analyses were performed to detect dusp , phosphorylated p (p -p), total p , phosphorylated iκbα (iκbα-p), total iκbα, phosphorylated irf (irf -p) and total irf . infection was monitored using anti-sev or anti-rsv antibodies (n proteins are shown). detection of actin was used as a loading control. the data are representative of three different experiments. samples that are compared derive from the same experiment. [ ] [ ] [ ] [ ] . a main function of jnk and p in the host defense against virus infection is to regulate the activation of ap- (atf- /c-jun) that participates in the enhanceosome structure controlling ifnβ expression and contributes to the regulation of proinflammatory cytokine expression , , . the observation that jip prevents jnk from being dephosphorylated by dusp suggests a model in which ap- -dependent functions would be protected from the negative regulation of dusp during the infection. to confirm that jnk/p were involved in virus-induced ap- activation and downstream cytokine expression, a cells were infected with sev in the absence or presence of jnk (sp ) and p (sb ) inhibitors. immunoblot analysis showed that inhibition of jnk and p significantly decreased the levels of phosphorylated c-jun and atf- (fig. a) . accordingly, inhibition of jnk and p significantly impaired the induction of ifnβ, ccl , cxcl and il- by sev, quantified using luminex-based assays ( fig. d and supplemental figure a ). these results confirmed the role of the jnk/p -ap- signaling cascade in the regulation of sev-mediated cytokine induction and therefore prompted us to evaluate the impact of ectopic expression of dusp on this pathway. although ectopic expression of dusp prevents jnk and p phosphorylation, sev-and rsv-induced phosphorylation of c-jun and atf- remained similar to control cells (fig. b,c) . analysis of the expression profile of a panel of cytokines using luminex-based multiplex assays during sev infection revealed that none of them were significantly altered following dusp ectopic expression ( fig. e and supplemental fig. b) . additionally, silencing of dusp did not affect the levels of ifnβ, ccl , ccl , cxcl and il- induced by sev ( fig. f and supplemental fig. c ). measurement of sev-induced ifnb, cxcl and ccl expression levels revealed no impact of dusp ectopic expression, excluding a role of dusp in the transcriptional regulation of cytokines (supplemental fig. ). similar results were observed during rsv infection, with silencing of dusp having no impact on the induction of ifnβ, ccl , ccl , cxcl and il- ( fig. g) . altogether these observations point to a selective role of dusp in the negative regulation of jnk and p during sev and rsv infection, leaving the ap- pathway and downstream cytokine production intact. dusp promotes sev-induced apoptosis independently of jnk/p inhibition. jnk/p mapk pathways are also known to play a critical role in the regulation of apoptosis, being either pro-or anti-apoptotic depending on the context [ ] [ ] [ ] [ ] [ ] [ ] . a mavs/jnk pathway has been shown to be indispensable for sev to induce apoptosis , . therefore, we sought to assess the impact of dusp on the jnk/p -dependent apoptosis triggered by sev. a cells were transfected with sictrl or sidusp followed by treatment with dmso (vehicle) or sp and sb to inhibit jnk and p before sev infection. quantification of annexin v+ cells, corresponding to early and late apoptosis, was performed by flow cytometry analysis of annexin v/pi stained cells (fig. a) . confirming a moderate role of jnk/p in sev-induced apoptosis, inhibition of jnk/p reduced the number of annexin v+ cells by %. unexpectedly, dusp silencing also similarly decreased sev-induced apoptosis by %. importantly, when dusp silencing and jnk/p inhibition were combined, sev-induced apoptosis was decreased by %, showing an additive effect of the two pathways. sev-induced apoptosis involves the intrinsic pathway leading to caspase and activation . therefore, to confirm the impact of jnk/p inhibition and/or dusp silencing on sev-induced apoptosis, caspase and cleavage was monitored by immunoblot. confirming the results of annexin v, both inhibition of jnk and p and silencing of dusp significantly impaired sev-induced caspase cleavage (fig. b) . additionally, caspase cleavage that was induced following sev infection confirming the engagement of the intrinsic pathway, was reduced in the absence of dusp compared to control cells (fig. b) . these findings confirmed that a jnk/p signaling module is involved in the induction of apoptosis during sev infection, but they also argue against a negative regulation of this pathway by dusp . rather, the observations support the hypothesis that dusp is required for induction of the intrinsic apoptotic pathway independently of jnk and p . in contrast to sev, rsv does not induce significant apoptosis at early time of infection ( and data not shown) when dusp dephosphorylates jnk/p . this prompted us to evaluate the role of dusp is alternative functions during rsv infection. compelling evidence has implicated jnk/p in the positive regulation of cell migration in several cell types . phenotypic observation of a cells during rsv infection following dusp silencing showed obvious formation of pseudopods that were not observed in control cells (supplementary movie ). this pointed to a propensity of infected cells to stretch out and migrate in the absence of dusp . to investigate the role of dusp in cell migration during rsv infection, d cell migration dynamics was assessed by single-cell tracking using time-lapse video-microscopy in subconfluent a cell cultures either left uninfected or infected with rsv following sictrl or sidusp transfection (supplementary movie ). as hypothesized, cell trajectories and migration rates were altered following dusp silencing. in the absence of dusp , rsv-infected cells were migrating further from their origin compared to control cells that rather adopted a trajectory in circles around their point of origin (fig. ) . these observations demonstrate a critical negative role of dusp in the regulation of cell migration during rsv infection. dusp is expressed in various cell types and tissues and mainly acts as a critical negative regulator shaping the duration, magnitude and spatiotemporal profile of p and jnk mapks, and to a lesser extent erk, activation following physiological and pathological stimuli . as such, dusp has been extensively shown to negatively regulate the innate immune anti-bacterial defense and the cellular response to allergens , . in these contexts, dusp mainly inhibits proinflammatory cytokine expression , [ ] [ ] [ ] [ ] [ ] [ ] . the role of dusp in the innate response to virus infection is far less known and, to our knowledge, was not previously assessed in the context of infection by rsv or sev. in the present study, we show that dusp negatively regulates p and jnk phosphorylation induced by sev and rsv (figs and ) . analysis of virus replication, of the nf-κb and irf signaling pathways and of the induction of ifns levels excluded a role of dusp in the negative regulation of the antiviral defense (figs and ). the observation that pharmacological inhibition of jnk/p mapks during sev infection significantly decreased downstream atf- /c-jun activation and cytokine induction (fig. a,d) confirmed the previously documented existence of a jnk-p /ap- signaling axis critical for virus-induced cytokine production , , . unexpectedly, dusp -mediated inhibition of jnk/p phosphorylation had no effect on downstream phosphorylation of atf- /c-jun or on the levels of a wide panel of cytokines elicited during infection (fig. ) . we hypothesized that this observation might reflect the existence of different subsets of jnk segregated through the interaction with specific scaffold proteins. in the mapk signaling modules involving jnk, members of the jip family of scaffold proteins selectively enhance specific jnk-dependent functions by interacting with and linking the upstream kinases to jnk activation , . in our experimental model, we showed that jnk interacts with jip , but not with jip , at basal levels and during infection (fig. a) . importantly, we found that the jip /jnk interaction dampens the h (f,g) . in (a-c), levels of phosphorylated atf- (atf- -p), total atf- , phosphorylated c-jun (c-jun-p), total c-jun and dusp were analyzed by immunoblot. actin was used to verify equal loading. the data are representative of three independent experiments. samples that are compared derive from the same experiment and blots were processed in parallel. full-length blots are presented in supplementary figure . in (d-g), release of cytokines was quantified using luminex-based multiplex assays. heatmaps represent cytokine levels (pg/ml) logarithmically transformed, centered and scaled, measured in each biological replicates (n = in d,f and g, n = in e). scatter plots of cytokine levels are shown in supplemental figure . scientific reports | : | doi: . /s - - - capacity of dusp to dephosphorylate jnk (fig. b-d) . compelling evidence demonstrates that jip is essential for the activation of the jnk/ap- axis that controls cytokine expression [ ] [ ] [ ] [ ] . although we cannot exclude other mechanisms, it is reasonable to speculate that during sev and rsv infection, the sequestration of jnk through molecular interaction with jip protects jnk and downstream ap- and cytokine response from dusp negative regulation (fig. ) . further studies will be required to fully address this model. previous reports have shown that interaction with jip leads to the retention of jnk in the cytoplasm , . dusp is a nuclear phosphatase , and therefore predominantly dephosphorylates jnk and p in the nucleus . it is thus very likely that retention of jnk in the cytoplasm by jip contributes to protect jnk from dusp dephosphorylation. our results differ from previous reports showing dusp -dependent inhibition of proinflammatory cytokine expression following challenge with the viral dsrna mimetic poly i:c, coronavirus and vaccinia virus infection , , . additionally, dusp was shown to enhance vaccinia virus replication . thus, dusp appears to differentially regulate the cytokine response and virus replication depending on the virus. one might speculate that the jip /jnk interaction might be differently affected upon infection by distinct viruses. while we did not observe changes of the jip /jnk complex during sev and rsv infection, other viruses may interfere with this interaction thereby making jnk available for dephosphorylation by dusp to negatively regulate downstream cytokine release. although to date no virus was reported to dissociate the jip /jnk complex, it is interesting to note that vaccinia virus-encoded b r kinase interacts with jip leading to increased binding of jnk to jip and downstream activation of c-jun . amongst alternative functions driven by jnk and p mapk pathways are the regulation of cell proliferation and apoptosis . cell cycle analysis during sev and rsv infection following ectopic expression or silencing of dusp failed to demonstrate a role of dusp in cell proliferation (data not shown). instead, we found that dusp is required for induction of the intrinsic apoptotic pathway triggered during sev infection (fig. b) . confirming previous reports , , , we also found that a jnk/p pathway contributes to sev-induced apoptosis (fig. a) . the observation that inhibition of dusp and jnk/p have additive effect on the negative regulation of sev-induced apoptosis (fig. ) strongly hints at a model in which dusp regulates virus-induced apoptosis in a jnk/p -independent manner. further studies will be required to challenge this model. additionally, this also argues toward a model in which the jnk/p pro-apoptotic function is also protected from dusp dephosphorylation, possibly through the observed jnk/jip interaction. this model is consistent with the fact that mavs-mkk -jnk defines a pro-apoptotic pathway during sev infection and that jip specifically functions in the jnk pathway by tethering mlk (mapkkk), mkk (mapkk) and jnk , . moreover, the jip /jnk axis has also been shown to be pro-apoptotic in neurons exposed to stress , . rsv does not induce significant apoptosis at early time of infection ( and data not shown) when dusp dephosphorylates jnk/p , but dusp silencing resulted in formation of pseudopods that were not observed in controls cells (supplementary movie ). in the quest to characterize dusp -dependent jnk and p functions, we thus assessed the impact of dusp on cell migration during rsv infection. indeed, accumulating evidence implicates jnk and p in pro-migratory functions in various contexts . here, we demonstrate that in the absence of dusp , cell migration of rsv infected cells was highly enhanced (fig. ) pointing to a critical role of dusp in the negative regulation of jnk/ p -mediated cell migration during rsv infection. in contrary to rsv, sev induces high levels of apoptosis at early time of infection, which prevented an unbiased analysis of the migration parameters. regulation of apoptosis and cell migration are part of the arsenal of the host response that have the potential to not only influence the outcome of virus spreading, but also the extent of virus-induced tissue damage and repair thereby contributing to pathogenesis , . the implication of the observed dusp -mediated regulation of apoptosis and cell migration in sev and rsv spreading can be excluded based on the lack of effect of dusp ectopic expression or silencing on virus replication (fig. ) . induction of apoptosis and inhibition of cell migration by dusp rather suggests a role in the induction of cell damage and inhibition of tissue repair and thereby in virus-induced pathogenesis. altogether our results suggest a model in which dusp is a negative regulator of important host mechanisms that limit tissue damage and promote tissue repair. an exaggerated cytokine response can also contribute to host self-inflicted damages and thereby contribute to virus-induced pathogenesis . we found that the cytokine response remains intact upon manipulation of dusp expression due to protection by the interaction of jnk with jip (fig. a) . the observation that jip /jnk interaction prevents a potential negative regulation of the cytokine response by dusp opens avenues for specific therapeutic targeting. indeed, one can hypothesize that inhibition of the jip /jnk interaction might restore dusp -dependent inhibition of the ap- /cytokine axis during the infection, while leaving other jip -independent jnk functions unaffected. further studies will be required to evaluate this possibility. the jip /jnk interaction has long been considered an interesting specific target and this led to the development of small molecules and peptides that inhibit the interaction between jnk and jip and efficiently block jnk activity toward selective substrates, including atf- and c-jun [ ] [ ] [ ] [ ] . alternatively, direct inhibition of dusp , might be a strategy to improve tissue homeostasis, by reducing virus-induced apoptosis and restoring cell migration, during virus infection. cell culture. infections. subconfluent monolayers ( % confluency) of a cells were infected with sendai virus (sev, cantell strain, charles river laboratories) at - hemagglutinin units (hau)/ cells as indicated in serum free medium (sfm). at h post-infection, the medium was supplemented with % hi-fbs and the infection pursued for the indicated time. infection with rsv a (advanced biotechnologies inc), amplified and purified as described in , was performed at a moi of - as indicated in medium containing % hi-fbs. where indicated, a were pretreated with μm mg (calbiochem) or dmso (vehicle, sigma-aldrich) for h before infection. pretreatment with μm sb and μm sp (invivogen) or the corresponding vehicle dmso was performed for min prior to infection. plasmid transfection. plasmid transfection in a cells was achieved using the transit-lt transfection reagent (mirus). briefly, a total of μg of dna was transfected per mm plates of a cells at % confluency using a transfection reagent/dna ratio of : . transfection was pursued for h to h before further treatment, as indicated. qrt-pcr. total rna were extracted using the rnaqueous- isolation kit (ambion) and quantified. reverse transcription was performed using μg total rna using the quantitect reverse transcription kit (qiagen). specific mrna levels were quantified by qpcr using fast start sybr green kit (roche) for sev n (s: agtatgggaggaccacagaatgg, as: ccttcaccaacacaatccagacc). a reaction without rt and a reaction with h o were performed with each run to ensure absence of genomic dna contamination. fluorescence was collected using the rotor-gene real time thermal cycler (corbett research). results were analyzed by the ΔΔct method after normalization to s mrna levels (s: cgtctcgaccaagagctga, as: ggtccttctcatcaagcgtc). virus titration by plaque forming unit (pfu) assay. rsv infectious virions were quantified by methylcellulose plaque forming units assay. briefly, supernatant from infected plates was harvested and subjected to serial dilutions before being used to infect monolayers of vero cells. after h of infection, vero cells were washed and covered with % methylcellulose prepared in dmem containing % fbs. rsv plaques were immunodetected at days post-infection. after removal of the methylcellulose, cells were fixed in % methanol for min and air-dried. plates were incubated for min at room temperature (rt) in pbs . x ph . containing % milk and . % tween before being incubated with anti-rsv antibodies (#ab , chemicon international) for h at rt, washed times in pbs . x ph . containing . % tween and finally incubated with rabbit anti-goat antibodies (jackson laboratories) for h at rt. after more washes, plates were incubated with enhanced chemiluminescence substrate (ecl, perkin elmer life sciences) for min at rt and chemiluminescence signal was detected using a ccd camera-based apparatus (las mini, ge healthcare). quantification of plaques was performed using the imagequanttl software (molecular dynamics) and expressed as pfu/ml. rnai oligonucleotide (on-target sirna, dharmacon) transfection was performed using the oligofectamine reagent (invitrogen). a non-targeting sequence, described in , was used as control. for efficient dusp expression silencing, a mix of two sirna sequences, caguuauggugaugacuuauu and ccgacgacacauauacauauu, were used. cells were plated at % confluency and transfected for - h before further treatment depending on specific experimental design. co-immunoprecipitation experiments. wce were prepared as described in the immunoblot section. for co-immunoprecipitation studies, - . mg of wce were incubated with μg anti-flag m antibodies (#f , sigma-aldrich) linked to μl protein g sepharose beads (sigma-aldrich) for h at °c. beads were washed times with lysis buffer and the elution of immunocomplexes was performed by incubation with a solution of μg/ml flag peptide (#f , sigma-aldrich) prepared in lysis buffer for h at °c. immunoprecipitates were then denatured with : (v/v) x loading buffer and heated for min at °c. wce and immunoprecipitates were resolved by sds-page as described in the immunoblot section. scientific reports | : | doi: . /s - - - luminex-based quantification of cytokines. supernatants from infected a cells were collected and centrifuged for min at g followed by a min centrifugation at g to remove cell debris. rsv-infected supernatants were uv-inactivated for min before further analysis. cytokines were quantified by luminex xmap technology using the bio-plex pro tm human cytokine standard plex group i and group ii or custom assembled plex for ifnβ, il- , cxcl , ccl or ccl from bio-rad on a bio-plex system (bio-rad). analyses were performed using the bio-plex manager software version . . . . heatmaps were produced from the raw expression analysis data using r and the gplots package on logarithmically transformed, centered and scaled data. detection of apoptosis by flow cytometry (annexin v/pi staining). for detection of apoptosis, the supernatant of cell culture was collected and cells were harvested using . % trypsin-edta. supernatant and cells were pooled and centrifuged for min at g at °c. the cell pellet was resuspended in annexin v buffer ( mm hepes ph . , mm nacl, mm kcl, mm mgcl and . mm cacl ) and stained using μl of fitc-annexin v (# , bd biosciences) and μl of propidium iodide (pi, mg/ml, #p- , sigma-aldrich) per - × cells for min at °c. immediately after staining, acquisition of fluorescence was performed on a bd lsr ii flow cytometer (bd biosciences) using the bd facsdiva software (bd biosciences). a minimum of cells was analyzed for each sample. data were analyzed using the flowjo software (flowjo, llc). analysis of cell migration. two-dimensional cell migration of single cells ( cells/picture and pictures/ condition) was evaluated by single-cell tracking using live-video microscopy. images were captured at min intervals over an h period by digital camera connected to a zeiss microscope (axio observer.z ) as in . the cell trajectories were analysed using the imagej software (national institutes of health, bethesda, md, usa). quantitative results are represented as mean +/− sem. statistical comparisons were performed with prism software (graphpad) using the indicated tests. the following p-values were considered significant: p < . (*), p < . (**), p < . (***) or p < . 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statistical computing (r foundation for statistical computing gplots: various r programming tools for plotting data complementary roles of kca . channels and beta -integrin during alveolar epithelial repair was supported by graduate studentships from cihr, the fonds de la recherche du québec en santé (frqs) and the réseau en santé respiratoire du frqs. ag was recipient of a studentship from the natural sciences and engineering research council of canada (nserc). nz was recipient of a post-doctoral fellowship from frqs/inserm (québec/france) and da by a post-doctoral fellowship from frqs we thank members of the laboratory and of the immunovirology group at crchum for fruitful discussions. a.c.r., n.z., d.a., e.b. and n.g. conceived and designed the experiments. a.c.r., e.c., n.z., e.m., d.a., m.m., a.g. and a.f. performed experiments. a.c.r., n.z., d.a., e.b. and n.g. analyzed the data. a.c.r. and n.g. wrote the manuscript. all co-authors edited and approved the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -d qxfo d authors: trezza, alfonso; iovinelli, daniele; santucci, annalisa; prischi, filippo; spiga, ottavia title: an integrated drug repurposing strategy for the rapid identification of potential sars-cov- viral inhibitors date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: d qxfo d the coronavirus disease (covid- ) is an infectious disease caused by the severe acute respiratory syndrome-coronavirus (sars-cov- ). the virus has rapidly spread in humans, causing the ongoing coronavirus pandemic. recent studies have shown that, similarly to sars-cov, sars-cov- utilises the spike glycoprotein on the envelope to recognise and bind the human receptor ace . this event initiates the fusion of viral and host cell membranes and then the viral entry into the host cell. despite several ongoing clinical studies, there are currently no approved vaccines or drugs that specifically target sars-cov- . until an effective vaccine is available, repurposing fda approved drugs could significantly shorten the time and reduce the cost compared to de novo drug discovery. in this study we attempted to overcome the limitation of in silico virtual screening by applying a robust in silico drug repurposing strategy. we combined and integrated docking simulations, with molecular dynamics (md), supervised md (sumd) and steered md (smd) simulations to identify a spike protein – ace interaction inhibitor. our data showed that simeprevir and lumacaftor bind the receptor-binding domain of the spike protein with high affinity and prevent ace interaction. the world health organisation (who) declared the coronavirus disease (covid- ) outbreak a pandemic on march th , and as of may st, over , , cases and , deaths have been reported (https :// www.who.int/emerg encie s/disea ses/novel -coron aviru s- /situa tion-repor ts/). the severe acute respiratory syndrome-coronavirus (sars-cov- ) was identified as the viral agent causing the disease. sars-cov- is closely related to the sars-cov, which caused a pandemic in - , and it is believed to be the third member of the coronaviridae family to cause severe respiratory diseases in human . despite several ongoing clinical studies, there are currently no approved vaccines or drugs that specifically target sars-cov- . sars-cov- has a single-stranded positive-sense rna composed of , nt containing five genes, orf ab (codifying non-structural proteins), spike (s), envelope (e), membrane (m) and nucleocapsid (n) genes . the virus uses the s homotrimeric glycoprotein located on the virion surface to allow entry into the human cells . the s protein goes through major structural rearrangements to mediate viral and human cell membranes fusion. the process is initiated by the binding of the receptor-binding domain (rbd) of the s subunit to the peptidase domain (pd) of angiotensin-converting enzyme receptor (ace ) on the host cell . structural studies have shown that two s protein trimers can simultaneously bind to one ace dimer . this induces a conformational change that expose a proteolytic site on the s protein, which is cleaved by the cellular serine protease tmprss . dissociation of s induces transition of the s subunit to a post fusion conformation, with exposed fusion peptides , which allows endocytic entry of virus . wrapp et al. have shown that, despite sars-cov- and sars-cov sharing a similar cell entry mechanism, sars-cov- s protein binds ace with a -to -fold higher affinity than sars-cov s, which may be related to the higher person-to-person transmission of sars-cov- . s glycoprotein is highly immunogenic, and it is a promising target for drug design . we showed that a combination of four -mer synthetic peptides disrupting sars-cov s heterotrimer reduced or completely inhibited infectivity in vitro www.nature.com/scientificreports/ have potential for therapy . in fact, disruption of the binding of the s protein to ace prevents the virus from attaching to the host cell . the social and economic impact of covid- and the possibility of future similar pandemics are pushing for the rapid development of treatments. as such, targeting viral-host protein-protein interaction (ppi) may represent a promising way to prevent or reduce the spreading of the virus before a vaccine is available . in this study we performed an extensive analysis of the intrinsic dynamic, structural properties and drug targeting of sars-cov- rdb. in particular starting from the structure of rdb in complex with ace , we identified transient pockets on rdb on the ace interaction surface area. our data provide detailed information on the dynamic features of rdb that we exploited for docking studies. we carried out a virtual screening using fda-approved drugs to explore new therapeutic benefits of existing drugs. to take into account unique features of molecules, such as conformational flexibility, charges distribution, and solvent role in target recognition and binding, we performed an extensive molecular dynamics simulation analysis. by combining molecular dynamics simulations (md), supervised md (sumd), steered md (smd) and interaction energy calculations, we showed that simeprevir and lumacaftor bind rdb with high affinity and prevent ace interaction. overall, by adopting a robust in silico approach, our results could open the gates toward the development of novel covid- treatments. structural resources. d structure and fasta sequence of sars-cov- rbd in complex with human hace (pdb id lzg) were retrieved from the rcsb protein data bank . to avoid errors during the molecular dynamic (md) simulations, missing side chains and steric clashes in pdb files were adjusted through homology modelling, using pymod . and modeller v. . . d structures were validated using procheck . gromacs . with charmm -mar force field was used to resolve high energy intramolecular interaction before docking simulations, and cgenff was used to assign all parameters to ligands. structures were immersed in a cubic box filled with tip p water molecules and counter ions to balance the net charge of the system. simulations were run applying periodic boundary conditions. the energy of the system was minimized with . steps of minimization with the steepest descent algorithm and found to converge to a minimum energy with forces less than kj/mol/nm. a short ns classic molecular dynamics (cmd) was performed to relax the system. all the cmd simulations were performed integrating each time step of fs; a v-rescale thermostat maintained the temperature at k and berendsen barostat maintained the system pressure at atm, with a low dumping of ps − ; the lincs algorithm constrained the bond lengths involving hydrogen atoms. a ns cmd simulation was used, as described above, for the identification of transient pockets. transient pockets were identified by analysing md trajectories of sars-cov- rbd structure with epos tool , using parameters by default. the volumes of the transient pockets during the simulation were measured using povme . open pockets in close proximity to ace binding site were selected based on the depth and polarity of the cavity. a box with dimensions of , , and Å was created around the transient pocket using autodock tools . subsequently, a virtual screening using the fda-approved drug library available on drugbank was carried out on sars-cov- rbd using autodock/vinaxb . mgl-tools scripts and openbabel were used to respectively convert protein and ligand files and to add gasteiger partial charges. the full fda-approved library was downloaded from drugbank and a total of , molecules, corresponding to the number of structures successfully processed by openbabel, was used for the virtual screening. drugs names and structures used in this study are available on https ://githu b.com/fpris chi/suppl ement ary_fdali brary .git supervised molecular dynamics (sumd) simulations. sumd were used to sample the binding of hace to rbd and to probe the binding of hace to rbd-simeprevir/lumacaftor complexes. sumd methodology relies on a tabu-like algorithm that monitors the distance between hace and centre of mass of the rbd binding site during unbiased md simulations to sample a binding event in the range of nanoseconds . the protocol is based on performing a series of short unbiased md simulations, where after each simulation the distance points collected at regular time intervals are fitted into a linear function. if the resulting slope is negative, then hace is getting closer to the rbd binding site and the md steps are kept. if the slope is not negative, then the simulation is restarted by randomly assigning the atomic velocities. we used an sumd step of , ps with a constant temperature and pressure of k and atm respectively. when the distance between the hace and rbd reached Å or less, then the supervision was disabled and a ns cmd simulation was performed. the analysis was performed with an in-house written python and bash script. in order to evaluate the binding interaction between rbd and simeprevir or lumacaftor, the rbd-simeprevir/lumacaftor complexes were simulated to dissociate using a ps smd simulation by constant force pulling of kj/mol/nm. while the backbone of rbd was not allowed to move, simeprevir and lumacaftor experienced a constant force in x, y, z direction, specifically ( , , ) for both compounds. simeprevir and lumacaftor were pulled with an external force in the npt ensemble at atm and k with fs time steps. md analyses was performed with gromacs . package and displayed with grace. protein-ligand interaction energy. to quantify the strength of the interaction between the rbd and simeprevir/lumacaftor, we computed the nonbonded interaction energy. gromacs has the ability to decom-scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ pose the short-range nonbonded energies via the energygrps keyword in the .mdp file. the energy terms of interest are the average short-range coulombic interaction energy (coul-sr) and the short-range lennard-jones energy (lj-sr). the total interaction energy (ie binding ) is defined by: driven by polar interaction, with an overall ~ Å buried surface area. a close analysis of the interface reveals the absence of cavities on rbd in the interaction surface. we performed md simulations to account for the protein conformational flexibility and detected , transient pockets. based on the druggability features of the cavities, i.e. volume, depth, polarity, and proximity to the hace binding site, we detected a cluster of transient pockets. in order to identify possible ppi inhibitors the transient pocket that contained key residues involved in hace recognition and binding (fig. a ) was selected and used for the virtual screening of fda-approved drugs. this curated library of drugs, whose structures were freely downloaded from drugbank , represents a reservoir of bioactive molecules that could be repurposed for covid- treatment relatively fast, due to their pre-existing approval for use in humans. the best compounds (lumacaftor, paritaprevir, dihydroergotamine, trypan blue, midostaurin, dihydroergotoxine, simeprevir, lurasidone, spinosyn d, olaparib) showed high binding free energy scores (− . to − . kcal/mol) (fig. s ). the compound with the highest binding energy (− . kcal/mol) was lumacaftor, a cftr corrector that traffic the mutant protein to the plasma membrane . an analysis of the quality of interactions of the best compounds revealed that simeprevir had the highest number of polar bonds with side chains of residues in the rbd binding pocket. simeprevir, a second-generation hcv ns / a protease inhibitor , has been reported to be both a sars-cov- main protease inhibitor and a s protein-rbd interaction inhibitor . simeprevir forms an extended network of h-bonds with arg , lys , gln , gly and tyr , and forms van der waals interactions with tyr , tyr and tyr (fig. b) . differently, lumacaftor has a higher number of hydrophobic contacts, compared to simeprevir, specifically with tyr , leu , tyr , phe and tyr , with the potential formation of π-stacking using the cζ, of arg , and forms h-bonds with gln , lys and asn (fig. c) . analysis of the crystal structure of rbd in complex with ace reveals that the residues involved in the binding with the two drugs are key drivers of rbd and ace interaction . of particular interest are residues lys , leu and gln , which are not conserved www.nature.com/scientificreports/ in sars-cov and have been linked to the higher affinity of sars-cov- s protein for ace . taken together, these data show that simeprevir and lumacaftor are able to form clearly defined specific interactions with the sars-cov- s glycoprotein and are promising ppi competitive inhibitors. in order to understand if simeprevir and lumacaftor are able to interfere and prevent the binding between the s glycoprotein and ace , we ran a supervised molecular dynamics (sumd) simulations. using sumd it is possible to simulate the full binding process of ace to rbd in presence of simeprevir or lumacaftor in an unbiased way (i.e. independently from starting relative positions), taking into account hydration patterns and drug binding-unbinding events. we first validated the sumd protocol by simulating the binding process of rbd with ace . the resulting relative position of ace bound to rbd is comparable to that in the crystal structure (fig. s ) . the interaction between ace and rbd is established after ns of productive trajectory and is mediated by key residues in the receptor binding motif (rbm). specifically, sars-cov- tyr , asn , tyr , gln , asn and tyr form h-bonds with ace , whereas sars-cov- phe interacts with ace via van der waals forces. outside the rbm, we see the formation of the salt bridge between sars-cov- lys and ace asp in line with published data suggesting that this key interaction contributes to the difference in affinity between sars-cov and sars-cov- s proteins for ace . using the same approach we then simulated the binding of ace to rbd bound to simeprevir or lumacaftor. during the sumd simulation ace did not displace the drugs and did not form interactions with the s glycoprotein even after ns of simulation. this is very likely due to the drugs interacting with the side chains of the key residues lys , tyr , asn and tyr , which prevent ace target recognition. taken together these data show that simeprevir and lumacaftor prevent ace recognition and binding to the s glycoprotein. simeprevir and lumacaftor binding stability. during the sumd drugs were allowed to move and find a more energetically favourable pose in the binding pocket. we noticed very limited movements of simeprevir and lumacaftor and, to confirm binding stability, we performed ns cmd simulations of rbd alone and in complex with the drugs. the pose of simeprevir and lumacaftor did not change significantly during the simulation, and the rmsd average was . Å and . Å respectively (figs. s and a) . in order to exclude presence of artefacts in our analysis, we monitored the protein structural integrity during the simulations. we noticed limited differences between the rmsd of the apo protein ( . Å) and the rmsd of rbd bound to simeprevir or lumacaftor ( . and . Å respectively), which excludes presence of different protein structural rearrangements in the three cmd simulations (fig. b) . to quantify the strength of the interaction between simeprevir and lumacaftor on rbd, we computed the interaction energy between the protein and the two drugs. the total interaction energy for simeprevir and lumacaftor was − . ± . kj/mol and − . ± . kj/mol respectively. taken together these data suggest that simeprevir and lumacaftor bind spontaneously to the target with high affinity. www.nature.com/scientificreports/ to further characterise the recognition process of the two drugs to the s glycoprotein we performed steered molecular dynamics (smd) simulations. we ran a ps smd simulation on rbd in complex with both simeprevir and lumacaftor, and the time-averaged force profiles during the unbinding simulation of complexes is shown in fig. a . both drugs have a steady increase of the applied forces on the first ~ and ~ ps of the simulation, respectively for lumacaftor and simeprevir, until they reach the maximum, which corresponds to the rupture force of lumacaftor and simeprevir unbinding along this dissociation pathway. the force then quickly decreases and stays constant until the end of the simulation. in the first step, between and ps of the simulation for simeprevir and and ps for lumacaftor, the two drugs slowly detach and move away from the transient pocket; in the second step, between and ps of the simulation for simeprevir and and ps for lumacaftor, they move away from the protein and enter the solvent region (fig. b,c) . the comparable rupture forces reflect similarity in the unbinding from rbd in line with our binding energy data. www.nature.com/scientificreports/ discussion sars-cov- invades human cells via ace , a transmembrane protein expressed on the surface of alveolar cells of the lungs. upon binding of ace , viral and host cell membranes fuse and the virus enters into the host cell. this results in the development of an infectious disease, called covid- , which is associated with a major immune inflammatory response. deaths are caused by respiratory failure, which have been linked to a cytokine storm with high serum levels of pro-inflammatory cytokines and chemokines . there are currently no approved vaccines or drugs that specifically target coronavirus infection, and, despite several ongoing clinical trials, treatment options have been based on different clinical approaches with limited background testing. an exponentially growing number of computational studies have tried to provide molecular data in support of these novel potential covid- treatments , [ ] [ ] [ ] . the aim of this proof of principle study was to propose a robust in silico protocol that overcame limitations of classic virtual screening studies . the role of hydration patterns in target recognition and binding is completely absent in docking simulations. furthermore, in most virtual screenings, while the ligand is flexible, proteins are only semi-flexible, which affects both the resulting pose of the ligand and the scoring system . more reliable information can only be obtained by md simulations, which, despite being computationally expensive, allow to take into account macromolecules' unique features, such as conformational flexibility, charge distribution, and hydration patterns in target recognition, drug binding, and drug unbinding , . in this study we coupled docking with cmd, sumd and smd to identify a spike protein-ace interaction inhibitor. transmission electron microscope image of sars-cov- have shown how the viral envelope is densely populated by the s protein, which, due to its role in pathogenesis, is the main target of neutralizing antibodies and vaccines . an analysis of the crystal structure of the rbd with ace reveals that the rbd of the s protein has a relatively flat surface, which would be unsuitable for drug targeting. previous studies have shown that the analysis of protein dynamics allows for the identification of transient pockets where small molecules can bind proteins . we identified a transient pocket with druggability features on the rbd which may represent a hot spot . indeed, comparison with the structure of sars-cov s protein in complex with a neutralising antibody isolated from a sars-cov survivor shows that the pocket we identified lies on the same surface recognised by the cdrs of the antibody . we retrieved the structure of the protein with an open pocket from the trajectory of the md simulation and we used it for a virtual screening of fda-approved drugs. the advantage of focusing on fda-approved drugs is that the safety issues are all within suitable bounds and are well understood, meaning that they could proceed to clinical trial reasonably quickly. the compounds showing high binding energies and forming a network of specific interaction with side chains of residues in the rbd binding pocket were simeprevir and lumacaftor. simeprevir, a direct-acting antiviral agent for the treatment of hcv infections, is a second generation of orally available ns / hcv protease inhibitor . lumacaftor is a cftr corrector that stabilises the first transmembrane domain of cftr, resulting in an improved maturation of cftr mutants . the two drugs were also selected for their reported minimal off-targeting, suggesting a lack of binding to other human proteins , . furthermore, kadioglu et al. also identified simeprevir as a potential s protein-ace interaction inhibitor. in a similar attempt to overcome limitations of classic in silico docking, they adopted a complex approach combining virtual drug screening, molecular docking and supervised machine learning techniques . while their results strongly reinforce the findings of our study, our strategy provided comprehensive information about the ability of compounds to interfere with the spike protein binding to ace by combining cmd, sumd and smd. virtual screening and in vitro studies suggested that lumacaftor and simeprevir are also sars-cov- main protease inhibitors . interestingly, several in silico and in vitro studies have identified antiviral agents targeting hcv infection (single-stranded negative-sense rna virus) as promising treatments for covid- , which include hcv approved inhibitors of the viral rna synthesis, the cl protease and the helicase activity . antiviral agents against hcv infections have also been studied for their promising ability to interfere with other viral infections caused by rna viruses, i.e. sars-associated coronavirus , mers , enterovirus a , herpes simplex virus type and zika virus . this would suggest the possibility to use and/or develop simeprevir into broad-spectrum antiviral drugs . simeprevir and lumacaftor are also promising for their potential ability to inhibit multiple steps of the sars-cov- infection by interfering with the s protein binding to the ace receptor and by inhibiting the sars-cov- main protease, essential for processing the polyproteins that are translated from the viral rna . the concept of multi-target drugs that inhibit several proteins simultaneously has been successfully used for the treatment of many diseases. for example, the anti-hiv drug cosalane was developed to inhibit binding of the hiv gp envelope glycoprotein to cd and simultaneously to inhibit the cytopathic mechanism of hiv- . while writing this paper, several drug repurposing studies targeting the s protein have been published. interestingly, several papers , , , carried out virtual screenings on the same surface we identified as a transient pocket. binding energies of proposed compounds are however lower than the one we observed for simeprevir and lumacaftor. this is very likely linked to the protein structures used for virtual screening and/or a binding pocket not being in the optimal open conformation, highlighting the strength of our in silico approach. our results show the importance of taking into account the full structural features of a protein-ligand complex and how a combination of md simulations may help predict the validity of a proposed inhibitor. our work suggests that simeprevir and lumacaftor could be potential initial compounds able to prevent and treat sars-cov- infection. received: may ; accepted: july the proximal origin of sars-cov- severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges the outbreak of sars-cov- pneumonia calls for viral vaccines structure, function, and antigenicity of the sars-cov- spike glycoprotein structural biology: structure of sars coronavirus spike receptor-binding domain complexed with receptor structural basis for the recognition of sars-cov- by full-length human ace sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor structure, function, and evolution of coronavirus spike proteins characterization of spike glycoprotein of sars-cov- on virus 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pockets with pass ps:surface, sasa, cavity, software, hole, channel, tunnel povme . : software for mapping binding pocket flexibility software news and updates autodock and autodocktools : automated docking with selective receptor flexibility drugbank . : a major update to the drugbank database implementation of xbsf, new empirical halogen bond scoring function, into autodock vina open babel: an open chemical toolbox supervised molecular dynamics (sumd) approaches in drug design two small molecules restore stability to a subpopulation of the cystic fibrosis transmembrane conductance regulator with the predominant disease-causing mutation direct anti-hcv agents gc- , and calpain inhibitors ii, xii inhibit sars-cov- viral replication by targeting the viral main protease in silico molecular dynamics docking of drugs to the inhibitory active site of sars-cov- protease and their predicted toxicology and adme trials of anti-tumour necrosis factor therapy for covid- are urgently needed 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impaired in their conformational adaptability by a pathogenic loop mutation and dynamically stabilized by lumacaftor multisociety consensus quality improvement revised consensus statement for endovascular therapy of acute ischemic stroke identification of novel compounds against three targets of sars cov coronavirus by combined virtual screening and supervised machine learning pharmacoinformatics and molecular dynamic simulation studies reveal potential inhibitors of sars-cov- main protease clpro therapeutic options for the novel coronavirus ( -ncov) -bis-arylmethyloxy- -hydroxychromones with antiviral activity against both hepatitis c virus (hcv) and sars-associated coronavirus (scv) quantitative structure-activity relationship and molecular docking revealed a potency of anti-hepatitis c virus drugs against human corona viruses from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design novel paradigms for drug discovery: computational multitarget screening molecular investigation of sars-cov- proteins and their interactions with antiviral drugs a.t. conceived the original idea of the work and performed, analysed and interpreted of data and reviewed the manuscript. d.i. created new algorithms used in the work. f.p. made substantial contributions to the design of the work, data interpretation and wrote the manuscript. a.s. and o.s. reviewed the paper and provided positive opinion for this work. all authors approved the submitted version. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to f.p. or o.s.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -xzionafe authors: chang, chia-yu; cheng, ivan-chen; chang, yen-chen; tsai, pei-shiue; lai, seiu-yu; huang, yu-liang; jeng, chian-ren; pang, victor fei; chang, hui-wen title: identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diarrhoea virus spike protein date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xzionafe since , newly identified variants of porcine epidemic diarrhoea virus (pedv) have caused high mortality in neonatal piglets which has devastated the swine industry. the spike (s) glycoprotein of pedv contains multiple neutralizing epitopes and is a major target for pedv neutralization and vaccine development. to understand the antigenicity of the new pedv variant, we characterized the neutralizing epitopes of a new genotype b pedv isolate from taiwan, pedv pintung (pedv-pt), by the generation of neutralizing monoclonal antibodies (nmabs). two nmabs, p b- , and e e- – that recognized the ectodomain of the full-length recombinant pedv s protein and exhibited neutralizing ability against the pedv-pt virus were selected. recombinant truncated s proteins were used to identify the target sequences for the nmabs and p b- was shown to recognize the c-terminus of co- k equivalent epitope (coe) at amino acids (a.a.) – of the pedv s. interestingly, e e- – could recognize a novel neutralizing epitope at a.a. – within the s (a) domain of the pedv s protein, whose importance and function are yet to be determined. moreover, both nmabs could not bind to linearized s proteins, indicating that only conformational epitopes are recognized. this data could improve our understanding of the antigenic structures of the pedv s protein and facilitate future development of novel epitope-based vaccines. determination of neutralizing antibodies. after purification and quantification of mabs, the neutralizing ability of each mab against the g b taiwan pedv-pt strain was assessed using the virus neutralizing assay. the starting concentration for each mab in the neutralizing assay was μg/ml, with a two-fold serial dilution to obtain a final neutralizing concentration of . μg/ml. the mabs, p b- , and e e- - , possessed potent neutralizing ability that completely blocked the cpe of the taiwan pedv-pt strain under μg/ml. on the contrary, the mabs, r h- , and unk- , showed no neutralization ability against pedv-pt even at μg/ml. synthesized codon-optimized complete gene sequence of the pedv-pintung s protein (pedv-pt; genbank accession no. kp ) was used to amplify dna amplicons of varying lengths that encode different truncated forms of the s protein genes. using the primer pairs listed in supplementary table , dna sequences of different lengths were amplified namely s - ( bp), s - ( bp), s - ( bp), s - ( bp), s - ( bp), s - ( bp) and the full-length s ectodomain ( bp) of the pedv-pt strain. these dna fragments were digested with restriction enzymes and ligated into the pcdna . -v -his vector. seven plasmids containing the v -tag, namely pcdna . -pedv s - -v -his, pcdna . -pedv s - -v -his, pcdna . -pedv s - -v -his, pcdna . -pedv s - -v -his, pcdna . -pedv s - -v -his, pcdna . -pedv s - -v -his, and pcdna . -pedv s-v -his were constructed. we have shown the construction of the full-length spike ectodomain of the taiwan pedv-pt strain in a previous publication . the expression levels of various truncated recombinant s proteins, s - , s - , s - , s - , s - , and s - of the pedv-pt strain, were successfully detected using icc staining with the anti-v tag antibody ( fig. ) , as well as western blotting (fig. ) . the v tag-positive hek cells for each construct occupied % to % of the total population. the positive ratio of each construct was adjusted between % and % by mixing with untransfected hek cells, which were used as internal negative control in the icc staining. the corresponding molecular weights of s - , s - , s - , s - , s - , s - , and the full-length spike protein were detected to be approximately , , , , , , and kda, respectively (fig. ) . mapping the epitopes of the pedv-pt by icc staining. as summarized in table , the two nmabs, p b- and e e- - and the non-neutralizing mab, r h- , all exhibited the ability to recognize full-length pedv spike-expressing hek cells but were unable to recognize pedv s - -expressing hek cells in icc staining. more precisely, p b- recognized pedv s - expressing hek cells but did not recognize the hek cells expressing s - , s - , s - , s - , s - of pedv-pt (fig. ) . in other words, p b- was unable to bind to the amino acids upstream of a.a. of the spike protein of pedv-pt. therefore, the targeting epitope of p b- must be located between a.a. - on the pedv spike protein. surprisingly, e e- - exhibited good binding affinity for hek cells expressing pedv proteins s - , s - , s - , s - , and s - , but with reduced binding affinity for the pedv s - . this evidence obtained from the icc staining confirmed this nmab recognizing the novel epitope present within the - a.a. region of pedv-pt spike protein (fig. ) . in contrast, the non-neutralizing mab, r h- , that recognized only the full-length pedv spike-expressing hek cells, was unable to bind to the pedv spike protein upstream of a.a. (fig. ) . these results suggest that the targeting epitope of r h- is downstream of a.a. of the pedv spike protein. elisa results showing reactivity of mabs to various truncated pedv s proteins. to further confirm the reactivity of each mab to the targeting epitopes, elisas were performed using plates coated with www.nature.com/scientificreports www.nature.com/scientificreports/ various truncated pedv spike proteins. the pedv nmabs (p b- and e e- - ) and the non-pedv recognizing mab (unk- ) were all serially diluted to conduct the elisa. as shown in fig. , p b- had a binding affinity with appropriate dilution effects toward the purified full-length pedv spike protein, as well as pedv s - , but had its affinity towards purified pedv proteins, s - , s - , s - , s - and s - was weak. however, e e- - , which targets a novel neutralizing epitope as shown with icc staining, was also capable of binding to the full-length pedv spike protein as well as truncated pedv proteins, s - , s - , s - , s - , and s - with appropriate dilution effects. similar to the results obtained by icc staining, e e- - showed no binding ability www.nature.com/scientificreports www.nature.com/scientificreports/ toward pedv s - by elisa and therefore, an nmab targeting a novel epitope of the new variant of pedv specifically at a.a. - in the s region was confirmed. the non-pedv recognizing mab, unk- showed no binding affinity towards the various truncations of the spike protein and hence, it was used as the external control for the elisa assay. binding affinity of nmabs to linearized epitopes. to further determine whether the binding of neutralizing epitopes require conformational integrity, immunodot blotting was performed by denaturing the various truncated pedv spike proteins. the conformational proteins as well as the denatured proteins of s - , s - , s - , s - , s - , s - , and the full-length spike of pedv-pt were dotted onto the membrane. as shown in fig. (a-c), p b- was able to detect the conformational s - and the full-length spike protein on the membrane but was unable to detect the proteins upstream of a.a. of spike, consistent with the findings from the elisa and icc staining. the other nmab, e e- - , could bind to s - , s - , s - , s - , s - and the full-length spike proteins in conformational structures. the r h- , a spike-recognizing but non-neutralizing mab, was only capable of binding to the conformational full-length spike protein. p b- , e e- - , and r h- were unable to stain the denatured target proteins on the membrane, indicating the loss of binding affinity after protein linearization. moreover, to ensure all the proteins were equally dotted on the membrane, the results of mab-stripping and re-probing with anti-v tag antibodies are shown in fig. (d-f). a molecular model of recombinant pedv s ectodomain protein was established using swiss model and re-edited using ucsf chimerax molecular modelling system. as shown in fig. , the monomer of pedv s protein was divided into s (a.a. - , in light-blue) and s (a.a. - , in grey). the novel neutralizing epitope (a.a. - of the s protein), characterized by the e e- - nmab labelled in yellow in fig. (a) , was inferred to be at the c terminal region of the s a domain. the two mutations, v i, and mab s - s - s - s - s - s - s www.nature.com/scientificreports www.nature.com/scientificreports/ s a that were different from the cv strain were highlighted in red sphere; and the one mutation, e q that was different from the taiwan historic pedv hc -s strain was highlighted with a green sphere. on the other hand, the region of the neutralizing epitope (a.a. - of the s protein; labelled in yellow) characterized by the p b- nmab was deduced at the c-terminal region of the s b domain in fig. (b) . the a.a. mutation (g s) different from the cv strain was highlighted with a purple sphere; and the a.a. mutations, d e and l f, different from the taiwan historic pedv strain hc -s were highlighted with pink spheres. the mutation, q e that was different from both cv and taiwan historic pedv strain hc was highlighted with an orange sphere. the six truncated as well as full-length spike protein of pedv-pt were coated into separate wells and probed with two-fold serially diluted mabs, p b- and e e- - . the non-pedv recognizing mab, unk- , was added as the external control for elisa under the same dilution conditions. the two-fold serially diluted anti-v tag antibody was used as the standard to show the dilution effect of the binding affinity assay. the data is represented as the s/p ratio. the wells incubated with only secondary antibodies and the wells incubated with anti-v tag antibody at , ng/ml were used as negative and positive controls for s/p ratio respectively. the results of the elisa for the various truncations of the spike proteins are shown as follows: www.nature.com/scientificreports www.nature.com/scientificreports/ since , outbreaks of infections caused by new pedv variants have been correlated to viral mutations in critical neutralizing epitopes of the spike protein , . although several sequences of the new variant pedv spike genes have been decoded , and multiple domain architectures of the pedv s protein have been predicted by three-dimensional structures of alphacoronaviruses , the effects of genetic mutations on the antigenicity of pedv remains poorly understood. a thorough evaluation of the neutralizing epitopes and antigenicity of the new pedv variants is therefore needed. in the present study, we generated a panel of mabs using pedv-pt viral particles. by selecting mabs that exhibited high binding affinity to full-length spike ectodomain-expressing hek cells, we successfully identified two representative nmab against pedv-pt. the targeting epitopes identified by these two nmabs were located at a.a. - and a.a. - of the pedv spike protein, respectively, as shown by icc staining and elisa. further, none of these nmabs were able to bind to the linearized forms of the recombinant ectodomain of s protein of pedv-pt strain was modelled using swiss model and edited using ucsf chimerax. the s protein was present as monomer and the s and s regions of pedv were displayed in light-blue and grey, respectively. the conformational neutralizing epitope regions characterized by the mabs are given as follows: (a) e e- - mab labelled in yellow; two mutations (v i and s a) on the epitope different from the cv strain were highlighted in red sphere and one mutation (e q) on the epitope different from the taiwan historic pedv strain (hc -s) was highlighted in green sphere. (b) p b- mab was labelled in yellow; the g s mutation on the epitope different from the cv strain was highlighted in a purple sphere; mutations, d e and l f, on the epitope different from the taiwan historic pedv strain (hc -s) were highlighted in pink sphere. the orange sphere represented the common mutation (q e) noted on the epitope of pedv-pt s protein that was different from both of cv and taiwan historic pedv (hc -s) strains. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ the truncated spike proteins, indicating that they recognize the integral epitopes with appropriate conformation. taken together, we discovered a novel conformational neutralizing epitope in the s a domain at a.a. - , whose function has not been clearly determined in coronaviruses. in addition, we also discovered a conformational neutralizing epitope close to the c-terminus of the coe domain, at a.a. - . the neutralizing epitopes of the historic strains of pedv on the s protein that have been previously published include the co- k equivalent epitope (coe epitope; a.a. - in the brl strain; a.a. - in the pedv-pt strain) , c epitope (a.a. - in the cv strain; a.a. - in the pedv-pt strain) , and s d (a.a. - in the cv strain; a.a. - in the pedv-pt strain) . in case of the new variants of pedv, the domain b (a.a. - in the pedv gdu and the pedv-pt strain) and domain in the s region (a.a. - in the pedv gdu and the pedv-pt strain) are demonstrated to have neutralizing epitopes . furthermore, okda et al. also discovered several linear epitopes at the n-terminus of s . previously, the identification of neutralizing epitopes of the historic pedvs led to the proposal that several single nucleotide polymorphisms (snps), such as three serine amino acid substitutions in the coe epitope and two serine amino acid substitutions in the s d epitope, may be related to newly-occurring global outbreaks , . in the present study, the nmab, p b- , was able to recognize the epitope within a.a. - on the s protein, which overlaps with the c-terminus of the coe epitope, echoing the findings of previous studies . as the s b domain consists of the main coe neutralizing epitope of pedv and is the receptor binding domain for the majority of the coronaviruses, its importance has been emphasized in many studies , , . li et al. have demonstrated that the binding affinity of neutralizing antibodies could be dramatically altered by exchanging one amino acid on the s protein of pedv . hence, mutations in s b domain, especially in neutralizing epitopes of the pedv s protein, are speculated to represent the evolution of viral escape from antibodies, like the one evoked by cv -based vaccine immunization of a historic pedv strain infection , . interestingly, the other nmab, e e- - , presented a high binding affinity toward a.a. - of the s protein that precludes the coe epitope and locates in the s a domain has also been identified in our study. to our knowledge, the function of the s a domain of the pedv s protein has not been well studied. the s a domain of human coronavirus hcov-nl is speculated to interact with heparan sulfate proteoglycans on the host cells to mediate the virus anchoring and infection . the n-terminus of s a domain of bovine coronavirus has been shown to have two sugar binding loops which can recognize the carbohydrate receptor on the host cells . moreover, heparan sulfate has also been demonstrated as an attachment factor for pedv . however, as the actual topography of the pedv trimeric s protein remains to be solved and the function of s a domain is yet to be determined, the actual interaction of the s a domain targeting mab with these sugars or the heparan sulfate proteoglycans-binding domains on the pedv s glycoprotein remains to be further evaluated. visualization of the topology and interaction of pedv s glycoprotein with these neutralizing antibodies for guiding future immunogen and therapeutics design is an important future goal. epitopes for antibody recognition can generally be divided into two main classes: linear and conformational forms. linear epitopes are formed by a continuous sequence of amino acid in a protein, while conformational epitopes consist of amino acid sequences that are discontinuous in the protein but are brought together upon three-dimensional protein folding. it has been demonstrated that % of b cell-recognizing epitopes are conformational epitopes that result from the antigen internalizing process and special antigen-recognizing ability , . many conformational epitope-targeting antibodies, including neutralizing antibodies, are particularly difficult to determine because the antibody-antigen complex is formed solely in the native structure of the protein. over the past several decades, with the use of phage-display peptide probing, the neutralizing epitope of cv , named c has been identified . using elisa or pepscan assays, several linear neutralizing epitopes have been discovered on the s glycoprotein subunit of new pedv variants . additionally, two b cell epitopes, named ss and ss , in the region of the s d neutralizing epitope were evaluated using a combination of phage-display peptide probing and pepscan , . however, these epitope mapping approaches focus mainly on linear epitopes . using icc staining, elisa, and denatured immunodot blotting, all of the b cell-recognizing epitopes identified in the present study were found to be conformational epitopes. thus, these affinity-binding assays could serve as valuable platforms for studying the conformational epitopes of nmabs. in conclusion, the nmabs and various truncated s proteins constructed generated in the present study could contribute to a better understanding of the antigenicity and immunogenicity of highly virulent pedvs, especially in revealing the antigenic role of s a domain, and the pathogenesis of immune escape that leads to an outbreak of pedv. moreover, our study may provide important fundamental information for the future development of novel epitope-based vaccines. collection (atcc) no. crl- ) as previously described . after freezing and thawing, the harvested viral supernatant was centrifuged at rpm for min to remove cell debris and filtered through a . µm ultrafiltration cup (corning, new york, usa). the viral supernatant was then added onto a % sucrose (sigma, missouri, usa) cushion, purified by ultracentrifugation at , × g for . h. the viral pellet was re-suspended in phosphate buffered saline (pbs) (gibco, gaithersburg, usa), and then applied to a - % sucrose-tne ( mm tris-hcl (ph ) (sigma), mm nacl, mm edta (sigma)) gradient, and centrifuged at , × g for . h in an optima ™ l- xp preparative ultracentrifuge using an avanti j- rotor (beckman coulter, sykesville, usa). purified virions were diluted in tne buffer, pelleted by centrifugation at , × g for . h to remove the sucrose and then, re-suspended in tne buffer. www.nature.com/scientificreports www.nature.com/scientificreports/ mab production. three balb/c mice were intramuscularly (im) immunized with μg purified pedv viral particles mixed with μl complete freund's adjuvant (sigma). after two weeks, two im booster injections were administered using μg purified pedv viral particles with μl incomplete freund's adjuvant (sigma) at intervals of weeks. three days before sacrifice, mice were immunized with μg purified pedv viral particles in pbs (gibco) via intrasplenic (is) injection. serum antibody titres at each immunization were monitored using a complete pedv viral particle elisa and the mouse with the highest titre was sacrificed for hybridoma preparations. hybridoma preparation. splenocytes were isolated from the mice immunized with purified pedv particles. after gentle washing with brief centrifugation, splenocytes were fused with sp myeloma cells at a cell ratio of approximately : using % polyethylene glycol (sigma). hybridomas were seeded onto -well culture plates in rpmi- medium supplemented with % foetal bovine serum (gibco), mg/ml streptomycin, and iu/ml penicillin (sigma), and incubated overnight at °c in a humidified incubator with % co . after incubation, approximately % medium was removed from each well, and a selective hat rpmi- medium (hat-rpmi) (sigma) was added to achieve a final concentration of % foetal bovine serum (gibco), mg/ml streptomycin, iu/ml penicillin, mm hypoxanthine (sigma), mm aminopterin (sigma), and mm thymidine (sigma). wells containing growing hybridoma cells were screened for antibody production by icc staining using pedv-infected vero cells or hek cells (atcc crl- ™ ) expressing the full-length pedv s protein. positive clones were isolated for limiting dilution and incubated in selective ht rpmi- medium without aminopterin. after two limiting dilutions, the supernatant from each line was further tested for anti-pedv s-specific antibodies by icc staining using pedv-infected vero cells or hek cells expressing the full-length recombinant pedv s protein. mab purification and quantification. to purify mabs from cultured supernatants, pierce ™ protein l magnetic beads (thermo fisher scientific, waltham, usa), which selectively bind to mouse immunoglobulin were utilized following the manufacturer's instructions. the beads were mixed with ml supernatants of each mab and incubated for h after which the antibody-bound beads were collected using a magnetic stand. after washing thrice wash buffer (tris-buffered saline (tbs), . % tween- detergent), the mabs were eluted with μl elution buffer ( . m glycine, ph . ) for min and then, alkalized pbs buffer (ph . ) was added for neutralization. the concentrations of each purified mab were determined by the pierce ™ bca protein assay kit (thermo fisher scientific). of the full-length pedv s protein of the taiwan pedv-pintung- (pedv-pt) strain (genbank: ky . ) was performed as previously described . to prepare recombinant truncated pedv s proteins, regions of the s gene coding for amino acids - (s - ), - (s - ), - (s - ), - (s - ), - (s - ), - (s - ) were amplified using specific primers (supplementary table s online) and the resultant proteins were purified as described previously . the amplicons encoding different truncated genes were subcloned into a bamhi-noti restriction site in the pcdna tm . /v -his topo ® vector (invitrogen, carlsbad, ca, usa), transfected into hek cells and selected by using μg/ml geneticin (g , gibco). after two weeks of selection, cells stably expressing the truncated pedv s proteins were subjected to icc staining and western blotting. purification and western blot detection of truncated spike proteins. large-scale purification of the truncated s proteins of pedv-pt was performed as previously described . the protein expressing hek- cells were harvested, resuspended, and cultured in freestyle expression medium (gibco) for one week. after supernatant collection and removal of cell debris through a . μm filter, the proteins were banded with hispur cobalt resin (thermo fisher scientific) following the manufacturer's protocol. the purified proteins were subsequently concentrated with a kda vivaspin ® (ge healthcare life sciences, ny, usa), complete ™ edta-free protease inhibitor cocktail (roche molecular biochemicals, quebec, canada) was added and the concentrations were measured using the pierce ™ bca protein assay kit. the purified proteins were denatured in a buffer containing nupage ® lds sample buffer and nupage ® reducing agent, and heated for min at °c. using bio-rad mini-protein ® electrophoresis system(bio-rad, hercules, ca, usa), the samples were separated using % sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis (page) cast in a gradient t-pro ez gel solution (t-pro biotechnology, taiwan) according to the manufacturer's protocol. following this, proteins were wet-blotted onto a polyvinylidene difluoride (pvdf) membranes (bio-rad)blocked with % skim milk in tris-buffered saline with . % tween (tbs-t) buffer at room temperature (rt) for h. membranes were incubated with a : diluted anti-v tag antibody for h at rt. after washing with tbs-t, horseradish peroxidase (hrp)-conjugated goat anti-mouse igg secondary antibody ( : , dilution in blocking buffer; jackson immunoresearch laboratories, philadelphia, usa) was added and incubated at rt for h, the signals were visualized using clarity ™ western ecl blotting substrates (bio-rad) and detected with a chemidoc ™ xrs+ imaging system (bio-rad). neutralizing assay for the pedv-pt strain. a neutralizing assay was conducted as previously described with some modifications . next, μl suspended vero cells (atcc no. crl- ) were seeded onto -well culture plates at × cells/ml and incubated overnight at °c in a humidified incubator with % co until cells reached % confluence. purified mabs were two-fold serially diluted from μg/ml to . μg/ml in post-inoculation medium (pi medium) containing dulbecco's modified eagle medium (gibco) supplemented with tryptose phosphate broth ( . %) (sigma, missouri, usa), yeast extract ( . %) (acumedia, ca, usa), and μg/ml trypsin (gibco). fifty microliters of diluted culture supernatant from each hybridoma clone were mixed and incubated with an equal volume of tcid /ml pedv-pt passage (pedv-pt-p ) at °c in % co porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis journal of veterinary diagnostic investigation: official publication of the american association of veterinary laboratory diagnosticians a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea phylogenetic analysis of the spike (s) gene of the new variants of porcine epidemic diarrhoea virus in taiwan assessment of the economic impacts of porcine epidemic diarrhea virus in the united states porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus emerging and re-emerging infectious diseases of livestock cellular entry of the porcine epidemic diarrhea virus receptor usage and cell entry of porcine epidemic diarrhea coronavirus identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (pedv) neutralizing epitopes identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies the s glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes efficacy of heat-labile enterotoxin b subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains new variant of porcine epidemic diarrhea virus evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains receptor recognition mechanisms of coronaviruses: a decade of structural studies structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy crystal structure of bovine coronavirus spike protein lectin domain porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor mapping epitope structure and activity: from one-dimensional prediction to four-dimensional description of antigenic specificity continuous and discontinuous protein antigenic determinants prediction of residues in discontinuous b-cell epitopes using protein d structures evaluation and comparison of the pathogenicity and host immune responses induced by a g b taiwan porcine epidemic diarrhea virus (strain pintung ) and its highly cell-culture passaged strain in conventional -week-old pigs display of porcine epidemic diarrhea virus spike protein on baculovirus to improve immunogenicity and protective efficacy ucsf chimera-a visualization system for exploratory research and analysis washing six times, μl of abts ® peroxidase substrate system (kpl, seracare) was used to obtain a positive signal. the coloration procedure was stopped after min by adding μl stopping solution (kpl, seracare). the signals were detected at nm wavelength by emax plus microplate reader (molecular devices, crawley, uk). the final data is shown as a sample to positive ratio (s/p ratio), representing the difference between the od values of the sample and the negative control divided by the difference between the od value of the positive and negative controls. the od value of the positive control was obtained from the result of , x diluted anti-v tag antibody, and the od value of the negative control was obtained from the result of the secondary antibody only wells.linear epitope mapping by immunodot blotting and western blotting. to confirm the conformational significance of the neutralizing epitopes, we also denatured the six purified truncated spike and the full-length spike protein of pedv-pt by mixing them with nupage ® reducing agent (thermo fisher scientific) and heating for min at °c. the purified proteins and the denatured proteins were separately dotted on nitrocellulose membranes (merck millipore, ma, usa) at ng. the reducing agent mixed with water was also dotted on the membrane as a negative control. following a -h block in % skim milk, the membranes were stained with nmabs (p b- and e e- - ) and non-neutralizing mab (r h- ) at . μg/ml diluted in tbs-t buffer at rt for h. after washing three times, hrp-conjugated goat anti-mouse igg secondary antibody ( : , dilution, jackson immunoresearch laboratories) was added and incubated at rt for h. protein signals were visualized using the clarity ™ western ecl blotting substrates (bio-rad) and detected with a chemidoc ™ xrs+ imaging system (bio-rad) as previously described. to ensure the denatured proteins were correctly dotted on the membrane, after the first run of probing, the mabs were stripped with stripping buffer (thermo fisher scientific) and the membranes were separately re-probed by anti-v tag antibodies (invitrogen). a full-length homology model of the ectodomain of recombinant pedv s protein was generated by swiss-model and built with promod version . . (https://swissmodel.expasy. org). the manipulated protein sequence of the pedv s ectodomain was constructed using the trimeric human coronavirus nl spike structure (pdb accession no. szs) as a template. the images were edited using ucsf chimerax molecular modelling system . the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - qvwjejv authors: gera, tamás; nagy, eszter; smausz, tamás; budai, judit; ajtai, tibor; kun-szabó, fruzsina; homik, zsolt; kopniczky, judit; bozóki, zoltán; szabó-révész, piroska; ambrus, rita; hopp, béla title: application of pulsed laser ablation (pla) for the size reduction of non-steroidal anti-inflammatory drugs (nsaids) date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: qvwjejv we studied the application of pulsed laser ablation (pla) for particle size reduction in non-steroidal anti-inflammatory drugs (nsaids). grinding of the poorly water-soluble nsaid crystallites can considerably increase their solubility and bioavailability, thereby the necessary doses can be reduced significantly. we used tablets of ibuprofen, niflumic acid and meloxicam as targets. nanosecond laser pulses were applied at various wavelengths (krf excimer laser, λ = nm, fwhm = ns and nd:yag laser, λ( ) = nm/λ( ) = nm, fwhm = ns) and at various fluences. ftir and raman spectra showed that the chemical compositions of the drugs had not changed during ablation at nm and nm laser wavelengths. the size distribution of the ablated products was established using two types of particle size analyzers (smps and opc) having complementary measuring ranges. the mean size of the drug crystallites decreased from the initial – µm to the submicron to nanometer range. for a better understanding of the ablation mechanism we made several investigations (sem, ellipsometry, fast photography) and some model calculations. we have established that pla offers a chemical-free and simple method for the size reduction of poorly water-soluble drugs and a possible new way for pharmaceutical drug preformulation for nasal administration. | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ any chemical decomposition. in recent years, researchers have also started to use lasers for particle size reduction in drugs in liquid environment (plal) [ ] [ ] [ ] [ ] [ ] [ ] . in this work, three nsaids (ibuprofen, meloxicam and niflumic acid) with different chemical structures but similar solubility, dissociation constant, particle size and crystallinity were selected as targets to study the effect of a high-energy pulsed laser beam on the chemical degradation and particle size distribution of the ablated drug particles. being selective cyclooxygenase (cox- ) inhibitors, these model drugs are used in acute pain therapy where a basic requirement is rapid absorption through the gastric mucosa . since these drugs have a weak acidic character, their solubility in gastric juice (ph = . ) is very poor. on the other hand, ablated particles dissolve faster and can produce rapid analgesic effect. our aim was to study the validity of the pla method for particle size reduction in different drugs with low dissolution rates, in ambient gas at normal pressure. ibuprofen, meloxicam and niflumic acid are frequently used, poorly water-soluble nsaids, representing three different molecular derivatives. we also wanted to explore the mechanism of pla when drug targets with different thermal and optical properties are ablated under various experimental conditions. to cover the uv-vis-ir regime with ablating laser wavelengths, a krf excimer laser (λ = nm) and a nd:yag laser system (λ = nm, λ = nm) were used. we adjusted laser fluences at each wavelength to the optical and mechanical properties of the target materials. ablated products were collected at normal ambient pressure using n gas flow. we studied the chemical composition and particle size distribution of the ablated particles, the optical absorption of the target drugs and made fast photography measurements too. our motivation was to show that pla, a simple and chemical-free particle grinding method, has a great potential in pharmaceutics. laser ablation produced drug particles can be preferable during the preparation of medicines for per os, nasal and pulmonary drug administration. pulmonary administration of anti-inflammatory drugs like meloxicam or ibuprofen can be especially significant in the treatment of pneumonia or severe acute respiratory syndrome caused by viral infections (e.g. coronavirus). it has been shown that only sub-micrometer size particles can get to the lower lung area (alveolar region) . therefore, size-reduced drug particles produced by pla may form the basis of a new type of pulmonary drug formulation for the fast and effective treatment of the hard-to-reach alveolar region of the lung. ibuprofen. ibuprofen (α-methyl- -(isobutyl) phenylacetic acid) was obtained from sigma-aldrich ltd., (saint louis, missouri, usa). it is a white powder with a particle size of . μm (d( . )). nsaid classification: propionic acid derivatives. thermal properties: melting point t m = - °c; boiling point t b = °c; degradation temperature t dec = to °c. meloxicam. meloxicam( -hydroxy- -methyl-n-( -methyl- -thiazolyl)- h-benzothiazine- -car-boxamide- , -dioxide) was obtained from egis ltd., (budapest, hungary). it is a yellow powder with a particle size of . μm (d( . )), % crystalline. nsaid classification: enolic acid (oxicam) derivatives. thermal properties: melting point and decomposition temperature are the same, t m = t dec = °c. niflumic acid. conventional niflumic acid (nif) ( -[[ -(trifluoromethyl)phenyl] amino] - -pyridinecarboxylic acid) was purchased from g. richter pharmaceutical factory, budapest, hungary. the particle size is . μm (d( . )). nsaid classification: anthranilic acid derivatives (fenamates). thermal properties: melting point and decomposition temperature are the same t m = t dec = °c. pulsed laser ablation (pla) of drug tablets at different laser wavelengths. we applied three different laser wavelengths for pulsed laser ablation (pla) of various drug tablets. for uv irradiation a krf excimer laser (llg twinamp, fwhm = ns, λ = nm, f = hz) was used, while for vis and ir ablation we applied the first and second harmonics of a nd:yag laser (quantel, fwhm = ns, λ = nm/ nm, f = hz), respectively. we varied the number of laser pulses and adjusted the fluence from . to j cm − . in order to achieve a significant ablation yield, we used fluences slightly above the ablation threshold. the highest applied fluence was limited by the mechanical fracture of the target tablets. the tablets were produced from commercially available drug powders using a hydraulic compactor at mpa pressure. our experimental setup is shown in fig. . the tablets were placed on a rotating sample holder at the bottom of the reaction chamber. the laser beam was focused on the target surface at a grazing angle of ° by a fused silica (uv) and an n-bk (vis-ir) fused silica plano convex lens (focal length = . cm). to be able to direct and control the ablated particles, we created a flow field of n gas along the chamber. a constant flow rate ( . - l/ min) was maintained by a t-element at the entrance of the gas jet. the ablated particles were either collected for chemical analysis on a filter (pore size ~ μm, merck millipore ltd. omnipore membrane filter), or they directly entered the particle size analyzer at the exit of the gas stream. theoretically, the pore size of the membrane filter is μm, yet smaller elements can also be collected as the pores fill up with particles reaching the filter first. chemical composition characterization methods. fourier transformed infrared spectroscopy (ftir) . the preservation of the medical effect is an essential requirement during particle size reduction in pharmaceutical substances. to ensure that the chemical composition of the ablated products is identical with that of the initial drugs, the ablated particles were analyzed by ftir spectroscopy (thermo nicolet avatar , labx midland, on, canada). the particles were collected from the filters and mixed with kbr powder for pellet formation. ftir spectra were recorded in the - cm − range, at a resolution of cm − , using the average of scans. particle size analysis. pla provides an efficient way for grinding; however, the size distribution of the ablated particles spans a wide range, wherefore we used two types of size analyzers, having complementary, slightly overlapping measuring ranges. scanning mobility particle sizer (smps). we investigated the particle size distribution in the - nm range with smps (grimm system, aerosol technik, germany, type smps + c). we set the scanning time to min and repeated the measurements three times. the gas flow was . l/min during the scans. optical particle counter (opc). to establish if particles larger than nm were also produced, we used an opc (grimm system, aerosol technik, germany, type . ). opc can reliably measure particles in the nm to μm size range. we set the scanning time to min and took at least scans during each measurement. the gas flow in the equipment was l/min. to better understand the particle formation mechanism during ablation, we investigated the laser treated tablet surfaces, the morphology of the laser irradiated areas and the backscattered particles nearby using scanning electron microscopy (hitachi s- ). prior to imaging, the samples were sputter-coated with gold (bio-rad sc ). sem images were recorded at a magnification of ×. ellipsometry. the optical absorbance of the target material plays a key role in laser ablation. since there is no relevant and reliable information about the optical absorbance ([α] = /nm) of the studied drugs in the - nm wavelength range, we determined the relevant values from ellipsometric data. ellipsometric measurements were performed at different points on the tablets using a woollam m f rotating compensator ellipsometer. data were collected at ° angle of incidence, applying focusing optics. the ellipsometric data measured in the - nm wavelength range could be directly transformed to complex refractive index values, from which the absorption coefficients were determined according to the α = *π*κ/λ expression. fast photography of the ablation process. we also set up a commonly used pump and probe system ( fig. ) for the visual observation of the ablation process. the pump laser was the same nd:yag laser (quantel, fwhm = ns, λ = nm/ nm) as for the ablation experiments. as probe lasers, we built two types of nitrogen laser induced dye lasers corresponding to the ablating wavelengths. in case of λ ablating = nm we used coumarin as a laser active material in the dye laser (λ = nm, fwhm = ns), while in case of λ ablating = nm we had to change the laser active dye to rhodamine g (λ = nm, fwhm = ns) in order to avoid an overlap between the pump and probe signals. the dye laser beams were transported to the experimental field by an optical fiber and collimated with an n-bk focusing lens (f = cm). a ccd camera (the imaging infrared spectroscopy (ftir) of ablated drug particles. ftir spectra obtained from the collected particles are shown in fig. a . some contamination of the sample was inevitable while the collected particles were scraped or peeled off the filter. therefore, we subtracted the ftir spectrum of pure millipore filter as background spectrum. the two peaks between and cm − are related to the h o and co content of the samples and give no relevant information for this investigation. ftir spectra of ibuprofen particles generated at different wavelengths and fluences are shown in fig. . it can be seen that all laser fluences applied at the uv wavelength ( nm) caused significant chemical damage to the particles. however, the persisting characteristic peaks of ibuprofen [e.g. cm − (h-bonded c=o); cm − (h-bonded co-h); cm − (c=o)] in the ftir spectra indicate that no chemical changes occurred at vis ( nm) and ir ( nm) wavelengths. we had similar observations in the case of niflumic acid [e.g. cm − (c-o); cm − (benzene ring); cm − (c=o/oh); cm − (c-f)] , and meloxicam [e.g. cm − (n-h); cm − (thiazole ring); and cm − (sulfone)] , (fig. ) . during ir ablation of niflumic acid, . j cm − and j cm − laser fluences yielded a sufficient amount of particles for ftir analysis. since we wanted to avoid chemical modification of the pharmaceutical compounds, we used only vis and ir wavelengths for further investigations. www.nature.com/scientificreports/ raman spectroscopy of generated drug particles. we recorded several spectra at different places from each ablation product to obtain information about their homogeneity. for simplicity, in fig. , only those spectra are compared with the original pharmaceuticals' spectra that were recorded from the particles ablated by the highest laser fluences (i.e., may have suffered the most severe chemical changes) at both wavelengths. we marked some characteristic bands of ibuprofen ( , and cm − ) (fig. a) , niflumic acid ( , and cm − ) (fig. b) and meloxicam ( , and cm − ) (fig. c) , and found no discrepancies in the spectra of the original drugs and their ablation products produced at and nm. particle size analysis. using both smps and opc, we investigated the size distribution of the ablated particles in a very wide range, from nm to μm. for each fluence, the repetition rates of the laser shots were adjusted to prevent the suction tube from getting clogged by ablated particles. during evaluation, we normalized the particle numbers to hz repetition rate for data comparison. size distribution (measured by smps) of ibuprofen particles produced by laser ablation at nm and nm wavelengths can be seen in fig. . the mode values in both distributions were below nm. the ablation yield of niflumic acid was considerably higher than for ibuprofen, especially at nm (fig. ) . the broadest size distributions with the highest mode values were obtained for the ablated meloxicam particles (fig. ) ; the full widths at half maximums (fwhms) of the distributions ranged from to nm and the mode values for higher fluences exceeded nm. we fitted one or more lognormal curves to the measurements. in case of high ablation yields data could be fitted to a single curve, while we had to cumulate two or more curves for small amounts of ablated particles. www.nature.com/scientificreports/ all measurements above were also repeated with the opc system to see if particles larger than nm were also produced. since opc uses a higher gas flow than smps, the number of particles had to be normalized to the flow rate of smps and hz repetition rate. it must be noted that the two systems give different size values due to the different detection mechanisms (smps-by electrical mobility; opc-by optical scattering). hence the differences between them are minor and the values are mainly the same . we did not find many particles larger than nm in either case. figure shows the particle size distributions obtained by both smps and opc for the ablation of niflumic acid. we chose this plot to demonstrate a common overlap of the distributions. the overlaps are the same for the other ablating parameters too. the main experimental results are summarized in table . there is no sign of clear relationship between laser fluences and size distributions in the investigated fluence range. however, we can establish that the number of ablated drug particles grows with the applied fluence at any wavelength. the growth in ablated particles can clearly be seen at λ = nm ablation. in case of ibuprofen and niflumic acid, the most abundant particles are smaller at ir ablation than at vis with equivalent fluences. in the case of meloxicam, there were no overlaps in the fluence values due to different ablating outcomes (thresholds, cracks). nevertheless, we can still claim that at higher laser fluences the modes of meloxicam particle size distributions belong to the same size range for ir and vis wavelengths. it can be clearly stated that in any case the efficiency of ablation (i.e. the number of ablated particles) is the highest for meloxicam, slightly lower for niflumic acid, while it is the lowest for ibuprofen. sem investigation of ablated surfaces. sem images taken from the intact and ablated drug tablet surfaces are shown in fig. . laser irradiations at vis and ir wavelengths resulted in similar surface structures: ellipsometry. absorption spectra of the investigated drugs were recorded in a wide optical range, from ultraviolet to near infrared. the smallest absorption coefficients were obtained in the ir, while the biggest ones in the uv range (fig. ) . the increased absorption in the uv is most noticeable for meloxicam and niflumic acid. this is in accordance with our observation that drug molecules degrade during ablation by uv light. . this wavelength falls outside our measurement range ( - nm), therefore we could not detect it. we estimated the laser induced temperature rise at different depths in the target drugs by simple model calculations in which we varied the laser wavelength and fluence for each drug. ignoring heat conduction, we applied the beer-lambert law (eq. ) to calculate the temperature change at the surface and at the penetration depths: where α is the wavelength dependent absorption coefficient, x is the distance from the surface, f is the applied fluence of the laser beam, ρ is the density and c is the specific heat of the target material. we used the mean specific heat values between room temperature and the decomposition temperatures of the drugs in the absence of reliable data in the literature and since c is temperature dependent. we also calculated the reflexivity of the drugs for different incident angles and polarizations. since reflexivity was under % for all wavelengths, we neglected it in the calculations. the parameters and the calculated penetration depths (d = /α) are summarized in table . in all three medicines the highest temperatures occur at the uv wavelength. the temperature values are close at vis and ir wavelengths, hence the differences between the absorption coefficient values are minor. at these two wavelengths, during ibuprofen ablation the estimated temperatures (depending on the fluences) exceed several thousand kelvins ( - , k) at the surface where the temperature values are the highest. this is followed in magnitude by meloxicam, where these values are higher and reach , - , k. surface temperatures are the highest in niflumic acid, where these values reach , - , k. the temperatures at the laser penetration depths always significantly exceed the decomposition temperatures of the materials. therefore, chemically preserved particles cannot originate from the volume element (v e )-defined by the laser spot size and penetration depth (v e = a spot *d)-and these particles must be produced by another mechanism. fast photography. successive stages of the ablation process were captured by fast photography. this information is valuable for us in representing and understanding the dynamics of particle generation. figure shows fast photography images deemed essential for the important stages of particle generation. it can be clearly seen that the ablation mechanism is the same for all three studied drugs. the generated shock wave is closely followed by a cloud of non-condensed state of matter expanding at a slightly lower speed than the shock front. after that, solid particles increasing in size with time start to exit the surface, which means that bigger and heavier particles have lower velocities. it can be easily observed that particles of all sizes were generated during the ablation of drug tablets, although we could not detect a sufficient number of particles above μm with the particle sizer methods. this can be explained with the mass dependence of the mean free path and the phenomenon that the magnitude of gas flow limits the sizes of transportable particles. ( ) figure . absorption coefficients of meloxicam (black), niflumic acid (red), and ibuprofen (blue). www.nature.com/scientificreports/ to estimate the significance of photomechanical effects during ablation, we calculated the pressures produced in the target material at the shock fronts . first we calculated the propagation velocity of the shock front by measuring its travel distance as a function of time, and then the pressure was estimated according to eq. : where p is the pressure produced at the shock front, ρ is the density of the medium, v is the velocity of the shock front and γ is the proportion of specific heats (c p /c v ) of the medium. in fig. some examples of the calculated pressures are shown. the recoil pressures-which are equivalent with the initial pressures at the shock front-are - atm. these relatively high pressure values confirm our assumption, that the photomechanical effects have a significant contribution to the ablation process. in the course of pla, two successive and significantly different material ejection mechanisms can be distinguished: (i) prompt evaporation of the target material directly from the center of the focused laser beam spot, where very high temperatures are created close to the surface. (ii) material ejection induced by the photomechanical effect due to pressure pulse and shock wave formation in the bulk material as a result of fast evaporation. according to our approximate calculations, temperatures as high as k to k can be created at the www.nature.com/scientificreports/ target surface. some of the imparted energy is transferred inside the target material by heat conduction, and in the volume element (v e ) the temperature exceeds a critical value and fast vaporization processes occur with the formation of high-pressure ionized plasma. in this nonequilibrium plasma micro-explosions take place, and the pressure pulse initiates the propagation of a high velocity shock wave outwards from the excited volume element. then vaporized and highly decomposed products burst out as shown in fig. a . however, a major part of the target material is ejected via photomechanical processes. the shock wave induced recoil forces and mechanical stresses result in the ejection of small and large fractures from the surface. the photomechanical ablation mechanism is more significant in case of nanosecond laser pulses . the fractured particles leave the target at different velocities in accordance with their size and mass. this is in good agreement with our fast photography results (fig. ) where submicron sized particle ejection occurs at nanosecond and few microsecond time scales. larger, micron-sized and irregularly shaped particles are ejected in the microsecond scale [ ] [ ] [ ] [ ] . the above observations apply mainly to homogeneous bulk materials. in case of drug tablets where the targets are highly porous and consist of micrometer-sized grains, the pla process is slightly different. the uneven surface of the ablation holes, which can be clearly seen in the sem images (fig. ) and the pressure calculations in indicate strong photomechanical effects. thus, the subsequent laser pulses reach irregularly shaped target surfaces. the effect of target inhomogeneity is pictured in fig. , as multiple shock waves are developing after one laser pulse. consequently, the ideal homogenous optical absorption mechanism does not hold for our porous drug tablet targets, where lower temperature changes and photomechanical effects occur. furthermore, since the drug grains are more loosely bound together in compacted tablets than in a bulk material, much higher ablation yields can be attained. occasionally the recoil forces rip out large pieces (several microns in diameter) from the tablets. these phenomena explain the random fluence dependency in the size distribution of the ejected particles (table ). in bulk materials, the average size of the ablated particles and the fwhms of the particles' size distribution increase with the fluence , . according to the ftir investigations, pla at uv (λ = nm) laser wavelength is not an adequate method for creating chemically preserved particles. this conclusion is supported by the high absorption coefficients of drugs measured in the uv regime (see fig. ). our calculations, which were based on the absorption coefficients, show that the uv laser beam creates the highest temperature in the excited volume element, and it has the lowest penetration depth in the target material (see table ). therefore, the secondary photomechanical effects, which can lead to the ejection of chemically non-degraded particles, are less significant. moreover, the energy of the photons (e p (λ = nm) = ev) exceeds the energy of several chemical bonds in the drug molecules, enhancing the probability of direct photochemical reactions like photolysis or photo dissociation. we concluded that by pla at λ = nm, the ejected particles consist mainly of degraded drug molecules, similarly to polymers seen before [ ] [ ] [ ] . at higher wavelengths, i.e. at lower photon energies [e p (λ = nm) = . ev; e p (λ = nm) = . ev], the photochemical effects are less significant, but at the same time more drastic photomechanical effects can be expected. due to the lower temperatures induced in the target material and the larger penetration depths of the vis/ir laser beams, the secondary material ejection mechanisms are more prominent at nm and nm laser wavelengths. this allows for the production of chemically preserved drug particles . with the use of a laser beam at nm considerably higher ablation yields could be achieved even at lower fluences than at nm (table ). this can be related to the higher (but still moderate) absorption coefficient at nm. according to our observations, at nm laser wavelength the mechanical degradation (photo disruption) of the tablets was so severe that the tablets rapidly broke into pieces, greatly decreasing the ablation lifetime and ablation yield. a more elaborate evaluation of the ablation yields requires the consideration of the thermal and mechanical characteristics of the target materials too. it has been shown that there is a strong relationship between the mechanical properties (density, mechanical hardness) of the target and the ablation yield . we suspect that the mechanical properties of the drug tablets influenced our experiments too. this can explain our observation that the ablation yields are higher for meloxicam than for niflumic acid although meloxicam's absorption coefficients are lower at both wavelengths. we must make two additional remarks. one concerns the absence of absorption bands in the ftir spectra, which can be related to the degraded molecules. we suppose that at temperatures of thousands of kelvins in the evaporation zone the material is practically atomized/ionized, and therefore cannot be filtered out from the gas jet. this means that if there are any chemically modified but not completely atomized molecules, they must represent a negligible proportion of the whole ablation mass. our other comment is that during the size distribution measurements (fig. ) no particles above ~ μm in diameter were detected, although the formation of bigger particles can evidently be seen in the fast photography pictures (fig. ) . most probably, these bigger and heavier particles simply fell back to the surface, since even the highest adjustable gas flow was unable to transport them. conclusion pla can be applied for the size reduction of poorly water-soluble nsaids. in the case of meloxicam, ibuprofen and niflumic acid, submicron to nanometer size particles can be produced by careful selection of the laser parameters, reducing the initial mean average sizes by orders of magnitude. we have found that ablation without any chemical destruction of the initial pharmaceutical compounds can only be performed with laser beams having wavelengths in the vis and ir range. higher ablation yields are obtained at nm than at nm laser wavelength, and the yields are generally increasing with the applied fluence for the studied drugs. without aiming to conduct a thorough investigation we could also establish the influence of the absorption coefficients and the structure of the target drug tablets on the ablation mechanism. the smps/opc measurements provided particle size data precisely from that fraction of the laser ablated aerosol which can be pumped through a nasal delivery device and inhaled by the patient. | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ we concluded that pla offers a chemical-free, verifiably reproducible and simple method for the size reduction of poorly water-soluble drug crystallites. the achieved submicron size range can be especially advantageous for the development of new pulmonary drug formulations for the medication of the lower lung area in pneumonia or severe acute respiratory syndrome. the adjustable laser parameters and thereby the fine tuning of the grinding mechanism during pla can be advantageous over other commonly used methods when formulating drug delivery systems for sensitive compounds. drug solubility: importance and enhancement techniques a literary endorser solubility enhancement techniques-a review improvement in solubility of poor water-soluble drugs by solid dispersion production of ibuprofen in crystalline and amorphous forms by pulsed laser deposition (pld) enhanced solubility of non-steroidal anti-inflammatory drugs by hydroxyl terminated s-triazine based dendrimers nanocrystals: industrially feasible multifunctional formulation technology for poorly soluble actives particle size reduction of biomaterials using cryogenic milling process an investigation on the application of cryogenic ball milling to ibuprofen particle and its characteristics combinative particle size reduction technologies for the production of drug nanocrystals overview of milling techniques for improving the solubility of poorly water-soluble drugs methods of size reduction and factors affecting size reduction in pharmaceutics analysis of submicron-sized niflumic acid crystals prepared by electrospray crystallization laser ablation synthesis in solution and size manipulation of noble metal nanoparticles size reduction of gold nanoparticles by pulsed laser ablation and re-irradiation in water media laser ablation of inorganic and organic materials a study of particle generation during laser ablation with applications production of meloxicam suspension using pulsed laser ablation in liquid (plal) technique laser fragmentation as an efficient size-reduction method for pulmonary drug discovery: proof-of-concept study of beclomethasone dipropionate ultrafast laser processing of drug particles in water for pharmaceutical discovery nanoparticle formation by laser ablation of perylene microcrystals in an aqueous solution of triton x- reagglomeration mechanism of drug nanoparticles by pulsed laser deposition indomethacin nanoparticles directly deposited on the fluidized particulate excipient by pulsed laser deposition modifying the physicochemical properties of nsaids for nasal and pulmonary administration air quality criteria for particulate matter conformational stability of ibuprofen: assessed by dft calculations and optical vibrational spectroscopy spectral studies of niflumic acid aggregation in dissolved, solid and adsorbed states structural characterization of ambazone salt with niflumic acid mechanochemical preparation of organic-inorganic hybrid materials of drugs with inorganic oxides physicochemical characterization, the hirshfeld surface, and biological evaluation of two meloxicam compounding pharmacy samples a method for segregating the optical absorption properties and the mass concentration of winter time urban aerosol detection of ibuprofen and ciprofloxacin by solid-phase extraction and uv/vis spectroscopy approximate spherical blast theory including source mass permalloy nanoparticles generated by laser ablation particle generation by ultraviolet-laser ablation during surface decontamination laser application of polymers wavelength dependence of pulsed laser ablation of calcified tissue t.g. and e.n. carried out the ablation of drugs and ftir measurements. r.a. and p.sz.r. supported the pharmaceutical background of the article. t.s.k., t.g. and z.h. was responsible for fast photography measurements and calculations. t.a., f.k.s., and z.b. made the particle size measurements and prepared relevant figures. j.b. did the ellipsometry measurements. t.g., j.k. and b.h. wrote the main manuscript text. all authors reviewed the manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to t.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -xwedgllw authors: korabecna, m.; zinkova, a.; brynychova, i.; chylikova, b.; prikryl, p.; sedova, l.; neuzil, p.; seda, o. title: cell-free dna in plasma as an essential immune system regulator date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xwedgllw the cell-free dna (cfdna) is always present in plasma, and it is biomarker of growing interest in prenatal diagnostics as well as in oncology and transplantology for therapy efficiency monitoring. but does this cfdna have a physiological role? here we show that cfdna presence and clearance in plasma of healthy individuals plays an indispensable role in immune system regulation. we exposed thp cells to healthy individuals’ plasma with (np) and without (tp) cfdna. in cells treated with np, we found elevated expression of genes whose products maintain immune system homeostasis. exposure of cells to tp triggered an innate immune response (iir), documented particularly by elevated expression of pro-inflammatory interleukin . the results of mass spectrometry showed a higher abundance of proteins associated with iir activation due to the regulation of complement cascade in cells cultivated with tp. these expression profiles provide evidence that the presence of cfdna and its clearance in plasma of healthy individuals regulate fundamental mechanisms of the inflammation process and tissue homeostasis. the detailed understanding how neutrophil extracellular traps and their naturally occurring degradation products affect the performance of immune system is of crucial interest for future medical applications. to catch and destroy the infectious microorganisms. these nets are subsequently cleared from the circulation by dnase i and their insufficient clearance can result in occlusion of blood capillaries, leading to impaired microcirculation, enzymatically damaging tissues and further progression of inflammation . the elevated formation of nets was reported in most comorbidities worsening the clinical course of covid- . the hyperactivated neutrophils and monocytes-macrophages are the usual initiators of the cytokine storm responsible for the most serious consequences of coronavirus sars cov- infection . bacteria and protozoa are able to convert the nets using their own enzymes into the dna degradation product such as deoxyadenosine which is toxic for macrophages and causes their apoptosis , . despite all these findings and the massive cfdna-based diagnostic technique developments, there are surprisingly few studies focused on the fundamental biological function of cfdna in healthy individuals. one study treated human monocytes either with the plasma of dialyzed patients or healthy individuals, both containing cfdna. only the plasma from dialyzed patients stimulated the production of pro-inflammatory interleukin il in target cells . it was also revealed that the cfdna isolated from individual tumor cell lines or complex tumors injected into animal blood circulation caused tumor transformation, referred to as genometastasis . the biological character and function of unmethylated fetal cfdna was explored and the increased proportion of fetal cfdna in maternal circulation during pregnancy was found , .this fetal cfdna stimulated a maternal immune response against the placenta, resulting in the proper timing of labor . the influence of cfdna on human macrophages was tested using isolated cfdna from various blood products with different storage time to emulate the conditions immediately after transfusion. the cells were cultivated in the presence of calf serum bearing its own cfdna. the study found increased expression of genes involved in the innate immune response (iir), including chemokines and their receptors in macrophages, and concluded that cfdna contained in the stored blood products might interfere with the immune system of transfusion recipients . the authors reported the elevated expressions of cxcl and ddit in experiments, but the results may be affected by the presence of calf serum in all experiments. this raises the following question: what is the fundamental role of cfdna in healthy organisms? we assumed that we could find it by exposing identical cell lines to plasma with and without cfdna and excluding any other factors, such as calf serum or potential damage of cfdna complexes by isolation procedure. we performed all stimulatory experiments using the thp cell line as a representative of primary human monocytes to show the fundamental role of cfdna in healthy organisms. the experiments were conducted in duplicates using plasma containing cfdna (np) and the reference one with cfdna removed by dnase (tp) to recognize the effect of plasma cfdna on transcriptome and proteome of monocytes. we used native human plasma samples obtained from healthy volunteers with no animal serum addition to the cultivation medium in order to avoid the presence of uncharacterized animal cfdna and dnases in the experiments. in the discovery phase, we used six plasma samples, searching for differences in transcriptomes related to treatment with np and tp using genechip human gene . st array strip (thermo fisher scientific). we detected significant differences ( fig. a ; supplementary table ) , which were further validated using single-target quantitative pcr (qpcr) and another ten plasma samples ( fig. b-d) . to differentiate between the effects of buffers alone and the effect of dnase turbo treatment, we performed a set of experiments ( supplementary fig. ). the addition of activation buffer containing divalent cations is necessary for the activity of dnase turbo in plasma originally treated with ethylenediaminetetraacetic acid (edta). the addition of this buffer alone led after incubation to the decrease of cfdna in the plasma sample to . % of its original level due to the activation of plasma endogenous dnase i. the incubation of plasma sample with this buffer and dnase turbo resulted in the detection . % of cfdna original amount when measured after its isolation from plasma using qpcr. in this set of stimulation experiments, we tested the effects of the complete procedure allowing the dnase turbo activity in plasma and its subsequent removal but without the addition of dnase turbo itself and compared the results with np and tp samples. all these experiments were performed using a plasma sample obtained from an identical donor. we examined the expression of all validated genes and concluded that the activation of endogenous dnase i was mostly sufficient to produce the basic differences which could be in some cases further strengthened with subsequent dnase turbo treatment ( supplementary fig. ). the procedure serving for the removal of divalent cations was applied during the processing of all treated plasma samples to avoid the elevated concentrations of these ions in samples due to the dnase turbo activation buffer addition. we received the results identical with the results of validation experiments in eight out of eleven examined genes as we explored the stimulatory capacity of only one differentially treated plasma sample. the expressions of didt and sesn were significantly elevated in cells treated with np samples in validation experiments but decreased in cells treated with np sample of this donor. the ccl expression was significantly increased in cells stimulated with tp samples in validation study but elevated when treated with np sample of this individual. the inconsistencies found in the expressions of ddit , sesn and ccl may thus reflect the individual variability deserving of further study. we used the validation phase results to perform a direct comparison of signaling pathways activated in cells as a consequence of their treatment with np or tp samples (table a , b) using the database reactome. this analysis demonstrated the critical importance of the presence/absence of the intact cfdna for the expression profile of cells and regulatory pathways activation. this fact was also documented by the reactome analysis of results obtained by mass spectrometry used for proteome examination of thp cells treated with np or tp (table c) . the presented work documented that the cfdna and its clearance in plasma is under physiological conditions indispensable for immune system performance. we demonstrated that monocytes in intact cfdna presence (np) upregulated the central pathways responsible for immune system homeostasis, especially notch signaling | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ www.nature.com/scientificreports/ and unfolded protein response (table a) while the degraded cfdna in plasma (tp) resulted in upregulated cxcl expression (table b) . this mrna should translate into interleukin (il ), a pivotal protein involved in the direct activation of iir and also regarded as a marker of cellular senescence , but its elevated level was not found in proteomic analysis after our three-hour-long cultivation experiments. nevertheless, we detected the upregulation of proteins contributing to the activation of complement (table ; supplementary table ) , confirming the inflammatory state of these cells. the software ingenuity pathways analysis (ipa) was used to explore all obtained data (supplementary table ). the ipa results ( fig. a , b) confirmed the findings of reactome analysis (summarized in fig. b inset). in thp cells cultivated with tp, multiple pathways involved in immune response were detected among significantly changed canonical pathways ( fig. a) and overrepresented disease-specific pathways (fig. b) . ipa also predicted the accumulation of granulocytes, leucocytes, myeloid cells, phagocytes, and complement activation as the consequence of events signalizing the presence of cells that are confronted mainly with degraded cfdna (fig. ). we developed and tested an experimental workflow which allowed us to compare the effects of cfdna pool in native plasma and cfdna in plasma degraded by endogenous dnase i and additionally with dnase turbo on the thp cells. the native plasma samples were fixed by edta as a potent indirect dnase i inhibitor. in the edta treated plasma samples, the activity of the endogenous dnase i is completely stopped . the activation of endogenous dnase i and the subsequent activity of dnase turbo were allowed by addition of an activation buffer containing divalent cations to the edta treated plasma samples. to inactivate these divalent cations, the dnase turbo inactivation procedure was applied according to the protocol provided by the manufacturer for the aqueous solutions of isolated rnas. our results are of course limited by the fact that we are working with such a complex sample as human blood plasma. it is not possible to design the experiments to exclude completely the influence of changing divalent cations concentrations , during the entire experimental procedure, the presence of cfdna hidden in plasma exosomes , or in supramolecular complexes and on cell surfaces . nevertheless, when we quantified the cfdna isolated from np, from the sample exposed to the entire cfdna removal procedure but without addition of dnase turbo and from the tp sample, we found striking gradual decrease in cfdna levels toward the last sample. the changes in expression profiles of selected validated genes were detectable after the decrease of cfdna levels to . % of its original native concentration as the result of endogenous dnase i activity ( supplementary fig. ). therefore we could speculate also about the role of cfdna www.nature.com/scientificreports/ degradation products in the induction of these expression changes. the significance of differences in expression profiles of cells treated with np and tp was statistically proven in validation experiments. we detected namely the differences in immune system regulatory pathways discussed in the next paragraphs. the inflammatory response in mammalian cells is regulated by notch signaling pathways acting through four different notch receptors (notch - ) transducing extracellular signals ; the notch is involved in myeloid lineage differentiation leading to monocytes . the constitutive tonic activity of notch signaling pathways was described in non-activated immune cells . we found that the monocytes treated with np expressed hes mrna by ≈ . × more than the ones treated with tp (fig. d) . this elevated expression documents the notch pathways' activity in these cells. the pathways leading from toll-like receptors (tlrs) may provide additional signals to the notch ligands . in primary macrophages, it has been shown that the notch target genes, like hes , can also be induced exclusively by tlr stimulation . in such a model, the presence of cfdna itself could ensure the constitutive tonic notch signaling via tlr as a receptor specialized in dna sensing. different types of cellular stress lead to unfolded protein accumulation in the lumen of the endoplasmic reticulum. this accumulation activates signal-transduction cascade known as unfolded protein response (table a) . it has been demonstrated that this cascade plays the central role in the modulation of immune system functions regarding both innate and adaptive responses . in our experiments, the expression of sesn (sestrin ) is upregulated in cells treated with np (fig. b) . sesn is well-known as a stress-inducible protein suppressing inflammasome activation by the induction of mitophagy. sesn plays a crucial role in this unique regulatory mechanism of mitophagy activation which is pivotal for the maintenance of immunological homeostasis and protection from sepsis . we found the increased expression of arrdc and irf genes in cells treated with np (fig. b) additionally to the genes involved in the pathways detected by reactome ( table ). the role of arrestin domain-containing (arrdc ) in iir was recognized but only partially understood . this gene is transcribed in monocytes in healthy individuals; after a viral infection, its levels were increased and correlated with concentrations of interleukins in serum. interferon regulatory factor- (irf ) is a transcription factor expressed at low levels in immune system cells and induced by different cytokines. it controls the transcription of its target genes in different types of immune cells . cultivation of thp cells with tp led to the change of expression pattern. apart from increased cxcl expression, we found upregulated expression in mtts , gpr , and ccl (fig. c) . metastasis suppressor- (mtss ) was originally identified as a metastasis suppressor in the carcinoma cell line. nowadays, it is known that this multifunctional cytoskeleton scaffold protein regulates the cytoskeleton dynamics and inhibits cell migration . g protein-coupled receptor (gpr ) is a member of the signaling pathway leading to repression of notch signaling . ccl codes for eotaxin , which is produced by activated monocytes and attracts lymphocytes, basophils, eosinophils, and monocytes to the site of inflammation. it has been reported that eotaxin induced apoptosis of thp cells . significantly higher expression of proteins belonging to complement cascade is evident at the proteomic level upon the treatment of cells with tp in comparison with cells treated with np (table c ). the soluble complement www.nature.com/scientificreports/ proteins detected in plasma are synthesized mainly in the liver, but their local production by circulating immune cells including monocytes is well described . we documented that the monocytes without contact with physiological cfdna concentrations in plasma activated emergency mechanisms and began to initiate iir. the normal functions of these cells were downregulated (notch signaling), potential migration was inhibited, and the genes for attractants of immune cells (cxcl and ccl ) were overexpressed (fig. b inset and fig. ) . previously, we found elevated expression of another key member of the cytokine network, namely tumor necrosis factor-alpha (tnf-α) in tp treated cells , . tnf-α functions as a master regulator of inflammation and ensures tissue homeostasis . we reported the statistically significant differences validated in qpcr experiments earlier on two different occasions-in our study in which plasma samples of healthy volunteers were used for stimulation of thp cells and in the report studying the stimulatory capacity of plasma samples obtained from patients with celiac disease . under the stringent conditions set by us for the evaluation of genechip experiments, the tnf-α was not reported as differentially expressed, but its expression was higher in cells treated with tp per our previous results. to date, the regulatory mechanisms keeping the cfdna concentrations in plasma at physiological levels are not well understood. natural regulatory mechanisms are balancing the nets production and their clearance. these mechanisms may involve the negative closed feedback loop system, which is widely spread in biology and also used by pharmacologists for drug delivery systems . the failure of the feedback loop mechanism might cause exacerbated dnase i production, resulting in iir. some bacteria are equipped not only with dnases but also with their own ´-nucleotidases. the activity of these enzymes destructs nets and converts their degradation products into deoxyadenosine which is toxic namely for macrophages and induces their apoptosis . different ectonucleotidases are found on the surfaces of different human cells , , their participation in nets degradation and toxic product formation cannot be excluded. our results suggest that the exposition of cells to relatively elevated concentration of cfdna degradation products can evoke and promote the inflammatory state as the consequence of clearance of high cfdna concentrations associated with tissue damage or with the affected clearance of the products of netosis . elevated deregulated netosis is reported in common comorbidities not only in dialyzed patients but it is also typical in most comorbidities characterized as risk factors predisposing to serious complications of sars cov- infection . here we provided evidence that the cfdna in human plasma and its clearance represents an essential natural tool for regulation of innate immune response. we used the thp cell line as a model of human monocytes to demonstrate that cfdna plays an indispensable role in the immune system homeostasis. the cells treated with the native plasma of healthy volunteers expressed genes whose products maintain immune system homeostasis. however, the cells treated with identical plasma samples with degraded cfdna directly activate iir with elevated production of mrna for interleukin at the transcriptomic level. they also upregulated the complement compounds at the proteomic level. the role of intact and degraded cfdna and their sensing by cells seems to be one of essential aspects of immune system performance; therefore, further studies focusing on this subject are highly topical. it is of utmost importance to understand the mechanisms of cfdna release, the clearance and mechanisms of the homeostasis maintenance, as well as the role of different types of cfdna sequences and their chemical modifications in cfdna mediated regulatory events, in addition to the recognition of all aspects of cfdna presence sensed by the cells. the detailed knowledge of mechanisms involved in immune system regulation by the levels of circulating cfdna may lead to new clinical applications, especially concerning the complete understanding of the pathogenesis of sepsis, covid- and therapeutic dnase treatment. monitoring the cfdna level can serve as an actual tool for early well-advanced diagnoses of several diseases such as cancer, sepsis and covid- , as well as labor timing. this study is the first to show the fundamental role of cfdna and its clearance in plasma of healthy individuals in the regulation of innate immune response, thus warranting further research in this direction. subjects. plasma donors were selected from healthy volunteers who satisfied the following criteria: . they were taking no medication . they had no chronic illness . they had not recovered from an infectious disease in the last two weeks . they were in very good physical and psychical status. the subset of samples described in our previous study was utilized for expression arrays experiments. five women and five men aged between and years with age of ( . ± . ) year (mean ± standard deviation from samples) were selected for validation of quantitative pcr (qpcr) experiments. the ethical committee of the st faculty of medicine of charles university and general faculty hospital in prague, czech republic, approved our study. we obtained informed consent from all study participants. all methods were performed per the relevant guidelines and regulations. preparation of plasma samples. whole blood was collected into vacuette tubes containing k edta (greiner bio-one). we separated blood plasma using the following steps: centrifugation rate, time and temperature set to , rpm, min and °c, respectively; then the transfer of plasma into ml low binding collection tubes and centrifugation set to rate of , rpm for min to remove the residual cells. finally, we transferred the plasma into clean ml dna lobind tubes (eppendorf). the samples were stored at a temperature of − °c. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ before cell experiments, the plasma sample thawed and split into two identical aliquots. the first one was used at its native state (np), and the second one was treated with turbo dna-free dnase (thermo fisher scientific) according to the manufacturer's recommendation (tp). each μl of the plasma sample was treated with μl turbo dnase, μl water and μl × turbo dnase buffer. this mix was incubated at °c for min and then turbo dnase was inactivated by adding μl dnase inactivation reagent, mixed and incubated at room temperature for min. dnase inactivation reagent was removed by centrifugation at an acceleration force of , g for . min and the supernatant was transferred into the new tube. in order to evaluate the influence of individual protocol steps on the alteration of expression profiles, the following experiments were performed: the plasma sample was handled according to the protocol for dnase turbo treatment but dnase turbo was supplemented by water to recognize the effect achieved by this enzyme. inactivation buffer was added either immediately or after incubation step to recognize the influence of sample incubation at °c with activation buffer. identical experiments were performed with dnase turbo addition. all these plasma samples were used for stimulation experiments with thp cells and expressions of all validated genes was determined as described below. for determination of cfdna levels in plasma before and after treatment with turbo dna-free kit components, the qpcr at quantstudio k flex real-time pcr system (applied biosystems, usa) was performed using the cfdna samples isolated by qiaamp circulating nucleic acid kit. powerup sybr green pcr master mix (life technologies, usa) constituted one half of μl reaction, the total cfdna amount was measured using primers for the gene b and the standard curve dilution had range from ng to . ng per reaction. cell cultivation and stimulation. we used the thp cell line for stimulation experiments. after the recovery of cells from a deep-frozen state, we used the protocol published earlier , . the cells were stimulated for h in the rpmi medium (sigma aldrich) containing % of a plasma sample. the cells were collected after h of cultivation; after centrifugation, the supernatant was removed, and the cells were preserved in lysis solution (sigma-aldrich) and stored at − °c. the results were multiplied by a factor of to increase resolution and presented in arbitrary units (au). statistical analysis was done in graphpad prism . . software (graph-pad software). first, we performed the d´agostino-pearson normality test. in the case of parametric data distribution, the t-test was used; the wilcoxon matched-pairs signed-rank test was applied for non-parametric data distribution. the statistical significance was set to level ≤ . for all comparisons. sample preparation for mass spectrometry. frozen thp- cell pellets containing ≈ , cells were resuspended in μl p phosphate-buffered saline and lysed using buffer composed of % sodium dodecyl sulfate (sigma-aldrich) in mm -( -hydroxyethyl)- -piperazineethanesulfonic acid buffer with ph . (carl-roth) supplemented with × complete ultra protease inhibitor cocktail-ethylenediaminetetraacetic acid (roche) and shortly sonicated. mixtures were heated for min at °c and subsequently cooled by placing samples on ice. twenty-five units of benzonase nuclease (sigma-aldrich) was added into each tube, and the tubes were incubated at °c for min to degrade chromatin. the obtained protein mixtures were centrifuged to remove cell debris, and the supernatant was determined by the bca method using the nanodrop uv-vis spectrophotometer (thermo fisher scientific). | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ proteins in the lysate ( μg) were reduced in the protein lobind tubes (eppendorf) by the addition of dithiothreitol solution (sigma-aldrich) to a final concentration of mm and incubated for min at °c. alkylation was performed by the addition of iodoacetamide to a final concentration of mm and incubation for min at °c in the absence of ambient light. reactions were quenched by the addition of μl of m dithiothreitol per tube. then the protein solution was acidified by % formic acid to reach ph value between and . in the same tube, μg of suspension of paramagnetic carboxylate-modified microparticles sera-mag speedbeads μm (ge healthcare) was resuspended in the protein sample solution. acetonitrile was immediately added to obtain fifty percent solution, and sample suspension was mixed and incubated for min at °c. next, the tube was placed into a magnetic stand, and paramagnetic microparticles with captured proteins were washed twice using ml of % ethanol for s. finally, microparticles were dried by μl of acetonitrile for s and resuspended in ≈ μl of ≈ mm triethylammonium bicarbonate buffer ph . . proteins were eluted by on-bead digestion after the addition of trypsin/lys-c protease mixture (promega) in enzyme to substrate ratio of : and overnight incubation at °c . the next day, the obtained peptide eluate was discharged from microparticles, which were again washed twice using μl of mm triethylammonium bicarbonate ph . . the pooled peptide mixture was acidified by μl of % trifluoroacetic acid and then desalted using omix c pipette tips (agilent) according to the user manual. the clean peptide sample was evaporated on the vacuum concentrator (eppendorf) and stored at − °c in protein lobind tubes. nanolc-ms analysis. nano reversed-phase columns (easy-spray column, cm × µm id, pepmap c , µm particles, nm pore size) were used for liquid chromatography/mass spectrometry analysis. mobile phase buffer a and b was . % formic acid in water and acetonitrile, respectively. samples were loaded onto the trap column model c pepmap with μm particle size and dimension of μm × mm (thermo scientific) for min at . μl min − . the loading buffer was composed of water, % acetonitrile and . % trifluoroacetic acid. peptides were eluted with the mobile phase b gradient from to % b in min. eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a thermo orbitrap fusion modelq-ot-qit (thermo fisher scientific). survey scans of peptide precursors from to m z − were performed at k resolution at m/z with a × ion count target. tandem ms (ms ) was performed by isolation at . th with the quadrupole, hcd fragmentation with a normalized collision energy of , and rapid scan ms analysis in the ion trap. the ms ion count target was set to , and the maximum injection time was set to ms. only those precursors with charge states from to were sampled for ms . the dynamic exclusion duration was set to s with a ppm tolerance around the selected precursor and its isotopes. monoisotopic precursor selection was turned on. the instrument was run in top speed mode with s cycles . ms data analysis. all data were analyzed and quantified with the maxquant software (version . . . ) . the false discovery rate (fdr) was set to % for both proteins and peptides, and we specified a minimum peptide length of seven amino acids. the andromeda search engine was used for the ms spectra search against the human as downloaded from current uniprot human database. enzyme specificity was set as c-terminal to arg and lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. dithiomethylation of cysteine was selected as fixed modification and n-terminal protein acetylation and methionine oxidation as variable modifications. the match between runs feature of maxquant was used to transfer identifications to other lc-ms runs based on their masses and retention time with a maximum deviation of . min; this was also used in quantification experiments. quantifications were performed with a label-free algorithm in maxquant . data analysis was performed using perseus . . . . software . bioinformatic analysis. the sets of differentially transcribed genes and differentially expressed proteins were analyzed using reactome dababase . the sets were also subjected to ingenuity pathway analysis 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transition via notch inhibition eotaxin- induces monocytic apoptosis in patients who have undergone coronary artery bypass surgery and in thp- cells in vitro regulated by thrombomodulin production of complement components by cells of the immune system cell-free dna from human plasma and serum differs in content of telomeric sequences and its ability to promote immune response immunoregulatory properties of cell-free dna in plasma of celiac disease patients-a pilot study checkpoints in tnf-induced cell death: implications in inflammation and cancer advances in bioresponsive closed-loop drug delivery systems infection: microbial nucleases turn immune cells against each other nucleotidase activities in soluble and membrane fractions of three different mammalian cell lines the ectonucleotidases cd and cd : novel checkpoint inhibitor targets neutrophil dysfunction, immature granulocytes, and cell-free dna are early biomarkers of sepsis in burn-injured patients: a prospective observational cohort study molecular mechanisms regulating netosis in infection and disease netosis provides the link between activation of neutrophils on hemodialysis membrane and comorbidities in dialyzed patients ultrasensitive proteome analysis using paramagnetic bead technology the one hour yeast proteome maxquant enables high peptide identification rates, individualized ppb-range mass accuracies and proteomewide protein quantification accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed maxlfq the perseus computational platform for comprehensive analysis of (prote) omics data the reactome pathway knowledgebase causal analysis approaches in ingenuity pathway analysis m.k. designed the experiments, interpreted the results and wrote the manuscript; a.z. and i.b. performed preparation of plasma samples, cell cultivation and qpcr experiments; b.c. performed microarray experiments; p.p. conducted proteomic analysis and data interpretation; l.s. and o.s. conducted ipa analysis; p.n. was involved in results interpretation and manuscript writing. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to m.k.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -y z pwgn authors: ding, nai-zheng; xu, dong-shuai; sun, yuan-yuan; he, hong-bin; he, cheng-qiang title: a permanent host shift of rabies virus from chiroptera to carnivora associated with recombination date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: y z pwgn bat virus host shifts can result in the spread of diseases with significant effects. the rabies virus (rabv) is able to infect almost all mammals and is therefore a useful model for the study of host shift mechanisms. carnivore rabvs originated from two historical host shifts from bat viruses. to reveal the genetic pathways by which bat rabvs changed their host tropism from bats to carnivores, we investigated the second permanent bat-to-carnivore shift resulting in two carnivore variants, known as raccoon rabv (rrv) and south-central skunk rabv (scskv). we found that their glycoprotein (g) genes are the result of recombination between an american bat virus and a carnivore virus. this recombination allowed the bat rabv to acquire the head of the g-protein ectodomain of the carnivore virus. this region is involved in receptor recognition and binding, response to changes in the ph microenvironment, trimerization of g proteins, and cell-to-cell transmission during the viral infection. therefore, this recombination event may have significantly improved the variant’s adaptability to carnivores, altering its host tropism and thus leading to large-scale epidemics in striped skunk and raccoon. bat virus host shifts have resulted in the emergence of several serious diseases in humans and animals, such as sars , ebola , and rabies viruses. to predict and control the outcome of these host shifts, it is crucial to determine the genetic pathways by which these viruses rapidly adapt new host species, which are poorly understood for most host-virus systems . the rabies virus (rabv; genus lyssavirus, family rhabdoviridae) is one of the most significant pathogens threatening animal and human health, exhibiting a high fatality rate. this virus has the capacity to infect almost all mammals. although rabv is usually maintained in distinct host species-associated transmission cycles, typically within the carnivora and chiroptera [ ] [ ] [ ] , host shift events are still of public health concern due to increasing risks of human exposures . recent host shifts between carnivores include the re-emergence of rabv in wild taiwan ferret-badgers , as well as the outbreaks of domestic dog-associated rabv in ethiopian wolves and of striped skunk-associated rabv in gray foxes in california . it has also been reported that multiple host shifts between bat and mesocarnivores have occurred with the big brown bat-associated rabv in arizona . in south america, the vampire bat, a reservoir of rabv, is resposible for the high number of rabies cases among terrestrial mammals in the rural cycle . therefore, rabv is a useful model for studying the mechanisms of bat virus host shifts . multiple factors may be involved in rabv host shifts, including viral adaptability, host barriers, ecological factors, and human interventions . of these, viral adaptability may play a significant role. interestingly, although evolutionary analyses have demonstrated that bat-to-carnivore host-shifting viruses accumulate few adaptive mutations , , , in none of these examples did bat rabv establish permanent transmission cycles in the new host species . to date, there have been two permanent host shifts from the chiroptera to the carnivora in the history of lyssaviruses, with disastrous results . the first occurred thousands of years ago and has resulted in worldwide rabies epidemics in terrestrial mammals through the present . the second permanent host shift appears to have occurred in north america, producing a lineage including two variants, raccoon rabies virus (rrv) and south-central skunk variant (scskv). scskv mainly circulates in striped skunks and sporadically in other terrestrial animals, such as wolf. the rrv variant was first found in raccoons in northern america and has seriously phylogenetic analysis. rabv phylogenies were reconstructed with the neighbor-joining (nj), maximum parsimony (mp), and maximum likelihood (ml) methods utilizing mega v . the best nucleotide substitution model was determined using the model selection program implemented in mega v , and the gamma parameter was also estimated for the nj method. the robustness of each monophyletic group was assessed with the bootstrap method with replicates. a monophyletic group with > % bootstrap support was considered a robust lineage. a shimodaira-hasegawa test implemented in the treetest program (http://aix .uottawa.ca/~sarisbro/) was employed to determine whether the phylogenetic trees were significantly different . recombination analysis. the putative recombinant sequence and its parent sequences were identified using the simplot and rdp . software packages, as previously reported . analyses were also carried out with the bootscan program in simplot using the putative recombinant sequence as a query. mosaicism is suspected when high levels of phylogenetic relatedness are obtained between the query sequence and more than one reference sequence in different genomic regions. finally, recombination breakpoints were analyzed by maximizing χ , employing a combination of simplot and rdp . . due to genetic mutations and restrictions on protein function, convergent evolution may alter the topology of a phylogenetic tree inferred from gene sequences. because adaptive selection for convergent evolution operates mainly at the amino acid sequence level of a protein , it can be identified by comparing phylogenetic trees inferred from nucleotides at the third codon position with those based on first and second codon positions for the gene of interest. different topologies in the two resulting trees suggests that convergent evolution has occurred during evolutionary history. we reconstructed the phylogenetic histories of the recombinant region of the g gene using third vs. first and second codon positions. estimates of the time to the most recent common ancestor (tmrca). the time to the most recent common ancestor (tmrca) of each lineage was calculated to date the recombination event using the bayesian markov chain monte carlo (mcmc) method implemented in beast (version . . ) . bayesian mcmc analyses were performed with gtr nucleotide substitution models with invariant sites and a gamma distribution of four rate categories (Γ ). each bayesian mcmc analysis was run for million states and sampled every , states. posterior probabilities were calculated with a burn-in of million states and checked for convergence using tracer (version . . ) (http://tree.bio.ed.ac.uk). nearly half of the g gene of scskv and rrv is inherited from a carnivore rabv. sequences of scskv and rrv g genes were collected from several previous reports (n = , see additional file ) and reference rabvs from genbank (n = , see additional file ). as previously reported , , phylogenetic analysis suggested that they fell in the bat clade (see additional file a). we also retrieved worldwide rabv g genes from genbank in order to identify their positions in the rabv phylogenetic tree (see additional file b). next, we further investigated the g gene sequences from three representatives of the scskv variant, the carnivore lineage, and the bat lineage: a - , an american wolf isolate belonging to the scskv genotype; ca, isolated from a north american skunk of the cosmopolitan clade virus; and lah , isolated from a north american bat (lasiurus borealis/cinereus), which shares the highest similarity to scskv among available bat rabvs. we initially compared all of the variable sites in the a - , ca, and lah g genes and discovered two crossover sites. between the two crossover sites, a - shared higher similarity with the carnivore virus, while outside of this region, a - exhibited higher sequence identity with the bat rabv (fig. a ). comparison of a complete rrv genome with the bat and carnivore rabvs also demonstrated a similar g gene pattern (see additional file ). bootscan analysis of the g gene with an outgroup (lagos bat viruses nga and sen) indicated that this discordant sequence likely derives from hr. two crossover sites were also identified in the bootscan analysis (fig. b) . between the two crossover sites, a - was found to be homologous to carnivore rabvs, while it was more similar to bat rabvs outside of this region. taken together, these results suggest that the g gene of scskv likely underwent a recombination event, resulting in the two breakpoints. using the three representative isolates, the recombination event was further examined through seven recombination analysis methods implemented in the rdp software package, which properly allows for statistical analysis of the probability of recombination (fig. c) . to determine the two putative breakpoints, we next analyzed informative sites using fisher's exact test (fig. d ). two putative breakpoints were discovered at positions (χ max = . , p < . ) and (χ max = . , p < . ) of the g gene open reading frame (orf), suggesting that over half of the g gene ( / nucleotides = . % of the scskv g gene) may be derived from an american carnivore rabv. the gold-standard approach for confirming the presence of recombination is a set of statistically incongruent phylogenetic trees . inside the breakpoints, they were nested within the clade of carnivore rabvs with % (of replicates) bootstrap support ( fig. a) . outside of the two breakpoints, however, scskv and rrv clustered into the american bat lineage with % (of replicates) bootstrap support (fig. b) . the topologies of these two trees were significantly different (shimodaira-hasegawa test, p < . ). to assess whether the significant discrepancy in the g gene's phylogenetic history may have resulted from convergent evolution, phylogenies of the incongruent region were reconstructed using nucleotides from codon position (fig. a) and from positions and (fig. b ). the topologies of the two trees are identical, indicating that hr caused the phylogenetic difference seen in the rrv and scskv g gene, rather than convergent evolution. the recombination event may have occurred several hundred years ago. depending on a molecular clock model based on genetic divergence of rabies virus variants in bats of different species, the common ancestor of north american bats lived around to . to estimate the timing of the recombination event, we also calculated the tmrca of the recombinant lineage according to each sequence region delimited by the two breakpoints using a bayesian evolutionary analysis method. the incongruent origins of the recombinant lineage were also reflected in the different topologies of the two bayesian trees (fig. ) . the resulting trees estimated that the tmrca of the recombinant was ( % highest probability density, hpd: - ; fig. a ) or ( % hr resulted in rapid evolution of the bat rabv g protein. finally, we compared the amino acid sequences of the g proteins of the recombinant lineage and its parent lineages and found positions between the two putative breakpoints where the recombinant is more similar to the carnivore rabv (see additional file ). we also assessed d n /d s ratios employing datamonkey and found no positively selected sites along the stem branch of the recombinant lineage at a % significance level. however, in the recombinant sequence of rabv following the host switch, the non-synonymous substitution rate is up to % (versus . % in the same region of the virus without recombination and remaining associated with bat host). these results indicate that the recombination event induced rapid changes to the bat rabv g protein. the recombinant region derived from the carnivore rabv g gene encodes three of the four functional g protein domains: the lateral domain, the trimerization domain, and the ph domain (fig. a) . these constitute % of the amino acids in the head region of the trimeric g protein hairpin conformation that is exposed on the outside of the virus (fig. b ). we found that the bat virus resulting in the second permanent rabv host shift was derived from an hr event hundreds of years ago between a parental american carnivore strain and a parental bat strain. the bat strain provided most of the mosaic genome except for half of the g gene (see additional file ). in the recombinant g gene orf, the region from positions to ( nucleotides) derives from a carnivore rabv, while the rest of the gene derives from the bat rabv. all examined recombinants shared an identical recombination signal in their g genes (fig. ) , suggesting that these viruses descend from a single ancestor that experienced the recombination event. we are confident that the sequence data included in this study are reliable enough for recombination analysis. first, all available recombinant family members (> isolates) exhibited a similar recombination signal in the g gene, and they were isolated and sequenced by independent research groups, discounting the possibility that the signal derives from artificial pcr or sequencing errors. secondly, we identified two statistically significant breakpoints in the gene. finally, sequence identities and phylogenic histories were distinctly different on either side of these apparent breakpoints. recombination may be the most economic genetic strategy for altering the host tropism of a virus, allowing a spillover virus to rapidly obtain key genetic material from viruses familiar with the environment of the new host species and thereby reducing the phylogenetic barriers between distant host species. for example, influenza a viruses are notorious for causing frequent pandemics because chicken, pig, and human viral strains can exchange their genomic segments through genetic reassortment, a type of recombination seen in viruses with segmented genomes , . in retroviruses, hr between viruses from different primate hosts is associated with the emergence of the human immunodeficiency virus . recombination has also led to changes in host tropism in positive-strand rna viruses, such as coronaviruses (covs). the cov that causes severe acute respiratory syndrome (sars) may have arisen from a recombination event between a bat cov and another virus before infecting human and carnivore hosts . a middle east respiratory syndrome (mers) cov recombinant lineage has been dominant since december and subsequently led to human outbreaks in . despite such examples in other viruses, hr is commonly thought to be rare or absent among negative-strand rna viruses (nsrvs) , . therefore, recombination has not typically been considered as a genetic mechanism behind nsrv host shifts . recombination between rabvs is also controversial. it has been proposed that hr between rabies viruses does not occur , . however, we recently isolated two rabv strains with mosaic n genes in china . interestingly, this study provides robust evidence supporting the fact that hr is an important genetic mechanism driving rabv evolution. despite this, hr between bat and carnivore rabvs may be uncommon, as it may be rare that the two viruses meet in the same host neuron. this would be consistent with the fact that there have been only two permanent host shifts observed over the long time-span of lyssavirus evolution . in order for a recombination event to take place, co-infection with the parental viruses is necessary. spillover infections of rabv can occur in the american bat and terrestrial mammals , providing the opportunity for recombination between bat and carnivore rabvs. the most likely hosts for the recombination event derive from natural rabv vectors, where viruses can persist and undergo vertical transmission, thereby increasing the probability of recombination. to date, these mosaic viruses mainly circulate in striped skunk and raccoon, although the ball and stick model of d structure of the mosaic trimeric g protein ectodomain. the d structure is inferred from that of vesicular stomatitis virus g protein (pdb id: i m). the amino acid residues obtained from skunk rabv and bat rabv are displayed in bright green and black, respectively. (c) schematic diagram of the origin of the recombinant. on the left, the two parent viruses are shown with red and black denoting regions derived from the skunk and bat rabvs, respectively. in the center, the two viruses come into contact and recombine inside a skunk cell. on the right, the mosaic offspring with a carnivore rabv-derived g-protein head circulates in carnivores. they can sporadically be isolated from other species, suggesting that other animals may only represent dead-end hosts. the american bat, striped skunk, and raccoon are the natural reservoir hosts of rabv . therefore, we propose that the recombination event may have occurred in one of these three species. moreover, these three rabv natural vectors live within the same environmental niche, increasing the opportunity for co-infection and recombination. since recombinant offspring have not yet been found circulating in bat, the recombination may have occurred instead in a terrestrial animal infected by the carnivore rabv parent when the bat virus (the major parental strain) infection occurred. considering that scskv is more closely related to the mrca of the recombinant variant than the raccoon sub-lineage (fig. ) , an american striped skunk is the most likely candidate host for the co-infection and recombination (fig. c) . future studies should attempt to determine which particular cell types are the most likely host cells for the recombination event in striped skunk. for a virus to establish a permanent host shift, four stages are necessary: exposure to a new host, infection of the host, onward transmission, and permanent, sustained transmission in the new species . since bat rabvs are potentially able to infect carnivore species , , , , we also propose that recombination may function as the third and fourth stages of establishment of a permanent host shift by significantly enhancing the adaptability of a bat virus in terrestrial mammals. the g protein plays a dominant role in the host tropism of rabvs. after binding to a receptor, rabv enters the host cell through the endocytic pathway and subsequently fuses with a cellular membrane within the acidic environment of the endosome . both receptor recognition and membrane fusion are mediated by the g protein, which is trimeric and forms spikes that protrude from the viral surface . in the mosaic g protein, almost all functional sites directly related to host tropism and infectivity are located in the region derived from the carnivore rabv (positions to of the g protein). for example, neuroinvasiveness and receptor binding is known to be associated with lysine (lys) , and arginine (arg) , residues present at positions and , respectively. in addition, a recent study suggested that positions , , and affect the efficiency of cell-to-cell transmission of the viral infection . interestingly, we found that arg of the carnivore rabv replaced lys of the putative major parental strain, lah (see additional file ). these changes may lead the recombinant offspring to be better adapted to the new host species than to bats. the three-dimensional ( d) structure of the mosaic g protein may allow for a better understanding of the influence of the recombination event on the permanent host shift of the recombinant lineage. although a d structure of a rabv g protein is not available, three of the four major antigenic sites/epitopes (i/cr , iii, and iv) are derived from the american carnivore rabv, reflecting the fact that the recombinant region is exposed on the outside of the g protein d structure (fig. a ). in addition, the mosaic protein structure can also be estimated by studying the g protein of a bullet-shaped virus family member, vesicular stomatitis virus (fig. b) . among the four functional domains of the mosaic g protein ectodomain (i, lateral domain; ii, trimerization domain; iii, ph domain; and iv, fusion domain), the parental bat rabv only provided domain iv (residues - , fig. a ), which mainly constitutes the stem of the hairpin conformation of the trimeric g protein (fig. b ). in contrast, almost all of domains i, ii, and iii are derived from the parental carnivore rabv (fig. a) , forming % of the g protein head (fig. b) . these domains are involved in receptor recognition and binding, response to ph changes, and g-protein trimerization . it has been reported that mutations in the ph domain affect the structural transition of the g protein and therefore may alter the ability of rabv to escape antibody neutralization and to infect cells . thus, after acquiring these key genetic elements from skunk rabv, the mosaic offspring could have rapidly adapted to permanent residence in the new host species. in conclusion, we showed that hr likely resulted in increased genetic diversity and rapid evolution of rabvs. through this mechanism, a bat rabv strain may have acquired the key genetic information from a carnivore rabv to overcome the phylogenetic barrier between chiroptera and carnivora hosts, 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mutant of rabies virus is unable to infect motoneurons in vivo and in vitro characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus amino acid substitutions at positions , and in rabies virus glycoprotein affect spread of viral infection crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g identification of amino acids controlling the low-ph-induced conformational change of rabies virus glycoprotein biological differences between vesicular stomatitis virus indiana and new jersey serotype glycoproteins: identification of amino acid residues modulating ph-dependent infectivity this work was supported by national natural science foundation of china ( ) and ji'nan city college independent innovation project ( ). h.c.q. and d.n.z. conceived and designed the experiments; h.c.q. wrote the manuscript; h.c.q., d.n.z., x.d.s., s.y.y., and h.h.b. performed the study. supplementary information accompanies this paper at doi: . /s - - - competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- -ewp m jp authors: churchward, colin p.; al-kinani, ali a.; abdelkader, hamdy; swinden, julian; siwoku, opeoluwa; varnakulasingam, thinuba; alany, raid g.; snyder, lori a. s. title: monocaprin eye drop formulation to combat antibiotic resistant gonococcal blindness date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ewp m jp neisseria gonorrhoeae bacteria are acknowledged as an urgent threat to human health because this species has developed resistances to all of the antibiotics used clinically to treat its infections. n. gonorrhoeae causes the sexually transmitted disease gonorrhoea, but also causes blindness when the bacteria infect the eyes. infants are particularly susceptible, acquiring the infection from their mothers at birth. we have shown that the monoglyceride monocaprin rapidly kills n. gonorrhoeae and other bacterial species and is non-irritating in ocular assays. here we show that the physical and chemical properties of monocaprin make it ideal for use in a thickened eye drop formulation to combat eye infections. monocaprin-containing formulations were assessed using analytical techniques and for antimicrobial activity in vitro and in ex vivo infections. monocaprin-containing formulations retained activity after three years and are non-irritating, unlike preparations of povidone iodine in our assays. a recommended formulation for further development and investigation is . % monocaprin in % hpmc with % polysorbate . once a drug to combat infection has been discovered, then formulation is required so that the drug can effectively be converted to a form that could be used to clear a particular infection from a patient. monocaprin has been proposed as a treatment and prophylaxis for neisseria gonorrhoeae and other bacterial eye infections [ ] [ ] [ ] . n. gonorrhoeae is a bacterial species that causes the sexually transmitted infection gonorrhoea and as a result can be transferred to the eyes of infants during childbirth causing ophthalmia neonatorum . autoinnoculation can also result in adult eye infections . progressive development of resistance to all antibiotics used against n. gonorrhoeae clinically presents a problem for prophylaxis and treatment of ophthalmia neonatorum and treatment of adult gonococcal eye infections. prophylaxis for ophthalmia neonatorum has involved the use of substances applied within an hour of birth that have prevented the establishment of gonococcal infection , [ ] [ ] . silver nitrate was the first prophylaxis and significantly reduced cases of gonococcal blindness, however it has been discontinued in most areas due to issues with chemical conjunctivitis and toxicity . some cases of ophthalmia neonatorum still developed with silver nitrate, which is unable to treat an active infection . antibiotics erythromycin and tetracycline, in ocular ointments, have largely replaced silver nitrate as prophylaxis, however resistance to these antibiotics emerged in n. gonorrhoeae in the 's . although povidone iodine has been suggested for prophylaxis it is not currently recommended by the cdc and has similar issues as silver nitrate, both in terms of ocular irritation and inability to treat active infections [ ] [ ] . the world health organisation recommendations for treatment of gonococcal eye infections are: mg/kg ceftriaxone; mg/kg kanamycin; or mg/kg spectinomycin, each as a single im dose, noting that "the choice of treatment may depend on the cost and quality of the medicines in different settings and on equality considerations . " the , british association for sexual health and hiv national guidelines for the management of infection with n. gonorrhoeae recommend treatment of gonococcal conjunctivitis with g ceftriaxone intramuscularly as a single dose . this advice is based on successful treatment of adults in and there is no mention of an alternative suggestion in cases of ceftriaxone resistant infections. given cases of ceftriaxone resistant n. gonorrhoeae and wide-spread multi-drug resistance , an alternative is needed to prevent blindness from these bacteria. in , before the advent of antibiotics, up to % of children in institutions for the blind had suffered from gonococcal ophthalmia www.nature.com/scientificreports/ neonatorum at birth . the infection can progress rapidly, with the bacteria perforating the globe of the eye and causing blindness within h [ ] [ ] . we have identified that monocaprin is able to kill n. gonorrhoeae, including clinical isolates of n. gonorrhoeae, the related species neisseria meningitidis, which can also cause eye infections, and other keratitis causing bacteria, including chlamydia trachomatis, another major cause of ophthalmia neonatorum , , . monocaprin has been shown to retain its antimicrobial activity in artificial tear fluid, is devoid of corneal and conjunctival irritation, kills all bacteria present within min, and n. gonorrhoeae have been unable to develop resistance to monocaprin - . a monocaprin-containing prophylaxis would prevent the establishment of infection, while a treatment would clear an established infection. the rapid bactericidal activity of monocaprin indicates a single formulation is likely to achieve both outcomes. considerations for a monocaprin-based ocular formulation. to be effective, a formulation against ocular infections must distribute to the corneal surface, conjunctiva, and tear drainage ducts and remain there long enough to exert the antibacterial activity. in seeking an antimicrobial agent, killing activity of min was selected rather than a longer time period, such as - h, because this is a realistic exposure time achievable on the eye . the hydrophilic-lipophilic balance (hlb) value for monocaprin is . , calculated via the davies' method . it is a monoglyceride that is a waxy solid powder at room temperature, with a melting temperature of - °c, which is poorly water soluble, but highly soluble in ethanol. direct analytic detection of monocaprin for development of pharmaceutical products has previously used high performance liquid chromatography (hplc) based mass spectrometry detection to identify monoglycerides down to - ppm . eye ointment formulations. currently in use are eye ointments as prophylaxis, containing erythromycin or tetracycline , . such semi-solid dosage forms are retained on the eye for longer periods than eye drops, therefore improving ocular drug levels . lack of aqueous solubility of monocaprin is not an issue for this type of formulation, as the ointment base, white petrolatum usp, is non-aqueous. the mechanical properties of commercially available % (w/w) chloramphenicol ophthalmic ointment was investigated and compared with different blends of light mineral oil and white petroleum (fig. a) . this data indicated that the mechanical properties of the commercial ophthalmic ointment can be replicated with a blend of two parts light mineral oil to ten parts white petroleum. the initial measure of parts for light mineral oil was by volume and the measure of parts for white petroleum was by weight. as light mineral oil has a density of . g/cm , this means that this mixture is equivalent to approximately % by weight light mineral oil and % by weight white petroleum. the texture analysis graph for chloramphenicol ointment (fig. b ) and the blends of the light mineral oil and white petroleum match in terms of the mechanical properties of hardness (positive peak height), cohesiveness (positive peak area), and adhesiveness (negative peak area; fig. b ) are presented. monocaprin concentrations of . %, . %, . %, and . % by weight were incorporated in the developed ointment base with no sign of phase separation or precipitation upon storage. eye drop formulations. eye drops are sterile liquid dosage forms, which may be unsuitable carriers for monocaprin given its poor solubility in water. however, solubility can be improved through the addition of excipients . in addition, viscosity enhancers can be added to eye drops to prolong the time they are retained on the eye, such as carboxymethylcellulose (cmc, e number e ) and (hydroxypropyl)methyl cellulose (hpmc, e number e ), both long chain polymers derived from cellulose and commonly used in food and pharmaceutical products . ophthalmic viscosity enhancing polymers cmc and hpmc are both widely used in ophthalmic preparations and have good safety records [ ] [ ] . monocaprin concentrations in the liquid dosage formulations were . %, . %, . %, and . %, comparable to the ointment. to enhance solubility of monocaprin, it was first dissolved in propane- , -diol, which would improve the aqueous solubility of the monocaprin and would be less likely to cause ocular irritation than other solvents. the monocaprin was then combined with the viscosity enhancing polymer. however, monocaprin precipitated in cmc-based formulations and a cloudy layer formed at the top of the vessel just below the meniscus in hpmc-based formulations (fig. ). the addition of . % (v/v) polysorbate (tween ) improved monocaprin solubility (fig. ) . at this concentration of polysorbate , the monocaprin was completely in solution in the hpmc polymer-based formulations, but this was not the case in the cmc-based formulations (fig. ). this indicates that hpmc is the polymer of choice for further formulation development. antimicrobial activity of formulations. thickened eye drop and ointment dosage formulations were assayed for bacteriostatic activity and bactericidal activity via bacterial growth inhibition assays and bacterial log reduction assays, respectively. ointments produced the smallest zones of inhibition at . mm for . % to . mm for . % monocaprin, likely due to a slower diffusion rate of the monocaprin from the well into the agar compared to the liquid dosage formulations (fig. a) . the hpmc-based formulations, producing zones of inhibition of . mm for . % to . mm for . % monocaprin (fig. c) , performed slightly better than cmc-based formulations, with zones of . mm for . % to . mm for . % monocaprin (fig. b) . it was revealed that polysorbate , included in the eye drop formulations, had some antimicrobial activity against n. gonorrhoeae on its own. the mean zone of inhibition with polysorbate alone was mm, whereas the zones of inhibition for the cmc and hpmc-based monocaprin formulations started at and mm, respectively ( www.nature.com/scientificreports/ based on producing larger zones of inhibition ( fig. c ) and being the polymer of choice for solubility ( fig. ) , the hpmc-based thickened eye drops were assessed in log reduction assays, demonstrating that they are effective at killing n. gonorrhoeae, with over log activity, the threshold needed for bactericidal activity (fig. d ). to evaluate if the observed antimicrobial activity of polysorbate was bactericidal it was assessed alone at concentrations of . %, . %, . %, %, %, and %. even at the highest concentration tested, no bacterial killing was observed, showing that the action of the surfactant in the formulation is bacteriostatic. surprisingly, however, the higher the concentration of polysorbate in the formulations the lower the anti-gonococcal properties of the monocaprin (fig. d) . a similar effect has been reported in a previous study investigating polysorbates and antimicrobials . this inhibition of the monocaprin by the surfactant appears to be concentration dependant; the greater the monocaprin concentration in the formulations the more polysorbate is required to inhibit its action. to achieve a log reduction of or greater, a maximum concentration of . % polysorbate should be used for . % and . % monocaprin formulations, . % polysorbate for . % monocaprin formulations, and % polysorbate for . % monocaprin formulations. it is plausible that polysorbate and monocaprin form mixed micelles, decreasing the availability of the free monocaprin and consequently reducing the anti-gonococcal properties of the formulation. both the semi-solid and liquid dosage forms had strong bacteriostatic activity, however only the liquid dosage forms were bactericidal. this could mean that the ointment, when applied to the eye, may be able to stop the growth of gonococci but may not kill gonococcal cells in the tear fluid. this may be sufficient for a prophylaxis, but not for a treatment. it appears that release of the monocaprin from the ointment base is insufficient to reach a level where it would actively kill the bacterial cells. in comparison, the thickened eye drop formulation killed all of the gonococcal cells present in the simulated tear fluid, therefore making it a preferred candidate for future clinical studies because it can be used for both prophylaxis to prevent establishment of infection and treatment of active infections. the thickened eye drop and ointment formulations were assessed using an ex vivo corneal infection model to evaluate the viability of n. gonorrhoeae on the surface of an eye following treatment. excised bovine corneas were maintained in mem and infected with approximately gonococcal cells. saponin was used to lyse the cells of the epithelial layer of the corneas to release attached and internalised gonococci. a reduction in bacteria recovered from the corneas (fig. a ) and in bacteria printed onto gc agar from the corneal surface (fig. b) was seen in all corneas treated within an hour of infection, as would be the case for prophylaxis. reduction in gonococci is also seen with the application of a % monocaprin, . % polysorbate hpmc-based formulation www.nature.com/scientificreports/ (fig. ) , which suggests that the surfactant is contributing to reduction in bacteria. in addition to the antimicrobial activity observed, the surfactant may also interfere with the adhesion of the gonococci to the epithelial layer. therefore, inclusion of polysorbate in the formulation is beneficial both to improve the solubility of the monocaprin and also to contribute to the prevention of gonococcal infection. using this model system, it has been possible to demonstrate complete clearance of infection of n. gonorrhoeae from the eye (fig. ) . monocaprin antimicrobial activity and detection following storage.. following nine months of storage at room temperature in the dark, the hpmc-and cmc-thickened eye drop formulations were still as antimicrobially effective as newly made formulations, assessed by growth inhibition assays, as previously. to assess the stability of the monocaprin in the formulations after nine months, the published hplc method for monocaprin detection was used , with a minor modification. as the monocaprin peaks were wide and www.nature.com/scientificreports/ gave poor resolution at lower concentrations, a longer mm column based on solid core particles ( µm in diameter) was used, which increased the resolution. the final retention time of monocaprin using this modified method was approximately . min. the test performed well in regression analysis with a r value of . , with good inter-day and intra-day repeatability, precision, and accuracy. the international council for harmonisation of technical requirements for registration of pharmaceuticals for human use (ich) guidelines only give a brief outline of how to set the limits of detection (lod) and limits of quantification (loq) as there are a few methods that can be used . the lod and loq here were determined to be . and . µg/ml, respectively. using this method, the formulations stored for nine months had no detectable decrease in monocaprin nor the presence of any degradation products. at months after preparation of the formulations, antimicrobial activity was still evident via growth inhibition assays. www.nature.com/scientificreports/ comparison with povidone iodine. comparison of these formulations with povidone iodine, which has been proposed as an alternative prophylaxis for ophthalmia neonatorum, demonstrates that the monocaprinbased thickened eye drops are superior. the recommended concentrations of povidone iodine for prophylaxis resulted in signs of irritation using the bovine corneal opacity and permeability (bcop) assay, whereas the monocaprin formulation did not (fig. ). credé's silver nitrate prophylaxis has been discontinued and is unavailable in much of the world due to its reported ocular irritation and resulting chemical conjunctivitis, as well as treatment failures . cases of toxic chemical conjunctivitis and treatment failures are reported with povidone iodine , , , consistent with the irritation results seen here (fig. ) . these results suggest that monocaprin-containing formulations are a superior choice and a promising alternative for ophthalmia neonatorum prophylaxis. although a monocaprin ophthalmic formulation still needs to be assessed in humans, it is envisaged that for treatment, patient eyes would be rinsed or wiped with normal saline to clear excess discharge and non-adherent bacteria, then the monocaprin eye drop would be instilled. it is not known if more than one application will be necessary, however complete killing of n. gonorrhoeae can be achieved at . % and . % monocaprin in our models (figs. d, ) . as with other forms of prophylaxis, it is envisaged that the monocaprin formulation would be applied within an hour of birth or as soon as possible after accidental autoinoculation in the case of adult exposures. a single dose application should be sufficient for prophylaxis due to the relatively lower bacterial load and extent of dissemination to ocular tissues versus treatment of an active infection. we propose single-use applicator packaging for the purposes of infection control. the eye drop formulations are relatively easy to make, from inexpensive components, which could be extemporaneously prepared in areas of urgent need where antimicrobial resistant n. gonorrhoeae incidence rates are high. during formulation investigations, it was found that at the final concentrations determined to be best for the hpmc-based thickened eye drop, all components combined readily. given the activity of monocaprin against c. trachomatis , n. meningitidis, and other bacteria capable of causing ocular infections , this formulation has strong potential to replace current prophylaxis and treatments. in addition to its antibacterial activity, monocaprin also has known antiviral activity against enveloped viruses, including herpes simplex virus type (hsv- ), human immunodeficiency virus type (hiv- ), vesicular stomatitis virus, visna virus, respiratory syncytial virus (rsv), and parainfluenza virus type , which are all enveloped viruses like sars-cov- , , [ ] [ ] [ ] . indeed, it has been proposed that antiviral fatty acids may have a role in aiding to decrease morbidity and mortality in covid- . therefore, there may be use for an ocular formulation such as this, for example to supplement ppe for health care workers in high-risk areas, where use during and/or at the end of shifts working with covid- positive patients may reduce transmission or reduce the initial viral load. bovine corneal opacity and permeability (bcop) assay reveals povidone iodine irritation makes monocaprin more suitable for prophylaxis. monocaprin has previously been shown to be non-irritating in several assays . povidone iodine, however, is known to cause irritation , , . comparison with the sodium hydroxide control, a strong ocular irritant, the bcop assay indicates povidone iodine is a slight ocular irritant, according to the scoring scheme , where opacity is observed (white arrows in fluorescein staining), yet it is not confluent. this irritation increases with concentration (bottom three rows). here a concentration of monocaprin × higher than proposed in the eye drop formulation is shown to be non-irritating ( . % monocaprin) and indistinguishable from the negative control (top row). images on the left are under white light and those on the right are with a cobalt light filter following fluorescein staining to highlight corneal damage to the area of application of the substance to the surface of the eye. scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ in conclusion, a thickened eye drop formulation of monocaprin in hpmc with polysorbate has been demonstrated to have rapid and complete killing activity of n. gonorrhoeae in vitro and ex vivo, is biocompatible according to ocular irritation assays, and is stable and retains its antimicrobial activity. the recommended formulation comprise . % monocaprin in % hpmc with % polysorbate . at these concentrations all components dissolve effectively in sterile distilled water forming a simple aqueous solution, ready to use eye drop. methods texture analysis. blends of light mineral oil and white petroleum were made by adding one, two, three, four, or five parts light mineral oil ( . g/ml at °c; sigma-aldrich) to parts white petroleum blends (vaseline; sigma-aldrich). the light mineral oil was mixed into the white petroleum on a black pill tile and sheered together using two cm spatulas until an even consistency was obtained. to compare these to commercially available ophthalmic ointments, % (w/w) chloramphenicol ophthalmic ointment (goldeneye, typharm limited) was used. texture analysis of semi-solid samples used ml wide-necked glass vials placed on a stable microsystems ta.xtplus texture analyser testing stage and clamped into position. the probe tested the sample twice, automatically, before the sample was removed, the surface flattened, and tested again. each sample was thus tested five times. stable microsystems exponent lite (version . . . ) software was used to obtain data and to combine each of the five replicates into representative plots. preparation of ophthalmic ointments. monocaprin was warmed at °c until melted, mixed with light mineral oil warmed to °c, and added to the required amount of white petroleum on a black pill tile. the mineral oil was mixed and then sheered into the white petroleum using two cm spatulas until an even consistency was obtained and the drug was evenly distributed throughout the ointment. ointments were stored in ml plastic pots (sterilin ltd) at room temperature until required. gonorrhoeae strain nccp was cultured as previously overnight on a gc plates (oxoid) with kellogg's supplements at °c, % co . a cell suspension ( . mcfarland standard) was streaked onto gc plates and a sterilised number cork borer was used to make a well in the agar with a diameter of mm. approximately µl of the test formulation was placed in the well. for testing of ointments, the semi-solid material was used to fill the well until full. plates were incubated at °c, % co for - h. the diameters of the inhibition zones were measured. assessing bactericidal properties of formulations via log reduction. log reduction assays were conducted as previously with slight modifications; µl of a double concentration of bacteria suspension ( × ) was added to . g of the test formulation. all hpmc-based formulations were tested for two minutes, as previously . formulations without monocaprin were used as negative controls. ex vivo infection of corneas. assessment of clearance of bacteria from the corneal surface used corneas prepared similarly to those used for herpes simplex virus infection studies [ ] [ ] . excised bovine corneas (abp, guildford, uk) supported by scaffold medium of . g agarose in minimum essential media (mem, gibco), were used for infection studies within h. corneal surfaces were infected with µl of n. gonorrhoeae strain nccp at approximately cfu/ml and incubated for h at °c, % co . corneas were then washed five times with sterile phosphate-buffered saline to remove non-adherent bacterial cells before application of µl of the thicken eye-drop formulations containing . % hpmc, plus varying concentrations of monocaprin and polysorbate : % monocaprin and . % polysorbate (no drug control); . % monocaprin and . % polysorbate ; . % monocaprin and . % polysorbate ; . % monocaprin and . % polysorbate ; and www.nature.com/scientificreports/ . % monocaprin and . % polysorbate . after min, formulations were washed off the corneas by dipping them in sterile pbs ten times. colony forming unit counts of surviving gonococcal cells were determined by incubating corneas in % saponin at °c for min. a clean scalpel blade was passed over the epithelial layer and then the saponin was used to wash the epithelial layer of any loose cells. saponin was recovered, serially diluted in gc broth, and plated as for the log reduction assays. plates were incubated at °c, % co for h before counting. additionally, treated and washed corneas were pressed against a gc plate for s. plates were incubated at °c, % co for h before assessing them for bacterial growth. high performance liquid chromatography monocaprin detection. the hplc-based monocaprin detection method was developed based on a previous method using a perkin elmer series hplc system with a series auto-sampler and series ep diode array detector (uv/vis). data was acquired using perkin elmer totalchrom workstation (version . . ). a . × mm c column packed with µm kinetex solid core particles (phenomenex) was used to analyse samples. an isocratic mobile phase of : (v/v) of acetonitrile/water was used at a flow rate of ml/min. samples were injected at a volume of µl. monocaprin was detected by uv absorption at nm. a stock of mg/ml monocaprin was made in acetonitrile. this was diluted in acetonitrile to monocaprin concentrations of , , , , , , and . µg/ml. five injections of each of these standards was made on the optimised method followed by five injections of acetonitrile (blank). for the intra-day and inter-day testing, monocaprin concentrations of , , , and . µg/ml were tested. three injections of a , µg/ml monocaprin (standard) followed by a blank and then a sequence of the test samples with each being tested in triplicate and a standard and blank being run after every six injections. the limits of detection (lod) and quantification (loq) were determined by a signal-to-noise approach. a signal-to-noise ratio of : was used to determine the lod and : used for loq . bovine corneal opacity and permeability (bcop) assay. the bcop assay was performed as previously . bovine eyes (abp, guildford, uk) without visible damage were placed in weigh boats in a water bath at °c for min. an mm rubber o-ring was placed on the centre of the cornea into which was added one drop of sterile saline before returning to the °c water bath for five minutes. the saline was carefully removed and replaced with µl of test substance, left of the eye for s before washing with ml of sterile saline and incubating for min in the °c water bath. after sodium fluorescein treatment ( % (w/v) ph . ), the cornea was examined under a cobalt blue filter ( - nm, peak nm) and the eyes were scored for opacity . mean opacity was given a score between and ( , no opacity; , slight opacity; , marked opacity; , severe opacity; opaque opacity) . each of the test substances was tested in triplicate using three different bovine eyes on the same day. monocaprin was tested at times the concentration proposed for the eye drop formulation, demonstrating no irritation even at increased dosage. povidone iodine was prepared in distilled water at %, . %, and %, the latter two being concentrations cited in the literature [ ] [ ] . a negative control of hpmc and positive control of . m sodium hydroxide were conducted. the datasets generated during and/or analysed during the current study are available from the corresponding author on l.snyder@kingston.ac.uk. prevention of ophthalmia neonatorum caused by neisseria gonorrhoeae using a fatty acid-based formulation mutations in neisseria gonorrhoeae grown in sub-lethal concentrations of monocaprin do not confer resistance the growing threat of gonococcal blindness challenges in the management of neisseria gonorrhoeae keratitis prevalence of gonococcal conjunctivitis in adults and neonates gonorrhoea treatment failure caused by a neisseria gonorrhoeae strain with combined ceftriaxone and high-level azithromycin resistance silver nitrate ophthalmic solution and chemical conjunctivities prophylaxis of ophthalmia neonatorum comparison of betadine, erythromycin and no prophylaxis antimicrobial resistance in neisseria gonorrhoeae in the st century: past, evolution, and future cdc sexually transmitted diseases treatment guidelines a controlled trial of povidone-iodine as prophylaxis against ophthalmia neonatorum who guidelines for the treatment of neisseria gonorrhoeae british association for sexual health and hiv guidelines for the management of infection with neisseria gonorrhoeae treatment of gonococcal conjunctivitis with single-dose intramuscular ceftriaxone epidemiology and control of gonococcal ophthalmia neonatorum hydrogels containing monocaprin have potent microbicidal activities against sexually transmitted viruses and bacteria in vitro a quantitative kinetic theory of emulsion type. i. physical chemistry of the emulsifying agent. gas/liquid and liquid/ liquid interfaces enrichment and quantification of monoacylglycerols and free fatty acids by solid phase extraction and liquid chromatography-mass spectrometry ophthalmic ointments the science of dosage form design current eu approved additives and their e numbers effects of polysorbates on antiviral and antibacterial activity of monoglyceride in pharmaceutical formulations validation of analytical procedures: text and methodology. q (r ) development and evaluation of microbicidal hydrogels containing monoglyceride as the active ingredient virucidal activities of medium-and long-chain fatty alcohols and lipids against respiratory syncytial virus and parainfluenza virus type : comparison at different ph levels can bioactive lipids inactivate coronavirus (covid- )? neisseria gonorrhoeae. i. virulence genetically linked to clonal variation activation of checkpoint kinase is critical for herpes simplex virus type replication in corneal epithelium inhibition of ataxia telangiectasia mutated (atm) kinase suppresses herpes simplex virus type (hsv- ) keratitis eye irritancy screening for classification of chemicals we would like to acknowledge the generous funding and support of the sparks charity, who have provided for this work with project grants kin and kin to support colin churchward and this research. we also thank kingston university for support of the host:pathogen interactions laboratory, funding, and other facilities used in this work. we would like to acknowledge contributions to experimental work on povidone iodine made by vajiharan sriskantharajah. raid g. alany and lori a. s. snyder are co-inventor of uk patent gb . (pending)/eu patent . - (pending). the rest of the authors declare to have no competing interest. correspondence and requests for materials should be addressed to l.a.s.s.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - bv aak authors: schneider, annika; kurz, sandra; manske, katrin; janas, marianne; heikenwälder, mathias; misgeld, thomas; aichler, michaela; weissmann, sebastian felix; zischka, hans; knolle, percy; wohlleber, dirk title: single organelle analysis to characterize mitochondrial function and crosstalk during viral infection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: bv aak mitochondria are key for cellular metabolism and signalling processes during viral infection. we report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies. mitochondria are crucial for cellular energy metabolism, critically involved in the coordination of signalling processes within cells and orchestrate induction of apoptotic cell death , . besides this, cell-autonomous defence mechanisms during viral infection link innate immune sensing of infection and inflammation at the level of mitochondria , . the research in the recent years has expanded our knowledge about the different roles of mitochondria. for the different functions mitochondrial shape and motility, but also size, are important and are highly dynamic processes . mitochondrial shape and size are continuously changed during the dynamics of mitochondrial fusion and fission and mitochondrial turnover is controlled by mitophagy . viruses modify the host cell to create an ideal ambience, which includes metabolic support for viral gene expression and replication. such modifications of cellular metabolism and structure of viruses can also affect mitochondria. there are more and more reports about viruses known to influence mitochondrial dynamics. viruses known to enhance mitochondrial fission are hepatitis b virus (hbv), hepatitis c virus (hcv) and epstein-barr virus [ ] [ ] [ ] [ ] . viruses, which interfere with or enhance mitophagy are hbv, hcv and measles virus [ ] [ ] [ ] . sars coronavirus is reported to enhance the fusion of mitochondria . but the influence of viral infection on mitochondrial membrane potential and stress response has not been addressed in detail because of methodological constraints. so far, analysis of mitochondria and their functions relied mostly on bulk analysis of mitochondrial populations analysed ex vivo. in infected tissues where both, infected and non-infected cells are simultaneously present, it is very difficult to discriminate between mitochondria from infected versus healthy non-infected cells. this may be achieved by serial tissue sections analysed by electron microscopy, where viral particles could be visualized. however, this is a very time demanding process yielding results with little statistical power. we therefore aimed to develop a technology, where high numbers of single mitochondria and their function can be analysed in the context of viral infection in order to characterize changes induced by viral infection. we chose the liver, and more specifically hepatocytes, as viral infection increases size and mitochondrial fragility of liver mitochondria. we first aimed to determine the influence of viral infection of the liver on the size of liver mitochondria by flow cytometry. to that end, we established a reference curve using polystyrene microparticles with defined sizes ( . µm, , µm and µm). forward scatter analysis of these polystyrene microparticles revealed clear demarcation of the differently sized microparticles and a direct linear correlation of forward scatter results with microparticle size (r = . ) ( fig. a) , consistent with earlier reports that forward scatter measurements directly correlate with microparticle size down to . µm , . the flow cytometric analysis revealed that mitochondria isolated from healthy non-infected liver ranged in size from . µm up to . µm (fig. b ) assuming that isolated mitochondria are spherical in morphology, which is indicated by electron microscopy (see fig. b ). since mitochondria from hepatocytes are much larger than those from non-parenchymal liver cells or immune cells, we assume that mitochondria ≥ . µm in size are derived from hepatocytes. mitochondria purified from virus-infected livers had a slightly higher mean size compared to healthy liver ( . ± . µm compared to . ± . µm, respectively) and ranged in size from . µm up to µm (fig. b) . infection with recombinant replication-deficient adenoviruses is a well-established preclinical model system to study hepatotropic infections [ ] [ ] [ ] . however, to confirm the results we repeated the experiments by infection with wildtype replication-competent lymphocytic choriomeningitis virus (lcmv). also after lcmv-infection, we detected an increase of mitochondrial size confirming the results obtained after adenoviral infection (supp. fig. a ). in order to investigate whether innate immunity generated during viral infection, was responsible for this increase in mitochondrial size, we induced a type i interferon response by application of poly i:c . flow cytometric analysis of mitochondria isolated after poly i:c application did not reveal any differences in their size compared to the control groups suggesting other mechanisms (supp. fig. b) . the exact determination of the size of single mitochondria now opened the possibility to use this information for further analysis. next, we evaluated mitochondrial functionality by determining the mitochondrial membrane potential using the potentiometric dilc ( ) fluorescence dye. dose titration experiments of the dilc ( ) dye demonstrated a dose-dependent increase in fluorescence intensity in purified mitochondria (supp. fig. c ). upon addition of the electron chain uncoupling agent cccp, we found a profound reduction in dilc ( ) fluorescence (fig. c ) demonstrating that flow cytometric determination of changes in dilc ( ) fluorescence reflected mitochondrial membrane potential. by flow cytometric analysis we observed a significant decrease in the mitochondrial membrane potential of mitochondria isolated from virus-infected vs. healthy livers after either adenoviral or lcmv infection compared to healthy controls ( fig. d and supp. fig. d ). in contrast, we did not detect changes in the membrane potential after innate immune stimulation by poly i:c (supp. fig. e ). yet, the size of mitochondria may influence dilc ( ) signal intensity. indeed, we found a direct correlation between mitochondrial size and dilc ( ) staining ( fig. e and supp. fig. f ) suggesting that larger mitochondria purified from virus-infected livers should show higher dilc ( ) fluorescence intensity. we therefore compared mitochondria with the same size isolated from healthy or virus-infected livers. such direct comparison demonstrated that mitochondria of the same size from healthy vs. virus-infected livers showed a remarkable decrease in the membrane potential of mitochondria from infected livers (fig. f ) and suggested that viral infection caused changes in mitochondrial functionality. mitochondria also function to take up calcium from the cytosol and thereby coordinate cellular function , which can also serve as a stress test. when challenged with high concentrations of calcium ( µm), mitochondria isolated from virus-infected livers are much more fragile shown by time-dependent loss of membrane potential and change of their morphology indicated by decrease in side-scatter (fig. f ). this accurately detects mitochondrial swelling after loss of membrane potential following ca + challenge which is also detected by bulk analysis with a classical stress test by adding ca + and detection of loss of membrane potential by rh -fluorescence and swelling by measuring optical density at nm (supp. fig. g ) . consistent with the loss of membrane potential and changes in side-scatter signals, we detected loss of mitochondrial integrity after www.nature.com/scientificreports www.nature.com/scientificreports/ calcium challenge. number of viable mitochondria detected per second by flow-cytometry declined after calcium challenge, consistent with loss of mitochondrial integrity, and did so much faster in samples from virus-infected livers (fig. f ). comparing mitochondria with different sizes, it became evident that larger mitochondria are more fragile and disappeared more rapidly after ca + -challenge (fig. f ). taken together, here we detected an increase in size and a decrease in membrane potential as well as mitochondrial fragility of liver mitochondria after viral infection. however, since both, non-infected as well as infected hepatocytes are present in livers after adenoviral infection (see fig. b ), current protocols for isolation and analysis yield a mixture of mitochondria derived from healthy as well as infected hepatocytes. this makes it necessary to develop a methodology, by which mitochondria from healthy and virus-infected hepatocytes can be separated in order to characterize changes in mitochondrial function specifically in virus-infected cells. we generated a recombinant adenovirus expressing the fluorescent protein dsred fused to a mitochondrial localization sequence (ad-cmv-mitorl) that accumulates and selectively labels mitochondria within infected cells (supp. fig. ). we combined this mitochondrial labelling in infected cells with a multispectral flow cytometric single organelle measurement of isolated mitochondria. upon infection of hepatocytes with ad-cmv-mitorl in vitro we detected mito-dsred-fluorescence in mitochondria using confocal microscopy (fig. a) . since ad-cmv-mitorl also codes for luciferase, we detected in vivo bioluminescence of the liver after infection, thus www.nature.com/scientificreports www.nature.com/scientificreports/ confirming successful infection of hepatocytes in vivo (fig. b ). this allowed us to test whether mitochondria from ad-cmv-mitorl-infected hepatocytes (mito-dsred + mitochondria) could be distinguished from those of non-infected hepatocytes (mito-dsred − mitochondria) within the same liver. after density-gradient purification, mitochondria isolated from virus-infected livers were counterstained with mitotracker green and analysed by flow cytometry allowing discrimination of mito-dsred + mitochondria from mito-dsred − mitochondria from the same liver (fig. c ). mito-dsred + mitochondria from virus-infected hepatocytes had a mean size of . ± . µm as compared to mito-dsred − mitochondria from healthy hepatocytes with a mean size of . ± . µm (fig. d ). this confirmed the results obtained from mitochondria isolated from non-infected livers and further demonstrated a more pronounced size difference when mitochondria from virus-infected could be distinguished at the single organelle level from those of healthy hepatocytes. this was most likely related to a relative underestimation of size for mitochondria from virus-infected livers due to contamination with mitochondria from non-infected cells that are smaller than hepatocyte mitochondria. yet, we cannot formally exclude that mito-dsred localizing to mitochondria after infection with ad-cmv-mitorl may have contributed to the size difference. the almost identical forward scatter results and size of mito-dsred − mitochondria compared to mitochondria isolated from non-infected livers (fig. d) indicated that there was no influence of viral infection in neighbouring hepatocytes on mitochondrial size after isolation. differences in size of mitochondria may have also an influence on other parameters detected by flow cytometry and we therefore systematically measured mitochondrial autofluorescence from to nm using a spectral flow cytometer. as expected, we detected increased fluorescence at nm in mito-dsred + mitochondria, where the maximum of dsred fluorescence emission ( - nm) is expected . interestingly, we detected higher autofluorescence signals between and nm as well as above nm in mito-dsred + mitochondria (fig. e) . as the strength of autofluorescence may be influenced by the size of mitochondria, we analysed autofluorescence signals against the size of isolated mitochondria (fig. f ). we found that autofluorescence intensity between and nm directly correlated with mitochondrial size, which may explain the higher autofluorescence observed in larger mito-dsred + mitochondria. together, these data demonstrate the usefulness of single-organelle analysis by flow cytometry in combination with in vivo mitochondrial labelling in virus-infected hepatocytes to exactly determine physical parameters such as size or autofluorescence. mitochondrial crosstalk enables changes in membrane potential. since discrimination of mitochondria isolated from virus-infected compared to non-infected hepatocytes was reliably achieved using flow cytometry, we proceeded to test for changes in mitochondrial functionality upon infection. we assumed that the difference in membrane potential detected between mitochondria isolated from virus-infected livers compared to non-infected livers (see fig. ) has previously been underestimated, and that our method would allow to more specifically discriminate mitochondria from virus-infected hepatocytes compared to non-infected hepatocytes. we determined whether mito-dsred + differed from mito-dsred − mitochondria with respect to dilc ( ) fluorescence intensity. to our surprise, we found that the dilc ( ) signal was similar for all sizes of mito-dsred + compared to mito-dsred − mitochondria (fig. a ). since dilc ( ) fluorescence was homogenous in all mitochondria isolated from ad-cmv-gol infected livers (see fig. ), although they consisted of a mixture of mitochondria from infected and non-infected hepatocytes, we wondered whether there was an exchange of molecules between mitochondria. therefore, we mixed dilc ( )-labelled mitochondria with non-labelled mitochondria and by time-dependent flow cytometric analysis found that dilc ( ) fluorescence decreased in pre-labelled and increased in un-labelled mitochondria reaching an equilibrium of intermediate fluorescence intensity within seconds (fig. b) . however, mito-dsred was not exchanged between mitochondria, because we found clearly distinct dsred staining of mitochondria isolated from ad-cmv-mitorl-infected livers, and mito-dsred − mitochondria showed the same absent dsred fluorescence intensity as mitochondria isolated from non-infected livers. in order to further evaluate mitochondrial functionality, we challenged mitochondria with ca + as stress test and performed time kinetic measurements of dilc ( ) fluorescence and side-scatter of mito-dsred + and mito-dsred − mitochondria isolated from ad-cmv-mitorl infected livers. remarkably, the differences in mitochondrial characteristics observed when comparing mitochondria isolated from infected livers to mitochondria from non-infected livers (see fig. f ) where not present any more when comparing mito-dsred + to mito-dsred − mitochondria originating from the same liver. in fact, loss of dilc ( ) fluorescence, decrease in side scatter and mitochondrial events were the same for mito-dsred + mitochondria as compared to mito-dsred − mitochondria (fig. c) . when in direct physical contact with mito-dsred + mitochondria, also mito-dsred − mitochondria showed the same fragility as mitochondria from virus-infected hepatocytes. there, was still a small difference in the large mitochondrial group after calcium stimulation and flow cytometric analysis of the ssc and dilc ( ) which could be explained by the fact that to minutes after calcium stimulation the number of events was drastically reduced. only approximately % from the initial number of events are still detectable (shown by number of events/s). because of the statistical variation the conclusions at later time points has to be taken with caution. interestingly, also mixing of dilc ( ) labelled mitochondria isolated from either ad-cmv-golor lcmv-infected with those from healthy uninfected livers yielded in rapid loss of mitochondrial membrane potential to that measured in mitochondria from infected livers (fig. d and supp. fig. ) taken together these data demonstrate that mitochondria which are in close physical proximity exchange information leading to changes in mitochondrial membrane potential but not in mitochondrial size. here, we describe the influence of viral infection on the phenotype and function of mitochondria employing a new methodology combining spectral flow cytometry with virus-encoded markers to simultaneously evaluate multiple mitochondrial parameters at the level of single organelles. most studies involve confocal microscopy to detect mitochondria, which is also available in an automated manner to quantify large datasets of mitochondria . www.nature.com/scientificreports www.nature.com/scientificreports/ while most of these microscopic studies are performed in cell cultures to explore mitochondrial dynamics at the level of single cells, there are only few reports specifically detecting tagged mitochondria in tissues for ex vivo or in vivo analysis , . since in vivo microscopic analysis of mitochondria requires a complex experimental setup, is rather time consuming and does not allow for analysis of large numbers of mitochondria, we aimed to establish a methodology to evaluate mitochondria directly ex vivo following isolation from virus-infected tissue. so far, most of available methods analyse properties of mitochondria ex vivo at the level of mitochondrial populations, www.nature.com/scientificreports www.nature.com/scientificreports/ such as extracellular flux analysis, western blot analysis, calcium uptake or swelling assays. beyond visualization by microscopy, flow cytometry has emerged as technology to characterize mitochondria , , . however, mitochondria isolated from virus-infected tissues can be derived from both, virus-infected cells as well as healthy cells, which may skew the experimental results. we therefore generated recombinant adenoviruses containing a mito-dsred expression cassette to selectively label mitochondria of infected cells. fusion of a fluorescent marker to mitochondrial target sequences has previously been reported to reliably and specifically label mitochondria as shown by confocal microscopy , . we combined virus-encoded mito-dsred labelling of mitochondria to separate mitochondria of virus-infected cells from those originating from healthy cells, with the power of multi-parameter analysis by spectral flow cytometry. using this methodology, we provide evidence that mitochondria can be reliably separated from virus-infected cells and that viral infection led to an increase in size as well as a decrease of mitochondrial membrane potential. such changes in biophysical and functional properties of mitochondria were not triggered by innate immunity following recognition of infection through microbe-associated pattern recognition receptors indicating other reasons for these changes, which still have to be defined. time kinetic measurements of single mitochondria by flow cytometry further allowed us to detect a previously unknown mitochondrial cross-talk that involves rapid exchange of small molecules like the potentiometric dye dilc ( ) . such exchange of molecules among mitochondria required physical contact, occurred within seconds and did not include mitochondrial matrix-embedded proteins. this indicates a dynamic regulation of mitochondrial properties by cell autonomous mechanisms that require further investigation. taken together, the combination of mitochondrial labelling through mito-dsred together with single organelle analysis using spectral flow cytometry is ideally suited to further unravel biophysical and functional properties of mitochondria as well as mechanisms and consequences of mitochondrial interconnectivity in virus-infected cells. given the important role of mitochondria in cellular metabolism, anti-viral defence, cell signalling and cell death, the multiparametric analysis of single mitochondria opens new avenues to explore these complex mitochondrial functions in more detail in virus-infected cells. mice. c bl/ j mice were purchased from charles river (sulzfeld, germany). mice were maintained under specific pathogen-free (spf) conditions in the central animal facility of the klinikum rechts der isar, in accordance with the guidelines of the federation of laboratory animal science association. animal experiments were approved by the animal care commission of bavaria. male mice between the ages of - weeks were used. the expression cassette for cloning into recombinant adenovirus consists of the genes for the fluorescent protein dsred linked to a mitochondrial targeting sequence and cbg -luciferase separated by p a linker sites from the porcine teschovirus followed by a bgh poly(a) signal. gene expression was driven by the ubiquitous minimal cmv-promoter (ad-cmv-mitorl). ad-cmv-gol generation has been reported before . recombinant second generation serotype adenoviruses were generated using the gateway ® technology from thermofisher as described before . briefly, expression cassettes with cmv promotor, dsred linked to the mitochondrial targeting site, cbg -luciferase and the bgh poly(a) signal were synthesized (eurofins genomics, germany) and cloned into gateway ® pentr ™ dual selection vector (thermofisher scientific, germany). recombination of pentr ™ with expression cassette into pad/pl-dest ™ gateway ® vector (thermofisher scientific, germany) was performed in vitro via the lr clonase ® enzyme mix (thermofisher scientific, germany). the obtained pad/pl-dest ™ with expression cassette was linearized using the paci restriction enzyme and the resulting adenoviral dna was transfected with lipofectamine (thermofisher scientific, germany) into hek cells (crl- ™ ; atcc, usa). cell debris and supernatant were harvested when complete detachment of the cells occurred. this suspension was freeze/thawed, centrifuged and used for further infection of hek cells. cells from several cell culture dishes were harvested and resuspended in tris-buffer and freeze/thawed three times. cell debris was removed by centrifugation and supernatant purified by a two-step cscl gradient ultracentrifugation. the band containing adenovirus was harvested and dialyzed. virus titer was determined via adenovirus hexon titration. hek cells were infected with serial dilutions of purified adenovirus. after to hours, cells were fixed with methanol, and virus infected cells were stained with anti-hexon antibody (anti-hexon hrp, acris, germany) and detected via dab (dako, usa). the infected cells were counted and the titer was calculated. bioluminescence imaging. imaging of luciferase expression in infected mice was monitored by ivis lumina lt-series iii instrument (perkinelmer las, germany). five minutes before measurement mice have been anesthetized with . % isofluran and treated intraperitoneally with mg/kg bodyweight d-luciferin-k-salt (pjk gmbh, germany). isolation of mitochondria from murine liver tissue. heparin/nacl ( u/ µl) was injected i.p. into the mouse minutes prior to preparation. mice were sacrificed and livers were perfused via portal vein for minute with pbs to remove blood. liver was removed and weighed, and the liver was rinsed with isolation buffer www.nature.com/scientificreports www.nature.com/scientificreports/ ( mm mannitol, mm sucrose, mm hepes, mm edta, ph . ). the whole isolation procedure was performed on ice and in ice-cold isolation buffer. the tissue was rinsed with ml isolation buffer and cut with a blunt end scissor into small pieces. the liver fragments were resuspended in ml isolation buffer supplemented with . % bsa and protease inhibitor (protease inhibitor cocktail, edta-free, roche, switzerland) per . gram of weighted organ and homogenized in a potter-elvehjem with strokes at rpm. the homogenate was transferred to cooled ml falcon and centrifuged at x g for minutes to remove nuclei, intact cells and cellular debris. the supernatant was transferred to a glass tube and centrifuged at x g for minutes to sediment mitochondria. the received crude pellet was gently dislodged with a glass pestle from the side of the glass tube. mitochondrial purification by density gradient centrifugation. mitochondria were purified as previously described , . in brief, a discontinuous percoll density gradient was used for mitochondrial purification. crude mitochondria were resuspended in ip-buffer ( mm sucrose, mm tes, . mm egta, ph . ), loaded on a percoll density gradient ( %, % and % diluted in ipp buffer: mm sucrose, mm tes, . mm egta, . % w/v bsa, ph . ) and separated at × g for minutes. the phase containing mitochondria between % and % percoll-layer was recovered with a glass pipette and transferred to a ml glass tube, resuspended in ml ip-buffer and centrifuged for further minutes at × g. the pellet was washed again in ml ip-buffer and centrifuged at × g for minutes to get rid of remaining percoll. the supernatant was removed and mitochondrial pellet was dislodged from the side of the glass tube. the received mitochondria were resuspended in µl ip-buffer and kept on ice. determination of protein concentration. the protein content in the mitochondrial preparations was determined using the dc tm protein assay kit (bio rad laboratories, germany). the assay was performed according to the manufacturer´s protocol. four different bsa-dilutions reaching from . mg/ml to . mg/ml in ip-buffer were used as standards. the optical density was measured at nm with a multiplate reader (infinitem pro, tecan, germany). determining mitochondria by flow cytometry. mitochondria were diluted to µg protein per µl in ice-cold mitochondrial staining buffer msb ( . m saccharose, mm mops-tris, mm succinate, mm phosphoric acid, µm egta). the different mitochondrial probes were diluted in msb, mixed with the mitochondrial dilution in a : ratio and incubated at room temperature for minutes. the cell permeable carbocyanine-based mitotracker green probe (mtg, nm), which contains a mildly thiol-reactive chloromethyl moiety, was used to selectively stain all undamaged mitochondria regardless of the membrane potential. dilc ( ) ( nm), a cationic carbocyanine dye, was used to measure the membrane potential of isolated mitochondria. mitochondria were pelleted at x g for minutes and washed once in ice cold pbs. mitochondrial pellet was resuspended in msb to a final concentration of µg/µl and stored on ice for analysis. immediately before analysis, samples were diluted in ice-cold and filtered pbs to the final analysis concentration of . µg/µl. samples were analysed using the spectral cell analyzer sp (sony biotechnology inc, japan). the sample flow rate was set to record about events per second. as mitochondrial uncoupling by the protonophore cccp is well known to dissipate mitochondrial membrane potential (mmp), µm cccp (sigma-aldrich, st. louis, missouri, usa) was used as a positive control for membrane potential dependence of diic ( ) (biotium, hayward, usa). the mitochondrial permeability transition (mpt), a process characterized by a large increase of permeability of the inner mitochondrial membrane (imm), leading to an influx of solutes with a molecular weight less than . kda and water into the mitochondrion, is a ca + -induced process. the influx of solutes and water leads to swelling of mitochondria. in mpt-measurements µm ca + in msb was added to induce swelling and samples were analyzed immediately after administration and every following minutes for minutes in total. cyclosporina (sigma-aldrich, st. louis, missouri, usa) inhibiting mpt and thereby reversing the effect of ca + , was added at a concentration of µm. mitochondrial size was determined using polystyrene particle size standard beads (flow cytometry grade, spherotech) in three sizes: . μm, . μm and μm. beads of each size were separated via ultrasound, vortexed and beads/size were added per ml filtered pbs. immediately before analysis, mitochondria were diluted in bead mixture to the final analysis concentration of , µg/ml. data were analysed using flowjo software (version , flowjo, oregon, usa). western-blot. µg of protein per sample was loaded onto - % mini-protean ® tgx stain-free ™ precast gels (bio rad laboratories, münchen) and separation was performed within a gel chamber filled with x sds electrophoresis buffer at v for to hours. after separation, proteins were blotted using the trans-blot ® turbo ™ mini pvdf transfer packs (bio rad laboratories, germany). proteins were transferred onto membranes at . a for minutes using the trans-blot turbo ™ (bio rad laboratories, germany). membranes were blocked with % milk in tbs-t (tbs + . % tween- ) for hour at room temperature, washed three times with tbs-t and incubated with primary antibodies in % bsa in tbs-t overnight at °c. the membranes were washed three times with tbs-t and incubated for hours at room temperature with hrp-coupled secondary antibodies in % milk powder in tbs-t. blots were washed three times and developed using cheluminate-hrp picodetect (applichem gmbh, germany), which was evenly distributed on the membrane. the luminescence was detected for up to minutes using the imaging-system chemidoc tm xrs (bio rad laboratories, germany). to visualize www.nature.com/scientificreports www.nature.com/scientificreports/ several proteins on the same blot, primary and secondary antibodies were removed by incubating membranes for minutes at °c in stripping buffer containing ß-mercaptoethanol. subsequently membranes were washed three times with tbs-t and incubated as previously described with primary and secondary antibodies. following primary antibodies were used: adenine nucleotide translocator (ant) (santa cruz biotechnology usa), cytochrome-c-oxidase (cox iv), cytochrome-c (cyt-c), glyceraldehyde -phosphate dehydrogenase (gapdh), glucose-regulated-protein (grp ), histon b (h b), voltage dependent anion channel (vdac) (all cell signaling technology, usa), lysosome-associated membrane protein (lamp ) (thermo fisher scientific, usa), peroxisomal membrane protein (pmp ) (origene technologies, usa), following secondary antibodies were used: rabbit anti-goat hrp (santa cruz biotechnology, usa), mouse anti-rabbit hrp, goat anti-mouse hrp (jackson immunoresearch, uk). histology. mouse livers were fixed for hours in % paraformaldehyde. dehydrated livers (leica asp s, germany) were embedded in paraffin. serial µm-thin sections were prepared with a rotary microtome (hm s, thermofisher scientific, usa) and subjected to histological and immune-histochemical analysis. hematoxylin-eosin (he) staining was performed on deparaffinized sections with eosin and mayer's haemalaun according to standard protocol. immunohistochemistry was performed using a bondmax rxm system (leica, wetzlar, germany, all reagents from leica) with primary antibody against egfp (a- , diluted : in antibody diluent, invitrogen, thermofisher scientific, usa). slides were deparaffinized, pre-treated with epitope retrieval solution for minutes. bound antibody was detected with a polymer refine detection kit without post primary reagent and visualized with dab as a dark brown precipitate. counterstaining was done with hematoxyline. electron microscopy. tissues were fixed in . % electron microscopy grade glutaraldehyde in . m sodium cacodylate buffer ph . (science services, munich, germany), postfixed in % aqueous osmium tetraoxide , dehydrated in gradual ethanol ( - %) and propylene oxide, embedded in epon (merck, darmstadt, germany) and cured for hours at °c. semithin sections were cut and stained with toluidine blue. ultrathin sections of nm were collected onto mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (zeiss libra plus, carl zeiss nts gmbh, oberkochen, germany). pictures were acquired using a slow scan ccd-camera and item software (olympus soft imaging solutions, münster, germany). statistics. student's t tests were calculated using graphpad prism software. significance was set at p < . and denoted as *p < . , **p < . , ***p < . and ***p < . . all results are expressed as the mean ± standard deviation (sd). the data within this manuscript are available from the corresponding author upon reasonable request. mitochondrial control of cellular life, stress, and death mitochondrial signaling pathways: a receiver/integrator organelle mechanisms of mavs regulation at the mitochondrial membrane identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf mitochondria: dynamic organelles in disease, aging, and development hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis hepatitis c virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy epstein-barr virus latent membrane protein- a alters mitochondrial dynamics promoting cellular migration mediated by notch signaling pathway mitophagy enhances oncolytic measles virus replication by mitigating ddx /rig-i-like receptor signaling sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/ traf /traf signalosome living in the liver: hepatic infections correlated morphometric and biochemical studies on the liver cell. i. morphometric model, stereologic methods, and normal morphometric data for rat liver bioluminescence imaging allows measuring cd t cell function in the liver isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses analysis of mitochondria by flow cytometry real-time flow cytometry analysis of permeability transition in isolated mitochondria flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death measurement of mitochondrial mass by flow cytometry during oxidative cell sizing: a light scattering photometer for rapid volume determination overcoming limitations of microparticle measurement by flow cytometry tnf-induced target cell killing by ctl activated through cross-presentation outcome of anti-viral immunity in the liver is shaped by the level of antigen expressed in infected hepatocytes perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis reduced type i interferon production by dendritic cells and weakened antiviral immunity in patients with wiskott-aldrich syndrome protein deficiency calcium uptake mechanisms of mitochondria biochemistry, mutagenesis, and oligomerization of dsred, a red fluorescent protein from coral deep analysis of mitochondria and cell health using machine learning multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo in vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system flow cytometry of isolated mitochondria during development and under some pathological conditions why to compare absolute numbers of mitochondria analysis of mitochondrial dynamics and functions using imaging approaches strategies for imaging mitophagy in high-resolution and high-throughput a semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies progressive stages of mitochondrial destruction caused by cell toxic bile salts a chrome-osmium fixative for electron microscopy this work was funded by the deutsche forschungsgemeinschaft (dfg, german research foundation) -projektnummer -trr to d.w and p.k. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -fyvr tc authors: santiago, césar; mudgal, gaurav; reguera, juan; recacha, rosario; albrecht, sébastien; enjuanes, luis; casasnovas, josé m. title: allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: fyvr tc cell surface aminopeptidase n (apn) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. apn participates in tumor cell expansion and motility, and is a target for cancer therapies. small drugs that bind to the apn active site inhibit catalysis and suppress tumor growth. apn is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. three crystal structures that we determined of human and pig apn ectodomains defined the dynamic conformation of the protein. these structures offered snapshots of closed, intermediate and open apn, which represent distinct functional states. coronavirus envelope proteins specifically recognized the open apn form, prevented ectodomain progression to the closed form and substrate hydrolysis. in addition, drugs that bind the active site inhibited both coronavirus binding to cell surface apn and infection; the drugs probably hindered apn transition to the virus-specific open form. we conclude that allosteric inhibition of apn functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking apn with disulfides. blocking apn dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme. the apn ectodomain structure. as the apn protein is a type ii membrane protein, ectodomain expression required deletion of the n-terminal cytoplasmic and transmembrane domains, and introduction of a secretion signal sequence, as well as a hemagglutinin (ha) tag to allow protein detection and purification ( supplementary fig. s a ). as the n-terminal and middle portions of the hapn and papn ectodomains are heavily glycosylated, we produced them in cho cells (see methods). the purified proteins generated distinct crystal forms under different crystallization conditions (table and methods). in the past we reported a papn ectodomain crystal structure in complex with a cov spike (s) fragment (pdb code f c) , here we show three new structures for apn (table ). in the four structures, the n-terminal ha tag and ~ ectodomain residues were very disordered, indicating a large degree of flexibility in the membrane proximal polypeptide. the ectodomains have a hook-like conformation formed by domain i to iv and contained a zinc ion at the active site in domain ii (fig. ). the exposed convex side of domain iv mediates similar protein dimerization in the distinct crystals. approximately Å of each monomer is buried at the dimer interface ( table ) , indicative of a stable protein-protein interaction. domain iv is the largest apn domain and the most divergent in the m aminopeptidase family. in apn, domain iv has seven helix-turn-helix heat repeats and a single arm repeat formed by three alpha helices (α -α ) . the arm repeat is the most variable domain iv region in the hapn and papn structures, and can contact the peptide substrate bound to the active site (see below). although the dimeric assembly of human and pig apn ectodomains was preserved in various crystals, the conformation of each monomer differed among crystal forms, such that the distance between the n-terminal region of the ectodomains that formed the dimer varied from to Å in the structures ( table and supplementary fig. s b) . each crystal captured a single apn conformation, with all the monomers in the same form. these structures identified three distinct apn conformations, based on active site accessibility, which we termed closed, intermediate and open forms (fig. a) . as reported for other m family members , , the observed apn structural diversity indicated ectodomain dynamics in solution and on the cell surface. the active site accessibility at domain ii differed among crystal forms because of interdomain adjustments in the apn. the contacts between domain iv and other domains in the monomers varied among the structures, whereas the domain iv-iv buried surfaces in each monomer at the dimerization interfaces were preserved ( table ) . domain iv contacts with domain i or iii changed markedly less (~ - Å buried surface) than with domain ii ( Å ); domain ii-iv interaction thus mainly stabilized the closed apn conformation. there were no notable differences in the other interdomain contacts in the distinct apn forms (table ) . domains i-ii are distant from domain iv in the open conformation of the apn monomer (fig. a,b) , where the zinc ion at the catalytic site is more accessible to the solvent (fig. c) . the domain i to iii module swings ° toward domain iv, closing the active site (fig. b,c) . the hapn structure adopts an intermediate conformation in the crystals (fig. a) ; the distance between the n terminus of each monomer in the dimer is Å, and the angle difference of fig. a and table ). on the cell surface, the domain i to iii module must swing over domain iv, which is fixed by dimerization (fig. b, supplementary video s ). the module movement must be facilitated by the flexible ~ -residue polypeptide that links domain i to the transmembrane domain ( supplementary fig. s a ), although polypeptide length probably limits the interdomain movement shown here with apn ( °), which is less pronounced than that reported for erap- ( °, determined as in table ). the type of interdomain movement also differs between erap- and the apn. the domain iii-iv module moves together relative to domain i-ii in erap- , whereas domain i to iii swings over domain iv in the apn. in addition, the erap- hinge region is at the domain iii n terminus, whereas that of apn is in the domain iv n terminus. domains i, ii and iii can pivot at the beginning of the first (α ) or third (α ) domain iv helix, which are perpendicular to the swing angle (fig. b) . these differences in apn motion compared to other aminopeptidases are probably related to dimer formation, which is not observed in other m family members. the domain ii buried surface increases due to its interaction with domain iv when the conformation changes from open to closed (table ) , thus reducing accessibility of the active site cavity (fig. c) . m aminopeptidase dynamics is thought necessary for catalysis, and the closed the met residues of the papneh were replaced by seleno-met (see methods). highest-resolution shell is in parentheses. favored, allowed and outlier residues (%) in the ramachandran plot, as well as number of ectodomains in the asymmetric unit (asu) are shown. statistics for the papn-rbd crystal structure discussed here have been reported earlier (pdb code f c) . structure representations in supplementary fig. b . (fig. a) . in the closed papn, the side chain of a phenylalanine (phe ) at domain iv was placed at about . Å from the hydrolyzable peptide bond, whose carbonyl group is coordinated to the zinc ion. the phenylalanine was located in the loop that connects α and α in the single domain iv arm repeat of human and pig apn (fig. a) ; it penetrated the active site groove in the closed conformation and locked the peptide, ready for hydrolysis. domain iv residues that precede phe in the α -α loop contacted domain ii in the closed papn. a similar loop conformation is seen in a closed hapn structure (pdb code fys) . the phenylalanine side chain in closed apn probably hinders peptide release or translocation for further processing after p hydrolysis. it is likely that binding of the p ′ residue to the zinc ion required removal of the phenylalanine plug by domain ii displacement away from domain iv. the phenylalanine adopted a distinct conformation in the intermediate and open apn conformations (fig. a) . domain ii movement was accompanied by a conformational change of the α -α loop in domain iv (supplementary video s ), which became more solvent-exposed; the phenylalanine side chain faced into domain iv in the intermediate and open conformations and the peptide plug was removed from the active site. these changes would facilitate release of the n-terminal residue after hydrolysis. the small interdomain movement of the intermediate apn structure would be sufficient for peptide processing (fig. a) . we previously described in detail the cov spike rbd-papn binding interface . the porcine cov spike rbd binds to a papn region that is distant from the catalytic site ( supplementary fig. s b) . a critical cov receptor-binding motif, which bears an exposed tryptophan, penetrates a narrow cavity formed by domain ii and iv (fig. b) . the tryptophan aromatic side chain stacks onto papn domain iv residues his -pro , and is trapped by domain iv residues asn -pro on one side and domain ii residues gln -ser on the other (fig. b) . the main chain of domain ii residues is in close contact ( . Å) with the tryptophan side chain, and its imino nitrogen forms a hydrogen bond with the domain iv asn main chain carbonyl. domain iv-based superposition of the open papn with bound rbd and that of closed papn showed a shift in the domain ii main chain region that contacts the rbd; this region collides (< . Å) with the cov tryptophan (fig. b) . closing of the ectodomain would hinder penetration of the viral tryptophan between the papn domain ii and iv. cov binding to apn would lock the protein in its open conformation (fig. b) , preventing the ectodomain movement probably necessary for peptide hydrolysis (fig. a) . we analyzed the catalytic activity of soluble human and pig apn ectodomains in the presence of porcine cov s fragments bearing the rbd (fig. ) . the soluble s proteins specifically inhibited papn-mediated catalysis, measured as the hydrolysis of the l-pna substrate, and had no effect on hapn activity. the tgev (transmissible gastroenteritis coronavirus) spike does not bind hapn because it lacks the n-linked glycan recognized by porcine cov in papn , . the isolated rbd was sufficient to inhibit papn catalysis (fig. a) ; inhibition was dependent on rbd concentration. a high rbd:papn ratio was needed to achieve maximum inhibition ( - %; fig. b ), which decreased slowly after min (fig. c) drugs that bind the catalytic site inhibit cov binding to apn. non-hydrolyzable drugs that bind the apn catalytic site inhibit catalysis and prevent angiogenesis and tumor growth , , , . they appear to restrict ectodomain conformational changes, as shown by reduction in the number of some apn conformation-specific fig. s b) , with residues that contact the rbd in sticks with carbons in yellow (domain ii) and green (domain iv). the same residues are shown for the superposed closed structure (carbons in grey). the rbd motif that penetrates the papn cavity is shown with a grey surface and with residues as sticks (carbons in cyan or in magenta for trp). scientific reports | : | doi: . /srep mab epitopes . on the cell surface, active site epitopes recognized by the my mab decrease in the presence of actinonin, which indicates apn closure. crystal structures of m aminopeptidases in complex with these drugs show preferential adoption of a closed state , , , . drug binding would thus not only compete with substrates for active site binding, but might also restrict the aminopeptidase dynamics needed for peptide processing. the structure of the papn-rbd complex indicates that porcine cov would be specific for the open conformation (fig. b) . restriction of apn ectodomain opening by active site-binding drugs would thus have an allosteric effect on cov binding. to test this hypothesis, we studied tgev rbd binding to cell surface papn in the presence of drugs that bind to the active site (fig. ) . in flow cytometry, we determined the binding of an rbd-fc fusion protein to cells that expressed papn or an active site mutant (papn-hh/aa), alone or with various drugs (fig. a,b) . we analyzed the effect of the natural apn inhibitors actinonin and bestatin ; both reduced rbd-fc binding to cell surface papn (fig. a, left) . we then evaluated four synthetic amino-benzosuberone (abs) derivatives that bind with high affinity and selectivity to apn (supplementary fig. s a) ; all four abs molecules prevented rbd binding to papn and its effectiveness increased with apn-binding affinity (fig. b, left) . the bulkier abs and abs compounds, which contain a phenyl group and bind with the highest affinity to apn, more efficiently blocked binding of the tgev rbd to papn on the cell surface. the inhibitory molecules bind to the apn active site , , which is distant from the apn region recognized by cov (supplementary fig. s b) . to further determine whether the inhibitory effect was linked to drug binding to the papn active site, we analyzed rbd binding to the papn-hh/aa mutant, which lacks the two histidines (h and h ) that coordinate the zinc ion ( supplementary fig. s ) . staining for the rbd-fc protein was similar in cells expressing the mutant, alone or with the drugs (fig. a right and b right) , which showed that compound binding to the papn active site was necessary to prevent rbd binding to a distant site. in addition, inhibition of rbd binding to papn was drug concentration-dependent (fig. c) , and the amount of compound needed to reach % inhibition (ic ) decreased with compound affinity for apn (~ μ m for bestatin (ki ~ μ m), ~ μ m for actinonin (ki ~ μ m), ~ . μ m for abs (ki ~ . nm) ). these results show that drugs that bind to the cov cell entry and infection . we nonetheless found that active site-binding molecules hindered cov s protein binding and might inhibit virus infection. studies with low affinity binding drugs such as bestatin show no reduction in tgev infection . virus particles have high receptor-binding avidity, and these drugs might not have sufficient affinity to maintain most apn molecules closed. the selective compounds abs - have high affinity for apn and, at - μ m concentrations, inhibit capillary tube formation in cell cultures, with no cytotoxicity . in our cultures, we observed no toxicity at abs concentrations < μ m (not shown). we therefore analyzed the tgev-mediated cytopathic effect for each of the four abs molecules and actinonin at a μ m concentration and monitored inhibition of virus infection with abs ( log) and abs ( log) (fig. a) ; at the same concentration, the lower-affinity abs and abs compounds or actinonin did not inhibit. abs has a bromo substituent that is predicted to interact with the phenylalanine that plugs the substrate in the closed conformation ; this interaction likely helped maintain the closed ectodomain and efficiently prevented virus binding. tgev is a representative, extensively studied animal cov that use papn for cell entry , . to further determine whether abs inhibition of virus infection was linked to cell entry, we analyzed tgev replication at h post-infection and found that virus entry decreased with the abs concentration (fig. b ). abs addition after virus absorption at °c did not inhibit virus growth (not shown), which indicates that it prevented virus binding to cells. in addition, we studied the effect of abs concentration on tgev cell infection, and we observed that the tgev cytopathic effect was reduced and cell survival increased at higher abs concentrations (fig. c) . abs compounds are selective for apn molecules and designed to inhibit apn catalytic activity and tumor growth; here we show that they also prevent cov cell infections. to further analyze the importance of apn dynamics, we engineered disulfide bonds to bridge domains ii and iv and restrict ectodomain motion. we replaced the papn domain ii ser and domain iv ser and/or ser with cysteine to lock the ectodomain in the closed form with interdomain disulfide bridges. the ser main chain cα is ~ and ~ Å, respectively, from ser and ser in the closed form, but ser moves ~ Å away in the open form ( supplementary fig. s ). disulfide bond formation between papn cys and cys or cys should thus prevent ectodomain motion. we expressed the papn-cysteine mutants (c -c ) on the t cell surface and compared their catalytic and cov binding activity with that of the wild type papn (fig. ) . the papn cysteine mutants showed reduced catalytic activity (fig. a) and tgev rbd binding (fig. b ) relative to the wild type protein in t transfectants that express similar protein amounts. the higher activity of the papn c than the c or c mutants suggested that the cys -cys disulfide bond was more labile than the cys -cys bond, probably because cys is in a flexible loop (supplementary fig. s ). treatment with a reducing agent restored catalysis and rbd binding in the cysteine mutants and did not affect wild type papn binding activity (fig. ) . reducing the disulfide bonds fully restored rbd binding, but catalysis was partially recovered in the papn c and c mutants. substrate hydrolysis is proposed to close the ectodomain (see above), which would facilitate rebuilding the disulfides. locking the closed form and the phenylalanine in the domain iv arm repeat inside the active site probably impeded substrate processing (fig. a) . the papn cysteine mutants bound markedly less rbd than the wild type protein, which confirmed that cov s protein binding to the closed papn was sterically hindered (fig. b) , and that cov recognized the open form. overall, these results validate the functional relevance of the apn ectodomain conformations and its motion. structural dynamics is an intrinsic property of aminopeptidases. the apn crystal structures reported here indicate the dynamic conformation of its ectodomain, and functional studies show its relevance in catalysis and virus infection. distinct ectodomain regions mediate these functions, but agents that bind to one region prevent activities linked to the other. these allosteric effects with ligands are probably caused by restrictions in apn conformational dynamics, as confirmed with disulfide bond mutants. they demonstrated that preventing ectodomain motion and locking apn forms inhibits its functions. apn ectodomain movement is less pronounced and differs from that reported for other m aminopeptidases. these differences could be due to the apn dimeric conformation and its linkage to the cell surface. dimerization only engages the domain iv region, and we found that the dimer is conserved in all apn structures, closed, intermediate and open. apn domain iv thus does not move as described for erap- or f , , proteins that do not form dimers. the fixed conformation of the apn dimer determines that the domain i to iii module swings over domain iv (supplementary video s ), with the hinge at the domain iv n-terminal region. the length of this movement is less marked in apn ( °) than in erap- ( °), although the two proteins have very similar closed conformations. displacement of the apn domains i, ii and iii must be limited by the length of the flexible polypeptide that links domain i to the transmembrane region, whose movement is restricted by membrane fluidity. the extent of apn movement nonetheless appears to be sufficient for release of the hydrolyzed peptide n-terminal residue, which is not plugged by domain iv in the open or in the intermediate apn conformations (fig. a) . it is not clear how each monomer in the dimer moves, whether their movement is random or synchronized in the same or inverse directions. experiments with hapn antibodies and those shown here with the tgev rbd (fig. ) suggest that ~ % of the molecules adopt different forms; these data imply that each apn monomer maintains a distinct conformation (supplementary video s ). the crystal structures reported here provide snapshots of apn dynamic conformation, and also guided experiments that demonstrate its role in virus entry into cells and catalysis. the switch between a proteolytic active (closed) and an inactive (open) conformation has been proposed for several m aminopeptidases , , , . this dynamics is thought to be important for peptide hydrolysis and release from the aminopeptidase active site. the region that joins α and α in the domain iv arm repeat penetrates the active site groove in closed pig and human apn structures reported here and elsewhere (fig. a) , and a conserved phenylalanine in this region locks the substrate coordinated to the zinc ion, permitting hydrolysis. further peptide processing likely requires removal of the phenylalanine lock by opening the apn ectodomain, which facilitates n-terminal residue release and peptide translocation, both sterically hindered in the closed conformation (fig. a) . the inherent flexibility in the domain iv arm repeat that we demonstrate here is linked to interdomain arrangements might also enable substrate processing, and indicate how ectodomain movements participate in peptide hydrolysis. local changes in a conserved tyrosine (tyr in papn) at the active site of m aminopeptidases are also suggested to be important , , . among apn forms, the absence of conformational switches in active site residues at domain ii ( supplementary fig. s ) nonetheless indicates that tyrosine movement is not linked to interdomain motion. disulfide bonds that lock the apn closed conformation or drugs that prevent opening of the ectodomain inhibited cov protein binding and cell infection, whereas porcine cov s proteins probably hinder apn transition to the closed form and peptide hydrolysis. our results verify the critical role of apn dynamics in cov infection and catalysis, and demonstrate that the open apn structure is inactive in peptide hydrolysis. anti-apn antibodies that inhibit apn activity and reduce tumor growth , likely block ectodomain movements. the allosteric inhibition of apn functions shown here using viral proteins and drugs is likely to be due to suppression of apn transient conformational states, as shown for other enzymes . blocking apn movement prevents its functions, and suggests a new approach for the development of drugs that target this protein. small molecules or conformation-specific antibody inhibitors of ectodomain motions can bind to the active site or interact with distant sites, as shown here with cov spike fragments. high affinity drugs designed to inhibit catalysis and tumor growth prevent cov infections, which indicates that targeting apn ectodomain dynamics can be a valuable approach to block apn functions related to cancer progression and virus infections. . catalysis was determined at min as absorbance at od nm (see fig. and methods). relative rbd-fc binding to transfected cells determined from mean fluorescence intensity computed by flow cytometry as in fig. c . domain ii and iv residues replaced by cysteine are indicated at bottom (see supplementary fig. s ). mean ± sd (n ≥ ). . a papn protein with met residues replaced by seleno-met (se-met papn) was produced using methionine-and glutamine-free dmem (invitrogen) supplemented with % dialyzed fetal calf serum (fcs) and l-seleno-methionine (both from sigma). apn proteins secreted to culture supernatants were purified by affinity chromatography with anti-ha ac mab (roche), followed by size exclusion chromatography in hepes-saline buffer ( mm hepes, mm nacl) ph . . preparation of most soluble cov s proteins used here has been described , . s h and s h proteins were derived from the hol porcine cov s glycoprotein and bear the papn-binding domain. soluble tgev rbd was derived from the s glycoprotein of the tgev sc strain (genbank acc. n° aj ). it contains s residues to , an n-terminal ha peptide, and a flag sequence (monovalent rbd variant) or human igg fc (bivalent rbd-fc variant) at the c-terminal end. cov s proteins were produced in cho-lec or t cells and purified as described . analysis of apn catalysis. apn catalytic activity was determined using leucine p-nitroanilide (l-pna) (sigma) in a standard spectrophotometric assay in -well plates with soluble proteins or transfected cells. to study cov protein inhibition of apn catalysis, soluble apn ectodomains ( μ g/ml; ~ nm) were added to duplicate wells, alone or with soluble cov s protein variants, followed by the l-pna substrate ( mm) in μ l final volume ( °c). plates were incubated at room temperature and od at nm was measured at different times. background od of wells without apn was subtracted to determine specific catalytic activity. similar procedure was used with t cells ( × ) expressing papn hr after transfection. od of well with mock-transfected cells were taken as background. cell samples expressing various amounts of papn at the membrane were used to normalize the activity of the papn cysteine mutants. relative activity of the mutant to wild type was determined as the ratio of the papn mutant to the wild type od from samples with the same protein expression, as monitored by flow cytometry (see below). cov protein binding to apn. stably transfected bhk -papn, cho-papn and cho-papn mutant cells or transiently transfected t cells were used. the papn contained the ha peptide at the c terminus to monitor cell surface expression in cho and t cells. in the papn-hh/aa mutant, the two active site histidines were replaced with alanines, whereas in the papn cysteine mutants, the domain ii ser and the domain iv ser and/or ser were substituted by cysteine. we analyzed the effect on rbd binding to papn of two natural inhibitors of apn enzyme activity, bestatin and actinonin (sigma) , as well as four synthetic abs compounds . bestatin and actinonin were dissolved at mm in pbs, whereas abs compounds were used at mm in dmso. in wild type or mutant papn-expressing cells, we used flow cytometry to monitor tgev rbd binding to cell surface papn, essentially as reported , . cells were washed three times with cold pbs and resuspended ( cells/ml) in pbs supplemented with . % heat-inactivated fcs and . % bovine serum albumin (bsa; binding buffer); μ l of cell suspension were added to -well plates (nunc), cells were sedimented and resuspended in μ l of - μ g/ml rbd-fc solution alone or with inhibitors at indicated concentrations ( min, °c). an unrelated fc fusion protein was used as control. cells were washed and incubated with anti-human igg fluorescein isothiocyanate (fitc)-labeled secondary antibody ( min, °c). the mean fluorescent intensity was determined in a beckman coulter epics xl ; background cell staining with the fc protein was subtracted to determine the specific rbd-fc binding to cell surface papn. in parallel, the amount of cell surface papn expression was determined by flow cytometry with the anti-ha ac mab (roche) and an anti-mouse fitc-labeled secondary antibody (invitrogen). analysis of the papn cysteine mutants binding activity was normalized by the cell surface protein amounts as explained above for the catalytic activity. by qrt-pcr (quantitative reverse transcription polymerase chain reaction). stable transfected bhk -papn or bhk cells ( × cells/well) in dmem (dulbecco's modified eagle's medium) with % fcs were plated in -well plates ( h). plates were transferred to °c, medium was removed and μ l binding buffer alone or with apn-binding drugs or rdb protein were added to wells; after min, the solution was replaced with μ l virus inoculum at a multiplicity of infection (m.o.i) of , alone or with inhibitors in binding buffer. after virus adsorption at °c, cells were washed three times with binding buffer, and incubated in dmem with % fcs ( h, °c, % co ). cells were detached and lysed with μ l tri reagent (sigma) for rna extraction, and cdna was generated from μ g rna using the high capacity cdna reverse transcription kit (applied biosystems). real-time pcr reactions ( μ l) were performed in triplicate using μ l cdna sample, μ l of x hot firepol evagreen qpcr mix plus (rox) (solis biodyne) and . μ l of specific primers for mouse β -actin or for the tgev s gene, in a real time pcr system (applied biosystems) using a standard protocol. data were analyzed with software using the comparative ct method (Δ Δ ct). tgev s expression relative to β -actin was determined and the ratio of values alone and with inhibitor used as relative cell entry. infection or cytopathic effect of tgev was inhibited in porcine st cells. one day after seeding ( . × cells/well) in -well plates, cells were transferred to °c and pre-incubated with μ l binding buffer alone or with inhibitors, in duplicate. solutions were replaced with μ l of serial -fold dilutions of virus inoculum with inhibitors or with dmso (≤ . %) as control. after incubation ( h, °c), cells were washed three times with dmem with % fcs and incubated alone or with inhibitors for two days at °c. to determine cell survival after infection, medium was removed, cells were formalin-fixed, stained with crystal violet and viability determined by optical density (od) at nm. ratios from wells with and without virus were determined to calculate cell survival (see supplementary fig. s b ). crystallization and diffraction data collection. the endoglycosidase h-treated ( h, °c) papn (papneh) ectodomain was crystallized by the sitting drop technique with a crystallization solution of % polyethylene glycol (peg)- and % peg- (ph ~ ) and a mg/ml protein sample. alternatively, native glycosylated papn ectodomain crystals were prepared with a crystallization solution of % peg- and mm sodium acetate ph . . the hapn ectodomain ( mg/ml) was crystallized with a solution of % peg- , mm imidazole-hcl ph . . crystals were frozen in crystallization solutions containing % ethylene glycol for diffraction data collection at the european synchrotron radiation facility (esrf; id and id ) and swiss light source (sls; pxii) beamlines. diffraction data were processed with xds and scaled with scala programs . for statistical data, see table . structure determination. the structure of se-met papneh protein was solved by a combination of molecular replacement (mr) and single-wavelength anomalous dispersion (sad) methods. the crystals contained two molecules in the asymmetric unit (table ) . a partial structure was obtained by mr using the phaser program and domains i to iii of the tricorn-interacting factor f (pdb code z w), which share ~ % residue identity with papn. the phaser llg value for the best mr solution was , whereas rfz values were . and . , and tfz values of . and . . we then used the mrsad protocol in the auto-rickshaw server to determine the complete papneh structure, starting from the partial mr structure and using se-met papneh crystal diffraction data collected at the selenium peak wavelength. the final structure included the two papn molecules of the asymmetric unit, which were adjusted manually and refined with phenix.refine using data extending to . Å resolution (for statistics, see table ). the papneh structure comprises residues to and the zinc atoms coordinated in the enzyme active site. the other apn ectodomain structures (table ) were determined by the mr method using the papneh structure as search model. two ensembles including domain i, ii and iii or isolated domain iv were used for mr structure determination with phaser. structures were refined with phenix.refine (statistics in table ). in all structures, the engineered tags and - residues of the n-terminal ectodomains were very disordered and are not included in the final models. electron density maps of active site residues and of n-linked glycans are shown in supplementary figs s and s , respectively. structure representations prepared with pymol (pymol.org). aminopeptidases: structure and function families of zinc metalloproteases the moonlighting enzyme cd : old and new functions to target aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev 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endoplasmic reticulum aminopeptidase erap aminopeptidase-n/cd (ec . . . ) inhibitors: chemistry, biological evaluations, and therapeutic prospects a novel amino-benzosuberone derivative is a picomolar inhibitor of mammalian aminopeptidase n/cd determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site exploring s plasticity and probing s ′ subsite of mammalian aminopeptidase n/cd with highly potent and selective aminobenzosuberone inhibitors mt - , a fully humanized antibody raised against aminopeptidase n, reduces tumor progression in a mouse model allosteric inhibition through suppression of transient conformational states antigenic modules in the n-terminal s region of the transmissible gastroenteritis virus spike protein collaborative computational project, n. the ccp suite: programs for protein crystallography pushing the boundaries of molecular replacement with maximum likelihood on the combination of molecular replacement and singlewavelength anomalous diffraction phasing for automated structure determination phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics we thank the esrf for provision of synchrotron radiation facilities through bag-madrid projects, as well as the swiss-sls facility, s. rodríguez for technical support, and c. mark for editorial assistance. gm was a recipient of a la caixa fellowship. jr was supported by the juan de la cierva program and rr by nih grant p ai - a . the work was supported by grants from the spanish ministry of science (bfu - and bio - -r to jmc). key: cord- -dsysiefx authors: király, kornél; kozsurek, márk; lukácsi, erika; barta, benjamin; alpár, alán; balázsa, tamás; fekete, csaba; szabon, judit; helyes, zsuzsanna; bölcskei, kata; tékus, valéria; tóth, zsuzsanna e.; pap, károly; gerber, gábor; puskár, zita title: glial cell type-specific changes in spinal dipeptidyl peptidase expression and effects of its inhibitors in inflammatory and neuropatic pain date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dsysiefx altered pain sensations such as hyperalgesia and allodynia are characteristic features of various pain states, and remain difficult to treat. we have shown previously that spinal application of dipeptidyl peptidase (dpp ) inhibitors induces strong antihyperalgesic effect during inflammatory pain. in this study we observed low level of dpp mrna in the rat spinal dorsal horn in physiological conditions, which did not change significantly either in carrageenan-induced inflammatory or partial nerve ligation-generated neuropathic states. in naïve animals, microglia and astrocytes expressed dpp protein with one and two orders of magnitude higher than neurons, respectively. dpp significantly increased in astrocytes during inflammation and in microglia in neuropathy. intrathecal application of two dpp inhibitors tripeptide isoleucin-prolin-isoleucin (ipi) and the antidiabetic drug vildagliptin resulted in robust opioid-dependent antihyperalgesic effect during inflammation, and milder but significant opioid-independent antihyperalgesic action in the neuropathic model. the opioid-mediated antihyperalgesic effect of ipi was exclusively related to mu-opioid receptors, while vildagliptin affected mainly delta-receptor activity, although mu- and kappa-receptors were also involved. none of the inhibitors influenced allodynia. our results suggest pathology and glia-type specific changes of dpp activity in the spinal cord, which contribute to the development and maintenance of hyperalgesia and interact with endogenous opioid systems. and different extracellular matrix proteins like collagen and fibronectin . dpp is also known as cell surface antigen cd on t-lymphocytes , and as a receptor for coronaviruses . incretins are the most familiar substrates of dpp since these hormones are major regulators of postprandial insulin secretion. inhibition of dpp increases the incretin levels and prolongs the postprandial insulin action. therefore dpp has become a major target for the therapy of type ii diabetes. application of newly developed dpp inhibitors revealed several physiological and pathological processes such as lipid metabolism, myocardial, renal and liver functions, atherosclerosis and inflammation in which dpp is involved , . control of chronic pain associated with tissue injury, inflammation or ongoing diseases have made no progress for decades. current analgesics are at best moderately effective and associated with intolerable side effects. therefore, development of novel therapeutic interventions for pain relief is one of the chief challenges for medical sciences. it is well established that altered pain sensations such as hyperalgesia (an increased response to noxious stimuli), allodynia (painful response to normally innocuous stimuli) and spontaneous pain are characteristic features of various pain states . previously we have demonstrated dramatic reduction of mechanical hyperalgesia following spinal application of dpp inhibitors (ipi and vildagliptin) in subacute inflammation and this action was naloxone reversible suggesting an opioid receptor-mediated effect. none of the inhibitors changed the nociceptive threshold in acute nociceptive tail-flick test . analgesic and anti-inflammatory effects of dpp inhibitors were also showed in chronic inflammatory models in mice . machinery of the endogenous opioid system has been intensely investigated and clarified in recent decades. although inducing/regulating the endogenous opioid machinery would provide a powerful tool to control pain propagation, this possibility has remained largely unexploited. here, we identify dpp in the spinal dorsal horn, show that its expression changes during pathological conditions, and demonstrate that it shapes opioid signalling in a receptor-and treatment-specific manner. although synaptic dpp may have a key role in neuronal mechanisms of pain propagation, we identify glial cells as inducible dpp -batteries, in this way playing a role in hyperalgesia and opioid signalling. dpp transcripts in the rat spinal dorsal horn in physiological, inflammatory and neuropathic states. taqman qpcr detected dpp mrna in the dorsal horn of l spinal segments taken from control, inflamed and neuropathic rats. neither carrageenan treatment nor neuropathic condition caused significant alteration in the dpp mrna levels (relative quantities in control, carrageenan-induced inflammation and neuropathic groups: . ± . vs. . ± . vs. . ± . , one-way anova p = . ; fig. a) . dpp mrna showed a low expression by in situ hybridization in the spinal dorsal horn of l -l segments. while the grain density observed in sections hybridized using the sense probe was equal to the background, a significant signal was detected with the antisense probe (antisense: n = , median = . , % at . and % at . ; sense: n = , median = . , % at . , % at . ; mann-whitney rank sum test p < , ). dpp mrna was distributed evenly within the dorsal horn, and no significant differences among the experimental groups were detected (control: n = , . ± . ; inflamed: n = , . ± . ; neuropathic: n = , . ± . ; one-way anova, p = . ; fig. b) . the data obtained from both qpcr and in situ hybridisation studies clearly demonstrated that dpp mrna exists in the spinal dorsal horn and its quantity does not change either in inflammatory or neuropathic pain states. dpp protein expression in physiological condition and its changes during inflammation and neuropathy in the spinal dorsal horn. western blot banding pattern of dpp originated from rat spinal dorsal horn appeared very similar to that one which was taken from human white (pre)adipocytes using an antibody different from the one used in the current experiments . western-blot analysis in this study demonstrated an elevated protein level in the inflamed spinal cord being significantly different from those found in naïve and neuropathic spinal cords (control: . ± . ; inflamed: . ± . ; neuropathic: . ± . ; one-way anova, holm-sidak method, p = . ; fig. c -d). dpp immunoreactivity in the spinal cord appeared in naïve, inflamed and also in neuropathic animals. densitometry of the dpp immunolabelling showed a significant increase in the medial two third of the dorsal horn (corresponding to the hind paw) during inflammation compared to naïve and neuropathic conditions (control: n = , . ± . ; inflamed: n = , . ± . ; neuropathic: n = , . ± . ; one-way anova p = , ; fig. e ). in contrast to dpp mrna, the overall amount of dpp protein detected by western-blot and immunohistochemistry significantly increased in the spinal dorsal horn during inflammation and did not change in neuropathy compared to the physiological state. dpp immunoreactivity and its changes in individual cell types during pathology. punctate immunostaining was detected in neuronal cell bodies (fig. a) and also in axon terminals in naive animals. puncta representing dpp were embedded in synaptophysin stained elements suggesting close relationship between synaptic and dpp activity in many cases (fig. ) . dpp -immunopositive dots that appeared on the surface of map labelled dendrites were always associated with synaptophysin positivity (fig. a) suggesting that dendrites did not express the enzyme but received synapses from dpp -containing boutons. the enzyme appeared both in vesicular glutamate transporter (vglut ) immunolabelled excitatory (fig. c) and in vesicular gaba transporter (vgat) positive inhibitory axon terminals (fig. d) , as well as in cgrp stained primary afferent boutons (fig. b) . dpp labelling also occurred in gfap-positive astrocytes (fig. b) and iba -stained microglia cells (fig. c) . in naive rats, the density of the enzyme staining was the highest in astrocytes and differed significantly from that in other cell types. dpp density was also significantly higher in microglia than in neurons (astrocyte: n = , median = . kruskal-wallis one-way anova on ranks, dunn's method at p < . , in all cases n means the number of the analysed optical sections of neuronal or glial elements). it should be noted that dpp immunolabelling existed not only in the membranes of the different cell types but also in their intracellular compartments. taking the qualitative and quantitative data together ( table , fig. ), dpp immunostaining was detected in neuronal cell bodies and also in excitatory and inhibitory axon terminals in naïve animals. densitometric analysis showed that in naïve animals, microglia and astrocytes expressed dpp protein with one and two orders of magnitude higher than neurons, respectively. furthermore, dpp expression significantly increased in astrocytes and did not change in other cell types during inflammation. in neuropathic conditions, dpp immunoreactivity significantly increased in microglia, decreased in neurons and remained unchanged in astrocytes. to challenge the involved opioid receptor types, selective opioid receptor antagonists were applied spinally together with two different dpp inhibitors in carrageenan-induced subacute inflammation. i.t. application of nmol/rat ipi and nmol/rat vildagliptin eliminated . ± . % and . ± . % of mechanical hyperalgesia measured by the randall-selitto test in intraplantar carrageenan-induced inflammation. co-administration of the mu-opioid receptor (mor)-selective inhibitor ctap reduced the antihyperalgesic effect of ipi to − . ± . %, while following co-application of kappa-receptor (kor) antagonist gnti and the delta-opioid receptor (dor) antagonist tipp[Ψ] the antihyperalgesic effect of ipi remained at . ± . % and . ± . %, respectively. following co-administration of mu-and kappa-antagonists, antihyperalgesic effect of vildagliptin was . ± . % and . ± . %, respectively, while the delta-antagonist tipp[Ψ] completely blocked the antihyperalgesic effect of vildagliptin by reducing its antihyperalgesic effect to − . ± . % (fig. ) . these results show that both ipi and vildagliptin resulted in robust but distinct opioid-dependent antihyperalgesic effects during inflammation. the antihyperalgesic effect of ipi was fully blocked by the selective mu-opioid receptor antagonist and no decrease was observed by the selective kappa-and delta-antagonists. in case of vildagliptin, the antihyperalgesic effect of this inhibitor was completely blocked by selective delta-receptor antagonist while selective mu-and kappa-receptor antagonists also decreased the effect significantly. effects of dpp inhibitors in neuropathy. different modalities of hyperalgesia and allodynia appear not only in inflammatory conditions but also in neuropathic pain states. none of the tested dpp inhibitors had significant effect on mechanical and cold allodynia, while both i.t. ipi and vildagliptin had a significant mechanical antihyperalgesic effect measured with the randall-selitto test one week after partial sciatic nerve ligation with mpe values of . ± . % and . ± . %, respectively. in contrast to inflammatory states, the nonselective opiate antagonist naltrexone (ntx) did not affect the antihyperalgesic action of the dpp inhibitors signifficantly in neuropathic conditions suggesting completely different actions of the enzyme on hyperalgesia in the two pain states (fig. ) . a high level of dpp expression in the developing brain and spinal cord was described, which dramatically decreased in adults, but persisted in leptomeningeal cells and capillary endothelial cells of the choroid plexus [ ] [ ] [ ] . dpp mrna was detected in cortical areas in adult naïve animals and its level did not change after cerebral ischemia. in contrast, dpp immunoreactivity was not found in the same regions in physiological state but its expression appeared in microglia, neurons and astrocytes in different time points during cerebral ischaemia . we are first to show that transcripts as well as the protein of dpp are detectable in the adult mammalian spinal dorsal horn. similarly to the effects of ischemic injuries in the brain, our q-pcr and in situ hybridization analysis showed that dpp mrna level was not influenced by inflammation or neuropathy in the spinal dorsal horn. the same treatments, however, caused marked changes of dpp protein level in this region. inflamation caused a five-fold increase in dpp protein level suggesting a very potent posttranscriptional control of dpp expression during quickly developing inflammation and ischemia in neurons and glia. our understanding of dpp molecular regulation is far from being complete. studies on lymphocytes have demonstrated that while retinoic acid and interferon administration result in an increase of dpp transcription, il upregulates only dpp translation and tnfα merely decreases the cell surface expression of dpp , suggesting a very complex mechanism of the regulation of dpp activity. high resolution confocal laser scanning imaging revealed that neuronal dpp was typically confined to presynaptic, and also to somatic domains, with significantly decreased densities in neuropathy. in contrast, astrocytes were amply decorated with dpp -immunoreactive profiles, with significantly increasing density in inflammatory but not in neuropathic states. finally, a third-party involvement in spinal neuropathic mechanisms was likely figure . dpp immunoreactivity in various types of axon terminals. dpp immunolabelling on map stained dendritic surfaces was associated with synaptophysin (syn) indicating that not the dendrites but the axon terminals express the receptor (row a). dpp is expressed by the majority of peptidergic (cgrpcontaining) c primary afferent terminals (row b) and coexpression of dpp with syn and vglut (row c) or vgat (row d) suggests that dpp is present both in excitatory and inhibitory nerve endings. all the images are single optical sections. scalebar: μm. reflected by the increase of dpp expression in microglia which showed an obvious protein expression level in control animals, too. detectable levels of the dpp protein in healthy spinal cord, in contrast to brain tissue, could be due to the quick on-demand regulatory role of the moonlight protein in nociceptive processes where sp and npy offer ample and typical substrates for dpp . the presynaptic location of dpp in neurons suggests a possible role for this protein in synaptic physiology. on the other hand, glial expression of dpp which is significantly upregulated in a pathology and cell type specific manner opens further aspects to detect alternative roles of this protein in different pain states. previously, we have demonstrated that dpp inhibitors do not change the nociceptive threshold in acute nociceptive condition . in contrast to this, robust antihyperalgesic effects of different dpp inhibitors such as ipi, vildagliptin and sitagliptin in carrageenan-induced and complete freund's adjuvant (cfa)-provoked chronic inflammatory models have been reported previously , . in addition to the mechanical and heat hyperalgesia, mechanical and heat allodynia may also exist in the carrageenan-induced inflammatory model . the two modalities of allodynia were not measured in this study since the effects of the non-noxious heat or mechanical stimuli cannot be detected very precisely because of the extreme large paw swelling. in the other hand, touch sensitivity measured by dynamic plantar aesthesiometry (dpa) in cfa-induced chronic inflammatory model showed no and neuropathic (c) animals demonstrate that dpp is expressed predominantly by glial cells (arrows: microglia, arrowheads: astrocytes). all the images are single optical sections. scalebar: μm. integrated density values (d) demonstrate that the majority of dpp -immunopositivity is related to gfap-labelled astrocytes. a proportion of dpp labelling belongs to microgila and very few dpp is expressed by neuronal cell bodies. inflammation significantly increased the dpp expression on astocytes but not on the other cell types. in contrast to inflammation dpp density was significantly higher on microglia and lower in neurons during neuropathy (kruskal-wallis one way anova on ranks, dunn's method, p < , ). the antihyperalgesic action of ipi and vildagliptin appeared opioid-mediated in the carrageenan-induced inflammation since the nonselective opioid receptor antagonist naloxone/naltrexone reversed their effects , . in this study, we examined the opioid receptors involved by using selective antagonists against mor, dor, and kor and measuring mechanical hyperalgesia in carrageenan-induced acute inflammation. surprisingly, the antihyperalgesic effect of the ipi exclusively related to mor, while vildagliptin affected mainly dor but had also effect on mor and kor. it has been demonstrated previously that ipi does not activate mors directly . both ipi and vildagliptin are inhibitors of the dpp but ipi with a penultimate proline is also a substrate of the enzyme and its competitive inhibition is a kinetic artefact . although both inhibitors target the active site of the enzyme, the extent of inhibition depends on the residual interaction between drug and active site residues. x-ray crystallography analysing the co-crystal structure of different inhibitors with dpp demonstrated that the inhibitors, but not the substrates could bind well beyond the s subsite to increase their inhibitory activity . taking all these together supports that different residual interactions of the two inhibitors can affect the dpp activity in different ways. endogenous opioids, especially enkephalins, dynorphins and endorphins, are released from spinal and supraspinal sites during acute inflammation, but are degraded very quickly by extremely high enzymatic activity . a common feature of these opioids is that they can activate each opioid receptor with different potencies. inhibitory effects of opioid antagonists on ipi and vildagliptin related antihyperalgesia are summarized on bar graphs (g) and (h) constructed from data recorded min after i.t. drug application. comparisons were made with two-way anova, bonferoni post hoc test; + p < . ; +++ p < . (a-f) and one-way anova followed by dunnett's post hoc test. ***p < . (g and h). asterisks always indicate significant differences between the time-matching points of dpp inhibitor and dpp inhibitor + opioid antagonist curves. data on each curves and bars are given as mean and sem. since both ipi and vildagliptin are very selective dpp inhibitors, it is very unlikely that it has inhibitory effect on opioid degrading enzymes. on the other hand, it has been demonstrated that glial cells express opioid receptors and can synthesize endogenous opioids [ ] [ ] [ ] . these processes are at least partly regulated by inflammatory mediators including il- β . the interaction between the two systems is mutual, since the endogeneous opioids also have effect on the production of inflammatory mediators released by glial cells . glucagon-like peptide- (glp ) receptor activation results in β-endorphin release from microglia and blocks inflammatory nociception and mechanical allodynia in the spinal nerve ligation model , . in this study, we have found an increase in the expression of dpp both in microglia and astrocytes that can facilitate the degradation of many peptides including glp . systemic increase of glp peptide has been detected during inflammation and ipi/vildagliptin blocking dpp activity may increase further the glp level in the spinal cord and induce β-endorphin release from microglia. in this study, we have found a substantial increase in the expression of dpp in astrocytes during inflammation. it has been also demonstrated that astrocytes can synthesize both proenkephalins and dynorphins , that makes them good candidates for the source of these peptides. it has been shown that purinergic and toll-like receptor (tlr) activation results in dynorphin-a and -b releases from this glia type and proenkephalin release was also detected in cultured astrocytes . however, the conditions in which astrocytes release proenkephalin in vivo have not been determined. recent in vitro studies in monocytes demonstrated that dpp inhibitors suppressed tlr mediated upregulation of proinflammatory cytokines including il- β, il- , nlrp inflammasome which is a key molecule to figure . antinociceptive effects of the dpp inhibitor ipi and vildagliptin in chronic neuropathic condition induced by partial sciatic nerve ligation. dpp inhibitors were ineffective in dynamic plantar aesthesiometer (a and d) and noxious cold sensitivity (b and e) tests. in randall-selitto test, both ipi and vildagliptin had antihyperalgesic effect which was not antagonized by ntx (c and f). maximal possible effects (mpe%) of dpp inhibitors alone or in combination with subtype specific opioid antagonists is given on bar graphs (g-i). comparisons were made with two-way anova, bonferoni post hoc test; + p < . ; ++ p < . ; +++ p < . (a-f) and one-way anova followed by dunnett's post hoc test; *p < . **p < . (g-i). data on each curves and bars are given as mean and sem. scientific reports | ( ) : | doi: . /s - - - process and release of il- β and il- , and extracellular-regulated kinase (erk) activation that has also critical role in expression of inflammatory cytokines . these data suggest an important interaction between tlr and dpp activity which may exist also in glial cells and regulates the synthesis and release of inflammatory mediators and endogenous opioids. partial nerve injury is one of the main root causes of causalgiform pain disorders in human. partial ligation of the sciatic nerve is a model of this pain state resulting in mechanical hyperalgesia together with mechanical and cold allodynia , . although the altered pain sensation is a characteristic feature of both inflammatory and neuropathic pain states, very distinct anatomical and molecular mechanisms, as well as signalling pathways could be activated . therefore, dpp inhibitors were also tested in this model. both ipi and vildagliptin produced a significant decrease in mechanical hyperalgesia, but this effect was not opioid-dependent. comparing the extent of the antihyperalgesic effect of dpp inhibitors in inflammation and neuropathy may suggest that dpp inhibitors are more effective in inflammatory conditions then in neuropatic states. however, it must be emphasized that neuropathic pain and hyperalgesia are very difficult to treat and efficacy of dpp inhibitors is comparable to the adjuvant analgesic reference drugs used in the clinical practice in doses not causing sedative side-effect and motor incoordination . therefore, this moderate, but significant antihyperalgesic action should indeed be considered remarkable. in addition to the mechanical hyperalgesia, mechanical and cold allodynia also appeared in this neuropathy. none of the dpp inhibitors affected either types of allodynia. several models of different types of allodynia and hyperalgesia have been proposed with the common feature that the two phenomena based on different mechanisms , - . the major issue is whether the specially designed behaviour tests for mechanical allodynia or mechanical hyperalgesia reflects these phenomena or not. in this study, the threshold-decrease determined with the randall-selitto test that applies a basically painful pressure stimulus on the rat hind paw is expected to show mechanical hyperalgesia. the touch stimulus applied by the dpa that utilizes a blunt-end metal needle is considered to be non-painful under intact, normal conditions, therefore its threshold-decrease is expected to reflect mechanical allodynia. since the spinal application of the two inhibitors, resulted in significant changes in the randall-selitto test but not in dpa, our data indicate that the two tests measure two different processes. therefore, we can conclude that dpp inhibitors selectively affect hyperalgesia, but not allodynia. our results indicate the contribution of the dpp to the development and maintenance of mechanical hyperalgesia in this model of neuropathy, but the underlying mechanisms are unknown. the increased expression of the enzyme was most obvious in microglia suggesting the role of this cell type in this process. the contribution of microglia to the development of allodynia in neuropathic condition is intensively studied [ ] [ ] [ ] , but its role in the induction and maintanance of hyperalgesia and its relationship to the dpp requires further studies. in this study, we demonstrated that dpp is expressed in the spinal cord and, similarly to that in higher brain area, its expression significantly changes during pathological conditions. inhibitors of the enzyme do not affect acute nociceptive processing and allodynia but can selectively block glial mechanisms that contribute to the development and maintenance of hyperalgesia both in inflammatory and neuropathic conditions. this raises the possibility that dpp inhibitors targeting the central nervous system could be an important antihyperalgesic and anti-inflammatory component of new analgesics for the treatment of severe and persistent pain without serious side effects. animals. nociceptive threshold measurements were carried out on male wistar rats weighing - g and received from the breeding colony of the semmelweis university that were used for carrageenan induced hyperalgesia model, or on animals weighing - g at the start of the partial sciatic nerve ligation experiments (seltzer model painful pressure stimulus on the rat hind paw shows mechanical hyperalgesia. the touch stimulus applied by the dynamic plantar aesthesiometry (dpa) using a blunt-end metal needle is considered to be non-painful under intact, normal conditions, therefore, its threshold decrease reflects mechanical allodynia. the changes in withdrawal latency from ice-cold water reflects cold allodynia . carrageenan-induced subacute inflammation. inflammation was induced by intraplantar injection of μl l% λ-carrageenan dissolved in saline (sigma-aldrich, ) into the right hindpaw. nociceptive threshold to pressure was determined by the randall-selitto method , , using a type analgesimeter (ugo basile, comerio, italy). rats were lightly restrained and an evenly increasing force was applied onto their paws inserted between the cone-shaped clamps of the apparatus. at the moment of paw withdrawal, the actual force was recorded as the nociceptive threshold. the baseline nociceptive threshold was initially determined on both hindpaws (at − min), then carrageenan was injected into the right hindpaw (at min). the nociceptive threshold was measured again at min and i.t. injection of vehicle, dpp inhibitors alone (ipi or vil) or in combination with subtype specific opioid receptor antagonists (ctap, tipp[Ψ] or gnti), was performed. cardinal signs of the inflammation such as redness, swelling and heat of the paw had been obvious before the second measurement started. nociceptive threshold readings were repeated at , , and minutes. five rats were involved in each vehicle experiment, while - animals were used for drug and drug combinations. time-matching data sets on different ipsilateral curves were compared with two-way repeated measures anova followed by bonferroni post hoc test. percentage maximum possible antihyperalgesic effects were calculated according to the following equation: mpe (%) = × (ipsilateral threshold min after i.t. drug application − hyperalgesic baseline)/(contralateral threshold at the same time − hyperalgesic baseline), where hyperalgesic baseline was defined as the nociceptive threshold of the inflamed hindpaw min after intraplantar carrageenan injection, then comparisons were made with one-way anova followed by dunnett's post hoc test , . partial sciatic nerve ligation-induced chronic neuropathic pain model (traumatic mononeuropathy). partial ligation of sciatic nerve results in mechanical hyperalgesia together with mechanical and cold allodynia , . accordingly, baseline nociceptive thresholds were determined on two consecutive days using: dynamic plantar aestesiometry (dpa, mechanical allodynia), noxious cold stimulation (cold allodynia) and randall-selitto test (mechanical hyperalgesia). under deep pentobarbital anesthesia ( mg/kg i.p. euthasol, produlab pharma, raamsdonksveer, netherlands), the sciatic nerves of rats (n = ) were tightly ligated high in the thigh unilaterally using a braided silk suture (mersilk - , ethicone) so that approximately / - / of the diameter of the nerve was trapped in the ligature. the wound was closed afterwards with - silk sutures and the animals were allowed to recover for one week. on the th postoperative day, nociceptive threshold measurements were repeated for each animal at short intervals and percentage hyperalgesia/allodynia values were calculated for the nerve-injured paws with the following formula: hyperalgesia/allodynia (%) = × (preoperative − postoperative values)/(preoperative values). only animals that developed a minimum of % decrease of threshold with each method were included in treatment groups (n = ). rats were arranged into groups having similar degree of hyperalgesia/allodynia and received ( ) i.t. vehicle or ( ) dpp inhibitor or ( ) dpp inhibitor min after s.c. ntx pretreatment. for i.t. vehicle and dpp inhibitor experiments, - animals were used in each group, while groups undergoing i.t. dpp inhibitor application following s.c. ntx pretreatment consisted of - rats. nociceptive measurements from each animal were carried out - min after i.t. injection starting with dpa at min, followed by the randall-selitto test at min and finishing with noxious cold stimulation at min. during dynamic plantar aesthesiometry, rats were placed into an observation chamber positioned on a metal mesh surface. the touch stimulator unit was placed under the animal's paw and increasing upward force ( g/s) was exerted until the rat removed its paw. withdrawal thresholds were measured times in turns for each hindpaw and the mean values were used for statistical analysis. if no withdrawal occurred, the preset maximum ( g) was used in the evaluation. randall-selitto test was performed as detailed above. to measure noxious cold sensitivity, hindpaws of lightly restrained rats were immersed into a °c water bath and the latency to paw withdrawal was recorded. the cut-off time was set to seconds. withdrawal thresholds recorded before nerve ligation, then on the th postoperative day before and after drug applications were compared with two-way repeated measures anova followed by bonferroni post hoc test. percent maximum possible effects were calculated according to the following formula: mpe (%) = × (withdrawal threshold after drug application − withdrawal threshold before nerve ligation)/(withdrawal threshold before drug application − withdrawal threshold before nerve ligation), then comparisons were made with one-way anova followed by dunnett's post hoc test. carrageenan was injected into both hindpaws of rats and partial ligation of both sciatic nerves was performed in animals. survival time was hours and days, respectively. development of inflammatory or neuropathic hyperalgesia was confirmed by the randall-selitto test, and then rats were sacrificed by decapitation, with a further animals used as controls. l -l spinal segments were removed and frozen on dry ice. rna was isolated using the rneasy lipid tissue mini kit (qiagen) from spinal cord samples according to the manufacturer's instructions. the purity and concentration of the rna were analyzed using a smartspec plus spectrophotometer (bio-rad, uk). reverse transcription was performed with μg of rna to convert the total rna to cdna using the high-capacity cdna reverse transcription kit (applied biosystems by life technologies). concentration of the generated cdna was determined using the qubit . commercially available taqman probe (rn _n ) on -ng cdna template in duplicates. glyceraldehyde- -phosphate dehydrogenase (rn _s ) was used as a housekeeping gene, and its expression did not vary between the experimental groups. for statistical analyses, qpcr data were expressed as relative quantification values (rq; mean ± sem) and compared between groups by one-way anova. in situ hybridisation. the - bp long fragment of the rat dpp cdna (gene bank accession #nm_ ) was purchased from blue heron biotechnology inc. (bothell, wa, usa), subcloned into pbc ks + (addgene, cambridge, ma, usa) vector, and verified by sequencing. in situ hybridisation (ish) was performed as described earlier . riboprobes in sense and antisense directions were prepared by in vitro transcription (maxiscriptkit, life technologies, carlsbad, ca, usa) and labelled using [ s]utp-(per-form hungaria kft, budapest, hungary). carrageenan was injected into both hindpaws of rats and partial ligation of both sciatic nerves was performed in animals. survival time was hours and days, respectively. development of inflammatory or neuropathic hyperalgesia was confirmed by the randall-selitto test, and then rats were sacrificed by decapitation, with a further animals used as controls. the spinal dorsal horn of l -l segments were removed and frozen on dry ice. serial coronal sections were cut in a cryostat and mounted onto positively charged superfrost plus slides (life technologies). slides were hybridized overnight in humid chambers at °c with cpm/slide of the radioactively labelled probes, washed and dehydrated. slides were dipped into ntb nuclear track emulsion (carestream health deutschland gmbh, stuttgart, germany) for weeks. emulsion-coated slides were developed using kodak dektol developer and fixer (sigma-aldrich kft, budapest, hungary). sections were counter-stained with . % giemsa solution (sigma), air dried and coverslipped using depex mounting medium. dark-field images of three samples per animal were captured by a bx olympus microscope (olympus corporation, hamburg, germany) attached to a qicam (qimaging, surrey, bc, canada) camera. the grain density indicating the level of the dpp mrna expression was calculated as the area percent occupied by the silver grains within a given region of interest (roi; × pixel ) using the image j . j program. in each section, rois from the background (area outside of the tissue) and rois from the dorsal horn were measured and averaged. then the background was subtracted from the value that was obtained from the tissue. the sense and antisense signals were compared using student's t-tests with sigmastat . program (systat software, inc. san jose, ca, usa). one way anova was used for comparing the antisense in situ hybridization signals among the groups. western blotting. five rats with unilateral carrageenan-induced hindpaw inflammation, rats undergoing unilateral partial nerve ligation one week earlier with further naïve rats were tested with the randall-selitto method as it was described above. after confirming the obvious decrease of nociceptive thresholds, animals were sacrificed by decapitation, and the spinal dorsal horn of l -l segments were removed and snap-frozen on dry ice. samples were homogenized in tne buffer containing . % triton x- (sigma), mm naf, μm na vo and a cocktail of protease inhibitors (completetm, roche) and briefly sonicated. cell debris and nuclei were pelleted by centrifugation ( g, min at °c). protein concentrations were determined by bradford's colorimetric method . samples were diluted to a final protein concentration of μg/μl, denatured in x laemmli buffer, and analysed by sds-page on a % resolving gel. after transferring onto immobilon-fl polyvinylidene difluoride membranes (millipore), membrane-bound protein samples were blocked in % bsa and . % tween- diluted in tbs for . h, and subsequently exposed to the goat dpp primary antibody ( table .) overnight at °c. signal detection was achieved by using a hrp-conjugated donkey anti-goat secondary antibody (jackson; : , ). densitometric data were normalized to β-actin and for quantification three blots per treatment were used. image acquisition and analysis were performed on a bio-rad xrs + imaging platform. immunofluorescent labelling. antibodies. polyclonal goat dpp antibody was raised against the synthetic peptide c-pphfdkskkyp representing the internal region of dpp according to np_ . and labelled one band at approx. kda in rat lung lysate in western blot experiments (for details see the supplier's datasheet). in our western blot experiments rat lung and pancreas lysates were used as positive controls (fig. c) . monoclonal mouse dpp antibody was produced against the full length rat cd protein. to test the specificity of the two dpp antibodies double immunofluorescent staining was carried out . both antibodies labelled the same profiles in neurons, astrocytes and microglia (fig. ) . mouse anti-neuron-specific nuclear protein (neun) was used for labelling neuronal somata together with neurotrace / blue-fluorescent nissl stain (thermo fischer scientific -invitrogen, cat#: n- ; : ). dendrites were identified with monoclonal mouse antibody produced in mice against microtubule-associated protein (map ) , . antibody stained one single lane at kda in western blot experiment using rat brain extract. ionized calcium-binding adaptor molecule- (iba ) was used as specific microglia/macrophage marker and anti-iba antibody was isolated from the serum of rabbits immunized with a synthetic peptide corresponding to c-terminus of iba . according to the provider antibody stains one single lane in western blot experiments at around kda. monoclonal mouse antibody against glial fibrillary acidic protein (gfap) was used for specific labelling of astrocytes. guinea pig antibody against vesicular glutamate transporter (vglut ) was used to identify excitatory axon terminals. the antibody is raised against a amino acid long sequence of the rat protein and western blot analysis on rat brain lysate showed kda lane as it is seen on the provider's datasheet related to lot#: ng . this vglut antibody is heavily used and well characterized . inhibitory boutons were labelled with polyclonal antibodies against vesicular gaba transporter (vgat) raised in rabbits immunized by the synthetic peptide aeppvegdihyqr (amino acids - in rat vgat) coupled to key-hole limpet hemocyanin via an added n-terminal cysteine. based on the supplier's data sheet this antibody is knock-out verified. synaptophysin (syn) was used as a synaptic marker and monoclonal mouse antibody was raised against synthetic peptide corresponding to a region near to the c-terminal end of the full peptide. syn antibody validation by immunohistochemistry and western blot is found in the human protein atlas (http://www.proteinatlas. org). calcitonin gene-related peptide (cgrp) antibody raised in guinea pig and used for labelling peptidergic unmyelinated primary afferents recognizes identical structures to those detected by well characterized rabbit and goat antibodies against rat α-cgrp . for detailed specifications of antibodies see table . secondary antibodies were all raised in donkey: alexa fluor (af) - labelled anti-goat, af- conjugated anti-mouse, af- labelled anti-rabbit (all from thermo fischer scientific-invitrogen-molecular probes; : ) and rhodamine red x-anti guinea pig, cyanine -anti mouse, cyanine -anti rabbit (all from jackson immunoresearch; : ). general immunofluorescent staining protocol. transcardial perfusion of deeply anesthetized ( mg ketamine and . mg xylazine; i.m.) rats ( naive, unilateral carrageenan treated, and unilateral nerve ligated as described above) was initiated with % (para)formaldehyde and completed with % (para)formaldehyde containing % sucrose. l -l spinal segments were removed and immersed overnight into % sucrose dissolved in pbs. segments were frozen with liquid nitrogen and then µm thick sections were cut on a vibratome. endogenous peroxidase activity was blocked for minutes with % hydrogen peroxide diluted in phosphate buffer (pb), then sections were transferred into phosphate buffered saline (pbs) with % normal horse serum (nhs). after the blocking procedure they were incubated overnight in the dpp antibodies then reacted with alexa fluor labelled species specific secondary antibodies. sections were incubated for hours in mixtures of the other primary antibodies and were reacted overnight with fluorescently labelled species specific secondary antibodies. all the primary and secondary antibodies were dissolved in pbs. in some sets of experiments, sections were immersed into neurotrace fluorescent nissl dye (invitrogen-molecular probes; : in . m phosphate buffer) for half an hour before mounting. after rinsing, sections were mounted in vectashield (vector laboratories) and scanned on a confocal laser scanning system (zeiss, lsm ). densitometry of dpp immunostaining. l and l spinal cord segments taken from control, carrageenan-treated and nerve-ligated rats were used for quantitative analysis. three to six spinal cord sections were taken from each segment on the basis of the gray matter shape and to confocal optical sections were scanned from each section. dpp staining was not viewed prior to selecting any sections, rois or cell profiles. the quantitative analysis was carried out by an independent observer, who was blind to the experimental conditions and was not involved in the scanning either. to determine dpp immunoreactivity and its alteration under different circumstances fields containing the whole dorsal horn were scanned through a x lens of the confocal microscope to produce z-stacks with z separation of µm. the scanning parameters were selected and optimized in sections from control animals and were used further in sections of treated rats. single optical sections containing black and white images were selected from each z-series and analysed using the image j program (rasband ws, image j, nih, bethesda, maryland). the medial two third of the dorsal horn containing the first four laminae (the area receiving inputs from the sciatic nerve) was drawn in each section and used as region of interest (roi). the threshold was adjusted and the density of the immunostaining was calculated as the area percentage occupied by the immunostained dots within a given roi. data were compared among groups by one-way anova. to analyse the density of the dpp immunoreactivity in individual cell types, non-overlapping fields of µm × µm within the medial two third of the spinal dorsal horn were scanned through a x oil immersion lens to generate z-stacks with a z-separation of . µm. the same optimized parameters were used for all types of sections. iba -, gfap-and dpp -immunolabellings (for glial cells) or dpp immunolabelling with fluorescent nissl staining (neuronal cell bodies) were imaged in different colour channels sequentially. outlines of microglia and astrocytes were determined automatically by using the autothreshold plugin of imagej in iba and gfap image channels, respectively. to exclude non-specific labelling of glial cells and nuclei by the fluorescent nissl dye, contours of randomly selected neurons ( neuron/field) were drawn manually. then integrated density values from dpp image channel were measured in previously delineated glial or neuronal profiles to detect changes in protein expression due to different treatments. the density values were subjected to statistical analysis. statistical analysis. statistical methods used are detailed at each experiment individually. analyses were made with sigmastat . program (systat software, inc. san jose, ca, usa) and curves/bar graphs were created with the graphpad prism . software (graphpad software inc., la jolla, ca, usa). in general, data were represented as mean ± sem when the population was normally distributed or as median with % and %, otherwise. in both cases, p < . was considered as statistically significant. data availability. the datasets generated and analysed during the current study are available from the corresponding author on reasonable request. the multifunctional or moonlighting protein cd /dppiv -peptidase iv (cd )-role in the inactivation of regulatory peptides purification and characterization of a peptidyl dipeptidase resembling angiotensin converting enzyme from the electric organ of torpedo marmorata major metabolites of substance p degraded by spinal synaptic membranes antagonize the behavioral response to substance p in rats binding of adenosine deaminase to the lymphocyte surface via cd a collagen-binding glycoprotein on the surface of mouse fibroblasts is identified as dipeptidyl peptidase iv expression of the rat cd antigen (dipeptidyl peptidase iv) on subpopulations of rat lymphocytes soluble cd /dipeptidyl peptidase iv enhances human lymphocyte proliferation in vitro independent of dipeptidyl peptidase enzyme activity and adenosine deaminase binding molecular basis of binding between novel human coronavirus mers-cov and its receptor cd glycemia lowering and risk for heart failure: recent evidence from studies of dipeptidyl peptidase inhibition models and mechanisms of hyperalgesia and allodynia intrathecally injected ile-pro-ile, an inhibitor of membrane ectoenzyme dipeptidyl peptidase iv, is antihyperalgesic in rats by switching the enzyme from hydrolase to synthase functional mode to generate endomorphin 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analgesia in rats are diprotin a (ile-pro-ile) and diprotin b (val-pro-leu) inhibitors or substrates of dipeptidyl peptidase iv? a comparative study of the binding modes of recently launched dipeptidyl peptidase iv inhibitors in the active site acute inflammation induces segmental, bilateral, supraspinally mediated opioid release in the rat spinal cord, as measured by mu-opioid receptor internalization the non-peptide glp- receptor agonist wb - blocks inflammatory nociception by stimulating beta-endorphin release from spinal microglia the interleukin- beta-mediated regulation of proenkephalin and opioid receptor messenger rna in primary astrocyte-enriched cultures primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mrna opioid and nociceptin receptors regulate cytokine and cytokine receptor expression activation of spinal glucagon-like peptide- receptors specifically suppresses pain hypersensitivity glp- secretion is increased by inflammatory stimuli in an il- -dependent manner, leading to hyperinsulinemia and blood glucose lowering cultured astrocytes release proenkephalin spinal astrocytes produce and secrete dynorphin neuropeptides dpp- (cd ) inhibitor alogliptin inhibits atherosclerosis in diabetic apolipoprotein e-deficient mice dpp- inhibitors repress nlrp inflammasome and interleukin- beta via glp- receptor in macrophages through protein kinase c pathway dpp- (cd ) inhibitor alogliptin inhibits tlr -mediated erk activation and erk-dependent mmp- expression by u histiocytes a novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury stability of neuropathic pain symptoms in partial sciatic nerve ligation in rats is affected by suture material gabapentin enhances anti-nociceptive effects of morphine on heat, cold, and mechanical hyperalgesia in a rat model of neuropathic pain identification of spinal circuits involved in touch-evoked dynamic mechanical pain allodynia and hyperalgesia in neuropathic pain: clinical manifestations and mechanisms identification of spinal circuits transmitting and gating mechanical pain mechanical allodynia p x r+microglia drive neuropathic pain microglia control neuronal network excitability via bdnf signalling microglia in the spinal cord and neuropathic pain a method for measurement of analgesic activity on inflamed tissue chronic repeated restraint stress increases prolactin-releasing peptide/tyrosine-hydroxylase ratio with genderrelated differences in the rat brain a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding specificity of immunoreactions: the importance of testing specificity in each method a quantitative study of neurons which express neurokinin- or somatostatin sst a receptor in rat spinal dorsal horn nerve stretch injury induced pain pattern and changes in sensory ganglia in a clinically relevant model of limblengthening in rabbits clusters of secretagogin-expressing neurons in the aged human olfactory tract lack terminal differentiation differential localization of map- and tau in mammalian neurons insitu inhibitory interneurons that express gfp in the prp-gfp mouse spinal cord are morphologically heterogeneous, innervated by several classes of primary afferent and include lamina i projection neurons among their postsynaptic targets cocaine-and amphetamine-regulated transcript peptide (cart) is present in peptidergic c primary afferents and axons of excitatory interneurons with a possible role in nociception in the superficial laminae of the rat spinal cord. the european journal of neuroscience we thank professor a. j. todd for helpful discussion and advice, ms. katalin keserű góglné, dóra pintér Ömböliné, andrea németh, judit szalay for the professional technical assistance. we also say thanks to csaba dávid for his technical advices to image analysis. the work was supported by national research, development and innovation (nkfi) fund: pd-otka , nkfi k , the national brain research programme b: ktia_nap_ - - and ktia_ _nap_a_i/ , hungary, kornél király was supported by "jános bolyai fellowship" from the hungarian academy of sciences: bo / . supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ds rhbkv authors: lani, rafidah; hassandarvish, pouya; chiam, chun wei; moghaddam, ehsan; chu, justin jang hann; rausalu, kai; merits, andres; higgs, stephen; vanlandingham, dana; abu bakar, sazaly; zandi, keivan title: antiviral activity of silymarin against chikungunya virus date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ds rhbkv the mosquito-borne chikungunya virus (chikv) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. although there are several studies to evaluate the efficacy of drugs against chikv, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. here, we investigated the antiviral activity of three types of flavonoids against chikv in vitro replication. three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against chikv using a chikv replicon cell line and clinical isolate of chikv of central/east african genotype. a cytopathic effect inhibition assay was used to determine their activities on chikv viral replication and quantitative reverse transcription pcr was used to calculate virus yield. antiviral activity of effective compound was further investigated by evaluation of chikv protein expression using western blotting for chikv nsp , nsp , and e e proteins. briefly, silymarin exhibited significant antiviral activity against chikv, reducing both chikv replication efficiency and down-regulating production of viral proteins involved in replication. this study may have important consequence for broaden the chance of getting the effective antiviral for chikv infection. phylogenetic analysis shows that chikv has three genotype variants; west african, east/central/ south african (ecsa) and asian . malaysia had its first outbreak in late at the suburb klang in the state of selangor and the chikv was identified as asian genotype. localized outbreaks and clustered cases were reported in larut matang, lama, perak. the ecsa genotype virus caused outbreaks after . in early , together with the neighboring singapore, the epidemic in johor began. this spread throughout states in peninsular malaysia and in , many cases occurred in selangor, melaka, negeri sembilan and pahang states and peaked in kelantan, kedah, terengganu, perlis and sarawak , , . from april until march , over , cases were reported in these nationwide outbreaks, although there were no fatalities . the main mosquito vectors for chikv are a. aegypti and a. albopictus , , . a. aegypti was a primary vector for the outbreak that happened in kenya, comoros, africa and asia, but the ' asian tiger mosquitoe' a. albopictus was the main vector for the reunion and subsequent indian ocean outbreak . vector switching to a. albopictus became predominant due to the substitution of alanine residue to valine residue in the chikv e protein (a v) . this convergent mutation seems to be due to the selective pressure as the impact of different and new environment or ecosystem. extensive studies continue around the world to find an effective cure, safe antiviral treatment or vaccine for chikv infection. in one study it was shown that chloroquine phosphate, an antimalarial drug, can decrease the intensity or duration of pain, and number of joints involved. however, the effectiveness of chloroquine to treat chronic symptoms could not be evaluated due to the lack of adequate control patients with these symptoms . the synergistic effect of ribavirin with interferon alpha b against chikv infection was reported only in an in vitro study . it has been shown that arbidol strongly interferes with the early stages of chikv infection by targeting the cellular membrane. however, it is only a potent inhibitor against in vitro chikv infection . flavonoids are polyphenolic compounds that are present in different plants, foods and drinks. flavonoids are well known for their different biological properties including: antimicrobial activity, anti-inflammatory activity, anti-allergic activity, and cytotoxic antitumor activity . quercetin has antiviral activity against herpes simplex virus (hsv) type- , respiratory syncytial virus, pseudorabies virus, parainfluenza virus type and sindbis virus, a type member of genus alphavirus. the mechanism of actions of quercetin includes: enhancing the antiviral activity of interferon, binding to viral proteins, and interfering with viral nucleic acid synthesis by binding to the viral polymerases. another flavonoid, kaempferol is active against hsv, human coronavirus and rotavirus replication . silymarin, extracted from milk thistle (silybum marianum), was shown to inhibit hepatitis c virus (hcv) in both in vitro and in vivo by inhibiting hcv entry, rna synthesis, viral protein expression and infectious virus production; in addition it also acts by blocking of the virus cell-to-cell spread . it becomes our concern to explain the definition for the nomenclature of 'milk thistle extract' , 'silymarin' and 'silibinin' since the findings that will be discussed further involve these three important terms. in brief, the initial extract of the crushed milk thistle seeds made up of - % silymarin and - % fatty acids, such as linoleic acid. silymarin itself, is a complex of more than flavonolignans (silybin a, silybin b, isosilybin a, isosilybin b, silychristin, isosilychristin and silydianin) and flavonoid whereas silibinin is a semi-purified, commercially available fraction of silymarin, with an approximate : mixture of diastereoisomeric compounds, silybin a and silybin b which are also referred to in the literature as silibinin a and b . here we evaluated in vitro antiviral activity of quercetin, kaempferol and silymarin against a clinical isolate of chikv. our results support the antiviral properties of silymarin as a promising compound for further investigations towards the development of an anti-chikv drug. cytotoxicity of flavonoids. the mts assay was used to determine cytotoxicity of each flavonoid for vero and bhk cells. the cc value of each compound was calculated (table ). there was no observed antiviral activity assay. primary screening. different non-cytotoxic concentrations of silymarin, kaempferol and quercetin were tested on chikv-infected vero cells to find the effective compound. a cpe inhibition assay was used at hpi. it was shown that μ g/ml of silymarin significantly inhibited the chikv-cpe presentation (p = . ) (fig. a) . this finding was not unexpected as silybin (also known as silibinin) is one of the components of silymarin and has been shown to possess anti-chikv activity . in contrast, the highest concentrations of quercetin and kaempferol only exhibited % cpe inhibitory activity (fig. a) . the results of cpe inhibition assay were further confirmed by using mts assay (fig. b) . silymarin inhibits early post-entry stages of chikv replication. from analyzed compounds only silymarin was able to suppress chikv mediated cytotoxicity. therefore it was chosen for a time-of-addition assay performed with aim to determine which stage of virus infection cycle this compound affected. as it is shown in fig. the inhibition was most efficient when silymarin was added at hpi. the time-dependence of inhibitory effect of compound is coherent with hypothesis that its anti-chikv activity may be due inhibition of some early, but probably post-entry, step of chikv replication cycle. anti-entry assay has also been performed and the result has clearly shown that there is no antiviral activity of silymarin against chikv at the anti-entry phase (fig. ). cell line contained non-cytotoxic replicon of chikv which, in addition to the virus replicase proteins, expresses also puromycin acetyl transferase, egfp and rluc markers . in this system the rluc activity is proportional to viral replicon rna replication. it was found that μ g/ml of silymarin suppressed the activity of rluc marker expressed by the chikv replicon by . % (fig. ) . the inhibition was highly significant (p = . ) confirming that silymarin affects post-entry steps of chikv infection. this data also indicates that silymarin can interfere with chikv rna replication by affecting the viral replicase system. interestingly, in previous study ic of silybin, one of the major components of silymarin, was estimated as . μ m (approximately μ g/ml). compared to this silymarin was somewhat more efficient as nearly three-fold inhibition was observed at μ g/ml indicating that other components of silymarin likely contributed to its anti-chikv activity. to confirm the post-entry antiviral activity of silymarin against chikv a virus yield assay using qrt-pcr was used. the chikv rna copy number in supernatants of cells treated with each concentration of inhibitor and that from the control cells was interpreted and depicted (fig. ). there is a significant reduction of chikv replication as treated with increasing concentrations of silymarin (p = . ), silymarin exhibits a statistically significant (p < . ) dose-dependent inhibition on the rluc activity produced by bhk-chikv-nct replicon. the rluc activity was measured at h post treatment. vehicletreated ( . % dmso) cells were used as control (" ") concentration). data from triplicate assays were plotted and analyzed from a non-parametric correlation (spearman) two-tailed test (graph pad prism version , graph pad software inc., san diego, ca). as compared to the vehicle control treated cells. based on this data it was calculated that silymarin had ic = . μ g/ml with selectivity index (si) of . . taking into account that adding silymarin hpi showed significant inhibitory effect against chikv replication confirms that silymarin acts at the post-entry stage of chikv infection. in contrast, in similar assay neither kaempferol nor quercetin showed any significant inhibitory effect against chikv replication (fig. ) . as it is shown in fig. , μ g/ml of silymarin can inhibit the chikv yield by more than %. to evaluate whether the silymarin also affects the chikv protein synthesis, a series of western blot analyses were performed using lysates of the chikv infected and silymarin treated vero cells (fig. ). as it is evident from constant levels of β actin, silymarin treatment did not result in protein degradation and, at used concentrations, the compound was not toxic for the vero cells. in contrast, dose-dependent reduction of amounts of nsp , nsp and e proteins was observed (fig. ) indicating that silymarin limited chikv replication and virus-encoded protein synthesis within the treated cells. silybin interferes with early post-entry of chikv infection in dose-dependent manner. questioned by whether the silymarin effects on the chikv replication are influenced by the fractions it is made up of, since the study done by pohjala l. et al., has proved that the silybin (one of the silymarin fraction) can suppressed the activities of rluc marker gene expressed by the chikv replicon, we have evaluated the effect of silybin against chikv intracellular replication. hence, silybin was tested for its early post-entry activity and we confirmed its anti-chikv activity in a dose-dependent manner with the ic of . µg/ml, which is higher than the ic of silymarin. this result makes us to conclude that the activity of silymarin against chikv replication might also be enhanced by other components or fractions that made it up. considering the crippling pains that persist for weeks and even years due to chikv infections and the absence of antiviral treatment for the virus, the search for effective antiviral compounds is imperative. due to their known broad spectrum anti-viral activity and low toxicity, we evaluated three flavonoids as viable candidate compounds . from these compounds only silymarin was identified as the flavonoid with significant anti-chikv activity. it was considered that it has potential for future development as its use results in ≥ % reduction of chikv infection based on cpe inhibition and mts assays (fig. ) . remarkably, the effective concentration of silymarin was much lower than mntd. in contrast, neither kaempferol nor quercetin showed significant antiviral activity against in vitro replication of chikv in both assays. to quantify the copy number of viral rna released (most probably in form of genomic rna packed into virions) from infected cells were used qrt-pcr and amplified part of nsp encoding region of chikv genome. since nsp which is involved in the negative-strand and subgenomic rna synthesis, the quantification of the negative-strand rna reflects the active viral replication, we selected the nsp as the gene target for qrt-pcr to quantify the virus load during replication [ ] [ ] [ ] [ ] . our data demonstrated that treatment with silymarin results in effective reduction of number of released viral genome indicating reduction of viral rna synthesis and/or virion formation and release. in post-adsorption assay silymarin taken at μ g/ml resulted in ≈ % inhibition of chikv replication. we therefore performed a time-of-addition assay using this concentration of silymarin to determine the stage of infection where the silymarin treatment results in biggest antiviral effect. the result was consistent with our data from post-adsorption assay; hence it can be concluded that silymarin is most effective if it is added shortly after virus infection ( hpi). the reason for lower anti-chikv activity of pretreatment with silymarin could be due to the half-life of silymarin which based on previous bioavailability study is less than hours , although, there is no data available on in vitro half-life or stability of silymarin which might be interesting for future studies. as our findings were consistent with hypothesis that silymarin effect on post-entry stages of chikv dose response analysis was also performed using a chikv replicon cell line where no virus entry or exit takes place. again, it was found that silymarin is able to suppress the activity of rluc marker expressed by chikv replicon. this finding is consistent with that from previous assay and confirms that silymarin suppresses chikv rna replication and does it in dose-dependent manner. the rluc, expressed by chikv replicon, is fused to nsp protein of the virus. accordingly, reduction of rluc activity indicates reduced amounts of nsp -rluc protein in chikv replicon cell line. to further confirmation of anti-chikv activity of silymarin, we have shown the effect of silymarin on viral protein synthesis in chikv infected cells (fig. ) . again decrease of viral protein synthesis was observed. this effect could be due to inhibition of chikv rna replication and/or transcription. however, as in virus expression and replicon cell lines the synthesis of viral rnas and proteins are coupled further study is necessary to evaluate the direct effect of silymarin on inhibition of newly synthesized chikv proteins. it is possible that some of non-structural proteins of chikv represents direct target for silymarin. the possibilities include nsp protein, which is involved in the synthesis of the negative strand of viral rna and rna capping, and nsp protein, that is another component of the viral replicase complex. down regulation of e expression may represent consequence of suppression of replication (directly or via inhibition of ns-protein(s)). however, this down regulation is clearly relevant from point of view of development of effective antiviral as e protein is one of the important virion glycoproteins and is essential for receptor binding. interestingly, a previous study showed that silymarin can inhibit the expression of ns b of hepatitis c virus (hcv) which is catalytic subunit of hcv replicase . although, the hepatoprotective property of silymarin in the context of blocking hcv infection based on in vivo studies is another important criteria for this compound to be considered as a therapeutic candidate for hepatitis c , , . this study represents an important first report on the anti-chikv properties of silymarin. previously other flavonoids such as apigenin, chrysin, naringenin and silybin, which are also one of components of silymarin, are known to be inhibitors of viral replication mainly through affecting important cellular elements for chikv replication . however, inhibitory properties of silymarin are somewhat different from that of silybin. thus our data for silymarin warrants further mechanistic studies to characterize the antiviral properties of all its components and other flavonoid compounds and head-to-head comparison of their antiviral effects at lower concentrations/ different conditions so that the best option of anti-chikv can be determined. in summary, our study showed that silymarin exhibited significant in vitro antiviral activity against chikv. it suppressed post-entry stages of viral replication, most likely chikv rna replication significantly with a dose dependent manner. coherent with this the expression of proteins, needed for rna replication, and also expression of viral structural e protein were down-regulated. in contrast to silymarin it was found that quercetin and kaempferol are unable to suppress chikv replication and accordingly are not good candidates for anti-chikv drugs. these findings warrant future mechanistic, in vivo anti-viral, toxicity and pharmacokinetic studies as part of the process for evaluation of silymarin as a potential anti-chikv therapeutic. virus and cells. the chikv isolate used in this experiment was a clinical isolate from an outbreak in johor in coded as my/ / /fn . it belongs to the ecsa genotype and has the a v mutation in e protein , . bhk- (baby hamster kidney) and vero (african green monkey kidney) cells from the american type culture collection (atcc) were used in this study. vero cells have been widely used in the in vitro antiviral research for example in the antiviral testing for the chikungunya virus, human enterovirus and coxsackievirus a , . both cell lines were cultured using eagle's minimum essential medium (emem, gibco, ny, usa) containing % inactivated fetal bovine serum (fbs) and penicillin-streptomycin, and incubated at °c with % co . the chikv my/ / /fn strain was propagated in bhk- cells and harvested after full cytopathic effect (cpe) was observed. virus stock titer was determined by the tissue culture infectious dose (tcid ) methods ; then the obtained stock was aliquoted and stored at - °c. during the time of virus propagation and antiviral assay the fbs concentration of the cell culture medium was reduced to %. vero cell line was used for further experiments such as mts assay and antiviral assays. the chikv replicon cells were grown and maintained in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) supplemented with % fetal bovine serum (fbs), % tryptose-broth phosphate and penicillin-streptomycin, and incubated at °c with % co . flavonoids. silymarin, quercetin and kaempferol were purchased from sigma-aldrich corporation (sigma-aldrich, st. louis, mo, usa) and stored in − °c for future use. stock solution was diluted with emem and was filtered through a syringe filter with . μ m pore size (millipore, ma, usa) at the time of use; dmso concentration in working solutions prepared on emem was kept at . %. ( -( , -dimethylthiazol- -yl)- -( -carboxymethoxyphenyl)- -( -sulfophenyl)- h-tetrazolium) assay (promega, wi, usa) was performed to evaluate the cytotoxicity of tested compounds against vero cells and bhk cells according to the manufacturer's protocol. briefly, monolayers of vero cells were grown in -well plate and were treated with different concentrations of each compound in triplicate together with negative control (media containing . % dmso). the plate was then incubated at °c with % co for hours before the mts assay was performed. treated and control cells were kept for two days at °c, under similar conditions and duration until used for antiviral activity assay. after two days post-treatment, mts solution was added to the cells and incubated for hours at °c with % co prior to absorbance detection at nm wavelength using infinite pro multiplate reader (tecan, männedorf, switzerland). all experiments were conducted in triplicate. the half maximal cytotoxic concentration (cc ) for each compound was determined through this assay using graph pad prism (graph pad software inc., san diego, ca, usa, ). antiviral activity assays. primary assay for antiviral activity. a monolayer of vero cells were grown in -well plate in emem containing % inactivated fbs. the tested compounds were added to the wells in triplicate together with chikv at an moi = . the plate was then incubated at °c with % co for hours. the assay was conducted in duplicate for each concentration of each compound. after two days, the plate was viewed under the microscope and the degree of cytopathic effect (cpe) as measure of virus replication inhibition was expressed as the percent yield of virus control (% virus control = cpe experimental group/cpe virus control × ). the results were confirmed by performing the mts assay (promega, wi, usa) according to the manufacturer's protocols. all experiments were repeated three times independently. the statistical analysis on percentage of cpe inhibition was performed by using the gaussian populations (pearson) two-tailed assay. anti-entry assay. the procedure of the anti-entry assay was modified and performed according to lee rch et. al., . monolayers of vero cells were grown in -well plate with emem supplemented with % inactivated fbs. the vero cells were then infected with chikv and the plate was incubated for hour at °c. non-adsorbed virus was then washed with xpbs. tested compound was added in different concentrations and incubated at °c with % co for hours. the plate was again washed with xpbs and treated with citrate buffer (ph = ) to inactivate the non-internalized virus, before the plate was again washed with xpbs. the emem supplemented with % inactivated fbs was added into every wells and the plate was incubated for hours at °c with % co . chikv replicon cell line based assay. monolayers of chikv replicon cells were prepared in -well white plate (corning inc, ny, usa) and treated with different concentrations of tested compounds. after hours incubation at °c with % co the activity the renilla luciferase (rluc), expressed by chikv replicon, was detected using renilla luciferase assay (promega, wi, usa) performed according to the manufacturer's protocols. the luminescence signal was then measured using the glomax / luminometer (promega, wi, usa), plotted against the log transformation of the concentrations of compounds and a sigmoidal curve fit with variable slope was created to obtain the half maximal inhibitory concentration (ic ) value for each compound by graph pad prism (graph pad software inc., san diego, ca, usa, ). data were represent as the means ± standard error of the mean (sem) from triplicate assay from three independent experiments. the statistical analysis to determine the correlation between the concentration of silymarin and the rluc activity was performed by using non-parametric correlation (spearman) two-tailed assay. time-of-addition assay. in this experiment, a monolayer of vero cells were grown in -well plate in emem containing % inactivated fbs. the wells (in triplicate) were designated as − , − , , , , and -hour, that they are named according to the time of chikv (moi = ) infection. as for − hour, vero cells were treated with μ g/ml of silymarin. the plate was then incubated for one hour at °c with % co . one hour later, − hour wells were also treated with μ g/ml of silymarin. at the hour, all wells except the control wells were infected with chikv and again incubated for one hour at °c with % co . after -hour incubation and for every -hour after incubation at °c with % co , μ g/ml silymarin were added to − , , and hour wells respectively. the plate was then incubated at °c with % co for hours. the supernatant of the treated and control wells (treated with . % dmso was used as vehicle control) were collected two days post-treatment and analyzed using qrt-pcr. the half maximal effective concentration (ec ) of silymarin was calculated using graph pad prism (graph pad software inc., san diego, ca, usa, ). all data are from triplicate assay from three independent experiments. quantitative reverse transcription pcr(qrt-pcr). a qrt-pcr assay was used to quantify the chikv rna copy number. for this amplification of base region of nsp encoding sequence was performed as described by chiam and colleagues . the primers were nsp -f( '-gcgcgtaagtccaagggaat- ') and nsp -r ( '-agcatccaggtctgacggg- '). the cdna was first generated from the rna extracted from the supernatants (qiagen, germany) of the previous assays plate, using nsp -r primer and superscript iii reverse transcriptase (life technologies, usa) according to the manufacturer's protocol. the unincorporated primers were then digested with u of exonuclease i (new england biolabs, usa). the qrt-pcr was performed with a step-oneplus real-time pcr system (life technologies, usa) with × power sybrgreen pcr master mix (life technologies, usa), following the manufacturer's protocol, and using serially diluted standards. cycling parameters were °c for min, cycles of °c for s and °c for min. the amplified product was verified by melting curve analysis. the statistical analysis to determine the correlation between the rna copy number and the silymarin concentration was performed by using the gaussian populations (pearson) two-tailed assay. western blotting. vero cells at the density of × cells were seeded into a cm flask in emem containing % fbs and penicillin-streptomycin. the next day, each flask was infected with chikv inoculum at an moi = , placed on a rocker for minutes, and then incubated at °c with % co for h. then each flask was treated with different concentrations ( , , , . , . μ g/ml) of the trial compound, control flasks were treated with solutions containing . % dmso. all the flasks were then incubated at °c with % co until the appearance of cpe in the vehicle control. once cpe was observed, cells were scraped, washed with pbs and lysed using μ l of % triton x (sigma-aldrich, st. louis, mo, usa) containing complete protease inhibitor cocktail (sigma-aldrich, st. louis, mo, usa) at °c for min. cellular debris was pelleted out by centrifugation at , × g for min. a micro bca tm protein assay kit (thermo scientific, rockford, il) was used to quantify the protein concentration for each sample. lysate containing μ g of protein were denatured using sds-loading buffer and proteins were separated using sds-page in % gels. the gels were then equilibrated in towbin buffer ( . m tris, . m glycine % methanol) for min and proteins were transferred to a pvdf membrane using the bio-rad wet transfer system (bio rad, san francisco, ca). for detection of nsp and nsp , membranes were blocked with x pbs % casein blocker (bio rad, san francisco, ca) for h at room temperature on a shaker. the blots were rinsed three times with x pbs tween before being incubated with primary anti-chikv nsp , anti-chikv nsp or anti-chikv e rabbit polyclonal antibodies in % casein solution. the blots were then washed three times with x pbs tween for min each time. this was followed by incubation with the secondary goat anti-rabbit igg (abcam, cambridge, uk) antibodies conjugated with horseradish peroxidase (hrp) for h at room temperature on an orbital shaker. membranes were washed three times with pbs containing tween for minutes each time. for the loading control, separate blots containing the same samples were incubated with primary anti-β-actin mouse monoclonal antibody conjugated with hrp (cell signaling technology, ma, usa) dissolved in % casein for hour at room temperature on shaker. the blots were then washed three times with x pbs tween for minutes each time. membranes were developed by the colorimetric method using appropriate substrates (thermo scientific, rockford, il). a potentially emerging epidemic? biology and pathogenesis of chikungunya virus comparison of chikungunya virus infectivity in baby hamster kidney (bhk- ) cell line and c / , an aedes albopictus cell line 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activity of propolis multiple effects of silymarin on the hepatitis c virus life cycle silymarin for hcv infection inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays flavonoids as nutraceuticals: a review use of a highly sensitive strand-specific quantitative pcr to identify abortive replication in the mouse model of respiratory syncytial virus disease a structural and functional perspective of alphavirus replication and assembly quantitative analysis of dengue- virus rna during extrinsic incubation period in individual aedes aegypti regulation of sindbis virus rna replication: uncleaved p and nsp function in minus-strand rna synthesis, whereas cleaved products from p are required for efficient plus-strand rna synthesis plasma concentrations of free and conjugated silybin after oral intake of a silybin-phosphatidylcholine complex (silipide) in healthy volunteers multiple effects of silymarin on the hepatitis c virus lifecycle inhibition of t-cell inflammatory cytokines, hepatocyte nf-kappab signaling, and hcv infection by standardized silymarin chikungunya virus of asian and central/east african genotypes in malaysia outbreak of chikungunya in johor bahru, malaysia: clinical and laboratory features of hospitalized patients structure-activity relationship study of arbidol derivatives as inhibitors of chikungunya virus replication antiviral effects of phyllanthus urinaria containing corilagin against human enterovirus and coxsackievirus a in vitro a simple method of estimating fifty per cent end points mosquito cellular factors and functions in mediating the infectious entry of chikungunya virus real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus we would like to thank ministry of higher education ( key: cord- -x cwyvb authors: puenpa, jiratchaya; suwannakarn, kamol; chansaenroj, jira; nilyanimit, pornjarim; yorsaeng, ritthideach; auphimai, chompoonut; kitphati, rungrueng; mungaomklang, anek; kongklieng, amornmas; chirathaworn, chintana; wanlapakorn, nasamon; poovorawan, yong title: molecular epidemiology of the first wave of severe acute respiratory syndrome coronavirus infection in thailand in date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: x cwyvb the coronavirus disease pandemic caused by severe acute respiratory syndrome coronavirus (sars-cov- ) is a major global concern. several sars-cov- gene mutations have been reported. in the current study associations between sars-cov- gene variation and exposure history during the first wave of the outbreak in thailand between january and may were investigated. forty samples were collected at different time points during the outbreak, and parts of the sars-cov- genome sequence were used to assess genomic variation patterns. the phylogenetics of the samples were clustered into l, gh, gr, o and t types. t types were predominant in bangkok during the first local outbreak centered at a boxing stadium and entertainment venues in march . imported cases were infected with various types, including l, gh, gr and o. in southern thailand introductions of different genotypes were identified at different times. no clinical parameters were significantly associated with differences in genotype. the results indicated local transmission (type t, spike protein (a t)) and imported cases (types l, gh, gr and o) during the first wave in thailand. genetic and epidemiological data may contribute to national policy formulation, transmission tracking and the implementation of measures to control viral spread. in december patients presenting with viral pneumonia of unknown cause were reported in wuhan, china. in january a novel human coronavirus provisionally named ' novel coronavirus' was identified via next-generation sequencing , . the international committee on taxonomy of viruses subsequently changed the official name of the virus to 'severe acute respiratory syndrome coronavirus ′ (sars-cov- ) , and the disease it caused was dubbed 'coronavirus disease ′ . on january the world health organization declared the sars-cov- outbreak a so-called 'public health emergency of international concern' , and on march it declared the outbreak a pandemic . as of may more than million people had been infected with sars-cov- , and there had been more than , deaths . sars-cov- belongs to the family coronaviridae, the subfamily coronavirinae, and the order nidovirales. the genome of coronaviruses consists of positive-stranded rna of approximately to kb in length, including to open reading frames (orfs) and untranslated regions at the ′ and ′ ends of the rna . based on their genomic diversity coronaviruses are divided into four genera; alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. prior to the emergence of sars-cov- , in the last two decades six other coronaviruses have been detected in humans. two are alphacoronaviruses (human coronavirus nl and scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ human coronavirus e) that usually cause mild upper respiratory disease . the other four are betacoronaviruses, including the weakly pathogenic human coronavirus oc and human coronavirus hku , and the highly pathogenic severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). sars-cov- is the seventh human betacoronavirus, and its genome is closely related to sars-cov which emerged in and . as of may there were more than , sars-cov- genome sequences in the gisaid database (https ://www.gisai d.org/), contributed by numerous laboratories around the world. during the early period of the outbreak genome sequencing revealed two types of viruses based on differences in two single nucleotide polymorphisms in orf ab and orf ; l type and s type . another analysis categorized sars-cov- into three types, a, b, and c, based on amino acid changes . as of may there were seven clades identified in the gisaid database, s, l, v, g, gr, gh, and o. sars-cov- genetic variation may be associated with differences in viral replication , though more evidence is needed to verify any putative associations between mutations and pathogenesis in humans. in thailand the ministry of public health reported the first laboratory-confirmed case of sars-cov- in a -year-old chinese traveller who had arrived from wuhan on january . this was reportedly the first recorded case outside of china . by the end of january two confirmed cases had been reported, both thai nationals. of those, one was a -year-old woman from nakhon pathom province who had recently returned from china. the first unequivocally domestically contracted sars-cov- infection in thailand was documented on january when a taxi driver who had not travelled outside thailand tested positive. in january the percentage of confirmed cases in which the patients were travellers from other countries was . % ( / ) , but the corresponding percentage in february was . % ( / ). on february sars-cov- was designated a dangerous communicable disease under the communicable disease act. the act stipulates that all infected people must be hospitalized. on march covid- spread within a boxing stadium and drinking venues in bangkok, then it spread throughout thailand . notably however, most cases confirmed in april were people who had returned from a mass religious meeting in indonesia, thai workers from malaysia, and immigrants at the detention centre in songkla province , . similar to china, the incidence of covid- in thailand decreased dramatically when the thai government prohibited social gatherings after the first wave of the rapid spread of sars-cov- . on march the thai government announced that the thai new year's national holiday (songkran) between the th and th of april would be postponed indefinitely . on march the thai government began implementing a social distancing policy, including the mandatory closure of all schools and universities, entertainment and sporting venues, and all stores except food markets . subsequently the thai government announced that a nationwide p.m. to a.m. curfew would commence on april . as of june there had been , hospitalizations and deaths in thailand, equating to a fatality rate of < % . in the current study the genotypes of the sars-cov- strains involved in the outbreak in thailand during the first wave from february to april were investigated. based on the genome sequences available in giasid, nucleotide variation in four regions of the sars-cov- genome was used to conduct viral tracking and identify sites of origin of outbreaks in thailand. ethics statement. the research proposal was approved by the institutional review board of the ethics committee of the faculty of medicine, chulalongkorn university, thailand (irb number / ). the institutional review board of the ethics committee for human research waived the need for consent because all samples were anonymous. all methods were performed in accordance with the relevant guidelines and regulations. study population and sample collection. the study was conducted using anonymized sars-cov- -positive specimens collected from a diagnostic service except for the first specimens from the chinese traveller from wuhan (provided from the department of medical science, ministry of public health, thailand). patient identifiers including personal information and hospitalization number were removed from the samples to ensure patient confidentiality. the demographic data recorded included sex, age, vital signs and exposure history. the specimens were collected from bangkok, nonthaburi, samut prakan, songkla, ubon ratchathani and yala ( figure s ). the samples included a record of the date they were procured, and the putative location of infection (boxing stadium, specific drinking venues, a mass religious meeting from indonesia, thai workers from malaysia, and an immigrant detention centre, among others) (fig. ). a total of positive nasopharyngeal and/or throat swab samples (except the first specimens from the chinese traveller from wuhan) were confirmed to be sars-cov- -positive via two separate multiplex real-time reverse transcription polymerase chain reaction (rt-pcr) assays. in the first multiplex assay the allplex -ncov (seegene, seoul, republic of korea) incorporating primers and probes specifically targeting rdrp, n and e genes was used. the second assay used the lightmix modular sars and wuhan cov (tib-molbiol, berlin, germany) incorporating primers and probes corresponding to the rdrp and e genes. viral rna was extracted from µl of sample using a maglead gc instrument (precision system science, chiba, japan) with a maglead consumable kit (precision system science, chiba, japan) in accordance with the manufacturer's instructions. all rna specimens were transferred to the center of excellence in clinical virology at chulalongkorn university for conventional pcr and sequencing, and sars-cov- rdrp, s, n and e genes were amplified via a primer set specific for sars-cov- (table s ). www.nature.com/scientificreports/ the one-step rt-pcr reactions were conducted using the superscript iii platinum one-step rt-pcr system with platinum taq dna polymerase (invitrogen, carlsbad, ca). briefly, the pcr reaction mixture contained - μl of rna, . μm of each primer, . μl of x reaction mix (invitrogen) and μl of ssiii rt/platinum taq mix, and was adjusted to a final volume of μl with nuclease-free water. amplification was conducted in a thermal cycler (eppendorf, hamburg, germany) via a protocol including reverse transcription at °c for min, initial denaturation at °c for min, cycles of s of denaturation at °c, s of primer annealing at °c, s of extension at °c, and further extension for min at °c. pcr products were separated on a % agarose gel with a -base pair dna ladder and visualized on an ultraviolet trans-illuminator. pcr products were gel-purified using the hiyield gel/pcr dna fragment extraction kit (rbc bioscience co, taipei, taiwan). dna sequencing was performed by first base laboratories sdn bhd, selangor, malaysia. www.nature.com/scientificreports/ phylogenetic analysis. the seqman ii component of the dnastar software (v. . ) was used for nucleotide sequence assembly. genome sequences were aligned using clustalw, implemented via the bioedit program (v. . . ). the mega program (v. . ) was used for phylogenetic tree construction, which was performed via the neighbour-joining method with , bootstrap replicates. evolutionary distances were calculated using the maximum composite likelihood method. representative sequences from different areas of the world available in the genbank and gisaid databases were utilized in phylogenetic analysis. sequences were deposited in gen-bank (accession no. mt -mt ; table s ). statistical analysis. statistical analysis was conducted using the statistical package for social sciences v. . (spss inc., chicago, il). the chi-square test was used to analyse demographic patient factors, and p < . was deemed to indicate statistical significance. first wave of sars-cov- outbreak in thailand. the first wave of sars-cov- outbreak started in early march and peaked between the th and th of march . as the thai government implemented incremental public health measures to mitigate viral transmission, there was a marked overall decline in the sars-cov- cases since march (fig. ) . ) were selected to analyse genetic variations. phylogenetic analysis of concatenated sequences of worldwide sars-cov- isolates revealed five main clusters (fig. a) . based on genetic variations and amino acid changes the clusters were defined as l, s, g, v and o types. the types and patterns of nucleotide substitution are shown in fig. b . type l originated in china during the first period of the outbreak. wuhan-hu- (nc_ ) is the reference type l strain. type s was detected during the early period of the outbreak, and it has nucleotide substitutions at position , in orf ab (c t) and , (t c) in orf . types g and v evidently branched off from type l. type g has single nucleotide changes in orf b (c t) and s (a g). there is also a gr strain in the present study wu was identified as type l, which was closely related to wuhan-hu- . ten strains were identified as type g, including seven gh strains and three gr strains. two strains were identified as type o. most of the samples ( / ) were clustered within the distinct type s branch. this branch was defined as type t based on a nucleotide substitution in the s gene (g a) and an amino acid change in the spike protein (a t). therefore, type t was the most prevalent in the samples collected in thailand in the present study. variation patterns and exposure history. in the current study exposure history was divided into three categories; imported, boxing stadium, and nightclub. 'imported' was defined as any cases in which the patient had returned from an endemic area or had been in contact with travellers who had returned from an endemic area. it also included the aforementioned migrant worker and religious pilgrimage cohorts. 'boxing stadium' was defined as cases in which the patient contracted sars-cov- from the boxing stadium in bangkok, or had been in contact with anyone who was most likely infected at that boxing stadium. the boxing stadium group was the largest cluster in the march outbreak in bangkok. 'nightclub' was defined as cases in which the patient most likely contracted sars-cov- from a nightclub, entertainment venue, or restaurant in bangkok, or had been in contact with a person who was most likely infected at a nightclub. the wu strain was clustered with the l type detected in january during the first period of the outbreak in thailand. this isolate was closely related to wuhan-hu- . the likely source of exposure was an imported case from wuhan, china, the first endemic area. samples in the imported group also belonged to types gh, gr, and o. two samples isolated in march were classified as o types. the travel histories of the patients these samples were derived from indicated that one of them had recently returned from outside of thailand, and the other was a member of the cohort from the southern part of thailand who had recently undertaken a religious pilgrimage. the sars-cov- samples derived from the above-described group of migrant workers in may were identified as type gr. all samples in the boxing stadium group were identified as type t. these samples were obtained during the large outbreak in march in thailand. the sars-cov- samples derived from the nightclub included gh, gr and t types. most of the type t samples in the nightclub group were collected in march , and the gh and gr type samples in that group were collected in march and april . thus, there were multiple sars-cov- types circulating during this period. to assess associations between sars-cov- types and clinical signs, the results of genetic variation analysis were compared with clinical data. clinical data pertaining to samples was missing, and these samples were excluded from this analysis. by way of this, the one type l sample identified in the present study was excluded from this component of the analysis. clinical symptoms and sars-cov- types are shown in table . most samples were derived from patients with common symptoms of upper respiratory infection such as fever, coughing, and a sore throat. eight of the patients had pneumonia, and of the samples from these patients were sars-cov- type t. one patient with type t sars-cov- required admission to the intensive care unit (icu). one patient reported diarrhoea, and none of the patients died. there were no significant associations between sars-cov- type and clinical symptoms. table . clinical symptoms and the type of viral variation. there were four samples of which no clinical data were available. the missing data were excluded from this table. age (average age) viral genome sequencing data have been used to investigate viral transmission and factors associated with it, in a field known as 'genomic epidemiology' . several reports describe the use of genomic data to track viral transmission [ ] [ ] [ ] . many reports have described sars-cov- genome variation and the use of complete genome data to track its transmission , , . however, analysis of polymorphic viral genome segment may be helpful in rapidly identifying patterns of epidemiology and viral genetic cluster. in the current study four regions of the sars-cov- genome were sequenced to identify genetic variants. in january and february the confirmed sars-cov- cases were identified as having been imported from china. genetic variations of l and s types were identified during the early period of the outbreak in china . one sample in the current study collected in january was closely related to the sars-cov- strain circulating in china at that time identified as type l. the first outbreak in thailand was evidently associated with a boxing stadium and entertainment venues in bangkok during march . all the samples associated with that outbreak analysed in the present study were type t. type t branched off from type s, which originated from china, but type t has not been identified in other countries. this indicated local transmission in bangkok. interestingly, after the first outbreak in march type t was detected less frequently. this may have been a result of intervention policies such as mandatory closure of sporting and entertainment venues (fig. ) . the mandatory closure of public places may help to control local transmission. notably however, there were several cases of patients who had recently returned from outside of thailand testing positive for sars-cov- . they included multiple genetic variants such as types gh, gr and o. in march the patients classified as imported cases-including returned travellers and the group who had undertaken a religious pilgrimage from the southern part of thailand -were identified as having type o. after the land border closure and suspension of all international flights, the number of cases decreased (fig. ) . these interventions may help to limit imported cases. a new cohort of imported cases identified in may included a group of migrant workers in the southern part of thailand with type g sars-cov- . this indicated multiple introductions of sars-cov- , and that there may be an outbreak in the southern part of thailand. in the current study no specific clinical signs were significantly associated with any specific sars-cov- types. upper respiratory infection, fever, coughing, sore throat, and runny nose were the most common symptoms in covid- patients, as has been frequently previously reported [ ] [ ] [ ] [ ] . clinical outcomes may associate with host factors such as age, lymphocytopenia, and cytokine responsiveness rather than sars-cov- genetic factors . as of may only one of the forty patients involved in the present study had been admitted to the icu, and all had been discharged from hospital. in the patients analysed in the present study the clinical course of covid- was generally mild. the percentage of patients admitted to the icu was . %, the percentage with concurrent pneumonia was . %, the percentage who were asymptomatic was . %, and there were no fatalities, suggesting that sars-cov- does not usually lead to severe disease, unlike sars-cov and mers , . these clinical data are similar to reports derived from china in which approximately % of confirmed cases were considered mild, % of confirmed cases were diagnosed as severe with pneumonia, and approximately % were deemed critical cases . the reported case-fatality rate of covid- in thailand ( . %) is lower than that of sars ( %) , as it is in several countries including italy ( . %), iran ( . %), spain ( . %), the uk ( . %), the netherlands ( . %), france ( . %), china ( . %), and the usa ( . %) . reports suggest that elderly covid- patients are at higher risk of hospitalization, pulmonary complications, and death , , , as are elderly sars and mers patients , . the multiple origins of sars-cov- transmission into thailand identified in the current study via phylogenetic analysis are similar to the pattern identified in shanghai . our study had some limitations. we did not analyse the whole-genome sequence for all samples due to time and cost. therefore, we may have missed some genetic polymorphisms, which could potentially be of interest and reveal novel subclades to sars-cov- . due to the small sample size, a significant correlation between clinical symptoms and the viral genetic variation was not obtained. the samples we obtained encompassed the peak outbreak activity in thailand at that time, so although they are limited in numbers, we believe that they may be sufficiently representative despite the relatively low sample size compared to other studies. in summary, in the present study sars-cov- tracking and sites of origin were investigated in thailand via genetic analysis. most patients exhibited mild febrile illness without sequelae, but multiple origins of sars-cov- were evident. understanding viral genetic and transmission patterns may facilitate more accurate prediction of future trends, and assist the development of more informed intervention policies. received: june ; accepted: september a novel coronavirus from patients with pneumonia in china a 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sars-cov pneumonia coronavirus disease (covid- ) in the eu/eea and the uk -eight update viral and host factors related to the clinical outcome of covid- summary of probable sars cases with onset of illness from middle east respiratory syndrome coronavirus (mers-cov) infection: epidemiology, pathogenesis and clinical characteristics characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention cross-country comparison of case fatality rates of covid- /sars-cov- . osong public health res coronavirus disease in elderly patients: characteristics and prognostic factors based on -week follow-up the epidemiology of severe acute respiratory syndrome in the hong kong epidemic: an analysis of all patients occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update j.p. collected data and drafted the manuscript. k.s. analyzed data and drafted the manuscript. j.c. performed sequence analysis and edited the manuscript. p.n., r.y. and c.a. performed genome sequencing and analyzed data. r.k., a.m. and a.k. collected and provided specimens. n.w., c.c. and y.p. supervised the study and finalized the manuscript. all authors reviewed the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to y.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -a wue e authors: cohen, isaac v.; makunts, tigran; moumedjian, talar; issa, masara a.; abagyan, ruben title: cardiac adverse events associated with chloroquine and hydroxychloroquine exposure in years of drug safety surveillance reports date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: a wue e chloroquine (cq) and hydroxychloroquine (hcq) are on the world health organization’s list of essential medications for treating non-resistant malaria, rheumatoid arthritis (ra) and systemic lupus erythematosus (sle). in addition, both drugs are currently used off-label in hospitals worldwide and in numerous clinical trials for the treatment of sars-cov- infection. however, cq and hcq use has been associated with cardiac side effects, which is of concern due to the higher risk of covid- complications in patients with heart related disorders, and increased mortality associated with covid- cardiac complications. in this study we analyzed over thirteen million adverse event reports form the united states food and drug administration adverse event reporting system to confirm and quantify the association of cardiac side effects of cq and hcq. additionally, we identified several confounding factors, including male sex, nsaid coadministration, advanced age, and prior diagnoses contributing to drug related cardiotoxicity. these findings may help guide therapeutic decision making and ethical trial design for covid- treatment. , and nct . despite limited efficacy data, hcq and cq are currently being administered as part of first-line treatment for sars-cov- in various hospitals across the world , . as previously mentioned, cardiac complications attributed to hcq and cq have been described in various reports. a systematic review of the literature published in june examined reports including individual cases and short series from an online database search. the report identified patients with cardiac events attributed to hcq and cq administered in the context of various inflammatory disorders with a median daily dose of mg for cq and mg for hcq . recent disproportionality analysis study observed an elevated prevalence of torsade de pointes and qt prolongation reports in cq and hcq . the cardiac aes of these therapeutics are of increased concern since a subset of patients infected with covid- present with cardiac injury, suggesting a relevant cardiovascular involvement in the pathophysiology of the disease . in a single-centered cohort study from wuhan, china, shi et al. examined the incidence of cardiac injury in hospitalized patients with covid- . among those, of the patients demonstrated cardiac injury, and higher in-hospital mortality rates were seen in patients with cardiac issues ( . %) compared to those without ( . %) . guo et al. described similar findings in a retrospective single-center case series analysis in which out of hospitalized patients suffered from myocardial injury. the mortality rate in those patients was . % compared to . % in those without cardiac injury . in support of these findings, recent studies have shown that major cardiac outcomes associated with cardiomyopathy ( %) and cardiac injury ( %) are common in critically-ill patients , . similar lines of evidence are also followed by an italian case report of a -year-old woman with lab-confirmed covid- who was admitted to the hospital for severe lv dysfunction and acute myopericarditis. this case highlights that sars-cov- can impact the cardiovascular system even in the absence of major respiratory tract involvement . other studies have observed covid- cardiac complications such as fulminant myocarditis, ventricular tachycardia [ ] [ ] [ ] . the goal of this study is to reanalyze the extensive clinical data of cq and hcq cardiac aes collected during the last years to derive the strength of the associations and, more importantly, contributing risk factors. the findings may improve the safety of these therapeutics for covid- treatment in patients that are already at higher risk of cardiac complications. fda adverse event reporting system. the study used over thirteen million ae reports available from the united states food and drug administration adverse event reporting system (faers) and its older version, adverse event reporting system (aers) data sets. at the time of the study the faers/aers set contained reports from years - , all available online at: https ://www.fda.gov/drugs /quest ions-and-answe rs-fdas-adver se-event -repor ting-syste m-faers /fda-adver seevent -repor ting-syste m-faers -lates t-quart erly-data-files data preparation. faers/aers reports are collected through voluntary reporting to the fda through medwatch and stored in quarterly data subsets with their respective parameters (age, sex, drug, ae etc.), and common case identifiers. faers data format has had changes historically, requiring each quarterly set to be individually downloaded and modified into consistent data tables [ ] [ ] [ ] . since the faers/aers set has reports from all over the world with their respective drug brand names, unique terms were recognized and translated into single generic cq and hcq names. the final data set contained , , ae reports. sle, ra, and malaria were considered for possible sources of reports, however due to low sample size of malaria treatment with hcq or cq in faers, only sle and ra were included in the analysis. cohort selection and data cleaning. , reports were obtained from the fda faers database to form three cohorts for analysis by logistic regression: cq cohort, hcq cohort and control cohort. the control cohort for both drugs was defined by reports with ra and sle patients where hcq and cq were not used (n = , ). the cq and hcq cohorts were defined by reports with ra and sle indication with cq (n = ) and hcq (n = , ) was used in addition to other therapeutics. rstudio (version . . ) and r (version . . ) were employed for data cleaning and logistic regression modeling. faers/aers data sets historically include a small fraction of duplicate reports. the set was scanned for these entries with the r package "dplyr" "distinct" function and were removed as appropriate (when records were found to have matching age, sex, weight, submission date, country of origin, drugs, indications, and outcomes). a summary of the records demographic factors is made available in table . in order to define the list of possible cardiac aes in this database a table was generated and manually checked for errors by the investigators. for a copy of this table of all aes considered cardiac related see supplementary table s . similarly, a list of non-steroidal anti-inflammatory drugs (nsaids) was generated for use in our analysis and is made available in supplementary table s . note that the number of nsaids did not include aspirin, as this was modeled separately due to expected divergent effects such as higher cardiovascular risk in the patient demographics. during the data cleaning stage, age was limited to a range of to years. for the purpose of our analysis, only sex values of "f" or "m" from fears/aers were analyzed. the r package "dplyr" function "mutate" and "str_detect" were employed for counting the number of nsaids and cardiac aes observed in each report. a subsample of the original database that included reports with non-empty values for age and sex was also prepared. for a summary of the sample size of the subgroups see supplementary table s . measured outcomes. the primary outcome of interest was the report of any cardiac ae as defined by one of terms listed in supplementary table s . this outcome was chosen over other more granular outcomes, such as qt prolongation, in order to increase the sensitivity of the analysis. the r package "glm" was employed scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ for logistic regression modeling via the "binomial" family function. cardiac aes were the outcome of interest in logistic regression modeling and were coded as a binary (" " if occurred in the report or " " if not). covariates explored in the modeling included age (as a continuous variable expressed in years), sex (as a binary), disease state (coded as a binary of either sle or ra), nsaid usage (as a integer variable equal to the number of nsaids observed in the record), and aspirin usage (as a binary). nsaids were selected as a covariate of interest due to their high utilization among sle and ra patients. aic (akaike information criterion) and n number of records included are reported for each of the eight models presented. note that due to absence of some values in the raw data from the fda, models including age and sex have lower sample size than those without these covariates (supplementary table s ). subject weight was not used for model building due to extreme paucity of the data. models a, b, a, and b are built using the control and cq cohorts (described above and presented in tables and , and supplementary table s ). similarly, models a, b, a, and b are built using the control and hcq cohorts (described above and presented in tables and , and supplementary table s ).f coefficient estimates, standard error, adjusted odds ratio (aor), % confidence intervals ( % ci), and p values are reported (tables and ) . p values that meet the significance threshold of less than . are marked in tables with an asterisk (*). the adjusted odds ratio is defined as an odds ratio that controls multiple predictor variables table s ). these numbers of records are large, in particular for hcq, they cover a range of demographic parameters, and are sufficient to evaluate the contributors to the cardiac aes with logistic regression. the last row of table indicates an elevated number of cardiac side effectcontaining reports for both cq and hcq, . % and . % respectively, compared to . %. interestingly, the most common individual adverse effects (not grouped by the category) for each cohort were calculated and they did not include cardiac aes (see supplementary fig. s ). chloroquine. several logistic regression analyses were performed in order to evaluate whether cardiac aes were related to cq. binary logistic regression was employed to determine the confounding variables contributing to the apparent effects of the drugs on occurrence of cardiac side effects. in the simplest analysis model a ( hydroxychloroquine. hcq cardio ae analysis reveals a similar pattern to the results presented in table . table explores the same effects due to hcq in ra and sle patients. sle, nsaid use, aspirin use, male sex, and advanced age were all shown to be important factors in predicting cardiac aes (table ). in the simplest model a, hcq was shown to significantly increase cardiac aes by a modest adjusted odds ratio of . ( % ci . - . ) . interestingly, in model b this effect was lost after controlling for nsaid and aspirin use (p = . ). surprisingly, after adding age and sex into the model this effect's significance was regained (model b, aor . [ % ci . - . ]). although modest, hcq has been shown to have significant effects on reporting of cardiac adverse reactions. these trends have also been shown to be exacerbated by both clinical and demographic factors (table ). in our study we analyzed , fda adverse event reports divided into cq, hcq and control cohorts to determine their association with cardiac aes when taking into account other factors. although both drugs were significantly associated with increased cardiac ae reports, it was observed that this effect was substantially larger for cq than hcq (fig. ) . additionally, age, sex, concurrent nsaid use, and disease state were identified to contribute to cardiotoxicity reporting with both therapeutics. after controlling for these factors, it was observed that the deleterious effects of cq and hcq on cardiac aes remained significant (tables and ) . we expanded the current safety surveillance evidence by using a more comprehensive list of cardiac aes (supplementary table s ), to avoid diluting the safety signal with many individual terms. additionally, we performed multivariate analysis and identified several at-risk populations. the results of the multivariate binary logistic regression were validated by prior knowledge from literature: ( ) sex, when compared females, males are more often diagnosed with myocardial infarction, fatal coronary heart disease, among other cardiac diseases ; ( ) age, cardiovascular disease (cvd) has been shown to increase with age across multiple populations ; ( ) www.nature.com/scientificreports/ nsaids, especially preferential and selective cyclooxygenase- inhibitors, increase the risk of cvd , . two other essential factors were shown to be necessary to correct for, due to their association with cardiac side effects in the patient population used for our analysis and the current utilization of cq and hcq: ( ) sle: sle patients have been shown to have a greater risk of cardiac aes than patients with ra. this makes sense, as it is well known that cvd is one of the major complications and is one of the leading factors in mortality in patients living with sle . additionally, the host inflammatory response seen in sle may be similar to that in covid , . ( ) aspirin: it may be expected for aspirin to present a protective effect, however the opposite effect was observed. use of aspirin in itself does not increase cvd risk, in fact it is mainly used for preventing cardiac events . aspirin use in our study population is likely heavily associated prior cardiac related medical history, that is not listed in fears, and could not be otherwise accounted for in the model. aspirin increases the robustness of the model by correcting for prior treatment of cardiac disease or prevention in high risk patients. the regression model was instrumental to exclude this aspirin association from the quantification of the direct cardiac side effects of cq and hcq, which remained significant after adjustment ( generalizability of results to covid- treatment with cq and hcq. this analysis revealed a trend of increased cardiac ae reporting propensity associated with cq and hcq that likely apply to a wide range of patients. although our study was not performed on reports from covid- patients, due to absence of such reports at the time of the study, the results may still be of value because of our mode of evaluation. additionally, the cardiac complications and related mortality of sars-cov- patients are mainly attributed to the inflammatory nature of the infection , , which is also seen in sle pathophysiology , . in fact, several case reports describe the heart related mortality being associated with inflammation of the myocardium , . cq and hcq use in sle and ra cohorts for the study was a beneficial coincidence, since they are also inflammatory conditions affecting the cardiovascular system that are also treated with cq and hcq , . further clinical studies are needed to subdivide the cardiac ae's in to clinically relevant subcategories, such as arrythmia or myocardial infarction. the attempts to split the cardiac outcomes into subcategories with the current dataset met the obstacle of insufficient numbers of reports for significant conclusions. clinical studies with prospective methods may benefit from targeted monitoring of the important induvial clinical outcomes. and hcq with respect to other therapeutics used for ra and sle. the association remained significant when demographic parameters and concurrent medications were accounted for in the analysis. it may be beneficial to closely monitor patients for cardiac complications. hcq may be safer for use than cq in patients at higher risk of cardiac complications. although, when compared to cq, hcq use was associated with a lower reporting of these events, the association was still statistically significant. when available, alternative therapeutics may be safer to use for sars-cov- patients who are already at higher risk of cardiovascular complications due to age, pre-existing cardiovascular issues, concomitant medications, and the sars-cov- infection itself. due to the voluntary nature of the faers/aers reports, actual population incidences of the adverse events cannot be derived. medwatch reporting may also be biased by newsworthiness and legal variables. the safety surveillance data misses comprehensive medical records and medication history limiting the scope of the analysis. as with any association study, causality may not be derived from association, since the cases were not uniformly evaluated for causality by clinical specialists. however, the postmarketing surveillance data analysis of over , reports provides population scale evidence which can be used to identify safety signals that might go unnoticed in small scale studies. while our approach with multivariate logistic regression controls for important biases in the data, such as sex, age, and concomitant drug use, some unaccounted-for factors may remain. the outcome modeled in this study (presence of any cardiac ae) is broad; further study is necessary to investigate more granular outcomes such as arrythmias. new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? chloroquine for the novel coronavirus sars-cov- targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hhs accepts donations of medicine to strategic national stockpile as possible treatments for covid- patients remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro nct ) hydroxychloroquine treatment for severe covid- pulmonary infection (hydra trial) (hydra) nct ) hydroxychloroquine chemoprophylaxis in healthcare personnel in contact with covid- clinicaltrials.gov. 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manifestations of coronavirus (covid- ) fulminant myocarditis due to covid- we thank members of the abagyan lab for support during this project. we also thank da shi for the contribution to processing the faers/aers data sets. i.v.c. and t.ma. performed the experiments. t.ma. i.v.c., and r.a. designed the study, i.v.c., t.m., m.a.i., t.ma and r.a. drafted the manuscript and reviewed the final version. r.a. processed the data. i.v.c. and t.ma. contributed equally to this work. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to r.a.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -npefpo t authors: yinda, claude kwe; zeller, mark; conceição-neto, nádia; maes, piet; deboutte, ward; beller, leen; heylen, elisabeth; ghogomu, stephen mbigha; van ranst, marc; matthijnssens, jelle title: novel highly divergent reassortant bat rotaviruses in cameroon, without evidence of zoonosis date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: npefpo t bats are an important reservoir for zoonotic viruses. to date, only three rva strains have been reported in bats in kenya and china. in the current study we investigated the genetic diversity of rvas in fecal samples from straw-colored fruit bats living in close contact with humans in cameroon using viral metagenomics. five (near) complete rva genomes were obtained. a single rva strain showed a partial relationship with the kenyan bat rva strain, whereas the other strains were completely novel. only the vp and vp genes showed significant variability, indicating the occurrence of frequent reassortment events. comparing these bat rva strains with currently used human rva screening primers indicated that most of the novel vp and vp segments would not be detected in routine epidemiological screening studies. therefore, novel consensus screening primers were developed and used to screen samples from infants with gastroenteritis living in close proximity with the studied bat population. although rva infections were identified in % of the infants, there was no evidence of zoonosis. this study identified multiple novel bat rva strains, but further epidemiological studies in humans will have to assess if these viruses have the potential to cause gastroenteritis in humans. rva/bat-wt/ken/ke / /g p [ ] , and possesses the following genotype constellation: g -p[ ]-i -rx-c -mx-ax-n -t -e -h . two other bat rvas were found in china in a lesser horseshoe bat (rhinolophus hipposideros) and a stoliczka's trident bat (aselliscus stoliczkanus) named rva/bat-tc/chn/mslh / / g p [ ] and rva/bat-tc/chn/myas / /g p [ ] , respectively , . phylogenetic analysis showed that strains mslh and myas , although sampling sites were more than km apart, shared the same genotype constellation (g -p[x]-i -r -c -m -a -n -t -e -h ) except for the p genotype which was p [ ] for mslh and p [ ] for myas . to further study the genomics of rva in bats and their zoonotic potential in humans, we screened stool samples of straw-colored fruit bats (eidolon helvum) living in close proximity with humans in the south west region of cameroon (fig. ) , as well as samples from infants with gastroenteritis. our choice of this region is due to the fact that bats are considered a delicacy and the species sampled are the most commonly eaten bat species in these localities. sample characterization. a total of pools of - bat fecal samples were constituted, enriched for viral particles and sequenced. illumina sequencing yielded between . and . million reads per pool, and diamond classification of the obtained contigs indicated that five pools contained a significant amount of rva sequence reads. the percentage reads mapping to rva in each pool ranged from . - . % (table ) . partial segments were completed by regular pcr and sanger sequencing, to obtain at least the entire orf for each of the obtained variants. obtained sequences were used for phylogenetic comparison with a selection of representative members of each genotype. the rva strains discovered in this study were named rva/bat-wt/cmr/batli / / g p [ ] , rva/bat-wt/cmr/batli / /g p [ ] , rva/bat-wt/cmr/batli / /g p [ ] , rva/bat-wt/ cmr/batly / /g p [ ] and rva/bat-wt/cmr/batly / /g p[ ] hereafter referred to as batli , batli , batli , batly and batly , respectively. all the obtained sequences were highly divergent from established genotypes and were therefore submitted to the rotavirus classification working group (rcwg) for novel genotype assignments (see below) and to genbank (accession numbers: kx -kx ). phylogenetic analysis. the vp gene of batly was % identical (on the nucleotide (nt) level, supplementary data s ) to the kenyan bat rva strain ke counterpart, which had been previously classified as a g genotype (fig. ). batli and batli were % identical and also clustered closely with strain batly ( % similar). this cluster was only distantly related to all other known vp rva sequences as well as to strain batli , which also formed a unique long branch in the phylogenetic tree. both clusters only show similarities below % with established genotypes (fig. ) . the vp of these strains (batli , batli , batly and batli ) did not belong to any of the established rva g-genotypes, according to the established criteria , and were assigned genotypes g (batli , batli and batly ) and g (batli ) by the rcwg. for vp , vp and vp , all five cameroonian bat rvas strains were distantly related to other known rva strains, including the kenyan and chinese rva strains and were therefore assigned to novel genotypes according to the rcwg classification criteria (fig. ) . the vp gene of strains batli , batli and batli (representatives of the novel genotype p[ ]) were almost - % identical to each other and only - % identical to any other p-genotype. that of strains batly and batly had % nt dissimilarity to each other and their nt identity ranged from - % with other p-genotypes and therefore were assigned the genotypes p[ ] and p[ ], respectively. the vp and vp genes of batli , batli , batli and batly were nearly identical (nt identity range - %) and clustered together but distinct from other established r and m-genotypes, thereby representing the new genotypes r and m , respectively (fig. ) . the vp and vp genes of batly were only distantly related to the other four cameroonian bat rvas ( - % nt identity) and are the sole member of the newly assigned genotypes r and m , respectively (fig. ) . the vp , vp , nsp , nsp and nsp gene segments of of our strains (batli , batli , batli and batly ) were distantly related to their counterparts of other mammalian and avian rvas fig. ) . for all the strains, these gene segments clustered together and were - % identical to each other and consequently they constitute new genotypes for the different gene segments (i , c n , t and h , respectively). the vp , vp , nsp , nsp and nsp gene segments of batly phylogenetically clustered together with the kenyan bat rva strain ke in the previously established i , c , n , t and h genotypes, respectively (fig. ) . for nsp , the cameroonian bat strains batli , batli , batli and batly clustered closely together ( - % nucleotide sequence identity) in the novel genotype a , and showed only % nucleotide similarity to strain batly (a ). these new nsp gene segments were only - % identical to that of the most closely related established nsp genotype a and a (from cow) (fig. ) . the nsp gene segments of all the rvas discovered in this study were quite divergent to those of other known bat rotaviruses (at most % nucleotide sequence identity) and other rvas (approximately - % nucleotide similarity) forming two distinct clusters. the nsp gene segments of strains batli , batli , batli and batly (genotype e ) were % identical but all were - % divergent from that of batly (e , fig. ). batli (p ) batlyp (p ) batli (p ) batli (p ) batlyp (p ) bat rotaviruses in humans? several different primer pairs are currently being used to detect human rva vp and vp gene segments, to determine the g-and p-genotypes using sequencing or multiplex pcr assays [ ] [ ] [ ] [ ] . in order to find out if the currently used human rva screening primers would detect the bat rva strain from this study in case of zoonosis, we compared these primers with their corresponding sequences in the respective gene segments (table and supplementary data s ). overall, the similarity percentages for the vp forward and reverse primer between the bat rva sequences and the human primers were . - % and . - %, respectively. for vp , the percentage similarity ranged from . - . % and . - . % for the forward and reverse primers, respectively. vp forward primers beg , sbeg and con -l showed a (near) perfect match with batly -g , whereas strain batli -g and batly -g (first nt are missing for this strain), showed up to and nucleotide mismatches at the ′ end of the primers, respectively. vp forward primer con -l showed a perfect match with all the genotypes (g , g and g ). considering the vp reverse primers enda, vp -rdeg, end and rvg , batly -g did not show a perfect match as there were , , and mutations, respectively. the mismatches with enda, vp -rdeg and rvg were near the middle or at the ′ end of the primer, whereas of those of end were close to the ′ end. comparing the same vp reverse primers with strain batli -g and batli -g also showed mismatches. for enda and vp -rdeg maximum mismatches are located in the middle or near the ′ -end, whereas for end and rvg, there were multiple mismatches of which and mismatches, respectively were right at the ′ -end. for vp forward primer vp - additionally, to determine if any of these bat rvas could cross species and infect humans, we designed primers (rva-vp _ f and rva-vp _ r) from an alignment of both human and bat rva vp segments to screen diarrheic infant samples (infants living around the same region where the bat samples were collected). thirty-six percent of human samples were positive for rva, however, none of them was of bat rva origin. they all possessed the typical human genotype i and were % identical to the gambian, senegalese, belgian and brazilian wa-like g p [ ] (table ) . screening human samples for these bat rvas indicated no interspecies transmissions and primer comparison showed that not all the strains can be picked up with the currently used screening primers. bats have been proven to harbor several human pathogenic viruses including sars, mers-related coronaviruses, as well as filoviruses, such as marburgvirus, or henipaviruses, such as nipah and hendra virus [ ] [ ] [ ] , but bat rvas [ ] , were isolated from a lesser horseshoe bat, and a stoliczka's trident bat in china, respectively , . to better understand the spread and diversity of rva in bats, we performed an rva screening in cameroonian bats, after trapping both male and female, young and adult bats close to human dwellings in muyuka, limbe and lysoka localities of the south west region of cameroon (fig. ) . using an unbiased viral metagenomics approach, we identified divergent novel bat rva strains, of which were genetically similar to each other. the fifth strain was related to the kenyan bat strain. interestingly, all these rvas were identified in adult (both female and male) straw-colored fruit bats (eidolon helvum) which is in contrast to human and other animals whereby rva (symptomatic) infections occur mostly in juveniles . also, diarrhea or other obvious signs of sickness were not noticed in these bats. this may suggest that bats may undergo active virus replication and shedding without obvious clinical signs , which potentially could increase human exposure. even though there exists a considerable genetic divergence between bat rva and human rva, suggestions have been made about potential interspecies transmission of chinese and kenyan bat rva strains. the two table . nucleotide comparison between the sequence of human rva screening primers for vp (beg , sbeg , con -l, enda, vp -rdeg, end and rvg ) and vp (vp - - f, con and con ) with their corresponding region of the new bat rva genotype. black shaded nucleotides indicate dissimilar nucleotides between a strain segment sequence and primer and bold numbers at the beginning and end of sequence indicate nucleotide positions for different strains. for clarity the reverse complement sequence of the reverse primers is used for comparison with bat rva sequences. chinese rva strains are genetically quite conserved (all segments of both strains have the same genotype except for their vp gene). based on genome comparisons of chinese bat and partial human rva strains from thailand (cmh and cmh ) and india ( m, m and mcs ), xia and colleagues speculated that asian bat rvas may have crossed the host species barrier to humans on a number of occasions . in addition, the unusual equine strain e shares the same genotype constellation with either myas and/or mslh in all segments except vp . this data therefore suggests that this equine rva strain most likely share a common ancestor with asian bat rvas. furthermore, the genotype constellations of these asian bat rva (table ) are reminiscent to the au- -like genotype backbone of feline/canine-like rva strains, as well as to the genotype constellation of two unusual simian rvas (rrv and tuch) , suggesting that interspecies transmissions might have also occurred in the distant past. moreover, an unusual ecuadorean human rva, ecu is closely related to bat sequences from brazil recently submitted to genbank. similarly, possible interspecies transmission trends were also suggested by he and colleagues between bovine strain rva/cow-wt/ind/rubv / /g p [ ] and the bat strain mslh . given the novelty of the bat rva strains described here, it is questionable if the currently used human rva screening primers (for vp and vp ) will pick up these divergent strains in case an interspecies transmission from bats to humans would occur. comparisons (table and supplementary data s ) of these primers with the corresponding sequences showed that the primer combination con -l and vp -rdeg , , would most likely detect both g and g rva strains. also, the combination of either beg or sbeg with end or rvg might be successful in amplifying g , but the same combinations might not be able to pick up the novel g and g genotypes in pcr screening assays. furthermore, the primer combination beg /sbeg and enda are likely to detect g , but will be unsuccessful in case of g and g especially if the forward primer beg is used. considering vp , both forward primers, vp - - f and con in combination with the reverse primer con will be sub-optimal in detecting any of the p genotypes. generally, with the exception of strain g , detection of most of the bat strains will be sub-optimal or not successful at all for the different available primer combinations. therefore, zoonotic events of bat rva strains could easily be missed with the current screening primers depending on the primer combinations, pcr conditions and/or circulating zoonotic strains. in order to investigate the possibility of bat rva infecting humans who are living in close contact with bats, we used novel primers (rva-vp _ f and rva-vp _ r) designed from an alignment of both human and bat vp rva segment to screen infant samples from patients with gastroenteritis, living around the same region where the bat samples were collected. interestingly, % of human samples were positive, however, none of these were positive for bat rvas. all were of the typical human rva genotype i and therefore there is no evidence for interspecies transmissions of bat rva to humans. however, this result is not conclusive as only a small sample size was considered here. sampling a larger number of subjects and from different localities around the region might result in more conclusive answers with respect to the zoonotic potential of these bat rva strains. the genotype constellations of the two chinese bat rvas showed clear indication of recent reassortment event(s) because they possessed different p genotypes (p [ ] for myas and p [ ] for mslh ), and some gene segments were nearly identical whereas others were not , . their genotype constellation differs markedly from the kenyan straw-colored fruit bat strain (ke ). although this strain showed a unique genotype backbone, some of its segments were similar to some human and other animal rvas . moreover, ke share the same genotypes in several gene segments (vp , vp , vp , nsp , nsp and nsp ) with our bat rva strain batly indicating possible reassortment events between different bat rva strains, as well as a large geographical spread of this virus. furthermore, batli , batli , batli and batly had conserved genotype constellations (in vp , vp -vp , nsp -nsp ) with - % nucleotide sequence similarity except for the vp of batli and vp of batly , again confirming reassortment events within bat rvas. the high genetic divergence and partial relatedness of most of the segments of the different bat rva strains and the ones identified in this study indicate the frequent occurrence of reassortment events in the general bat population and those of cameroon in particular. also, with the current knowledge of the genetic diversity, there seems to exist several true bat rva genotype constellations, as has been previously described for humans, and cats/dogs , . however, this needs to be further confirmed by identification of a larger number of rvas from bats from different age groups and different geographical locations. ethical authorization. ethical authorization for the use of human samples was obtained from the cameroon national ethics committee, yaoundé. all human experiments were performed in accordance with the ministry's national ethics committee guidelines. ethical authorization for the protocol and the use of animal samples was also obtained from the cameroon national ethics committee, yaoundé. all animal experiments were performed in accordance with the ministry's national ethics committee guidelines. all experimental protocols used in this study were approved by cameroon national ethics committee. administrative authorization was obtained from the delegation of public health for south west region, cameroon. informed consent was obtained from human subjects or their parents or guardians. bat sample collection. bat samples were collected between december and may using a previously described method . briefly, bats were captured in different regions (lysoka, muyuka and limbe) of the south west region of cameroon (fig. ) using mist nets around fruit trees and around human dwellings. captured bats were retrieved from the traps and held in paper sacks for - min, allowing enough time for the excretion of fresh fecal boluses. sterile disposable spatulas were used to retrieve feces from the paper sacks, and placed into tubes containing ml of universal transport medium (utm, copan diagnostics, brescia, italy). labeled samples were put on ice and then transferred to the molecular and cell biology laboratory, biotechnology unit, university of buea, cameroon and stored at − °c, until they were shipped to the laboratory of viral metagenomics, leuven, belgium where they were stored at − °c. each captured bat was assessed to determine species, weight (g), forearm length (mm), sex, reproductive state, and age. all captured bats were then marked by hair clipping to facilitate identification of recaptures, and released afterwards. trained zoologists used morphological characteristics to determine the species of the bats before they were released. no clinical signs of disease were noticed in any of these bats. sample preparation for ngs. eighty-seven fecal samples were grouped into pools each containing three to five samples and treated to enrich viral particles as follows: fecal suspensions were homogenized for min at rpm with a minilys homogenizer (bertin technologies, montigny-le-bretonneux, france) and filtered consecutively through μ m, μ m and . μ m membrane filters (merck millipore, massachusetts, usa) for s at g. the filtrate was then treated with a cocktail of benzonase (novagen, madison, usa) and micrococcal nuclease (new england biolabs, massachusetts, usa) at °c for h to digest free-floating nucleic acids. nucleic acids were extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions but without addition of carrier rna to the lysis buffer. first and second strand cdna synthesis was performed and random pcr amplification for cycles were performed using a whole transcriptome amplification (wta) kit procedure (sigma-aldrich), with a denaturation temperature of °c instead of °c to allow the denaturation of dsdna and dsrna. wta products were purified with msb spin pcrapace spin columns (stratec, berlin, germany) and the libraries were prepared for illumina sequencing using the nexteraxt library preparation kit (illumina, san diego, usa). a cleanup after library synthesis was performed using a . ratio of agencourt ampure xp beads (beckman coulter, inc., nyon, switzerland) . sequencing of the samples was performed on a hiseq platform (illumina) for cycles ( × bp paired ends). partial sequences were completed using rt-pcrs with specific primers (supplementary s ). for gene segments lacking the ′ and/or ′ ends of the orf the single primer amplification method (primers in supplementary s ) was used as described previously . sanger sequencing was done on an abi prism genetic analyzer (applied biosystems, massachusetts, usa). lysoka local clinic and kumba district hospital of the south west region of cameroon ( fig. ) after informed consent was obtained from patients or their parents or guardians. the patients were either diarrheic or came into contact with bats directly (by eating, hunting or handling) or indirectly (if family member is directly exposed to bats). the samples were put in utm containing tubes and stored the same way like the bat samples. screening primers (supplementary data s ) were designed from a consensus sequences of human and bat vp rvas and a total of samples from infants ( - years) who had diarrhea were screened by reverse transcriptase polymerase chain reaction (rt-pcr) using the onestep rt-pcr kit (qiagen). the products of positive samples were sequenced using sanger sequencing method. genomic and phylogenetic analysis. raw illumina reads were trimmed for quality and adapters using trimmomatic , and were de novo assembled into scaffold using spades . scaffolds were classified using diamond in sensitive mode . contigs assigned to rva were used to map the trimmed reads using the burrows-wheeler alignment tool (bwa) . open reading frames (orf) were identified with orf finder analysis tool (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) and the conserved motifs in the amino acid sequences were identified with hmmer . amino acid alignments of the viral sequences and maximum likelihood phylogenetic trees were constructed in mega . , using the gtr + g (vp , vp , nsp and nsp ), gtr + g + i (vp -vp , vp and nsp ), hky (nsp ) and t (nsp ) substitution models (after testing for the best dna/protein model), with bootstrap replicates. nucleotide similarities were also computed in mega by pairwise distance using p-distance model. phylogenetic analyses were performed using appropriate reference strains in addition to the rva discovered in this study. determinants of rotavirus host range restriction-a heterologous bovine nsp gene does not affect replication kinetics in the pig virus taxonomy: ninth report of the ictv candidate new rotavirus species in sheltered dogs exotic rotaviruses in animals and rotaviruses in exotic animals full genome-based classification of rotaviruses reveals a 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rotateq genotype constellation and evolution of group a rotaviruses infecting humans metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis g rotavirus strains isolated in the democratic republic of congo belong to the ds- -like genogroup trimmomatic: a flexible trimmer for illumina sequence data spades: a new genome assembly algorithm and its applications to single-cell sequencing fast and accurate short read alignment with burrows-wheeler transform hmmer web server: interactive sequence similarity searching molecular evolutionary genetics analysis version . . molecular biology and evolution r: a language and environment for statistical computing. r foundation for statistical computing geographic analysis and modeling with raster data this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- - o scan authors: hisada, shohei; murayama, taichi; tsubouchi, kota; fujita, sumio; yada, shuntaro; wakamiya, shoko; aramaki, eiji title: surveillance of early stage covid- clusters using search query logs and mobile device-based location information date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: o scan two clusters of the coronavirus disease (covid- ) were confirmed in hokkaido, japan, in february . to identify these clusters, this study employed web search query logs of multiple devices and user location information from location-aware mobile devices. we anonymously identified users who used a web search engine (i.e., yahoo! japan) to search for covid- or its symptoms. we regarded them as web searchers who were suspicious of their own covid- infection (wssci). we extracted the location of wssci via a mobile operating system application and compared the spatio-temporal distribution of wssci with the actual location of the two known clusters. in the early stage of cluster development, we confirmed several wssci. our approach was accurate in this stage and became biased after a public announcement of the cluster development. when other cluster-related resources, such as detailed population statistics, are not available, the proposed metric can capture hints of emerging clusters. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ devices (i.e., smartphones and tablets) and approved use of their location information for the research purpose. subsequently, we counted the number of wssci in each day and each area based on their location information. a previous study demonstrated that symptom-related search queries have an advantage of capturing early signals of infectious diseases. in addition, some studies [ ] [ ] [ ] attempted to utilize search queries for forecasting or predicting influenza epidemic. a recent study reports that both search queries and social network data can produce precise and usable estimation of the influenza development by investigating which kind of data source leads to better results. a recent work-in-progress paper also uses web search queries to predict the country-level covid- epidemic . to evaluate the transferability of the prediction model, these authors investigated the italian prediction model in the other seven countries without having experienced any of them yet. in contrast, we utilized web search query logs per user to detect wssci and gather their location histories to identify locations they visited or passed through, resulting in covid- cluster detection. we believe that our approach is suitable for covid- cluster detection because smartphone applications are widely used nowadays. this study investigates the feasibility of our approach through case studies of the covid- clusters that occurred in february , in hokkaido, japan. the covid- pandemic indeed made us realize that obtaining reliable information on the current status during a pandemic crisis is challenging. however, even in such a low resource condition, smartphone users are still available and can be regarded as a type of social sensors who voluntarily report current events in real time,in most cases, without realizing it. to take advantage of social sensors, it is essential to examine the validity of the wssci-based approach in advance. we focused on the area of hokkaido where two covid- clusters were reported in march , according to cluster maps , released by the mhlw. figure a shows the timelines of the clusters based on official announcements by the hokkaido government , kitami cluster and sapporo cluster . figure b shows the spatial distribution of population in hokkaido. figure c shows the total number of wssci per half grid (wssciphg) between january and march , in hokkaido. figure a -c show the spatio-temporal distributions of wssci in the areas of hokkaido, kitami, and sapporo, respectively. in fig. a , wssci hot spots existed in the two cluster areas before they were reported, suggesting that wssci could be a clue for the cluster build-up. however, there is no cluster in several wssci hot spots near the central and the southern part of hokkaido. this suggests that not all hot spots are clusters, exposing the limitation of our approach. to check the correlation between wssci and the number of new patients in the areas of hokkaido, kitami, and sapporo, respectively, the cross-correlation (ccf), mentioned in "methods" section, was calculated. as a result, in hokkaido as a whole, the ccf between wssci and the number of new patients showed . , with a time lag of days. the ccf informed us about temporal trends in the areas where the clusters occurred as shown in fig. . in the kitami area (fig. a) , the wssci has a maximum cross-correlation of . , determined days after the change in the number of new patients. in the sapporo area (fig. b) , the wssci has a maximum cross-correlation of . , estimated days after the change in the number of new patients. these results suggest that the wssci have also increased in response to the increase in the number of new patients in each area. cluster i: kitami cluster. the first cluster comprises more than cases related to the participants of an exhibition and a dinner party in kitami between february and , . eight participants have been confirmed to be infected in this event, as shown in the upper part of fig. a before the breaking news of the first patient infected with covid- in kitami on february , , we could confirm several wssci between february and , , in fig. b , which were not confirmed at all in the previous week, january to february , . after the breaking news on february , the wssci spread across this area. from this fact, it could be inferred that the appearance of wssci might indicate the cluster existence, demonstrating the feasibility of the wssci-based cluster prediction. the lower part of fig. b shows the spatio-temporal distribution of wssci; we can see some hot spots of wssci on february , . considering the initial status of this area between january and february , , that is the nonexistence of wssci, as shown in fig. b , these hot spots might provide us hints about the cluster existence or the possibility of cluster occurrence in the near future. cluster ii: sapporo cluster. in another cluster in hokkaido, as shown in the lower part of fig. a , eight people have been confirmed to be infected in a live music bar in sapporo. starting with a store clerk, no. , who was tested positive on february , , after visiting a medical office, other two clerks (nos. and ) were also positive on march , . the bar remained open until february , , when the first person tested positive. two guests (nos. and ) visited the bar on february , , one from otaru and the other from kitami. the other three guests (nos. , , and ) who visited the bar on february , , have been confirmed to be infected also. in addition, the infection spread to a total of close contacts of the people who had been in the live music bar. in sapporo, the cluster occurrence was first reported on march , . unlike the kitami cluster, the first patient who tested positive in sapporo was not directly related to the sapporo cluster. furthermore, there were already patients who tested positive in sapporo, and patients tested positive in the entire hokkaido before february , , when the first case of the sapporo cluster was confirmed. therefore, although we can confirm that wssci clearly increased and spread across sapporo during the week when the first positive case of sapporo www.nature.com/scientificreports/ cluster (february -march , ) was confirmed, many wssci were already spreading across the area before the week of february - , as shown in fig. c . to investigate more detailed trends of wssci, the lower part of fig. c shows the daily spatial distribution of wssci in sapporo from february to february , . on february , , several wssci had already existed, indicating that this area is a part of densely populated city in terms of both population statistics as shown in fig. b and wssci as shown in fig. c . on february , , the occurrence of the first case of a suspected www.nature.com/scientificreports/ patient in this area made national headlines. however, there was no cluster at that time yet (unlike cluster i: kitami cluster). in such a situation, many wssci had appeared after the report of the suspected patient on february , . the reason for this sudden burst of wssci is that many people attempted to obtain more information about covid- even though they did not have any symptoms. this indicates that the wssci-based approach could be easily biased by media coverage. www.nature.com/scientificreports/ advantages. this study attempted to detect early clusters by identifying people who suspected their own covid- infection using web search query logs and by extracting their location information. the wsscibased approach is capable of identifying potential covid- -positive patients using web search query logs. although statistics of inpatients and outpatients have been reported, covid- patients with mild symptoms who do not go to the hospital are difficult to identify. in fact, even patients with mild symptoms can transmit the virus to others; it is therefore important to identify these patients. fortunately, our approach can identify such patients through their location information. an advantage of the wssci-based approach is the real time nature of the users. in reality, it is difficult to follow up the population size accurately, as people can move from one city to another to avoid covid- . for example, remote-working enables people to work from anywhere, so that some move to less populated areas and others go back to their hometowns and live with their parents , . it might cause a dynamic change of population distribution as shown in fig. c . even in such a situation, the wssci-based approach can capture ongoing local migrations. additionally, we realized that it might be effective to support a real-time cluster surveillance in developing countries, where we can safely assume at least the existence of web search traffic in comparison to other smartphone applications (e.g., location-sharing social network services). limitations and future direction. this study on the ongoing pandemic crisis has the advantages described above, but there are limitations that should be considered to apply syndromic surveillance. one limitation is that an area with high concentration of wssci does not always indicate the existence of a cluster. in fact, among several hot spots in fig. b , the number of clusters officially detected was only two, causing an overestimation of clusters. however, as mentioned above, it would be helpful to narrow down potential areas that might be clusters in the early stages. another limitation is a bias caused by media coverage. whenever any mass media report the location of a covid- -positive patient, many people who are in areas adjacent to the reported location submit covid- -related queries to acquire additional information. therefore, media could change the nature of wssci, suggesting that our approach would be more effective to detect potential cluster areas before any national headlines. we also investigated another factor, news media, represented by the number of news articles that may affect wssci. the ccf between wssci and the number of news articles showed the highest ccf = . , whose lag is days; the ccf between the number of patients and the number of news articles also presented a similar result that the ccf is . , whose lag is days. in other words, the number of news articles rapidly reflects with the patient number, but it is not that accurate ( . ). in contrast, there is a lag of days between wssci and the number of new patients, but it has a similar correlation ( . ). here, one important question arises as to whether the wssci increase was caused by mass media reports or the occurrence of news about the patients. based on this result alone, however, we could not answer the question. although we could not determine the actual causal relations among the three factors, we could conclude that the wssci are highly correlated with the previous ( days before) patient number. investigating the news biases to wssci is one of the remaining future tasks. [bottom] ccf between the two-time series. the x-axis indicates lag ( is the highest correlation day). the y-axis indicates correlation. in (a) kitami, its ccf is . , whose lag is days. in (b) sapporo, the ccf value is . , whose lag is days. both show a significant correlation (p< . ). www.nature.com/scientificreports/ the cross-correlation analysis indicates the difficulty of the wssci-based real-time cluster detection because there is a lag of days. strong news bias made it hard to observe the precise effect of wssci. still, several practical applications are possible because the wssci detected the increase in the number of new patients days later. even if it would be challenging to develop a nation-wide surveillance system, which would be rarely required at the early stage of this crisis, a government could detect the patient increase in any local cities, leading to rapid local support, including the arrangement of medical facilities, specialist management, and so on. material. we utilized web search query log data and location information provided by yahoo japan corporation as signals for syndromic surveillance. yahoo japan corporation hosts a wide variety of over services of wide variety, including web search, news, maps, weather forecasts, online shopping, and knowledge search on a famous portal site called the "yahoo! japan" and a popular mobile operating system application called the "yahoo! japan app". according to the report , these services are used among a wide range of users of all ages, gender, areas, occupations, and annual income, with little difference based on any of these categories. the number of monthly active users is approximately . million ( . million users on smartphones, which is about % of the smartphone users in japan and . million users on pcs, which is about % of the pc users in japan) and its market share is second to that of google . the yahoo! japan search engine stores more than billion search queries on all devices to their server. we extracted search query logs related to covid- for the identification of infection suspecting searchers as described in the following subsection. more than one million of all "yahoo! japan app" users who explicitly approved the use of their mobile device location information for research purposes, constituted the study sample. this app stores the gps location information to the server. due to the privacy policy of yahoo japan corporation, we could not utilize data that could identify an individual user. instead of individual tracking, we investigated area-based statistics data. infection suspecting searcher identification using web search query logs. we leveraged the search logs on all devices from yahoo! japan search to identify search users who were possibly affected by the disease and were looking for information about the characteristic features of its symptoms to check whether their symptoms corresponded to typical cases of the disease. queries submitted by search users were matched against predefined patterns, consisting of three singleterm patterns and double-term patterns. single-term patterns included japanese short phrases, literally meaning "likely to be corona", "likely to be corona-virus", or "likely to be new type pneumonia". double-term patterns included one of three main terms succeeded by a facet term out of patterns. the main terms were "corona%", "new type", or "new type pneumonia", where % denoted wild card matching, as used in the "like" operator of the sql language. the facet terms included japanese phrases meaning one of the symptoms, namely, "cough", "diarrhea", "coughing up phlegm", "slight fever", "headache", "cold", "fevered", "no fever", "without fever", "high fever", "develop fever", "runny nose", "chills", "throat", "chest", "phlegm", and "feel tired" or "weariness", or two phrases meaning "designated hospitals" (which means hospitals validated by the local health authorities as specialized for treatments of covid- -infected patients) or "advice" (which was presumably intended for special consultation services in charge of advising those who suspected to be infected with the virus). these terms in double patterns were and-combined; therefore, their order was irrelevant. a query was matched against a pattern if and only if all terms were matched against any terms in user queries. therefore, for queries matched against one of the aforementioned patterns, anonymized user ids of searchers were stored only when they had already approved the use of their smartphone location information for research purposes. these searchers were defined as web searchers who are suspicious of their own covid- infection (wssci). user location information extraction. among the app users who explicitly approved the use of their location information, we mapped the number of wssci using the symptom suspected queries described in the previous subsection into each half grid square ( m × m) according to the location information. concerning the privacy, we used neither the ids of each searcher nor their exact location information, but only the number of searchers in each day in the areas defined by the half grid square code system , which we called the number of wssci per half grid (wssciphg). the number of searchers was counted only when the location information of the searchers was stored during their stay in the grid area. to determine whether the wssci could predict clusters or not, we calculated the crosscorrelation function (ccf) between the wssci and the number of new patients for each area, sapporo and kitami. to investigate the relationship between wssci and the number of new patients, we also examined the cross-correlation and lag between them, which indicate a hint for their causal relations. to investigate a bias caused by the news broadcast, we examined the relationship between wssci, the number of new patients, and the number of news articles related to covid- in the target area, hokkaido. to do so, we used news articles that contain at least one of the keywords "coronavirus" or "pneumonia" from a news article archive site . given two time series of data, the ccf identifies a similarity and chronological difference between them by incrementally shifting one time series vector and repeatedly calculating the correlation between two signals. a chronological difference is called a lag, which represents how many days apart the two time series are. if the peak correlation is at the center (lag = ), this indicates that the two time series are most synchronized at that time. to compute the ccf, we used one of the numpy modules, numpy.correlate www.nature.com/scientificreports/ ethics statement. all participants provided written (or electronically displayed) informed consent before participating in this study and agreed to the terms and conditions of the "use of services" provided by yahoo! japan services when they disclosed their location. this study did not require the participants to be involved in any physical and/or mental intervention. participants' information was unlinkable, anonymized, and deidentified prior to analysis. this research did not obtain identifiable private information, meaning that it was exempt from institutional review board approval according to the ethical guidelines for research of the japanese national government. yahoo japan corporation has approved the use of the participant data in this study. although the data that support the findings of this study are not allowed to be publicly available according to the privacy policy and data disclosure policy of yahoo japan corporation and the japanese privacy law, the corresponding author can comply with a reasonable request. l/ - - -state ment-on-the-secon d-meeti ng-of-the-inter natio nal-healt h-regul ation s-( )-emerg ency-commi ttee-regar ding-the-outbr eak-of-novel minister of health, labour and welfare. a map showing clusters of covid- infections in japan (as of march clinical management of severe acute respiratory infection (sari) when covid- disease is suspected: interim guidance version . . tech. rep. who/ -ncov/clinical/ . , world health organization detecting influenza epidemics using search engine query data advances in nowcasting influenza-like illness rates using search query logs predicting seasonal influenza epidemics using cross-hemisphere influenza surveillance data and local internet query data accurate regional influenza epidemics tracking using internet search data comparing social media and google to detect and predict and predict severe epidemics tracking covid- using online search minister of health, labour and welfare. a map showing clusters of covid- infections in japan (as of march outbreak of new coronavirus infection in hokkaido outbreak of new coronavirus infection in the city outbreak of new coronavirus infection in the city these places will pay remote workers to move there to boost covid- recovery coronavirus: more japanese to swap urban life for the countryside search engine market share standard grid square and grid square code used for the statistics syndromic surveillance using search query logs and user location information from smartphones against covid- clusters in japan the custom codes in these analyses are available .received: april ; accepted: october s.f. and k.t. provided the user data to this study. s.h., t.m., and s.f. conceived the experiment(s), s.h., t.m., and k.t. conducted the experiment(s), and all authors analyzed the results. all authors contributed to the study design, wrote the main manuscript text, and reviewed the manuscript. this study received no funding. the authors had full access to all data in the study and had final responsibility for the decision to submit for publication. the authors have no competing interests. s.f. and k.t. are current employees of yahoo japan corporation who provided the user data to this study. preprint of this paper is available . correspondence and requests for materials should be addressed to e.a.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -saqoft p authors: heffner, kelley; hizal, deniz baycin; majewska, natalia i.; kumar, swetha; dhara, venkata gayatri; zhu, jie; bowen, michael; hatton, diane; yerganian, george; yerganian, athena; o’meally, robert; cole, robert; betenbaugh, michael title: expanded chinese hamster organ and cell line proteomics profiling reveals tissue-specific functionalities date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: saqoft p chinese hamster ovary (cho) cells are the predominant production vehicle for biotherapeutics. quantitative proteomics data were obtained from two cho cell lines (cho-s and cho dg ) and compared with seven chinese hamster (cricetulus griseus) tissues (brain, heart, kidney, liver, lung, ovary and spleen) by tandem mass tag (tmt) labeling followed by mass spectrometry, providing a comprehensive hamster tissue and cell line proteomics atlas. of the unique proteins identified, high similarity was observed between cho-s and cho dg and included increases in proteins involved in dna replication, cell cycle, rna processing, and chromosome processing. alternatively, gene ontology and pathway analysis in tissues indicated increased protein intensities related to important tissue functionalities. proteins enriched in the brain included those involved in acidic amino acid metabolism, golgi apparatus, and ion and phospholipid transport. the lung showed enrichment in proteins involved in bcaa catabolism, ros metabolism, vesicle trafficking, and lipid synthesis while the ovary exhibited enrichments in extracellular matrix and adhesion proteins. the heart proteome included vasoconstriction, complement activation, and lipoprotein metabolism enrichments. these detailed comparisons of cho cell lines and hamster tissues will enhance understanding of the relationship between proteins and tissue function and pinpoint potential pathways of biotechnological relevance for future cell engineering. proteomics sample preparation. samples for proteomics were thawed on ice and lysed in a solution of % sodium dodecyl sulfate (sds) in µl of cell lysis buffer supplemented with . mm phenylmethylsulfonyl fluoride (pmsf) and mm ethylenediaminetetraacetic acid (edta), ph - . lysates were sonicated x times for s at % amplitude followed by a s pause. protein concentration was measured by a bicinchoninic acid (bca) protein assay. one hundred micrograms of each sample were reduced in mm tris( -carboxyethyl) phosphine (tcep), ph - , at °c for h on a shaking platform. iodoacetamide was added to alkylate the sample to a final concentration of approximately mm and incubated for min in the dark. next, samples were passed through kda filters to reduce the sds concentration as described in the filter-aided sample preparation (fasp) method . during the fasp method, tetra-butyl ammonium bicarbonate (teabc) was added after the urea washes to increase protein recoverability from the filters. the samples were finally digested using trypsin/lysc enzyme mix (promega v a) at an enzyme to substrate ratio of : , overnight at °c on a scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ shaking platform. after digestion, peptides were cleaned up by c cartridges and labeled with tmt reagents. all tmt labeled samples were combined and vacuum centrifuged to dryness removing the entire liquid. labeling. in order to compare protein expression, samples were labeled in duplicate (biological replicates) using two tmt- plex labeling kits (thermofisher scientific, waltham, ma, usa). triplicates were used for ovary tissue and cho-s. each of the reagents has the same nominal mass and chemical structure, so that for each sample a unique reporter mass ( - da) was used to relate protein expression levels. specifically, we included technical and biological replicates of cho-s (two samples in tmt and one sample in tmt ) to aid in comparisons between and within the tmt experiments. all other samples contained only biological replicates, which were randomly assigned to one of the tmt- plex kits. the tmt labeling design is provided in table . following protein digestion, tmt reagents were thawed, and acetonitrile was used to dissolve the reagents. one reagent tube was added to each sample and then incubated at room temperature for h. hydroxylamine was subsequently added to quench the reaction before the tubes were combined. all tmt labeled samples were combined and vacuum centrifuged to dryness, removing the entire liquid. each tmt- plex was subjected to analysis by two-dimensional liquid chromatography tandem ms ( d lc/ms/ms). protein identifications were made using proteome discoverer software with a high confidence cutoff [< % false discovery rate (fdr)]. the protein intensities were evaluated by fold change, using the cho-s cell line technical replicate in each tmt as the basis. protein accession numbers were mapped to gene symbols using the biological database network (https ://biodb net-abcc.ncicr f.gov) for functional analysis by gene ontology (go). for go annotation, gene symbols were mapped to biological processes, using the go-cho platform (https ://ebdru p.biosu stain .dtu.dk/gocho ). all programming for the hypergeometric test were calculated in matlab version vb (https ://www.mathw orks.com/produ cts/matla b) and rstudio (https ://www.rstud io.com). enrichment and depletion p-values were calculated using the hygecdf and hygepdf functions in matlab. these values were this study was undertaken to compare protein expression of various cho cell lines and hamster tissues, resulting in the most comprehensive multi-tissue analysis of the cricetulus griseus proteome (fig. a) . this multi-tissue and multi-cell line analysis aims to improve our understanding of the chinese hamster as the original tissue www.nature.com/scientificreports/ source for cho cell lines. additionally, since cho is the dominant biopharmaceutical production host in biotechnology, this comparison elucidates similarities and differences across cells and tissues. an overview of the proteomics workflow is shown in fig. b . following ms identification, the protein accession numbers were determined using the annotated chinese hamster genome . protein accession numbers were converted to gene symbols for functional analysis . for missing gene symbols, the accession numbers were searched against mouse and human databases using the online database, biodbnet . in this study, gene ontology (go) and ingenuity pathway analysis (ipa) were used for functional analyses of the differentially expressed proteins. go analysis converts gene symbols to their molecular function, cellular component, and biological processes in order to evaluate the relationship between the molecular activities of gene products, location of activity, and pathways comprising the activity of multiple gene products, respectively . in another functional analysis, each gene symbol was mapped to the relevant ipa pathways , suggesting enriched and depleted pathways for comparisons between different tissues and cells. for both go and ipa, the enrichment and depletion values were determined based on p-values that were calculated via the hypergeometric distribution, with p < . set for evaluating significance. protein identification and total protein intensity differences. pca comparison. for each sample, the number of unique proteins and peptides identified are listed in table along with the number of spectra obtained. the complete list of identified proteins and fold change ratios are listed in supplementary table and are searchable online through the nist database (https ://pepti de.nist.gov). over proteins were identified in each sample with at least two unique peptides per protein; this corresponded to a total of unique proteins containing a false discovery rate (fdr) of % for protein identification when the tmt samples were combined and the duplicates were removed ( table ) . between the two tmt experiments, there were common proteins identified in both sets ( fig. a) . a total of proteins were unique to tmt and proteins were unique to tmt ( fig. a) . protein intensity fold change ratios were initially evaluated through principal component analysis (pca) of proteins identified in all samples, as shown in fig. b . the pca distribution represents differences in protein expression for those proteins identified in all samples and is influenced by proteins highly expressed in one tissue versus another. not surprisingly, as shown by pca, the cell samples clustered together and were distinct from all the tissues. for the tissue samples, the expression of proteins in spleen, liver, and heart clustered together. similarly, protein expression in the lung and kidney clustered together. interestingly, the first cluster (spleen, liver and heart) comprises tissues with dense connective tissue and capillary systems . in contrast, the second cluster (lung and kidney) is specialized for transport, with tubular systems and thin insterstitium [ ] [ ] [ ] [ ] . ovary and brain were clustered separately from the other organs. next, the two replicates from the same tissue were plotted to examine their consistency. fitting a curve to the scatter plot and calculating the r value assessed the linearity of the two replicates. shown in fig. c -f are the plots for the tissue replicates with the highest r value for each cluster, specifically lung (from the lung/kidney cluster) and heart (from the heart/kidney/spleen cluster), plus brain and ovary. for the following analyses, we studied these representatives from each of the clusters; additional data on all proteins is provided in supplementary table . to compare between cell lines and tissues, protein intensity was averaged between replicates and plotted in fig. . the data was log transformed in order to ensure a normal distribution for each sample. a student's t-test was performed to determine the likelihood of significant differences between samples from cells and tissues. a summary of the statistical analysis, performed using student's t-test as a means comparison, is shown in table . when comparing the cell lines to each other, no significant differences were observed between the cho-s and cho dg cell lines (table and fig. a ) at a p-value of . . we identified proteins with significantly higher expression in cho-s relative to cho-dg and proteins with significantly higher expression in cho dg relative to cho-s, representing the lowest number of total outliers for any comparison. a comparison between cells and tissues is shown in fig. b through i. for these comparisons, the total number of proteins with low and high expression (fold change < . or > . , respectively) is approximately %. for the cell to tissue comparison, all tissues show a statistically significant difference as compared to either the cho-s or cho dg cell lines. the proteins expressed at significantly higher levels in cells over tissues include proteins related to dna replication, transcription, translation, and controlling cell apoptosis as expected to maintain rapid cell growth in exponential culture. among the most highly expressed proteins in cho-s and cho dg are dna-directed rna polymerase ii, eukaryotic translation initiation factor, histone h . t, general transcription factor c, s ribosomal protein, and apoptosis inhibitor . www.nature.com/scientificreports/ in comparison to ovary tissue (fig. e ,i respectively, p < . ), cho-s and cho dg cells exhibited differences in expression patterns. this is somewhat surprising considering that cho cells were derived from a mixture of ovary and the surrounding connective tissue. in fig. e ,i, the proteins colored blue and yellow represent those with higher expression in ovary or cho cells, respectively. in addition to ovary, statistical differences in protein expression were observed for cho-s and cho dg cells and to lung tissue (fig. d,h, p < . ) . similar to the comparison against ovary, an increase in upregulated proteins with higher expression was observed in the lung tissue in comparison to the cell lines. this suggests that tissue-specific functions may contribute to differences in expression patterns with cells regardless of the type of tissue. next, both cho cell lines show statistically significant differences in outliers as compared against heart tissue (fig. c ,g, p < . ). similar to the ovary and lung comparison, there are a greater number of proteins with higher expression in the heart tissue when compared to cell lines. over % of the cells in the heart are cardiac fibroblasts, which contributes to the specificity of this organ . in addition, the heart has endothelial, smooth muscle, and pacemaker cells. this high degree of specialization is likely influential for the differences in proteins between cho and heart. finally, both cho-s and cho dg also show a difference in expression www.nature.com/scientificreports/ when compared to the brain, which is also likely related to the high degree of specialization required for brain cells such as neurons and glia (fig. b ,f, p < . ). amongst the tissues, wide differential regulation, both upregulation and downregulation, can be observed when comparing tissues against each other (fig. j-o) . there is a greater difference in terms of the total percentage of outliers in the brain versus heart comparison (~ % total outliers for brain and ~ % total outliers for heart). interestingly, only brain and heart tissues were found to have a statistically significant difference between each other (table ). all other tissue to tissue comparisons were found to have insignificant differences in the percentage of outliers. one reason for the relatively high number of outliers in brain tissue may be attributed to the distinct separation in terms of embryonic development from the other organs. the brain originates from the ectoderm whereas the circulatory system (heart), epithelial layer of the lungs, and ovary develop from the table ) are provided in the appendix. we also examined some of the top upregulated proteins in each tissue. hierarchical clustering of protein expression is depicted in the center plot of fig. in which the color pink represents highly expressed proteins table . means comparison using student's t-test for total percentage of outliers. *p-value < . indicates that the outlier comparison is statistically significant. coloring is shown from low (green) to high (pink) abundance. distinct clusters are shown for brain, lung, heart, and ovary tissues. from top to bottom: the protein functions exhibiting high expression for each tissue are shown relative to other tissues for ovary (a), brain (b), heart (c), and lung (d) plotted using genesis software. www.nature.com/scientificreports/ for each specific tissue. for each sample, the top upregulated proteins, corresponding to approximately % of the total proteins in a tissue, were identified in order to highlight tissue specificity (supplementary table ). for example, disintegrin and metalloproteinase domain-containing proteins (adam , , ) were highly upregulated in hamster ovary (supplementary table ). indeed, adam can control follicle formation by regulating the recruitment of ovarian follicle supporting cells . disintegrin would be useful for control of the extracellular matrix as agrees with results from the human ovary-specific proteome . similarly, highly upregulated brain proteins include amyloid beta a protein, neuron navigator (nav ), calcium and integrin binding protein, and serine/threonine protein kinase (supplementary table ). nav is specifically targeted to the nervous system . in contrast, the heart is a strong muscle that must contract continually and is predominantly composed of cardiomyocytes and fibroblasts. the human heart tissue proteome was found to have upregulated proteins , including retinol dehydrogenase (rdh ) which was also significantly upregulated in our hamster heart proteome (supplementary table ). previous studies have indicated that the knock-down of rdh led to abnormal neural crest cell migration and an abnormal heart loop in mutant embryos, signaling its importance in heart tissue . we also observed high expression of proteins including pleckstrin homology domain-containing family f member -like, actin-related protein, glutathione s-transferase, protein o-glycosyltransferase, and ras gtpase-activating protein (supplementary table ) . we also identified the top upregulated proteins in hamster lung tissue (supplementary table ). examples include branched chain aminotransferase, glycine n-methyltransferase, integrin alpha- , and vesicle-associated membrane protein, indicating the importance in the lung of key metabolic and membrane process. as a comparison, the human proteome identified genes highly expressed in the lung including similar membrane and secretory proteins . indeed, the lung is particularly adept at expressing membrane and secreted proteins such as surfactants and solute carrier proteins . analysis of the most abundant proteins for each tissue or cell line can, at least in some cases, relate to key function and roles of specific tissues and provide targets of opportunity for genetic engineering of cho cell lines. ipa pathway analyses. next, the proteins were annotated with gene symbols in order to perform pathway analysis. proteins with fold change values of less than . or greater than . were used for each comparison in order to determine downregulation and upregulation of pathways in the ipa software. as shown in fig. , protein intensity hierarchical clustering was used to identify proteins with significantly higher expression in a specific tissue. protein functions identified in ipa that are enriched in the brain include g /s phase arrest, metabolism of acidic amino acids, and organization of the golgi apparatus, and transport of phospholipids (fig. b) . the golgi plays a central role in cholesterol and other lipid metabolism; almost % of the human body's unesterified cholesterol is present in the brain . additionally, glycosphingolipids are abundant in the nervous system, and are synthesized in the endoplasmic reticulum and completed in the golgi apparatus . acidic amino acids, including glutamate and aspartate, serve important signaling functions in the brain, so an increase in their metabolic activity would be expected, especially since these metabolites are not readily obtained from the diet , . pathways enriched in the lung include catabolism of branched chain amino acids (bcaas), metabolism of reactive oxygen species (ros), trafficking of vesicles, and synthesis of lipids (fig. d) . the lung shows high secretory capacity and thus trafficking of the secretory products through vesicles is important . furthermore, the lung is the source of surfactants, composed of % lipids; thus, lipid synthesis will be an important component of its function , . the lungs are also particularly sensitive to hypoxia occurring at high altitude. the lung responds to these hypoxic conditions through signaling, including the release of ros species to trigger hypoxia-inducible factor (hif) . thus, the capacity for ros metabolism may be critical for lung function and adaptation to changes in oxygen levels in different environments. similarly, in the heart, enriched pathways include metabolism of alpha-amino acid, sorting of protein, and translation (fig. c ). amino acid metabolism was studied in rat heart with amino acid levels rising up to fivefold higher than plasma levels , accompanied by increases in ribosome activity and translation . finally, protein functions enriched in the ovary include development of extracellular matrix, docking of vesicles, and exocytosis (fig. a) . the matrix of ovary tissue is important for numerous physiological activities, including growth, migration, and differentiation, and the composition changes during ovulation , critical to the fertility process . further, malfunctions of the matrix are observed during ovarian cancer . high levels of extracellular matrix components in chinese hamster ovaries are in agreement with our previous research examining the hamster ovary tissue proteome using label-free proteomics approaches . for go functional analysis, gene symbols were annotated for biological processes in order to group proteins with similar biological relevance. the biological process go category was analyzed to determine enrichment and depletion p-values for the cell-to-tissue and tissue-to-tissue comparisons. the appendix lists the top most enriched biological processes for cho-s (supplementary table ) and cho dg (supplementary table ) comparisons with tissue. enrichment is determined by hypergeometric distribution, with a p-value of < . used for significance. biological processes, representing a biological function involving the gene product, complements the pathway analysis in "ipa pathway analyses" shown above . in both cho-s and cho dg , the most common biological processes enriched involve dna and mrna processing, and metabolism. some of the most common enriched processes in cho-s include mrna processing and splicing, dna replication and repair, transcription, cell cycle, chromatin modification, and chromosome condensation. in comparison, signaling, transport, and adhesion were significantly enriched across the different hamster tissues. enriched brain biological processes relative to the cho cell lines include ion transport, axon guidance, www.nature.com/scientificreports/ synaptic transmission, and metabolic process, among others . ion transport helps to maintain the stability of cerebral function due to the key roles that ions play in currents and synaptic transmission . abnormal distributions of ions in the brain can lead to defects in neuronal function including seizures and depression. enriched biological processes in the heart compared to the cho cell lines show functions related to circulation and heart function such as vasoconstriction, sodium-independent organic ion transport, complement activation, blood coagulation, and lipoprotein metabolism. for example, complement activation involves mannosebinding lectin and complement components, c , c , and cd . the complement cascade can be activated during heart disease or failure, especially in cases of myocardial ischemia and reperfusion , . biological processes that are enriched in the lung as compared to the cho cell lines include g-protein coupled receptor signaling pathway, innate immune response, vesicle-mediated and transmembrane transport, and signal transduction. indeed, vesicle transport is an important component of secretory pathway machinery. unraveling the complexities of lung secretions may yield new insights into ways by which secretion differs in tissues and cell lines. signaling through g-protein coupled receptors and other pathways is an important proinflammatory response in lungs, which can undergo modifications leading to lung cancers , . not surprisingly, protection of the lungs using the host innate immune response is critical as this organ is exposed to a variety of pathogens including bacterial, fungal, and viral, during breathing , . finally, enriched biological processes in the ovary highlight differences between the cells and tissue around the region from which cho cells were derived. enriched ovarian biological processes include transmembrane transport, protein transport, vesicle-mediated transport, cell adhesion, and cell death. when vesicle production rate was quantified in the ovary, the turnover indicated high vesicle recycling across the endomembrane system , which is consistent with these proteomic results. cell adhesion is also important to ovary function and follicle maturation through interactions with the extracellular matrix and direct cell-cell contacts. mutation of the ovarian surface is causative of approximately % of malignant ovarian tumors , . the results from the chinese hamster proteome provide new insights into global protein expression across a wide variety of tissues and multiple cell lines. these differences highlight the role of tissues in executing key organ functions which require a specific metabolic processes, such as transport and communication, in comparison to cho cells, which are focused on replication and gene expression, characteristics useful for rapid growth and the production of biologics. because of their relevance to biomedicine and the biotechnology industry, we compared the tissue proteome to cho cell lines and each other in order to identify functional differences in expression across tissues and cell lines. specifically, we observed enrichment of many physiological pathways in tissues that were not enriched in cells, such as ion, protein, and vesicle transport, signal transduction, and cell adhesion. often, these differences correlated with specific tissue functions while the activities in cell lines were often correlated with dna replication, cell cycle, or rna processing. furthermore, some of the proteins with high expression in lung, ovary, or other tissues versus cho, such as vesicle-mediated and protein transport, provide significant opportunities for cho cell engineering going forward. in this way, the study expands on our cho and chinese hamster tissue knowledge base by virtue of establishing an atlas to differentiate proteins across cells and tissue for this critically important biotechnological and biomedical host species. indeed, this comparison has enabled us to appreciate the changing proteomic landscape across cells and tissues and furthermore to recognize how the expressed proteins from different cell types can represent signatures for some of their key physiological or biotechnological functions. the complete list of identified 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navigator , a novel microtubule-associated protein involved in neuronal migration retinol dehydrogenase, rdh l, is essential for the heart development and cardiac performance in zebrafish site-specific differences in gene expression of secreted proteins in the mouse lung: comparison of methods to show differences by location cholesterol metabolism in the brain the role of glycosphingolipid metabolism in the developing brain dietary amino acids and brain function amino acid precursors of monoamine neurotransmitters and some factors influencing their supply to the brain mass spectrometry-based secretome analysis of non-small cell lung cancer cell lines intermediary metabolism of the lung the role of lipids in pulmonary surfactant lung cell hypoxia: role of mitochondrial reactive oxygen species signaling in triggering responses regulation of protein synthesis in heart muscle. i. effect of amino acid levels on protein synthesis characterization of the human heart mitochondrial proteome the roles of the ovarian extracellular matrix in fertility alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy ion regulation in the brain: implications for pathophysiology complement activation in heart diseases. role of oxidants effects of complement activation in the isolated heart. role of the terminal complement components common pathways for activation of proinflammatory gene expression by g protein-coupled receptors in primary lung epithelial and endothelial cells pathogenesis of lung cancer signalling pathways: roadmap for therapies innate immunity in the lungs estimation of golgi membrane flow rates in ovary glands of aptenia cordifolia using cytochalasin b cell-cell adhesion in the normal ovary and ovarian tumors of epithelial origin; an exception to the rule cell biology of human ovarian surface epithelial cells and ovarian carcinogenesis the authors acknowledge the financial support of astrazeneca for funding this project. in addition, this material is based upon work supported by the national science foundation graduate research fellowship program under grant no. dge- and grant no. dge- . any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the national science foundation. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to m.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - t hg wi authors: bernard-stoecklin, sibylle; nikolay, birgit; assiri, abdullah; bin saeed, abdul aziz; ben embarek, peter karim; el bushra, hassan; ki, moran; malik, mamunur rahman; fontanet, arnaud; cauchemez, simon; van kerkhove, maria d. title: comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from to date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: t hg wi since its emergence in , , cases and deaths due to middle east respiratory syndrome coronavirus (mers-cov) have been reported to the world health organization. most cases were due to transmission in healthcare settings, sometimes causing large outbreaks. we analyzed epidemiologic and clinical data of laboratory-confirmed mers-cov cases from eleven healthcare-associated outbreaks in the kingdom of saudi arabia and the republic of korea between – . we quantified key epidemiological differences between outbreaks. twenty-five percent (n = / ) of mers cases who acquired infection in a hospital setting were healthcare personnel. in multivariate analyses, age ≥ (or . , %ci: . – . ) and the presence of underlying comorbidities (or: . , % ci: . – . ) were associated with increased mortality whereas working as healthcare personnel was protective (or . , % ci: . – . ). at the start of these outbreaks, the reproduction number ranged from . to . ; it dropped below within to weeks. this study provides a comprehensive characterization of mers hca-outbreaks. our results highlight heterogeneities in the epidemiological profile of healthcare-associated outbreaks. the limitations of our study stress the urgent need for standardized data collection for high-threat respiratory pathogens, such as mers-cov. such large healthcare-associated (hca) outbreaks have mainly been limited to the kingdom of saudi arabia (ksa) and the united arabian emirates (uae) until the spring , when a single imported case of mers returning from the middle east initiated a cluster of cases in the republic of korea (rok) across at least hospitals and much of the country . super spreading events in healthcare settings has been described for several previous mers outbreaks, including an outbreak in al-hasa governorate in and during the outbreak in rok, where approximately % of the transmission events were epidemiologically linked to five mers cases , , . superspreading events in health care facilities have been observed in similar high threat respiratory disease pathogens, such as severe acute respiratory syndrome (sars) in canada, china, singapore [ ] [ ] [ ] . while more than half of the laboratory confirmed mers-cov infections reported globally to date are associated with human-to-human transmission in healthcare settings , there has been little human-to-human transmission reported in household settings . outbreak investigations and scientific studies conducted during or after mers hospital outbreaks have identified that aerosol-generating medical procedures with improper or inadequate personal protective equipment place medical personnel and patients sharing wards with mers patients and family visitors at higher risk for mers-cov infection , , with exposure to infectious droplets being the likely source of contamination. although close unprotected contact with a mers patient is generally considered necessary for human-to-human transmission , several studies have revealed that mers-cov particles can persist on surfaces as long as several days, raising the possibility of a role of fomites in transmission , . fomite transmission is further supported by observed viral spreading between rooms that were clearly separated , and outbreaks that occurred in hemodialysis units , . factors leading to healthcare-associated outbreaks include overcrowding in emergency departments, slow triage and isolation of suspected patients and inadequate compliance to infection prevention and control procedures , , . however, few studies have described or compared the characteristics of hca-outbreaks as a whole in terms of their size, epidemiologic factors , , or the role of interventions to stop transmission , . here, we provide the largest comprehensive study of eleven healthcare-associated outbreaks that occurred between and june . we carried out a comparative analysis of these outbreaks in terms of epidemiological profiles, demographic characteristics and clinical outcome. study design. we analyzed epidemiological datasets of laboratory-confirmed mers patients and focused our study on eleven healthcare-associated outbreaks that were reported in ksa and rok since , when policies and procedures for case identification and comprehensive contact identification and follow up became systematic and were implemented by affected countries. the data used documented mers-cov infections reported to who under the international health regulations ( ). we only included clusters of cases/outbreaks that were linked to healthcare facilities. supplemental rok case-based data were provided as a detailed line list of the korean mers cases included in a published study . we defined laboratory-confirmed mers-cov infection as following who guidelines , . we defined a hca-outbreak as the occurrence of or more laboratory-confirmed mers-cov infections with reported epidemiologic links between cases and during which the human-to-human transmission events were documented within a single healthcare facility, with no more than days apart between cases symptom onset. the mers outbreak in the republic of korea in is treated as a single outbreak. individual-level variables included information on age, sex, nationality, occupation (healthcare personnel (hcp) yes/no), dates of symptom onset, date of notification to who, presence of any pre-existing co-morbid conditions, and clinical outcome. in case of missing or conflicting information and when information from the country was not available, we considered the data as missing. statistical analysis. descriptive analysis was performed by hca-outbreak (outbreak-level analysis) using aggregated data, and for all cases (individual-level analysis). all analyses were conducted using stata, version (college station, tx: statacorp lp), microsoft excel (version . , jones, chicago usa) and r. outbreak-level analysis. we calculated the duration, size and case fatality ratio for each outbreak. the duration of an outbreak was calculated as the number of days between the date of symptom onset of the first reported case to the date of symptom onset of the last reported case. we obtained weekly smoothed estimates of the case reproduction number based on the approach developed by wallinga and teunis , using the r package. we assumed that the serial interval of mers-cov had a gamma distribution with a mean of . days and a standard deviation of . days, as described elsewhere . individual-level analysis. we summarized case characteristics as frequencies and proportions for categorical variables, as median and interquartile ranges (iqr) for continuous variables. chi-square tests were used to compare subgroups of cases when appropriate. a p value of less than . was used to indicate statistical significance. univariate analysis identified variables significantly associated with fatal outcome, which were included in a multivariable model. model selection was performed using a multilevel mixed-effects logistic regression with backwards selection taking into account clustering of individuals by outbreak. for the variable "age", the cut-off was fixed at , based on the results of the univariate analysis. variables with p-values < . were retained in the final model. all data used in these secondary analyses were de-identified data obtained from who or datasets from peer-reviewed literature. as such, these data were deemed exempt from institutional review board assessment. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ general characteristics of hca-outbreaks. since january to october , , laboratory-confirmed mers-cov infections have been reported to who. figure a illustrates the global epidemic curve since mers was first identified in humans. each peak is associated with a health care associated outbreak (colored lines, fig. a ). from , affected countries implemented systematic contact tracing and follow up (including laboratory testing), investigation and data collection of mers suspect cases , . in our analysis, a total of laboratory-confirmed mers cases from eleven distinct hca-outbreaks during - were included ( table ). the eleven hca-outbreaks varied in terms of duration, size and epidemiological profile ( table , fig. b) . the median number of total reported cases per outbreak was (interquartile range, [iqr] - ), ranging from to cases. the median duration was days (iqr - ), ranging from to days. three outbreaks began with sporadic cases during the first two to five weeks of the outbreak, while the other eight displayed a rapid increase to the peak. the median time between onset of symptoms of the first reported case and the peak of incidence was weeks (iqr - . ), ranging from to weeks. the case fatality ratio (cfr) in outbreaks was % ( reported deaths among cases), compared with the global overall cfr of . % ( reported deaths among , cases reported as of october (table ) . during hca outbreaks, cfr ranged from to % (p < . ) and cfr was significantly lower among hcp mers-cov infections compared to non-hcp mers-cov infections ( % vs. % p < . ). demographic and clinical characteristics. the demographic and clinical characteristics of the cases from hca outbreaks included in our analyses are summarized in table . the median age was (iqr, - ), and significantly varied by outbreak (p < . ). five outbreaks had a median age < and the other outbreaks had a median age ≥ . the majority of cases were male ( %, n = / ), and the sex ratio among cases differed significantly between outbreaks (p < . ). the overall proportion of hcp was % (n = / ). this proportion varied significantly by outbreak (p < . ), from % to % (table ) . median age was significantly lower among hcp than non-hcp cases ( iqr - vs iqr - , p < . ) and the proportion of females was higher among hcp than non-hcp ( % vs %, n = , p < . ). more than half ( %, n = / ) of cases had at least one underlying co-morbid condition (table ) , and this was significantly lower among females compared to males ( % vs %, respectively, n = , p < . ) and among hcp compared to non-hcp ( % vs %, n = , p < . ). sixteen percent (n = / ) of cases were asymptomatic at time of reporting (table ). this proportion varied significantly between outbreaks ranging from % to % (n = , p < . , fig. c ). median age of asymptomatic cases was (iqr, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the majority of whom were females ( %, n = / ) and had no underlying co-morbid conditions ( %, n = / ). the proportion of hcp among asymptomatic infections was high ( %, n = / ), and the cfr was null. the median duration between symptom onset and case notification to who was days (iqr - ). risk factors associated with fatal outcome. in univariate analysis, fatal outcome was significantly associated with age (p < . ), presence of underlying comorbidities (p < . ), non-hcp status (p < . ), and male sex (p < . , estimation of time-varying reproduction number. at the start of each hca outbreaks, the case reproduction number r (t) ranged from . ( % ci . - . ) to . ( % ci . - . ) ( table and fig. ). estimates of r (t) dropped below within to weeks from the first reported case in the outbreak (n = outbreaks, median weeks, iqr - ). year of outbreak period of time* www.nature.com/scientificreports www.nature.com/scientificreports/ we provide here a comparative characterization of mers hca-outbreaks and report substantial heterogeneity between hca-outbreaks illustrating the complexity of the factors contributing to the emergence of a cluster of cases associated with nosocomial transmission. the duration and epidemic profiles of outbreaks varied; some started with an apparent sharp increase in incidence while others began more slowly with isolated cases emerging intermittently for a few weeks before a cluster of cases appeared in a healthcare facility. some outbreaks had a sharp decline in cases, while others experienced a long tail lasting several weeks after the peak. the median estimates of the reproduction number r(t) in the early stages of outbreaks included in our analyses reached as high as . in the republic of korea, as has been found by others , likely facilitated by multiple superspreading events at two hospitals . what is perhaps most informative from a public health perspective is the length of time it took to bring the outbreaks under control. all of outbreaks reached r t values below within to weeks after the first cases were identified, highlighting that the time frame in which hospital and ministry officials can implement control measures to stop nosocomial outbreaks. factors explaining differences in hca outbreak size and duration might include variations in the speed in which cases were suspected and timing of interventions implemented in healthcare settings, including contact identification, management and isolation of patients, improved infection prevention and control measures and in some cases, the requirement to close departments [ ] [ ] [ ] [ ] [ ] . in this study, we were not able to evaluate the impact of interventions in these outbreaks. prevention of large hca outbreaks since (fig. a) , may be, in part, explained by improvements in contact tracing policies implemented in . in , contact tracing became more systematic with the identification and follow up of high (close, unprotected contact) and low risk contacts (protected hcw). in affected countries, national ministries of health and hospital staff comprehensively list all contacts of known mers patients, including healthcare workers at all facilities/departments the patient visited, patients who shared wards/ rooms with mers patients, family and visitors and occupational contacts. follow up of contacts includes the testing of all high-risk contacts, regardless of the development of symptoms. recommendations stated that positive contacts are placed in quarantine (home or hospital isolation for asymptomatic or symptomatic secondary cases, respectively) until they test negative , [ ] [ ] [ ] . additionally, affected countries enhanced infection prevention and control procedures education, and training, and implemented visual triage systems to reduce delays in testing, isolation and care of suspected mers-cov patients. this has again been recently illustrated by the lack of secondary cases following the identification of a confirmed case of mers in korea in september was due to the rapid and comprehensive isolation, treatment and management of contacts of the patient. the variation in outbreak size and duration is also affected by superspreading events early in some outbreaks, during which a limited number of cases generated a disproportionately large proportion of the secondary cases under specific conditions in hospitals, occurring in some outbreaks [ ] [ ] [ ] . two super spreading events have been documented in ksa and in the republic of korea. in the republic of korea, the practice of "doctor shopping", extended stays in overcrowded emergency departments, cultural practices of large numbers of family members visiting sick relatives, and environmental contamination amplified transmission from some patients to others , , , . during the outbreak in ksa in at the ministry of the national guard hospital, a high number of secondary cases were among hcp very quickly after the hospitalization and a surgical procedure of the index case . these events triggered comprehensive review ipc in hospitals, emergency department layout, movements of patients, triage of respiratory visits, duration of emergency department stay, training of hospital staff and disinfection of healthcare facilities. our study confirmed that age and presence of comorbidities are linked to increased risk of death, similar to previously published results , whereas being hcp was protective. the protective effect of hcp could be explained by the fact that hcp are more likely to be younger (< years old) and have fewer underlying medical conditions than hospitalized patients, but also that they are likely to be identified earlier or seek medical care soon following contact with a confirmed patient. the proportion of asymptomatic secondary cases identified during outbreaks has increased since . there is no evidence to suggest that this represents changes in virus pathogenicity, epidemiology or transmission www.nature.com/scientificreports www.nature.com/scientificreports/ patterns of mers in recent years. however, the increase in the number of reported asymptomatic cases is hypothesized to be due to earlier detection efforts from more aggressive contact identification and testing during hca-outbreaks since as testing policies adopted and implemented by ksa and other countries have changed following the large outbreaks in jeddah/riyadh in , , . in , - % of the laboratory confirmed hcp secondary cases experienced no symptoms and were detected as part of a policy to test all contacts irrespective of symptoms (table ) . we believe that the identification of hcp asymptomatic cases, and their subsequent isolation, has had a strong impact on prevent further human to human transmission in health care settings. this is visually demonstrated in fig. c by the outbreak labelled sau _ , which included (of www.nature.com/scientificreports www.nature.com/scientificreports/ reported cases) asymptomatic cases. while this is a large number of secondary cases, we argue that the early identification, isolation and recovery of these asymptomatic/mildly symptomatic cases effectively stopped human to human transmission. our study has several limitations due to variability in the completeness and quality of case-based data provided to who since and also due to the lack of detailed information on the timing specific interventions were implemented in relation to each outbreak. without detailed information on the timing of interventions in each health care facility it was not possible in our analyses to determine which intervention or combination of interventions had the greatest impact on stopping the mers outbreaks. moreover, prior to , contacts without symptoms were not tested for mers-cov infection, thus the rate of identification of secondary cases was drastically different prior to , which complicates the comparison of data collected before and after . the improvements in data reporting on cases (e.g., more systematic reporting of underlying conditions, reported exposures, contacts between patients) from allowed us to perform better analyses with less missing values. we continue to encourage the policy of identifying, following and testing of all high risk contacts of mers patients in hca-outbreaks , , . the natural history of asymptomatic infection and role of asymptomatic or mildly symptomatic hcp in transmission of the virus between patients, requires detailed scientific studies to better understand their potential role in transmission . the sharing of outbreak experiences between affected hospitals within and between countries and a detailed evaluation of the impact of non-therapeutic interventions is critical to our understanding and for the prevention of nosocomial outbreaks of respiratory pathogens. health care professionals and hospitals currently have tools to limit the extent and impact of such events, which include early identification and isolation of suspect patients and strict adherence to standard infection prevention and control measures. these are the hallmark of effective mers-cov control. a combination of interventions including the efficient triage of patients with respiratory symptoms at hospital entry; limiting wait times and overcrowding in waiting areas; isolation of suspected and confirmed cases; appropriate use of droplet personal protection equipment by hcp; basic hand hygiene; increased protective aerosol precautions for hcp during aerosol-generating medical procedures; efficient surface and environmental decontamination of areas with mers patients, and extensive contact tracing, can prevent human to human transmission in 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reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -j ewxk q authors: lin, jing-wen; sodenkamp, jan; cunningham, deirdre; deroost, katrien; tshitenge, tshibuayi christine; mclaughlin, sarah; lamb, tracey j.; spencer-dene, bradley; hosking, caroline; ramesar, jai; janse, chris j.; graham, christine; o’garra, anne; langhorne, jean title: signatures of malaria-associated pathology revealed by high-resolution whole-blood transcriptomics in a rodent model of malaria date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: j ewxk q the influence of parasite genetic factors on immune responses and development of severe pathology of malaria is largely unknown. in this study, we performed genome-wide transcriptomic profiling of mouse whole blood during blood-stage infections of two strains of the rodent malaria parasite plasmodium chabaudi that differ in virulence. we identified several transcriptomic signatures associated with the virulent infection, including signatures for platelet aggregation, stronger and prolonged anemia and lung inflammation. the first two signatures were detected prior to pathology. the anemia signature indicated deregulation of host erythropoiesis, and the lung inflammation signature was linked to increased neutrophil infiltration, more cell death and greater parasite sequestration in the lungs. this comparative whole-blood transcriptomics profiling of virulent and avirulent malaria shows the validity of this approach to inform severity of the infection and provide insight into pathogenic mechanisms. scientific reports | : | doi: . /srep offers a feasible alternative as it is one of the "highways" of the immune system via which naïve, and activated or primed immune cells travel between lymphoid organs and the tissues affected by the infection. by profiling global transcriptomes of whole blood, insights can be obtained into the complex changes in systemic or even local host responses brought about by an infection, and thus inform more targeted mechanistic studies. to investigate the use of genome-wide transcriptomic profiling of the whole blood in identifying pathology signatures in malarial infection, and to gain insights into the mechanisms underlying pathology, we used the well-establised p. chabaudi chabaudi rodent malaria model to study malarial immunology and pathology , . using two strains of p. c. chabaudi, as and cb, that differ in virulence in c bl/ mice, we performed high-resolution comparative whole-blood transcriptomic analysis throughout the acute phase of the blood-stage infection, and identified several transcriptomic signatures associated with severe malarial pathology before the onset of pathology or disease. the virulent cb strain of p. c. chabaudi induces more severe pathology in the acute phase of a blood-stage infection compared with the avirulent as strain. infection of c bl/ mice was initiated by intraperitoneal inoculation of infected red blood cells (irbc) of p. c. chabaudi as or cb. infection with the cb strain gave rise to a more severe infection, resulting in % (range - %) of mice reaching the humane end points (more than % weight loss, persistent laboured breathing and severe hypothermia), while all as infected mice survived the infection without showing severe pathologies (fig. a) . as and cb infected mice showed comparable irbc loads (parasitemia multiplied by total rbc numbers), despite the fact that higher peak the total numbers of infected red blood cells (irbc) per ml of blood (b) and the parasitemia (percentage of infected irbc) (c) in the mice infected with as or cb parasites. (d,e) the change in rbc numbers during the infection in as or cb infected mice (d) and the hemoglobin (hgb) concentration in the blood of the infected mice (e). (f,g) the percentage change in temperature (f) and weight (g) during the infection in as or cb infected mice. data were pooled from mice in independent experiments. graphs show mean with sem, mann-whitney u test was performed (* p < . , **p < . , ***p < . ). scientific reports | : | doi: . /srep parasitemias were observed in the acute cb infection (fig. b,c) . a more severe rbc loss with a significantly lower hemoglobin concentration was observed in cb infection at days post infection (dpi) compared to that in as infected mice, agreeing with previous observations, which showed more severe anemia in cb infected balb/c mice . moreover, the rbc loss in cb infected mice is longer lasting, even after the peak of infection at dpi (fig. d,e) . in addition, at dpi, cb infected mice showed greater temperature and weight loss (fig. f,g) . virulent cb and avirulent as strains of p. c. chabaudi induce distinct responses in host whole blood transcriptome. to investigate whether as and cb parasites induce different host responses that might contribute to the differences in severity of the blood-stage infection we carried out genome-wide transcriptomic analyses of whole blood during the acute phase of infection. peripheral blood was collected into tempus tubes via cardiac puncture from c bl/ mice infected with as or cb at , , , , , and dpi. blood samples collected from age-matched uninfected animals at day and day were used as naïve controls to exclude transcriptional changes due to time. the total rna was extracted, depleted of globin mrna and analysed using illumina mouse wg- v . beadarrays. spearman's rank correlation coefficient (r s ) analysis of unfiltered transcripts normalised across the median of all samples, revealed high levels of similarity amongst naïve and , dpi samples in both as and cb infections (fig. ai ,ii, r s ranging from . to . ), while from dpi onwards the whole blood transcriptomes diverge significantly from the earlier time points (r s ranging from . to − . compared to naïve controls). when comparing as and cb infections at each of the time points, lower correlation values were observed between - dpi (fig. aiii , r s = . , . , . , respectively). this indicates that as and cb infections induce different host responses at these times of infection. as infected mice and those cb infected mice that survived this phase of infection showed similar profiles at dpi (fig. aiii , r s = . ). differential expression analysis (anova unequal variance with post-hoc hsd test, fdr < . ) was performed on transcripts normalised to the median of their respective naïve controls (supplementary table ). at any of the post-infection time points examined, there were differentially expressed transcripts with a greater than -fold change in the infected mice compared to the naïve mice (fig. b, supplementary fig. a) . consistent with correlation analyses of unfiltered transcripts (fig. a) , only a few transcripts were differentially expressed at and dpi (fig. b) . strikingly, at dpi, a large majority of transcripts were down-regulated compared to naïve controls in both as and cb infections. however, the number was greater in cb infected mice compared to as (as out of transcripts, cb out of transcripts) (fig. b) . the number of down-regulated transcripts increased steadily from to dpi in as infection (fig. b) . while down-regulation of transcripts was also observed at - dpi in the cb infection, the level of down-regulation was significantly lower at dpi and there was a sudden up-regulation of transcripts occurred only in cb infected mice at this time point (fig. b , supplementary fig. a ), a critical time point when infected animals were either recovering from acute infection or suffering from severe pathologies leading to death (fig. ) , indicating that these up-regulated transcripts may relate to the manifestation of severe pathology in the cb infection. indeed this higher level of transcriptional up-regulation was further confirmed in cb infected mice that had reached humane end points at dpi ( supplementary fig. b ). this up-or down-regulation of gene expression in whole blood are due to both the leukocyte population changes and transcript regulation during the infection ( supplementary fig. c ). in the blood associated with severe pathology. a hierarchical clustering of the differentially expressed transcripts of whole blood revealed clusters of transcripts comprised of genes that were significantly more up-regulated in cb infected mice between and dpi than in the blood of as infected mice ( supplementary fig. , genes in fig. a) , during which severe clinical signs were manifested (severe hypothermia, weight loss and host death) (fig. ). ingenuity pathway analysis (ipa) diseases and function analysis showed that some of these genes were significantly enriched in 'functions' of inflammation and myeloid cell movement (fig. b) . interestingly, genes (fig. a , indicated in red) were enriched in 'disease' of severe acute respiratory syndrome (sars) (fig. b) . these genes have been shown to be amongst the top up-regulated genes in transcriptomic analysis of pbmcs (peripheral blood mononuclear cells) isolated from sars patients suffering from severe lung inflammation (fig. c) . importantly, in of the cb infected mice that had reached humane end points at dpi, these genes showed an even higher level of up-regulation compared to naïve controls (fig. c) , indicating possible lung pathology in these cb infected mice. to identify further pathology-related blood transcriptomic signatures, we included the microarray data of blood isolated from the cb-infected mice that had reached the humane end points, and followed the same differential expression analysis as described above, yielding a total of differentially expressed transcripts (supplementary table ). a self-organising map (som) clustering method was used to generate clusters of co-expressing transcripts ( supplementary fig. , supplementary table ) . the cluster expression level was defined as the average fold-change of all transcripts within each module compared to that of naïve controls (fig. d) . the clusters were annotated with ipa diseases and function analysis and manually curated. several clusters, c , c , c , c , c and c showed greater up-regulation in cb infection during - dpi, indicating their association with lethality/severe pathology in cb infection (fig. d , indicated in red). modular analysis identified an early platelet aggregation signature only associated with the virulent p. c. chabaudi cb infection. clusters c and c , were exclusively up-regulated in the cb infection, especially in the mice that had reached humane end points at dpi (fig. d ). c showed a maximum average of . -fold up-regulation and c showed a maximum average of . -fold up-regulation in cb infected mice that were dying from the infection. twenty-seven genes within these clusters are associated with platelet aggregation/pro-coagulation, a common feature of severe malaria infection (fig. a) . they are significantly enriched in canonical pathways integrin signalling (−log(p value) . , z score ) and actin cytoskeleton signalling (−log (p value) . , z score ). importantly, this set of genes was significantly up-regulated in all cb infected mice as early as dpi (fig. b) , at the time of which very few genes were differentially expressed compared to naïve controls (fig. b) . modular analysis identified a more pronounced and longer-lasting anemia signature in the virulent p. c. chabaudi cb infection. two clusters, c and c contained genes related to anemia (fig. a ). for most of these genes, their down-regulation is associated with anemia, and up-regulation is associated with alleviation of anemia ( supplementary fig. ). the anemia signature was already present at dpi in both infections ( fig. a ), prior to clinical observation of rbc and hemoglobin loss at dpi (fig. ). the mean normalised intensity of these genes was significantly down-regulated compared to naïve controls, and cb infected mice had even significantly lower values (fig. b) . the peak activation z scores calculated by ipa for this anemia signature of cb infection was . -fold higher compared with that of as (fig. c , . vs . , dpi), agreeing with the more severe rbc loss (area under the curve analysis) in cb than as infection ( . -fold, . vs . ) (fig. d) . interestingly, at dpi, out of the genes within this signature were already up-regulated in as infections compared to naïve controls; by contrast, the majority ( ) of these genes were still down-regulated in the blood of cb infected mice (fig. a, supplementary fig. ). the activation z score was − . in as infected mice indicating anemia alleviation at this time point, compared to that of . in cb infected mice (fig. c ), which indicates a longer-lasting anemia. this is consistent with the more severe rbc and hemoglobin loss in cb infection at dpi compared to as (fig. d,e) . moreover, in support of this data, ipa canonical pathway analysis of heme biosynthesis ii pathway also showed that genes in this pathway were more down-regulated or less up-regulated in cb than in as infected mice at dpi compared to naïve controls (fig. d ). together, these data suggest a deregulated or stressed erythropoietic response in cb infected mice, leading to a stronger and prolonged severe anemia (fig. d ,e). the lungs of p. c. chabaudi cb infected mice. in addition to the genes we identified from hierarchical clustering, the som analysis revealed a further genes in clusters c and c related to sars, and the majority of these genes were up-regulated only in cb infected mice that reached human end point at dpi ( supplementary fig. a ). we therefore investigated whether this lung inflammation signature was reflected by more severe lung pathology in cb infected animals. lungs isolated from systemically perfused cb infected animals were of a darker coloration than lungs of uninfected or as infected mice (fig. a) . examination of bronchoalveolar lavage fluid showed significantly higher levels of igm in the lungs of mice infected with as or cb parasites compared to naïve mice (fig. b) . the hematoxylin and eosin (h&e) stained sections of perfused lungs a) were even more highly up-regulated compared to naïve controls in the blood extracted from cb infected mice that had reached humane end points at dpi compared to randomly selected mice infected with as or cb parasites at dpi. fold changes in sars patients were from ref. . (d) the twenty-four modules identified by using self-organising map (som) method were presented with the average fold change of all differentially expressed transcripts within each module compared to naïve controls. red represents positive mean fold change above zero (white) and blue indicates negative mean fold change. the clusters were annotated with ipa diseases and function analysis with manual curation. clusters indicated in red showed greater up-regulation in cb infection during - dpi. from both as and cb infected mice showed signs of leukocyte infiltration. in the lungs of some cb infected mice, patches of dense leukocyte accumulation between epithelial walls were observed in close proximity to hemozoin (hz) crystals ( supplementary fig. b) . we also observed more cell death in the lung tissues of cb infected mice (greater than two fold) compared to as infected mice by tunel staining (fig. c) . flow cytometry analysis confirmed leukocyte infiltration in the lungs of both as and cb infected mice compared to that in naïve mice (fig. d , gating strategy in supplementary fig. ). we further characterised the cell populations within the infiltrating leukocytes in the lungs of infected animals. although cd + t cells, cd + b cells and cd − cd − innate cell numbers all increased in both infections, they did not significantly differ between as and cb infections; however, there was a trend towards higher t cell numbers in the as infection, and more innate cells in the cb infection ( supplementary fig. b,c) . within the innate cell populations, both the percentage and the cell numbers of ly g + cd b + neutrophils were significantly increased in lungs of cb infected mice compared to as infected mice (fig. e) , whereas other myeloid cell populations did not significantly differ between the two infections ( supplementary fig. d ). this observation of neutrophil infiltration is consistent with the ipa 'disease and function' analysis on the sars signature, which was up-regulated more in cb infection compared to as infection, indicating myeloid cell movement (fig. b) , especially neutrophils (z score . ). one of the top up-regulated genes, s a (mrp , myeloid-related protein ) , indicates that neutrophils may be involved in the lung pathology of the cb infection. we therefore investigated whether the up-regulation of mrp transcript in the blood is associated with higher protein level and neutrophil infiltration in the lungs. we analysed the concentration of mrp protein in the serum at dpi. while high level of mrp protein was also detected in sera of all ( ) cb infected mice, it was below detection limit by elisa in the sera from out of as infected mice and all ( ) from naïve mice . f) . we next measured the amount of mrp in the whole lung lysates, and found that cb infected mice contained more than twice the amount of mrp protein compared to that of lungs of as infected mice at dpi; moreover, this upregulation was observed as early as dpi (fig. g) . ifng, il , kc (cxcl ) and lix (cxcl ) were higher in the lungs of cb infected mice, indicating a heightened proinflammatory response in the cb infection ( supplementary fig. a ). immunohistochemical staining of mrp on lung sections showed more mrp + cells present in the lungs of cb infected mice compared to naïve and as infected mice ( supplementary fig. b) , and flow cytometry analysis confirmed a greater than two fold increase of mrp hi ly g + neutrophils in cb compared to as infected mice (fig. h) . together, these data show that the blood transcriptomic signature of lung inflammation is linked to an mrp -associated neutrophil response in the lungs of mice infected with the more virulent cb strain of p. c. chabaudi compared with the avirulent as strain. amount of hz accumulated in the lungs of cb infected mice compared to as infected mice (fig. a) . this observation suggested a higher level of sequestration/accumulation of irbc in the lungs of cb infected mice. to investigate the level of sequestration of irbcs in p. c. chabaudi as and cb infected mice, we generated transgenic parasites, pccasluc p and pcccbluc p , expressing luciferase constitutively throughout the plasmodium life cycle under the control of eef a promoter (supplementary fig. ). at day , and dpi, the total parasite load was determined by measuring luciferase activity from μ l tail blood when the parasites were at late trophozoite stage , and the level of sequestration in different organs was investigated during schizogony. consistent with the peripheral load of irbc (fig. b) , there were no significant differences in luciferase activity between pccasluc p and pcccbluc p infected mice at dpi either in peripheral blood or by whole body imaging (fig. b) . after intensive systemic perfusion, the luciferase activities in isolated organs were measured and relative ratio of sequestration was calculated as the level of luciferase activity per organ (total flux per second) relative to the total parasite load measured in peripheral blood before schizogony (relative light unit, rlu) (fig. c) . consistent with previous findings , both as and cb irbc, sequester/accumulate mainly in the spleen, liver and lungs, with no significant signals observed in the kidney or brain. the relative levels of sequestration/accumulation in the spleen and liver were similar between as and cb infections. by contrast, a significantly higher level of sequestration in the lungs occurred in the cb infection at dpi, and the trend was still maintained at dpi (fig. c) . the higher level of sequestration/accumulation of schizonts in the lungs is consistent with the observation of greater amounts of hz in the lungs of cb infected mice compared to as infected mice (fig. a ). host genetics and immune status play important parts in the outcome of an infection with plasmodium , . however, there is an increasing amount of evidence showing that genetic diversity of the parasite also contributes to the varying severity of malarial disease. in this study we used a top-down systems analysis of peripheral blood to investigate whether transcriptomic signatures could be identified that would indicate or predict severity of acute blood-stage malaria caused by strains of p. c. chabaudi of differing virulence. using high-resolution (c) bar charts showing the relative ratio of sequestration in different organs, which was quantified as the level of luciferase activities in the perfused ex vivo organs relative to the total parasite load measured in peripheral blood at late trophozoite stage (b, left). all data in (b,c) were pooled from independent experiments (n = - in total). in all bar charts, median values are shown and each dot represents an individual mouse. mann-whitney u test was performed, p values are provided when significant difference was observed. profiling of the whole blood transcriptomics over multiple time points during the acute phase of infection, and data-driven modular analysis, we investigated the involvement of biological processes rather than specific genes, and uncovered several transcriptomic signatures related to severe pathologies in the virulent cb infection. these include distinct signatures for platelet aggregation, anemia and lung inflammation, which can be seen at different time points and distinguished the two infections. this analysis also revealed several signatures common between avirulent as and virulent cb infections, but they occurred at different time points or were of different magnitude. this highlights the value of studying pathological factors in the host induced by parasites over the course of the infection and not at a single time point. the platelet aggregation signature was highly up-regulated in all cb infected mice that had reached humane end points. this was the earliest pathology signature identified in this study and similar to the anemia signature was detected before the onset of severe disease. this set of genes was up-regulated as early as days post infection in all cb infected mice regardless of eventual survival. it has been shown that in severe p. falciparum infections, platelets mediate irbc clumping and adhesion , . these observations of association between infection severity and platelets aggregation suggest that similar mechanisms underlie pathology in both the p. c. chabaudi model of malaria and in human infections, and the experimental model may be useful to explore the underlying mechanisms. it would be of great interest to analyse the platelet aggregation signature in human malarial infections and investigate whether this transcriptomic signature could be used as an early marker to predict development of severe pathology. the anemia signature identified was present in the whole blood transcriptome ahead of the clinical onset of rbc loss in both avirulent as and virulent cb infections, but it was stronger and lasted longer in the cb infection. this transcriptomic signature predicted the more severe and longer-lasting anemia we have observed in cb infections . both the anemia signature and the heme biosynthesis ii pathway analysis indicate a deregulated or stressed host erythropoietic response in the more severe cb infection. in addition to the platelet aggregation and anemia signatures, we identified a lung inflammatory signature in cb infected mice. although sequestration of p. c. chabaudi as parasites in lungs has been documented , lung damage has not been previously reported for this experimental model. we confirmed that this sars-related lung inflammation signature in the blood was indeed associated with a more severe pulmonary neutrophilic infiltration and more cell death in the lungs in cb infections. furthermore, it was linked to a higher level of sequestration of cb irbc in this organ. both p. falciparum and p. vivax can sequester within the pulmonary microvasculature and cause lethal malaria-associated acute respiratory distress syndrome (ma-ards) . members of the pfemp family (p. falciparum erythocyte membrane protein- ) of variant surface-expressed parasite proteins have been shown as parasite ligands mediating parasite cytoadherence . although pfemp is lacking in other plasmodium parasites, another multigene family pir (plasmodium interspersed repeat) is present in most, if not all, species of plasmodium; and there is evidence that some pirs in p. vivax bind to icam- endothelial receptor in vitro . it is possible that differential pirs expression between p. c. chabaudi as and cb is responsible for this differential pulmonary sequestration ability. however, it is also possible that as parasite is removed more effectively from the lung than cb due to the higher inflammation caused by cb infection. the higher level of cb pulmonary sequestration leaves greater amounts of hemozoin compared with the as parasite. there is evidence that hz can directly induce pulmonary proinflammatory responses . in addition it has been shown that parasite-derived microparticles can induce macrophage activation in a tlr (toll-like receptor )-myd dependent manner . in our study, cb infection induced higher level of inflammation (ifng, il- and mrp ) in the lungs of infected mice. mrp (s a ) is one of the top up-regulated genes identified in the lung inflammation signagure. together with s a (mrp ), mrp / forms a heterodimer complex that has previously been shown to be a potent chemotactic factor for myeloid cells, especially neutrophils . mrp / are tlr ligands and are recognized as damage-associated molecular pattern molecules (damp) involved in many inflammatory diseases and infections . for example, in tuberculosis and influenza infection, mrp / is shown to exacerbate pro-inflammatory responses, cell-death and pathogenesis , . of relevance here, mrp protein is significantly increased in p. falciparum and p. vivax infected patients , . interestingly, in our p. c. chabaudi mouse model, mrp was detectable in all mice infected with the virulent cb strain; by contrast, it was detectable in only % of mice infected with the avirulent as strain. moreover, when mrp was detected in as infected mice, it was present at significantly lower level than that in cb infected mice. this coincided with a significantly higher number of mrp hi ly g + neutrophils in the lungs of cb infected mice. it is possible that mrp + cells respond to the microparticles upon rupture of sequestered cb schizonts, leading to proinflammatory response and recruiting more mrp + neutrophils. our analysis offers evidence that different parasite strains, exhibiting different sequestration tendencies, can lead to different levels of lung inflammation and damage. deciphering the complex host immune responses during acute malaria is extremely challenging. here we demonstrate that whole blood transcriptomic signatures can help to reveal severe malaria-associated pathologies, often preceding clinical observations. our data demonstrate the potential in searching further transcriptomic signatures in human malaria for severity diagnosis and prognosis. furthermore, these blood signatures can also provide crucial information about the pathogenic processes taking place in organs or tissues during infection, as demonstrated here with the neutrophil-related lung inflammation signature. this unbiased modular analysis of blood transcriptomic data also offers a promising method to search for protective mechanisms in mouse and human malarial infections. this is particularly important for p. vivax infections of humans, because of its greater genetic diversity , and the recent surge in reports of severe and fatal p. vivax malaria , . mice. female c bl/ aged - weeks from the spf unit at the francis crick institute mill hill laboratory were housed under reverse light conditions (light . - . , dark . - . gmt) at - °c, and had continuous access to mouse breeder diet and water. core body temperature was measured with an infrared surface thermometer (fluke); body weight was calculated relative to a baseline measurement taken before infection; and erythrocyte density was determined on a vetscan hm haematology system (abaxis). this study was carried out in accordance with the uk animals (scientific procedures) act (home office licence / and / ), and was approved by the francis crick institute ethical committee. walliker, university of edinburgh, uk and subsequently passaged through mice by injection of infected red blood cells (irbc) at the mrc national institute for medical research, uk and cryopreserved as described . for experimental work, infections were initiated by intraperitoneal (i.p.) injection of irbc derived from cryopreserved stocks. the course of infection was monitored on giemsa-stained thin blood films by enumerating the percentage of rbc infected with asexual parasites (parasitemia). the limit of detection for patent parasitemia was . % infected erythrocytes. mice were culled upon reaching humane end points by showing the following signs: emaciation (more than % weight loss), persistent labored breathing, severe hypothermia (body temperature below °c), inability to remain upright when conscious or lack of natural functions, or continuous convulsions lasting more than min. p. c. chabaudi as and cb expressing luciferase under the control of the constitutive promoter eef a were generated by transfection with the construct ppc-luc p, targeting the neutral p p locus (pchas_ or pchcb_ ). transfection and cloning of transgenic p. chabaudi parasites were performed as described previously , and integration was verified by southern blot analysis of chromosomes separated by pulsed field gel (pfg) as described . the construct ppc-luc p was modified from ppc-luccam by replacing the p. chabaudi ssu targeting region with p targeting region, chab - , generated by gene synthesis (genewiz llc, nj, usa). female c bl/ mice aged between - weeks were intraperitoneally infected with irbc of p. c. chabaudi as or cb. at , , , , and days post infection, . ml of blood was collected via cardiac puncture into ml tempus rna stabilising solution (applied biosystems). naïve samples were also collected at day (the day of infection) and day (the end of the experiment) and used as controls. samples were snap frozen on dry ice and stored at − °c until rna isolation. total blood rna was extracted using perfectpure rna blood kit ( prime) and globinclear kit (ambion) was used to remove globin mrna according to the manufacturer's instructions. crna samples were prepared from μ g globin reduced blood rna using illumina totalprep rna amplification kit (ambion) and hybridized to illumina mouse wg- v . beadarrays according to the manufacturer's protocols. at each step, the quantity and quality of the rna samples was verified using nanodrop spectrophotometer (thermo fisher scientific) and caliper labchip gx (caliper life sciences). microarray data analysis. illumina beadstudio/genomestudio software was used to subtract background and scale average samples' signal intensity and genespring gx . software (aigent technologies) was used to perform further normalization and analyses as described previously . first, all signal intensity values less than ten fluorescent units were set to equal ten, log transformed and per chip normalised using th percentile shift algorithm. transcripts were further normalized to the median across all samples or to the median of control samples. transcripts were first selected if they were present (cut off . ) in ≥ % of all samples, and further filtered with a minimum of -fold expression up-or down-change compared with the median intensity across all samples. all microarray data are deposited in geo under accession number gse . genespring software was used to perform statistical tests, anova unequal variance with post-hoc tukey's hsd (honest significant difference) test, followed by benjamini-hochberg multiple test correction (fdr < . ); fold change was further performed on the combined list of transcripts differentially expressed either in as or cb infection, with -fold cut off compared to naïve controls. the set of transcripts was defined as differentially expressed transcripts and was used for further analyses. hierarchical clustering of samples at different infected time points compared to naïve were performed using pearson uncentred correlation with an average-linkage-clustering algorithm that organizes all transcripts according to their trend of expression across all samples. the hierarchical clustering on the transcripts differentiall expressed in as or cb (supplementray table ) across the acute phase of infection was performed using euclidean distance metric with ward's linkage rule. for the pathological modules (in fig. ) , prediction was performed on the transcripts differentially expressed in as or cb infection including the samples collected at dpi (supplementray table ) using the self-organising map algorithm in genespring. euclidean distance was used for similarity measurement and the maximum number of iterations was set at e . the initial learning rate was set at . and initial neighbourhood radius , the number of grids was tested from × , till less than % of clusters have similarities above %, at a final grid of × ( clusters). ingenuity pathways analysis (ipa) (qiagen) was used to identify enriched disease and functions and networks. the significance of the association between the dataset and each analysis was measured using fisher's exact test, z score cut-off and/or p value cut-off . . this program was also used to map the canonical pathways and overlay it with expression data from the dataset. module annotation was determined using disease and function analysis in ipa. to obtain bronchoalveolar lavage fluid (balf), mice were terminally anaethetised, and lungs were cannulated and inflated with μ l pbs. the liquid was retrieved and spun at g for min at °c. supernatants were obtained and kept at − °c till further analysis. igm levels in balf were quantified using a sandwich elisa. (southern biotech). detection of mrp and cytokines in sera and lung lysate. mouse serum was collected via cardiac puncture, clotted in room temperature for min and collected by centrifuging twice at , g for min at °c. lung proteins were extracted in ripa lysis buffer with protease inhibitor cocktails (sigma) and homogenized with polytron homogenizer (kinematica) on ice. protein levels were quantified by pierce bca protein assay (thermo scientific) as per the manufacturer's instructions. all plates were read on a safire ii plate reader (tecan). mouse s a elisa kit (r&d systems) was used to determine the level of s a /mrp in serum and lung lysate samples following manufacturer's instructions. cytokine concentrations were determined using cytometric bead array (biolegend) following the manufacturer's manual. histology and immunohistochemical analyses. the lungs were extensively perfused with ml pbs and then inflated by injection of ml of % neutral buffered formalin (nbf) through the tracheal cannula. tissue was then fixed overnight in % nbf, and transferred into % ethanol until embedded in paraffin and sectioned. each lung specimen was stained with haematoxylin and eosin (h&e). for each mouse, the number of hemozoin crystals were quantified from randomly selected fields on h&e stained slides under leica light microscopy ( × ). immunohistochemical staining was performed to examine the expression of mrp on paraffin-embedded lung sections with anti-mrp antibody (clone b ). tunel staining was performed using apoptag ® fluorescein in situ apoptosis detection kit (merck millipore) following the manufacturer's protocol. imaging of slides was performed on a vs slide scanner (olympus) with a vc camera, a uplsapo lens, at a magnification of × or × . images were processed and analysed using olyvia image viewer . (olympus) and fiji . and tunel positive cell numbers were quantified in an area of nm using olyvia image viewer . . in vivo imaging and luciferase assay. mice were infected intraperitoneally with rbc infected with pccasluc p or pcccbluc p parasites; and at each time point μ l of heparinized tail blood was collected before sequestration . bioluminescence was assessed with the luciferase assay system (promega) according to the manufacturer's protocol and quantified with the tecan safire plate reader and magellan software (tecan). under these conditions, bioluminescence intensity is proportional to the amount of parasites in this blood volume , which reflects the total parasite load before sequestration. at the time of maximum sequestration ( . - . h gmt, reverse light) , d-luciferin ( mg/kg, caliper life sciences) was injected subcutaneously min before whole-body and organ imaging. mice were terminally anaesthetized and systemically perfused by intracardiac injection of ml pbs . the brain, lungs, liver, spleen, left kidney and gut were removed immediately and luciferase assessed using in vivo imaging system ivis lumina (xenogen), with a cm field of view, a binning factor of , and an exposure time of s. bioluminescence (total flux per second) was quantified with the software living image (xenogen) by adjusting a region of interest to the shape of each organ. to account for the influence of total parasite load on the number of parasites sequestered in the organs, bioluminescence in the organs was normalized to total parasite load. luciferase activities measured in the organs were divided by parasite load in μ l blood (see above), allowing comparison between mice with different parasite burdens. quantifying genetic and nongenetic contributions to malarial infection in a sri lankan population malaria infection changes the ability of splenic dendritic cell populations to stimulate antigen-specific t cells disruption of il- signaling affects t cell-b cell interactions 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during tuberculosis damp molecule s a acts as a molecular pattern to enhance inflammation during influenza a virus infection: role of ddx -trif-tlr -myd pathway mrp / as marker for plasmodium falciparum-induced malaria episodes in individuals in a holoendemic area up-regulated s calcium binding protein a in plasmodium-infected patients correlates with cd (+ )cd (+ ) foxp regulatory t cell generation the malaria parasite plasmodium vivax exhibits greater genetic diversity than plasmodium falciparum is plasmodium vivax malaria a severe malaria?: a systematic review and meta-analysis evidence and implications of mortality associated with acute plasmodium vivax malaria vector transmission regulates immune control of plasmodium virulence transformation of the rodent malaria parasite plasmodium chabaudi high-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite plasmodium berghei detectable changes in the blood transcriptome are present after two weeks of antituberculosis therapy fiji: an open-source platform for biological-image analysis were made in graphpad prism, each dot represents an individual biological replicate and p-values were derived from mann whitney u test or multiple t-test. we would like to thank thibaut brugat, audrey vandomme and barbara capuccini for critical reading of the manuscript. we are grateful to the brf at mill hill for animal husbandry, to the high-throughput screening (hts), flow cytometry, experimental histopathology and microscopy facilities at mill hill for excellent technical support. this work is supported by the francis crick institute, which receives its funding from the uk medical research council (grant u ), cancer research uk, and the wellcome trust (grant wt ma). key: cord- -tls i authors: leidenberger, s.; schröder, ch.; zani, l.; auste, a.; pinette, m.; ambagala, a.; nikolin, v.; de smit, h.; beer, m.; blome, s. title: virulence of current german pedv strains in suckling pigs and investigation of protective effects of maternally derived antibodies date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: tls i porcine epidemic diarrhea (ped) has caused tremendous losses to the united states pig industry since . from , outbreaks were also reported from central europe. to characterize the central european pedv strains regarding their virulence in suckling piglets, and to assess the protective effect of maternally derived antibodies (mda), four trial groups were randomly assigned, each consisting of two pregnant sows and their litter. to induce mda in a subset of piglets, two sows received a cell culture-adapted pedv strain, and another two sows were inoculated with field material from german ped outbreaks. four sows stayed naïve. subsequently, all piglets were inoculated with the corresponding pedv strains at an age of to days, and virus shedding, clinical signs and occurrence of specific antibodies were assessed. piglets without mda showed a morbidity of % and low lethality, while almost all mda-positive piglets stayed clinically healthy and showed considerably lower virus shedding. taken together, the central european pedv strains showed rather low virulence under experimental conditions, and pre-inoculation of sows led to a solid protection of their offspring. the latter is the prerequisite for a sow vaccination concept that could help to prevent ped induced losses in the piglet sector. clinical and pathological observations. clinical signs upon inoculation of sows. two sows, which were inoculated with a cell culture-adapted pedv strain (pedv eu, group a ) showed slight depression over a period of four days, accompanied by reduced feed intake. in contrast, the two sows of group b (inoculated with german field pedv, de) showed diarrhea and vomiting upon inoculation. moreover, both sows were anorectic for one day. thereafter, one of the sows (ear tag ) was still showing slight depression over four days whereas the other was free of clinical signs again. all four sows of groups a and b , which stayed naïve prior to farrowing, remained healthy during the pre-farrowing time. clinical signs upon inoculation of piglets. sows which were naïve before farrowing and got infected via their inoculated piglets during the trial, showed more severe clinical signs according to our standardized score system for sows (table ) than sows, which were directly inoculated four weeks before farrowing (see above). almost complete anorexia, watery diarrhea, and decrease in milk production was observed from h after the inoculation of the piglets in sows of groups a and b (see supplementary fig. f ). the lack of milk production led to a more severe situation for their unprotected infected piglets (malnutrition). sows, which were inoculated before farrowing (sows of groups a and b ), did not show any clinical signs indicative for ped upon challenge of their piglets. one sow of group a died from septicaemia two days after farrowing (necropsy revealed severe peritonitis after uterine rupture and intestinal invagination). the respective piglets (which had all taken colostrum) were reared by the remaining sow of group a . as can be seen in fig. , none of the mda-positive piglets showed any severe clinical signs indicative for ped upon challenge inoculation. very mild signs were discontinuously observed in some animals (pasty feces). one litter (group b , sow ) showed general weakness and remittent shivering leading to clinical scores prior to inoculation. a viral genesis could not be detected by routine pcrs (porcine enteroviruses (pev), suid alphaherpesvirus- , classical and african swine fever virus, atypical porcine pestivirus). piglets without mda and inoculated with the cell culture-adapted virus (pedv eu, group a ) started vomiting hours post inoculation (hpi), and showed diarrhea from hpi. the clinical signs remained over to days with diarrhea as the leading sign (see fig. , upper graph). mda-negative piglets challenged with the german field material (de), started to show signs indicative of ped at hpi, also beginning with vomiting followed by diarrhea. clinical signs persisted over to days (fig. , lower graph) . as the respective sows showed an almost complete cessation of milk production, piglets showed signs of malnutrition. two piglets had to be euthanized with an endpoint clinical score because of dehydration and general weakness. necropsy of the euthanized piglets showed gas-filled intestines. all other piglets were without specific results indicative for ped at the time point of necropsy (end of the trial). clinical score values differed significantly between groups with and without mda from to days post inoculation (dpi). in addition, a statistically significant difference was observed at dpi between groups a and a , and to dpi between groups b and b . the above mentioned condition in one litter of group b led to a significant difference at the day of challenge that was however not ped related. interested in feed without clear feed intake watery feces, reddened anal region, vomiting strong depression, almost entirely resting, decreased milk production, abortion total anorexia watery feces with blood or fibrin added, highly reddened anal region, vomiting table . clinical score system for sows in ped infection studies. the humane endpoint was defined as a cumulative score > . moreover, euthanasia was carried out after abortion or other signs indicative for inacceptable suffering. shed virus from or days post inoculation (dpi) for to days, while the group inoculated with the field virus (b ) was pcr-positive from dpi for to days. all naïve sows were negative prior to the inoculation of piglets. pedv shedding of piglets. detection of pedv genome occurred in all groups but to a much lesser extent in the mda-positive animals (see fig. , pedv eu in the upper graph, field material de in the lower). in general, the amount of detectable pedv genome was influenced by the faecal load on the swab. however, as this phenomenon occurred in all groups, it was regarded as systematic error. all below mentioned genome copy numbers refer to µl rna extracted from µl faecal suspension (all swabs were submerged in ml medium, irrespective of their load). piglets of groups a and a showed viral rna in rectal swabs beginning dpi until dpi, and the genome load was markedly lower in piglets of group a with mda. significant differences were found among groups a and a on , , , and dpi with higher genome loads in the animals without mda (a ). on the other days, the genome loads were still higher in group a , but not significantly different. it was quite the same in group b. in this case, genome loads were significantly higher on to dpi and to dpi in piglets without mda (b ). in more detail, all animals of group a were found positive at dpi with genome loads between and genome copies per µl, whereas only low virus shedding could be detected in group a with genome copy numbers ranging from . to per µl (see fig. , upper chart). at dpi, a comparable picture was seen at lower level. however, single results varied considerably (genome copies ranging from to in group a and to in group a ). only low copy numbers were detected for all animals at dpi (no significant differences). up to dpi, mainly low genome loads were detected with single animals showing considerable shedding (up to copies per µl for a mda negative piglet at dpi). at dpi all but one piglets of group a still showed genome loads of . to genome copies per µl. animals of group a showed generally lower genome loads with . to genome copies per µl. only one piglet of this groups shed higher genome loads with genome copies per µl. thereafter, shedding decreased, with faster decline in the mda positive groups (see fig. , upper graph). compared to group a , mda negative animals of group b showed higher initial virus shedding (up to copies per µl) at and dpi. only a few animals of the mda positive group were detected positive for a few days with genome loads ranging from . and genome copies. from dpi, virus shedding decreased gradually with scattered positive findings and high variability. from dpi, all mda positive animals (b ) were negative while shedding still occurred in mda negative animals of group b (see fig. , lower graph). detection of other relevant pathogens. the inoculum of the sows was tested negative for transmissible gastroenteritis virus and porcine delta coronavirus. positive results were obtained for porcine circovirus , rotavirus a, and porcine enteroviruses (pev). the piglet inoculum was tested positive for pev genome (cq value: ) and showed negative results for all other pathogens. however, no pev-shedding could be detected in piglets, which showed signs that might have been indicative for pev infection (group b ; litter of sows and ). the cell culture adapted virus (pedv eu, groups a and a ) was free of all tested pathogens. prior to the study, sows were tested for pedv-specific antibodies by three commercial indirect igg isotype antibody elisa. all but one animal showed clear negative results. the serum sample of the remaining sow which had shown inconclusive results was retested by indirect immunofluorescence with clear negative results. all sows, which were inoculated with pedv prior to farrowing (groups a and b ), showed igg isotype antibodies in blood and colostrum samples at the day of farrowing using the elisa assays mentioned above. in general, the used elisa kits seemed to vary regarding specificity and sensitivity. nevertheless, the overall tendency of antibody levels in the different groups was comparable (see fig. a -c). colostrum samples were also tested positive for pedv s protein specific iga isotype antibodies using an in-house elisa. in those pre-inoculated animals, antibody levels in blood decreased till the end of the trial. sows in groups a and b had no detectable pedv specific igg isotype antibodies in blood or colostrum at the day of farrowing but developed rising levels till the end of the trial once they got exposed to pedv from their offspring. blood samples of all but two piglets received from pre-inoculated sows (both in group a ) were shown to contain pedv antibodies prior to inoculation (see fig. a -c, upper right chart for pedv eu and lower right chart for de). at the end of the trial . % (b /b ) to . % (a /a ) of the piglets and all of the sera from the sows were positive in the igg-elisa (see fig. a -c). all milk samples collected at the day of slaughter or death were negative for pedv-specific igg, but showed varying amounts of iga isotype antibodies. this was also found in samples of sows which were naïve prior to farrowing (groups a and b ) (see supplementary fig. f ). as can be seen in fig. a -c, a clear increase of serum antibodies was observed over the course of the trial in mda negative piglets of groups a and b , whereas antibody levels markedly decreased in the mda positive groups a and b . bacteriology. the fecal swabs taken at dpi and dpi did not show any growths of pathogenic bacteria. the bacterial flora was similar in the samples at and dpi. following the disastrous ped outbreaks in the us, re-introduction was also confirmed in central europe , , [ ] [ ] [ ] [ ] . with the exception of the highly virulent strains occurring in ukraine , all recent european pedv strains were so-called s-indel variants with an expected lower virulence than the us-type non-indel pedv variants. overall, the ped impact on pig industry seemed to be low, but sporadic cases with high mortality and long persistence of the disease in continuously producing enterprises was observed . follow-up investigations showed that the reported virulence of the german pedv strains varied considerably among breeding herds (suckling pigs) while only mild diarrhoea was observed in fatteners. interestingly, the causative strains were highly identical, even on the full-genome level . detailed molecular analyses did not reveal any significant impact of viral and bacterial co-infections or viral variants . in order to assess strain-related virulence and disease dynamics among suckling pigs in the presence and absence of maternally derived antibodies, the presented study was carried out under controlled laboratory conditions. in a nutshell, the pedv strains obtained from recent german outbreaks (both cell culture and field material) showed indeed a low virulence with only two mda-negative piglets that had to be euthanized at the humane endpoint. all remaining piglets recovered completely and were able to compensate the weight loss by the weaning age. however, piglets with mda were almost completely protected from clinical disease. in direct comparison, the field material induced more severe signs in naïve piglets than the cell culture isolate. possible reasons include attenuation by cell culture adaptation but also differences in inoculation dose and secondary pathogens. regarding the inoculation dose, it can be stated that the genome load was higher in the cell culture material than in the field material. provided that this gives at least an indication for the virus titer, we can assume that the virus titer of the field material was at least not considerably higher than that of the cell culture material. testing of the field materials for secondary pathogens showed low rotavirus a and enterovirus loads. the secondary materials used for the inoculation of the piglets in groups b and b contained pedv and low pev genome loads. the latter was however not found in any of the piglets. despite the observed differences, % morbidity and long-term shedding of viral rna was still observed for both variants in mda-negative piglets. in contrast to the study with a cell culture adapted us-type pedv, published by poonsuk et al. in , all sows that were naïve prior to farrowing showed diarrhea after challenge of their piglets. disease progression in these sows was worse than the disease course in the sows following direct inoculation prior to farrowing (groups a and b ). diarrhea and depression even led to a severe decrease of milk production and thus inadequate nutrition of piglets. this underlines the importance of the sow's health status for the fate of ped-diseased piglets. the increased severity could probably be explained by the more fragile immune system during the farrowing and lactating period, but also with the higher virus loads in the stable and ongoing contact to pedv shed by the infected piglets . shedding of viral rna in both groups was detected over three weeks and beyond, which is in line with former studies , , and field observations. the variability of individual results was probably due to the different content of fecal material on the swab. however, also the desquamation of intestinal cells and hereby the amount of virus at rectal swabs might vary. in order to gain individual samples, pooled faecal samples were not considered appropriate. the long-term shedding of virus could be a most important issue for disease control and elimination. with shedding over such a long time, it is possible that clinically healthy but still shedding piglets are sold and brought to another holding for subsequent production steps where they pose a risk for naïve stable/pen mates. the risk of disease transmission by those piglets would however need further investigation. most importantly, our study clearly shows that maternally derived immunity against pedv is able to protect piglets in the most vulnerable phase after birth. this is prerequisite for a sow vaccination strategy which should be explored in light of the tremendous impact of ped in the us and also farms in the ukraine. with neonatal piglets being born agammaglobulinemic and possessing limited, undeveloped lymphoid tissues and no effector and memory t-lymphocytes , it is absolutely necessary that they receive maternal cell-mediated and humoral immune components through the ingestion of colostrum and milk . it is already shown for tgev that natural oral infection of sows leads to a more effective maternal derived lactogenic protection for suckling pigs than systemic immunization , . this effect is mainly based on a larger amount of secretory iga in colostrum and milk because of stimulation of the gut-mammary-axis of the sow. systemic immunization usually induces high levels of igg in both the blood and colostrum, but iga titers in blood might stay on a low level offering no reliable lactogenic protection against infection of piglets . this is in line with the results of the provided study, in which high amounts of igg could be detected in colostrum but not in milk samples, whereas iga could be found in both -colostrum of pre-infected sows and milk of all sows. thus, also the sows that stayed naïve prior to farrowing seroconverted upon challenge of their piglets and low levels of iga could be detected in milk samples at the end of the trial. when revisiting ped vaccination, this route-dependent issue should be kept in mind as it could explain the unsatisfactory protection against virus shedding and morbidity of piglets from sows that were only parenterally immunized using different types of vaccines . therefore, a sow vaccination concept against pedv should probably prefer oral or other mucosal immunization routes for proper protection of suckling pigs if possible , , . regarding the duration of immunity in both sows and piglets, we could confirm that the antibodies are rather short-lived. sows pre-exposed prior to farrowing showed already decreasing levels of igg at the end of the trial (with elisa results still being positive). with a high antibody titer in the blood being a requirement for solid and protective antibody titers -especially secretory iga -in colostrum and milk, it seems that sows would have to be re-vaccinated/re-infected prior to each farrowing to ensure piglet protection. moreover, immunization is needed close to the farrowing date. from literature it is known that antibody levels of igg and iga in serum of primiparous and multiparous sows can remain stable up to months post field infection as measured by high titers in elisa and virus neutralization assay . the respective sows were obtained from commercial breeding farms that had reported previous ped outbreaks. thus, booster/reinfection seems to play a role for the duration of immunity. with regard to piglets, field studies observed antibody levels in piglets that lasted up to days , . for the piglets/young weaners, the source of antibodies clearly influences the duration of detection. while a decay of mda is visible already after a few weeks, antibodies induced by active immunization or infection might last much longer. in our study, mda-positive animals had only low antibody levels by the end of the trial (at the age of weaning). in these animals, protection in later stages of pig production would have been questionable and this could explain field observations that showed reinfection of finishing pigs that had been exposed to pedv as suckling pigs. these aspects also need consideration when designing a vaccination concept. another important fact is the reliability of commercial elisa assays. in this study three commercial elisa assays were compared and variations in specificity and sensitivity were found as already shown in other studies . therefore, the combined use of different diagnostic tools to investigate the serological status of an animal should be taken into account. in conclusion, the tested pedv strains were of moderate to low virulence and natural infection of sows during gestation led to an effective lactogenic immunity in piglets. this is a promising outcome when vaccination concepts are discussed. as a spin-off of our trial, a simple and standardized score system was implemented that worked well even with limited clinical signs. the presented score system (see table ) is less detailed than others that were previously described . nevertheless, it seems sufficiently reliable and will be used for subsequent immunization-challenge experiments. study design. in total, eight sows and their offspring were used in this study. the multiparous sows were purchased in late pregnancy from a commercial breeding farm with a high veterinary hygiene standard (bundes hybrid zucht programm, bhzp, dahlenburg-ellringen, germany). they were transported to the high containment facilities at the friedrich-loeffler-institute greifswald, insel riems, germany, five weeks before farrowing. according to eu legislation, the animals were kept in open pens in sets of two before they were moved to commercial farrowing pens one week before farrowing. two farrowing pens were integrated in one stable room so that each experimental group could be kept together. all animals had access to water ad libitum and were fed with commercial feed for breeding sows and after farrowing for lactating sows. all applicable animal welfare regulations, including eu-directive / /ec and institutional guidelines, were taken into consideration. the animal experiment was approved by the competent authority, landesamt für landwirtschaft, lebensmittelsicherheit und all animals were tested prior to arrival with negative pcr-results for pedv and antibodies against pedv. after arrival, the animals were randomly assigned to four treatment groups. two sows (ear tags and ) were orally inoculated with cell culture adapted pedv (pedv eu) four weeks before farrowing to induce mda-positive piglets (group a ). another two sows (ear tags and ) were treated the same way orally with german field material (organ suspension and fecal homogenate; de) containing pedv to also obtain mda-positive piglets (group b ). the remaining four sows stayed untreated to obtain mda-negative piglets (group a : ear tags and ; group b : ear tags and ). all piglets born alive were individually ear tagged prior to challenge inoculation. piglets born to sows of groups a ( piglets of sow and piglets of sow ) and a ( piglets of sow and piglets of sow ) were orally challenged with pedv eu at an age of three to six days whereas piglets born to sows of groups b ( piglets of sow and piglets of sow ) and b ( piglets of sow and piglets of sow ) received the german field material (de). during the whole trial, daily rectal swabs (copan plain cotton swabs without medium) were taken of all animals for real-time reverse transcription polymerase chain reaction (rt-qpcr) analyses. samples of days to post inoculation (pi) as well as days pi and pi were tested. moreover, samples were tested from day pi till the end of the trial. additional rectal swabs were taken of four randomly chosen piglets of each sow prior to inoculation and two days afterwards for bacteriological examination. moreover, clinical signs indicative for ped were recorded using a standardized score system (see table ). blood samples were taken at the day of inoculation and the day of slaughter or euthanasia. additional blood and colostrum samples were collected from all sows during farrowing. furthermore, milk samples were collected from all but two sows at the day of slaughter (samples missing from sow which died from septicemia, and sow which milk production had already ceased). at the end of the trial, all remaining piglets were euthanized and seven sows were slaughtered (electro-stunning and subsequent exsanguination). all animals were necropsied and samples of the small intestine were taken. for inoculation of animals of groups a and a , cell culture adapted pedv was provided by the boehringer ingelheim veterinary research center (pedv eu). this cell culture isolate was obtained from a ped outbreak in northern germany with high mortality rates in suckling piglets. it is sharing over % nucleotide identity with the u.s. strain oh and recent central european pedv strains. the initial isolate was obtained using the following (standard) protocol: × vero cells were seeded into -wells plates. after h of incubation at °c, confluent monolayers were obtained and the cell culture medium was removed. all wells were washed three times with ml phosphate-buffered saline (pbs). thereafter, cells were inoculated with µl of filtrated ( . µm) homogenate from piglet guts. after the addition of ml of pedv media containing µg/ml trypsin, cultures were incubated for - h at °c. the cultures were checked daily for the presence of characteristic fusion formation, and positive materials were further passaged. for the purpose of our study, the titer was defined by end point titration and recorded as tissue culture infection dose (tcid )/ ml. the stock titer was . × tcid /ml (rt-qpcr: cq value ). each sow of group a received ml of this stock diluted with ml pbs. the solution was orally fed using a ml syringe. piglets of groups a and a were also orally inoculated. in this case, each piglet received ml of a : diluted viral stock (titer . × tcid / ml) using ml syringes. to inoculate animals of groups b and b , pedv-pcr-positive field material (de) from recent clinical cases in south-western germany was used. in these cases mainly fattening pigs were affected by rather mild clinical signs. the material was a pool of different fecal samples and intestines. to obtain the inoculum, intestines were homogenized with sterile sea sand using mortar and pestle. faecal samples were mixed and also homogenized. each sow of group b received ml of the organ suspension (rt-qpcr: cq value ) and ml of the faecal homogenate (rt-qpcr: cq value ) orally using ml syringes. all piglets of groups b and b received a faecal homogenate obtained from the sows of group b at dpi and dpi (rt-qpcr: cq value ). the piglets were inoculated orally with ml of this faecal homogenate using ml syringes. sample preparation and nucleic acid extraction. rectal swabs were submerged in ml dulbecco's modified eagle medium with standard antibiotics and antimycotics (antibiotic-antimycotic ×, gibco) and incubated for hour at room temperature. viral rna was extracted using either the manual qiamp viralrna mini kit (qiagen) or the nucleomagvet-kit (macherey-nagel) in combination with the kingfisher extraction platform (thermo scientific). the rna was stored at − °c until further use. blood samples were centrifuged at × g for minutes at room temperature to obtain serum. the resulting serum was aliquoted and stored at − °c. colostrum and milk samples were aliquoted and stored at − °c until further use. to detect pedv genome in fecal swabs, a rt-qpcr system targeting the s-gene of pedv was used as described previously . cq values above were considered negative and the amount of pedv genome copies was calculated by using a standard curve. in addition, the inoculum for sows and piglets was tested for porcine circovirus , porcine enteroviruses (including enteroviruses, teschoviruses, and sapeloviruses), rotavirus a, transmissible gastroenteritis virus, and porcine delta coronavirus using specific in-house rt-pcr assays (primer sequences and reaction conditions are available from the authors upon request). swabs of animals which showed central nervous signs that could have been indicative for infection with teschoviruses were also tested in the pev pcr assay (concerns the litters of sows and , group b ). plots and statistics. creation of different plots and charts was performed using sigmaplot for windows version . (systat software). shapiro-wilk test was used for normality testing and a mann-whitney rank sum test was conducted as implemented in the software package. statistical significance (p <= . was considered significant) was tested using sigmaplot software. veterinary treatments unrelated to ped. six sows (all except animals and ) received cloprostenol (estrumate, intervet) to induce farrowing. moreover, all sows were treated with antibiotics (riketron n, ani-medica) and anti-inflammatory drugs (metacam, boehringer) for a mild metritis-mastitis-agalactia-syndrome over days. piglets of sow no. (group b ) received baytril orally over days because of mild enteritis prior to inoculation. where needed, piglets received anti-inflammatory drugs (metacam, boehringer) for lameness (arthritis, panaritia). antibody detection. three commercial indirect elisa (swinecheck ped indirect, biovet, gc kerkrade, the netherlands; ingezim pedv, ingenasa, madrid, spain; id screen pedv indirect, grabels, france) were performed with all sera according to the producer's manual. colostrum and milk samples were tested in the same manner after initial validation. while elisa kits of biovet and idvet are using recombinant nucleoprotein, the ingenasa elisa plates are coated with recombinant spike protein. in cases of ambiguous results, the respective samples were tested in indirect immunofluorescence assays using commercial pedv fa substrate slides (vmrd, pullman, washington) following the manufacturer's instructions. the slides were screened for specific fluorescence with a standard fluorescence microscope (zeiss axio vert.a , oberkochen, germany). in addition, colostrum and milk samples were tested for pedv specific iga antibodies using an in-house indirect elisa (assay specifications are available from the authors upon request). data availability statement. the datasets generated and analysed during the current study are available from the corresponding author on reasonable request. isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive conventional neonatal and weaned pigs comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc a in conventional -day-old nursing piglets vs. -day-old weaned pigs ratification vote on taxonomic proposals to the international committee on taxonomy of viruses an apparently new syndrome of porcine epidemic diarrhoea pig farming porcine epidemic diarrhoea: new 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shedding and antibody response in swine farms: a longitudinal study inter-laboratory study to characterize the detection of serum antibodies against porcine epidemic diarrhoea virus experimental infection of young pigs with an early european strain of porcine epidemic diarrhoea virus and a recent us strain this research was supported by boehringer ingelheim. we thank our colleagues from boehringer for sharing knowledge and discussion of research results. our special thanks goes to svenja mamerow who helped us throughout the trial. we are also grateful to all of our colleagues in the laboratory and stable. n.v., des. h., b.m. and b.s. designed the experiment. l.s., s. ch., au. a., b.s., and z.l. carried out the animal trial and collected clinical samples. sample analyses were performed by l.s., z.l., au. a., l.s., m.p. and a.a. analysed data. the manuscript was written by l.s., b.s. and b.m. all authors took part in discussion and interpretation of results. all authors read, advised and approved the final manuscript. supplementary information accompanies this paper at doi: . /s - - -w competing interests: the authors were part of an industrial funded research project from boehringer ingelheim. the pedv cell culture isolate was kindly provided by boehringer ingelheim. no other conflicts of interest exist.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - obh i authors: sande, charles j.; mutunga, martin; muteti, jacqueline; berkley, james a.; nokes, d. james; njunge, james title: untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: obh i the upper airway – which consists mainly of the naso- and oro-pharynx - is the first point of contact between the respiratory system and microbial organisms that are ubiquitous in the environment. it has evolved highly specialised functions to address these constant threats whilst facilitating seamless respiratory exchange with the lower respiratory tract. dysregulation of its critical homeostatic and defence functions can lead to ingress of pathogens into the lower respiratory tract, potentially leading to serious illness. systems-wide proteomic tools may facilitate a better understanding of mechanisms in the upper airways in health and disease. in this study, we aimed to develop a mass spectrometry based proteomics method for characterizing the upper airways proteome. naso- and oropharyngeal swab samples used in all our experiments had been eluted in the universal transport media (utm) containing significantly high levels of bovine serum albumin. our proteomic experiments tested the optimal approach to characterize airway proteome on swab samples eluted in utm based on the number of proteins identified without bsa depletion (total proteome: protocol a) and with its depletion using a commercial kit; allprep, qiagen (cellular proteome: protocol b, ci, and cii). observations and lessons drawn from protocol a, fed into the design and implementation of protocol b, and from b to protocol ci and finally cii. label free proteome quantification was used in protocol a (n = ) and b (n = ) while commercial tmt plex reagents were used for protocols ci and ii (n = ). protocols ci and ii were carried out under similar conditions except for the elution gradient: h and h respectively. swab samples tested in this study were from infants and children with and without upper respiratory tract infections from kilifi county hospital on the kenyan coast. protocol a had the least number of proteins identified ( ) while b produced the highest number of protein identifications ( ). when protocol b was modified through sample multiplexing with tmt to enable higher throughput (protocol ci), the number of protein identified reduced to . modification of protocol ci by increasing the peptide elution time generated protocol cii that substantially increased the number of proteins identified to . the coefficient of variation among the tmt runs in protocol cii was < %. there was substantial overlap in the identity of proteins using the four protocols. our method was were able to identify marker proteins characteristically expressed in the upper airway. we found high expression levels of signature nasopharyngeal and oral proteins, including bpifa / and amy a, as well as a high abundance of proteins related to innate and adaptive immune function in the upper airway. we have developed a sensitive systems-level proteomic assay for the systematic quantification of naso-oro-pharyngeal proteins. the assay will advance mechanistic studies of respiratory pathology, by providing an untargeted and hypothesis-free approach of examining the airway proteome. the human naso-oro-pharynx constitutes the first line of mechanical and immunological defence against infectious pathogens and particulate pollutants in the air. pathogens and commensal microorganisms constantly interact with the cells of the upper airway [ ] [ ] [ ] and in order to maintain homeostasis, humans have evolved complex and dynamic defence mechanisms that maintain a functional mucosal barrier . this barrier regulates microbial colonisation and maintains a potent immunological defence against pathogenic micro-organisms . the effector functions that underpin this regulatory role are a product of the airway proteome, which is derived mainly from epithelial cells as well as innate and adaptive immune cells [ ] [ ] [ ] . as part of these regulatory mechanisms, incipient antigens are initially expelled by non-immunological mechanisms such as mucus entrapment, enzymatic degradation and through mechanical egress via mucociliary action [ ] [ ] [ ] [ ] . endosomal degradation by phagocytes such as neutrophils and macrophages as well as other non-specific immunological mechanisms such as complement activation may be employed for antigens that persist. the final layer of immunological defence in the upper airway consists of adaptive immune responses -such as antibody responses -that are specifically tailored to specific pathogens . in spite of its crucial role in the maintenance of airway health, the proteome of naso-oro-pharynx remains poorly studied, particularly in infants and children with serious respiratory infections. the naso-oro-pharynx and its secretions provide an excellent resource for identifying potential biomarkers of disease and could contribute substantially to the understanding of mechanisms of airway diseases. mass spectrometry-based proteomics combined with computational analysis has become a powerful tool for large-scale systematic investigation of biological processes in an untargeted and hypothesis-free approach . proteomics has been employed to profile the nasal cavity and mucous proteome in general [ ] [ ] [ ] [ ] [ ] [ ] , as well as investigate airway diseases such as allergic rhinitis , [ ] [ ] [ ] [ ] [ ] [ ] , chronic rhinosinusitis [ ] [ ] [ ] , and cystic fibrosis , . it is additionally notable that mass spectrometry platforms used to investigate protein expression in these studies may have previously lacked sufficient sensitivity necessary for extensive proteome coverage that would be helpful in elucidating important biomarkers of mechanisms or prognosis. in this study, we developed a high-throughput swab proteomics workflow that can be used to investigate the airway proteomes of infants and children with serious respiratory illnesses especially from hospitals and field samples. study population and sampling procedures. naso-oro-pharyngeal samples were collected using nasal and oropharyngeal (npop) swabs (copan diagnostics, usa) and were eluted in ml of universal transport media (utm, copan diagnostics, usa) that had been supplemented with ul of a protein/dna/rna preservative, allprotect (qiagen, germany). upon collection, samples were stored at − °c. the study population consisted a total of infants and young children admitted to kilifi county hospital, in coastal kenya, with respiratory infections and control infants who were sampled from home and did not exhibit any respiratory symptoms (protocol a: n = , protocol b: n = , protocol c: n = ). diagnosis of respiratory infections was done using a multiplex pcr that was designed to detect respiratory pathogens: respiratory syncytial virus (rsv -a & b), rhinovirus, parainfluenza virus ( , , & ) adenovirus, influenza (a, b & c), coronavirus (oc & e ), human metapneumovirus and mycoplasma pneumoniae. written informed consent was sought from the parents or legal guardians of the recruits prior to sampling while ethical clearance for the conduct of this study was provided by the kenya medical research institute (kemri) scientific and ethical review committee (seru). all methods were performed in accordance with good clinical laboratory practice (gclp) guidelines. overview of protocol development for analysis of the upper-airway proteome. our proteomic experiments tested the optimal approach to characterize airway proteome on swab samples eluted in utm based on the number of proteins identified. protocols were developed and tested in a stepwise sequence, with observations and lessons drawn from one protocol, feeding into the design and implementation of the next. the first protocol that was tested (a) was based on six randomly selected patient samples which were lysed with urea, peptides generated using a standard workflow and analysed by label free quantification. in the second protocol (b) nasal samples from four randomly selected children were selected and cells were obtained by centrifugation. the cells were then lysed, total protein extracted from the lysate using a commercial kit (allprep, qiagen), peptides generated and analysed by label free quantification. in the third and fourth protocols (ci and cii) samples were selected from children. in protocol c, proteins and peptides were prepared in the same manner as protocol b, after which peptides were labelled with isobaric mass tags (tmt plex). protocols ci and ii were carried out under similar conditions except for the elution gradient: h and h respectively. a schematic outline of all the protocols is shown in fig. while a detailed description of the individual protocols is outlined in supplementary methods. mass spectrometry analysis. peptides ( μl) were loaded using a dionex ultimate nano-flow ultra-high-pressure liquid chromatography system (thermo scientific, usa) on to a µm × cm c trap column (thermo scientific, usa) and separated on a µm × cm c reverse-phase analytical column (thermo scientific) at heated at °c. for lfq protein quantification; elution was carried out with mobile phase b ( % acetonitrile with . % formic acid) gradient ( to %) over min at a flow rate of . μl/min. for tmt protein quantification, two gradients were employed: in protocol c i, the gradient was similar to that used for lfq protein quantification, while the gradient employed in protocol c ii was modified to elute peptides for min. each lc run was finished by washout with % b for min and re-equilibration in % b for min. five blanks of min each were run on the column between each injection comprising of two wash cycles with % b and an equilibration phase of min to avoid sample carryover. peptides were measured using a q exactive orbitrap mass spectrometer (thermo scientific, usa) coupled to the chromatography system via a nano-electrospray ion source (thermo scientific). on the q exactive, the ms^ settings for peptides were: resolution, ; agc target, e ; maximum it, ms; scan range, - m/z; while the ms^ settings for fragmentation spectra of peptides were: resolution, ( for labelled peptides); agc target, e ; maximum it, ms; isolation scientific reports | ( ) : | doi: . /s - - - window, . m/z. ms data were acquired by data dependent acquisition where the top ( for labelled peptides) most intense precursor ions in positive mode were selected for ms^ higher-energy c-trap dissociation fragmentation which were subsequently excluded for the next s following fragmentation event. charge exclusion was set to ignore peptide spectrum matches that were unassigned, singly charged, and those with ≥+ charges. for lfq protein quantification. cysteine carbamidomethylation was set as a fixed modification and n-terminal acetylation and methionine oxidations as variable modifications. the false discovery rate (fdr) was set to . for both proteins and peptide-spectrum matches and was determined by searching a fasta protein database comprising target and reversed target sequeces (decoy) derived from the organism being studied, by switching the amino-carboxyl orientation of a protein's amino acids to generate sequences that do not exist in nature . enzyme specificity was set as c-terminal to arginine and lysine with trypsin as the protease. a maximum of two missed cleavages were allowed in the database search. peptide identification was performed with an allowed initial precursor mass deviation of up to ppm and an allowed fragment mass deviation of up to ppm. a minimum peptide length of amino acids and a maximum peptide mass of da was allowed for the searches. the label free quantification (lfq) algorithm in maxquant was used to obtain quantification intensity values. for labelled protein quantification. the plex tmt was specified under isobaric labels for reporter ion ms^ and reporter mass tolerance was set at . da. carbamidomethylation(c) and tmt- plex labeled n-terminus and lysine were set as a fixed modification while oxidation (m) and acetylation (protein n-term) as variable figure . a schematic overview of the naso-oro-pharyngeal proteomics experiments: naso-and oropharyngeal swabs were collected from infants and eluted in universal transport media supplemented with a protein/dna/rna preservative. in one set of samples (protocol a) cells in utm were lysed with urea, reduced, alkylated and digested with trypsin. peptides were then separated by hplc and eluted in a h gradient prior to ms analysis. in another set of samples (protocols b & c) cells in utm were pelleted by centrifugation, lysed and proteins extracted using a commercial extraction kit. proteins were then reduced, alkylated and digested with trypsin. in protocol b, peptides were separated by hplc and eluted on a h gradient. in protocol c peptides were labelled using tmt isobaric mass tags and pooled together prior to hplc separation. in protocol ci, proteins were eluted on a h gradient, while in protocol cii, proteins were eluted on a h gradient prior to ms analysis. scientific reports | ( ) : | doi: . /s - - - modifications with both types of modifications being used for protein quantification. the plex reporter ion intensity matrix for the study participants was extracted from the maxquant proteingroup matrix file and batch corrected using an in house platform. assay-performance. the performance of the four test protocols was based on the number of proteins identified in each protocol as well as by the presence of marker proteins that are known to be expressed by cells of the upper respiratory tract. of the four protocols, protocol b -in which proteins were extracted from the patient samples using a commercial protein/dna/rna extraction kit, followed by label free quantification -resulted in the highest number of protein identifications ( fig. a) while protocol a -where samples were lysed with urea and then reduced, alkylated and digested, followed by label-free quantification -resulted in the lowest number of protein identifications. protocol c, which comprised two sub-protocols, distinguished by the elution gradient, resulted in an intermediate number of identifications, although, protocol cii -where proteins were extracted using the commercial extraction kit, peptides labelled with tmt tags and subsequently eluted on a -minute gradient -yielded a substantial increase in protein identifications over protocol ci. of the three protocols that yielded the highest number of protein identifications (protocols b, ci & cii) there was a substantial overlap in the identity of proteins detected by the three methods as shown in fig. b . further analysis of assay stability and reproducibility was restricted to samples from protocol cii, which was deemed to be the most practical for routine application due to its capacity for multiplexing and superior performance in protein identification compared to protocol ci. the inter-batch correlation of the total proteome of the pooled control sample was carried out by calculating pairwise pearson's correlation coefficients between batches. as shown in fig. a , there was a high degree of correlation between batches, with a median pearson's r value of . (fig. b) . a random selection of proteins (acat , gcc , rpl & dsp) whose median expression levels were spread out across the entire dynamic range of protocol cii, showed that the expression levels of these proteins remained relatively stable across all experimental batches (fig. c) . cell-specific marker protein analysis. in order to further characterise the proteomes identified in protocol cii, the expression of signature proteins that are typically expressed by upper-airway cells was assessed. high expression levels of the signature nasopharyngeal proteins bpifa and bpifa was noted (fig. a) . the coefficients of variation for these proteins as well as other proteins in the pooled control was less than % (fig. b) . in addition, mucins -muc and muc -major components of the mucinous secretions that ubiquitously coat mucosal surfaces of the respiratory tract, were expressed at high levels in the nasopharyngeal secretions of all infants in the study (fig. a & d) . the two most highly expressed proteins in the naso-oro-pharyngeal proteome were the signature mucosal epithelia keratins, keratin (krt ) and keratin (krt ) - fig. d . in addition to these nasopharyngeal marker proteins, we also noted high expression levels of definitive oral/salivary proteins such as salivary amylase in all samples that were analysed - fig. d . other proteins such as the polymeric immunoglobulin receptor (pigr), a protein that mediates active trans-cytosis of secretory immunoglobulins onto the luminal surface of the respiratory tract -were expressed at high levels in all patient samples analysed - fig. d . in addition to proteins whose origins could be attributed to resident epithelial cells, we noted the presence of proteins secreted by non-epithelial cells. we evaluated the expression levels of different sets of proteins stratified by their putative origins, including acute phase proteins (app), antimicrobial peptides (amp), immunoglobulin proteins, milk proteins and proteins associated with the effector functions of phagocytic cells. there was a high degree of heterogeneity in the expression level of acute phase proteins, with the median expression level of haptoglobin (hp) being the highest in this category while c-reactive protein (crp) had the lowest median expression level among the apps analysed - fig. a (first panel) . a similarly diverse pattern of expression was also observed among antimicrobial peptides, with cathepsin g (ctsg), being the most abundant amp while the alpha defensin, defa , was expressed at much lower levels - fig. a (second panel) . in addition to apps and amps, extremely high expression levels of immunoglobulin heavy chains (alpha, gamma and mu) were observed in the naso-oro-pharyngeal secretions of all the children in the study, although, the iga heavy chain, igah , was expressed at much lower levels compared to the other immunoglobulin subclass heavy chains - fig. a (third panel) . the final set of immune-related proteins was associated with the effector functions of phagocytic cells such as neutrophils and macrophages - fig. a (fifth panel) . three proteins in this category, lactotransferrin (ltf), s calcium and zinc binding protein s a , a component of calprotectin (s a / ) and myeloperoxidase (mpo) were among the most highly expressed proteins in the entire naso-oro-pharyngeal proteomes - fig. d -with their respective median expression levels being exceeded only by the cytoskeletal and microfibrillar mucosal keratins (krt and krt ) and beta-actin (actb). in addition to these host proteins, we also identified high levels of non-host proteins in the respiratory tract. the most highly expressed of these were three milk proteins: lactalbumin alpha (lalba), beta casein (csn ) and kappa casein (csn ) - fig. a (fourth panel) . validation of whether these milk proteins were of human or bovine origin was not carried out. to determine the age correlates of these proteins, the respective expression levels of the top three proteins in each functional category was plotted against the age of the study participants - fig. b . with the exception of milk proteins, no systematic changes in protein expression level could be associated with increasing age. in the case of the milk proteins, all three appeared to be most abundant in the first - months of life, after which a precipitous decline in later years was observed - fig. b (fourth panel) . finally, we investigated the expression of selected t-cell associated pro-inflammatory proteins. interferons (ifn-α/β/γ), t-cell phenotypic markers (cd / / ) or their associated effector proteins (il / / / / , granzyme) were not detected in the naso-oro-pharyngeal proteomes of any of the study subjects. using the data from protocol cii, we undertook ordination analysis to visualize whether the proteome of children with respiratory infections of varying severities and healthy controls sampled from home varied. using nonmetric multidimensional scaling, we found that the airway proteomes of children admitted to hospital with severe respiratory infections and those with mild infections that did not warrant hospitalisation, largely overlapped (supplementary figure ) . however, the proteomes of healthy control children who were sampled from home, clustered separately from those with respiratory infections. we describe the development of a comprehensive method of analysing the naso-oro-pharyngeal proteome of infants and young children with respiratory illnesses. of the four protein identification methods reported, the highest number of protein identifications was achieved using protocol b, a method in which proteins in naso-oro-pharyngeal samples were extracted using a commercial extraction kit, followed by label-free quantification. however, unlike the methods described in protocol c, this method is limited by the fact that only one sample can be analysed at a time, limiting its practicality for high-throughput applications. in protocols ci and cii, peptides from different patient samples were labelled with tmt plex reagents, allowing for sample multiplexing prior to ms analysis, followed in-silico de-multiplexing after analysis. this analytical design not only increases sample throughput, but concurrently limits the inter-sample variance that is inherent to single-sample analysis. for this reason, we limited further analysis of assay sensitivity, stability and reproducibility to protocols ci & cii. we found high levels of correlation between control samples run on different assay batches as well as limited inter-batch variance, suggesting that the methods developed were sufficiently robust for the quantitative analysis of the upper-airway proteome. in order to increase the sensitivity of protein identification, we experimented on the effect of different elution gradients on protein identification. in the initial set of experiments in protocol ci, peptides were eluted for minutes, resulting , protein identifications, however when the elution time was increased to minutes -(protocol cii) -a further proteins were identified, bringing the total number of proteins identified in protocol cii to , . compared to previous mass-spectrometry-based proteomics studies of the upper airway, the number of proteins identified in protocol cii, vastly exceeded the number of proteins reported in previously published reports , , , , , , , . further characterisation the proteomes identified in protocol cii, was done by evaluating the expression levels of marker proteins of resident naso-oro-pharyngeal epithelial cells. in-line with recent studies of the upper airway transcriptome we found consistently high expression of the signature upper-airway proteins bpifa and bpifa . these proteins have a unique expression profile, which is restricted to the trachea, nasopharyngeal epithelia and salivary gland and are therefore considered to be the signature proteins of the upper airway. in addition to these definitive upper-airway proteins, we found a consistently high level of expression of mucins (muc & ) -the main component of respiratory tract mucus -and mucosal-associated keratins, krt and krt . these keratins were the most abundantly expressed of all proteins detected in the naso-oropharyngeal proteome. krt and krt are both differentiation keratins of the oral mucosa and therefore the high levels at which they were detected is consistent with the anatomical locations that were targeted for sampling. in addition to these upper-airway differentiation proteins, we found high expression levels of a number of antibody related proteins, including the polymeric immunoglobulin receptor, pigr. pigr is a type i, membrane-spanning antibody fc receptor that is expressed at high levels by mucosal epithelial cells and facilitates the active transcytosis of secretory immunoglobulins . the expression of pigr was linked to the equally high expression of the immunoglobulin heavy chains alpha(iga), mu(igm) and gamma(igg), an association that aligns with the known biological function of pigr and likely indicates its role in maintaining high levels of these antibodies on the mucosal surface. we also examined the secretome of immune cells in the airway. we found a large number of proteins that are associated with innate immunity; which in most cases, were expressed at very high levels. for example, the expression levels of the neutrophil antimicrobial effector proteins ltf, s a and mpo, were not only among the highest of the entire naso-oropharyngeal proteome, but they were also present in the upper-airway proteome of each child in this study. the observed overabundance of proteins these proteins, as well others also related to antimicrobial immunity like ctsg, camp,defa / and htn is most likely related to the need to prevent bacterial overgrowth and maintain homeostatic balance in the upper respiratory tract. previous studies have provided compelling evidence of the functional role of proteins such as ltf, camp, ctsg and others in limiting bacterial replication , and the presence of these proteins at the high levels at which they were detected, supports the notion, that they play a key role in regulating the levels of commensal microbiota hence preventing bacterial overgrowth that could result in pathology. interestingly, we also noted high levels of milk proteins such as lactalbumin alpha (lalba) and alpha & kappa caseins (csn & csn ). these proteins were present at the highest levels in the first months of life and declined in the second half of the first year of life. most children over the age of months had relatively low levels of milk proteins in their upper airways. taken together, these observations are consistent with the fact most infants under the age of six months are predominantly breast-fed and the decline milk-protein levels after six months is most likely associated with weaning. the precipitous decline observed after the first year of life most likely reflects the complete cessation of breast feeding. finally, using ordination analysis, we compared the airway proteomes of children with different clinical manifestations of respiratory illness to that of healthy children who were sampled from home. the proteomes of children with respiratory infections clustered separately from those of well children. this distinction is most likely attributable host innate inflammatory responses mounted by the host in order to address an ongoing infection. these responses would be absent in well controls, in whom the absence of any clinical symptoms at the time of sampling, indicates the absence of an intense inflammatory response. our search of t-cell associated marker and effector proteins, did not identify any such proteins in the upper-airway proteomes of any of the children in this study. t cell effector cytokines and chemokines such as il- , ifn-g, il- , il- and other mediators have been widely reported in studies of paediatric upper airway secretions using sensitive immunoassays such as luminex and msd mesoscale. the absence of these mediators in this study, suggest a possible limitation of the methods reported in this paper: the failure to identify very low abundance proteins in the upper airway proteome. in spite of this shortcoming, ms-based methods reported here offer great advantages over targeted approaches by providing an unbiased, hypothesis-free overview of the upper-airway proteome, thereby increasing the likelihood of identifying novel associations with disease pathology. data analysis was done using r. cluster analysis to determine differences in protein expression between children with respiratory infections and healthy controls was done using nonmetric multidomensional scaling analysis. the accession number for the data reported in this paper is proteomexchange: pdx . the airway microbiome and disease the developing hypopharyngeal microbiota in early life oropharyngeal microbiota in frail older patients unaffected by time 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expression pattern the catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces cathepsin g and neutrophil elastase play critical and nonredundant roles in lung-protective immunity against streptococcus pneumoniae in mice this study was supported by fellowship funding to cjs from the wellcome trust (wt ma). conducted data analysis and interpretation. all authors has input into the manuscript and have approved the manuscript for publication. supplementary information accompanies this paper at https://doi.org/ . /s - - - . publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - t v ipt authors: forni, diego; filippi, giulia; cagliani, rachele; de gioia, luca; pozzoli, uberto; al-daghri, nasser; clerici, mario; sironi, manuela title: the heptad repeat region is a major selection target in mers-cov and related coronaviruses date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: t v ipt middle east respiratory syndrome coronavirus (mers-cov) originated in bats and spread to humans via zoonotic transmission from camels. we analyzed the evolution of the spike (s) gene in betacoronaviruses (betacovs) isolated from different mammals, in bat coronavirus populations, as well as in mers-cov strains from the current outbreak. results indicated several positively selected sites located in the region comprising the two heptad repeats (hr and hr ) and their linker. two sites (r and v ) were positively selected in the betacovs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. during the most recent evolution of mers-cov, adaptive mutations in the hr (q/r/h ) arose in camels or in a previous host and spread to humans. we determined that different residues at position establish distinct inter- and intra-helical interactions and affect the stability of the six-helix bundle formed by the hrs. a similar effect on stability was observed for a nearby mutation (t n) that increases mers-cov infection efficiency in vitro. data herein indicate that the heptad repeat region was a major target of adaptive evolution in mers-cov-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function. camel to human transmission rather than vice versa . therefore, the most likely scenario envisages that a bat-derived mers-cov spread to humans via the zoonotic transmission from dromedary camels. coronaviruses use their spike (s) protein to bind a host receptor and to promote membrane fusion. the spike protein assembles as a trimer on the viral surface and belongs to the class i fusion protein family . class i fusion proteins are found in many other virus genera including retroviruses, orthomyxoviruses, paramyxoviruses, and filoviruses and share similar domain organization, as well as common functional properties . host proteases cleave the cov spike protein into two functionally distinct domains: the n-terminal region (usually referred to as the s subunit) contains the receptor binding domain (rbd), whereas the c-terminal portion (s subunit) includes the fusion peptide, two heptad repeats (hr and hr ), and the transmembrane (tm) domain (see fig. a ). following receptor binding, membrane fusion is mediated by a major conformational rearrangement that exposes the fusion peptide and results in the formation of a six-helix bundle ( hb) , . the core of the hb is a triple-stranded coiled coil formed by the hr s of the three spike subunits forming the trimer; the hr elements pack within the grooves of the coiled coil in an antiparallel direction , . because of its central role in membrane fusion, a number of antiviral peptides that interfere with the hb formation have been developed as potential therapeutic compounds against hiv , ebola virus , sars-cov , , and mers-cov . although the rbd of spike proteins is generally considered the major determinant of host range, several reports have suggested that variation in the c-terminal portion of spike proteins, particularly in the hr and hr , determine host range expansion . moreover, recent works indicated that mers-cov and ty-batcov hku bind dpp both of human and of bat origin [ ] [ ] [ ] . in particular, although mers-cov binds human dpp with higher affinity than ty-batcov hku , which shows a preference for the bat receptor, the rbds of the two viruses engage human dpp via a similar binding mode . these observations suggest that mers-cov and related viruses have the potential to shift host range with little adaptation of the rbd. motivated by the notion that evolutionary analyses can provide information on the molecular events that underlie host shifts and, more generally, host-pathogen interactions , we investigated the evolutionary history of s proteins in mers-cov and related betacovs. specifically, we aimed to determine whether natural selection drove the evolution of specific regions and sites that may contribute to variation in host range or replication efficiency. thus, using different strategies, we analyzed mers-cov strains isolated from human and camels, as well as mers-cov-related viruses from other mammals. data indicate the hr to hr region as a major target of adaptive evolution in these viruses. positive selection shaped the evolution of clade c betacov spike protein. we first investigated whether positive selection drove the evolution of mers-cov-related coronavirus spike proteins. previous phylogenetic analyses of s genes of viruses isolated from humans/camels (mers-cov), bats, and hedgehogs (supplementary table. s ) indicated that the s and s regions display different tree topologies (fig. b) , possibly as a result of recombination . because recombination can inflate estimates of positive selection , we separately analyzed the two regions. we pruned the s and s multiple sequence alignments (msas) from unreliably aligned codons and we screened them for evidence of additional recombination events using gard (genetic algorithm recombination detection) , which detected no breakpoint. the saturation of substitution rates represents a major problem in the detection of positive selection among distantly related sequences. computation of the nonsynonymous (dn) and synonymous (ds) substitution rates over whole phylogenies allows breaking of long branches, resulting in improved rate estimation. thus, evidence of episodic positive selection was searched for by using the codeml branch-site test , which is relatively insensitive to the saturation of substitution rates . in the s and s regions, and branches yielded statistically significant evidence of positive selection under different codon frequency models (fig. b , table and supplementary table s ). positively selected sites along these branches were detected using the beb (bayes empirical bayes) procedure and validated using the mixed effects model of evolution (meme) . one positively selected site was found in the s region, sites in the s subunit (fig. a , table ). the r selected site (in s ) almost corresponds to two mutations that independently arose in the sars-cov spike gene as a result of in vitro adaptation of zoonotic strains to primate cells (fig. a) . in s , most selected sites are located in the hr , hr , and in the intervening linker. remarkably, position is the almost exact counterpart of aminoacid changes that expand the host range or cell-type tropism of infectious bronchiolitis virus (ibv, l f) and murine hepatitis virus (mhv, e d) ( fig. a) , . no positively selected site was found to be located in the rbd (fig. a) . nevertheless, the pruning of unreliably aligned codons operated on the msa left a minority of rbd sites available for analysis. we thus repeated the branch-site test on a subset of more closely related sequences (fig. b) . this procedure decreased divergence and pruning in the rbd and allowed analysis of most codons; even with this procedure, no positively selected site was detected (not shown). indicated that positive selection targeted the spike protein and particularly its s region . nevertheless, recombination was not accounted for in that analysis. we thus analyzed the sequence alignments of the hong kong isolates for the presence of recombination breakpoints using gard. the algorithm detected recombination breakpoints for the pi-batcov hku alignment (fig. a) and none for ty-batcov hku . the ty-batcov hku spike gene was therefore analyzed using the codeml site models, which test the hypothesis that a subset of codons evolve with dn/ds > . no evidence of positive selection was found, even using the relatively non-conservative model m /model m comparison (supplementary table s ). as for the pi-batcov hku spike gene, rampant recombination prevents application of a similar approach. we thus resorted to the simultaneous estimation of selection and recombination using omegamap . this analysis confirmed high recombination along the whole gene ( supplementary fig. s ) and detected a single positively selected site in the s region (fig. c ). the same site (codon , position relative to the mers-cov spike sequence) was also detected by meme, which was run by incorporating the alternative phylogenies detected by gard. most of the previously reported selected sites were not detected using other methods that account for recombination (supplementary table s ). we thus consider that robust inference of positive selection in pi-batcov hku spike genes can only be made for position , which lies outside the rbd (fig. a ). positive selection in the mers-cov heptad repeat . we next wished to determine whether positive selection occurred at the spike gene of mers-cov viruses circulating in the recent outbreak. a previous study suggested that positive selection drove the evolution of two codons in the mers-cov spike gene (positions and ) . nevertheless, in that study only one method was used to infer selection and sequences isolated from camels were not included. we thus retrieved fully or almost fully spike sequences of mers-cov isolated from camels or humans (supplementary table s ). alignments for the s and s regions were separately analyzed and screened for the presence of recombination. no breakpoint was detected and the codeml site models were applied. for the s region, two models of gene evolution that allow a class of codons to evolve with dn/ds > (nssite models m a and m ) showed better fit to the data than the null models (nssite models m a and m ), strongly supporting the action of positive selection ( table , supplementary table s ). no evidence of selection was detected for the s portion ( table , supplementary table s ). in s , both beb and meme detected one selected site: position in the hr (fig. a) . interestingly, three different residues are observed at this site in both camel-and human-derived viruses (fig. a) . mers-cov is thought to have spread from camels to humans; the presence of the three alternative residues in viruses isolated from camels suggests that adaptive evolution at this site occurred prior to the infection of humans. interestingly, the variant is in proximity to a mutation (t n) (figs a and a) that arose during tissue-culture adaptation of mers-cov (strain emc ) and increases replication efficiency . through its side chain, the q residue forms hydrogen bonds with d and interacts with m in hr ( fig. a,b) . replacement of the glutamine residue with histidine or arginine (observed in the camel-and human-derived viruses) results in loss of side chain interactions with m and variably affects hydrogen bonds with d (fig. b) . to gain further insight into the effect of adaptive evolution at position , we performed a stability computational analysis after in silico mutagenesis. q was replaced with all other possible aminoacids: even if changes of different magnitude in Δ g were obtained using three different methods - , trends were very consistent (fig. c ). in particular, replacement with histidine or arginine residues resulted in mild destabilization (fig. c) . as a comparison, the same analysis was performed for position . replacement of the threonine residue with asparagine, which was previously associated with increased replication efficiency in vitro , resulted in a similar level of destabilization as observed for q h/r (fig. c ). we analyzed the evolution of the s protein in betacovs, in bat ty-batcov hku and pi-batcov hku viral populations, as well as in mers-cov isolates from the current outbreak. different strategies were applied, as appropriate depending on divergence and recombination. results indicated that several adaptive changes are located in the s region, with fewer in the s domain and none of these within the rbd. it should be noted, though, that in all analyses we applied quite conservative approaches and we intersected two different methods to declare a site as positively selected. whereas this approach was meant to limit the false positive rate it may have yielded some false negative results. in particular, the branch-site test we used to analyze the s and s regions of betacovs is robust to saturation issues and has a minimal false positive rate, but lacks power . moreover, due to the high divergence and the consequent need of alignment pruning, analysis of the rbd was performed on a shallower phylogeny compared to the other regions. this procedure is expected to reduce power, but is nonetheless necessary. in fact, alignment errors, together with unrecognized recombination, inflate estimates of positive selection and represent major sources of false positive results in evolutionary analyses , . consistently, when we accounted for recombination in pi-batcov hku sequences most previously described selection signals disappeared, including those in the rbd . thus, whereas we cannot exclude that adaptive variants in the rbd of betacovs were missed by our approach, we conclude that the more recent evolution of mers-cov, ty-batcov hku , and pi-batcov hku was not driven by positive selection in this domain. conversely, as previously shown for sars-cov , our data support a role for the s region separating the rbd and fusion peptide as a determinant of betacov host range expansion. analysis of both betacovs and mers-cov strains revealed evidence of positive selection in the s region. most positively selected sites were found to be located either in the heptad repeats or in the intervening linker. among these sites, position is particularly interesting, as it almost corresponds to substitutions that modify the host range and/or cell tropism in mhv and ibv , . both viruses belong to the coronavirus genus. the beaudette strain of ibv has been adapted to embryonated chicken eggs; following passages in culture, the strain was further adapted to infect vero cells (from african green monkey) and primary chicken kidney cells . the l f mutation was shown to represent a major determinant of the fusogenic activity in these cell types table . likelihood ratio test statistics for models of variable selective pressure among sites in mers-cov isolates. m a is a nearly neutral model that assumes one dn/ds (ω ) class between and , and one class with ω = ; m a (positive selection model) is the same as m a plus an extra class of ω > . m is a null model that assumes that < ω < is beta distributed among sites; m (positive selection model) is the same as m but also includes an extra category of sites with ω > . Δ lnl: twice the difference of the natural logs of the maximum likelihood of the models being compared. positions are relative to the mers-cov sequence (emc/ ). mutation was recovered after passages in mouse liver and was shown to contribute significantly to the hepatotropism and hepatic virulence of a previously attenuated strain (mhv-a ) . additional variants in the hr and fusion peptide of mhv strains cooperate with changes in the s region, resulting in a broadening of receptor usage (to heparan sulfate) and, consequently, an extension of the host range . finally, in the mhv-a strain the ability to bind human caecam receptors is strongly influenced by mutant residues located in the fusion peptide, hr and hr /hr linker . similar observations have been reported for viruses that do not belong to the coronavirus genus, but that use a class i fusion protein. for instance, one single mutation in the hr of a simian-human immunodeficiency virus (shiv) strain (kb ) increases by two-to three-fold infection efficiency in cells expressing the marmoset cellular receptors , whereas a nearby hr change in siv contributes to macrophage tropism . overall, these findings pinpoint the relevance of changes in the hr and hr as modifiers of host range and cell-type tropism. the molecular mechanisms underlying the altered phenotype of hr and hr mutants remain to be determined in all these instances, although changes in conformational structure have been suggested as a possible explanation. unfortunately, no coronavirus s protein fusion intermediate or pre-fusion state has been solved to date, hampering investigation of molecular interactions. we therefore analyzed the effect of variation at the position of mers-cov on the stability of the hb in the post-fusion conformation . the presence of a q had previously been suggested to confer higher stability to the mers-cov hb compared to sars-cov . indeed, replacement of this residue results in loss of intraand inter-helical interactions. in line with these observations, three different methods used for stability analysis were concordant in showing that the alternative arginine and histidine residues at position result in a moderate and similar level of destabilization. although the observed Δ Δ g is relatively small, it was calculated on the single monomer, and is expected to be multiplied in the trimer. the observation whereby mildly destabilizing variants are favored by selection may seem counterintuitive. nonetheless, we show that a similar level of destabilization is observed for a mutation in mers-cov hr (t n) that increases infection efficiency, at least in vitro . mutagenesis of hr in retroviral type i fusion proteins has indicated that, whereas strong destabilization of the hb (as measured by circular dichroism) almost inevitably results in reduced infectivity, a minor stability decrease is not necessarily associated with defects in cell fusion and infection efficiency [ ] [ ] [ ] . for instance, different aminoacid replacements at the same hr position in hiv- gp result in decreased stability, but unaffected or even increased infectivity . in the case of eiav (equine infectious anemia virus), destabilized hr mutants were found to display different infection phenotypes depending on temperature . this observation may be extremely interesting in the context of mers-cov, as both bats and dromedary camels display adaptive heterothermy (i.e. sensible daily or season variation in body temperature). coronavirus spike proteins are highly exposed on the virus surface and represent major targets for antibody response , raising the possibility that adaptive evolution of the s protein is driven by the host immune system. this hypothesis is difficult to address due to the paucity of information concerning the specific mers-cov epitopes recognized by human antibodies. recently, different studies identified human antibodies againts mers-cov from non-immune human antibody libraries: all of them were directed against the rbd, suggesting that this region represents a major target for the host immune system . nevertheless, data on the humoral immune response to mers-cov in infected subjects are presently lacking, whereas such information is richer for sars-cov. indeed, analysis of antibodies derived from a patient who recovered from sars indicated that some of them recognize epitopes in the hr region . their neutralizing effect was ascribed to interference of the interaction between hr and hr . whether antibodies against the s region also arise in human subjects (or other mammalian hosts) infected with mers-cov and related betacovs remains to be determined; if this were the case, some of the selected sites we identified may be under selective pressure to evade recognition. finally, it is worth noting that hrs have been studied in different viruses because synthetic peptides interfering with hb formation are promising antiviral molecules [ ] [ ] [ ] [ ] . this is also the case for mers-cov, and hr -like peptides were recently shown to be effective in vitro . these peptides were tested against a mers-cov strain carrying q and all include the interacting m residue . these antivirals may display decreased activity depending on the mers-cov strain and its aminoacid status at the selected position. sequences and alignments. virus sequences were retrieved from the ncbi database and a list of accession numbers is provided as supplementary table s . sequences of ty-batcov hku and pi-batcov hku isolated in hong kong were derived from a previous work . errors in the inferred multiple sequence alignment (msa), which may be common when highly divergent sequences are analyzed, can inflate estimates of positive selection. we therefore used prank for building the msa and guidance for filtering unreliably aligned codons (i.e. we masked codons with a score < . ), as suggested . detection of recombination and positive selection. to detect positive selection at the s gene of clade c betacovs we applied the branch-site test from the paml suite . the test compares a model (ma) that allows positive selection on one or more lineages (foreground lineages) with a model (ma ) that does not allow such positive selection. twice the difference of likelihood for the two models (Δ lnl) is then compared to a χ distribution with one degree of freedom . specifically, the internal branches of previously reconstructed bayesian phylogenies of the s and s regions were set as the foreground lineages in independent tests. a false discovery rate (fdr) correction was applied to account for multiple hypothesis testing (i.e. we corrected for the number of tested branches), as suggested . positively selected sites were identified through the beb analysis (with a p value cutoff of . ), which calculates the posterior probability that each site belongs to the site class of positive selection on the foreground branch(es). sites were validated using meme (with the default cutoff of . ), which allows the distribution of dn/ds (also referred to as ω ) to vary from site to site and from branch to branch at a site, therefore allowing the detection of episodic positive selection . the site models implemented in paml were applied -independently-for the analysis of hku and mers-cov sequences, which display very limited divergence and do not suffer from saturation problems. to detect selection, site models that allow (m a, m ) or disallow (m a, m ) a class of sites to evolve with ω > were fitted to the data . trees were generated by maximum-likelihood using the phyml program with a gtr model of nucleotide substitution and γ distributed rates. positively selected sites were identified using the beb analysis (from model m ) . again, sites were validated using meme. to assure consistency, all models were run using the f × and the f codon frequency models. msas were screened for the presence of recombination using gard. recombination breakpoints were considered significant if the hk (kishino-hasegawa) p value was < . . simultaneous inference of selection and recombination for analysis of positive selection was performed using omegamap , a program for detecting natural selection and recombination based on a model of population genetics and molecular evolution. the model uses a population genetic approximation to the coalescent with recombination. this latter is estimated from patterns of linkage disequilibrium assuming that recombination events occur only between codons and not within them. omegamap applies reversible-jump markov chain monte carlo (mcmc) to perform bayesian inferences of both ω and the recombination parameter ρ , allowing both parameters to vary along the sequence. an average block length of and codons was used to estimate ω and ρ , respectively. to determine the influence of the choice of the priors on the posteriors, analyses were repeated with alternative sets of priors (supplementary table s ). three independent omegamap runs, each with , iterations and a , burn-in iteration, were compared to assess convergence and merged to obtain the posterior probability estimate. the rel (random effects likelihood) analysis models variation in both dn and ds across sites according to a predefined distribution with different rate classes; positively selected sites are identified through an empirical bayes method . the default criterion of a bayes factor > was used to identify positively selected sites. for gard, meme, and rel the nucleotide substitution models were chosen using a genetic algorithm implemented in the datamonkey suite . all analyses were performed through the datamonkey server (http://www.datamonkey.org). in silico analysis of hr variants. the crystal structure of mers-cov hr and hr region was obtained from pdb (pdb id: mod). histidine or arginine residues were introduced at positions and suitable rotamers were sampled through the rapid torsion scan utility in maestro (maestro. . ; schrodinger). intraprotein interactions were calculated with pic (protein interaction calculator) . because programs that calculate stability changes achieve only moderate accuracy , we used three different methods to assure reliability. these approaches are based on different principles. specifically, popmusic uses statistical potentials and takes into account amino acid volume variation upon mutation ; foldx uses an empirical force field and evaluates the energetic effect of point mutations and the interactions contributing to the stability of proteins . finally, i-mutant . is based on a neural network approach to evaluate the free energy change after a single point mutation with incorporation of information on the three-dimensional structure of the protein . in foldx and i-mutant the Δ Δ g values are calculated as follows: ΔΔg = Δg mutant − Δg wild-type . in foldx and i-mutant Δ Δ g values > kcal/mol indicate mutations that decrease protein stability, whereas in popmusic Δ Δ g values > kcal/mol are mark of mutations increasing protein stability. therefore, popmusic Δ Δ g values were multiplied by − to obtain homogeneous results. in the analysis carried out with foldx d, the three-dimensional structure of the protein was repaired using the < repairpdb> command. mutations were introduced using the < buildmodel> command with < numberofruns> set to and < vdwdesign> set to . temperature ( k), ionic strength ( . m) and ph ( ) were set to default values and the force-field was used to predict the water molecules on the protein surface. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs prevalence and genetic diversity of 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coronavirus the foldx web server: an online force field popmusic . : a web server for the estimation of protein stability changes upon mutation and sequence optimality : predicting stability changes upon mutation from the protein sequence or structure improving the performance of positive selection inference by filtering unreliable alignment regions cooperative involvement of the s and s subunits of the murine coronavirus spike protein in receptor binding and extended host range amino acid substitutions in the s subunit of mouse hepatitis virus variant v encode determinants of host range expansion adaptation of the human immunodeficiency virus type envelope glycoproteins to new world monkey receptors mechanisms for adaptation of simian immunodeficiency virus to replication in alveolar macrophages detailed mechanistic insights into hiv- sensitivity to three generations of fusion inhibitors the fusion activity of hiv- gp depends on interhelical interactions structural and biochemical insights into the v/i t mutation found in the eiav gp vaccine strain development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge an algorithm for progressive multiple alignment of sequences with insertions guidance: a web server for assessing alignment confidence scores multiple hypothesis testing to detect lineages under positive selection that affects only a few sites paml : phylogenetic analysis by maximum likelihood estimating maximum likelihood phylogenies with phyml accuracy and power of bayes prediction of amino acid sites under positive selection not so different after all: a comparison of methods for detecting amino acid sites under selection codontest: modeling amino acid substitution preferences in coding sequences datamonkey : a suite of phylogenetic analysis tools for evolutionary biology pic: protein interactions calculator performance of protein stability predictors host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein vice rectorate for graduate studies and scientific research in king saud university (ksu) key: cord- -w hsr m authors: jiang, lili; lee, vernon jian ming; cui, lin; lin, raymond; tan, chyi lin; tan, linda wei lin; lim, wei-yen; leo, yee-sin; low, louie; hibberd, martin; chen, mark i-cheng title: detection of viral respiratory pathogens in mild and severe acute respiratory infections in singapore date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: w hsr m to investigate the performance of laboratory methods and clinical case definitions in detecting the viral pathogens for acute respiratory infections (aris) from a prospective community cohort and hospital inpatients, nasopharyngeal swabs from cohort members reporting aris (community-ari) and inpatients admitted with aris (inpatient-ari) were tested by singleplex real time-polymerase chain reaction (srt-pcr), multiplex rt-pcr (mrt-pcr) and pathogen-chip system (pathchip) between april and december . community-ari and inpatient-ari was also combined with mild and severe cases of influenza from a historical prospective study as mild-ari and severe-ari respectively to evaluate the performance of clinical case definitions. we analysed community-ari and inpatient-ari episodes ( inpatient-ari excluded because multiple pathogens were detected), involving and samples respectively. detection by pcr declined with days post-onset for influenza virus; decrease was faster for community-ari than for inpatient-ari. no such patterns were observed for non-influenza respiratory virus infections. pathchip added substantially to viruses detected for community-ari only. clinical case definitions discriminated influenza from other mild-ari but performed poorly for severe-ari and for older participants. rational strategies for diagnosis and surveillance of influenza and other respiratory virus must acknowledge the differences between aris presenting in community and hospital settings. influenza and other respiratory viruses such as respiratory syncytial virus, rhinovirus, parainfluenza virus, adenovirus and human metapneumovirus are common causes of respiratory infections . while often manifesting as a mild illness, these viruses can result in serious complications , , hospitalisations and deaths . identifying the viral aetiology of respiratory infections has applications in both clinical management and surveillance. early diagnosis may allow timely initiation of appropriate treatment , , and where warranted, rapid confirmation of an outbreak can lend itself to control and mitigation efforts for influenza , . and while specific therapeutic or preventive measures for most viral respiratory agents (other than influenza) are lacking, diagnosis can still help in ruling out other causes of respiratory illness and facilitate implementation of appropriate infection control measures in healthcare settings . moreover, with increasing concerns about the spread of new and dangerous viral respiratory pathogens such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) viruses, we need to better appreciate how we can optimally combine clinical information with routine as well more complex and expensive laboratory testing technologies to diagnose and conduct surveillance for unusual pathogens . current approaches to diagnosis and surveillance rely heavily on clinical case definitions and a variety of laboratory assays. however, overlapping symptoms makes existing clinical case definitions for respiratory infections inadequate for diagnosis of specific infections , . reverse transcriptase-polymerase chain reaction (rt-pcr), which detects viral nucleic acid by use of amplification techniques, is now considered as the gold standard assay for detection of respiratory viruses, and also has the advantage of short turn-around times as compared to scientific reports | : | doi: . /srep methods based on virus culture and isolation . moreover, multiplex rt-pcr (mrt-pcr) assays, which allow for rapid detection of multiple types of known viral agents, are now widely available . studies have evaluated the performance of pcr-based assays for diagnosing respiratory viruses [ ] [ ] [ ] , but were mostly restricted to either outpatients or inpatients, or to at-risk groups such as the elderly and young children. moreover, large-scale pathogen detection technologies, like the genome institute of singapore (gis) pathchip, have recently been developed , and there is also a need to rationalize how these could be integrated with routinely available assays. our study had two key objectives. firstly, we investigated the performance of the singleplex rt-pcr (srt-pcr) and mrt-pcr, as well as explored the use of the gis pathchip for the detection of viral respiratory pathogens for mild and severe acute respiratory illness (ari). secondly, we evaluated how well clinical case definitions might differentiate influenza from other causes for mild and severe aris. we collected samples and clinical data from both a community cohort and adult inpatients to address the first objective while we combined this with data from mild and severe influenza cases prospectively recruited during the influenza a(h n )pdm pandemic of to support the second objective. of participants from the community enrolled into a cohort, reported having ari episodes. we also enrolled inpatients from tan tock seng hospital (ttsh), and retrieved influenza cases from a previous study (historical-flu). community participants with ari episodes did not differ significantly from the underlying cohort (table ) . compared to community participants with ari, inpatients were older, more likely to be male and to have chronic medical conditions. similarly, historical-flu participants with medical indications were older, and more likely to have chronic medical conditions as compared to those who were admitted for public health indications. historical-flu participants with medical indications were fairly similar to the inpatients although they were slightly younger and less likely to have copd and heart disease. one hundred and thirty episodes were reported from community participants (community-ari) with , , and individuals reporting , , and episodes respectively. for the inpatients, there were episodes with only individual having and another having episodes (inpatient-ari). viruses detected by srt-pcr and mrt-pcr in community-ari and inpatient-ari episodes. on using srt-pcr with mrt-pcr, . % of the community-ari episodes were diagnosed as panel virus positive (i.e. positive for at least one of the viruses on the mrt-pcr panel used), including ( . %) influenza virus positive episodes ( a(h n ), a(h n )pdm and influenza b) and ( . %) non-influenza panel virus positive episodes. two viruses were more common than influenza in community-ari, with ( . %) episodes positive for rhinoviruses and ( . %) for coronaviruses (table ). there were no co-infections detected by mrt-pcr in either community-ari episodes or influenza negative inpatient-ari. however, influenza positive inpatient-ari episodes were positive for more than one pathogen, including dual-pathogen (influenza a + influenza b [ ] , influenza a + rhinovirus, and influenza b + respiratory syncytial virus) of the and samples from community-ari and inpatient-ari episodes, and respectively were for influenza positive episodes (where multiple samples were collected for each episode). the time between episode onset and the first sample for inpatient-ari was longer than for community-ari (median of days vs day respectively, p < . ); however, no such difference was observed between influenza versus other ari episodes either for community-ari or inpatient-ari (p = . and p = . respectively). we compared the detection of influenza and non-influenza panel viruses by days post-onset. mrt-pcr detected influenza in only . % of the samples that were positive for influenza by srt-pcr. for influenza positive episodes, the proportion positive by either assay decreased as days post-onset increased for both the community and inpatient samples (fig. a ,b). the srt-pcr ct values also increased with days post-onset in both groups, but the ct value for community samples increased more sharply than for the inpatients. the gee model suggested that inpatient-ari had a higher starting ct value than community-ari (p = . , table ). ct value increased as days post-onset increased (p < . ), but the increase in ct value with every additional day post-onset for inpatient-ari was lesser than that for community-ari (p < . for interaction term between participant type and days post episode onset). for non-influenza panel virus episodes, changes in the proportion of positive samples showed no consistent pattern by days post-onset in both community-ari and inpatient-ari (fig. c ,d). amongst (no samples were sent for pathchip analysis for pcr negative inpatient-ari episodes) pcr negative episodes, pathchip did not detect any viruses in episodes. in episodes, multiple viruses (both respiratory and non-respiratory) were detected; these were excluded from further analysis. episodes were positive to mrt-pcr panel virus; all were from community-ari, with positive for rhinovirus, for influenza a and another for metapneumovirus. more episodes ( community-ari, inpatient-ari) were positive for viruses including coxsackievirus ( ), human enterovirus( ), human parainfluenza virus ( ) and influenza c virus ( ) where ari is a recognised presentation . episodes had only non-ari viruses detected (human herpesvirus , human papillomavirus, molluscum contagiosum virus and human t-lymphotropic virus ). the pathchip results increased the proportion of community-ari episodes positive for respiratory viral pathogens from . % to . %, and from . % to . % for episodes meeting febrile respiratory illness (fri) criteria (i.e. ari with self-reported fever, regardless of body temperature measurement). due to the small number of influenza positive episodes in community-ari, we combined community-ari and inpatient-ari episodes with influenza episodes identified from a previous study (described further in supplementary material). clinical features of historical-flu admitted for public health indications were fairly similar to influenza cases identified amongst community-ari, which justified grouping them together as mild-ari (i.e. community-ari + historical-flu, public health indications); likewise historical-flu with medical indications for admission was fairly similar to table . non-influenza mono-infection episodes in community-ari and inpatient-ari episodesvirus identified. a as proportion of all community-ari episodes. b as proportion of community-ari episodes positive for non-influenza viruses. c for inpatient-ari, this excludes episodes where there was a co-infection between influenza and non-influenza viruses on mrt-pcr. d as proportion of inpatient-ari monoinfection episodes positive for non-influenza viruses. e includes hcov e/nl and hcov oc . influenza from inpatient-ari, and were designated severe ari (i.e. inpatient-ari + historical-flu, medical indications, also see supplementary table s ). none of the respiratory symptoms assessed were significantly more common in influenza, and for mild-ari, sore throat and runny nose were significantly less common in those testing positive for influenza (table ). however, for both mild and severe ari, fri and influenza-like illness (ili) table . outcome of the gee model a evaluating the factors on the dynamics of ct values. a generalized estimating equation model which included participant type (community cohort or inpatient), days post episode onset, age, gender, comorbidities (comparing those who reported any comorbidities with those who didn't report any comorbidities, with the comorbidities included being diabetes, asthma, copd, heart disease, cancer and other significant conditions), as well as the interaction term between participant type and days post episode onset. case definitions as well as temperature cut-off points showed significant discriminatory value for influenza over non-influenza episodes or any panel viruses positive over viruses negative episodes. figure a shows that in mild-ari, while only . % was influenza positive by srt-pcr, this rose to . %, . % and . % for fri, ili-u (us centers for disease control ili definition) and ili-w (world health organisation ili definition) respectively, with lr+ > for ili case definitions and temperature cut-off points ≥ . °c. while using ili-w increased the proportion positive for any mrt-pcr panel viruses to . %, this represented a relatively small improvement over the . % positive in all mild-ari, with lr+ of only . (fig. b) . for severe-ari, use of more specific case definitions also improved the likelihood of being positive for influenza and viral respiratory infection, but the lr+ only ranged from to . . notably, for severe-ari, ili-w performed best, with . % and . % of such episodes positive for influenza and any panel viruses respectively (fig. c,d) . we performed a sensitivity analysis excluding the historical-flu cases and the results were essentially the same other than for the wider confidence intervals (due to the reduced sample size, see supplementary fig. s ). there were insufficient numbers of mild-ari with age ≥ years for age-stratified analysis, but influenza was significantly more likely than non-influenza episodes to meet fri, ili-u and ili-w criteria for both mild-ari and severe-ari in those aged < years (table ). however, case definitions had poorer discriminatory value in severe-ari and older ages. for instance, in those aged < years, severe influenza was significantly more likely than mild influenza to meet ili-w criteria ( . % vs . % respectively, p = . ), but non-influenza causes of severe-ari were also more likely to have febrile presentations, with . % meeting ili-w criteria (vs . % for non-influenza mild-ari, p < . ). in addition, comparing severe influenza between age groups shows that episodes in older individuals were also significantly less likely than to meet ili criteria (e.g. for ili-u, . % for severe-ari in those aged < years versus only . % for those aged ≥ years, p = . ). our study concurrently assessed the role of routine laboratory diagnostics, and usefulness of the novel pathchip platform as well as ili case definitions in identifying respiratory virus infection in a community cohort and hospital inpatients from a broad range of age groups ( to , and to years respectively), to reflect what may be encountered in either community or primary care (mild-ari) as well as tertiary care settings (severe-ari) in a tropical environment with less distinct seasonal patterns. our study clarifies the role of singleplex and multiplex rt-pcr in the respective populations. we observed imperfect sensitivity of mrt-pcr for influenza ( . %) as compared to srt-pcr, but better performance was reported in two outpatient studies ( - %) , , possibly related to the longer time between onset and sample collection for inpatients in our study, as the recovery of samples positive for influenza and sensitivity of mrt-pcr for influenza was dependent on the time between onset and sample collection. post episode onset, the proportion positive for influenza decreased (ct value increased) faster in community-ari as compared to inpatient-ari, but no such pattern was observed for non-influenza panel viruses; likewise, others have found slower viral clearance in inpatients compared to outpatients for influenza a(h n )pdm , and a lack of association between days post-onset and ct values for adenovirus, human metapneumovirus, parainfluenza virus - . pcr-based assays may thus retain greater value for detecting influenza amongst inpatients that present late than in the community where episodes presenting more than days post-onset would likely be influenza negative. our study also explored the application of newer platforms like the pathchip for detecting respiratory virus infections. in contrast to multiplexed pcr-based technologies which rely on a set of primers that target a finite number of specific pathogens, the pathchip is designed to detect a wide array of pathogens simultaneously by detecting signatures in the pathogen genome sequences. while costly, our results suggests that it has the potential to complement existing technologies as a diagnostic tool. simões et al previously evaluated the diagnostic value of the pathchip using paediatric nasal wash samples and reported variable sensitivity ranging from . % to . % and reasonable specificity from . % to % . in our study, the pathchip was a valuable addition in community-ari where it substantially increased the proportion positive for respiratory etiological agents, so that only about % of febrile episodes did not have a viral aetiology identified. notably, the vast majority of additional viruses detected were those known to cause ari. it may thus help in ruling out more sinister causes of ari if added to mrt-pcr for surveillance in community settings; it may also help detect unexpected but dangerous infections such as sars and mers-cov following further validation on actual patient samples with such infections. however, due to the high cost of the assay (about four times mrt-pcr) and the imperfect sensitivity for some pathogens (as low as . % for adenovirus), we opted to test only the rt-pcr negative samples with the pathchip. as such, we are unable to comment on its sensitivity for samples from adults, which may be inferior to the previously published results which used samples from paediatric subjects, who are known to have higher viral loads for some viruses , . however, given its current cost, variable sensitivity and slower turn-around time as compared to mrt-pcr (about hours for pathchip), we believe this technology is most appropriately used in the way we designed our study, which is to detect additional respiratory viral pathogen positive episodes as an adjunct to more widely available mrt-pcr panels designed to cover the most common pathogens from community-based samples. finally, our study highlights how the same clinical case definitions, which have been used in both community and inpatient settings , may actually perform differently in the two settings for distinguishing influenza from other respiratory infections, identifying influenza cases, and increasing the yield of diagnostic assays. we found that, while respiratory symptoms themselves are poor in distinguishing influenza from other ari causes, clinical case definitions using fever or temperature cut-off points demonstrated good discriminatory value in our mild-ari episodes, which should reflect what can be anticipated in primary care settings. however, for ari severe enough to require hospitalisation, the case definitions had poorer discriminatory value. this was partly because, while a good majority of older individuals ( . %) with severe influenza had self-reported fever (as in fri), only about half met the temperature criteria used for ili case definitions ( table ). the use of such high temperature cut-off points would hence substantially reduce sensitivity for detecting influenza in the elderly. also, non-influenza episodes in ari severe enough to require hospitalization are more likely to also have a higher temperature than non-influenza episodes in mild ari; this further reduces the ability of ili case definitions to distinguish influenza from other respiratory causes in the inpatient setting. the choice of an appropriate case definition hence depends on the objectives and setting of the application. if the aim is to diagnose as many influenza cases as possible (e.g. for identifying patients for antiviral treatment or outbreak management), ili case definitions have inadequate sensitivity, particularly in older age groups. however, the discriminatory value of ili criteria finds application when aiming to reduce background noise during syndromic surveillance, either in community type settings or even a healthcare worker population . the ili case definitions are also useful for optimizing the yield of influenza viruses from samples tested, and particularly efficient in settings where milder ari presentations are encountered (fig. a) ; and if the aim is to identify all types of respiratory viruses for surveillance, ili criteria would also modestly improve the probability of obtaining a positive result as compared to sampling all ari in both mild as well as severe presentations (fig. b,d) . a key limitation we acknowledge is the less than ideal number of influenza infections identified through our community cohort, which led us to supplement our concurrent community and inpatient studies with data from our older prospective study of influenza cases. while not ideal, supplementary table s suggests that influenza cases from this historical dataset which were classified as mild were reasonably similar to those from the community cohort, and likewise those classified as severe were similar to those from the later inpatient study. a sensitivity analysis without the influenza cases from our historical dataset gave a similar result. however, even with these additional influenza cases, the numbers remained inadequate to assess the performance of the case definitions for mild influenza in older age groups, and we are also unable to assess performance in paediatric subjects. secondly, the panel of viruses tested on the multiplex pcr assay was not comprehensive; while this was supplemented by the pathchip, there is no means of ascertaining what proportion of the remaining ari episodes are truly not due to an infectious viral aetiology. we also recognise that our study population, in particular the community cohort, may not be representative of the general community, which was further complicated by fluctuations in the rate at which participants notified us of ari episodes, as well as intra-seasonal variations within each year and sporadic outbreaks of particular respiratory pathogens . scientific reports | : | doi: . /srep the performance of various technologies and case definitions for viral respiratory pathogens presenting with ari differs substantially between community and hospital-based settings. pcr-based assays may still be relevant for detecting influenza amongst inpatients that present late but less so for community ari episodes with delayed presentations. the pathchip may add value for respiratory virus detection in samples negative by multiplex rt-pcr, but only for community-ari. finally, us-cdc and who ili case definitions had similar performance for mild ari, but performed less adequately amongst presentations severe enough to require hospitalisation, including in older individuals. rational strategies for diagnosis and surveillance of influenza and other respiratory viruses must acknowledge the differences between these two populations. study population. we recruited a community cohort and inpatients from ttsh between april and december . the community cohort was recruited by contacting community-dwelling adults who participated in pre-existing prospective cohort studies conducted by the national university of singapore (nus) saw swee hock school of public health. consenting individuals were then enrolled through home visits, where we also opportunistically recruited other members of the household, including children. at enrolment, participants contributed demographic and health information through a baseline interview at enrolment, and then followed-up for up to . years, with instructions to notify the study team within hours on developing ari symptoms (community-ari). once notified, research staff would obtain nasopharyngeal swabs and symptom data from the participant on the next working day. for inpatients, on each working day, we would screen through the list of adults admitted to ttsh within the last hrs who had undergone routine diagnostic testing (by srt-pcr) for influenza. we then enrolled and collected nasopharyngeal swabs from consenting inpatients fulfilling the same ari criteria (inpatient-ari) used for the community cohort. to better characterise influenza, we intentionally oversampled influenza to obtain a ratio of approximately influenza positive to influenza negative patients. additional swabs were taken on days to and days to post-onset (community-ari) or post-enrolment (inpatient-ari) for influenza positive episodes where possible. in addition, we retrieved historical influenza data from a prospective study of admissions to ttsh testing positive for influenza between may and september (historical-flu). this included ari patients with medical indications who were admitted alongside ari cases referred to ttsh for public health indications (clinically suspected to have influenza a(h n )pdm based on epidemiological risk factors like travel and contact history). ethics approvals were obtained from nus institutional review board and the national healthcare group (singapore) domain specific review board, in accordance with relevant guidelines and regulations. a written informed consent was signed by each study participant. laboratory analysis. three assays were performed. srt-pcr and mrt-pcr assays were conducted at the nphl on all available samples while pathchip assays were performed at gis using rt-pcr negative samples. srt-pcr. this assay was only used to detect influenza virus types a and b at a higher sensitivity than might be achieved using multiplexed pcr assays, and to further subtype influenza a positive samples. the protocols had been adopted from the studies by spackman et al. and krafft et al. and, respectively, with cycle threshold (ct) values documented for positive samples (samples with a ct value less than were considered as positive). influenza a positive specimens were then subtyped with subtype specific primers and probes that targeted at haemagglutinin (ha) gene of a(h n ) , and ha and nucleoprotein (np) genes of a(h n )pdm pathchip. this assay was used to detect additional viruses beyond what was covered by the primers within the mrt-pcr panel used. for each sample, the cdna was amplified from extracted nucleic acid. the novel platform from the genome institute of singapore (gis) then automatically detects which pathogens' recognition signatures are present, based on a proprietary algorithm constructed based on genetic sequences of viruses clinically relevant to humans (downloaded from the ncbi taxonomy data-base, http://www.ncbi.nlm.nih.gov/taxonomy/ taxonomyhome.html/) to evaluate how the gis-pathchip might add to detection above routinely available mrt-pcr assays, viruses were grouped into panel viruses (i.e. virus group represented in seegene rv ), non-panel viruses for which ari is a common presentation and non-ari viruses. in our analysis on the combined performance of clinical case definitions and laboratory assays, we grouped all community-ari episodes (none of which required hospitalisation) with historical-flu admitted for public health indications; these were designated as "mild-ari" since these would ordinarily not be sufficiently severe as to require hospitalisation. inpatient-ari was grouped with historical-flu cases admitted for medical indications ("severe-ari"). this increased the number of influenza cases available for evaluating the discriminatory value of clinical parameters and case definitions, which were: • ari: episode with acute onset with any key respiratory symptoms including cough, shortness of breath, sore throat, or runny nose. • febrile respiratory illness (fri): ari with self-reported fever, regardless of body temperature (t) measurement. • influenza-like illness defined by centers for disease control and prevention of the united states of america (ili-u): fever ≥ . °c together with cough and ⁄or sore throat in the absence of a known cause other than influenza . • influenza-like illness defined by world health organization (ili-w): fever of ≥ °c plus cough with onset within the last days . the proportion of episodes fulfilling different criteria was compared by viral pathogen status (influenza viruses, non-influenza panel viruses and negative), and we then investigated the discriminatory performance by calculating the positive likelihood ratio (lr+ ) and its % confidence intervals (ci) for positive detection of influenza and any viral infection. this was also done to investigate the effect of age (age < vs ≥ years). estimates of lr+ requires assumptions on the anticipated prevalence of influenza. for community-ari, this was assumed to be what was obtained in ari samples from the community cohort. for inpatient-ari, since influenza positive inpatients were intentionally oversampled (crude_flu), the proportion positive for influenza was estimated based on the proportion positive for influenza from routinely ordered tests of ari admissions to ttsh (adjusted_flu), which was then applied to derive the adjusted (adjusted_non-flu) from the crude proportion (crude_non-flu) positive for non-influenza viruses detected in mrt-pcr using the following formula: adjusted non flu crude non flu ( adjusted flu)/( crude flu) all analyses were conducted using r version . . 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pandemic and post-pandemic periods in singapore use of healthcare worker sickness absenteeism surveillance as a potential early warning system for influenza epidemics in acute care hospitals seasonal trends of viral respiratory tract infections in the tropics development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h and h hemagglutinin subtypes evaluation of pcr testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification who|who information for molecular diagnosis of influenza virus-update overview of influenza surveillance in the united states |seasonal influenza (flu)| cdc. available at who|who surveillance case definitions for ili and sari. who available at we would like to acknowledge funding from the national medical research council of singapore (ppg - ). supplementary information accompanies this paper at http://www.nature.com/srep the authors declare no competing financial interests. key: cord- -fegcf fu authors: oliveira-de-souza, deivide; vinhaes, caian l.; arriaga, maría b.; kumar, nathella pavan; queiroz, artur t. l.; fukutani, kiyoshi f.; babu, subash; andrade, bruno b. title: aging increases the systemic molecular degree of inflammatory perturbation in patients with tuberculosis date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: fegcf fu tuberculosis (tb) is a chronic infection that can affect individuals of all ages. the description of determinants of immunopathogenesis in tb is of tremendous interest due to the perspective of finding a reliable host-directed therapy to reduce disease burden. the association between specific biomarker profiles related to inflammation and the diverse clinical disease presentations in tb has been extensively studied in adults. however, relatively scarce data on profiling the inflammatory responses in pediatric tb are available. here, we employed the molecular degree of perturbation (mdp) score adapted to plasma biomarkers in two distinct databanks from studies that examined either adults or children presenting with pulmonary or extrapulmonary disease. we used multidimensional statistical analyses to characterize the impact of age on the overall changes in the systemic inflammation profiles in subpopulation of tb patients. our findings indicate that tb results in significant increases in molecular perturbation, with the highest values being detected in adult patients. furthermore, there were unique differences in the biomarker perturbation patterns and the overall degree of inflammation according to disease site and age. importantly, the molecular degree of perturbation was not influenced by sex. our results revealed that aging is an important determinant of the differences in quality and magnitude of systemic inflammatory perturbation in distinct clinical forms of tb. immunoassays. we evaluated two databanks containing plasma concentrations of a panel of cytokines, tissue remodeling mediators and matrix metalloproteinases to examine molecular degree of inflammatory perturbation using different immunoassays. the was no pre-selection of biomarkers. only biomarkers which concentrations were recorded in databank from both children and adults were used for the analysis. in the original study, biomarkers were measured in edta-treated plasma samples. biomarkers included were cytokines, acutephase proteins and tissue remodeling proteins. bio-plex luminex assay system (r&d systems) was employed to measure the cytokines analyzed. the list of cytokines included interleukin (il)- β, il- , il- p , il- , interferon (ifn)-γ and tumor necrosis factor α (tnf-α). moreover, ifn-α and ifn-β levels were quantified using the verikine serum elisa kit (pbl interferon source). plasma levels of vascular endothelial growth factor (vegf) was measured using the milliplex map kit system by merck millipore. concentration of extracellular matrix metalloproteinases (mmps)- , and , and of the tissue inhibitors of metalloproteinases (timps)- , , and were measured using luminex technology (r&d systems), according to the manufacturer's protocols. plasmatic hemoxygenase (ho- ) was measured by elisa (assay designs). data analysis. categorical data were presented as proportions and continuous data as medians and interquartile ranges (iqr). the fisher's exact test ( × ) or the pearson's chi-square test was used to compare categorical variables between study groups. continuous variables were compared using the mann-whitney u test (between groups) or the kruskal-wallis test with dunn's multiple comparisons ad hoc test. p-values were adjusted for multiple measurements using the holm-bonferroni's method. hierarchical cluster analyses (ward's method) of z-score normalized data were employed to depict the overall expression profile of indicated biomarkers in the study groups. dendrograms represent euclidean distance. profiles of correlations between biomarkers in different clinical groups were examined using network analysis of the spearman correlation matrices (with × bootstrap). in indicated analyses, only correlations with significant adjusted p-values (established cut-off was p-value < . ) were included in the network visualization. in such analyses, markers that exhibited similar correlation profiles were clustered based on a modularity , which infers sub-networks inside the of the correlation network profiles, and depicted using fruchterman reingold (force-directed graph drawing). an analytical algorithm using sparse canonical correlation analysis (cca) was employed to assess whether data on correlation between combinations of circulating biomarkers could discriminate between subgroups of patients. the cca model was chosen because many variables were studied. this model is able to perform dimensionality reduction for two co-dependent data sets (mdp biomarker profile and baseline characteristics profile, which were age and sex) simultaneously so that the discrimination of the clinical endpoints represents a scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ combination of variables that are maximally correlated. thus, trends of correlations between parameters in different clinical groups rather than their respective distribution within each group are the key components driving the discrimination outcome. in our cca model, simplified and adapted from previously reported investigations of biomarkers for tb diagnosis , - and of inflammatory pathways associated with pathogenesis of other infectious diseases . in the biomarker profile dataset, we included values of all the inflammatory marker. the diagnostic class prediction values obtained were calculated using receiver operator characteristics curve analysis. probability of being molecularly perturbed according to increases in age was calculated using kaplan-meyer curves. adaptation of the molecular degree of perturbation to examine plasma concentrations of biomarkers. the plasma measurements of both datasets were normalized equally with a log transformation and the batch effect within the different study datasets was corrected using combat algorithm from sva package . the combat algorithm is a widely used method for adjusting batch effects in microarray and rna-seq data associated with technical variance effects. the molecular inflammatory perturbation is based molecular degree of perturbation (mdp) method used in the present study is an adaptation of the mdh described by pankla et al. . in the present study, instead of using gene expression values as in prada-medina et al. , we inputted plasma concentrations of a defined set of biomarkers which were described in previously published studies from our group that investigated tb pathogenesis , . thus, herein, the average plasma concentration levels and standard deviation of a baseline reference group (healthy uninfected controls) were calculated for each biomarker. the mdp score of an individual biomarker was defined by the differences in concentration levels from the average of the biomarker in reference group divided by the reference standard deviation. essentially, the mdp score represents the differences by number of standard deviations from the healthy control group. the formulas used to calculate mdp in the present study are shown below: n = number of data points, x i = each of the value of data, x = mean of the data points, σ = standard deviation. in the present study, we applied the mdp scoring system using data on biomarkers measured from two distinct groups of patients, adults and children with active tb and healthy uninfected controls. the mdp transformation was used as an approach to normalize data cross experiments resulting in datasets with markers distributed in a similar scale. ethics statement. all clinical investigations were conducted according to the principles expressed in the declaration of helsinki. written informed consent was obtained from all participants or their legally responsible guardians. the study was approved by the institutional review board of the national institute for research in tuberculosis, chennai, india (nirt; protocol numbers nct and nct ). characteristics of study participants. baseline characteristics of the adult and pediatric participants have been described elsewhere . adults with pulmonary tb (ptb) and uninfected healthy controls were more frequently males than those with extrapulmonary tb (eptb) ( . % vs. % vs. . %, respectively; p = . ). in addition, adults with ptb were on average older than healthy controls (median (iqr) in years: ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) vs. . ( - ) , respectively; p = . ) but had median age similar to the group of eptb (supplementary table s ). in the pediatric study, patients with ptb were similar to those presenting with eptb or hc with regard age (median (iqr) in years: . ( . - . ) vs. ( . - ) vs. ( - ), respectively; p = . ) and sex (p = . ), with and overall high frequency of male individuals ( . % vs. . % vs. . %) (supplementary table s ). we next compared the adults and children and found that there were no significant differences in sex distribution in comparisons between the hc, the ptb as well as the eptb subgroups (supplementary table s ). increases in molecular degree perturbation of plasma cytokines and tissue remodeling mediators in adult and children with active tuberculosis. plasma levels of cytokines and tissue remodeling mediators were compared between ptb and eptb and uninfected healthy controls (hc) in adult and children from india, separately (concentration values are described in supplementary tables s , s ). in adults, compared to hc, the ptb or eptb groups exhibited higher levels of most parameters, except for ifn-β, timp- , il- p and timp- which were not statistically different (supplementary table s ). in the pediatric population, individuals with active tb (ptb or eptb) exhibited on average higher levels of fewer markers (ho- , mmp- , mmp- , timp- and timp- ) than controls (supplementary table s ). these results suggested that tb in adults may lead to more significant changes in the concentration levels of plasma biomarkers than what we observed in children with this condition. the data described above were originated from two different cohorts , . in order to directly compare the groups from the distinct studies, we calculated the overall mdp score values according to our previous publication and found that active tb was associated with a substantial increase in mdp scores compared to hc in both adults (ptb p < . , eptb p < . ; fig. a ) and children (ptb p < . , eptb p = . ; fig. b ). adult patients with ptb exhibited higher mdp values than those with eptb (p = . ; fig. a ), whereas mdp values in ptb and eptb were indistinguishable in children (p > . ; fig. b) . interestingly, adult patients fig. c ) suggesting that the impact of mtb on changes in the systemic molecular degree of perturbation is likely influenced by age. reinforcing this idea, we found no differences in mdp values between adults and children from the hc group (p = . ; fig. d ). plasma markers driving the overall molecular degree of perturbation in tuberculosis are distinct between adults and children. we examined the mdp expression values for each individual plasma cytokine and tissue remodeling mediators. an unsupervised hierarchical clustering was used to test whether perturbation values of all the markers combined were associated with specific changes that could characterize active tb (ptb or eptb) and hc in adults and children. we found that the overall expression profile was very distinct between adults with active tuberculosis and hc ( fig. a, left panel) . intriguingly, children showed heterogenous profile expression of mdp values of all the markers between active tb and hc (fig. b , left panel), without a clear separation in the cluster analysis. these findings indicate that the overall expression profile of the mdp values can be used to distinguish active tb from controls in adults but not in children. they also suggest that although the overall perturbation was substantially higher in children with active tb, perturbation of individual markers was not sufficient to reliably segregate those from the healthy control group. in addition, ptb could not be grouped separately from eptb in both adults and children ( fig. a ,b, left panels), indicating that active tb drives specific changes in mdp independent on disease site. univariate analyses comparing the mdp values for each marker between hc and ptb or eptb groups are shown in supplementary figs. s and s . furthermore, we employed a sparse canonical correlation analysis using mdp values to test whether the profile of correlations between the plasma markers, rather than their individual concentration values, could be used to distinguish ptb or eptb and hc in both adults and children. curiously, the markers with the strongest contributions for discrimination in adults with ptb vs. hc were ho- , ifn-γ, il- , il- β and timp- whereas in children such markers were timp- , ho- , vegf, timp- and mmp- ( fig. a ,b right panels). the canonical model also specified that the markers which contributed the most for the discrimination between eptb and hc in adults were il- β, ifn-γ, ifn-α, mmp- and ho- , whereas in children were il- , ho- , mmp- , il- β and tnf-α ( fig. a ,b right panels). of note, the mdp values of out of markers which were relevant to identify ptb in adults (ho- , ifn-γ and il- β) were also part of tb signature observed in adults with eptb. moreover, in children, only marker which was relevant to identify ptb (ho- ) was also relevant network correlation profiles of molecular degree of perturbation in active tb are distinct between adults and children. to understand the nuances between molecular degree of perturbation of individual markers and their direct effect on overall mdp values, we employed network analysis based on spearman correlation matrices, as previously described . using this approach, we found that the presence of pulmonary infection in adults was associated a greater number of correlations when compared with those that developed eptb (fig. a) . the group of adults with ptb was marked by several positive correlations, highlighting that the degree of perturbation in ho- , ifn-α, ifn-γ, il- , il- , il- β and timp- markers was directly associated with the overall mdp score values (fig. a , left panel). in addition, perturbation of mmp- levels was inversely correlated with the overall mdp values in this clinical group. furthermore, the top nodes exhibiting the highest number of significant correlations in the network of adults with ptb were the overall mdp score followed by mmp- , il- , ho- and mmp- . importantly, the correlation profile found in adults with eptb was distinct from ptb (fig. a) . indeed, only the degree of perturbation of ho- and ifn-γ were directly associated with the overall mdp values in the eptb group. node analysis of the eptb network demonstrated that mmp- , ifn-γ, overall mdp and ho- were the most highly connected parameters (fig. a, right panel) . when the network analysis was extended to the pediatric population, we observed a decreased number of statistically significant correlations in pulmonary infection when compared with extrapulmonary tb (fig. b) . interestingly, we found that mtb infection was associated with marked absence of negative correlations in children. node analysis of the ptb network indicated that il- p , il- β and tnf-α were the most highly connected markers (fig. b, left panel) . curiously, the overall mdp values, which were highly connected in the networks from adults, were statistically correlated only with perturbation of ho- and timp- . children with extrapulmonary tb exhibited tnf-α, followed by ifn-α, timp- and timp- as the most relevant nodes (fig. b, right panel) . the degree of perturbation of ho- , ifn-α, timp- and timp- was directly associated with the overall mdp values in children with eptb. furthermore, in children, there was a lack of correlations between the mdp values of markers described to be important in tb pathogenesis such as ifn-γ, il- β and tnf-α in the networks. these findings argue that age influences the correlation between specific molecular markers that drive the overall molecular perturbation in active tb. age directly influences the overall inflammatory perturbation profile in active tuberculosis independent of sex. the results described above suggested that age was associated with the molecular www.nature.com/scientificreports/ degree of inflammatory perturbation. to directly test this hypothesis, we grouped each individual based on age and performed an exploratory investigation using unsupervised hierarchical cluster analysis with z-score normalized values of the mdp calculated for each marker. we found that ptb did not exhibit a distinct biomarker mdp profile compared to eptb independent on age (fig. a, left panel) . of note, mdp values detected for many markers were relatively lower in children independent on the tb clinical presentation, except for timp- , timp- , timp- and timp- , which tended to be higher in those who were younger (fig. a, left panel) . we next tested direct correlations between age and the individual mdp values of each marker. spearman correlation analysis revealed that the degree of perturbation of ifn-γ, il- β, tnf-α, ifn-α, ho- , il- , mmp- , mmp- and vegf was directly correlated whereas timp- values were inversely correlated with age (fig. a , right panel). to determine association between age and probability of being molecularly perturbed (see methods for definition) in the entire population, we used a model adapted from the kaplan-meier survival curve (fig. b) . this approach revealed that increase in age was directly associated with higher probability of overall molecular perturbation. indeed, the overall mdp score values were positive correlated with age in all the clinical groups evaluated (supplementary fig. s ). we next analyzed the association between age and mdp values of each marker in our clinical groups, in adults or in children. we found that there were differences in regard to the relationship between aging and variation of mdp values of individual biomarkers in the distinct clinical groups in adults ( supplementary fig. s a ) as well as in children (supplementary fig. s b) . however, the vast majority of these differences were not statistically significant (adjusted p-values ≥ . ). finally, we examined the influence of sex on the association between age and inflammatory perturbation. overall mdp values were not different between male and female individuals stratified in the distinct clinical groups (fig. a, insert) . furthermore, using the www.nature.com/scientificreports/ kaplan-meier survival curve test, we found that, in general, there was no different in the curves of female and male participants (p = . , fig. a ). in fact, it was possible to notice that females tended to be less perturbed between and months of age, however with a considerable variation represented by large confidence intervals (denoting the low precision of the assessment) which overlapped with those from the curve of male participants (fig. a) . furthermore, spearman correlation analysis demonstrated that mdp values of most of markers were positively correlated with age in both male and female participants (fig. b) . timp- was the only marker that displayed a negative correlation in females. these findings suggested that sex does not substantially influence the higher probability of inflammatory perturbation with aging in tb. mechanisms of disease pathogenesis in tb have been extensively studied over the years, but despite that, the immunopathology of this infection in pediatric populations remains poorly understood. importantly, mtb infection is one of the main causes of childhood morbidity and mortality worldwide , , , and the field needs www.nature.com/scientificreports/ elucidation of the determinants of immunopathogenesis. in the present study, we used an adaption of molecular degree of perturbation , to estimate the level and quality of systemic inflammation in patients with active tb (ptb and eptb) according to age. our findings indicate that there are important discrepancies in the mdp values between adults and children with active tb, with adults exhibiting higher values, whereas the molecular perturbation was similar among individuals from the healthy control groups independent of age. thus, while the systemic inflammatory profile is similar between adults and children without tb, it becomes very distinct in patients with active disease. although we have not directly tested potential influence of maturation of immune system in the results, it is possible that the capacity to promote significant systemic inflammation in the context of tb may be affected by this process. mycobacterial infection is known to cause profound stimulation of both innate and adaptive immune response, in vitro and in vivo models , . as a consequence, differential activation of leukocytes, such as monocytes , and lymphocytes , , has been used to characterize the disease and even to serve as potential diagnostic biomarkers for ptb and eptb, as recently reported by our group . whether differential activation of immune cells in tissue or in peripheral blood upon mtb infection are the underlying factor dictating the systemic degree of perturbation described here is an assumption to be tested in future studies. our exploratory analyses characterized the systemic inflammatory response and indicated that mtb infection (pulmonary and extrapulmonary) was associated with overall increases in the mdp values in both adults and children. however, in either ptb or eptb groups, adult patients exhibited augmented mdp values compared to pediatric patients. this finding reinforces the idea that in the context of tb, adults are more prone towards presenting with a higher degree of inflammation than children. we expanded these analyses to show the degree of perturbation of each individual biomarker and found that the in adults, individuals with active tb exhibit a very distinct profile compared to those without. nevertheless, in the pediatric population, there was no clear combined biomarker profile that could distinguish tb from controls. hence, there are specific changes in the biomarker mdp profile that are age dependent. of note, using discriminant analyses based on a canonical correlation model, we identified that the top markers responsible for the discrimination between the clinical groups differed between adults and children. in adults, ho- , ifn-γ and il- β, markers which have been associated with tb pathogenesis [ ] [ ] [ ] were the top markers that contributed to discrimination between the disease groups (both ptb and eptb) and controls , , , whereas in children, timp- , il- and ho- were the markers that most contributed for discrimination between active tb patients and uninfected controls. of note, one important finding of this analytical approach was that the molecular perturbation of ho- could discriminate pulmonary and extrapulmonary tb from healthy controls in both adult and children populations. ho- is important marker of oxidative stress response, highly expressed in the lungs , , with a critical role in cytoprotection . these observations made us hypothesize ho- may be important in tb pathogenesis regardless of age. additional studies in more diverse populations are warranted to test this idea. the inflammatory process results from an intricate relationship between factors from the host and pathogen, and can be evaluated using network analysis , , . using this approach, we showed important differences in the correlation profiles between mdp values from each individual biomarker and the overall mdp values in adults and children with active tb. pulmonary infection with mtb in adults led to a coordinated inflammatory burst, which was read by detection of many statistically significant correlations between individual markers and overall mdp values. on the converse, in children presenting with ptb the augmented inflammation seemed less coordinated, meaning increases in molecular perturbation of a given marker were not followed by simultaneous increases of other markers or the overall mdp values. this suggests that pediatric ptb patients may have a decreased ability to mount a coordinated immune response. it is reasonable to hypothesize that such uncoupling of inflammatory activity may be a consequence of immune immaturity that leads to dissemination of bacilli in children, as previously demonstrated . curiously, in children with eptb, the networks were more complex, indicating a higher number of connections. such phenomenon could be consequence of extrapulmonary tissue damage, and maybe argue that despite the lower capacity to mount and sustain a coordinated response in pulmonary infection, eptb in children is marked by a more balanced interplay between innate and adaptive immune response . importantly, ifn-α, a relevant cytokine in the context of tb pathogenesis, was one of those most highly connected markers in children with eptb, being also connected to overall mdp expression values. this finding suggests that ifn-α may be a parameter influencing or being influenced by the pathological inflammation which characterizes eptb. moreover, in adults with eptb, the complexity of the inflammatory network was reduced, in agreement with the previous published evidence of probably less organized and an unfettered immune activation in adults with eptb . the determinants of the differences in correlations between concentrations of mediators of inflammation between distinct clinical forms of tb in adults and children are still unknown. an interesting result reported here was the association between increases in age and augmentation of the overall molecular degree of perturbation in both ptb and eptb patients. growing old has been associated with increased basal inflammatory state that alters susceptibility to many diseases, including tb . our results demonstrated that increase in age leads to rise in probability of a tb patient becoming more perturbed, independent on disease site (ptb or eptb), mainly after months. importantly, we described that degree of perturbation of many individual plasma markers correlated with age, reinforcing the idea that the capacity to induce systemic inflammation is proportional to age. in pediatric patients, immune activation has several peculiarities compared to that in adults, which result from a continuous evolution of the immune system from infancy to old age . fetal and neonatal immune adaptations facilitate intrauterine survival and provide early postnatal protection against extracellular pathogens, but they leave infants susceptible to intracellular pathogens such as viruses that are acquired perinatally . contrasting immune activation profiles between children and adults have been reported in the context of infection with mtb , helicobacter pylori , hiv , sars-cov- , and by several other pathogens. in these settings, the immune response described in children are frequently more modest, less intense than what is observed in adults. it is known that failure of alveolar macrophages to contain mtb and scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ recruit additional mononuclear cells to the site of infection helps to explain the more fulminant course of tb in early life . moreover, as far as in the current covid- pandemics, it has been reported that infected adults can develop different outcomes, from asymptomatic, mild, moderate to severe disease, and death. children, on the other hand, can also be infected by sars-cov- , but most cases with laboratory-confirmed infection are mild and severe disease is rare , . our results lead us to speculate that children infected with mtb do not substantially activate systemic inflammation as well as in adults. intriguingly, our findings suggest that the probability of being perturbed in the context of tb is dramatically increased after months of age. it is possible that, in highly endemic regions such as south india, there is an accumulation of exposures to mtb as well as to other infectious agents during the childhood. such gradual exposure could result in increased capacity to induce an augmented degree of systemic inflammation, which is proportional to the inflammatory perturbation assessed here through the mdp score. additional studies are necessary to really define if this is an intrinsic characteristic of pediatric patients of is conditioned to certain diseases. future investigations are also required to directly test whether the response of immune cells from individuals with tb at different age ranges are reflected by the differential probability of being molecularly perturbed. furthermore, our analysis showed that biological sex did not influence the probability of increase in mdp values, arguing that the potential effect of sex-related hormones on systemic inflammation may be superposed by the effect of aging in tb patients. in india, bcg is part of the national vaccination schedule and offered to all newborns. in our study, both pediatric and adult population exhibited the bcg scar and thus were vaccinated. therefore, it is difficult to discriminate the effect of bcg vaccination on the concentration values of plasma cytokines in such setting. the details on long-lasting effects of bcg on systemic levels of cytokines are not completely understood. two possible immunological mechanisms have been suggested to describe the nonspecific effects of bcg vaccination. the first mechanism is heterologous immunity, in which cross-protection is mediated by heterologous t-cell memory responses . the studies investigating this hypothesis fail to describe meaningful changes in plasma levels of cytokines; they rather show increased capacity of cells to respond in vitro upon stimulation . a second mechanism of protection has been recently proposed in the form of epigenetic reprogramming of immune cells, a phenomenon conferring nonspecific immune memory to innate immune responses and termed 'trained immunity' . indeed, bcg vaccination in healthy volunteers has been reported to induce epigenetic reprogramming of monocytes, leading to increased cytokine production in response to nonrelated pathogens for up to months after vaccination . in addition, such vaccine is capable of inducing boosted heterologous th /th responses, which could persist in up to year after vaccination . although to our knowledge there is no formal demonstration of the long-lasting effect of bcg on plasma levels of mediators of inflammation, it is possible that bcg vaccination had a potential impact on the circulating levels of cytokines in the pediatric participants. it is known that bcg triggers important effects on innate responses, mainly in macrophages and natural-killer cells , , leading to increases in il- β and tnf-α, aside from ifn-γ . our findings demonstrated slightly higher expression of il- β in healthy controls, higher tnf-α in children presenting with eptb and increased levels of ifn-γ in children with ptb, but such differences were not statistically significant. the mdp score values of these three important cytokines were all significantly higher in adults with eptb or ptb compared to their matched disease subgroups in children whereas the values between the hc groups were indistinguishable. future studies specifically testing the effects of bcg on systemic immune responses and whether such effects drive changes in the molecular degree of perturbation are warranted. our study has limitations. we were unable to test association between mdp and bacillary loads due to lack of data from the pediatric population. it is possible that higher mdp values detected in adults may be a consequence of the increased mycobacterial infection loads. in addition, the sample size of the group of children was relatively small, although the it was sufficient to result in a substantial study power. regardless, the extensive exploratory analyses performed revealed unique relationships between age and the systemic degree of inflammation. the results presented here shed light into the impact of aging on the systemic immune activation during tb. the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. resurgence of tuberculosis in children tuberculosis transmission among children and adolescents in schools and other congregate settings: a systematic review research agenda for childhood tuberculosis 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immune responses to hiv infection and vaccination the immune system of children: the key to understanding sars-cov- susceptibility? diagnosis, treatment, and prevention of novel coronavirus infection in children: experts' consensus statement no one is naive: the significance of heterologous t-cell immunity trained immunity: a memory for innate host defense bacille calmette-guerin induces nod -dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes long-lasting effects of bcg vaccination on both heterologous th /th responses and innate trained immunity heterologous effects of infant bcg vaccination: potential mechanisms of immunity non-specific effects of vaccines illustrated through the bcg example: from observations to demonstrations tuberculosis vaccines-rethinking the current paradigm the authors acknowledge study participants. this project was supported by the intramural research program of the niaid to s.b. and n.p.k. this study was also financed in part by coordenação de aperfeiçoamento de the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to b.b.a.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - h oqb k authors: guillén, lucía; padilla, sergio; fernández, marta; agulló, vanesa; garcía, josé alberto; telenti, guillermo; garcía-abellán, javier; botella, Ángela; gutiérrez, félix; masiá, mar title: preemptive interleukin- blockade in patients with covid- date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: h oqb k excessive interleukin- signaling is a key factor contributing to the cytokine release syndrome implicated in clinical manifestations of covid- . preliminary results suggest that tocilizumab, a humanized monoclonal anti-interleukin- receptor antibody, may be beneficial in severely ill patients, but no data are available on earlier stages of disease. an anticipated blockade of interleukin- might hypothetically prevent the catastrophic consequences of the overt cytokine storm. we evaluated early-given tocilizumab in patients hospitalized with covid- , and identified outcome predictors. consecutive patients with initial sequential-organ-failure-assessment (sofa) score < fulfilling pre-defined criteria were treated with tocilizumab. serial plasma biomarkers and nasopharyngeal swabs were collected. of patients admitted with covid- , met the inclusion criteria. after tocilizumab, ( . %) had an early favorable response. adjusted predictors of response were gender, sofa score, neutrophil/lymphocyte ratio, charlson comorbidity index and systolic blood pressure. at week- , . % of responders and % of non-responders had cleared the sars-cov- from nasopharynx. temporal profiles of interleukin- , c-reactive protein, neutrophil/lymphocyte ratio, nt-probnp, d-dimer, and cardiac-troponin-i differed according to tocilizumab response and discriminated final in-hospital outcome. no deaths or disease recurrences were observed. preemptive therapy with tocilizumab was safe and associated with favorable outcomes in most patients. biological and clinical markers predicted outcomes. the pandemic of coronavirus disease has become one of the greatest global health challenges. the novel severe acute respiratory syndrome coronavirus- (sars-cov- ) has spread to the five continents and caused more than , , new infections and , deaths. although the majority of infected people have a mild disease with good prognosis, a subset of patients develops bilateral interstitial pneumonia, diffuse alveolar injury and acute respiratory distress syndrome (ards), which carries a mortality higher than % and constitutes the main cause of death , . in contrast to other respiratory viral infections like influenza, a major pathogenic mechanism implicated in severe clinical manifestations of covid- is an aberrant host immune response resulting in an excessive cytokine and chemokine release known as "cytokine storm" or "cytokine release syndrome" , . among immune molecules contributing to the cytokine-mediated damage and inflammation, interleukin- (il- ) is one of the key factors . patients with covid- show high plasma levels of il- , which correlate with disease severity and multiorgan failure , . excessive il- signaling induces different biological effects that, in addition to the immune system and inflammation mediators, also involve the blood vessels or the myocardium, and result in organ damage , . due to the different pathogenic mechanisms implicated in covid- , combined therapeutic approaches including both antivirals and blockage of inflammatory pathways have been advocated for an optimal disease control . tocilizumab (tcz) is a humanized monoclonal anti-il- receptor antibody, subclass igg . because of the prominent role of il- in covid- , it was the first drug proposed for the treatment of the inflammatory complications in patients with severe disease . however, data are limited to a scarce number of studies that mostly included severely ill patients [ ] [ ] [ ] [ ] . several questions remain to be elucidated. first, whether tcz administration at an earlier stage of disease might hypothetically prevent the catastrophic consequences of the overt cytokine open infectious diseases unit, hospital general universitario de elche, camí de la almazara s/n, elche, alicante, spain. www.nature.com/scientificreports/ storm. as a counterpart, since il- can both facilitate but also suppress viral replication , , tcz could have a detrimental impact on viral clearance if given prematurely. therefore, information on the effect of tcz on sars-cov- replication might help understanding the benefits and risks of therapy. a third question refers to whether there could be predictors of a higher probability of response to tcz to select the most suitable candidates for therapy and to design alternative strategies for the anticipated non-responders. finally, mid-term effects and incidence of disease recurrences after treatment cessation, or complications derived from the tcz-associated immunosuppression in patients diagnosed with covid- remain to be determined. in march , the spanish agency of medicines and medical devices granted an emergency-use authorization for using tcz in the setting of covid- , and our center developed specific guidelines for treating patients requiring hospital admission. the institutional guidelines included the use of tcz at an early stage of disease, according to predefined criteria, evaluating clinical, biological and virological outcomes. in this article we describe the short and mid-term effects of early therapy with tcz and identify predictors of a higher probability of response. during the study period, adult patients were admitted with covid- . of them, received tcz, and met the inclusion criteria and were selected for the analysis; of them had sars-cov- infection confirmed by rt-pcr. overall cohort mortality ( % ci) was . ( - )%; . ( . - . )% in patients receiving tcz, and no deaths occurred in patients meeting the inclusion criteria. median (q -q ) sofa score was . ( ) ( ) , and median (q -q ) number of days from illness onset to hospital admission was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . baseline characteristics of included patients are shown in table . median (q -q ) age was ( - ) years, and ( %) were male. the majority ( %) of patients had coexisting comorbid diseases, mainly hypertension ( %), cardiovascular disease ( %), diabetes ( %) and chronic obstructive pulmonary disease (copd) ( %), with median (q -q ) charlson comorbidity index score of ( - ). antimicrobial and/or immunomodulatory therapy administered during hospital stay is detailed in table , p = . ), with no differences between groups in the frequency of treatment with methylprednisolone. there were no disease recurrences after tcz interruption, or additional bacterial infections as a complication of therapy during hospitalization or at the -week follow-up visit. among the patients meeting the inclusion criteria with confirmed sars-cov- infection by rt-pcr, there were ( . %) responders and ( . %) non-responders. when both groups were compared, the results were similar to those observed in the patients comprising the entire study sample (see supplementary table ) . table shows the multivariate logistic regression analysis to identify predictors of response to tcz. the model showed that male sex (or . ; % ci . - . ), a nlr > . (or . ; % ci . - ), higher sofa score (or . ; % ci . - . per unit increase), higher systolic blood pressure (or . ; % ci . - . per mmhg) and higher charlson comorbidity index (or . ; % ci . - . per unit increase), were associated with unfavorable outcome following tcz administration. in a sensitivity analysis including only the patients with confirmed sars-cov- infection by rt-pcr, the significant variables in the adjusted multivariate model were a nlr > . (or . ; % ci . - ), higher charlson comorbidity index (or . ; % ci . - . ) per unit, and higher sofa score (or . ; % ci . - . ) (supplementary table ). figure shows the temporal changes of several biomarkers in tcz responders and non-responders analyzed through local polynomial regression. non-response to tcz was associated with an increase in il- not observed in responders (p < . ), and the same occurred for the levels of d-dimer (p = . ), the nlr (p < . ), and the nt-probnp (p = . ). for the platelet/d-dimer ratio, initial levels were significantly lower in non-responders (p = . ), although trends were not significantly different between groups. the crp levels decreased in both groups, and the decrease tended to be faster in responders to tcz (p = . ). table shows roc analysis to identify plasma biomarkers and clinical variables that, measured at h after tcz initiation, best discriminated between favorable or unfavorable further clinical outcome. the best predictors among the biomarkers were the crp, the platelet/d-dimer ratio and the hs-cardiac troponin. at h of tcz initiation, a crp value above www.nature.com/scientificreports/ ( . - . ) copies/swab (p = . ). figure shows temporal changes of sars-cov- rna obtained from nasopharyngeal swabs among tcz responders and non-responders analyzed through local polynomial regression. at month after admission, / ( . %) of tcz responders and / ( %) of non-responders showed undetectable viral load (p = . ). in this cohort of patients admitted with covid- in whom tcz was used at an earlier stage of disease, there was a low rate of mortality. previous studies included severely-ill patients, and mortality rates between and % [ ] [ ] [ ] , with the exception of a study including patients, where no deaths occurred . our results support that prompt initiation of therapy with tcz is safe in the short and mid-term, and might be associated with a favorable outcome in patients hospitalized with covid- , as reflected by the low overall mortality observed in our entire cohort of patients admitted for covid- , which was lower than the . % mortality described in hospitalized patients in spain (https ://www.mscbs .gob.es/en/profe siona les/salud publi ca/ccaye s/alert asact ual/ncov-china /situa ciona ctual .htm). we identified early clinical and biological predictors of response to tcz, which might contribute to selecting alternative strategies for the anticipated non-responders. we found that non-response to tcz was associated with higher sofa score and systolic blood pressure on admission, as well as a higher charlson comorbidity index. baseline levels and the initial response after tcz administration of some biomarkers were also useful to predict the clinical outcome; a lower nlr on admission, and lower platelet/d-dimer ratio and hs-cardiac troponin i at h following tcz initiation were associated with favorable tcz response. likewise, the performance of several biomarkers after tcz initiation revealed to be useful to discriminate among responders and non-responders. specifically, the levels of il- , nlr, d-dimer, nt-probnp, and the platelet/d-dimer ratio showed an increase in non-responders that was not observed in responders. one of the concerns regarding anti-il- therapy is the potential risk of impaired viral infection control. il- regulates the immune response through differentiation of cd and cd t cells to induce a cytotoxic and cytolytic activity against pathogens . in experimental studies, il- has demonstrated to induce t cell and b cell responses against viruses for viral clearance, the phagocytic activity of macrophages and tissue remodeling , , and to suppress the replication of viruses like hepatitis b or hiv , . therefore, anti-il- therapy could potentially have detrimental consequences if given too early in patients with covid- , before the full effect of the antivirals has been established. however, data also exist pointing to a negative influence of il- on viral clearance by inhibition of cd t cell response, favoring viral persistence through a synergistic interaction with il- , leading to higher viral replication and higher virulence , . in our cohort, patients receiving tcz had a favorable outcome, and viral clearance was not significantly different among those with favorable or unfavorable response, although due to the low number of patients, our study cannot exclude a potential relationship between the worse clinical outcome and a negative effect of tcz on viral dynamics in the subgroup of non-responders. we neither observed additional bacterial infections as a complication of therapy during hospitalization or at the -day follow-up visit. we analyzed baseline and serial levels of biomarkers of inflammation, coagulation and myocardial function on an every-other-day schedule during hospital stay, and at month after admission. we found that the initial levels of a number of different biomarkers allowed the prediction of subsequent response to tcz. lower levels on admission of the nlr were associated with a favorable tcz response after adjusting for other predicting covariables. this biomarker reflects excessive inflammation and dysregulation of immune cells that play a central role in severity of disease in viral infections , and has been associated with mortality in patients hospitalized with covid- . the nlr has shown to be the most important factor for the early-stage prediction of disease progression in patients with covid- , and a useful tool for risk stratification . our findings support a similar central role of the nlr for the prediction of response to tcz, and also suggest that early initiation of therapy with tcz before the catastrophic hyper-inflammatory response has been triggered might improve the prognosis of patients with covid- . in addition to baseline levels, we found that the initial response of some biomarkers after tcz administration also predicted disease outcome. the levels of crp at h after tcz infusion with a cut-off value of mg/dl showed a high specificity and moderate-to-high sensitivity to predict favorable subsequent response. the same was observed with a cut-off value of for the platelets/d-dimer ratio and of . ng/ml for the hs-cardiac troponin i, although for the latter value the sensitivity was fair. the association of biomarkers of coagulation www.nature.com/scientificreports/ and myocardial function with outcome reflects the multifactorial pathogenesis of covid- , and its potential reversibility with anti-il- therapy before the overt disease is manifested. conversely, further immune-based or antiviral therapy should be considered in addition to tcz when the initial levels of the biomarkers predict poor response, in order to optimize treatment results. the performance of several biomarkers after drug administration was another factor contributing to characterize the responders to tcz. the levels of the inflammation markers il- and the nlr showed an increasing trend during the following days after tcz initiation in non-responders, and peaked several days afterwards; by contrast, they remained relatively stable in patients with favorable outcome. as a result, in some patients tcz would not be capable of inhibiting the cytokine storm, and this would be a predictor of poor outcome. il- showed a second peak during the th week after tcz, and the same was seen for crp, and again only in non-responders, probably reflecting more than one flare of disease in this subset of patients. in addition to inflammation, a biomarker of cardiac disease such as the probnp also showed a raising trend in non-responders, figure . temporal changes in the levels of several biomarkers in tocilizumab responders and non-responders analyzed through local polynomial regression. p values for the comparison in the temporal trends between responders and non-responders. *p value for the comparison of baseline levels of the biomarker between responders and non-responders. no significant differences observed in the temporal trends between groups. lod limit of detection. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ suggesting that myocardial dysfunction may contribute as well to the lung complications and adverse outcome. the same occurred with d-dimer, an independent predictor of hospital death in patients with covid- , that reflects a pro-coagulant state and increased thrombotic risk associated with severe disease in covid- , and which only showed a decreasing trend in patients responding to tcz. the role of il- in venous thromboembolism remains to be well defined. while il- has been associated with the resolution of thrombi through macrophage recruitment and the induction of proteolytic enzymes , other studies have demonstrated an association between the development of deep venous thrombosis and the inflammatory injury of vascular endothelial cells caused by the imbalance of cytokine expression and, particularly, il- was significantly upregulated in clinical and experimental studies of deep venous thrombosis, and blocking il- in mice with neutralizing antibodies inhibited the formation and development of thrombosis . the charlson comorbidity index was a predictor of response to tcz. our patients had a high frequency of hypertension, followed by cardiovascular diseases, diabetes and copd, as described in other series . the table . performance in roc analysis of serum biomarkers and clinical variables at h of tocilizumab initiation on clinical outcome. unfavorable response was defined as reaching a sofa score > during hospital stay, intensive care unit admission or death. il- interleukin , hs high-sensitivity, auc area under the roc curve, ci confidence interval, sp specificity (%), sn sensitivity (%). a composite score using il- , platelets, spo and spo /fio variables. www.nature.com/scientificreports/ four comorbidities are included in the charlson index, and have been associated with higher disease severity in patients with covid- . although the implicated pathogenic mechanisms have not been characterized, in patients with diabetes or underlying cardiovascular disease, the infection could induce additional damage due to direct or indirect effects related to inflammation, hypercoagulable state and hypoxia , which may aggravate the acute respiratory syndrome. the inverse association observed between the charlson score and the response to tcz supports the recent results of a nationwide cohort of hospitalized patients with covid- , where a greater number of comorbidities was associated with poorer outcome . blood pressure showed to be an independent predictor of response to tcz, and lower levels on admission were associated with a better response to tcz. sars-cov- binds to the angiotensin converting enzyme- (ace- ) receptor, and the expression of ace- is increased in patients treated with ace inhibitors and angiotensin ii type-i receptor blockers , . this may facilitate infection with sars-cov- , and might also lead to higher severity of disease and mortality. higher systolic blood pressure in our study could be a surrogate marker of hypertension, and might also be associated with raised filling pressures and contribute to pulmonary edema in patients with ards. interestingly, increased levels of hs-troponin i and nt-probnp were associated with non-response to tcz, which might suggest that the cytokine-related stress cardiomyopathy might not be reversed by tcz if it is not promptly initiated. this impaired myocardial function may be aggravated in patients with higher blood pressures and/or hypertensive heart disease. limitations of the study are the absence of a comparable control group, and the possibility of a selection bias in patients receiving tcz due to the observational nature of the study. the small number of non-responders to tcz does not allow to exclude a role of viral load as a predictive factor, and other potential contributors like the host immune response were not explored in the study. strengths are the distinctive characteristics of the population included, and the exhaustive sequential analysis of a high number of biomarkers following tcz administration which, in addition to clinical factors, allowed the characterization of the patients with a higher probability of response. in conclusion, early therapy with tcz is safe and it is associated with low mortality. our results suggest that a prompt use of tcz before the overwhelming secretion of cytokines has occurred might lead to better patient outcomes, and support confirmatory randomized clinical trials to assess the role of immune modulator therapies at an early stage of covid- . from all hospital admissions due to covid- at hospital universitario of elche, spain, between march th and april th, , consecutive patients treated with tcz with an initial sofa score < were included in the analysis. patients with confirmed or probable covid- according to european centre for disease prevention and control criteria (https ://www.ecdc.europ a.eu/en/covid - /surve illan ce/case-defin ition ) were included. microbiological confirmation was performed through real-time polymerase chain reaction (rt-pcr) from a nasopharyngeal smear sample or a bronchial aspirate. patients were managed according to a pre-defined protocol approved by hospital general universitario de elche, consisting in the collection of epidemiological, demographic, and clinical data, as well as serial nasopharyngeal samples for sars-cov- , chest x-ray, and routine blood tests including levels of interleukin (il- ), ferritin, lactate dehydrogenase (ldh), magnesium, procalcitonin, c-reactive protein (crp), n-terminal pro b-type natriuretic peptide (nt-probnp), highly sensitive (hs)-troponin i, d-dimer, and coagulation tests. comorbidities and the charlson comorbidity index were collected on admission. blood samples for lab tests, electrocardiogram (ekg), chest x-ray and nasopharyngeal samples for sars-cov- were obtained at different time points during hospital stay. in addition to sars-cov- , pcr analysis for influenza a and b viruses and for respiratory syncytial virus (rsv) were also performed from nasopharyngeal samples upon admission, as well as pneumococcal and legionella antigens from a urine sample, and serological test for hiv, hcv and hbv, and the quantiferon test. as stated before, therapy for covid- was given following institutional guidelines of hospital general universitario de elche. patients received antimicrobial and/or immunomodulatory therapy containing lopinavir/ritonavir, hydroxychloroquine, azithromycin or interferon-β- b ± methylprednisolone. according to guidelines, tocilizumab (tcz) was added to initial therapy on admission if any of the following criteria were met: a curb- > ; oxygen saturation < %; respiratory frequency > per min; a chest radiograph with bilateral multilobar infiltrates; one of the following biological markers: d-dimer ≥ . µg/l, il- ≥ pg/ml, lymphocytes < × /l, ferritin ≥ µg/l, fibrinogen > mg/dl or pcr > mg/l. the dose of tcz was mg iv if the weight was ≥ kg or mg if the weight was < kg. the patients were reevaluated on the following h. response to therapy was defined as resolution of fever, improvement in tachypnea, improvement in oxygen saturation by at least %, decrease in crp of at least %, or no radiological progression h after tcz administration. non-response was defined by the absence of any of h response criteria, or an increase in the sofa score > measured at - h or at day after tcz, icu admission or death. if no clinical response was achieved at h after tcz administration, a second dose of tcz ( mg) or intravenous corticosteroids were administered. contraindications to the use of tcz drug were a neutrophil count < × /l, platelets ≤ , × /l, ast/alt > × upper normal limit and documented sepsis by other pathogens. the protocol was approved by the ethical committee of the hospital general universitario de elche (spain) as part of the covid- @spain study. informed consent was obtained from all subjects. for rna extraction and rt-pcr analysis for sars-cov- , nasal and oropharyngeal flock swabs were placed together into ml transport medium (vicum, deltalab, rubí, spain). viral rna was extracted from µl of the medium using the rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions and eluted in a final µl nucleic acid elution sample. eight µl rna was used for detection of sars-cov- www.nature.com/scientificreports/ by rt-pcr, with a commercially available kit (allplex -ncov assay, seegene, seoul, korea) which targeted the e, rdrp and n genes. viral load measurements of nasal/throat samples (log copies/sample) were performed with a standard curve of ten-fold serial dilutions from an in vitro rna transcript (macrogen, seoul, korea). assay procedure was carried out in accordance with the manufacturer's protocol, in a cfx real-time thermocycler (bio-rad, california, usa). the success of rna extraction and pcr were assessed by the internal control included in the kit and negative and positive controls were used in each assay. continuous variables are expressed as median ± th and th percentiles (q , q ), and categorical variables as percentages. wilcoxon or student's t-test were used to compare continuous variables, and the chi-square or fisher's exact test for categorical variables comparison. normality of data samples was assessed using shapiro-wilk test. odds ratios (or) were estimated through generalized linear models with binary response and logit link function. to represent the temporal changes of the biomarkers levels, local polynomial regression models were employed using weighted least squares to estimate the performance of each biomarker according to the day since tzc was initiated. differences in temporal trends between biomarkers were analyzed through linear mixed models in which an interaction term of day since tcz initiation and response was included. confidence intervals for the area under the receiver operating characteristic curve (auc of the roc curve) were calculated through stratified bootstrap replicates with attempts. the cut-off values with the best sensitivity and specificity were selected. statistical analysis was performed using r-project version . . ( - - ) and the libraries ggplot for the figures . the datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. received: june ; accepted: september risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease pneumonia in wuhan, china why tocilizumab could be an effective treatment for severe covid- ? pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid- patients can we use interleukin- (il- ) blockade for coronavirus disease (covid- )-induced cytokine release syndrome (crs) clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan immunotherapeutic implications of il- blockade for cytokine storm role of interleukin in myocardial dysfunction of meningococcal septic shock plea for multitargeted interventions for severe covid- cytokine release syndrome in severe covid- : interleukin- receptor antagonist tocilizumab may be the key to reduce mortality effective treatment of severe covid- patients with tocilizumab tocilizumab for the treatment of severe covid- pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of patients in brescia, italy tocilizumab for the treatment of severe coronavirus disease tocilizumab treatment in covid- : a single center experience the role of interleukin during viral infections the role of cytokines including interleukin- in covid- induced pneumonia and macrophage activation syndrome-like disease il- ameliorates acute lung injury in influenza virus infection late interleukin- escalates t follicular helper cell responses and controls a chronic viral infection involvement of il- in the anti-human immunodeficiency virus activity of ifn-tau in human macrophages interleukin- (il- ) and il- synergistically promote viral persistence by inhibiting cellular apoptosis and cytotoxic t cell function tlr ligand induced il- counter-regulates the anti-viral cd (+) t cell response during an acute retrovirus infection increased virulence of an epidemic strain of vesicular stomatitis virus is associated with interference of the innate response in pigs experimental infection of ponies with equine influenza a (h n ) virus strains of different pathogenicity elicits varying interferon and interleukin- responses pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with covid- neutrophil-to-lymphocyte ratio predicts critical illness patients with coronavirus disease in the early stage clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study coagulation abnormalities and thrombosis in patients with covid- crucial involvement of il- in thrombus resolution in mice via macrophage recruitment and the induction of proteolytic enzymes il (interleukin)- contributes to deep vein thrombosis and is negatively regulated by mir- - p clinical features of cases with coronavirus disease in wuhan, china comorbidities and multi-organ injuries in the treatment of covid- cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) comorbidity and its impact on patients with covid- in china: a nationwide analysis are patients with hypertension and diabetes mellitus at increased risk for covid- infection? the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases elegant graphics for data analysis this work was supported by the rd / / project as a part of the plan nacional research + development + innovation (r + d + i) and cofinanced by instituto de salud carlos iii-subdirección general de evaluación y fondo europeo de desarrollo regional; instituto de salud carlos iii (fondo de investigaciones sanitarias [grant number pi / ; pi / ; cm / cov - ]). the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to f.g. or m.m.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -grs x l authors: matin, farhana; jeet, varinder; moya, leire; selth, luke a.; chambers, suzanne; clements, judith a.; batra, jyotsna title: a plasma biomarker panel of four micrornas for the diagnosis of prostate cancer date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: grs x l prostate cancer is diagnosed in over million men every year globally, yet current diagnostic modalities are inadequate for identification of significant cancer and more reliable early diagnostic biomarkers are necessary for improved clinical management of prostate cancer patients. micrornas (mirnas) modulate important cellular processes/pathways contributing to cancer and are stably present in body fluids. in this study we profiled cancer-associated mirnas in plasma collected before (~ % patients) and after/during commencement of treatment (~ % patients), from age-matched prostate cancer patients and healthy controls, and observed elevated levels of mirnas - mir- , mir- , mir- - p and mir- - p, which were validated in an independent cohort. the mirna panel was able to differentiate between prostate cancer patients and controls (auc = . ). analysis of published mirna transcriptomic data from clinical samples demonstrated low expression of mir- - p in tumour compared to adjacent non-malignant tissues. overexpression of mir- - p increased proliferation and migration of prostate cancer cells, suggesting a role for this mirna in prostate cancer pathogenesis, a concept that was supported by pathway analysis of predicted mir- - p target genes. in summary, a four mirna panel, including mir- - p which likely targets genes with key roles in prostate cancer pathogenesis, has the potential to improve early prostate cancer diagnosis. prostate cancer is one of the most commonly diagnosed cancers in men worldwide with a forecast of increased incidence over the following years based on current trends . measuring the levels of prostate specific antigen (psa; also known as human kallikrein- ) in serum is the standard first line test to indicate risk of prostate cancer and the most widely used biomarker for prostate cancer diagnosis. the advent of the psa test in the late s as a tool for prostate cancer diagnosis has enormously benefited prostate cancer patients for more than two decades , . however, inadequacies continue to exist over its suitability for use as a diagnostic tool for early detection of prostate cancer due to lack of specificity and high rates of over-diagnosis and over-treatment associated with psa testing , . therefore, there is an urgent clinical need for better diagnostic tools for prostate cancer. although prostate biopsy is the gold standard prostate cancer diagnostic tool, it is bound by several limitations . most prostate biopsies are routinely performed taking cores under transrectal ultrasound guidance , with an increase in the core number found to increase cancer detection rate by only . fold . however, the major drawback lies in the possibility of generating false negatives, as the samples are often taken randomly due to the unknown location of the tumour, and patients may require repeated biopsies under mri guidance or in combination with ultrasound for better sensitivity . over recent years, considerable research has led to the development of several molecular and genetic assays that have provided a prospective direction for the development of prostate cancer biomarkers . such assays utilise body fluid-or tissue-based biomarkers for the diagnosis, prognosis and risk stratification of prostate cancer . despite recent advances, it is still necessary to understand the role of these tests in the overall management of prostate cancer patients and the search for potential new biomarkers continues. expression of selected plasma mirnas in discovery cohort. to validate the findings from the screening study, we quantitated the candidate mirnas by qrt-pcr in individual plasma samples in the discovery cohort (n = ) consisting of plasma samples collected from age-matched patients (n = ) and controls (n = ). the clinical characteristics of the participants are summarised in supplementary table s . the up-regulated mirnas in the screening study were differentially expressed in the same direction. however, the down-regulated mirnas were up-regulated in individual patient samples; due to this discrepancy between the analyses, these candidates were discarded. of the remaining mirnas, mirnas -mir- , mir- , mir- - p and mir- - p, had a mean fold-regulation value of > and were all significantly altered compared to controls as determined by the unpaired mann-whitney u test followed by multiple testing using bonferroni correction, p ≤ . (fig. b/ validation of plasma mir- , mir- , mir- - p and mir- - p. to further validate our results from the discovery cohort, we assessed an independent set of patient and control samples (n = ). this validation cohort comprised of patients and controls. out of patients (i.e. . %) in the validation cohort had a gleason score of , in contrast to patients in the discovery cohort where the gleason score was in out of patients (i.e. . %). out of patients (i.e. . %) in the discovery cohort had a gleason score of , and out of patients (i.e. . %) in the validation cohort had a gleason score of . the mean age of patients in the two cohorts was similar (i.e. . ± . s.d. years and . ± . s.d. years) and the controls were age-matched accordingly (supplementary table s ) . differential expression analysis in the validation cohort confirmed that the levels of mir- , mir- , mir- - p and mir- - p were significantly altered in patients compared to controls using unpaired mann-whitney u test followed by multiple testing using bonferroni correction, p ≤ . (fig. ). diagnostic mirna signature for prostate cancer. given the association of mirna expression with prostate cancer, we performed univariate logistic regression analysis on the discovery (n = ) and validation (n = ) cohorts for each of the four mirnas ( table ). the regression coefficient (b) indicates an estimated increase in the odds of the outcome (i.e. prostate cancer) with increase in the value of the exposure (i.e. plasma mirna level). the % confidence interval (ci) was used as a measure of precision of the regression coefficient and to determine the presence of statistical significance which was confirmed by the p-value. mir- , mir- and mir- - p were found to be significantly associated with disease occurrence in the discovery cohort, except for mir- - p which did not reach statistical significance in this cohort (table ). all four mirnas were significant in the validation cohort (table ) . adjustments were made for each patient and control sample for varying starting concentration of rna using off-set correction during binary logistic regression analysis. we further evaluated, using multivariate binary logistic regression and roc analyses, the diagnostic capacity of the individual and combination of mir- , mir- , mir- - p and mir- - p in the discovery and validation cohorts as well as in both cohorts combined. roc analysis for each mirna in the panel was also performed for the discovery, validation and combined cohorts to allow comparison between the auc of each mirna and the auc of all four mirnas together as a measure of diagnostic accuracy (supplementary figure s ) . the auc for individual mirnas ranged from . - . for mir- , . - . for mir- , . - . for mir- - p and . - . for mir- - p respectively (supplementary figure s ) . the combined predictive probability of all four mirnas produced an area under the curve (auc) of . (p < . , % ci = . - . ) in the discovery cohort (fig. a) , auc = . (p < . , % ci = . - . ) in the validation cohort (fig. b) and auc = . (p < . , % ci = . - . ) in the combined cohort (fig. c) for the discrimination of prostate figure . screening of plasma mirna markers in pooled patient and control samples and validation in the discovery cohort. (a) scatter plot of cancer-associated mirnas in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miscript mirna pcr array. mirnas found to be differentially expressed between the patient and control groups are shown in black, and unchanged mirnas are shown in grey. a fold regulation cut-off of . was selected for the analysis. (b) scatter plot showing the differentially regulated mirnas re-analysed by qrt-pcr in patient samples in the discovery cohort (n = ). the mean fold regulation of each mirna across the patient and control samples was taken into account and those that were below the fold regulation cut-off were excluded from further analysis. the selected mirnas are shown in colour. (c) relative levels of mir- , mir- , mir- - p and mir- - p analysed by qrt-pcr as in (b) in patients vs healthy controls. statistically significant differences were assessed using a mann-whitney u test; p values are shown after bonferroni correction for multiple testing. each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation. relative expression of mir- , mir- , mir- - p and mir- - p in the validation cohort (n = ). statistically significant differences in mirna expression levels between the patients and control groups were assessed using a mann-whitney u test; p values are shown after bonferroni correction for multiple testing. each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation. scientific reports | ( ) : | doi: . /s - - -w cancer patients from healthy controls. despite some differences in the gleason score between the two cohorts, the diagnostic accuracy of the mirna signature was similar in the discovery, validation and combined cohorts. mirna expression in transcriptomic data from clinical samples. since there may be differences between the expression of mirnas in the circulation and in tumour tissues, we next determined whether the plasma levels of the identified mirnas reflected similar changes in tumour tissues. analysis of published mirna transcriptomic data from clinical samples in the tcga dataset demonstrated low expression of mir- - p in tumour compared to adjacent non-malignant tissues (p = . ) (wilcoxon test, p ≤ . ) (fig. ) figure s ) . our results suggest a significant (p = . ) increase in the proliferative potential of lncap cells upon overexpression of mir- - p using mirna mimics (fig. a) . we observed a substantial increase in cell confluence and change in cell morphology in mir- - p mimic treated cells when compared to non-targeting negative control treated cells over a period tables s and s ). the mirnet predicted targets are determined experimentally as part of larger experiments such as microarray, rna-seq, qpcr and par-clip, an immunoprecipitation method for identifying the binding sites of cellular rna-binding proteins (rbps) and microrna-containing ribonucleoprotein complexes (mirnps) and each identified target gene is supported by the experiment name and pubmed literature . based on the analysis of mirna expression in the tcga dataset and functional assays, mir- - p targets were used as an input for ingenuity pathway analysis (ipa). ipa generated canonical pathways for the targets of mir- - p (supplementary table s ). prostate cancer signalling was among the top ten canonical pathways consisting of deregulated target genes (highlighted in purple) i.e. fgfr , irs , sos , hsp aa , kras, cdkn b, ccnd and pten of mir- - p (−log p = . ) (fig. ) where fgfr and irs are closely related to pi k and hence shown as a group. analysis of the molecular function of these genes confirmed their involvement in cellular processes such as cell cycle, cell morphology, cell growth and proliferation and cell movement (supplementary table s ). the analysis also generated a list of upstream regulators of the target genes (supplementary table s ). of the target genes of mir- - p, six overlapped with gsea where prostate cancer was found to be the second most important pathway among the top kegg pathways (supplementary figure s ) . furthermore, oncomine data analysis of these genes showed significant upregulation of ccnd (p = . figure s ). circulating mirnas in body fluids may serve as clinically important biomarkers of various malignancies, including prostate cancer. in the present study, we identified an association of elevated plasma levels of four mirnas i.e. mir- , mir- , mir- - p and mir- - p in prostate cancer patients. initially, we analysed the plasma prostate cancer signalling was among the top ten canonical pathways consisting of eight deregulated mir- - p target genes (highlighted in purple) i.e. fgfr , irs , sos , hsp aa , kras, cdkn b, ccnd and pten where fgfr and irs are closely related to pi k and hence shown as a group. some of the targets for e.g. hsp , kras and sos exist as a complex or group of genes. the p value was represented as −log p = . for the analysis. mirnaome in age-matched prostate cancer patients and healthy controls using a mirna pcr array approach. we selected sample pooling for initial screening of mirnas because it minimised cost, reduced analytical run times and compensated for limited amounts of plasma samples enabling us to profile a large number of mirnas in patient and control samples in a cost effective way, and has been employed in previous studies , . many biological experiments have relied on pooling of biological specimens and the efficiency of this method has also been statistically investigated previously , . although sample pooling has its own advantages, it is not the optimum approach because while mitigating the loss of information, it does not take into consideration biological variation within individual samples. this may lead to the difference observed between mirna expression in pooled and individual samples. therefore, subsequent qrt-pcr in single samples from the discovery and validation cohorts was performed for validation of results and only those mirnas that were deregulated in the same direction in both pooled and individual samples were selected for further analysis by applying a stringent cut-off. the majority of previously conducted studies have used a higher c t cut-off to identify multiple mirnas, however, we have applied a strict cut-off to our analysis as our goal was to determine the most obvious candidates for rapid identification along the line of clinical translation. our identified mirna signature generated roc curves with auc values better than the previously reported auc value for psa , . similarly, other groups have assessed the diagnostic performance of plasma or serum mirnas in patients with localised or metastatic prostate cancer, bph and healthy controls, and in most instances the specificity and sensitivity of the mirna biomarkers have outperformed the accuracy of the psa test , , . although early detection and management of prostate cancer is a complex ongoing issue, there is insufficient evidence to support the benefits of population-based screening for prostate cancer in australia and in the united states using the psa test [ ] [ ] [ ] . the cancer council australia and u.s. preventative services task force recommends against the psa test, partly due to the over-diagnosis and over-treatment associated with it , . psa screening in healthy men is uncommon in australia and thus, we did not have the psa values for our control group which limited our ability to perform the mirna vs psa comparisons in our study. a pertinent limitation of our study is that some ( % and %, respectively) patient samples in cohort- and were collected after or during primary treatment. although mirna levels are affected by the treatment regimen of patients, we have tried to mitigate this effect by keeping the timing of sample collection consistent between the discovery and validation cohorts, using appropriate statistical methods and relying on the stability of mirnas in body fluids. our study has implicated mir- as a prostate cancer diagnostic biomarker for the first time. to date, only two studies investigating the role of mir- in congenital obstructive nephropathy as an exosomal biomarker which may provide further clues to explore its therapeutic potential in prostate cancer. mir- has been identified in a recent study by kristensen et al. as a member of a new -mirna classifier consisting of mir- - p, mir- - p and mir- that predicted biochemical recurrence (bcr) after radical prostatectomy in prostate cancer patients . the study was one of the largest mirna expression profiling studies in tumour tissue samples from patients in the discovery cohort and patients in the validation cohorts . however, an increased level of plasma mir- has not been previously reported as an early diagnostic marker for prostate cancer. mir- , as a member of a five mirna signature has been correlated with the survival of patients with hepatocellular carcinoma, and identified as one of three mirnas applicable for the diagnosis of these patients in a more recent study . recent studies have indicated the importance of mir- in colon , lung , liver and prostate cancer. meta-analysis of six mirna datasets revealed up-regulation of mirnas, mir- being one of them, in recurrent compared to non-recurrent prostate samples suggesting the predictive ability of mir- along with other mirnas . to the best of our knowledge, we are the first to demonstrate plasma mir- - p as a candidate for early diagnosis of prostate cancer. finally, mir- has previously been shown to be substantially decreased in higher gleason grade prostate cancer tissues and its low expression was correlated with advanced pathological stages , . the expression pattern is in line with our findings in the tcga dataset where mir- is lowly expressed in tumour compared with adjacent non-malignant tissues. a possible mechanism for the decreased expression is likely to be methylation of the mir- host gene promoter which prevents transcription of this mirna in tumour tissues possibly due to its tumour suppressive nature . another mechanism could be its release into the circulation to enable tumour growth at metastatic sites which may explain our results obtained through functional assays using mir- - p mimics. elevated levels of plasma mir- have been proposed as a diagnostic biomarker for other cancers such as lung, colorectal and breast cancer , and our findings suggest its potential to detect prostate cancer either alone or in combination with the other three identified mirnas. although higher levels of these mirnas may be indicative of prostate cancer pathogenesis, their mechanism of action is difficult to determine. studies have reported that circulating mirnas are involved in cell to cell communication and can be taken up by recipient cells to exert functional effects such as proliferation, invasion and angiogenesis . for example, tumour exosomal mirnas were shown to promote metastasis in other sites of the body by modulation of stromal cells in distant organs . we did not observe any significant effect of mir- - p overexpression on the proliferative ability of pc prostate cancer cells as previously suggested by zhu et al. . in contrast, we found that overexpression of mir- - p significantly increased proliferation and migration in lncap prostate cancer cells which suggests that regulation by the androgen receptor (ar) may account for the observed differences. this is contradictory to the findings from another study by theodore et al. where low expression of mir- has been correlated with an increase in prostate cancer metastasis . however, this may provide a clue to the metastatic role of this mirna at a distant site other than its site of production possibly via exosomal escape as mirnas are known to play a role in metastasis , and mir- - p has been previously reported as an exosomal mirna , . it is also believed that mirna secretion is a selective process and therefore, scientific reports | ( ) : | doi: . /s - - -w circulatory levels are not a true reflection of intracellular levels . on the contrary, some mirnas are released by tumour cells as argonaute (ago) bound complexes or from dead cells in apoptotic bodies , thus indicating the tumour as one of their sources. identification of mirna-target genes and pathways to understand the molecular basis of prostate cancer pathogenesis is a major challenge, as there are several risk factors and numerous pathways that drive cancer. of the four mirnas, mir- - p targets were selected for further analysis due to the significant differential expression of this mirna in the tcga dataset, and our findings from the functional assays. in depth analysis of mir- - p targets generated target genes through ipa and gsea in the prostate cancer signalling pathway with six common targets between the two analyses. the six genes were found to be important modulators of cellular processes involved in cancer progression. ccnd is a well-recognised oncogene involved in the direct phosphorylation of the retinoblastoma (rb) protein and promoting cell cycle transition from the g to s phase . ccnd is over-expressed in a considerable portion of human malignancies such as breast and prostate cancer , and it has been shown as a mirna target in breast cancer cells and primary prostatic epithelial cells . similarly, recent studies by lynch et al. and chakravarthi et al. demonstrated an inverse correlation between cdkn b and mirnas in prostate cancer cell lines and clinical prostatectomy specimens , . however, we did not observe any change in ccnd and cdkn b mrna expression in lncap cells with mir- - p treatment. kras is a well-established oncogenic gtpase protein playing an important role in cell division, differentiation and apoptosis. mirna targeting of the kras ′utr in a recent study induced cell apoptosis in vitro and exerted a tumour suppressive effect in vivo in xenograft mice models of colorectal cancer . this suggests the importance of the mirna-kras axis in prostate cancer where the mirna has been found to play a tumour suppressive role . on the contrary, our results suggest the upregulation of kras at the mrna level with mir- - p treatment in lncap cells which may explain the increase in cell proliferation and migration observed during our functional studies. hsp aa is a molecular chaperone that promotes regulation of specific proteins involved in signal transduction and cell cycle. a recent study by wilson et al. indicated the importance of histone methylation of ar binding sites of transcriptional targets, such as hsp aa in androgen signalling in prostate cancer progression . this may result in a positive regulation of hsp aa in metastasis as shown by our oncomine data analysis, whereas the expression of hsp aa possibly remains unchanged in localised cancer due to more stable complex formation between ar and hsp aa . we observed a decrease in hsp aa mrna in lncap cells after mirna treatment suggesting it as a possible target of mir- - p which in turn may play a role in the androgen signalling pathway. sos is a guanine nucleotide exchange factor involved in ras activation which stimulates a series of signal cascades crucial for malignant transformation. a recent study by alles et al. demonstrated that mirna overexpression in the hek- t kidney cancer cell line lead to reduced levels of endogenous sos . therefore, targeting of sos factors by mirnas may potentially inactivate ras signalling, however, many other factors are involved in signalling cascades resulting in cancer progression. pten is a lipid and protein phosphatase capable of regulating the pi k-akt pathway and suppress tumour growth in many cancers. pten expression and function is regulated by various mechanisms, including regulation by mirnas , and its co-inactivation has been reported to increase the neuroendocrine phenotype, a hallmark of prostate cancer progression during anti-androgen therapy . targeting of pten by mir- - p may therefore, result in increased cell proliferation and migration as observed in our functional assays, however, we did not observe any changes at the pten mrna level with mir- - p treatment. contrary to the previously reported role of pten, we also found that pten was up-regulated in both prostate cancer vs normal, and metastasis vs primary prostate cancer. the differential expression of pten was extracted in particular from the yu prostate dataset in the oncomine cancer microarray database . this dataset was selected over other datasets because it had the largest number of prostate cancer (primary/metastasis) and normal samples for comparison of all the target genes of interest. although pten is generally considered to be a tumour suppressor, it may act as a tumour promoter is some instances . it is also widely known that in addition to gene expression, genomic instabilities, mutations and copy number variation in the pten gene are often associated with prostate cancer , , and it is estimated that up to percent of primary prostate tumours have pten gene mutations . the yu prostate dataset in the oncomine cancer microarray database did not take these into account during data analysis and as a result, pten may be shown to be up-regulated. additional data analysis using other datasets in oncomine taking pten deletions and copy number variations into account revealed trends in the opposite direction. evidence suggests that the target genes discussed so far play a vital role in prostate cancer and their regulation by mirnas may be crucial in prostate cancer pathogenesis. some of these mir- - p targets are oncogenes while some are tumour suppressors and this suggests that mirna target modulation is dependent on various aspects such as genetic alterations, transcriptional/ post-transcriptional regulation, post-translational modification, target stabilisation, indirect regulation as well as cell type. moreover, validation of target genes and further functional studies are necessary to understand the mechanistic role of our identified mirnas in cancer progression. in conclusion, a major strength of our study is that it is one of the largest scale studies conducted on australian men to determine the potential of mirnas as diagnostic markers of prostate cancer. although it may have been ideal to include similar numbers of patients and controls, several published studies where the control group is smaller in number compared to the patient group have been reported previously . in addition, we have identified a novel plasma mirna panel consisting of four mirnas which have previously not been implicated for early diagnosis of prostate cancer. this was achieved by using higher than normal stringency to identify the most reliable candidates which is unique compared with many other studies. it was beyond the scope of our study to compare mirna expression levels in bph patients as analysed in several other studies as discussed in our review article . developments in the field of biomarker research are ongoing as stipulated by emerging mirnas and current tests that have a specific role in prostate cancer diagnosis and treatment. however, a critical need for scientific reports | ( ) : | doi: . /s - - -w robustness and reproducible data to implement such biomarkers in clinical practice still remains. undoubtedly, in the next few decades a substantial number of biomarkers will be available for clinical use in the overall management of prostate cancer. patients and blood collection. this is a retrospective study examining mirnas as risk/protection factors in relation to a diagnostic outcome. prostate cancer patients were recruited as part of the prostate cancer supportive care and patient outcomes project (proscan), a randomised controlled trial of a psychological intervention for newly diagnosed prostate cancer patients , for the discovery cohort (n = ) . the validation cohort of prostate cancer patients (n = ) were recruited as part of the australian prostate cancer bioresource (apcb) consisting of , patients including men recruited via collaborations with urologists, , men from the qld node of apcb, and men recruited in collaboration with the cancer council queensland . healthy male control participants for both the discovery and validation cohorts (n = ) were recruited through the electoral roll as part of the queensland men's health study (qldmen), a cross-sectional population-based study consisting of , men including age-and postal code-matched controls to complement participants in the proscan and apcb studies. the patient and control numbers were selected depending on the availability of samples from participants with similar clinical characteristics, as well as with reference to previous studies that we have discussed in our published review article . the patient group in each cohort is further divided into metastatic (≤ and > years survival) and non-metastatic patients. this subdivision ensured that each category of participants i.e. metastatic, non-metastatic and healthy in each cohort consisted of about samples. follow-up data for ~ years was available for the patients including those who survived for less than years after prostate cancer diagnosis. details of age, psa levels, clinical grade (gleason scores), pathologic state (tnm stages) and family history have been collected to document clinical characteristics of the disease. ~ - % of the patients and ~ - % of the healthy controls in each cohort had a family history of prostate cancer. . - . % patients and . - . % controls in both the cohorts did not have any family history of prostate cancer. blood samples were collected within - months of diagnosis and within - months of treatment, which included radical prostatectomy, androgen deprivation therapy (adt), brachytherapy and/or radiation therapy, from patients in the discovery cohort (n = ). similarly, for the validation cohort (n = ) blood was collected within - months of diagnosis and - months of any treatment, or at the time of radical prostatectomy for most of the patients, and for some collection was done after - months of hormone deprivation. although mirna levels are affected by the treatment regimen of patients, we have tried to mitigate this effect by keeping the timing of sample collection consistent between the discovery and validation cohorts, using appropriate statistical methods and relying on the stability of mirnas in body fluids . blood samples were collected in ethylene-di-amine-tetra-acetic acid (edta) containing tubes and processed within hour of collection. the resultant plasma samples were stored at − °c until rna extraction was performed, and transported on dry ice to the institute of health and biomedical innovation (ihbi) for rna isolation. all clinical samples were obtained from patients and controls after their written informed consent. the study was performed in accordance with the institutional ethics approval-approval numbers: h (proscan), (apcb), and (qldmen) from the queensland university of technology (qut) and the cancer council queensland (ccq). plasma rna isolation and cdna synthesis. prior to rna isolation, plasma samples in the discovery and validation cohorts were centrifuged for min at , × g at °c and all plasma samples were screened for haemolysis using a nanodrop nd- . for mirna screening (fig. ) , eight random plasma pools (two pools per group) were generated before rna extraction for the control group and three patient groups in the discovery cohort using a randomising web-based tool (https://www.miniwebtool.com/random-picker/). in this way µl of plasma required per pool was generated by dividing the total volume required (i.e. µl) by the number of samples per pool. for example, the control group consisting of healthy males was divided into two plasma pools of and controls. each of the controls in pool- contributed µl of plasma, while each of the controls in pool- contributed . µl of plasma. similarly the three patient groups consisting of , and samples were divided into × , × and × samples/pool respectively, and each patient per group contributed µl of plasma in pools- and , . µl of plasma is pools- and , and . µl of plasma in pools- and . therefore, total rna was extracted from µl of pooled plasma from each of the two pools per patient/ control group in the discovery cohort, and eluted in µl of rnase-free water using the mirneasy serum/plasma kit (qiagen) following the manufacturer's instructions. . µg of bacterial ribosomal rna (roche) was added to each sample for increased rna recovery, and . µl of mirneasy serum/plasma spike-in control i.e. cel-mir- (at . × copies/µl) (qiagen) was added to each sample as an internal control for plasma mirna expression profiling and normalisation of qrt-pcr data as previously described . µl of extracted rna from pooled plasma samples was reverse transcribed using the miscript ii rt kit with hispec buffer (qiagen) according to the manufacturer's instructions (fig. ) . mirna pcr array analysis. the resultant µl cdna, diluted in µl of rnase-free water, was applied to the -well miscript mirna pcr array (qiagen) containing forward primers for the detection of cancer-associated mirnas and duplicates of internal controls (fig. ) . the qrt-pcr was run on a viia system (applied biosystems) and the data analysed using the geneglobe data analysis software (qiagen). the cel-mir- spike-in control was used by the program to calibrate the data sets, and the mirna pcr array data was normalised by the global mean normalisation method . this method automatically calculated a global c t mean for the mirna targets that were commonly expressed in all the samples being analysed after an initial calibration to the exogenous cel-mir- spike-in control. for plasma mirna expression profiling in individual samples, the cel-mir- spike-in control was used as an internal control for normalisation of qrt-pcr data as previously scientific reports | ( ) : | doi: . /s - - -w described . fold regulation represents fold change values generated during mirna expression profiling in a biologically meaningful way. the fold change was calculated using the equation −∆∆ct . for fold regulation, the fold change values less than (meaning that the mirna is down-regulated) was transformed by calculating the negative inverse. therefore, the values remain the same while their representation is changed only. all c t (number of cycles required for fluorescent signal to cross the set threshold; inversely proportional to mirna levels) values > were considered as a negative call by the software. therefore, the lower limit of detection was ≤ c t to avoid false positives. the final values of mirna expression levels were generated in a biologically meaningful way as fold regulation i.e. the negative inverse of fold change. a . fold-regulation cut-off was used to shortlist deregulated mirnas. one of the challenges of mirna profiling from plasma samples is the lack of established housekeeping genes for data normalisation. in general, mirna profiling experiments use small noncoding rnas, such as small nuclear or nucleolar rnas (snrnas/snornas) for data normalisation . however, they may not serve as ideal reference genes because snornas and snrnas do not share the same properties as mirnas in terms of expression, transcription and processing . therefore, normalisation of data was performed using exogenous cel-mir- as an internal control and the global mean normalisation approach , . qrt-pcr validation. rna extraction from individual plasma samples (n = ) and cdna synthesis were performed as described previously and the resultant µl cdna diluted x in µl of rnase-free water. qrt-pcr of the shortlisted mature mirnas was performed using miscript primer assays (forward primers) (qiagen), universal primer (reverse primer) (qiagen) and the quantitect sybr green pcr master mix (qiagen) with µl cdna input for each µl reaction in triplicates. the mirna reverse transcription control (mirtc) primer assay (qiagen) was used as an internal control to determine the reverse transcription efficiency for each sample. the qrt-pcr data was analysed using the externally spiked-in cel-mir- (qiagen) as the housekeeping figure . study design and discovery and validation cohort study profiles. flow diagram summarising the methodology and statistical approach used to identify diagnostic mirnas followed by in silico target identification and pathway analysis, tcga data expression analysis and functional assays. scientific reports | ( ) : | doi: . /s - - -w gene with a fold-regulation cut-off used to identify deregulated mirnas. the results obtained from the discovery cohort were further validated in the validation cohort (n = ) using the same method as described above. statistical analyses. unpaired mann-whitney u tests were performed to identify differences in mirna expression levels between patients and healthy controls in cohorts and . the p-values were adjusted for multiple testing using bonferroni correction. univariate logistic regression was performed to determine the ability of individual mirnas to predict disease occurrence in prostate cancer patients. the rna concentration was set as an offset variable to account for the differences in the starting amounts of rna. the capacity of combined mir-nas to distinguish between patients and healthy controls was evaluated by roc curve analysis in the discovery (n = ), validation (n = ) and combined (n = ) cohorts to increase the predictive power of the analysis. for roc analysis of the mirna panel, binary logistic regression analysis was performed for all four mirnas at the same time enabling calculation of predicted probabilities of the mirna combination which was further used to generate auc values for the mirna panel. the auc is an effective and combined measure of sensitivity and specificity that describes the inherent validity of diagnostic tests. the roc curve corresponding to progressively greater discriminant capacity of diagnostic tests is located closer to the upper left-hand corner away from the diagonal reference line . a p-value of ≤ . was considered to be significant for all analyses. all statistical analyses were done using graphpad prism . , spss and rstudio in consultation with qualified statisticians from the queensland facility for advanced bioinformatics (qfab, uq) and the research methods group (rmg, qut). an overall summary of the methodology is presented in fig. . tcga data and cell line expression analysis. prostatic expression of the shortlisted mirnas was assessed in a publicly available cohort comprising of tumour and non-malignant prostate tissues from the cancer genome atlas (tcga). the expression of the significantly deregulated mirnas in prostate tumour tissues was evaluated in a panel of prostate cancer cell lines by qrt-pcr. rna was extracted from cell lysates using the mirneasy micro kit (qiagen) according to the manufacturer's instructions followed by cdna synthesis using the miscript ii rt kit with hispec buffer (qiagen). qrt-pcr using the miscript primer assays (qiagen) and quantitect sybr green pcr master mix (qiagen) was performed as described previously. cell proliferation and migration assays. for cell proliferation assays , lncap and , pc cells were plated per well in a well plate and allowed to grow for hours before transient transfection with mirna complexes as described above. for cell migration assays -well imagelock plates (essen bioscience) were coated with μl poly-l-ornithine (sigma) overnight. , cells/well were plated the next day and allowed to form a confluent monolayer for hours. the cells were treated with µg/ml aphidicolin (sigma) (an antimitotic reagent) for hour to minimise the effect of cell proliferation on migration. wounds were created using a -pin woundmaker (essen bioscience) and the growth media was aspirated to remove cell debris. the cells were treated with µl of mirna complexes per well for half an hour before replenishment with µl of rpmi containing % fbs. percentage confluence (for proliferation) and relative wound density (for migration) was measured every hours for hours using the automated incucyte live cell imaging system (essen bioscience) following the manufacturer's protocol. all treatments were performed with six replicates in three independent experiments. data analysis was performed using the incucyte software. pathway analysis. in silico network-based visual analysis was performed using the mirnet web-based platform to identify target genes and pathways potentially altered by the mirna signature . forward mapping, which allows users to map from mirnas to their targets, was performed by uploading the list of mirna ids of interest. after data processing, the results were presented as an interaction table with each row corresponding to one mirna and its target. the table also provided hyperlinks to the corresponding databases from where the mirna-target interactions were derived, together with references to pubmed literature. network visualisation was performed through functional annotations based on the kyoto encyclopedia of genes and genomes (kegg) pathway database, and the hypergeometric algorithm was used for enrichment analysis of input data to generate a list of mirna target genes. this was further used as an input for in depth analysis using ipa (qiagen) to determine top canonical pathways, upstream regulators and molecular and cellular functions of the mirna target genes. the association of the deregulated target genes with disease phenotype was also confirmed using the gene set enrichment analysis (gsea) computational method. the differential expression of the target genes in normal (n = ) vs prostate carcinoma (n = ) tissues were determined using the oncomine cancer microarray database and integrated data-mining platform . a p-value of ≤ . was considered to be significant for all the analyses. validation of in silico target genes of mir- - p. lncap ( , cells/well) were plated in a well plate for treatment with mir- - p and negative control mimics for hours as described above for the collection of cell lysates (norgen). this was followed by rna extraction (norgen), cdna synthesis (life technologies) and qrt-pcr (life technologies) to determine the relative fold expression of target genes of mir- - p using target-specific primers (sigma). data was normalised to the housekeeping gene rpl and further normalised relative to the non-targeting negative control to determine relative fold expression. the differences in target gene expression between the negative control and mir- - p treated cells were assessed using an unpaired t test, n = (*p ≤ . , **p ≤ . , ***p ≤ . and ****p ≤ . ). data availability statement. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. cancer statistics re: the prostate specific antigen era in the united states is over for prostate cancer: what happened in the last years? doctors' approaches to psa testing and overdiagnosis in primary healthcare: a qualitative study time trends and local variation in primary treatment of localized prostate cancer prostate cancer biomarkers: are we hitting the mark? mri-ultrasound fusion for guidance of targeted prostate biopsy cores prostate biopsies in the determination of prostate cancer and the importance of prostate 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micrornas -pre-analytic methodological considerations potential pitfalls in microrna profiling normalization strategies for microrna profiling experiments: a 'normal' way to a hidden layer of complexity? receiver operating characteristic (roc) curve analysis for medical diagnostic test evaluation this work is supported by the cancer australia priority-driven collaborative grant ( ), the ihbi mcr grant to a/prof jyotsna batra and the australian government department of health funding to the australian prostate cancer research centre-queensland (apcrc-q). fm is supported by the qut postgraduate research award (qutpra) and qut hdr tuition fee sponsorship. jac was supported by an nhmrc principal research fellowship. jb is supported by an nhmrc career development fellowship. the authors would like to thank patients and controls from the proscan and qldmen study who kindly agreed to participate in this study. the authors would also like to express their appreciation to the study participants who kindly donated tissue to the australian prostate cancer bioresource (apcb). the apcb is supported by the national health and medical research council of australia enabling grant ( ) and by a grant from the prostate cancer foundation of australia (pcfa). in addition, analysis of mirna transcriptomic data from clinical samples performed in this study is based on data generated by tcga, established by the nci and the national human genome research institute. the authors thank dr anne bernard (queensland facility for advanced bioinformatics, qfab, uq) and dr dimitrios vagenas (research methods group, rmg, qut) for their contribution to the statistical analyses. all authors confirm that they have contributed to the intellectual content of this paper. supplementary information accompanies this paper at https://doi.org/ . /s - - -w.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - yns hg authors: wu, gang; zhou, shuchang; wang, yujin; lv, wenzhi; wang, shili; wang, ting; li, xiaoming title: a prediction model of outcome of sars-cov- pneumonia based on laboratory findings date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: yns hg the severe acute respiratory syndrome coronavirus (sars-cov- ) has resulted in thousands of deaths in the world. information about prediction model of prognosis of sars-cov- infection is scarce. we used machine learning for processing laboratory findings of patients with sars-cov- pneumonia (including non-survivors and discharged patients). the maximum relevance minimum redundancy (mrmr) algorithm and the least absolute shrinkage and selection operator logistic regression model were used for selection of laboratory features. seven laboratory features selected in the model were: prothrombin activity, urea, white blood cell, interleukin- receptor, indirect bilirubin, myoglobin, and fibrinogen degradation products. the signature constructed using the seven features had % [ %, %] sensitivity and % [ %, %] specificity in predicting outcome of sars-cov- pneumonia. thus it is feasible to establish an accurate prediction model of outcome of sars-cov- pneumonia based on laboratory findings. www.nature.com/scientificreports/ all methods were carried out in accordance with relevant guidelines and regulations. study design and participants. this study was approved by the ethics commission of hospital (tj- - ). written informed consent was waived by the ethics commission of hospital. the author's center was the designated hospital for severe and critical sars-cov- pneumonia. patients underwent repeated rt-pcr tests to confirm sars-cov- . laboratory tests for sars-cov- pneumonia included: blood routine test, serum biochemical (including glucose, renal and liver function, creatine kinase, lactate dehydrogenase, and electrolytes), coagulation profile, cytokine test, markers of myocardial injury, infection-related makers, and other enzymes. repeated tests were done every - days for monitoring the patient's condition. oxygen support (from nasal cannula to invasive mechanical ventilation) was administered to patients according to the severity of hypoxaemia. all patients were administered with empirical antibiotic treatment, and received antiviral therapy. most of patients improved after treatment. however, a few critical patients continued to deteriorate and eventually died. data collection. fatal cases of sars-cov- pneumonia ( male, median age years) were collected by the electronic medical record system. discharged patients with sars-cov- pneumonia whose age and gender matched the non-survivors were selected ( male, median age years). the admission date of these patients was from feb , to mar , . we reviewed all laboratory findings for each patient. results of repeated tests were carefully compared to find the greatest deviation from normal value. in general, the greatest number in series of values was recorded. however, for platelets, red blood cell, lymphocytes, hemoglobin, calcium, total protein, albumin, estimated glomerular filtration rate (egfr), and prothrombin activity (pta), the minimum was recorded. laboratory findings at the day of mortality were not used. these recorded laboratory findings were considered as lab features of a patient. a initial data set of patients (non-survivor , discharge ) was thus built. there were patients who did not have the entire group of laboratory features, thus their data were deleted from the dataset. the remaining data of patients ( non-survivor, discharge) were analyzed by machine learning. statistical analysis and modeling. first, all the variables were compared between non-survivors and discharged patients using the mann-whitney u test for non-normally distributed features or the independent t test for normally distributed features , . features with p < . were considered significant variables and selected , . second, spearman's correlation coefficient was used to compute the relevance and redundancy of the features , . third, we applied the maximum relevance minimum redundancy (mrmr) algorithm to assess the relevance and redundancy of the features , . the features were ranked according to their mrmr scores , . fourth, the top features with high-relevance and low-redundancy were selected for least absolute shrinkage and selection operator (lasso) logistic regression model. the lasso logistic regression model was adopted for further features selection , . some candidate features coefficients were shrunk to zero and the remaining variables with non-zero coefficients were finally selected , . the model was used for calculating signature for each patient. mann-whitney u test was used for comparing signature between two groups , . receiver operator characteristic (roc), precision recall curve (prc) analysis and hosmer-lemeshow test were used for further evaluation of model. the statistical analyses were performed using r software (version . . ; https ://www.r-proje ct.org) , . the following r packages were used: the "corrplot" package was used to calculate spearman's correlation coefficient; the "mrmre" package was used to implement the mrmr algorithm; the "glmnet" was used to perform the lasso logistic regression model, and the "proc" package was used to construct the roc curve , . nine laboratory features were eliminated in the first step of feature selection because of non-significance. the remaining thirty-eight lab features were significantly different between two groups (p < . ), and then mrmr scores were obtained for them. there were seven features having non-zero coefficients after lasso algorithm, and were selected for the model. table shows the fifteen features with the highest mrmr scores. figure shows the correlation matrix heatmap of the thirty-eight significant features. figure shows the feature selection process with lasso algorithm. figure shows the contribution of the seven features to the model. figure shows the signatures of all patients, as well as roc. figure shows the prc for the model. non-survivors and discharged patients differed significantly in the signature derived from the model (p < . ). the auc was . [ % ci . , . ]. the sensitivity and specificity in predicting outcome of sars-cov- pneumonia were % [ %, %] and % [ %, %] respectively. the area under precision recall curve (auprc) was . . hosmer-lemeshow test showed good calibration (p = . ) for the model. the seven features included in the prediction model were as follows: pta, urea, white blood cell (wbc), interleukin- receptor (il- r), indirect bilirubin (ib), myoglobin, and fibrinogen degradation products (fgdp). all features had coefficients of positive number except pta. pta and fgdp are from coagulation profile. urea and ib are from renal and liver function respectively. wbc is from blood routine test. myoglobin is a marker of myocardial injury. il- r is related to immune response. the signatures derived from the model could be positive or negative numbers. www.nature.com/scientificreports/ non-survivors and discharged patients did not differ in age or gender (median age vs. , p = . ; percentage of males, % vs. %, p = . ). the comparisons of laboratory findings between non-survivors and discharged patients are shown in table . blood routine test. wbc and neutrophils were significantly higher in non-survivor group versus discharge group. lymphocyte, platelets and red blood cells were significantly lower in non-survivors. auc for them were . - . . table . the fifteen features with higher mrmr scores were selected for the step of lasso logistic regression. some candidate features coefficients were shrunk to zero and the remaining variables with non-zero coefficients were selected. mrmr maximum relevance minimum redundancy, lasso least absolute shrinkage and selection operator, pta prothrombin activity, wbc white blood cell, il- r interleukin- receptor, ib indirect bilirubin, tb total bilirubin, fgdp fibrinogen degradation products, hs-crp hypersensitive c-reactive protein, ldh lactate dehydrogenase, egfr estimated glomerular filtration rate. cytokine. il- r and il- were significantly higher in non-survivor group versus discharge group. auc for them were . - . . procalcitonin, high sensitive c-reactive protein, ferritin and n-terminal pro-brain natriuretic peptide (nt-probnp) were significantly higher in non- non-survivors and discharged patients with sars-cov- pneumonia differed significantly in thirty-eight laboratory findings. by using machine learning method, we established a prediction model involving seven laboratory features. the model was found highly accurate in distinguishing non-survivors from discharged patients. the seven features selected by artificial intelligence also indicated that dysfunction of multiple organs or systems correlated with the prognosis of sars-cov- pneumonia. the sars-cov- triggers a series of immune responses and induces cytokine storm, resulting in changes in immune components , . when immune response is dysregulated, it will result in an excessive inflammation, even cause death , . excessive neutrophils may contribute to acute lung damage, and are associated with fatality . higher serum level of il- r was found in non-survivors, indicating excessive immune response. in addition, high leukocyte count in sars-cov- patients may be also due to secondary bacterial infection , . liver injury has been reported to occur during the course of the disease , , and is associated with the severity of diseases. increased serum bilirubin level was observed in fatal cases. acute kidney injury could have been related to direct effects of the virus, hypoxia, or shock , . blood urea level continued to increase in some cases. non-survivors had higher blood urea compared to survivors. myocardial injury was seen in non-survivors, which was suggested by elevated level of myoglobin. the mechanism of multiple organ dysfunction or failure may be associated with the death of patients with sars-cov- pneumonia. some patients with sars-cov- infection progressed rapidly with sepsis shock, which is well established as one of the most common causes of disseminated intravascular coagulation (dic) . the non-survivors in our cohort revealed significantly lower pta compared to survivors. at the late stages of sars-cov- infection, level of fibrin-related markers (fgdp) markedly elevated in most cases, suggesting a secondary hyperfibrinolysis condition. a number of laboratory features were compared between non-survivors and discharged patients with sars-cov- pneumonia. the two groups differed significantly in as many as thirty-eight lab features. however, none of the futures provided adequate accuracy in predicting the outcome of sars-cov- pneumonia. thus, a novel prediction model involving multiple features was established in the study. with machine learning methods previously used in radiomics, a prediction model combining seven out of thirty-eight laboratory features was built for predicting the outcome of sars-cov- pneumonia. the mrmr algorithm was used for assessing significant features to avoid redundancy between features. the mrmr score of a feature is defined as the mutual information between the status of the patients and this feature minus the average mutual information of previously selected features and this feature , , . the top fifteen features with high mrmr scores were selected for the next step of modeling. the least absolute shrinkage and selection operator logistic regression model was used to processing the features selected by mrmr algorithm. lasso is actually a regression analysis method that improves the model prediction accuracy and interpretability . the signature calculated with the model can be positive or negative number, corresponding with poor and good prognosis respectively. our results showed that the auc of the signature was - % higher than that of a single feature. the modeling process is a black box; however, the choice of variables seems reasonable. pta can more accurately reflect the coagulation function compared to prothrombin time, and can also reflect the degree of liver injury. urea is a good index to reflect the degree of renal function damage. wbc can not only reflect immune www.nature.com/scientificreports/ it is suitable to start to use this model after three repeated laboratory tests (about weeks after admission), because doctors may have enough data at that time. lots of laboratory findings are generated in hospitalization. which are most important for predicting outcome? our study at least answered such a problem. seven laboratory features could be used to construct a new signature with the model. the new signature seems more useful than any single feature. we encourage such a simple-to-use model widely used in clinical practice. most of clinical factors are not continuous variables (such as underlying disease). we used a machine learning method similar to radiomics, which mainly deals with continuous features. our study focused on continuous laboratory variables. we had to exclude non-continuous clinical factor with the current machine learning method. by using other methods, a model that involves both continuous variables and category variables can be established. thus clinical factors raised as significant predictive factors (such as respiratory status or radiological features) could be included in the models. however, there are more than forty laboratory findings in our study, making establishment of model difficult. we felt it necessary to simplify laboratory features. thus we establish a sub-model based on lab findings. a new lab signature is thus created, and is proved highly valuable. in future study, the signature may be combined with clinical factors to establish a more complex model. our study has some limitations. first, this is a single-center retrospective study. multi-center large-sample studies are required to validate our prediction model. second, our model may not be directly used in other centers. however, they could easily establish a prediction model using their own data with machine learning method. third, some patients who did not have all the lab findings were excluded. selection bias must be present due to patients exclusion. other studies with more strict design were thus required to reveal the bias. fourth, statistical approach conducted in this study is not perfect. as lasso was used for variables, or more patients were needed. more patients should be collected in future study. in conclusion, it is feasible to establish a accurate prediction model of outcome of sars-cov- pneumonia based on laboratory findings. injury of liver, kidney and myocardium, coagulation disorder and excess immune response all correlate with the outcome of sars-cov- pneumonia. after publication, the data will be made available to others on reasonable requests to the corresponding author. received: march ; accepted: august identification of a novel coronavirus in patients with severe acuterespiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia the novel 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in nsclc patients using multimodal imaging and machine learning algorithms prediction model of aryl hydrocarbon receptor activation by a novel qsar approach, deepsnap-deep learning machine learning algorithms applied to a prediction of personal overall thermal comfort using skin temperatures and occupants' heating behavior nomogram based on shear-wave elastography radiomics can improve preoperative cervical lymph node staging for papillary thyroid carcinoma t -weighted image-based radiomics signature for discriminating between seminomas and nonseminoma dysregulation of immune response in patients with covid- in wuhan mers-cov infection in humans is associated with a pro-inflammatory th and th cytokine profile pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of novel coronavirus infection in china abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia liver injury during highly pathogenic human coronavirus infections pandemic influenza a in argentina: a study of patients on mechanical ventilation the clinical and chest ct features associated with severe and critical covid- pneumonia complement activation in human sepsis is related to sepsis-induced disseminated intravascular coagulation a new feature selection method based on symmetrical uncertainty and interaction gain machine learning-based analysis of mr radiomics can help to improve the diagnostic performance of pi-rads v in clinically relevant prostate cancer selection of important variables and determination of functional form for continuous predictors in multivariable model building we thank all patients and their families involved in the study. g.w., s.z. and y.w. collected the epidemiological and clinical data. t.w., w.l. and s.w. summarized all data. g.w., x.l. drafted the manuscript. t.w. and x.l. revised the final manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to t.w. or x.l.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -o uk if authors: tsay, calvin; lejarza, fernando; stadtherr, mark a.; baldea, michael title: modeling, state estimation, and optimal control for the us covid- outbreak date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: o uk if the novel coronavirus sars-cov- and resulting covid- disease have had an unprecedented spread and continue to cause an increasing number of fatalities worldwide. while vaccines are still under development, social distancing, extensive testing, and quarantining of confirmed infected subjects remain the most effective measures to contain the pandemic. these measures carry a significant socioeconomic cost. in this work, we introduce a novel optimization-based decision-making framework for managing the covid- outbreak in the us. this includes modeling the dynamics of affected populations, estimating the model parameters and hidden states from data, and an optimal control strategy for sequencing social distancing and testing events such that the number of infections is minimized. the analysis of our extensive computational efforts reveals that social distancing and quarantining are most effective when implemented early, with quarantining of confirmed infected subjects having a much higher impact. further, we find that “on-off” policies alternating between strict social distancing and relaxing such restrictions can be effective at “flattening” the curve while likely minimizing social and economic cost. www.nature.com/scientificreports/ resource availability and the extent to which social distancing is feasible. among works of this type, djidjou-demasse et al. investigated the optimal control of a single "intervention" input variable for an ode model. moore and okyere formulated a similar problem with additional inputs, including hospitalization rates and environmental spraying. both studies solved the optimal control problem using an iterative forward-backward sweep method and assumed the model parameters to be fully known. while optimal control of epidemiological models has been well-studied (e.g., biswas et al. , neilan and lenhart ), a limiting factor for implementing modeling and optimization concepts during early stages of an outbreak is that obtaining accurate estimates of key model parameters can be challenging. in this work, we report a novel and complete dynamic optimization-based approach to the entire epidemiological modeling and outbreak control workflow for the us covid- outbreak. we first formulate a dynamic optimization strategy for identifying both the values of time-invariant parameters and the historical trajectories of time-varying parameters (i.e., inputs) of an epidemiological model. we then investigate how optimal control of the inputs of the same model, which reflect social distancing and testing, relates to infection mitigation strategies. the optimal control problem is cast as a simultaneous dynamic optimization problem (i.e., reformulated as an algebraic problem) , enabling the natural use of deterministic global optimization technology . this in turn provides solutions that are proven to be globally optimal, whereas iterative schemes such as the above often only guarantee global optimality under certain conditions (the term "global" here refers to the notion that the best possible disease control policy is identified from a set of "local" solutions that otherwise satisfy optimality conditions. "global" in this context should not be interpreted as "world-wide"). furthermore, we show how state estimation should be used to update important hidden model states (e.g., the number unconfirmed infections) to reduce the impact of model inaccuracy. implementations of the parameter estimation and optimal control problems in open-source software are provided freely at https ://githu b.com/balde a-group /covid - . mathematical modeling. we consider the compartmental model shown in fig. , which brings a few key modifications to the conventional seir (susceptible-exposed-infectious-recovered) structure. importantly, the a(t) state is added to account for infected subjects that are not included in the count of confirmed cases, either because they are asymptomatic or because of insufficient testing. the virus is thought to be asymptomatic in - % of cases , and may be transmitted by asymptomatic carriers . we also include a p(t) state, which tracks the population that perishes due to the virus. the modified seair model has six compartments/states: s(t) represents the number of subjects that are susceptible to infection, e(t) the number that have been exposed to the virus, a(t) the number that are infected but asymptomatic/unconfirmed, i(t) the number with confirmed infections, and r(t) the number that have recovered from infection. the states are assumed to sum to a known total population n, and p(t) can be calculated algebraically at all times. note that n is assumed to be constant. this assumption is suitable since the number of deaths is much smaller than the initial population, and thus considering a time-varying total population would have little impact on our model at the expense of increased complexity. the model structure is shown in fig . the seair model is described by the following equations: www.nature.com/scientificreports/ where α a (t) and α i (t) are the rates of exposure to the virus from the population of asymptomatic/unconfirmed and confirmed infected subjects, respectively. these two rates of exposure are defined as time-varying and independent model inputs to reflect different measures taken during the course of the pandemic. specifically, α a (t) corresponds to exposure from asymptomatic carriers (a) and reflects social distancing and/or shelter-in-place strategies. on the other hand, α i (t) corresponds to exposure from infected subjects (i) and reflects quarantining of infected subjects. we also model κ(t) , or the rate at which unconfirmed cases become confirmed, as a timedependent input to reflect varying levels of screening and testing. the other parameters are assumed to be constant over the time horizons considered herein. t − latent is the inverse of the latent period of the virus, or the time before an exposed subject becomes infectious. we assume a value of t − latent = . days − based on peng et al. the parameter ρ describes the infectious period for subjects with unconfirmed infections, for which we assume a value of ρ = . days − , based on rocklöv et al. the rates at which subjects with confirmed infections recover and perish are described by β and µ , respectively. finally, γ describes the rate at which recovered subjects become susceptible to the disease again. since the immunity period for the virus is unknown, we assume a value of γ = . while these parameters largely describe the virus itself, they may change over longer time horizons, e.g., as new treatments are developed. a potential future avenue of research is to consider parameters β and γ to be time-varying, to reflect decision-making regarding budget allocation for medical supplies and equipment, as well as health care capacity expansion over time. in summary, the seair model ( )-( ) has three time-varying inputs ( α a (t) , α i (t) , and κ(t) ), three parameters ( t − latent , ρ , and γ ) whose values are based on available literature, and two parameters whose values must be estimated ( β and µ ). note that since e(t) and a(t) are unmeasured, their magnitudes relative to the infected population i are effectively set by (the bounds on) κ . as the prevalence of asymptomatic infection is established (e.g., bendavid et al. estimate up to % prevalence in santa clara county using serological testing) the magnitudes of e and a may be adjusted by scaling κ , and other parameters as relevant. parameter estimation results. this work primarily addresses the current covid- situation in the us, but the still relatively early stage of the outbreak in the us renders the available data insufficient to fit parameters reflective of prevention measures already in place. for this reason, we perform a comparative analysis by solving the parameter estimation problem for italy, spain, and germany, where, while the epidemic has not been fully contained yet, the daily number of new confirmed cases has been on a steady decline. model parameters for the us, italy, spain, and germany. figure shows the predicted values obtained by solving the parameter estimation problem and the historical data by country, retrieved by the center for systems science and engineering (csse) at johns hopkins university (https ://githu b.com/csseg isand data/covid - ; accessed april , ). day of the dataset corresponds to january , while day corresponds to april . the solid line in each state trajectory plot shows the mean predicted value. standard deviations for the predictions were estimated using bootstrapping, with the time-invariant parameters sampled from a multivariate normal distribution. the means and covariances of the estimated parameters for each country are provided in the supplementary information. plots corresponding to infected, recovered and perished (left plots in fig. ) were obtained by simulating ( )-( ) using the estimated trajectories of the time-varying inputs, shown in the right-most column of fig. . parameter estimation results provide several interesting insights. first we note that the mortality rates, while higher for the european countries, are comparable in magnitude for the four countries considered and are similar to prior estimates . the higher death rates in italy and spain are likely explained by demographic factors (e.g., age), as well as the medical resources available to treat the infected population. however, the simulated trajectories of p in italy and spain deviate slightly from the historical data, especially around day (fig. ) , suggesting that the death rate µ may be time-varying. while µ can vary in reality due to improved treatment, early detection bias, etc., it is difficult to anticipate future changes, and we therefore approximate it as a constant. from the trajectories of α a , α i , and κ , we observe that social distancing measures (i.e., lower values of α a ) were first implemented in europe, and more recently the us, which agrees with their true chronology. this insight is confirmed by the fitted values for e , with italy having the highest value and the us the lowest, which is representative of the beginning of the outbreak in each country. in general terms, the identified evolution of containment and testing measures follow similar trends for all four countries, which supports that the model www.nature.com/scientificreports/ structure is correct and can appropriately reflect (see "methods") different dynamics of the same underlying disease. further, we expect that the parameters obtained for the us, while being fitted using relatively premature information, are likely an adequate representation of the current covid- situation. for the estimated parameter values as described previously, we simulate the results of implementing two different simplistic control policies: (i) continuing with strict social distancing, quarantining, and testing, policies that result from continuing to lower the asymptomatic ( α a ) and infected ( α i ) exposures shown in fig. ; and (ii) a relaxed policy with more lenient measures and reduced testing, in this case the values of α a and α i are increased to . and . , respectively, while κ is decreased to . . the population levels resulting from implementing these two policies are shown in fig. , where the numbers of recovered subjects are omitted for the sake of brevity. we additionally show the number of new confirmed cases per day, as it is a commonly used metric to illustrate the current spread/containment of the virus. since the three european countries showed very similar trends under the two policies considered, we only compare the results for the us to those obtained for italy. on the one hand, from fig. it is evident that relaxing current control policies can result in an alarming number of infected cases and deaths, particularly in the us. on the other hand, continuing with the strict shelter-in-place measures and maximizing testing can result in earlier and substantially flatter pandemic peaks, with significantly lower numbers of casualties. while the latter approach is the most effective at preventing further exponential spread of the disease, it is also the most socially and economically disruptive policy. in light of this trade-off, we argue that by means of employing optimal control concepts it is possible to find effective policies that maintain the number of infected cases below a given threshold, while minimizing the extent of social and economic disruption. here, we consider the optimal control of the covid- infection in the us using the deterministic seair model ( )-( ) with the mean values of the time-invariant parameters as described above. as such, the optimization problem only considers the mean predicted values (e.g., solid lines in figs. and ). future work may consider a stochastic optimization approach that, at the expense of increased computational effort, leverages information about model uncertainty. optimization of future actions. we solve a dynamic optimization problem that minimizes a measure of socioeconomic cost, subject to keeping the peak (max) value of the infected population below a given number i peak . the lower bound of κ(t) is raised to . , from . in fig. www.nature.com/scientificreports/ figure depicts the solution of this problem for the cases where i peak = , subjects and i peak = , , subjects. the optimization problem was solved for the days following the end of the available dataset ( t = , t f = ), with the initial condition at t computed by simulating days - . although the optimization problem only considers the mean values (solid lines), two standard deviations are shown in the shaded areas to reflect parametric model uncertainty for the given inputs, computed using the same bootstrapping approach as above. considering the current status of the pandemic in the us, keeping the peak below , infected subjects is very challenging. note that the minimum feasible value of i peak corresponds to a peak of , infected subjects. therefore, maintaining i(t) ≤ , requires immediately decreasing α a and α i to their lower bounds for approximately the next days, during which the exposed population can decrease significantly. after this period, α a is relaxed to its upper bound for seven brief periods (fig. , left) . however, α i remains at its lower bound at all times, reflecting a strict quarantining policy. this policy reveals that the impact of α i on the infected population size is larger than that of α a . in turn, this suggests that quarantining of infected people is more important than social distancing, which mitigates exposure to unconfirmed cases. the optimization problem penalizes κ to account for the cost of testing, and κ is decreased during periods of decreased α a , suggesting that testing is less important during times of social distancing/lockdown. intuitively, when the exposure to asymptomatic subjects is already low due to social distancing, there is less benefit to testing asymptomatic subjects (and transferring them to the similarly quarantined infected population). on the other hand, κ is increased preemptively for each period of increased α a , suggesting that testing is most important in the days leading up to a period of relaxed social distancing. this testing moves asymptomatic subjects to the confirmed-infected population, which can remain quarantined. figure (right) depicts the solution obtained for a value of i peak = , , people. while α i again remains at its lower bound at all times, re-emphasizing the importance of quarantining infected subjects, there are more frequent and longer periods of increased α a . in this case, κ remains at its upper bound for most of the control horizon. in both cases, the optimization problem does not account for the effect(s) of the control policy after days, and growth of the infected population near the end of the time horizon may be concerning. this can www.nature.com/scientificreports/ be addressed, e.g., using a moving horizon control strategy, where policy measures are revised periodically, as discussed below. the lower bound used for α i is lower than estimated values for the us for the past (fig. ), corresponding to a new level of quarantining. furthermore, the current value of α a , or social distancing, may not be economically sustainable over longer periods. to investigate the effect of not achieving these proposed levels, we solve the same problem with i peak = , , people, using different lower bounds for the inputs. figure shows the optimal optimal containment and testing strategies to limit peak infections to , , in the next days for different constraints on α a and α i . top: α a (t) . bottom: κ(t) . the shaded grey area indicates the solution found using the normal bounds, replicated from fig. www.nature.com/scientificreports/ input profiles found for the cases where the lower bound of α a is doubled from . to . (dotted blue lines), and where the lower bound of α i is doubled from . to . (solid black lines). when the lower bound of α a is doubled (less social distancing is achieved during lock-down periods), the same infected population peak can be maintained by increasing the frequency of social distancing periods. however, when the lower bound of α i is doubled (less quarantining is achieved), maintaining the same number of peak infections requires social distancing at almost all times, closely resembling the control policy found for a lower peak (fig. , left) . in either case, the optimal value of α i remains at its lower bound (either . or . ) at all times and is therefore not shown. periods of social distancing are further increased in both frequency and duration if both bounds are doubled. compared to the decreases seen in fig. for the cases of continued control, the solutions in fig. seek to merely flatten the growth of the infected population. the associated optimal control policies resemble "bangbang" control (the inputs are always at the lower or upper bound), which can be expected from a systemstheoretic point of view for ( )-( ), as the equations are linear functions of the inputs α a , α i , and κ . this result supports the strategy proposed by the imperial college covid- response team , which involves periodic suppression measures. their strategy triggers the start of a social distancing period by when the number of weekly icu cases increases past an "on" threshold, and the end of the period when the same number decreases below an "off " threshold. figure shows that this type of strategy maintains a low exposed population, and therefore flattens the growth of the infected population while still allowing periods of social mobility. revisiting past actions. we examine the same optimal control problem with a time horizon starting from day (i.e., covering days in the past). rolling the time horizon of the optimization problem backwards to t = , allows us to investigate the optimal inputs for days - , corresponding roughly to the latter half of march and the first half of april. figure (left) depicts the solution obtained for a value of i peak = , people. the initial condition at t = was computed by simulating days - . the minimum historical values for α a and α i were used as their respective lower bounds for days - , such that the optimal control policy for days - only involves an extent of quarantining and social distancing already experienced by the general population. in this case, maintaining the number of infected subjects below , appears easy. the minimum feasible peak of infected subjects is , , compared to , when the optimization starts at day . therefore, maintaining i(t) ≤ , can involve extended periods of no social distancing α a = . . similar to the result in fig. (right) , testing and quarantine are important in this scenario: κ remains at its upper bound and α i its lower bound. the solution for an order-of-magnitude lower minimum peak, corresponding to , infected subjects is shown in fig. (right) . the optimal control policy for this scenario involves more frequent periods of social www.nature.com/scientificreports/ distancing. early implementation of testing and quarantining strategies clearly has an enormous effect, manifest in the large difference between the historical inputs (dashed) and optimal inputs (solid) in days - of fig. . estimation of hidden states and moving horizon control. the seair model ( )-( ) includes two hidden states, e(t) and a(t), which are not measured in practice (note that widespread serological testing may eventually reveal the true levels of the asymptomatic population a). nevertheless, solutions for the optimal control problem based on ( )-( ) are highly dependent on the values of e(t) and a(t), and therefore the initial conditions e(t ) and a(t ) . fig. (left) depicts the optimal control policies for i peak = , , people over the next days (similar to fig. , right) , where e(t ) and a(t ) are now underestimated by a factor of three. this is a practically motivated scenario, as the number of asymptomatic cases is considered largely uncertain , . the dashed-dotted lines show the state profiles predicted by the optimization problem (with incorrect initial conditions), while the solid lines show their true evolution for the given inputs. the error in the hidden states e(t) and a(t) causes the predicted and actual profiles to diverge over time. by the end of the days, there are . million actual infected subjects, almost % greater than the predicted . million. to account for discrepancies between the modeled and true systems, the optimal control inputs should be periodically updated. we propose to achieve this with a moving horizon decision-making strategy: after a given length of time, the dynamic optimization problem is re-solved, setting the measured values of the states as their initial conditions. the values of the un-measured, hidden states can be estimated using state estimation techniques, such as the kalman filter (the optimal linear estimator) or its nonlinear extensions. we consider a relatively long time span of days before each revision of policy decisions (and re-optimization), as planned suppression strategies likely cannot be altered quickly. figure (right) shows the optimal trajectories found for the same problem (initial conditions for e and a underestimated by a factor of three) using the moving horizon strategy. the vertical dotted lines indicate times when the optimization problem was resolved. values of the hidden states were estimated daily with the (discrete) unscented kalman filter , implemented using the pykalman library (https ://pykal man.githu b.io/; accessed april , ). measurements for state estimation and control updates were simulated by adding independent, normally distributed noise to the true state values. a standard deviation of , people was used for the measured states (i, r, and p) based on the size of residuals during parameter estimation. the predicted values (dash-dotted lines in fig. ) are first updated when the optimization problem is re-solved at t = days. the solutions to the re-optimization problems are shown in the supplementary information. the figure . optimal moving horizon control policy (right) to limit peak infections to , , , with e and a underestimated by a factor of three at t = , and comparison to the same situation without a moving horizon strategy (left). top: predicted (dash-dotted) and true (solid) population numbers. bottom: containment and testing profiles. the shaded grey area indicates past days, which were simulated using historical inputs (not optimized). the policies are updated every days with daily state estimation. scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ effect of this moving horizon strategy is quite significant, in comparison to the optimal policy shown in fig. (left). periods of social distancing are quickly increased after day to account for the larger-than-expected increase in infected population. testing levels, reflected by κ , are also updated accordingly. the moving horizon approach exhibits three clear benefits: • errors between the model predictions and reality are minimized by incorporating new measurements as they become available • the infected population does not grow exponentially at the end of the time horizon, as is the case when considering only a single time horizon. this work investigated dynamic optimization strategies to characterize and control the us covid- outbreak, by minimizing the socioeconomic cost associated with containment strategies and testing. the results provide several overarching conclusions. the quarantining of infected subjects is the most important of the considered mitigation strategies and should be maximized at all times. additionally, periods of social distancing/lock-down help to flatten the peak by preventing exposure from asymptomatic and unconfirmed cases. screening and testing for the disease are key immediately preceding periods of relaxed social distancing, in order to minimize the number of unconfirmed infections during periods of social mobility. early action has much larger effects than later interventions, even as the later interventions are more drastic. optimal policies are highly dependent on estimates of "hidden states, " i.e., the asymptomatic and unconfirmed cases. moving horizon (periodically updated) policies and state estimation should be used to mitigate inaccuracies in the model and counts of asymptomatic/unconfirmed cases, by accounting for new data as they becomes available. the "on-off " policies identified as optimal are characteristic of the class of problems considered (linear objective function and input-affine nonlinear model). further, these policies are likely the easiest to implement in practical scenarios and to convey to the general population (as opposed to a policy where the social distancing parameters would take values between their upper and lower bounds). their implementation would simply alternate between the strictest possible limitations, followed by periods of relative freedom of movement. the frequency of these periods may be restricted, e.g., by including rate-of-change constraints in the optimization problem, to account for the ability of a country or local authority to intermittently enforce social distancing policies. the model used in this work does not include population influx to and/or outflux from the given system. therefore, the optimal containment and testing strategies found do not account for new cases that may enter from outside the us. we also assumed that the recovery and death rates for the virus are constant and equal to their historical values for the next days. thus the results do not account for the possibility of, e.g., improved medical treatments, vaccine development, and/or viral mutation. similarly, the model does not account for possible surveillance/detection bias in the historical data (e.g., increased likelihood of testing for subjects with more severe symptoms). the impacts of these assumptions are topics for future work. also, while sameni determined fixed points and did linear stability and observability analyses for a similarly-structured seir model with timeinvariant parameters, another topic for future work is the extension of this mathematical analysis to the seair model with time-dependent parameters used here. parameter estimation. we follow nonlinear (least-squares) regression for parameter estimation, which can be cast as an optimization problem, where the objective is to find the parameter values that minimize the mean squared error (mse) between the predicted states and their measured values. the measured values correspond to data retrieved by the center for systems science and engineering (csse) at johns hopkins university (https ://githu b.com/csseg isand data/covid - ; accessed april , ) and are provided in the supplementary information for several countries. the data consist of the total number of infected ( î j ), recovered ( r j ) and dead ( p j ) reported subjects, where j represents each day during the time period from january to april , where the index j represents the time period each value was recorded. the mse is given by: we note that when solving the parameter estimation problem, the contribution of the term ρa(t) to the recovered state r(t), is removed from the system ( )- ( ) . this is because the data obtained only accounts for those www.nature.com/scientificreports/ recovered that were confirmed to have the disease, meaning the only contribution to the observed recovered is the term βi(t). the parameters with values/trajectories to estimate are α a (t) , α i (t) , κ(t) , β , and µ . furthermore, we must estimate the initial condition of the unreported states e and a at t = . we assume that there are initially no asymptomatic infections. the "seed" of the outbreak is therefore the initial number of exposed subjects e = e(t = ) , which we include as a parameter in the least-squares problem. we include bounds on the possible parameter values, based on values reported for similar models fitted to data from other regions . note that, while κ reflects the level of testing, it also affects the predicted asymptomatic ratio, which cannot be controlled. therefore, tighter bounds should be used for κ than for α a and α i . the estimated parameters and their bounds are summarized in fig. . to prevent over-fitting the available data, we restrict the time-varying inputs ( α a , α i , and κ ) to be piece-wise constant over five-day intervals. these constraints reflect the fact that policies implemented have significant time delays in steering the states towards the desired values, and therefore should not and practically cannot be manipulated too frequently before their effect is observed on the population. additionally, we constrain α a (t) and α i (t) to be monotonically decreasing over time, reflecting the enforcement of increasingly stricter disease control measures. similarly, κ(t) was constrained to be monotonically increasing over time to account for increased covid- screening availability. to easily interface with raw data sources, the least-squares regression problem was implemented in python using the pyomo modeling and optimization package . we discretized the dynamical system ( )-( ) with respect to the time domain using orthogonal collocation on finite elements , with one finite element per day. we used ipopt to solve the resulting nonlinear dynamic optimization problem to local optimality. standard deviations for the time-invariant parameters were approximated by fixing the time-varying inputs to their estimated trajectories, and solving a maximum likelihood problem for the time-invariant parameters using the model validation tool in gproms (general process modeling system) v . . . residuals were assumed independent and normally distributed, with variances estimated in the same maximum likelihood problem. the parameter covariances (see supplementary information) are then computed from the diagonal entries of the hessian of the objective function. optimal control. the aim of the optimal control problem is to find the trajectories of "handles" α a (t) , α i (t) and κ(t) , that minimize (or maximize) the value of a certain objective function. most response measures for the covid- outbreak seek to "flatten" the epidemic curve, that is, to contain the growth rate of the number of infected subjects via a combination of social distancing and testing. clearly, social distancing and isolation/ quarantining carry significant social and economic costs. we thus formulate the optimal control problem as a dynamic optimization problem, aiming to minimize a measure of social and economic cost subject to ensuring that the maximum number of infected subjects remains under a given peak value, i peak . the optimization problem is expressed mathematically as: where c(t) is the cost function, and κ is the relative cost of testing (increasing κ ). testing costs are assumed to be relatively small in comparison to isolation measures, and we selected κ = . . while the current cost function equally penalizes decreases in α a and α i , an additional weighting factor could be introduced to distinguish between the socioeconomic costs of social distancing and quarantining. the solution to ( ) can provide control policies over the full time horizon [t , t f ] as in the case of the initial studies presented in this work. alternatively, the policies can be updated after a shorter horizon (before t f is reached), as in the case of the moving horizon approach. in the latter case, the time window considered, [t , t f ] is shifted forward at a pre-determined time interval. note that t f is typically longer than the frequency at which the optimization problem is recomputed, and only the solution for the first time step(s) is implemented. for example, the moving horizon scenario presented here is solved with t f = days, but with policies updated after each -day period. while the problem ( ) identifies the minimum societal cost required to achieve a certain peak value of infections, a related, but different, problem could be formulated by minimizing i peak subject to an upper bound on c(t), i.e., finding the lowest achievable peak for a given total societal cost. for this study we investigate the relationship between c and i peak by solving ( ) for varying values of i peak , provided in the supplementary information. the optimization problem ( ) was again solved by discretization of the time domain using orthogonal collocation, with one finite element per day. to report the best possible solutions, the resulting algebraic optimization problem was implemented in gams and solved using the commercial global optimization solver baron v . . . problems were solved to a . % optimality gap (i.e., the reported solution is proven to have an objective function value within . % of that of the best possible solution). we additionally provide an implementation www.nature.com/scientificreports/ the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? world health organization declares global emergency: a review of the novel coronavirus (covid- ) lockdown in italy: personal stories of doing science during the covid- quarantine evaluation of the lockdowns for the sars-cov- epidemic in italy and spain after one month follow up the effect of human mobility and control measures on the covid- epidemic in china estimated effectiveness of symptom and risk screening to prevent the spread of covid- modeling infectious diseases in humans and animals data-based analysis, modelling and forecasting of the covid- outbreak epidemic analysis of covid- in china by dynamical modeling 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systems enterprise. general process modeling system (gproms a polyhedral branch-and-cut approach to global optimization models are available at https ://githu b.com/balde a-group /covid - .received: april ; accepted: june acknowledgements partial financial support from the national science foundation (nsf) through career award is acknowledged with gratitude. the authors also thank dr. richard pattison (apeel sciences) for his insightful comments throughout this study. all authors conceived the experiments; c.t. and f.l. implemented the optimisation models, conducted the computational experiments, and analysed the results; all authors prepared and reviewed the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to m.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -jspxlk a authors: homma, takujiro; ishibashi, daisuke; nakagaki, takehiro; fuse, takayuki; sano, kazunori; satoh, katsuya; atarashi, ryuichiro; nishida, noriyuki title: persistent prion infection disturbs the function of oct- , resulting in the down-regulation of murine interferon regulatory factor- date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: jspxlk a as a prompt response against invasion of various viruses, interferon regulatory factor- (irf- ) is initially phosphorylated to become activated and upregulates mainly type i interferons (ifn-i) in most cell types. we previously reported that irf- -dependent host innate immune responses partially interfere in infection of prions. here, we found that stable infection of prion suppressed irf- gene-expression. the decreased promoter activity of irf- was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of irf- transcription. we further investigated promoter activity of ′- flanking region of murine irf- using a luciferase reporter system and found that the nucleotides - to - were indispensable for the promoter activity. within this region, mutations in the oct- binding site significantly reduced the promoter activity and chromatin immunoprecipitation (chip) assay revealed that oct- indeed binds to the region. in addition, overexpression of oct- increased the promoter activity of irf- . intriguingly, oct- protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. taken together, we concluded that prion infection could interfere in the function of oct- , resulting in the down-regulation of irf- . i rf- is a key transcriptional factor involved in the signaling the pathway responsible for keeping the antiviral state of host cells. viral infection triggers phosphorylation of the irf- carboxyl-terminal region , and the phosphorylated irf- migrates to the nucleus, leading to the transcriptional activation of the type i interferon (ifn-i) genes. interestingly, it has also been reported that infection by some viruses enhances the degradation of irf- and/or inhibits its phosphorylation, resulting in the reduced response of ifn-i production and persistent infection by the virus [ ] [ ] [ ] [ ] [ ] [ ] . prion diseases are neurodegenerative disorders characterized by an aggregation of abnormal prion protein (prp sc ) . since prp sc itself seems to be an infectious agent and the protein is host-encoded, acquired immunity against prion disease should not be possible. conversely, we previously demonstrated that irf- plays a crucial role in the host's defense mechanism against prion infection. disrupted irf- genes developed prion diseases earlier compared to wild-type mice after inoculation. overexpression of irf- significantly reduced the amount of abnormal prion protein in persistently infected cells, indicating that irf- expression levels are closely related to the anti-prion state of the host cell , . although the innate immune responses against prion invasion are insufficient to stop disease progression, involvement of irf- inducible factors such as ifn-i may be one of the reasons why prion infection shows an extremely long incubation period. it would be of great benefit to understand how irf- inducible factors inhibit prion infection and how prions avoid this host-defense system. lowther et al. reported that a sufficient basal promoter activity of human irf- was found in the upstream region (nucleotides: nt; - to - ) of the transcription start site , however, the mechanism of the transcriptional regulation of irf- remains largely unknown. in this study, we analyzed murine irf- promoter activity in detail and its relationship to prion infection, and have shown that the octamer-binding transcription factor- (oct- ) positively regulates murine irf- , and the expression levels of oct- decreased in prion-infected cells. nucleotides - to - region are responsible for the murine irf- promoter activity. to determine the specific promoter region responsible for murine irf- induction, we generated a series of plasmids that included various sizes of the -flanking region of the murine irf- gene fused to the luciferase gene and transfected them into n a and t cells. as shown in fig. , a plasmid containing nt - to - relative to the transcription start site (pgl - /- ) showed approximately -fold (in n a cells) and -fold (in t cells) activity compared with the control (pgl basic), while plasmids containing nt - to - (pgl - /- ) and nt - to - (pgl - /- ) completely lost their responsiveness. plasmids containing nt - to - (pgl - /- ), nt - to - (pgl - /- ) and nt - to - (pgl - /- ) maintained similar levels of their promoter activity with the full-length promoter (pgl - /- ), suggesting that nt - to - was responsible for the promoter activity. irf- promoter activity is reduced in prion-infected cells. to examine the relationship between the irf- promoter activity and prion infection, we analyzed n a and scn a (n a cells persistently infected with l prion) cells. as shown in fig. a , mrna of irf- had a significant reduction in scn a cells. reduction of the promoter activity was confirmed in scn a cells which was transiently transfected with pgl - /- (fig. b) . furthermore, in the persistently other prion strain-infected cells by the mouse-adapted gerstmann-sträussler-schenker syndrome (gss) fukuoka- strain (fk-n a ), irf- mrna and the promoter activity were also significantly decreased (supplementary fig. s a and s b). next, we challenged with . % brain homogenates from linfected mice ( lbh) or normal mice (nbh) to n a cells and incubated for h. as expected, the promoter activity in lbhincubated cells was significantly decreased ( supplementary fig. s ), indicating that prion infection reduced the activity. furthermore, drug treatment was started after the initial seeding of the cells (at cm flask) and renewed every passage. the significantly reduced promoter activity in scn a cells increased after continuous treatment with congo red (cr, mm) or pentosan polysulfate (pps, mg/ml), which are both known as anti-prion drugs (fig. c ; cr: . -fold and pps: . -fold). the efficacy of the two drugs was evaluated by detection of pk-resistant prp using western blotting in cells after final passage (fig. d) . the activities of the uninfected control n a cells were not affected during continuous treatment ( supplementary fig. s ). oct- is responsible for irf- promoter activity. we identified putative transcription factor binding sites in nt - to - with the software tfsearch ver. . (http://www.cbrc.jp/research/db/ tfsearch.html) and found that this region contains a potential oct- binding site ( -atttgcat- , nt - to - ) and an acute myeloid leukemia protein (aml ) binding site ( -tgcggt- , nt - to - ). in addition, an e f transcription factor (e f ) binding site ( -tttcccac- , nt - to - ) was also conserved in murine (fig. a) . to determine whether the oct- site is important for promoter activity, an original and two different mutated plasmids from the oct- site (m and m ) were prepared (fig. b ) and the transiently transfected into n a and t cells. after h, we evaluated their respective promoter activity by reporter assay. the activities of pgl - /- (m ) were also significantly reduced compared with the original promoter activities in n a and t cells ( fig. c and d ). the activities of pgl - /- (m ) and pgl - /- (m ) were also significantly reduced ( supplementary fig. s ). we obtained similar results in the second mutated plasmid pgl - /- (m ) where activity was also significantly reduced in n a and t cells ( fig. e and f) . these results indicate that the oct- site might play a crucial role for murine irf- promoter activity. we next determined whether the e f site was important for the promoter activity. the original plasmid pgl - /- and the e f site mutated plasmid (m : fig. g ) were transiently transfected into n a and t cells. after h, we evaluated their respective promoter activity by reporter assay. the activities of pgl - /- (m ) were not significantly different compared to the original plasmid promoter activity in n a and t cells ( fig. h and i) , indicating the e f is not an important determining factor for the regulation of murine irf- promoter. since two different mutations in the aml site (m and m : shown in supplementary fig. s a ) had little effect on the promoter activity in n a cells (n a cells overexpress wild-type prion protein), indicating the aml is not also important ( supplementary fig. s b ). to confirm that oct- binds to the endogenous murine irf- promoter, chromatin immunoprecipitation (chip) assay was performed. pcr analysis revealed that the oct- antibody precipitated the promoter region (nt - to - ) from n a cells (fig. a) , while the negative control (anti-rabbit igg) did not exhibit the dna binding activity. these data suggest that endogenous oct- binds to the promoter region in vivo. furthermore, to investigate whether the promoter activity is affected by oct- , pcdna oct- -ha and pgl - /- were co-transfected into n a cells. ectopically oct- overexpression significantly increased the original promoter activities (pgl - /- mock vs pgl - /- pcdna oct- -ha), while oct- expression had no effect on the mutated promoter activities [pgl - /- (m ) mock vs pgl - /- (m ) pcdna oct- -ha], indicating exogenous oct- functions in the promoter region (fig. b) . we next examined the expression levels of oct- proteins in prion-infected cells. as shown in fig. a , oct- expression in scn a cells was significantly decreased compared with that in n a cells. similarly, we also analyzed the expression levels of oct- proteins in prion-infected mice brains. we intracerebrally inoculated l or fk- prion strains into ddy mice and analyzed the levels of oct- protein in their brains after becoming terminal, respectively. of note, the levels of oct- protein in the two distinct prion strains-infected mouse brains significantly decreased, suggesting that prion infection might repress endogenous oct- expression ( fig. b and supplementary fig. s c ). these results demonstrate that reduced irf- promoter activity in prion-infected cells is accompanied by decreased levels of oct- . we have investigated the promotion of the murine irf- gene and found promoter activity within the region nt - to - ( fig. a and b), which contains different promoter binding sites. it has been shown that e f negatively regulates human irf- gene expression . although the e f site was also present in the murine promoter, the e f site mutation had little effect on promoter activity ( fig. h and i ). mach et al. have shown that oct- also bound to the human irf- promoter . importantly, the putative transcription factor binding sites in flanking region are very similar between human and murine irf- promoter (supplementary fig. s ). however, the contribution of oct- in human irf- promoter remains controversial and elusive. in our experimental study, mutations in the oct- binding site lost their promoter activity (fig. c to f) and exogenous oct- enhanced the activity (fig. b) . in conclusion, oct- positively regulates the irf- promoter activity, while e f and aml are not involved in murine irf- gene regulation. intriguingly, oct- expression was significantly reduced in two distinct prion strains-infected cells and mice brains ( fig. and supplementary fig. s ). although we are not able to exclude yet the possibility that the accumulation of abnormal prion protein may directly impair the function of oct- , prion infection might alter the expression of oct- as a result of its destructive effect on protein synthesis in the host cell. it has recently been reported that prion infection could impair the host's ability to synthesize protein by means of inhibition of double-stranded rna-activated protein kinase (pkr)-dependent eif- a phosphorylation , . another study revealed that the global alteration of gene expression has occurred in prion-infected mouse brains by unknown mechanisms . in summary, our results demonstrated that oct- binds to the murine irf- promoter region and increases the transcription level and irf- expression is reduced by prion infection. although further investigations are required to elucidate how oct- is affected in prion infection, our novel findings on the alteration of host gene expression provide new insights into the molecular biology of prions and host responses against prion infection. (a) n a cell lysate was subjected to chip assay using anti-oct- (ip: oct- ) or normal rabbit igg (negative control) antibodies. after the dna was purified, the promoter region (nt - to - ) was amplified by pcr (see ''materials and methods'' in detail). (b) n a cells were co-transfected with either pgl - /- or pgl - /- (m ) and either pcdna oct- -ha (oct- ) or the empty plasmid (mock). results were normalized to the control renilla luciferase activity, and the value of (pgl - /- mock) was set to %. results representing the mean sd, n . p values were determined by one-way anova followed by tukey's multiple comparison (***: p , . , n.s.: not significant). scn a (persistently l scrapie prion infected n a cells) and fk-n a (persistently gerstmann-sträussler-scheinker syndrome-derived fk- prion infected n a cells) cells described as previously [ ] [ ] [ ] . all cells were cultured in a cell incubator and maintained at % co and uc. prion infection. the prepared % brain homogenates from l-infected terminally sick ddy mice ( lbh) or normal brain homogenates from healthy ddy mice (nbh) were treated with final . % concentration to n a cells and incubated for h. after replaced with the fresh medium to remove the prion from the cells, the cells were passaged at cm flask and cultured for d. the detailed methods were described as previously . generation of luciferase reporter plasmids. the information on primers used in this study is summarized in table . in order to analyze the -flanking region of the murine irf- gene for promoter activity, pcr products amplified from mouse genomic dna were subcloned into pgl -basic vector (promega) : pgl - /- (nt to - , where the first nucleotide of irf- exon has been designated ), pgl - /- (nt - to - ), pgl - /- (nt - to - ), pgl - /- (nt - to - ), pgl - /- (nt - to - ) and pgl - /- (nt - to - ). two different mutations (m : atttgcat to cgttgcat or m : atttgcat to ggggaacc) were introduced into the oct- site in the pgl - /- , pgl - /- and pgl - /- , generating pgl - /- (m ), pgl - /- (m ), pgl - /- (m ) and pgl - /- (m ). pgl - /- (m : tttcccac to gggggggg) was the e f transcription factor (e f ) sitemutated plasmid. pgl - /- (m : tgcggt to tgcgcg or m : tgcggt to tcgggt) were the acute myeloid leukemia protein (aml ) site-mutated plasmids. murine oct- cdna (ncbi reference sequence: nm_ . ) was amplified by rt-pcr from total mrna of n a cells and subcloned into pcdna . (invitrogen) to generate hemagglutinin (ha)-tagged oct- expression plasmid (pcdna oct- -ha). all generated plasmids were confirmed by sequencing. transient transfection and reporter assay. cells were seeded at densities of cells per well in -well plates and grown in a humidified incubator at uc and % co overnight to - % confluence. in basal promoter activity studies, cells were co-transfected with ng of reporter plasmids and ng of renilla luciferase control plasmid (prl-null vector; promega). for ectopic expression, cells were additionally co-transfected with ng of pcdna oct- -ha or pcdna (empty plasmid). transfections were performed using lipofectamine ltx (invitrogen) following the manufacturer's instructions. after h, cells were lysed using passive lysis buffer (promega) and the luciferase activity was quantified by the dual reporter assay system (promega) and mithras lb luminometer (berthold technologies) according to the manufacturer's instructions. results were normalized to the coexpressed renilla luciferase activity. western-blot analysis. whole cell lysates were prepared by the addition of cold lysis buffer ( mm tris-hcl ph . , mm nacl, . % triton x- , . % sodium deoxycholate, mm edta) containing protease inhibitors (nacalai tesque). the protein concentration was determined using the bio-rad protein assay kit. equal amounts of protein from each treatment were subjected to western-blot analysis using specific antibodies against oct- (santa cruz biotechnology, sc- ) or b-actin (mbl, pm ). for prp sc detection, samples were digested with mg/ml of proteinase k (pk; sigma-aldrich) at uc for min and subjected to western-blot analysis using anti-prp antibody (santa cruz biotechnology, m ). chromatin immunoprecipitation (chip) assay. n a cells grown in mm dish were incubated with % formaldehyde for min at uc to cross-link proteins to the dna and washed with cold pbs buffer twice before lysis with sds lysis buffer ( mm tris-hcl ph . , % sds, mm edta) containing protease inhibitors. cell lysates were sonicated on ice and the sheared chromatin was evenly divided and diluted in chip dilution buffer ( . % sds, . % triton x- , . mm edta, . mm tris/hcl ph . and mm nacl) containing protease inhibitors. a small portion of each diluted chromatin was collected, protein-dna cross-links were reversed, and samples were used as the input dna control. the rest of chromatin suspension was prepared with protein g-agarose slurry containing salmon sperm dna (millipore) and then incubated with either anti-oct- or normal rabbit igg antibodies ( mg) overnight at uc with continual rotation. the protein g-agarose slurry containing salmon sperm dna was added to each chromatin solution and incubated for another h at uc with constant rotation. the agarose beads were collected by centrifugation, washed and the antibody-bound chromatin was released from the agarose beads. then the cross-link was reversed, samples were digested with pk and finally purified by phenol/chloroform extraction and ethanol precipitation. the dna isolated from the immunoprecipitates was then subjected to pcr amplification. pcr was carried out as follows: cycle at uc for min; cycles at uc for s, uc for s, uc for s; and cycle at uc for min, using chip ( / ) primers that amplify the region between nucleotides and within the irf- promoter. then, nested pcr was carried out as follows: one cycle at uc for min; cycles at uc for s, uc for s, uc for s; and one cycle at uc for min, using chip (- /- ) primers that amplify the region between nucleotides to within the irf- promoter. the amplified bp dna fragment was separated on % agarose gel and visualized with ethidium bromide. drug treatment. n a and scn a cells were continuously treated with congo red (cr, mm) or pentosan polysulfate (pps, mg/ml) for days. the drug treatments were started after the initial seeding of the cells (at cm flask) and renewed every passage until three passages. after days from the last treatments, cells were reseeded in -well plates without drugs and transfected with pgl - /- and prl-null. after h, luciferase assay was performed as described above. in vivo infection experiments. four-week-old ddy mice were intracerebrally inoculated with ml of a dilution of brain homogenate prepared from mice terminally sick with l and fk- strains, respectively. as a control, age-and strainmatched mice were intracerebrally inoculated with phosphate buffered saline. the brains of the mice were removed at the terminal stage of disease. animals. all animal experiments were approved by the committee on the animal care and use committees of the nagasaki university, and were performed according to their recommendation. irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors classical swine fever virus npro interacts with interferon regulatory factor and induces its proteasomal degradation ubiquitination and proteasomal degradation of interferon regulatory factor- induced by npro from a cytopathic bovine viral diarrhea virus regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus human immunodeficiency virus type mediates global disruption of innate antiviral signaling and immune defenses within infected cells loss of interferon regulatory factor in cells infected with classical swine fever virus involves the n-terminal protease the infected cell protein encoded by bovine herpesvirus (bicp ) induces degradation of interferon response factor and, consequently, inhibits beta interferon promoter activity protective role of interferon regulatory factor -mediated signaling against prion infection protective role of myd -independent innate immune responses against prion infection cloning and functional analysis of the human irf- promoter sulfated polyanion inhibition of scrapie-associated prp accumulation in cultured cells characterization of the human irf- promoter and its regulation by the transcription factor e f the small rna gene activator protein, sphi postoctamer homology-binding factor/selenocysteine trna gene transcription activating factor, stimulates transcription of the human interferon regulatory factor- gene prion protein interaction with stress-inducible protein enhances neuronal protein synthesis via mtor sustained translational repression by eif alpha-p mediates prion neurodegeneration a systems approach to prion disease successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein prion straindependent differences in conversion of mutant prion proteins in cell culture biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures t.h. and d.i. designed the project, performed experiments, and wrote the manuscript. t.n., t.f., k. sano and k.satoh supervised and discussed the data. d.i., r.a. and n.n. revised the manuscript. supplementary information accompanies this paper at http://www.nature.com/ scientificreports competing financial interests: the authors declare no competing financial interests. key: cord- -kxivgxbg authors: haverkamp, ann-kathrin; lehmbecker, annika; spitzbarth, ingo; widagdo, widagdo; haagmans, bart l.; segalés, joaquim; vergara-alert, julia; bensaid, albert; van den brand, judith m. a.; osterhaus, albert d. m. e.; baumgärtner, wolfgang title: experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: kxivgxbg middle east respiratory syndrome (mers) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. in the current study, tissues of eight dromedaries receiving inoculation of mers-coronavirus (mers-cov) after recombinant modified-vaccinia-virus-ankara (mva-s)-vaccination (n = ), mva-vaccination (mock vaccination, n = ) and pbs application (mock vaccination, n = ), respectively, were investigated. tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. mers-cov infection in mock-vaccinated dromedaries revealed high numbers of mers-cov-nucleocapsid positive cells, t cells, and macrophages within nasal turbinates and trachea at day four post infection. double immunolabeling demonstrated cytokeratin (ck) expressing epithelial cells to be the prevailing target cell of mers-cov, while ck / and ck expressing cells did not co-localize with virus. in addition, virus was occasionally detected in macrophages. the acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase (dpp ), known to mediate virus entry. dpp was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. the present study underlines significant species-specific manifestations of mers and highlights ciliary loss as an important finding in dromedaries. the obtained results promote a better understanding of coronavirus infections, which pose major health challenges. in june a novel lineage c betacoronavirus (hcov-emc) was identified in a patient from the kingdom of saudi arabia who suffered from acute pneumonia and renal failure . subsequently, the virus was named middle east respiratory syndrome coronavirus (mers-cov) in accordance with the geographical area of its first description and main occurrence . until today, mers-cov represents an existential threat to global health since the virus spread to countries and caused more than laboratory confirmed cases in humans including fatal cases, which equals approximately one third of all affected patients (world health organization ( ) middle east respiratory syndrome coronavirus, available at http://www.who.int/emergencies/mers-cov/en/, accessed october , ) . the sequence of mers-cov was determined to be closely related to other betacoronaviruses isolated from bats and therefore a bat origin has been proposed early after genomic characterization [ ] [ ] [ ] [ ] [ ] [ ] . however, transmission of mers-cov to humans was suspected to occur via an intermediate mammalian host, since the majority of human middle east respiratory syndrome (mers) patients did not state any direct contact to bats prior to disease onset , . similarly, severe acute respiratory syndrome coronavirus (sars-cov), a betacoronavirus of the lineage b, originated from bats and spread from palm civets to humans in / . in , one year after the initial description of mers, serological investigations in livestock species suspected dromedaries (camelus dromedarius) to act as potential intermediate hosts for mers-cov . subsequent research on index cases , serological sampling , and virus isolation revealed a transmission from dromedaries to humans and confirmed dromedaries as the major reservoir for human infections . consequently, mers-cov infection in dromedaries was documented by serological surveys in large parts of the middle east, the canary islands, and africa, as previously summarized by hemida et al. and additionally in certain parts of southern asia and western africa . recently, mers-cov has also been detected in alpacas from qatar , but not in any other domestic livestock species . in recent animal trials with dromedaries, experimental mers-cov infection led to mild transient upper respiratory tract disease characterized by mild to moderate rhinitis with nasal discharge, tracheitis, and bronchitis accompanied by shedding of large quantities of virus from the upper respiratory tract. viral antigen was additionally detected in regional lymph nodes but not in any other extra-respiratory organ , . the serine exopeptidase dipeptidyl peptidase (dpp ; also known as cd ) has been identified as a major virus receptor in both humans and dromedaries , and is expressed in the lower respiratory tract of humans and the upper respiratory tract of dromedaries . more recently, carcinoembryonic antigen-related cell adhesion molecule (ceacam ) has been described as an additional cellular binding target for mers-cov that may facilitates virus entry . additionally, binding of mers-cov to sialic acids has been demonstrated in a newly published study by li and colleagues and is suggested to represent another important factor in viral host tropism . despite two recent animal trials , and the successful establishment of an orthopoxvirus-based vaccine against mers-cov in dromedaries , the virus infection triggered immune response and the exact cell tropism have not been evaluated so far, since valuable markers for immune cell phenotyping in dromedaries were hardly established. in order to facilitate such studies and other investigations in camels, a panel of antibodies for identification of different immune cell subsets has been recently tested . since dromedaries play a key role in transmitting mers-cov to humans, it was the aim of the present study to describe mers-cov associated lesions in the nasal epithelium and viral cell tropism of experimentally infected dromedaries in closer detail. experimental mers-cov infection of dromedaries leads to high virus load in nasal turbinates and trachea accompanied by necro-suppurative inflammation at day post infection. evaluation of hematoxylin and eosin (he) stained sections taken from respiratory epithelium of the nasal turbinates of mock-vaccinated animals at days post infection (dpi) revealed mild to moderate, segmental hyperplasia of the epithelium. moderate exocytosis of neutrophilic granulocytes, apoptosis and single cell necrosis of epithelial cells were observed within all layers of the epithelium and frequently accompanied by formation of small cavities in the apical epithelium (fig. a) . additionally, a partial loss of apical epithelial cells (erosion) was occasionally present within certain areas with marked inflammation (fig. b) . the epithelial surface was covered multifocally by large amounts of mucus, as well as viable and degenerated neutrophilic granulocytes and cellular debris. lamina propria and submucosa displayed mild to moderate edema and infiltration of moderate numbers of lymphocytes and macrophages as well as single neutrophilic granulocytes, which were predominantly located next to blood vessels. histology of trachea and bronchi revealed only mild lesions which were characterized by mild exocytosis of neutrophilic granulocytes and mild, segmental infiltration of lamina propria and submucosa by lymphocytes, macrophages, and low numbers of neutrophilic granulocytes (fig. c,d) . rarely epithelial erosion and accumulations of degenerated neutrophilic granulocytes within the apical epithelial layers (pustules) of the trachea were detected. the mva-s-vaccinated animals showed similar but less severe lesions at dpi in nasal turbinates, trachea, and bronchi which were rarely accompanied by degeneration, loss, and necrosis of single epithelial cells in the nasal turbinates. at dpi, very mild lesions were detectable in nasal turbinates, trachea, and bronchi of all investigated animals (mva-s-vaccinated and mock-vaccinated) characterized by mild, multifocal, lymphoplasmahistiocytic and neutrophilic infiltration of lamina propria and submucosa and rare exocytosis of neutrophilic granulocytes (data not shown). rarely, follicular aggregates of lymphoid cells were detectable in the lamina propria and submucosa of nasal turbinates and trachea in both groups and at all investigated time points. within pulmonary, tracheal and cervical lymph nodes as well as within tonsils mild to moderate follicular hyperplasia and single apoptotic lymphocytes were present. all other non-respiratory organs and the lungs did not show any significant lesions. in line with these observations, high amounts of mers-cov antigen were detected within the respiratory epithelium of the nasal turbinates of mock-vaccinated dromedaries at dpi by immunohistochemistry in areas scientific reports | ( ) : | doi: . /s - - - with most severe lesions ( fig. a) . rarely, mers-cov antigen was also present within single round cells located in the submucosa of the nasal turbinates, which were presumed to represent macrophages based on histological characteristics ( fig. a, insert) . in addition, single positive cells were detected in the epithelium of the trachea in both mock-vaccinated animals and in the epithelium of a large bronchus of one mock-vaccinated animal at dpi. in mva-s-vaccinated animals low numbers of positive staining cells were present in the epithelium of the nasal turbinates at dpi. no virus was detectable in trachea and bronchi of these dromedaries. at dpi, virus antigen was not detectable in the upper respiratory tract of mva-s-vaccinated animals but very rarely within the nose of mock-vaccinated animals (fig. b ,c). all these findings are in line with previous studies by the co-authors and other investigators . since high amounts of virus antigen were present within the nasal turbinates, and to a lesser extent also within the trachea of mock-vaccinated dromedaries at dpi, these tissues were further analyzed by additional immunohistochemistry using different antibodies. phenotyping of inflammatory cells revealed high numbers of cd + t cells in lamina propria and submucosa of nasal turbinates and trachea in mock-vaccinated dromedaries at dpi ( fig. a-c) . comparison of mock-vaccinated and mva-s-vaccinated animals at that time point revealed significantly increased numbers of t cells within the nasal turbinates, but not within the trachea of mock-vaccinated animals. at dpi, the numbers of t cells were scarce. similarly, increased numbers of cd + macrophages were detected at dpi compared to dpi in nasal turbinates and trachea, but no significant differences were present between both groups at the respective time points (fig. d-f) . the decrease of t cells and macrophages at dpi is in line with the significant reduction of virus antigen at the respective time point (fig. b,c) . immunohistochemistry for detection of cd + b cells did not reveal any differences between groups and time points in the nasal turbinates. however, at dpi numbers of b cells were significantly increased in the trachea of mva-s-vaccinated compared to mock-vaccinated animals and compared to the later time point (fig. g-i) . (fig. a,b) . to further distinguish epithelial subsets in the upper respiratory tract tissue of dromedaries, antibodies specific for certain ck of apical and basal cells have been established and were evaluated regarding their distribution (suppl. fig. s ). double immunofluorescence with these antibodies showed strong co-localization of mers-cov nucleocapsid with ck located in apical epithelial cells at dpi in nasal turbinates and trachea (fig. c,d) . there was no co-localization with ck / and ck , expressed by basal epithelial cells, in any localization (fig. e ,f). moreover, mers-cov nucleocapsid antigen was not identified in serous glands, located in the submucosa of the nasal turbinates, even if the apical proportion of these structures stained brightly positive for ck ( fig. c ). at dpi, localization of mers-cov nucleocapsid antigen in the nasal turbinates of mock-vaccinated remained basically identical to the distribution at dpi but the number of mers-cov positive staining cells was substantially reduced. in mva-s-vaccinated animals the amount of virus antigen detected by immunofluorescence was very low at dpi, but depicted similar co-localization with ck . (fig. c ). loss of dpp expression seems to be exclusively restricted to mers-cov-infected cells as adjacent cells staining negative for mers-cov antigen retained positivity for dpp (insert in fig. b ). for further characterization, staining of cilia was performed to evaluate whether multifocal lack of dpp was associated with concurrent ciliary loss. ciliary loss occurred frequently in association with infiltration of inflammatory cells into the epithelium but also in areas with rather mild inflammatory lesions restricted to lamina propria and submucosa, tunel assay was performed to determine whether the loss of cilia is directly related to virus-induced apoptosis of epithelial cells. since the tunel assay revealed some but not abundant apoptotic cells in the respiratory epithelium (suppl. fig. s ) cilia-specific alterations by mers-cov must be assumed. to finally elucidate the histogenesis of cells staining positive for mers-cov nucleocapsid antigen within the lamina propria of the nasal turbinates of mock-vaccinated dromedaries, additional double immunofluorescence labeling was performed. based on histological evaluation of he stained slides, mers-cov antigen was supposed to be located in macrophages or other inflammatory cells and double labeling with antibodies for detection of t cells, b cells, and macrophages was accomplished. staining of mers-cov nucleocapsid antigen with cd and cd antigen, respectively, did not reveal any co-localization, neither in the nasal turbinates nor in the trachea (fig. a-d) . however, single cells within the lamina propria of the nasal turbinates stained simultaneously positive for iba- and mers-cov nucleocapsid and were characterized by a macrophage-like morphology (fig. e) . in the trachea, double positive staining cell elements were absent (fig. f) . for further confirmation the reaction was repeated by use of another macrophage specific antibody. the anti-cd -specific antibody exhibited an intense co-localization of mers-cov nucleocapsid and cd in single cells in the same localization. overall, mers-cov nucleocapsid antigen was therefore not only detectable in ck + apical epithelial cells but also within the cytoplasm of single macrophages. however, co-localization of mers-cov nucleocapsid with iba- and cd , respectively, was not detected within the trachea, implicating that the presence of mers-cov in macrophages might be related to high virus loads in the respective localization. since dpp was only detectable within the apical brush border of the surface epithelium and submucosal glands, but not on the surface of inflammatory cells within lamina propria and submucosa of the nasal turbinates by immunofluorescence, dromedary and human lymphoid tissue were stained for comparison and control. whereas dpp was evident within lymphoid human tonsillar tissue (fig. a) , a positive signal was not observed in tonsils of mva-s-vaccinated and mock-vaccinated dromedaries using immunofluorescence. similarly, tonsillar tissue of the non-infected control animal did also not reveal any dpp expression. in human lymph node tissue dpp was rarely detectable by immunohistochemistry on round cells in the cortex and more frequently within the paracortex and medulla. in camels dpp was rarely demonstrable within the paracortex and medulla of lymph node but lacked expression in the cortex (fig. a) . in order to elucidate the subset of human immune cells expressing dpp , flow cytometry was performed and revealed dpp expression in human pbmc by cd + t cells and hardly by cd + b cells, cd + nk cells, and cd + monocytes (fig. b) . the s protein of mers-cov detected dpp + cells in both human and dromedary pbmc. lymphocytic and monocytic populations were differentiated based on their size and granularity. dpp was mainly expressed by lymphocytes in human pbmc and monocytes in dromedary pbmc even if the expression level was lower in dromedaries than in humans (fig. c) . it has recently been shown that dromedaries play a key role in the transmission of mers-cov at the animal-human interface . in addition, experimentally infected dromedaries serve as an important animal model for investigations on certain aspects of mers-cov pathogenesis , . the use of appropriate animal models is highly required, since human tissue samples from individual mers-cov cases are rarely accessible [ ] [ ] [ ] , which has in part been attributed to cultural reasons . two individual case descriptions detected viral particles via electron microscopy and immunohistochemistry in pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells, and macrophages within skeletal muscle. biopsies revealed necrotizing pneumonia, pulmonary alveolar damage, vascular disease, cardiac fibrosis, acute kidney injury, hepatitis, and myositis , . these reports from human tissue underline that the disease observed in dromedaries after natural and experimental mers-cov infection differs substantially from the human counterpart. whereas dromedaries develop only mild respiratory signs and lack overt pulmonary disease and systemic spread , , the disease in humans is often accompanied by acute respiratory distress syndrome, renal dysfunction, and lethal outcome . previous studies indicated that these differences are related to the fact that mers-cov predominantly replicates in the lower respiratory tract of humans but not of dromedaries that might, at least in part, be caused by differing expression patterns of the cell surface receptor dpp . whereas dpp is extensively expressed in the upper respiratory tract epithelia of dromedaries, its expression in the respiratory tract of humans is limited to alveolar epithelial cells and macrophages in the lower airways . in the present study, it has been shown that dpp is located on the apical brush border of ciliated ck expressing epithelia in the upper respiratory tract of dromedaries. in humans dpp dpp (red) is frequently detectable on lymphoid cells (white arrows) in the human tonsil but is completely absent in tonsillar tissue of dromedaries. immunofluorescence, x. in the lymph nodes of both human and dromedary, dpp (red) is detected in the paracortex and medulla (arrows), but in much smaller numbers in that of dromedaries (arrow). immunohistochemistry, x. in human pbmc, dpp is mainly expressed by cd + t cells and hardly expressed by cd + b cells, cd + nk cells, and cd + monocytes (b). s protein of mers-cov is used to detect dpp + cells in both human and dromedary pbmc. lymphocytes and monocytes population are differentiated based on their size and granularity. dpp is mainly expressed by lymphocytes in human pbmc while it is mainly expressed by monocytes in dromedary pbmc (c). data in figure b and c are visualized as the mean percentage. can be detected in the brush border of renal proximal convoluted tubules and enterocytes in the intestine but not within the upper respiratory tract . the present study demonstrates that acute mers-cov infection in dromedaries is accompanied by severe ciliary loss and concomitant lack of dpp on infected cells. adjacent cells in which mers-cov antigen is not detectable retain positive staining for dpp . ciliary loss and consequent disturbances of mucociliary clearance are a major issue in several viral infections and can foster the development of severe secondary bacterial disease . for instance, common cold in humans is accompanied by a massive loss of cilia and ciliated cells . similarly, human coronavirus infection of the upper respiratory tract has been described to be associated with migration of axonemes and basal bodies into the cell body (internalization) complemented by loss of cilia on the apical cell surface of infected cells. for the human disease it has been suggested that replicated virions are stored in apical vesicles before they are released. these vesicles may dislocate the basal body and withdraw the cilia into the cell . in dogs, canine respiratory coronavirus infection is also associated with loss and damage to tracheal cilia, accompanied by inflammation . similar mechanisms might also play a role in mers-cov in dromedaries and would at least explain the massive loss of cilia which appears not to be accompanied by massive cell death or other profound histological and ultrastructural alterations in the majority of affected epithelial cells. interestingly, ciliary loss is accompanied by lack of dpp , which serves as a cell entry receptor for mers-cov in dromedaries. therefore, the authors suggest that the mild and transient disease in dromedaries is, at least in part, attributable to the downregulation of its own cell entry receptor. further studies need to be performed to elucidate underlying mechanisms of dpp loss in mers-cov-infected ck positive staining cells of dromedaries. the remaining expression on adjacent mers-cov negative cells suggests a potential direct virus mediated mechanism. the detection of mers-cov nucleocapsid antigen in the cytoplasm of a limited number of cd /iba- positive staining cells within the lamina propria of the nasal turbinates of mock-vaccinated dromedaries at dpi is similar to previous investigations which detected viral antigen and rna in mononuclear cells and stellate cells of mediastinal lymph nodes in experimentally infected rhesus macaques and rarely within large mononuclear cells in the tracheal lymph node of infected dromedaries . moreover, infection of human monocyte-derived dendritic cells and monocyte-derived macrophages has been described in vitro and was accompanied by release of viral particles , . the infection of these cell types is supposed to be mediated by dpp , expressed on the cell surface of human macrophages , , and leads to suppression of the innate immunity by reduced expression of tumor necrosis factor (tnf) and interleukin- (il- ) . in contrast, it remains so far uncertain whether the intracytoplasmic detection of mers-cov nucleocapsid antigen in dromedary macrophages represents a true productive or abortive infection or whether it is related to phagocytosis of mers-cov fragments. since the number of positive staining macrophages was very low and dpp was not detectable on dromedary tonsils and hardly on dromedary lymphocytes and macrophages in lymph nodes it is not unlikely that the intrahistiocytic detection of mers-cov nucleocapsid antigen is related to phagocytosis of viral particles or infected cellular components. nonetheless the staining of the antigen within affected cells was brightly and diffusely distributed in the cytoplasm and the detection of viral antigen in phagosomes would be suspected to rather appear as discrete spots , . it might therefore been speculated that the viral antigen detection indeed represents virus infection of dromedary macrophages. however, further investigations have to elucidate whether it is a productive or abortive infection. the lack of detection of viral antigen in dromedary t and b cells is in contrast to the findings in humans where mers-cov is capable of infecting t cells derived from peripheral blood and lymphoid organs . on human t cells dpp is widely located on the cell surface and serves as a costimulatory molecule which contributes to t cell activation . contrarily, dpp has not been detected on the surface of dromedary lymphocytes in the tonsil and rarely on those located in lymph nodes of the upper respiratory tract in the present study. these results are in line with the lack of mers-cov antigen in infiltrated t and b cells within infected organs of dromedaries. in humans, abortive infection of lymphocytes induces apoptosis by activation of both the intrinsic and extrinsic pathway of apoptosis and is accompanied by massive downregulation of dpp on the surface of infected t cells . similarly, a loss of the dpp receptor has been detected on the surface of infected epithelial cells in the present study. taken together, obtained results suggest that the incapacity to invade t cells, alongside other factors, might contribute to the low pathogenicity of mers-cov in dromedaries. in summary, the present study highlights new and important differences between mers-cov infection in humans and dromedaries. most importantly, ciliary loss and reduction of dpp expression represent important features of the disease in dromedaries which will deepen our understanding of mers-cov. further investigations need to elucidate the underlying mechanisms of ciliary loss to gain insights into the pathogenesis of this emerging and life-threatening disease. just recently a novel alphacoronavirus has been detected in asian house shrews and future zoonotic transmissions of such novel and well-known coronaviruses will require a profound understanding of their pathogenesis in different host species to achieve better preparedness. animals and tissue sampling. all investigations were performed on archived postmortem tissue, which has been used in a formerly published animal trial pancreas, tonsil, palatum molle, intestine, abomasum, mesenteric lymph node and eyelid) were fixed in % neutral-buffered formalin and routinely paraffin-embedded for histology. serial sections were mounted on coated glass slides (superfrost plus ® , menzel co.) and stained with he or processed for immunohistochemistry and immunofluorescence, respectively. prior to embedding tracheal and nasal tissue were decalcified in % disodium-ethylenediaminetetraacetate (edta, serva electrophoresis gmbh) for h. as an additional control, nasal turbinate of a non-infected dromedary (collected during routine necropsy at the department of pathology, university of veterinary medicine hannover, germany) was used. euthanasia of the animal was performed due to non-respiratory disease. camel peripheral blood mononuclear cells (pbmc) were obtained from naïve animals from a previous experiment . human pbmc were obtained from healthy blood donors (sanquin bloodbank, rotterdam, the netherlands). the use of pbmcs for scientific research was approved by the sanquin bloodbank after informed consent was obtained from the blood donors. the human tonsillar tissue which was used for the establishment of the dpp -and ceacam -specific immunohistochemistry was kindly provided by one of the co-authors (is) and sampled during tonsillectomy. the human bronchiolar lymph nodes paraffin-embedded tissue samples were obtained from the erasmus mc tissue bank. these tissue samples were taken either from healthy donors or from patients with nonmalignant lung tumors. these tissue samples were residual human biomaterials that were collected, stored, and issued by the erasmus mc tissue bank under iso : standard operating procedures. use of these materials for research purposes is regulated according to human tissue and medical research: code of conduct for responsible use ( ). light microscopy, immunohistochemistry, immunofluorescence and tunel assay. he stained cross sections of all organs (mva-s-vaccinated and mock-vaccinated animals, and dpi) were evaluated by light microscopy for the distribution and characterization of lesions induced by mers-cov infection. in addition, all organs were screened for the presence of viral antigen by immunohistochemistry using a previously published monoclonal (mc) mouse antibody (sino biological inc.) . according to the results obtained by light microscopy and mers-cov-specific immunohistochemistry, nasal and tracheal tissues were further analyzed with respect to the immune response. immunohistochemistry was performed for the detection of cd + t cells (polyclonal rabbit anti-cd , dakocytomation gmbh), cd + b cells (polyclonal rabbit anti-cd , thermo fisher scientific), and cd + macrophages (monoclonal mouse anti-human macrophage scavenger receptor, human cd , biologo) as summarized in table . the used markers have been previously established for lymphoid dromedary tissue . additionally, epithelial cells were labeled by application of different antibodies (table ) directed against pan-cytokeratin (ck; monoclonal mouse anti-human cytokeratin, dakocytomation gmbh), ck / (monoclonal mouse anti-cytokeratin / , dakocytomation gmbh), ck (polyclonal rabbit anti-keratin , thermo fisher scientific) and ck (monoclonal mouse anti-cytokeratin , abcam). ceacam was investigated using a polyclonal rabbit anti-human ceacam antibody (abcam) and dpp was visualized using a polyclonal goat anti-dpp /cd -specific antibody (r&d systems). briefly, after dewaxing in roticlear ® (roth c. gmbh & co. kg) and rehydration in isopropanol and ethanol ( %, roth c. gmbh & co. kg), endogenous peroxidase activity was blocked by incubation of sections in % ethanol with . % h o (vwrtm international gmbh) for min at room temperature (rt). antigen retrieval was performed by incubating the sections in citrate buffer ( . g citric acid monohydrate in l distilled water, adjusted with naoh to ph = . ) for min in a microwave ( w). subsequently, sections were transferred to shandon coverplates tm (thermo electron gmbh) and nonspecific binding was blocked by inactivated % rabbit (dpp ) or goat serum (all other antibodies) diluted in phosphate buffered saline (pbs) including % bovine serum albumin (bsa; pbs/bsa) for minutes. afterwards sections were incubated with the respective primary antibody for min at rt. for appropriate negative controls, primary antibodies were replaced by ascites fluid from balb/c mice ( : ; cd , pan-ck, ck / , ck , mc mers-cov nucleocapsid), goat ( : ; dpp ) and rabbit serum ( : ; cd , cd , ck , ceacam ), respectively. subsequently, the appropriate secondary biotinylated antibody was added in a dilution of : with pbs (table ) . incubation for min at rt was followed by treatment with the avidin-biotin-peroxidase complex (vectastain abc kit standard, vectorlaboratories) according to the manufacturer's protocol. visualization of the reaction was achieved by the chromogen , -diaminobenzidine tetrahydrochloride (dab, . %, sigma aldrich chemie gmbh) and addition of . % h o . slides were finally slightly counterstained with mayer's hematoxylin (roth c. gmbh & co kg). subsequently, double immunofluorescence was implemented to determine the cell tropism of mers-cov in dromedaries. for this purpose, the two different mers-cov nucleocapsid-specific antibodies were used in combination with antibodies for detection of cd , cd , iba- (polyclonal rabbit anti-iba- , wako chemicals), cd , ck , ck , ck / , pan-ck and dpp , respectively. in addition, single immunofluorescence using anti-acetylated α-tubulin antibody (monoclonal mouse anti-α-tubulin, sigma-aldrich) was performed for visualization of the ciliary axoneme . after dewaxing and retrieval of antigens as described above, nonspecific binding was blocked by inactivated % horse (dpp ) or goat serum (all other antibodies), respectively, diluted in pbs/ . % triton x/ % bsa (pbs/triton x/bsa) for minutes. both primary antibodies were applied simultaneously in the respective concentrations (table ) diluted with pbs/triton x/bsa. for appropriate negative controls, primary antibodies were replaced by ascites fluid from balb/c mice ( : ; pan-ck, ck / , ck , cd , mc mers-cov nucleocapsid, α-tubulin), rabbit ( : ; ck , cd , cd , iba- , pc mers-cov nucleocapsid), and goat serum ( : ; dpp ), respectively. after incubation over night at °c both secondary antibodies were applied simultaneously ( : in pbs/triton x/bsa, table ) and were incubated for min at rt in the dark to visualize the respective antigens. nuclear counterstaining was performed with . % bisbenzimide (sigma-aldrich chemie gmbh) for minutes and sections were mounted with dako fluorescence mounting medium (dakocytomation gmbh). terminal deoxynucleotidyl transferase (tdt) dutp nick-end labeling (tunel) assay was performed to detect cells that undergo extensive dna degradation during the late stages of apoptosis on the camel nasal epithelium according to the protocol provided by the manufacturer (merck kgaa). tissues pre-treated with dnase were used as positive control while tissues stained with labelling solution only were used as respective negative control consistent with the manufacturer's recommendations. flow cytometry. in order to detect which subset of human immune cells expresses dpp , freshly isolated human pbmcs were incubated with a cocktail of fluorescence labelled monoclonal antibodies, i.e. anti-cd (clone sp - , bd biosciences); cd (clone mem , serotec); cd (clone b e , beckman-coulter); hla-dr (clone l , biolegend); cd (clone m e , bd biosciences); dpp (clone , r&d systems); and live/dead cell viability marker (thermo fisher scientific). appropriate ig isotype control antibodies were used as negative controls for each staining. s protein of mers-cov that recognizes dpp , used in a previous study , was applied to compare dpp expression in camel and human pbmc. cells were analyzed on a canto ii flow cytometer (bd biosciences) and using flowjo ® software (flowjo), then subsequently presented as mean percentage of positive cells. scanning electron microscopy. for scanning electron microscopy, formalin fixed samples of nasal mucosa, trachea, and bronchus of dromedaries infected with mers-cov were post-fixed in % glutaraldehyde (sigma-aldrich chemie gmbh) for hours. afterwards the samples were dehydrated in a series of graded ethanol, dried and coated in a sputter-coater (scd ; oerlikon balzers) with gold as described previously , . virus by immunohistochemistry five randomly selected images per section, antibody and animal were taken at x magnification from the epithelium and adjacent lamina propria and submucosa of nasal turbinates and trachea using an olympus bx- digital camera microscope (olympus optical co. gmbh) and the cell-d imaging software (olympus soft imaging solutions). afterwards, pictures were evaluated manually by counting immunopositive cells. statistical analysis was conducted using the statistical analysis software sas . and the enterprise guide . for windows (sas institute inc.). pair-wise comparison of groups (mva-s-vaccinated [n = high power fields] and mock-vaccinated [n = high power fields]) was performed by use of multiple mann-whitney-u-tests. results were considered statistically significant at p-value < . . column bars were generated with graphpad prism . (graphpad software, inc.). slides stained by immunofluorescence were evaluated for co-localization of antigens using an olympus ix inverted fluorescence microscope, the digital camera olympus dp (olympus life science) and the cell f imaging software (olympus soft imaging solutions gmbh, olympus optical co. gmbh). for visualization of the results of scanning electron microscopy a digital scanning microscope (dsm , carl zeiss jena gmbh) was used. per localization and time point post infection eight images were taken at x magnification and the percentage of ciliated area was estimated. data were analyzed using graphpad prism . 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syndrome corona virus spike glycoprotein suppresses macrophage responses via dpp -mediated induction of irak-m and pparγ evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice macrophage phagocytosis of foot-and-mouth disease virus may create infectious carriers middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways the costimulatory activity of the cd antigen requires dipeptidyl peptidase iv enzymatic activity discovery of a highly divergent coronavirus in the asian house shrew from china illuminates the origin of the alphacoronaviruses immunofluorescence staining of ciliated respiratory epithelial cells canine parainfluenza virus-induced encephalitis in ferrets intranasal infection of ferrets (mustela putorius furo) with canine parainfluenza virus the authors would like to thank bettina buck, petra grünig, kerstin schöne, caroline schütz, danuta waschke, and debby schipper for excellent technical assistance. the technical assistance of david solanes, xavier abad and ivan cordón and all animal caretakers from the cresa-irta biosecurity level laboratories and animal facilities is acknowledged. this research was performed as part of the zoonoses anticipation and preparedness initiative (zapi project; imi grant agreement no. ), with the assistance and financial support of imi and the european commission, and in-kind contributions from efpia partners. w. widagdo was supported by a top project grant ( ) funded by zonmw. cresa-irta thanks the funding through the cerca programme of the generalitat de catalunya (spain). supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - hrjzapj authors: chen, fangzhou; knutson, todd p.; rossow, stephanie; saif, linda j.; marthaler, douglas g. title: decline of transmissible gastroenteritis virus and its complex evolutionary relationship with porcine respiratory coronavirus in the united states date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: hrjzapj the epidemiology and genetic diversity of transmissible gastroenteritis virus (tgev) in the united states (us) was investigated by testing clinical cases for tgev by real time rt-pcr between january and november . prevalence of tgev ranged between . – . % and peaked during cold months until march , in which prevalence decreased to < . %. nineteen complete tgev genomes and a single strain of porcine respiratory coronavirus (prcv) from the us were generated and compared to historical strains to investigate the evolution of these endemic coronaviruses. sixteen of our tgev strains share unique deletions and distinct amino acid changes, which might greatly affect the biological characteristics of the variant tgev, and resulted in a “variant” genotype of tgev. the “variant” genotype shared similar unique deletions and amino acid changes with the recent prcv strain identified in this study, suggesting a recombination event occurred between the ‘‘variant’’ tgev and prcv. moreover, the results indicate the “variant” genotype is the dominant genotype circulating in the us. therefore, this study provides insight into the occurrence, origin, genetic characteristics, and evolution of tgev and prcv circulating in the us. is also associated with protease and adp-ribose "-monophosphatase activities . the functions of other nsps of tgev are unknown. the s glycoprotein attaches to the host cellular receptor porcine aminopeptidase n (papn) or sialic acid, induces cellular fusion, stimulates neutralizing antibodies, and has hemagglutination activity [ ] [ ] [ ] [ ] . the papn-binding domain of the s protein has two major antigenic sites, a and b . in addition, deletions in the orf gene led to attenuation and reduced pathogenesis of tgev and in vivo . in , porcine respiratory coronavirus (prcv), which is believed to have evolved from tgev since prcv and tgev shared a high nucleotide percent identity, was first identified in belgium. compared to the s gene of tgev strains, the s gene of prcv has - nt deletions at n-terminal, including in strains from asia . thus, the s gene is used to differentiate prcv from tgev. recently, a novel strain of prcv (oh ) was identified in the us, but the origin and evolutionary relationship to current tgev strains in the us is unknown. while the decline of tgev is believed to occur in response to partial immunity from prcv infections [ ] [ ] [ ] [ ] , the united states swine industry also made significant changes (increased biosecurity, site production model, etc.) to raise healthier pigs, which may have contributed to the reduction of tgev infections as well. the porcine enteric coronaviruses (including tgev, porcine epidemic diarrhea virus [pedv] , and porcine deltacoronavirus [pdcov]) cause similar clinical presentation, and co-infection of these enteric coronaviruses can occur . in , a highly virulent pedv emerged in china and later spread to the us in april . within less than a year, pdcov was identified in the us , . pedv quickly spread throughout the us , and by march , approximately % of the sow herds were infected with pedv . while the identification of the pedv lead to an increased biosecurity measure within the us swine industry, prevalence of pedv in the sow herds did not significantly decrease until july . within the past couple of years, a chimeric tgev and pedv virus (consisting of a tgev backbone and the spike of pedv) was identified in multiple countries in europe [ ] [ ] [ ] , illustrating the potential emergence of a chimeric tgev and pedv virus in the us. the occurrence and genetic diversity of tgev was investigated prior to and after the identification of pedv and pdcov in the us from the diagnostic cases submitted to the university of minnesota veterinary diagnostic laboratory since pedv recently emerged in the us, and a chimeric tgev and pedv virus was identified in europe. nineteen tgev strains (us, n = and mexico, n = ) and a single prcv strain from the us were sequenced, analyzed, and compared with other global tgev and prcv strains to characterize historical and currents strains. this research will further our understanding of the occurrence, genetic variability, and evolution of an endemic coronavirus in the us and will provide guidance for future efforts to prevent, monitor, and control endemic coronaviruses. decline of tgev positive cases from - . between january and november , , porcine enteric cases, distributed across states in the us and mexico, were tested for tgev by real time rt-pcr, and . % of the cases (n = ) were positive for tgev ( table ). the percentage of tgev positive cases was . % in , increased to . % in , and decreased to . % in (table ) . after the introduction of pedv in the us, the prevalence of tgev decreased further to less than . %. positive tgev cases were detected in the main pig raising regions (midwest, south-central, and southeast) of the us (fig. a) between january and november . most of the cases (n = , ) were from minnesota where . % (n = ) were positive for tgev. the percent of positive tgev cases per state ranged between . - . %, with the highest percentage found in tennessee. a seasonality trend occurred with the positive cases between winter and spring (november to april) compared to summer and fall (may to october) (fig. b) . genomic characteristic, entropy and recombination analyses. the genomic nucleotide sequence alignments of the tgev and pcrv strains revealed two main genotypes (traditional and variant genotypes) ( fig. a) there were major regions of insertion or deletion (indels) between the traditional and variant tgev strains. in the variant group, deletion regions occurred within nsp , deletion regions occurred between the s and orf a genes, deletion regions occurred in orf a genes, deletion region occurred between the orf a and orf b genes and a single deletion occurred in the m genes ( table and fig. b) . interestingly, the same orf a and orf b deletions were present in some of the historical tgev strains. however, these deletions were not present in the tgev strain purdue, which was used to create the attenuated tgev vaccine in the us. the variant tgev strain illinois / had a -nucleotide deletion in orf a, which resulted in the truncated protein compared with our variant tgev strains. the traditional tgev strains z/ , hb/ , and mex/ / shared the same nucleotide deletions in the s gene that were present in the tgev strain, purdue. surprisingly, the pcrv strains contained an assortment of these deletions in their genomes, and compared to the historical tgev strains, the tgev strains from our study (excluding z/ , hb/ and mex/ / ) contained deletions and amino acid changes similar to the recently reported prcv strain oh and the single prcv strain minnesota / from our study. www.nature.com/scientificreports www.nature.com/scientificreports/ to determine the level of nucleotide or amino acid variation across the tgev genomes, entropy analysis was conducted with an alignment of the complete genomes and concatenated amino acid sequences (fig. ) . based on the level of diversity in the dataset and previous work , entropy values greater than . were considered highly variable for the nucleotide and amino acid sequence alignments. the orf and s genes had the highest number of nucleotides with entropy levels above . (n = and n = , respectively) while the orf b, e and, orf genes lacked diversity in nucleotide positions (fig. a ). within orf , the nsp and nsp had the highest number of nucleotide positions with entropy values greater than . (n = and n = , respectively) while a single position was identified in nsp , nsp , nsp , nsp , nsp , and nsp . within the amino acid alignment, the m, n and orf genes lacked positions with entropy levels above . while orf and s proteins (n = and n = , respectively) had residues with entropy values higher than . (fig. ). there were high-entropy positions at orf b www.nature.com/scientificreports www.nature.com/scientificreports/ gene in the nucleotide sequence entropy value analysis while entropy value higher than . were lacking in the amino acid sequence entropy value analysis. recombination analysis was performed with the tgev and prcv strains. a single recombination event was detected by chimera, bootscan, maxchi, and siscan and rdp within the dataset between the recently identified variant tgev strains and the novel prcv strain minnesota / (fig. ) . the tgev variant oklahoma / shares a high nucleotide identity of the first , nucleotides (breakpoint) with prcv strain minnesota / while the remainder of the genome shares a high nucleotide identity with tgev strain minnesota / , indicating a complex nucleotide relationship between prcv and tgev. phylogenetic analyses of tgev and prcv strains. phylogenetic trees of complete genome sequence (n = ) and complete s gene (n = ) revealed two distinct genotypes, representing the traditional and variant tgev strains from the us (fig. a,b, respectively) . in the complete genome and s gene phylogenetic trees, the traditional genotype contained historical strains from the us, the recent strain from mexico, and historical and recent strains from china. from our study, the recent tgev strains from the us formed the variant tgev group, which share a common ancestor with the prcv strain ohio-oh / . also, the variant tgev strains were identified in the three main swine production regions of the us (midwest, south-central, and southeast) indicating substantial geographical distribution. interestingly, whole genome phylogenetic analysis of prcv strain minnesota / clustered within the variant genotype and not with historical prcv strains, indicating significant genetic diversity within prcv strains circulating in the us. since additional partial s gene sequences (first nt) were available from geographically different locations, a partial s gene phylogenetic tree was constructed to further investigate the global evolutionary relationship between tgev and prcv strains (fig. c) . the traditional tgev group consisted of tgev strains isolated from the us, china, japan, south korea, england and mexico. the variant tgev group consisted our us tgev (fig. a) . the different amino acid between traditional and variant tgev strains were highlighted in the predicted crystal structure of rbd ( fig. b-d) . four of eight amino acid substitutions within the rbd (l f, f l, v a, and d e) were exposed on the viral protein while two of the four amino acid changes in papn (d e and t a) were exposed on the viral protein ( fig. b-d) . porcine enteric coronaviruses (pedv, pdcov, and tgev) are significant, emerging pathogens causing severe enteric diseases in the global swine industry. while global historical strains of tgev caused severe enteritis and prcv is endemic in europe, asia, and the us, the mortality rate due to tgev infections has declined in these countries , , . however, a virulent tgev or prcv strain could emerge since the epidemiology of tgev and prcv is different in china. in , prcv was first reported in china and was not identified or reported in china afterwards while tgev is constantly reported as a significant viral pathogen for the chinese pig industry , , . the different clinical status of tgev in china and the western countries could be due to the stringent biosecurity measures in western countries compared to china. tgev was consistently detected in the us between and , but after the introduction of pedv into the us, the swine industry significantly increased their biosecurity to prevent and control pedv infections and the prevalence of pedv were reduced after march in us , , , which indirectly may have reduced the prevalence of tgev to less than . %. our study illustrates a seasonality pattern with tgev in the us with infection peaking during cold months, which has been identified in our swine pathogens as well , , . while tgev has been identified throughout europe , , , , asia , , , , and north america , , only the variant tgev genotype was detected in us pig farms since , suggesting that variant tgev is the dominant genotype currently circulating in the main pig raising midwest, south-central, and southeast regions. in addition, the first tgev genome from mexico was characterized, which helps us to understand the evolutionary relationship of tgev strains from different countries. surprisingly, the mex/ strain is phylogenetically related to the traditional tgev, and not the variant tgev strain. given the close geographical proximity of mexico and us and that the mexican and us pedv strains are phylogenetically related , the variant tgev strains could be circulating in mexico, but were not represented in our study since our study had only a single tgev strain from mexico. the circulation of tgev and pedv in the us indicates a chimeric virus could emerge in the us similar www.nature.com/scientificreports www.nature.com/scientificreports/ as the chimeric virus emerged in europe. characterizing of the current tgev strains will aid in understanding the emergence of virulent or chimeric tgev strains in the us, if such an event would occur in the future. prcv was first identified in belgium in and has been identified multiple countries including belgium , china , japan , uganda and the us . the evolutionary relationship between the recently identified prcv strain (ohio-oh / ) and our prcv strain (minnesota / ) was unclear given the limited number of globally available prcv strains. ideally, additional prcv strains would have been sequenced, but routine screen of prcv does not occur since prcv is not considered as a significant pathogen in swine. the lab accidently isolated the prcv strain, which was added to the study since the genetic and evolutionary relationship of prcv is unknown in the us. the variant tgev strains shared the same nucleotide mutations and amino acid deletions with the novel prcv strain ohio-oh / , and a recombination event between prcv and tgev was identified, illustrating the complex evolutionary relationship between tgev and prcv strains in the us. www.nature.com/scientificreports www.nature.com/scientificreports/ hopefully, future us studies will assess the genetic and evolutionary of prcv strains to fully elicit the complex relationship with tgev. historically, a partial gene fragment or a single gene of tgev was sequenced to differentiate viral strains due to limitations in technology and economic constraints. in this study, ngs technology generated the whole tgev genome, revealing a total of unique indels located in nsp , s, orf a, orf b and m protein, and these regions, excluding the m protein, had high entropy levels in our study (fig. a) . the nsp , s, orf a and orf b proteins of tgev are associated with enteric tropism, immunogenicity, neutralization, sialic acid and other receptor binding activity ability, virulence, protease and adp-ribose ″-monophosphatase activities [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and changes in these proteins might reduce indels efficacy to cause clinical disease. mutations in the spike gene in other coronaviruses has impact on initiation of membrane fusion , sialic acid binding activity , confer resistance to virus neutralization , and render trypsin independent for cell entry and fusion , which could be contribution to a reduction of the overall fitness of the indels. the attenuation of murine coronavirus in the natural host occurred due to deletion of the he, a/he, and / a genes , epidemics of feline infectious peritonitis were contributed to deletions in the orf a and orf b of feline coronavirus , , and disruption of functional expression of accessory protein occurred due to deletion of orf in human severe acute respiratory syndrome coronaviruses . given the results from the previous studies, we hypothesis the indels are significantly attenuated compared to the traditional strains of tgev. further studies are needed to confirm this hypothesis and investigate the effect of these deletions and mutations on the biological characteristics and fitness of the new tgev genotype. in conclusion, a variant genotype of tgev is the predominant in the us and evolutionary relationship between tgev and prcv is complex. the decline of tgev positive diarrhea cases after may in the us may be indirectly associated with the outbreak of ped in the us via increased biosecurity. compared with the www.nature.com/scientificreports www.nature.com/scientificreports/ traditional tgev strains, the unique amino acid indels might affect the biological characteristics of the variant tgev, which could lead to changes in pathogenesis or chimeric virus in the future. veterinarians routinely send samples to the university of minnesota veterinary diagnostic laboratory (mnvdl) to determine potential pathogenic agents contributing to disease and to promote the health of swine herds. the samples may represent clinical outbreaks of diarrhea or are for routine monitoring of enteric pathogens in swine herds. upon arrival, ownership of the samples belongs to the mnvdl and client(s) confidentiality is retained by removing identifiers associated with client(s) information. between january and november , a total of , samples, including fresh intestines and fecal samples from of clinical cases were submitted from us states and mexico and tested for tgev by real time rt-pcr under standard operation procedures (available upon request) and various bacterial and viral enteric pathogens dependent on the veterinarian's request. request may include beta-hemolytic escherichia coli, non-beta-hemolytic escherichia coli, rotavirus a, b, and c, pedv, and pdcov. randomly selected historical positive tgev samples from the mnvdl (n = ) were saved from previous enteric studies, two tgev samples from ohio (z and hb) were supplied from dr. linda saif. routine testing for prcv does not occur since it is not considered a major swine pathogen. however, a prcv isolate from mn was obtained from a nasal swab in during an attempted to isolate other viruses. all samples (n = ) were selected for whole genome sequencing using next generation sequencing as previously described . tgev prevalence information was exported from the mnvdl database and analyzed at the case-level with r software using the ggplot and maps . to investigate the differences in tgev strains and phylogenetic relationship with prcv, our sequences and available sequences from genbank (table s ) were aligned using clustalw in geneious v . . . nucleotide and amino acid entropy analyses of the concatenation orfs (orf a/b, s, orf a/ b, envelope, membrane, nucleocapsid and orf ) was performed using the matlab to determine regions of diversity within the alignment. entropy values higher than . in the nucleotide and amino acid alignments were identified as high variation positions . recombination analysis was performed using recombination detection program (rdp) v with rdp, bootscan, geenecov, siscan and maxchi algorithms (window size is bp). recombination event was represented using similarity plot, with oklahoma as the query strain. the similarity plot was implemented in the simplot, v. . . package , using the two-parameter (kimura) distance model with a sliding window of bp and step size of bp. whole genome (n = ), the whole s gene (n = ), and the partial s gene (first nt) (n = ) (table s ) phylogenetic trees were constructed using the maximum likelihood algorithm, with a gtr nucleotide substitution model (bootstrap analysis with , replicates) by mega v . . the s protein receptor binding domains (rbds) of tgev and prcv were modeled using the open-source modeling server swiss-model provided by the swiss institute of bioinformatics. predicted tertiary structure of the rbds of tgev and prcv were modeled using prcv rbd (pdb accession no. szs) reported in the previous study. spike monomer and trimer models were performed using human coronavirus nl model as a template to theatrically visualize the changes in the residues since tgev and prcv templates are lacking . illustrations were created using the python-based molecular viewer pymol . transmissible gastroenteritis virus (classical enteric variant) and transmissible gastroenteritis virus (respiratory variant) transmissible gastroenteritis and porcine epidemic diarrhoea in britain diseases of swine pseudorabies and transmissible gastroenteritis: a serological survey in south africa a transmissible gastroenteritis in pigs genetic evolution and tropism of transmissible gastroenteritis coronaviruses porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions current aspects in the etiology of viral diarrheas of swine: occurrence of infections with the epizootic viral diarrhea (evd) virus transmissible gastroenteritis in pigs in south east spain: prevalence and 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geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data rdp : detection and analysis of recombination patterns in virus genomes full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination molecular evolutionary genetics analysis version . swiss-model: modelling protein tertiary and quaternary structure using evolutionary information glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy the pymol molecular graphics system, version . schrödinger, llc the study was funded by the mnvdl. the authors thank the faculty and personal at the university of minnesota veterinary diagnostic laboratory (mnvdl) for their technical services. supplementary information accompanies this paper at https://doi.org/ . /s - - -z. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -wrzzb cy authors: brunner, jesse l. title: pooled samples and edna-based detection can facilitate the “clean trade” of aquatic animals date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: wrzzb cy the regional and international trade of live animals facilitates the movement, spillover, and emergence of zoonotic and epizootic pathogens around the world. detecting pathogens in trade is critical for preventing their continued movement and introduction, but screening a sufficient fraction to ensure rare infections are detected is simply infeasible for many taxa and settings because of the vast numbers of animals involved—hundreds of millions of live animals are imported into the u.s.a. alone every year. batch processing pools of individual samples or using environmental dna (edna)—the genetic material shed into an organism’s environment—collected from whole consignments of animals may substantially reduce the time and cost associated with pathogen surveillance. both approaches, however, lack a framework with which to determine sampling requirements and interpret results. here i present formulae for pooled individual samples (e.g,. swabs) and edna samples collected from finite populations and discuss key assumptions and considerations for their use with a focus on detecting batrachochytrium salamandrivorans, an emerging pathogen that threatens global salamander diversity. while empirical validation is key, these formulae illustrate the potential for edna-based detection in particular to reduce sample sizes and help bring clean trade into reach for a greater number of taxa, places, and contexts. www.nature.com/scientificreports www.nature.com/scientificreports/ context of screening u.s. service members for syphilis, pooling samples to reduce the number of tests required to detect rare infections is common in numerous contexts , including surveys for disease freedom. however, the formulae for inference from pooled samples generally assume sampling with replacement or, equivalently, very large populations, which does not align well with the problem of detecting infections in consignments or captive populations. recently a probability formula was developed for pooled samples collected from finite populations without replacement -but it does not accommodate imperfect tests. second, one might screen environmental dna (edna)-the genetic material shed into an organism's environment-for the pathogen or parasite of interest . the edna approach seems especially relevant to aquatic and semi-aquatic taxa, which make up a large portion of the wildlife trade , as edna can be simply filtered from water housing animals; they need not even be handled. (semi-aquatic species or life stages are often shipped in moist sphagnum moss or paper, but can then be placed in water to collect edna during a quarantine period .) environmental dna sampling is being used to detect pathogens in a growing number of settings, from natural environments [ ] [ ] [ ] [ ] to ballast water and even in trade [ ] [ ] [ ] . however, while the statistical framework for making proper inference from edna results is well-developed in certain contexts (e.g., the presence or absence of a target species in ponds using occupancy models , ), these approaches do not translate well to sampling small captive populations or consignments. while both approaches have the potential to reduce sample sizes, we lack the formal framework with which to determine sampling requirements and interpret results. in this paper i therefore develop formulae for imperfect tests of pooled samples in closed populations and edna, discuss the key assumptions and considerations in their application, and illustrate how edna may be especially useful for detecting infections in the live animal trade. i present these results in the context of emerging fungal pathogens that threaten amphibian diversity, for which trade appears to play a key role. the amphibian chytrid fungus, batrachochytrium dendrobatidis (bd), is the most devastating emerging pathogen on record, responsible for declines in over species, including that are likely extinct . it likely owes its global distribution in large measure to the international trade of african clawed frogs (xenopus laevis) used for pregnancy tests and research , american bullfrogs (lithobates catesbeianus) sold for food , , and myriad species involved in the pet trade , [ ] [ ] [ ] [ ] . more recently, the international pet trade aided the emergence of a novel chytrid fungus, b. salamandrivorans (bsal), that is particularly lethal to salamanders [ ] [ ] [ ] [ ] . bsal was introduced into northern europe via the pet trade from southeast asia , [ ] [ ] [ ] . while it has not yet been detected in the wild in much of europe - , it is already prevalent in and appears to have spread among private collections in europe [ ] [ ] [ ] . in north america, a hot-spot of salamander diversity, bsal is apparently absent from both wild and captive amphibians , - . however, the risk and potentially devastating consequences of its introduction via trade [ ] [ ] [ ] led to prohibitions on the importation of species of salamanders into the u.s.a. and all salamanders in canada . while laudable, such bans are inevitably crude. these restrictions were necessarily based on experiments with small samples sizes of few species, extended to others by taxonomic affiliation . moreover, it was quickly out of date since several other taxa, including frogs, are now known to carry bsal , . such bans may also promote black-market trade, which already occurs on a global scale similar to the black-market trade of narcotics . in response to these challenges, as well as the economic interests of the pet trade, there have been calls for a "clean trade" of amphibians [ ] [ ] [ ] . indeed, the european union recently mandated a program requiring screening, using a quantitative real-time pcr (qpcr) assay of skin swabs , or prophylactically treating all consignments of live salamanders into and between member nations of the eu . again, the scale of the international trade of live amphibians makes routine screening a formidable challenge. while the number of consignments of amphibians into or between eu member nations is not available in the literature, an estimated - million live amphibians are imported per year into the usa, alone , and , annually into the u.k. . since bsal is thought to occur at low prevalence in traded animals , the eu regulations require screening all or a substantial fraction of individuals in a consignment to ensure a reasonable chance of detection (fig. ) . while shipments of certain taxa or from certain sources might be excluded, even conservative assumptions about the volume of trade imply screening an enormous number of samples if this program were extended worldwide (e.g., consignments of salamanders each would require testing some , swabs). this scale of sampling is likely to place clean trade beyond the reach of many nations or interested parties (e.g., constituents in the pet trade industry). similar problems arise when considering other amphibian pathogens (e.g., ranavirus ) or those of fishes. the question in this paper is therefore: can pooling swab samples or using edna place routine surveillance in reach? the formulae presented below can account for false positives (i.e., less than perfect specificity, sp). it will be important to consider the sources and likelihood of false positives (e.g., from carry over of target dna from the water rather than infected hosts , vs. from contamination in a laboratory) as well as the consequences (e.g., slowing shipments, economic costs, loss of trust , ). in the case of bsal detection, i would hope that any positive test would be investigated further with additional, independent diagnostic tests. moreover, setting aside the issue of carry over contamination , which might be avoided by transferring animals to clean water prior to sampling, there is no evidence that false positive rates are higher in pools of samples or edna than typical individual samples. in fact, to the extent that fewer tests are conducted with pooling or edna one would expect fewer false positives at a given level of surveillance sensitivity. for these reasons i set aside the issue of specificity in the following discussion and focus on sensitivity. www.nature.com/scientificreports www.nature.com/scientificreports/ pooling individual samples. i extended the "pooled hypergeometric" distribution of theobald and davie to account for imperfect diagnostic tests (eq. ( )). the results are largely intuitive: when n individual-level samples are divided into m = n/k pools of size k, the number of pools that need be screened is reduced up to k-fold, assuming low prevalence of infection and high diagnostic sensitivity , . the gains in efficiency from pooling are reduced somewhat for less sensitive tests when infections are more common, but not markedly so. a key assumption of this formula is that diagnostic sensitivity is not affected by pooling. this implies on the one hand that the combined analyte (e.g., target dna) from multiple infected individuals does not increase diagnostic sensitivity. the probability of detecting a single infection in a pool of samples (i.e., swabs) is at most the diagnostic sensitivity of individual swabs. on the other hand, the formula assumes that the analyte is not overwhelmed or inhibited by non-target materials (e.g., pcr inhibitors in skin secretions, microbial dna) nor diluted below a detection threshold (e.g., as was observed with pools of fish screened for a megalocytivirus ). this later assumption is unlikely to hold with very large pools of samples (see laurin et al. for a review), but may be reasonable for small pools. it is an important assumption to test under realistic conditions, but one could simply use lower values of diagnostic sensitivity (se in eq. ( )) to account for target swamping or dilution. also, it is worth remembering that the same number of samples must be collected with or without pooling; efficiencies are only gained in the processing and screening steps. environmental dna. i developed a new formula for edna-based detection in small, closed populations (eq. ( )). the formula makes two key assumptions. first, it assumes that the edna shed into the water is homogeneously distributed in the water. while pathogen edna is likely clumped (e.g., bsal may be found principally in skin sheds), one could homogenize the water (e.g., with a blender) prior to taking samples to meet this assumption, at least in the smaller volumes used to house animals in shipments or captive populations. thus, replicate edna samples are essentially technical replicates in this framework. second, as with the formula for pooled samples, it assumes that test sensitivity is unaffected by the number of hosts in the water at the same time. that is, the target pathogen edna is not swamped by non-target host and microbial edna or other waste material leading to inhibition of the pcr reaction. this assumption needs to be empirically verified, but it may be possible to minimize pcr inhibition with dedicated kits or by diluting the eluted dna prior to pcr, although such methods risk reducing sensitivity. in any case, the formula i present could be extended to account for this swamping effect if needed. these assumptions have two important consequences for edna-based pathogen detection. first, every sample collects edna from the entire population. environmental dna avoids the issue of whether rare infections are included in any random sample of individuals-they are all sampled because the edna from all individuals is distributed homogeneously in the water. the question is only whether there is sufficient target edna in a sample to ensure a reasonable probability of a positive test. as a consequence, edna sampling can detect rare infections with many fewer samples than individual swabs, or pools of swabs, even when diagnostic sensitivity is very low (figs. and ). (i consider various factors that are likely to affect sensitivity in the next section.) this also means that the probability of detecting an infection is, all else being equal, independent of population size (fig. ) . thus, while individual-level swabbing can be more efficient than edna in small populations, in larger populations edna outperformed swabbing, even when pooled, over a wide range of parameter values (fig. ) . similarly, samples sizes for edna-based detection decrease with population size when prevalence is held constant because there are more infected individuals shedding pathogen edna, whereas they must increase with population size when using individual or pooled sampling to ensure infected individuals are included in any sample (fig. ). once again, it is essential to establish the actual performance of edna-based detection in real world contexts and test how it scales with population size and other variables. www.nature.com/scientificreports www.nature.com/scientificreports/ quantitative nature of detection. diagnostic sensitivity is usually defined in the context of a classification problem with binary infection status determined by a gold standard of infection (e.g., histopathology) or assigned in experiments. as such, sensitivity is generally assumed to be fixed; that is, all infections within a population or experiment are equally detectable. however, any test that reacts directly with the pathogen (e.g., pathogen dna or antigens) or host responses (e.g., antibodies) is necessarily dose-dependent ( fig. ; eq. ( )). the variation in the amount of the analyte between infected individuals or among laboratories, protocols, etc., may be small enough to ignore (but see e.g., rimmer et al. ), or the concentrations generally large enough that detection is assured (e.g., with clearly diseased animals), in which case it may be reasonable to assume diagnostic sensitivity is constant. at the other extreme, recently infected individuals, sub-clinical carriers , or otherwise inapparent infections tend to produce or shed little analyte and are thus much less easily detected than clinically infected animals. quantitative real time pcr of swabs, for instance, often fails to detect low-level bd infections )) of the per target copy detection rate, φ and the number of copies of the target dna or analyte, c. the arrows and dotted vertical lines show the consequences of increasing the volume of water in which animals are held ten-fold, thus diluting the analyte (blue) or increasing the number of infected animals from one to two, three, or four. note, however, that the concrete effect of such changes on the probability of a positive test depends on where one starts along the φc axis. www.nature.com/scientificreports www.nature.com/scientificreports/ have large effects on the pool-wide sensitivity . however, the the concentration-dependent nature of detection will be especially pronounced with edna sampling. the concentration of target dna in an edna sample co-varies with the type and intensity of infection and details of the sampling method (here volume sampled, pore size, storage conditions ), as with individual samples , but also depends on the conditions in which animals are held (e.g., temperature, volume, time in the water; fig. ). thus, one would expect a great deal more variability in sensitivity of edna between settings and studies, not to mention between taxa, than with traditional sampling directly from animals. empirical validation of edna-based pathogen detection should thus focus on understanding the causes and consequences of this variability across consignment types, holding conditions, and sampling approaches, and work to develop standards. equations ( )- ( ) are an initial attempt to explore these influences (fig. ) . they emphasize that equivalent changes in the number of infected animals, their shedding rates, the proportion of the environment sampled, or rates of degradation all have equivalent effects on the amount of analyte in a sample, and thus detection probability. it is worth noting, however, that researchers may have control over certain key variables, such as the duration and volume in which animals are held before collecting samples, allowing them to maximize sensitivity. for instance, if rates of edna shedding and degradation are fairly constant and fast relative to changes in the underlying infection intensity, one would expect edna concentrations in water to approach an equilibrium over time, at least as a first approximation (eq. ( )). the rate at which it asymptotes depends on the rate of degradation, and so water quality, temperature, ph, etc. are likely to be important , . however, unless rates of degradation are exceedingly rapid, one would expect concentrations of edna to accumulate over several days and so investigators might increase sensitivity by holding animals longer prior to sampling. while efficiency is not the only important criteria for a testing scheme (e.g., the expertise, specialized equipment, and costs involved must also be considered), routine pathogen surveillance and thus a "clean trade" becomes more feasible as the number of samples that must be analyzed are reduced. pooling swabs and sampling edna are both, in principle, viable approaches to reducing sample sizes, although to much different extents and with different drawbacks in their use. pooling and batch-processing samples collected from individual animals (e.g., swabs) can reduce several fold the number of diagnostic tests (e.g., dna extractions and pcr reactions) required to detect rare infections. while some n samples must still be collected, pooling them into groups of size k means that only n/k pools need be processed, which may substantially reduce the time and costs of surveillance. the critical question for pooling is the degree to which sensitivity, the probability of correctly identifying infections if present, declines as samples are pooled. laurin et al. found that sensitivity generally declined with pooling in aquatic pathogens, in part because moderate concentrations of analyte (e.g., pathogen dna) may be diluted below reliable detection limits in pools , but there are few studies that properly evaluate this. however, given the quantitative nature of detection (fig. ) and the large amount of variation in infection intensities and analyte in a sample, it may not be possible to provide simple, general estimates of sensitivity when pooling . in the case of bsal, sabino-pinto et al. found that diagnostic sensitivity of the typical qpcr assay was unaffected when dna was extracted from pools of up to four skin swabs. while the authors specifically recommend against pooling in trade and quarantine settings given the risks associated with the failure to detect a single infected individual , their results suggest pooling could reduce several-fold the number of dna extractions and qpcr reactions required to achieve the eu's requisite detection probability in a shipment ( figs. and ) . again, empirical tests in real-world settings, especially with low-level bsal infections, are needed to evaluate the actual sensitivity when pooling samples. hyatt et al. , for instance, found that diagnostic sensitivity of their qpcr assay for bd remained high in pools of five swabs, so long as "equivocal" results (one or two of three wells positive) were scored as positive-likely because of a dilution of target dna-but even here, one low-level infection went undetected in a pool. still, losses in sensitivity may be offset by increases in the fraction of a population or consignment that can be screened when samples are pooled, essentially increasing the chances that a rare infected individual is included in a sample. for instance, even if sensitivity is reduced from a probability of one with individual swabs to . with a pool of four swabs, many fewer assays need be run on the pools than the individual swabs to attain the same confidence of detecting an infection (fig. ) . equation ( ) and the included r code can be used to evaluate this trade-off. environmental dna offers a different trade-off. the samples collect genetic material shed into the water (or environment more broadly), so whatever material is shed, which may be minimal, is diluted in a larger volume and might be degraded. all of which means that fairly little target dna may end up in any given edna sample. thus, one would expect the diagnostic sensitivity of edna samples to be quite low relative to individual-based samples , (fig. ) . however, edna samples from the entire population, at least in the context of small captive populations or consignments of animals in water, perhaps with the aid of mechanical homogenization. this allows edna to circumvent the problem of including rare infected individuals in sample that is intrinsic to individual-level sampling (and pooling). as a result, even with low sensitivity, edna sampling can be much more efficient at detecting infections in large consignments than perfectly sensitive individual-based samples or pools of samples. moreover, sample sizes required to detect a rare infection do not increase with population or consignment size (fig. ) . sampling edna thus offers the promise of dramatically reducing the amount of sampling required to ensure disease freedom in the trade of live aquatic and semi-aquatic animals. the actual performance of edna-based detection likely depends on the nature of the consignment (e.g., volume, time animals have spent in water), collection and processing (e.g., volume filtered, presence and removal of pcr inhibitors), and biological realities (e.g., rates of shedding and degradation). it is important to evaluate these factors empirically so that the results can be properly interpreted. the simple model presented in eqs. ( )-( ) provides a useful starting point, illustrating how edna concentrations, and thus per sample detection www.nature.com/scientificreports www.nature.com/scientificreports/ probabilities, vary with volume, time, and rates of shedding and degradation (fig. ) . the proportion of holding water sampled and time animals spend in the water, shedding pathogen edna, are likely key parameters, but may vary substantially among taxa, life stages, and settings. a clever investigator, however, could use these factors to increase the sensitivity of edna-based samples, for instance by holding animals in small volumes of clean water for longer periods of time to ensure that target edna accumulates to readily detectable levels before collecting edna samples. perhaps the most fundamental question is whether pathogen edna is swamped by non target host or microbial dna or pcr inhibition increases with population size. while not unique to edna-pcr inhibition is common with swab-based detection of chytrid fungi-the issue maybe be magnified for environmental samples. if swamping or inhibition are a problem then the number of edna samples required to attain a particular detection probability would need to increase with population size and eq. ( ) adjusted accordingly. i must stress that it is only with a clear understanding of how these manifold conditions influence detection probabilities can edna-based detection be used reliably in the variety of settings and types of consignments that comprise the live animal trade. the goal of developing a "clean trade" may be the most realistic strategy for preventing the movement and introduction of pathogens such as bsal into new areas and naive hosts. but it is a daunting task given the immense number of animals involved in international and regional trade. the formulae developed here place sample pooling and edna in closed populations on a firm theoretical foundation. they suggest that pooling individual-level samples and, especially, collecting edna can substantially reduce sample sizes required to ensure rare, but important infections are found. moreover, sampling at key nodes in the distribution networks or crucial segments of the live animal trade (e.g., particular species, sources) can make surveillance even more tractable . it is my hope that these approaches can help bring clean trade into reach for a greater number of taxa, places, and contexts. however, diagnostic tests are rarely perfect. infected individuals are correctly detected with probability se (diagnostic sensitivity) and uninfected animals correctly test negative with probability sp (diagnostic specificity); false negatives and false positives occur with probabilities − se and − sp, respectively. cameron and baldock extended the hypergeometric model to include imperfect tests, such that the probability of observing x positive tests (t + ) is: ( ) often our goal is to determine the probability of at least one positive sample, given particular parameter values. consider a consignment of n = salamanders, some % (d = ) of which are infected, screened with individual swabs with a diagnostic sensitivity of se = . , matching the assumption in the eu regulations , and perfect specificity (sp = ). we see that the probability of detecting at least one of these infections is, using the equivalence p(t + ≥ x) = − p(t + = ): note that the part involving specificity simplifies to when se = . because x = , j is always zero and so this simplifies to: www.nature.com/scientificreports www.nature.com/scientificreports/ one can then try values of n such that p(t + ≥ ) ≥ . or any other threshold surveillance sensitivity. in this scenario the threshold is met when n ≥ . while this calculation can be done by hand, it becomes quite tedious. using the r code found below the same calculation can be achieved with pooledhyp_atleast (d= , n= , m= , k= , se= . , sp= ). note that that this function is written for pooled samples (see next subsection), but we can use it for individual samples with m = n "pools" (=samples) of size k = . pooling individual-level samples. when the n samples are divided into m groups of size k = n/m, which are then tested as pools, the probability that x of the m pools contain an infected individual in them (pool + ) is described by the "pooled hypergeometric" of theobald and davies : it is important to note that this formulation assumes diagnostic sensitivity and specificity are not influenced by the composition of a pool. a pool is equally likely to test positive if one or all of the individuals within it are positive, or if the pool is comprised of few or many samples. this assumption may be questionable for large pools, but may be reasonable with small k. returning to the example of a consignment of n = with se = . and sp = , but now pooling swabs into groups of size k = and assuming that the m pools is greater than the d = infections, we can find the probability of at least one pool testing positive as: , we obtain the following expression for the probably of observing x positive samples: note that because each edna sample collects material from all d infected individuals in the population, success is defined in the binomial as the probability of a positive edna sample [ − ( − se) d ], ignoring false positives, rather than the probability a sample includes an infected individual, as is the case when using the binomial to describe detection probabilities with individual samples taken with replacement. multiple infected individuals in a population simply increase the amount of target edna to be detected and thus the effective sensitivity of the edna test. both sensitivity and specificity are assumed to be constant and independent of the n − d uninfected animals in the population. finding the number of replicate edna samples to ensure ≥ ≥ . notice that both the binomial terms and the first term in brackets simplify to one since x = ; in other words, we only need to subtract the probability that all n samples test negative from one. ≥ ≥ . + p t ( ) edna when n ≥ . while this is simple to calculate by hand, the code, edna_atleast (d= , m= , se= . , sp= ) produces the same result. the quantitative nature of detection. any test that reacts directly with a pathogen (e.g., pathogen dna or antigens) or host responses (e.g., antibodies) is necessarily dose-dependent. focusing on dna-dependent detection methods (e.g., pcr), let us assume that every copy, c, of the target dna sequence in a sample has a small, fixed probability of causing a positive result in a reaction, φ. the probability of a positive test result can then be described by a "single-hit" model , : c which results in a typical dose-response relationship (fig. ) . (a logistic relationship between p(t + ) and log(c), which is commonly used in studies of analytic sensitivity, yields similar results, but requires an extra parameter.) if the c copies come from a single infected individual, then this function describes diagnostic sensitivity. the magnitude of c in a sample can therefore have a strong influence on sensitivity. as noted in the main text, one would expect greater variation in c with edna sample than with individual-level samples because edna is not collected directly from an animal, but from the environment, which can also play a role (although external swabs are also influenced by the environment). a simple model is useful in clarifying the factors that likely determine the number of target copies in an edna sample. in this model, the number of copies of target edna in a sample increase as the d infected individuals shed into the water at rate ψ, some portion, α, of which ends up in each edna sample, and decrease as edna degrades at rate δ: the solution is: where c is the initial concentration in the water (the first term on the right-hand side is zero when c = ). the solution asymptotes to c * = ψdα/δ over time at a rate dependent upon δ. substituting the equilibrium solution into the single-hit model (eq. ( )), one can see that detection probability depends on these rates in a straightforward way: while this model has several terms, they have clear biological meaning and could be estimated from experimental data [ ] [ ] [ ] . moreover, they enter the model as a product, with equivalent effects, at least at equilibrium. that is, doubling the volume of water collected in an edna sample is the same as doubling the number of infected individuals or halving the decay rate. this model also provides a way to link the many factors that can affect edna-based detection via their influence on target copy number, c (e.g., degradation, δ, is likely temperature-dependent ), or pcr efficiency (e.g., φ might be a function of ph and environmental inhibitors ). can be implemented in r using the "big integer" versions of multiplication and the binomial expansion found in the gmp package for greater precision: www.nature.com/scientificreports www.nature.com/scientificreports/ if(m*k>n) stop("individuals sampled (m*k) is greater than population size (n)") stopifnot(se<= , se>= , sp<= , sp>= , length(x)== ) innersums <− for(y in :m){ j<− :min(y,x) innersums[y+ ] <-pooledhyper(x=y, d=d, n=n, m=m, k=k) * sum(dbinom(j, size=y, prob=se) * dbinom(x-j, size=m-y, prob=( -sp))) } sum(innersums) # return sum of inner summations } note that when k= the "pools" are individual samples, as in eq. 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degradation in aquatic microcosms multiple precision arithmetic r package version . - i would like to thank kaare graesbøll and ming-wen an for interesting conversations as i was conceiving these ideas and jeb owen, christian yarber and other members of the brunner lab for helpful comments and discussion on earlier drafts, and suggestions from three anonymous reviewers. this work was supported by cgf j.l.b. conceived, developed, and and wrote the manuscript. the author declares no competing interests. correspondence and requests for materials should be addressed to j.l.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - lvue authors: pan, feng; zheng, chuansheng; ye, tianhe; li, lingli; liu, dehan; li, lin; hesketh, richard l.; yang, lian title: different computed tomography patterns of coronavirus disease (covid- ) between survivors and non-survivors date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: lvue this study aimed to compare the chest computed tomography (ct) findings between survivors and non-survivors with coronavirus disease (covid- ). between january and february , the records of consecutive patients diagnosed with covid- were retrospectively reviewed and divided into survivor ( / ) and non-survivor ( / ) groups. chest ct findings were qualitatively compared on admission and serial chest ct scans were semi-quantitively evaluated between two groups using curve estimations. on admission, significantly more bilateral ( . % vs. . %, p = . ) and diffuse lesions ( . % vs. . %, p < . ) with higher total ct score (median vs. , p < . ) were observed in non-survivor group compared with survivor group. besides, crazy-paving pattern was more predominant in non-survivor group than survivor group ( . % vs. . %, p < . ). from the prediction of curve estimation, in survivor group total ct score increased in the first days reaching a peak of points and then gradually decreased for more than other days (r( ) = . , p < . ). in non-survivor group, total ct score rapidly increased over points in the first days and gradually increased afterwards until ards occurred with following death events (r( ) = . , p < . ). in conclusion, persistent progression with predominant crazy-paving pattern was the major manifestation of covid- in non-survivors. understanding this ct feature could help the clinical physician to predict the prognosis of the patients. www.nature.com/scientificreports/ chain reaction (rt-pcr) test has a relatively high false-negative rate ( %) for covid- diagnosis, so chest computed tomography (ct) is recommended as the major screen modality with a higher sensitivity of % and faster performance [ ] [ ] [ ] [ ] . in hubei province, the centre of the outbreak in china, the clinical diagnostic criteria were only dependent on chest ct scan, instead of the rt-pcr test before february . however, the value of the consecutive ct scans for monitoring disease progression was still unclear. previous studies suggested a typical time course of ct findings in survivors with covid- , in which initial progression was followed by recovery, the latter starting after about weeks [ ] [ ] [ ] . case series have associated severe and critical covid- with more diffuse lung involvement, development of acute respiratory distress syndrome (ards), and multi-organ failure , [ ] [ ] [ ] . using a case-control design, this study aims to identify the differentiating ct features and compare the temporal evolution of pulmonary involvement between recovered and died patients with covid- . patients and groups. consecutive records of hospitalized patients with rt-pcr confirmed covid- were reviewed retrospectively for the period from january to february in this single-centre (union hospital, wuhan, china). the inclusion criteria included: ( ) with definite clinical outcomes (discharge or death events); ( ) no comorbidities which might impair the immune or pulmonary function, such as recent chemotherapy and chronic obstructive pulmonary disease; ( ) with more than three times of chest ct scans in the course for sufficient estimation of radiological patterns, unless fatal ards occurred resulting in impossibility to carry out the consecutive chest ct scans. eventually, patients were included and divided into two groups: survivor group (discharged patients, n = , including patients who were preliminarily reported in the previous study ) and non-survivor group (died patients, n = ) (fig. ) . clinical data (e.g. initial symptoms, past medical history, etc.) and serial chest-ct data in the follow-up (extended until march in survivor www.nature.com/scientificreports/ group) were retrieved through the institutional electronic patient database. diagnostic, isolation, grades of the disease severities (non-ards and ards), treatment, and discharge criteria were based on the published standard protocols from the continuously-updated national health commission of the people's republic of china . chest ct scan protocols. chest ct scans were performed using two commercial multi-detector ct scanners (philips ingenuity core , philips medical systems, best, netherlands; somatom definition as, siemens healthineers, erlangen, germany) during a single breath-hold. the low-dose mode was set up with a tube voltage of kvp and automatic tube current modulation. from the raw data, ct images were reconstructed as . mm thick axial slices and increment of . mm in transverse slice orientation with either hybrid iterative reconstruction (idose level , philips healthcare, best, the netherlands) or a pulmonary b f kernel (siemens healthineers, erlangen, germany). chest ct estimation. the abnormal radiological findings of ct reported using internationally standard nomenclature [ ] [ ] [ ] . ct abnormalities included ground-glass opacity (ggo), crazy-paving pattern, and consolidation. the distribution of abnormalities was also noted as being predominantly subpleural (involving mainly the subpleural one-third of the lung), random (without predilection for subpleural or central regions), or diffuse (continuous involvement without respect to lung segments) . a conventional semi-quantitative scoring system was used to evaluate the pulmonary involvement area of all these abnormalities , . there was a score of - for each lobe on the following: -no involvement; , < % involvement; , - % involvement; , - % involvement; , - % involvement; , > % involvement. the total ct score was the sum of the score of each lobe and ranged from (no involvement) to (maximum involvement). the analysis was performed using the institutional digital database system (vue pacs, version . . . , carestream health, oakville, canada) by two radiologists (cz and ly, who had and years of experience in thoracic radiology, respectively) and the decisions were reached in consensus. all radiologists were blinded to the groups and clinical progress of the patients to avoid information bias. statistical analysis. statistical analysis was performed using ibm spss statistics software (version ; ibm, new york, usa). quantitative data were presented as median with inter-quartile range (iqr) and qualitative data were presented as the percentage of the total unless otherwise specified. the comparisons of the quantitative data were statistically evaluated using the mann-whitney u test, according to the non-normal distribution assessed by the shapiro-wilk test. the comparisons of qualitative data were evaluated using the chi-square test or fisher's exact test. the dynamic total ct score with time from symptom onset was quantitatively assessed by using the spss curve estimation module . a p value of < . was defined as having statistical significance. basic characteristics. the median age of the patients was years (iqr - years) with an approximately equal male to female ratio ( : ). the median age of patients was significantly higher in non-survivor compared to non-survivors ( years vs. years, p < . ). the percentage of males was . % and . % in survivor and non-survivor groups, respectively (p < . ) ( table ) . non-survivors were also more likely to have a history of hypertension, diabetes, and coronary heart disease than survivors (p < . ) ( table ) . fever and cough were the most common initial symptoms ( . % and . %, respectively). chest distress was significantly more inclined to occur in non-survivors (p < . ) ( table ). there was no significant difference in the period of admission from symptom onset between survivor and non-survivor groups ( days vs. days, p = . ) ( table ). the median survival period of non-survivor group after admission was days (iqr - days) from admission, while the median hospitalized period in survivor group was days (iqr - days) (p = . ). the survivors underwent more times of chest ct scans than non-survivors ( vs. , p < . ) with a significantly longer duration ( days vs. days, p = . ) ( table ). all non-survivors aggravated to ards after a median of days ( - days) from symptom onset, while only one patient aggravated to ards in survivor group. multiple biochemical and haematological parameters differed significantly between the two groups such as lymphocyte count, neutrophil count, and c-reactive protein (crp) (p < . ) ( table ). comparison of major ct findings between two groups. all patients underwent a total of chest ct scans with a median interval between adjacent scans of days (iqr - days) ( on admission, bilateral lung involvement was more common in non-survivors than survivors ( . % vs. . %, p = . ) ( table ) . subpleural distribution was more inclined to be observed in survivors compared with non-survivor group ( . % vs. . %, p = . ), while diffuse distribution was more common in nonsurvivor group compared with survivor group ( . % vs. . %) (p < . ) ( table ) . ggo ( . %), consolidation ( . %), and crazy-paving pattern ( . %) were the major ct findings in both groups, while the crazy-paving pattern was more common in non-survivor group than survivor group ( . % vs. . %, p < . ) ( table ) . on admission, consolidation predominated in survivor group ( . %), but crazy-paving pattern predominated in non-survivor group compared with survivor group ( . % vs. . %, p = . ) ( table ) . besides, the total ct score was significantly higher in non-survivor group than survivor group (a median of vs , p < . ). dynamic estimation of pulmonary involvement between two groups. based interval between adjacent chest ct scans (days) (iqr) ( - ) ( - ) ( - ) . www.nature.com/scientificreports/ respectively; p < . , each) (fig. a,b ; si table s online). the optimal fitting equations were demonstrated in fig. c . from the optimal fitting, in survivor group the total ct score gradually increased in the first days with a peak value of and then gradually decreased afterwards lasting for more than another days (fig. c) . the typical ct manifestation was changed from subpleural ggo to enlarged consolidation with time which was gradually absorbed afterwards leaving residual ggo and parenchymal bands (fig. ) . but in non-survivor group, the total ct score rapidly increased in the first days and eventually approached until ards occurred (fig. c) . from the dynamic ct images, the persistently progressive pulmonary lesions from ggo with crazy-paving pattern to bilaterally extensive consolidation could be observed (fig. ). this study compared the temporal changes in ct manifestations between survivors and non-survivors with covid- . it demonstrated the pulmonary involvement of subpleural ggo and sequential consolidation gradually progressed reaching the peak after days since symptom onset in survivors. afterwards, the lesions started to be absorbed lasting for more than days. in contrast, non-survivors demonstrated more rapid and persistent www.nature.com/scientificreports/ progression with more extensive bilateral lesions until ards occurred. crazy-paving pattern was more predominant in non-survivors on admission compared with survivors. in accordance with the previous studies, old patients ( years, iqr - years) with more comorbidities such as hypertension, diabetes, and coronary heart disease were more inclined to develop fatal ards , . initial symptoms were similar between survivor and non-survivor groups, whilst chest distress was more common in non-survivor group. patients in non-survivor group underwent a progressive phase which culminated in the development of ards after a median period of three weeks from symptom onset. as a case-controlled study, the mortality rate of ards caused by covid- could not be evaluated, but from a previous study, it was reported mortality of . % . initial laboratory investigations on admission showed multiple haematological and biochemical abnormalities which were significantly different between survivor and non-survivor groups. this can be attributed to the systematic inflammation reaction and pulmonary vascular endothelial damage caused by a severe viral infection, similar to the systemic response seen in other types of severe pneumonia , - . it has been postulated that covid- could also damage t lymphocytes, thus, significant lymphopenia was probably a risk factor leading to the deterioration of patients' immune function and more rapid disease progression , , , . in addition, the increased levels of crp, lactate dehydrogenase, and d-dimer could also be indicators for development of ards, as reported in other types of pneumonia , , , . in the early stage of covid- , subpleural ggo was the predominant finding [ ] [ ] [ ] , . but in this study, patients were hospitalized after a median period of and days after the onset of symptoms in survivor and non-survivor groups, respectively, at which time the predominant findings in both groups corresponded with the progressive stage . thus, ggo was not the predominant finding in both groups but the consolidation and www.nature.com/scientificreports/ crazy-paving pattern. compared with the survivors, it demonstrated the predominant ct demonstration of crazy-paving pattern in non-survivor group on admission was a major difference except for more diffuse and bilateral distributions. pathologically, ggo may be an indicator of alveolar oedema and proteinaceous exudates . as the disease progresses, increasing alveolar oedema, exudates, and lymphocyte infiltrates fill the interstitial space leading to the radiological demonstration of diffuse "crazy-paving pattern" , , , . subsequent ards and potentially fatal respiratory failure developed as a result of diffuse alveolar oedema with loss of alveolar epithelium , . thus, it was speculated large area of crazy-paving pattern was probably a ct indicator of poor prognosis. considering the heterogeneities of the scan time among the patients, longitudinal comparisons were not appropriate. thus, the curve estimation was used to statistically compare the temporal evolution of the disease between two groups. being different from the static comparison of chest ct on admission using the logistic module, curve estimation could analyze the dynamic patterns of the pulmonary involvement with time , . thus, it could provide a more composite comprehension of the time course in covid- between survivors and non-survivors. as a result, it demonstrated a gradual resolution of abnormalities after a maximal total ct score of at days, longer than days reported in the previous report . it might be ascribed to a limited sample size in the previous study, which probably underestimated the recovery duration of covid- . compared with survivor groups, the total ct score in non-survivor group demonstrated a more rapid increase in the first days with a higher value of more than points. although the previous study showed the feasibility of making ct score as an indicator of prognosis, it did not demonstrate the dynamic changes of ct score in the whole course . this study revealed the total score persistently elevated to a higher level close to points www.nature.com/scientificreports/ without any decrease in non-survivor group, until the ards occurred with the following death events. from one pathological study in severe acute respiratory syndrome (sars), it found the long duration of illness resulted from the severe fibrosis and organization . considering the partial homology of sars and covid- , it might explain why the lesions were rarely absorbed in non-survivors with covid- . this is another major difference between the two groups in the course, associated with the refractory feature of the critical covid- under the present treatment protocols . this study has limitations. firstly, as a retrospective study, chest ct was used by the physician based on the clinical necessity and the status of the patient, so the heterogeneities of scanning time made it impossible to perform a conventional longitudinal comparison between two groups with regular intervals. second, ct was not clinically feasible for patients after developing ards so not enough ct information was provided in the course of ards. consequently, the majority of ct scans were performed in mild disease ( / , . %). to avoid data heterogeneity, the comparison of chest ct between two groups was only performed on admission due to a similar period from symptom onset and the curve estimation was used to evaluate the comprehensive trend of pulmonary involvement between two groups. third, the multi-variate regression or propensity matching involving the ct, clinical, and laboratory parameters was not performed owing to the limited sample size and a relatively large number of parameters with significant differences between the two groups. in summary, from comparisons between survivors and non-survivors, this study indicated that the presence of predominant crazy-paving pattern on chest ct with the high and rapidly increased ct scores may help to identify the patients at high risk of developing ards before clinical deterioration. a larger, prospective study is required to confirm these findings with the more accurate quantitative assessment modality of the ct images in covid- . ethical approval. this retrospective study was approved by the ethics of committees of union hospital, tongji medical college, huazhong university of science and technology (no. - ), and followed the helsinki declaration and its later amendments or comparable ethical standards. patient and other consents. informed consent/deceased patient permission form for this retrospective study was waived by ethics of committees of union hospital, tongji medical college, huazhong university of science and technology because only the anonymous data was collected and analyzed to facilitate better clinical decisions and treatment. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with novel coronavirus in wuhan situation report (geneva: world health organization clinical characteristics of coronavirus disease 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pathology of severe acute respiratory syndrome in toronto t-cell immunity of sars-cov: implications for vaccine development against mers-cov analysis of clinical features of patients with novel coronavirus pneumonia novel coronavirus pneumonia outbreak in : computed tomographic findings in two cases pulmonary pathology of early-phase novel coronavirus (covid- ) pneumonia in two patients with lung cancer crazy-paving appearance at thin-section ct: spectrum of disease and pathologic findings pathological findings of covid- associated with acute respiratory distress syndrome initial ct findings and temporal changes in patients with the novel coronavirus pneumonia ( -ncov): a study of patients in wuhan, china we express our sincere gratitude to the emergency services, nurses, doctors, and a lot of medical supports from other provinces for their efforts to combat the covid- outbreak in wuhan. besides, we are grateful to dr jiazheng wang (msc clinical & technical solutions, philips healthcare) and dr dandan zheng (msc clinical & technical solutions, philips healthcare) for many useful discussions through the formation and design of this work. the datasets generated in the current study are available from the corresponding author on request.received: april ; accepted: june the corresponding author declares that all authors have read the manuscript and they all gave permission to submit the work in its current version. f.p. and c.z. contributed equally to this article as the co-first authors. all authors have made substantial contributions to the conception and the design of the study, and/or acquisition of data, and/or analysis and interpretation of data, and the drafting of the article and its revision. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to l.y.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - s k guq authors: caldas, lucio ayres; carneiro, fabiana avila; higa, luiza mendonça; monteiro, fábio luiz; da silva, gustavo peixoto; da costa, luciana jesus; durigon, edison luiz; tanuri, amilcar; de souza, wanderley title: ultrastructural analysis of sars-cov- interactions with the host cell via high resolution scanning electron microscopy date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: s k guq sars-cov- is the cause of the ongoing covid- pandemic. here, we investigated the interaction of this new coronavirus with vero cells using high resolution scanning electron microscopy. surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed and h after infection. we showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of sars-cov- . | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ after internalization and rna release into the cytoplasm, a set of proteins is synthesized triggering the formation of vesicles that become a viral platform ensuring efficient replication and transcription of the rna , . new coronavirus particles are assembled in the endoplasmic reticulum and golgi complex. membrane budding between these compartments was reported in association with n protein and genomic rna along with m, e, and s proteins. the complete virions are delivered to the extracellular environment following a conventional secretory route [ ] [ ] [ ] . the research community has sought to better understand the genetic makeup of the virus and thus discover how to effectively treat it. social isolation for days is the main way to prevent the disease from spreading. quarantine and lockdowns were implemented in cities with high rates of infection and mortality . death is common in patients with severe symptoms including shortness of breathing, fever, lethargy, respiratory failure, and/or thrombosis , . understanding the virus-cell interactions is key to vaccines, treatments, and diagnoses. most microscopic studies of sars-cov- were performed with transmission electron microscopy. here, we used high resolution scanning electron microscopy (sem) to study inner cellular structures. the results offer evidence of infectioninduced cellular remodeling and the formation of a specialized region for viral morphogenesis. we also show intercellular extensions for viral cell surfing. these observations offer new insights into the transmission of sars-cov- . cells and virus. sars-cov- isolate (hiae- : sars-cov- /sp /human/ /bra (genbank accession number mt . ) was used in this work. the virus was grown in vero cells (monkey african green kidney cell line -atcc ccl- ) in the laboratory of molecular virology, at federal university of rio de janeiro, brazil. vero cells were maintained in dmem supplemented with % fetal bovine serum (fbs; gibco) at °c and % co . all work involving infectious sars-cov was performed in a biosafety level (bsl)- containment laboratory. infection assays. semi-confluent ( %) cells were grown on sterile glass coverslips in -well tissue culture plates infected with moi (multiplicity of infection -the rate of virus per cell) values of . , . , or using sars-cov- in free-serum medium. fresh medium containing % fbs was added after an absorption period of . h at °c and % co . cells were processed for electron microscopy or h post-infection (hpi). high resolution scanning electron microscopy. after or h post-infection (hpi), samples were fixed with . % glutaraldehyde in . m cacodylate buffer (ph . ) for h. the coverslips were washed with . m sodium cacodylate buffer and post-fixed for min in % oso with . % potassium ferrocyanide. after another washing cycle, the samples were dehydrated through a series of increasing concentration ( %- %) of ethanol. the samples were critical-point-dried in liquid co in a balzers cpd apparatus before monolayer scraping with a conventional adhesive scotch tape as in previous studies . this technique does not totally remove the plasma membrane, as happens when detergent extraction is used, but provides the exposing of large areas of the inner portion of the cells. they were then sputtered with a -nm thick platinum coat in a balzers apparatus. samples were observed using an auriga zeiss microscope operated between . and . kv. to identify alterations on the surface of sars-cov- -infected cells, we compared their morphology and the occurrence of surface projections (sp). while we did not detect any significant alteration in cell shape, the presence of sp increased on the surface of infected cells at hpi ( fig. a-c) . however, no viral particles were observed adhering to the cell surface or beneath these projections ( fig. d ). at hpi, we compared the surfaces of mock and infected cells (moi of . ) to highlight the presence of viral particles adhered to the smooth cell surface and to the sp (fig. e , f). at the same time, and with a moi of , viruses that egressed from a previous cycle of infection were observed during the process of attachment to the cell plasma membrane ( fig. a) . the corona-like features of the sars-cov- particles were discernible via sem (fig. b) , and the measurements showed sizes of approximately nm in diameter (fig. c) . removal of the host cell plasma membrane before platinum sputtering exposed the interior of the mock and infected cells. while mock cells displayed a diffuse distribution of organelles (fig. d) , infected cells exhibited a more polarized disposition of organelles and pit-coated vesicles approximately nm in diameter (fig. e, f) . with a moi of , cells at hpi showed a plethora of vacuoles ( . to µm; fig. a ). these were translocated to the cell plasma membrane presumably to perform exocytosis of viral particles (fig. b ). some of these vacuoles had their content revealed and were filled with immature viruses, amorphous materials, or a hemocyte-like format ( fig. c -e). although no virus-like particles could be distinguished in the er, bordering vesicles were observed on the vacuoles (fig. d) . cells at hpi also had viral particles near the cell surface membrane ruffles (fig. a ) and a filopodium-like structure (fig. b) . other viral particles were wrapped with thin (≃ nm) cellular projections that resemble nanotubes (fig. c) part of the challenge in controlling covid- is the innovative features of this coronavirus. new knowledge on virus genetics and morphology needs to be analyzed concurrently with viral "behavior" within the host cell as well as the dynamics that determine the fate of the particle. to approach sars-cov- /cell interactions, we investigate several steps of virus infection in vero cells at and hpi by sem. vero cells are a widely used model used in viral infection studies and is an adequately supports coronavirus replication , , , . this microscopic approach detailed virus-induced changes in the cell. our assays were performed using three mois ( . ; . and ), and we could discern the moi of . as the more adequate for this type of study. this moi allowed the best cell conditions and distribution and also allowed visualization of virions through the cell surface into the cell interior. the absence of virions adhered to the cells surface at hpi corroborates recent studies performed by belhaouari et al. in which sars-cov- particles were only observed at these loci after hpi. in contrast, www.nature.com/scientificreports/ sars-cov- particles were found lying on the cellular surface at hpi between surface projections and adhered to them. we also observed probable viral particles inside vacuoles suggesting a secretion route. these aggregates of cell organelles and components (fig. f ) may reflect the polarized release of virus previously described for sars-cov . all viruses measured by sem display a spiky round shape with a size of around - nm in diameter considering a platinum coating of nm. this agrees with the dimensions described in recent studies , , . viral particles adhered to smooth surface and microvilli-like surface projetions. the effects on the surface morphology of infected cells varies among viruses. infection by several viruses including htlv-iiib leads to a loss of cell sp that are then replaced by blebs . microvilli induction or increases were reported in several cases of dna or rna viral infection , . for rna viruses that egress by budding, e.g., influenza, the increase in sp of infected cells coincides with higher budding rates . similar to prior studies on sars-cov infection of vero cells , we also observed a ruffled host cell and thickened edges displaying a layered shape. these sites were appropriate to register the attachment of sars-cov- particles ( fig. a ) similar to transmission electron microscopy images of the same early step of sars-cov infection of vero cells . likewise, the proliferation of sp on the infected cells, especially at the apical region of these cells, is similar to sars-cov and sars-cov- . in addition, the abundance of sars-cov- particles held on sp, was recently showed and may facilitate the speed of viral propagation in the epithelium of conducting airways from the lumen of the respiratory superior tract because this environment is colonized by ciliated cells. www.nature.com/scientificreports/ vacuoles containing viral particles. cell scraping is a very useful expedient that is occasionally used in studies of host cell/parasite interactions , . infected cells are artificially devoid of plasma membranes and exposed to a myriad of vacuoles (fig. a ). drastic vacuolization due to viral infection was previously described for other rna viruses including sars-cov , . similar sites were recently reported as virus morphogenesis matrix vesicae (vmmv) . the particles observed in the interior of these vmmvs (fig. c -e) were previously described as doughnut-like particles when observed by electron microscopy , . sars-cov immature particles are presumed to bud into vesicles as part of the assembly process, and thus the observed particles were probably immature viruses devoid of the representative (corona) spikes of this virion. bordering vesicles were found in close association with the vacuoles (fig. d) , and thus we speculate that their role in viral pre-components leads to discharge into the compartments. studies with other coronaviruses identified large virion-containing vacuoles (lvcvs) where the complete particle would bud. there is correlation between these structures as observed by transmission electron microscopy and our data suggesting the occurrence of both phenomena. sive remodeling of cell membranes , ; the more condensed area depicted in the cytoplasm at hpi (fig. f ) may correspond to the main locus of viral morphogenesis. the proposed mechanism for the export of viruses to the extracellular space is via fusion of the transport compartment membrane with the cell plasma membrane . the size of the vacuoles we observed in the cell periphery was not compatible with the identified clathrincoated pits because the vacuoles measure approximately µm; clathrin-coated pits measure near nm in diameter. the presence of these endocytosis-associated players was recently reported in sars-cov- -infected cells. they are likely receptacles to the nucleocapsid after the incoming virus is uncoated . thus, our observations suggest that a boost in vacuoles is restricted only to a specific and more condensed part of the cytoplasm. this suggests translocation to the plasma membrane is required for release the viral particles by a fusion mechanism. cellular bridges containing viral particles. viral particles adhered to cell surface protrusions that were shown to connect two cells. this observation suggests viral "cell surfing" previously described by other enveloped viruses such as hiv and human metapneumovirus , . this mechanism is presumed to allow the in vivo penetration of virus in mucosal surfaces that display microvilli-rich cells. actin filaments play a fundamental role in viral extrusion by the cell for both rna and dna viruses. actin offers the strength to discharge the progeny virus particles to the extracellular medium, as occurs to some viruses www.nature.com/scientificreports/ that leave the cell by budding, including fowlpox and west nile viruses , . other examples include actin comets-these are an efficient form of poxvirus dissemination and cell-to-cell hiv spreading, which involves the direct engagement of gag proteins and f-actin , . previous studies have shown that the cytoskeleton network plays an important role in the maturation and, possibly, in the replication process of sars-cov . communication between the two cells in fig. c -d suggests the occurrence of a thin (< . µm) strand of f-actin containing tunneling nanotube (tnt). these intercellular membranous connections may provide the transference of molecular information especially viruses . 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viruses along filopodia precedes their entry into cells human metapneumovirus induces reorganization of the actin cytoskeleton for direct cell-to-cell spread morphogenesis and release of fowlpox virus actin filaments participate in west nile (sarafend) virus maturation process cryo electron tomography of native hiv- budding sites actin dynamics in host-pathogen interaction bridging the gap: virus long-distance spread via tunneling nanotubes /scientificreports/ reprints and permissions information is available at www.nature.com/reprints we thank professor kildare miranda (cenabio) for providing the microscopy facilities; dr. lorian cobra straker (cenabio) and flávio augusto ferreira martins bezerra (inmetro) for technical assistance. this work has been supported by fundação carlos chagas filho de amparo à pesquisa do estado do rio de janeiro-faperj, financiadora de estudos e projetos-finep and conselho nacional de desenvolvimento científico e tecnológico-cnpq. isolate preparations and virus growth were conducted by g.p.s. and l.j.c. infections were performed by l.m.h. and f.l.m. sample preparations and microscopy analysis were performed by l.a.c. and f.a.c.; analysis of the results was performed by l.a.c., f.a.c. and w.s. w.s. and a.t. contributed to the initial conception and design of this work. the first draft of the manuscript was written by l.a.c. and f.a.c. l.j.c., a.t. and w.s. commented on previous versions of the manuscript. e.l.d. provided sars-cov- isolate. all the authors were involved in reviewing and editing the manuscript. all authors read and approved the final manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to l.a.c.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - aotsb g authors: dong, jianbo; huang, betty; wang, bo; titong, allison; gallolu kankanamalage, sachith; jia, zhejun; wright, meredith; parthasarathy, pannaga; liu, yue title: development of humanized tri-specific nanobodies with potent neutralization for sars-cov- date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: aotsb g sars-cov- is a newly emergent coronavirus, which has adversely impacted human health and has led to the covid- pandemic. there is an unmet need to develop therapies against sars-cov- due to its severity and lack of treatment options. a promising approach to combat covid- is through the neutralization of sars-cov- by therapeutic antibodies. previously, we described a strategy to rapidly identify and generate llama nanobodies (vhh) from naïve and synthetic humanized vhh phage libraries that specifically bind the s sars-cov- spike protein, and block the interaction with the human ace receptor. in this study we used computer-aided design to construct multi-specific vhh antibodies fused to human igg fc domains based on the epitope predictions for leading vhhs. the resulting tri-specific vhh-fc antibodies show more potent s binding, s /ace blocking, and sars-cov- pseudovirus neutralization than the bi-specific vhh-fcs or combination of individual monoclonal vhh-fcs. furthermore, protein stability analysis of the vhh-fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against covid- . sars-cov- is a coronavirus that causes the human disease covid- , which is contagious and can rapidly spread to cause mild to severe infection, including death [cdc (https ://www.cdc.gov/coron aviru s/types .html) ]. the spread of this newly emergent virus has reached a pandemic level with a significant public impact on the world, leading to more than million infections and more than a . million deaths worldwide [world health organization (who) (https ://www.who.int/emerg encie s/disea ses/novel -coron aviru s- )]. in addition to threatening human health, covid- has also caused a significant socio-economic impact around the world [united nations (https ://www.undp.org/conte nt/undp/en/home/coron aviru s/socio -econo mic-impac t-of-covid - .html)]. although there are relatively successful diagnostic methods to detect the sars-cov- infection in humans, there are currently no successful therapies that can interfere with virus replication. the small antiviral molecule remdesivir (gilead) which inhibits the rna-dependent rna polymerase of sars-cov- decreases the recovery time in patients with covid- , but it most likely cannot completely stop or prevent sars-cov- infections in humans. another small antiviral molecule, grl- , shows promise in interfering with the sars-cov- replication by inhibiting the papain-like protease, however, it is yet to be tested in clinical trials . moreover, there are no fda-approved vaccines to prevent sars-cov- infections in humans, although several groups are currently in the pursuit such vaccines [who (https ://www.who.int/publi catio ns/m/item/draft -lands cape-of-covid - -candi date-vacci nes)]. therefore, rapid development of therapeutics and preventative strategies has become an essential and urgent need to fight the covid- pandemic. the trimeric spike (s) proteins that protrude through the envelope of the sars-cov- virion mediate virus entry into the host cells by interacting with the human ace receptor [ ] [ ] [ ] [ ] [ ] [ ] . therefore, a major target for anti-sars-cov- neutralizing antibodies in development are to block the interaction of sars-cov- s protein with ace . in particular, two popular strategies have been employed to discover and develop monoclonal igg antibodies that can recognize sars-cov- s protein mainly by binding to its receptor binding domain (rbd) [ ] [ ] [ ] [ ] [ ] [ ] . the first commonly used method is to clone the antibody v genes from the b cells of surviving covid- patients who have mounted a natural immune response against sars-cov- , , . this strategy has yielded a number of neutralizing monoclonal antibodies; however, it is important to note that the patients' antibody repertoire condition and the timing of blood sample collection play a critical role in its success. the other well-recognized and classic approach for antibody generation is by immunizing humanized mice . additionally, new sars-cov- identification of vhhs binding to different epitopes of sars-cov- s protein rbd. recently, we reported the identification of llama vhhs that bind to the sars-cov- s protein rbd . briefly, we used two llama vhh libraries (one naïve library and another humanized synthetic library derived from the naïve library) to screen for vhhs that bind to the sars-cov- s protein in-vitro . we identified a total of s protein binders, from the naïve and from the humanized libraries. out of the s protein binders, vhhs blocked the interaction between sars-cov- s rbd and ace receptor, with s/ace blockers identified from the naïve library and identified from the humanized library (data not shown). furthermore, we observed that the pairwise addition of some of the vhhs caused synergistic effects on sars-cov- s/ace blocking . we hypothesized that this synergistic effect is caused by binding of the vhhs to different epitopes within the s rbd. to test this, we performed epitope binning assays by biolayer interferometry (fig. a -c) or elisa (fig. d ) on a selected number of candidates. in the initial epitope binning assay (fig. a-c) , we used an s rbd sensor to capture a-fc, b-fc, or f-fc separately, followed by the incubation with our lead candidates b-fc, f-fc or a-fc. the vhhs were fused to human igg fc domains to render the fc effector functions against sars-cov- . this analysis showed that with the a-fc-loaded probe, the addition of f-fc further increased the signal compared to the a-fc control, while the addition of b-fc decreased the signal compared to the control (fig. a) . this indicates that f-fc does not compete with the a-fc site and it is likely that they bind to different s rbd epitopes. in contrast, b-fc competed with a-fc, indicating that they compete for binding to the same s rbd epitope (fig. a) . similarly, with the b-fc-loaded probe (fig. b) , f-fc increased the signal compared to the b-fc control. this shows that f-fc does not compete with b-fc. interestingly, a-fc also increased its signal compared to b-fc control. this suggests that although having a common binding region, a binds to a wider epitope than b (fig. b) . with f-fc-loaded probe, both a-fc and b-fc showed an increase of the signal compared to the f-fc control. this further shows that f-fc does not compete with either b-fc or a-fc, and likely bind to a different epitope (fig. c) . these results confirm our hypothesis and show that s/ace blocking vhhs bind to at least two separate unique epitopes within the s rbd. next, we performed an elisa-based epitope binning assay to assess five additional vhhs ( c, f, a, f, and g ) unfused to fc, but previously assessed to block the sars-cov- s/ace interaction . the assessment of more vhhs would allow us to categorize several of our other vhhs into binding groups, which will aid in multi-specific antibody design and construction. in this elisa, wells were coated with sars-cov- s and incubated with bi-specific vhh-fc b- a (based on previous data, b and a likely bind the same epitope) or monoclonal vhh-fc f-fc (based on previous data, this binds a different epitope than b or a) premixed with the vhh candidates. the resulting relative fluorescence signals obtained for each sample were calculated to reflect the percent difference from c, f, a, f, g , and controls ( f-fc and b- a-fc) signals, when the vhhs are combined with b- a-fc or f-fc (fig. d) . the results show that vhh-fcs c, f, f, as well as the b- a-fc control have almost % difference from b- a-fc, which highly suggest that they compete for the same epitope (highlighted in red). however, g (highlighted in light red) may partially compete with b- a-fc, whereas a does not likely compete for the same epitope (highlighted in green). additionally, these results show that a and the f-fc control may compete with f-fc (fig. d) , while other vhhs, including the b- a-fc control resulted in a lower percent difference. we also performed additional epitope binning assays using biolayer interferometry to assess the competition of the vhh-fcs c, g , and a to bind to s rbd. the vhh-fcs f and f poorly bound to the biolayer interferometry probes used for this assay and were excluded from analysis. this approach confirmed the results that we obtained by elisa and showed that c and g likely belong to group , and a belongs to group in terms of the binding competition ( supplementary fig. ). interestingly, g -fc shows competition with either b-fc and a-fc when it is loaded onto the probe first (data not shown). in contrast, reversal the of loading further increased its signal compared to both b-fc control and fig. ). taken together, we could categorize vhh blockers of s/ace interaction into major groups based on their epitope binding; group consist of vhhs, whereas group consist of vhhs (fig. e ). elucidation of epitopes on s rbd that bind to vhh-fcs. in an effort to elucidate the structural basis of the newly discovered epitope binding groups, we computationally generated structural models for b, f, and a vhhs and docked them with sars-cov- s rbd structure exported from pdb m j using schrodinger bioluminate software [ ] [ ] [ ] . for context, fig. a shows the sars-cov- s protein with the ace binding residues in red font. this approach generated an array of poses of each s rbd/vhh complex structure, which allowed us to further analyze the interfaces of those poses with a good piper cluster size and led us to identify five regions in the rbd which may interact with vhh b, a, and f, respectively (fig. a,b) [ ] [ ] [ ] . next, we generated different s rbd deletion mutants to validate the computationally mapped epitopes in-vitro to select the best docking model for molecular analysis. interestingly, these s rbd deletion regions have been shown to mediate the s rbd/ace interaction in recently published literature [ ] [ ] [ ] [ ] (table ) . we tested wild-type and all the s deletion mutants for their ability to bind to a tri-specific vhh-fc to check whether the proteins are folded and expressed on the cells. the results show that they are indeed expressed and folded as they all bind to the tri-specific vhh-fc, although the level of expression and/or folding might be different across the mutants based on the strength of the binding signals. the wild-type s rbd and the deletion (del ) shows stronger binding, whereas the deletions (del ), (del ), (del ) and (del ) show weaker binding to the tri-specific vhh-fc ( supplementary fig. ). then we assessed the binding profiles of the s rbd wild-type and the deletion mutants with selected vhh-fcs from group and group , as well as ace (fig. c,d) . the binding of vhh-fc candidates from both group and group , as well as ace to s rbd are affected following the removal of del . it is possible that this result is due to a conformational change or decrease of s protein expression following its deletion because based on crystal structure of the rbd/ace complex (pdb m j), the deleted domain is not part of the s rbd/ace interface. the del mutant, which is adjacent to a computationally-predicted epitope domain in region , does not prevent the binding of both group and group vhh-fcs to s rbd. in addition, it does not prevent the binding of ace to s rbd. the del , , and mutants all decreased binding of both group and group vhh-fcs to s rbd. however, these regions are more critical for group than (d) epitope binning of vhhs were assessed using an elisa method. briefly, the sars-cov- s protein was incubated with b- a-fc or f-fc, and binding competition was performed with the vhhs followed by the detection of the biotinylation. the experiment was performed in duplicates, and the average percent difference from the competing pairs relative to the vhh-fc alone signal are indicated in the table. the vhh associated percentages highlighted in red are likely high vhh competitors, in light red are partial competitors, and in green are likely non-competitors. (e) the two groups of vhhs categorized based on the binding to epitopes on s rbd. figure . elucidation of epitopes on s rbd that bind to vhh-fcs. (a) ace binding residues on sars-cov- s rbd were determined by schrodinger bioluminate based on the protein-protein interactions of protein data bank (pdb) m j. (schrödinger release - : bioluminate, schrödinger, llc, new york, ny, . https ://www.schro dinge r.com/produ cts/biolu minat e. requires permission to be used). the residues in red are predicted ace interactors. the deletions generated on sars-cov- s rbd are shown with the boxed regions. (b) deletion map schematics of the s rbd deletion mutants. (c) the binding of vhh-fcs and ace to expi cells expressing sars-cov- s wild type (wt) or mutant proteins (del -del ) were assessed by flow cytometry following fitc-conjugated secondary antibody treatment. an isotype control antibody and facs buffer were used as negative controls. the experiment was performed at least three times which yielded similar trends in results. a representative image of a single experiment is shown here. the graph was generated by the prism (graphpad) software (prism version . . . https ://www.graph pad.com/scien tific -softw are/prism /. requires permission to be used) (d) in the experiment shown in (c), the binding percentages relative to the s wt for each vhh-fc were calculated in the context of each deletion mutant. the group vhh-fcs and the values with binding differences that contributed to their categorization into that group are shown in red. those values for group vhh-fcs are shown in blue. (e) docking models between sars-cov- s rbd and the lead vhhs generated with schrodinger bioluminate software. (schrödinger release - : bioluminate, schrödinger, llc, new york, ny, . https ://www.schro dinge r.com/produ cts/biolu minat e. requires permission to be used). scientific reports | ( ) : | https://doi.org/ . /s - - -y www.nature.com/scientificreports/ group for their binding. in addition, these regions are critical for ace to bind to s rbd. taken together, the binding epitopes for group is more associated with del , and regions which are located at the interface of s rbd/ace , while at least part of the epitopes for group are shifted farther away from the s rbd/ace interface relative to the epitopes for group vhhs (fig. c,d) . based on the binding and epitope binning data, we constructed d docking models that predicted the interactions between sars-cov- s rbd, ace and lead vhh-fcs (fig. e) . these models show that predicted binding epitopes for group vhhs b and a are located at the s rbd/ace interface. in contrast, the epitope for group vhh f is located away from the s rbd/ace interface (fig. e) . interestingly, there are binding variations seen within group . the binding of a to del , del , del and del have decreased more than that of b. this shows that epitopes for a and b are not the same even though they compete with each other and were initially characterized to be within the same binding group (fig. c,d) . taken together, our analysis confirms that there are two major binding groups (group and group ) and we show the likely binding regions on the sars-cov- s protein for each vhh. tri-specific vhh-fcs show potent s rbd binding and s/ace blocking activity. next, we tested whether the combination of individual vhhs binding to different s rbd epitopes into bi-specific antibody molecules would yield synergistic effects in sars-cov- binding and s/ace blocking. as expected, the resulting bi-specific vhh-fc b- f showed superior binding to s rbd and s/ace blocking compared to individual component vhh-fcs . since sars-cov- s proteins formed trimers, we started to study whether trispecific antibodies with two binding units from group and another binding unit from group or vice versa would have better binding and blocking function than the bi-specific antibody [ ] [ ] [ ] . here, we only focused on tri-specific, as any larger multi-specific molecules will likely affect developability with fc fusion proteins. we selected the vhhs from both group and with the most favorable binding, functional and developability features, and constructed tri-specific vhh-fcs with the computer-aided antibody design using the software bioluminate (schrodinger) that enabled their effective construction and optimization [ ] [ ] [ ] . then, we tested the tri-specific, bi-specific and mono-specific vhh-fcs for their ability in-vitro for sars-cov- s protein binding and s/ace blocking (fig. a,d) . as expected, the multi-specific antibodies showed higher binding affinities to sars-cov- s protein rbd in-vitro, with the tri-specific vhh-fcs f- b- a (kd ~ . nm) and b- f- a (kd ~ . nm) showing more potent binding than bi-specific vhh-fc b- f ( fig. a -c,e). the binding affinities for tri-specific vhh-fcs were higher than that of individual component vhh-fcs b, f and a used in combination, and the binding affinity for b- f-fc was higher than that of individual component vhh-fcs b, and f used in combination (fig. a ). in addition, f- b- a and b- f- a showed potent blocking of the sars-cov- s/ace interaction, with ic values of . nm and . nm, and full inhibition around nm for both, respectively, that were far superior to using individual component vhh-fcs as combinations (ic of . nm and full inhibition around nm). in addition, f- b- a and b- f- a were more potent than bi-specific vhh-fc b- f in blocking sars-cov- s/ace interaction (fig. d) . interestingly, the tri-specific vhh-fc a- b- f had lower s/ace blocking ability showing the physical arrangement and/or binding orientation of the vhhs in a multi-specific antibody is important for its binding and blocking (fig. d) . taken together, this data indicates that the tri-specific vhh-fcs have a higher synergistic potency in both binding and blocking the s or s /ace interaction than bi-specific or mono-specific antibodies. tri-specific vhh-fcs have favorable developability features. during the computer-aided design process, we incorporated several development-enhancing features in the structures of our vhh-fcs. therefore, we analyzed the physico-chemical properties, using dls and dsf/sls methods, of our lead bi-and tri-specific antibodies to determine whether they possess favorable characteristics for large-scale manufacturing that is essential for the commercial development of the antibodies (fig. e) . our data revealed that the lead tri-specific vhh-fc f- b- a has lower aggregation potential based on the dls method and is thermostable based on the dsf/sls method (fig. e) . we tested the multispecific vhh-fcs for their ability to target sars-cov- in cell biological functional assays. first, we analyzed the virus neutralizing ability of our antibodies using a pseudovirus that expresses the sars-cov- s protein. the tri-specific vhh-fcs f- b- a, b- f- a, and the mono-specific combinations of vhhs ( b-fc + f-fc + a-fc) prevented the infection of human cells by the pseudoviruses (fig. a ). in accordance with (fig. a) . this pseudovirus data presented here confirm the synergistic effect of the tri-specific antibodies and most importantly, it suggests that it is likely effective in preventing the sars-cov- infection. as our vhh-fcs contain the fc domain of human igg , we expected it would be able to trigger the fcdependent functions to eliminate the viruses from the body. to test this, we used a cell line that transiently expresses the sars-cov- s protein. then, we assessed the ability of our multi-specific vhh-fcs to promote antibody-dependent cellular cytotoxicity (adcc) that is an fc-dependent function of the antibodies. in addition to our lead tri-specific vhh-fc antibody f- b- a, we also tested a- f- a-fc, another tri-specific antibody we constructed with similar s binding and s/ace blocking potency (supplementary fig. ). as expected, the vhh-fcs were able to induce adcc in the cells (fig. b) . this suggests that these vhh-fcs could bind to immune cells through their fc domain and elicit fc-dependent functions, thereby allowing multiple mechanisms of actions against sars-cov- , including binding sars-cov-s and blocking s /ace interactions. docking model for sars-cov- s rbd with tri-specific vhh-fc f- b- a was generated by bioluminate (schrödinger release - : bioluminate, schrödinger, llc, new york, ny, . https ://www.schro dinge r.com/produ cts/biolu minat e. requires permission to be used). in the software, the sars-cov- rbd spike protein trimer (pdb x a) was split into three monomeric forms (chain a, b and c). then, b/ f/rbd model structure was aligned with chain a of pdb x a to create group and a/rbd model structure was aligned with chain b of pdb x a to create group . then, group , group and chain c were merged to generate the final structure. the s rbd/vhh docking structure is represented with a surface structure (a) and ribbon structure (b). the enlarged s rbd/vhh docking structure is shown in right (c). in this study we developed and characterized llama-derived multi-specific nanobodies that yielded data that strongly suggests they will be effective against sars-cov- that causes covid- . the covid- pandemic has caused widespread health and social issues around the globe, and requires therapeutics that can effectively stop and prevent the infection of sars-cov- . several monoclonal antibodies against sars-cov- have been suggested and being tested as anti-viral therapies, either as individual agents or combination therapies; however, this is the first study that introduces and demonstrates the efficacy of multi-specific antibodies against sars-cov- to our knowledge [ ] [ ] [ ] [ ] [ ] [ ] , . to successfully design and construct multi-specific vhh binders, the epitope information for each individual vhh clone is necessary. here, instead of obtaining crystal structures for each antigen/antibody complex, we utilized a different method for epitope identification. we initially performed epitope binning with biolayer interferometry and categorized s/ace blocking vhhs into groups. the vhhs within each group competed, but there was no competition with vhhs from the other group, strongly suggesting that group and group vhhs are two separate binding groups. then, we computationally constructed vhh models and docked them separately to an s rbd structure obtained from a publicly-available crystal structure of sars-cov- s rbd/ ace , and utilized docking structures with higher pose cluster size to predict possible epitopes for the individual vhhs. to validate the involvement of predicted epitopes in vhh/s rbd binding, we compared the binding ability of each vhh to wild type s rbd and five deletion mutants with each predicted epitope deleted. as shown in fig. , both group and group vhhs likely bind to the regions del , del , and del which overlap with the ace binding interface of s rbd, however, at least part of the epitope for group is likely located more outwards of this region and has relatively less overlap with the ace binding interface of s rbd. currently, a number of structures of s rbd/antibody complexes have been published. the analysis of these structures show that there are likely main "hot" antibody binding regions in s rbd: one likely in the n-terminal region (del ) , , and the other likely in the ace binding interface (del , del , del ) , , . our selected vhh binders in tri-specific antibodies possibly cover both of these regions (fig. ). based on this information, we were able to define the lead tri-specific vhh-fc format, including the linker length and the order of the vhh binders. the tri-specific antibodies are advantageous as therapeutic agents because they simultaneously bind multiple epitopes within the s protein rbd that increase their antigen-binding affinity and avidity (fig. ) . the vhhs b and f that comprises the bi-specific antibody bind to two different epitopes in the s protein rbd . in our tri-specific antibody design, we incorporated the vhh a that has an almost identical epitope as b. these vhhs could bind in different orientations to the same or similar epitopes, or to a corresponding epitope in another s protein in the trimer, increasing the binding and blocking potency of the tri-specific vhh-fc. in fact, this phenomenon has been previously shown for other multi-specific antibodies. for example, the cd targeting t cell engager antibody cd -tcb (roche) with two cd binding domains ( : molecular format) has increased potency compared to other cd -binding bi-specific antibodies in clinical development . in agreement with this hypothesis, the resulting tri-specific vhh-fcs showed very potent characteristics in terms of the s binding and s/ace blocking efficacy, which are among the best in currently published anti-sars-cov- therapeutic antibodies ( table ) . because of these characteristics, the tri-specific vhh-fcs could be used at low concentrations for therapeutic applications that would potentially lower their toxicity in humans. in addition, the strong binding of the antibodies to the virions would minimize the risk of antibody-dependent enhancement (ade) that is caused by sub-optimal antigen-antibody interactions and promotes enhanced viral infections , . the multi-specific targeting approach also minimizes the loss of antibody binding to viral antigens due to the mutations of the viruses. the rna viruses are known to mutate, and in this sense coronaviruses could lose the binding to antibodies relatively easily due to structural changes in the viral components , . however, the vhh multi-specific antibodies would still bind to the mutated virus since the other vhhs in the tri-specific antibody would bind the unmutated epitopes of the virus. another advantage of the vhh multi-specific platform is the ability to target multiple viruses. for example, it is possible to adjoin vhhs that bind to other coronaviruses such as sars-cov and mers-cov, and construct pan-coronavirus tri-specific vhh-fcs that would be effective in preventing and treating a broad spectrum of coronaviruses. our multi-specific antibody design connects human igg fc domain to bi-or tri-specific vhhs. having the fc domain in the antibody structure confers fc-dependent cytotoxic functions such as adcc, complementdependent cytotoxicity (cdc) and antibody-dependent cellular phagocytosis (adcp) [ ] [ ] [ ] [ ] [ ] . these additional fc-dependent functions, in addition to blocking virus entry and possible virus aggregation, would equip the vhh-fcs with multiple mechanisms of action, making them more potent in neutralizing the coronaviruses. indeed, our lead tri-specific vhh-fc f- b- a show potent neutralization of sars-cov- pseudovirus infection in human cells. one of the questions in the field of antibody therapeutics is whether the effect of using multi-specific single molecule is better than using a combination of monoclonal antibodies that collectively target the same epitopes or not . here, we show that multi-specific antibodies are more effective in blocking host-virus interactions than a combination of monoclonal antibodies. our tri-specific vhh-fc f- b- a was much more potent in blocking the sars-cov- s/ace interaction than using vhh-fcs f, b and a individually as a combination. it is likely that physically combining the vhhs increases overall association constants (k on values) and decreases overall dissociation constants (k off values), producing lower binding constants, thus increasing antibody affinity towards antigens. it also increases the avidity of the antibodies, making them more effective in neutralizing viruses. one of the hallmarks of a successful therapeutic antibody is its developability features , . especially during pandemics such as covid- when rapid production of antibodies in high quantities is essential, the developability and manufacturability of the antibodies play even crucial roles. our design has the advantage of using scientific reports | ( ) : | https://doi.org/ . /s - - -y www.nature.com/scientificreports/ llama vhh nanobodies that have high stability , . indeed, the biochemical and biophysical characteristics of the multi-specific vhh-fc show that they can be purified in high quantity, have better aggregation resistance, and have favorable thermostability. in addition, our antibodies have high developability because the multi-specific design combines the individual vhhs into single molecules instead of combinations, making their manufacturing easier. an alternative strategy of increasing developability of the anti-sars-cov- multi-specific antibodies would be to combine vhhs without the addition of igg fc domain to construct tetra-specific vhhs. these molecules would have the added advantage of increased affinity and avidity towards sars-cov- s protein compared to bi-and tri-specific vhh-fcs, despite lacking the fc effector functions. these tetra-specific antibodies would be ideally suited as antibody prophylactic to prevent the sars-cov- infection in humans because their llama vhh-only structure would have increased thermostability, easier combination capability, and the possibility of easy large-scale manufacturing using cost-effective expression systems such as yeast . one of the key features of our therapeutic antibodies is the use of computer-aided design that greatly reduces their development time and enhances their optimization efficiency. for instance, from the inception of this project, it was possible to produce, optimize and test our lead tri-specific vhh-fcs in less than months. this shows that this strategy is powerful for producing novel therapeutic antibodies for time-sensitive unmet needs, and can be utilized for future outbreaks that would require rapid development of antibody therapeutics. cell lines and transfections. the cell lines used in this study were cultured in media as stated below. epitope binning (competition) assays. the initial assay was performed using gator system (probe life). after pre-wetting the sars-cov- s rbd sensors in q buffer (probe life), the sensor captured - µg/ ml of the first monoclonal vhh-fc for about s, then the loaded sensor captured µg/ml of the second monoclonal vhh-fc, either b, f, or a, which was quantified over time by gator. the follow-up assay for vhh-fcs b- a and f was performed by elisa. a -well plate was coated to a final concentration of µg/ml of sars-cov- s protein and placed overnight at ºc. to test the binding with vhhs c, f, a, f and g , the following method was used. b- a-fc or f-fc at µg/ml were premixed with each competing c-myc tagged vhhs from periplasmic supernatant at a : ratio. after another one hour of incubation, a biotinylated anti-c-myc antibody ( e ) was added and the samples were incubated for another one hour. then, streptavidin-hrp was added followed by the treatment with amplex red (thermo fisher scientific) and % h o containing development solution. the emitted signal for each sample was detected by using a fluorescence plate reader (spectramax gemini xps). to test the binding with vhhs b- a-fc and f-fc, the following method was used. b- a-fc or f-fc at µg/ml were premixed with competing biotinylated b- a-fc or f-fc at a : ratio. after one hour of incubation, the biotin-streptavidin detection system as described above was used to analyze their competition. the percent difference from the competing pairs versus the vhh-fc alone signal was calculated using the following formula; % difference from vhh-fc signal = ( − ((signal of competing pair − no antibody signal)/(signal of vhh-fc alone − no antibody signal)) × . cell binding assay. binding of vhh-fcs to sars-cov- s expressing cells was assessed by flow cytometry. briefly, cells were harvested and resuspended in pbs with % fetal bovine serum (fbs) and plated in v-bottom -well plates at × cells/well density. they were incubated for h at room temperature with µg/ml of indicated vhh-fcs, control antibodies or recombinant biotinylated human ace also dissolved in pbs with % fbs. then, the cells were washed twice with the same buffer, and incubated with fitc-conjugated goat anti-human igg (jackson immunoresearch) at : dilution or pe-conjugated streptavidin (thermo fisher, scientific reports | ( ) : | https://doi.org/ . /s - - -y www.nature.com/scientificreports/ for the detection of biotinylated ace ) at : dilution for min at room temperature. cells were washed again following the secondary antibody treatment. then they were analyzed by a facscalibur cytometer (bd biosciences). cell populations were visualized as forward vs side scatter and gated to exclude dead cells. cells treated with no antibodies were used to establish background fluorescence. the resulting facs data were analyzed by the software flowjo (bd biosciences) and the graphs were generated by the software prism (graphpad). in-vitro s protein binding assay. the -well elisa plates (greiner bio-one) were directly coated with sars-cov- s protein (acro biosystems) diluted in pbs at µg/ml and incubated overnight at °c. then, the plates were washed with pbs containing . % tween (pbst) and blocked with % bsa in pbs at room temperature for one hour. the plates were washed again with pbst and incubated with the test antibodies at room temperature for one hour. the antibodies were used at : serial dilutions. the plates were washed with pbst followed by the addition of anti-human-fc antibodies conjugated to horseradish peroxidase (hrp) (jackson immunoresearch) at : dilution in pbst and the plates were incubated at room temperature for h. following washing again by pbst, the plates were treated with elisa development buffer solution containing amplex red and % h o . the emitted binding signal for each sample was detected by using a fluorescence plate reader. the blocking was measured in relative fluorescence units (rfu) and the % inhibition was calculated as follows; % inhibition = ( − (mean experimental value/mean of no antibody control)) × . s/ace blocking assay. the -well elisa plates (greiner bio-one) were coated with sars-cov- s protein (acro biosystems) and incubated overnight as stated previously. then, the plates were washed with pbst and blocked with % bsa in pbs at room temperature for one hour. the plates were washed again with pbst and incubated with the test antibodies at room temperature for min. the antibodies were used at : serial dilutions. then, recombinant biotinylated-ace (acro biosystems) was directly added to the plates at . µg/ µl and incubated at room temperature for another min. the plates were washed with pbst followed by the addition of streptavidin conjugated to hrp at : dilution in pbst. the plates were incubated at room temperature for another min. then, they were washed with pbst and treated with elisa development buffer. the emitted binding signal for each sample was detected by using a fluorescence plate reader. analysis of physical characteristics of vhh-fcs. the purified vhh-fcs were analyzed by the uncle system (unchained labs) for their thermostability using differential scanning fluorimetry (dsf) and static light scattering (sls), and aggregation potential using dynamic light scattering (dls) assays. the dls was measured at °c and the data was analyzed using uncle analysis software. for dsf/dls assays, a temperature ramp of °c/min was performed with monitoring from to °c. sls was measured by uncle at nm and nm. tm and tagg were analyzed and calculated by the uncle analysis software. with genscript biotech (piscataway, nj). briefly, pseudovirus expressing luciferase and containing sars-cov- s as the envelope glycoprotein in a lentiviral vector was produced in hek t cells, and the virus titration was determined by elisa. hek cells expressing ace receptor and transmembrane serine protease (tmprss ) were used as the target cells, and were seeded in -well plates. then, pseudovirus with the serial dilutions of the antibodies were mixed with the target cells. the cells were incubated for h at °c and an amount of µl of the cell suspension was transferred to an assay plate. it was mixed with luciferase detection reagents from bio-glo™ luciferase assay system (promega) and incubated for - min at room temperature. then, the luminescence was measured by a plate reader. the background rlu was subtracted from the rlu of the experimental samples. the values for % inhibition were derived from rlu as follows; % inhibition = ( − (mean of experimental value − mean of cells treated only with buffer)/(mean of cells treated only with sars-cov- )) × . antibody-dependent cellular cytotoxicity (adcc) assay. target expi cells expressing s protein ( sprot) were washed with rpmi media containing % horse serum and ng/ml il- , and plated in -well plates at × cells/well density. then, they were mixed with antibodies at µg/ml of final concentration. then, nk- -cd cells expressing gfp were added to wells at × cells/well density (effector: target- : ) and the plates were incubated overnight °c, % co . then, the cells were washed twice and resuspended in dpbs with % fbs. they were assessed by flow cytometry using a facscalibur cytometer (bd biosciences). sprot and gfp-nk- -cd cells were each used as a reference to set up overall target cell gating and to establish the gfp positive nk- -cd populations, allowing differentiation between the nk- -cd effector cells and sprot target cells. the gfp negative sprot cell percentage was evaluated for all samples. then, cell death percentage was calculated as follows; % cell death = ( − (antibody treated cell percentage/average of isotype control percentage)) × . statistical analysis. the four-parameter non-linear regression analysis from prism software version . . was used for all binding and blocking curves, which also included the ic values for the blocking assays. all error bars represented in the data are based on standard deviation, unless otherwise specified. the data generated and/or analyzed during this study are available from the corresponding author on reasonable request. severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients remdesivir for the treatment of covid- -preliminary report papain-like protease regulates sars-cov- viral spread and innate immunity a pneumonia outbreak associated with a new coronavirus of probable bat origin the severe acute respiratory syndrome angiotensin-converting enzyme is a functional receptor for the sars coronavirus dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat 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structure determination using a combination of homology modeling, energy-based refinement, and loop prediction structure-based approach to the prediction of disulfide bonds in proteins applying physics-based scoring to calculate free energies of binding for single amino acid mutations in protein-protein complexes decoys as the reference state) potentials for proteinprotein docking an fft-based protein docking program with pairwise potentials the th meeting on the critical assessment of predicted interaction (capri) held at the mare nostrum a noncompeting pair of human neutralizing antibodies block covid- virus binding to its receptor ace the molecular biology of coronaviruses architecture of the sars coronavirus prefusion spike cryo-em structure of the -ncov spike in the prefusion conformation isolation of a human monoclonal antibody specific for the receptor binding domain of sars-cov- using a competitive phage biopanning strategy cd -tcb with obinutuzumab pretreatment as next-generation treatment of hematologic malignancies enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease the potential danger of suboptimal antibody responses in covid- rapid evolution of rna genomes complexities of viral mutation rates neutralization of virus infectivity by antibodies: old problems in new perspectives nk-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: biological evidence and clinical perspectives complement in monoclonal antibody therapy of cancer antibody-dependent cellular phagocytosis in antiviral immune responses fc-mediated antibody effector functions during respiratory syncytial virus infection and disease engineering multi-specific antibodies against hiv- developability assessment during the selection of novel therapeutic antibodies structure, heterogeneity and developability assessment of therapeutic antibodies we thank all ab studio inc. and ab therapeutics inc. members for their valuable support and input in this project. all authors of this study are employees of ab studio inc. yue liu also serves as the chief executive officer of ab therapeutics inc. supplementary information is available for this paper at https ://doi.org/ . /s - - -y.correspondence and requests for materials should be addressed to j.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -xflhxb authors: manso, carmen f.; bibby, david f.; mbisa, jean l. title: efficient and unbiased metagenomic recovery of rna virus genomes from human plasma samples date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xflhxb rna viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. new sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. through selective host rna depletion and compensatory protocol adjustments for ultra-low rna inputs, we are able to detect three major blood-borne rna viruses – hiv, hcv and hev. we recovered complete genomes and up to % of the genome from samples with viral loads of ( ) and ( ) iu/ml respectively. additionally, we demonstrated the utility of this method in detecting and characterising members of diverse rna virus families within a human plasma background, some present at very low levels. by applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of hcv genotype , and a co-infecting human pegivirus. this method builds upon earlier rna metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses. a complex host-enriched sample was prepared by diluting in negative human plasma (nhp, negative for each hiv, hcv, and hev) stored plasmas from four samples previously characterized by routine diagnostic testing to contain hcv (x ), hiv and hev (see table for details). nhp was obtained by centrifuging negative human blood for minutes at × g to remove cell debris. the final concentration of each virus in the primary panel sample was iu/ml (copies/ml for hiv -implied by iu henceforth for convenience), and three serial tenfold dilutions in nhp were prepared from this stock. virus multiplex reference (vmr) panel. a reagent comprising a suspension in pbs of rna viruses with different genomic and structural characteristics was provided by the national institute for biological standards and controls (nibsc, potters bar, uk). each viral component and its approximate relative concentrations is given in mee et al. . prior to extraction, the panel was mixed : with nhp. duplicate μl extractions were performed , as was genotyping of the hcv and hev clinical samples of indeterminate hcv genotype. four plasma samples collected from a patient between and were submitted to public health england (phe) for metagenomic analysis as previous genotyping results had been inconsistent. the most recent such test employed ns b sequencing , and reported the presence of a virus belonging to genotype but was unable to resolve the subtype with any further precision. rna extraction and quantification. before extraction, all samples were centrifuged for min at , × g to remove cell debris. triplicate, duplicate and single extractions were performed on the diluted vmr panel samples (referred to as 'vmr panel a/b/pbs'), the bbv panel samples (' - -a/b'), and the patient sample series, respectively. a negative control comprising μl of the same plasma used to dilute the panels was also extracted. the split rna extraction kit (lexogen) was used to extract μl of each sample input, according to the manufacturer's instructions. acidic phenol was used to preferentially recover the large rna fraction, which was eluted in μl of nuclease-free water. rna eluates were quantified using qubit rna hs assay kit (thermo fisher scientific), which is accurate for concentrations between pg/μl and ng/μl. depletion of ribosomal rna and dna digestion. ribosomal rna depletion and dna digestion was achieved using the riboerase kit (kapa biosystems). as all sample extracts were below the detection limit of the qubit quantification system, the total rna input was less than the recommended ng. the manufacturer's specifications were followed with the exception of using the entire μl of the extract, and after the dna digestion reaction clean up, eluting the residual rna was in μl of nuclease-free water. in the case of the bbv panel, two of the three extracts of each dilution were treated with the riboerase kit before rna library preparation. the third set of extracts remained untreated and was used to monitor the effect of the rrna depletion and dna digestion upon the subsequent library preparation and sequence analysis. in the case of the vmr panel (the two duplicates) and the negative control, rrna and dna depletion was performed on all extracts. in the case of the uncharacterized hcv strain, extracts from all four samples were treated with riboerase. an additional, untreated, extract of sample was included, again to monitor the process. rna library preparation with ultra-low rna input. libraries were constructed from μl of extracted rna or μl of rrna-depleted dnase-digested rna, using the nebnext ultra directional rna library prep kit (new england biolabs). as the protocol is designed to use a minimum rna input of ng, several modifications were made to adapt it to an ultralow rna input. these are listed in table . libraries were analysed for size distribution using the high sensitivity dna kit (agilent) on a bioanalyser instrument, and were quantified using the kapa sybr fast universal qpcr kit for illumina libraries (kapa biosystems) on a real-time pcr system (applied biosystems). to determine the relative abundances of viral inserts, libraries constructed from the bbv panel were analysed by qpcrs with primers and probes targeting each of the three viral components (refs - and supplementary table s ). reactions were performed using the quantitect virus kit (qiagen) according to the manufacturer's instructions. libraries labelled with different indexes were diluted to nm and pooled. sequencing was performed on an illumina miseq instrument using the miseq reagent kit v ( cycles) (illumina) according to the manufacturer's guidelines, with the following minor modifications. the library pools were denatured with . n sodium hydroxide for minutes rather than , diluted in kit reagent ht to produce a pm solution and then these were further diluted to pm. of this library pool dilution, μl were loaded onto the miseq cartridge. data analysis. all paired end fastq files were processed with trimmomatic v . , removing the illumina adaptor sequences, then trimming leading and trailing bases with phred scores below . reads were discarded where the length of either trimmed end was below bases. for the determination of genome sequences of blood-borne viruses, trimmed fastq sets were normalised using the normalise-by-median.py script in the khmer package (k = , c = ) and submitted to the spades de novo assembler without error-correction, applying the default kmer sizes of , , and . output contigs that matched each virus were identified with the nhmmer function of the hmmer v . b package using hidden markov models (hmms) built from alignments of each virus (detailed in supplementary table s ) . where necessary, the ends of contigs were trimmed to the whole genome alignment. bwa mem (v . . a, default parameters) was used to map the original trimmed fastqs to the genome sequence, and the sam files were converted to bam files using samtools v . . while discarding reads with either × and/or × flags set (i.e. retaining only fully-mapped paired-end reads). base frequencies at each nucleotide position within each component virus sequence were obtained from bam files using quasibam v . , an in-house c++ program that tabulates base frequencies at each nucleotide position within a reference and generates consensus sequences based upon user-defined depth and variant percentages . mapping of trimmed paired-end fastq to one or more virus reference genomes was also performed using bwa mem . . a. in each case, two independent mappings were performed, using as a reference the viral sequences, supplemented firstly by the march 'grch ' release of the human genome, and secondly by a set of human rrna sequences (nr_ . , nr_ . , v . , nr_ . , gij : - , and gij : - , as per malboeuf et al. ). the second file was used solely to derive counts for reads mapping rrna which would otherwise be subsumed into the human genome mapping results. from the filtered sam files, the numbers of reads mapping to each reference sequence were counted. counts for each of the constituent sequences of the human genome and rrna were pooled into a "human" count and an "rrna" count. quasibam was used to derive nucleotide frequencies from which depth and coverage data were calculated. a minimum depth of was required for inclusion in a derived consensus sequence for the bbv panel ( for the vmr panel). bbv and vmr panel sequences. the members of the multi-fasta reference file for the bbv panel were obtained by submitting fastq sets from the rrna-depleted sample with the highest virus concentrations to the spades-hmmer-mapping approach described in the previous paragraph. vmr panel references were derived from sequences obtained from genbank using accession numbers from mee et al. . additionally, the complete genome sequence of a human pegivirus (hpgv) present in the plasma diluent was discovered in the spades contigs file. a hmm profile was constructed from an alignment of genbank sequences (supplementary table s ). sample with uncharacterised hcv. to obtain full-length hcv genomes, each fastq set was submitted to the spades-hmmer-mapping process. where a complete genome was not obtained, hcv-matching contigs were aligned to the full-length genomes using mega . in addition, contigs with length > kb that did not align to the hmm profile were submitted to blast for identification. following this analysis, an additional pegivirus genome was derived in similar fashion to the hcv genomes, using the same hmm profile as above for the nhp pegivirus. when calculating the read percentages and coverage plots, both sample-derived full-length genome sequences (hcv and hpgv) were used as the reference sequence when mapping that sample's corresponding trimmed paired-end fastqs, as well where only incomplete hcv genomes were obtained. virus (bbv) panel was prepared, comprising two strains of hcv (genotypes a and b), and one each of hiv and hev diluted in nhp to iu/ml. three tenfold serial dilutions in plasma were made from this original panel. step manufacturer's recommendations protocol modification riboerase rna input . . ribosomal rna depletion was performed on two of each set of triplicate extractions prior to all three being subjected to the modified library preparation protocol. data from the most concentrated rrna-depleted samples were used to generate individual virus genome sequences for use in reference mapping. during this data analysis, an unexpected human pegivirus (hpgv) was found and traced to the nhp diluent. the full genome sequence of this hpgv was determined from the -b data and included in the mapping references. table gives the read counts, genome coverages and median depths for each virus-dilution combination, across each of the three samples per dilution ( - -untreated/a/b). each test sample yielded over , reads with the exception of -a, which gave just over , reads. with the exception of the two samples, in which only a very small volume of nhp was added to the clinical samples, the percentage of reads mapping to the hpgv remained relatively constant at - %. the exception is -b, in which the overall viral read percentage was lower than expected, with a corresponding elevation in reads mapping to the human genome suggesting possible incomplete dnase digestion during the rrna depletion step (supplementary table s ) . with increasing dilution, the total viral read percentages (excluding hpgv) decline from over % to . %. complete and near-complete genome coverages with depths greater than were achieved at and iu/ml for all four viruses. a few short regions in hiv had low coverages (< ) with -b, reflecting the reduced overall viral reads in this dataset, but at a minimum depth of , . % coverage was achieved with this sample, with only a -base sequence in pol having no coverage. at iu/ml, hcv a and hev continued to give . - . % coverage with median depths over . hcv b and hiv gave . - . % coverage ( - median depth) and . - . % coverage ( - median depth) respectively, and in -b, despite only . % of all reads mapping to the four viruses collectively, genome coverages of . - . % were achieved, with median depths up to . figure illustrates coverages and depths across target genome at each dilution, showing even distributions of reads across all four target genomes and hpgv. pooling duplicates consistently improved coverages (final column, table ). this is most clearly seen at the lower viral loads, where at iu/ml, three of the four viruses achieve combined coverages of > . % each, and . % in hiv. at iu/ml, the combined coverages for the four viruses are effectively what would be expected were the individual coverages independent, i.e. cov aub depletion of rrna substantially enhances the recovery of blood-borne virus sequences. the percentage of reads mapping to rna virus genomes in the rrna-depleted bbv panel samples was between and -fold higher than in corresponding untreated controls. individual target virus ratios decreased as they became more dilute, from over -fold for hcv in -a to . -fold for hiv at the lowest dilution. concomitantly, the ratio for hpgv rose markedly, from . -fold in -a to in -b, reflecting an effectively constant viral load against decreasing quantities of panel viruses (table and fig. ). genome coverage and median depth values were also much higher in the treated samples than untreated comparators. at the two highest virus concentrations, median depths were between -and -fold higher in the treated versus the untreated samples. only short fragments of hev were recovered from the untreated dilution, and almost no hiv or hcv sequences. by contrast, near complete genomes from all four target viruses were recovered from the treated comparators, with median depths of between and (as noted above, hiv in -a was an exception at . % coverage and a median depth of ). recovery of partial and complete genomes of diverse virus types from human plasma. the ability of our method to recover genome sequences from a range of rna viruses in the context of human plasma was evaluated using a virus multiplex reference (vmr) panel, putatively containing genomically and physicochemically diverse viruses. two plasma-diluted panels and one pbs-diluted panel were tested (table ). no reads from either of the three samples mapped to either of the two norovirus genomes, coronavirus e or influenza b virus. by the panel distributor's qpcr , the threshold cycle (c t ) of the coronavirus was > and the other three were not detected, hence these four targets were excluded from further analysis. notwithstanding influenza virus a h n and parainfluenza virus type also not being detected by the qpcr, we recovered reads from both, with genome coverages ranging from . % to . %. almost no reads belonging to the panel's dna viruses were found. sixty-nine percent of all reads obtained from the pbs-diluted panel mapped to vmr panel genomes, dropping to - % for the plasma-diluted samples, although the distribution of reads between targets was very uneven. parechovirus and rotavirus accounted for . - . % and . - . % of all viral reads respectively, with the other viruses collectively accounting for . - . %. depths and genome coverages showed some inverse correlation with the given c q values (fig. ) . as with the bbv panel data, coverage plots of the samples diluted in plasma were largely unbiased, giving pooled genome coverages close to those expected by independent distributions of reads between replicates ( table , final column). rotavirus and coxsackievirus were exceptions, where despite large numbers of mapped reads, almost identical patterns of read coverages and gaps were observed between their replicates, with minimal additive effect. the pbs-diluted sample gave larger read numbers, but their distribution was less even throughout the genomes, resulting in relatively lower coverages. characterisation of a new subtype belonging to hcv genotype and discovery of a second virus in a patient sample series. four plasma samples from a patient with hcv were used as starting material. all extracts were subjected to riboerase treatment; a second extract of sample remained untreated for comparison. de novo assembly analysis of fastq sets from samples , and each gave a full-length hcv genome sequence as a single contig. for sample , partially-overlapping contigs were obtained, covering % of scientific reports | : | doi: . /s - - - the hcv sequence. additionally, in all four samples, a single contig was obtained that was determined by blast and subsequent hmmer analysis to comprise an hpgv genome. the hcv and hpgv full genome sequences were combined in a single file to carry out reference mapping and nucleotide frequency determination on the four sample fastq sets (table ). samples , and had hcv read percentages ranging from . to . %, and gave complete genomes with median depths greater than . sample had the lowest viral load ( , iu/ml), had . % of reads mapping to hcv giving a genome coverage of % at a minimum depth of ( . % at depth ≥ ) and a median depth of . full coverage of the hpgv genome was obtained from all samples, with median depths over , , and read percentages ranging from . to . %. the depth plots in fig. again show unbiased and even coverages across both genomes, and the percentages of reads mapping to viral targets was again much higher in the rrna-depleted sample than in the untreated comparator ( -fold and -fold for hcv and hpgv respectively). table . detailed sequencing data from the bbv panel. for each of the three samples (untreated, a and b) at each dilution ( - ), the number and percentage of reads mapping to each virus are given, together with the genome coverages (depth ≥ ) and median depths. the final column gives these last two metrics from the combined data sets of both the a and b samples. included in the analysis are data for the hpgv discovered in the sample diluent. analysis of the hcv sequence showed it to belonging to a new subtype within genotype of which the details are presented in a separate manuscript (in preparation). the hpgv clustered with genotype strains, and is distinct from the nhp strain. including the nhp negative control were subjected to virus-specific qpcr for the detection and quantification of hcv, hiv and hev. all were detectable in the sample libraries, but were undetectable in the riboerase-treated negative control library (supplementary table s ). all samples were mapped against reference sequences that included human genome and human rrna sequences to evaluate the efficiency of riboerase treatment. the average ratio of the percentages of reads mapping to rrna in the untreated versus the treated samples was -fold with an approximate halving of the number of reads mapping to the human genome, across all panels (fig. ) . with the exception of the expected human pegivirus, mapping of the negative control fastq set against the reference sequences of the four bbv panel viruses, the two pegiviruses, the vmr panel and the patient hcv gave table . detailed sequencing data from the patient sample series. for each of the four samples - , the number and percentage of reads mapping to both the hcv and hpgv genomes are given, genome coverages (depth ≥ ) and median depths. the analysis of sample extracted without host rrna depletion is in the untreated column. very low numbers of reads mapped to viral genomes and no consensus sequences could be derived. further data for this section are found in supplementary tables s and s . in light of the large and ever-increasing number of human rna virus pathogens, it is perhaps unsurprising that standard serological assays and nucleic acid tests suffer from a lack of sensitivity to diverse variants of target viruses, overlook the presence of new or unexpected viruses, and provide only limited information about those targets they do successfully detect. hence the three main aims of metagenomic virology are to detect & identify known agents irrespective of their diversity, to discover novel agents of disease, and to obtain complete sequence information of detected viruses. most existing protocols achieve a maximum of two of these aims, but difficulties in selectively isolating viral rna species and short read sequences from those of the super-abundant host nucleic acid have limited the utility of metagenomic approaches in diagnostic virology. this study has addressed these limitations by establishing a novel methodology suitable for the agnostic detection and characterization of blood-borne rna viruses in plasma samples. by depleting host-derived nucleic acids and making modifications to an existing library preparation protocol to account for ultra-low rna input quantities, we have been able to reconstruct effectively full-length genomes of hcv, hev and hiv from plasma samples with viral loads of iu/ml (copies/ml for hiv) and substantial fractions of complete genomes at iu/ml. when applied to a series of clinical samples, we could elucidate simultaneously the full genome sequences of both a novel subtype belonging to hcv genotype and a hitherto-undetected human pegivirus. additionally, our system was able to recover viral sequences from a panel of diverse rna viruses diluted in human plasma, with a broad correlation between the genomic coverage and depth metrics and approximate concentration. although full genomes were not assembled in many cases, the independence of read distribution gave sufficient genome coverage for identification. the vast majority of rna molecules in a human plasma sample are host-derived, of which up to % comprises the six species of human rrna. their presence in our libraries was minimised by two key protocol steps in our modified protocol. firstly, we selected an extraction method that combined a phenol/chloroform step with a column format (lexogen split rna) which increased the amount of extracted viral rna by up to one log when compared to other extraction methods (data not shown). perhaps more importantly, by controlling the final precipitation step, small rna molecules below nt such as s rrna and trna are excluded from the eluates, as are the majority of molecules of human genomic dna. secondly, we employed dna probes complementary to human rrna such that hybridisation and subsequent digestion by rnase h dramatically reduced their frequency in the finished libraries. whilst this methodology has been successfully used in the detection and characterisation of two haemorrhagic fever viruses, the frequency of viral reads was often below % and an additional hybrid-capture step was employed to elevate read numbers . methods that do not deplete rrna generally give poor recovery of viral reads, yielding viral genome fragments that necessitate further work , , , , low read numbers even at viral loads over iu/ml [ ] [ ] [ ] , or at best, requiring dilution of both host and virus in pbs in order to recover full hiv genomes at low copy numbers . the resultant rrna-depleted sample extracts typically contain quantities of nucleic acid in the low picogram range. library preparation through hexamer-mediated reverse transcription followed by multiple displacement amplification constitutes an easy and effective means of amplifying very low amounts of dna , , , but in several studies (and in the authors' laboratory), significant amplification biases have been observed, leading to gaps in target genome coverage , [ ] [ ] [ ] . consequently, we adopted an approach using a standard rna library preparation kit, but with substantial modification to compensate for their minimum rna input requirements of at least ng and optimally ng- µg. we made key changes to the rna fragmentation and adaptor-ligation steps of the nebnext ultra directional rna library prep kit protocol. while prior rna fragmentation with heat and divalent cations improves sequence coverage, over-fragmentation of target genomes leads to the loss of material during the library preparation process . lower amounts of rna thus require shorter optimum fragmentation times and we found that minute at °c was optimal in terms of breadth of genome coverage. under standard kit conditions, our ultra-low rna inputs dramatically skewed the ratio of cdna to adaptor. the resulting adaptor excess led to the preferential amplification of adaptor dimers during the pcr step, and despite increasing cycle number to amplify low rna inputs, we were generally unable to generate sufficient quantities of target-specific material. accurate quantification and consequent equimolar pooling of libraries was compromised, as was the miseq clustering efficiency. we found that a reduced final adaptor concentration of . nm was crucial in reducing the amount of adaptor dimers in libraries from rrna-depleted samples whilst simultaneously extending the pcr cycle number. in the present study, serial dilutions of the blood borne virus panel were prepared in negative human plasma, reducing both the absolute quantity and relative frequency of the viral rna targets while maintaining the complexity of the sample in terms of host nucleic acid, thus mimicking that of a clinical sample. with rrna depletion, the number and diversity of viral reads was consistently high, with over % of all reads mapping to constituent virus genomes. throughout the three sample series, we obtained relatively high genome coverages of low-frequency viral targets. co-infections with multiple blood-borne viruses are common , so whilst we speculate that the depths and coverages of target viruses would be greater yet in these samples had it not been for the confounding effect of the unexpected human pegiviruses in both the plasma diluent and the patient sample series, it was reassuring to see the method performed well under such conditions. in our experiments using negative human plasma as sample diluent, we were able to recover levels of viral genomes comparable to previous work using pbs, both for bbv panel viruses and for the vmr panel , and we were able to recover from a patient sample a large percentage of the genome of a previously uncharacterised subtype of hcv genotype when present at × iu/ml, a diagnosis not possible using existing genotyping assays. the presence of an undiagnosed pegivirus in this sample further demonstrated the utility of the method in metagenomic analysis of blood-borne virus co-infections where the relative abundances of each virus can be highly variable . furthermore, in three of the four samples, depths greater than , were routinely obtained, which are likely to be sufficient to call minority variants for clinical resistance . a full description of the patient series and the new hcv strain are provided in a separate manuscript (in preparation). our approach can therefore not only accurately characterise rare or novel variants of existing viruses, but also generates the same level of information regarding unexpected viruses present in the sample. by comparison, vidisca , , and other random amplification-ngs techniques , have detected novel viruses in diverse clinical samples, but all have required further techniques to achieve full genome sequences. together with the vmr panel results, we were able to recover identifying sequence from both enveloped viruses (hcv, hiv, hev, influenza, and several paramyxoviruses), and non-enveloped viruses (several enteroviruses, astrovirus, rotavirus, and sapovirus). for the majority of viruses in the vmr panel, whilst dilution in plasma reduced the total percentage of reads recovered when compared to the panel diluted in pbs, a greater breadth of genome coverage was achieved. in the absence of any host nucleic acid background, it is possible that the pbs extracts had such ultra-low quantities of rna that despite the adjustments made to the library preparation protocol, the rna was over-fragmented, leading to a smaller number of genome fragments that were individually amplified to a greater extent than the larger array of fragments surviving the plasma extraction. in developing a similar approach, kohl et al. were only able to recover a percentage of reads exceeding % at a viral load over copies/ml. at an influenza a virus concentration of over copies/ml, this dropped to just . %, and at a reovirus concentration of - copies/ml, no viral reads were detected . with our method, whole genomes were obtained for those with the highest viral loads, and for minority viral targets, there was a correlation between ostensible quantity and coverage, including for two viruses undetectable by the panel distributor , a result superior to that recently obtained from influenza in clinical respiratory samples . again, the presence of high quantities of one or more target is likely to have inhibited the representation of the minority species such that if tested individually, superior depths and coverages would seem likely. with further reduction to the fragmentation time, or even its abolition, it may be possible to use this method to reconstruct genomes from old, partially degraded samples such as those recently used to re-evaluate the early hiv epidemic in the americas . our negative control data suggest that the level of contamination is low, with most viral reads therein belonging to the most abundant vmr panel member. nelson et al. identified a second source of contamination consisting of incorrect reads from other libraries that were sequenced during the same sequencing run due to truseq index misassignment (~ . % of reads, . % here). although cross-contamination between samples during the library preparation can be another source of contamination, the qpcr results suggest no bbv panel genomes were present after library preparation in the negative control sample. to conclude, by applying the three adaptations of selective large rna extraction, rrna depletion-dnase treatment, and the extensively modified library preparation in combination, ngs data sets can be produced from plasma samples that are rich in rna virus sequence data. complex bioinformatic processing has been employed to identify viruses within a metagenomic dataset , , , , , , but here, only simple bioinformatic processing is needed for detection and identification of known viruses, and by applying only moderately more advanced tools, an agnostic approach to virus detection can be taken, together with characterisation of the full genome even at low viral loads. the role of mutational robustness in rna virus evolution rna virus quasispecies: significance for viral disease and epidemiology ecological origins of novel human pathogens isolation of a novel coronavirus from a man with pneumonia in saudi arabia zika virus in the americas-yet another arbovirus threat reappearance of chikungunya, formerly called dengue detecting the emergence of novel, zoonotic viruses pathogenic to humans human viruses: discovery and emergence estimates of global, regional, and national incidence, prevalence, and mortality of hiv, - : the global burden of disease study global epidemiology and genotype distribution of the 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hepatitis b and hepatitis c viruses in sub-saharan africa highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data identification of a new human coronavirus identification of a contemporary human parechovirus type by vidisca and characterisation of its full genome evaluation of unbiased next-generation sequencing of rna (rna-seq) as a diagnostic method in influenza virus-positive respiratory samples patient ' hiv- genomes illuminate early hiv/aids history in north america analysis, optimization and verification of illumina-generated s rrna gene amplicon surveys a novel virus in swine is closely related to the human hepatitis e virus a modified rna-seq approach for whole genome sequencing of rna viruses from faecal and blood samples the authors would like to acknowledge the contribution of nibsc for kindly providing the viral multiplex reference panel and the blood borne virus unit of virus reference department, phe, for providing the hev sample. the genomic services unit at phe are acknowledged for running the miseqs. the authors are grateful to dr. brendan healy, dr. owen seddon and dr. nicola price from university hospital of wales for providing additional information regarding the patient sample series. this work was supported by funding from public health england. supplementary information accompanies this paper at doi: . /s - - - competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - ze w authors: büchel, beda; spanninger, thomas; corman, francesco title: empirical dynamics of railway delay propagation identified during the large-scale rastatt disruption date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: ze w transport networks are becoming increasingly large and interconnected. this interconnectivity is a key enabler of accessibility; on the other hand, it results in vulnerability, i.e. reduced performance, in case any specific part is subject to disruptions. we analyse how railway systems are vulnerable to delay, and how delays propagate in railway networks, studying real-life delay propagation phenomena on empirical data, determining real-life impact and delay propagation for the uncommon case of railway disruptions. we take a unique approach by looking at the same system, in two different operating conditions, to disentangle processes and dynamics that are normally present and co-occurring in railway operations. we exploit the unique chance to observe a systematic change in railway operations conditions, without a correspondent system change of infrastructure or timetable, coming from the occurrence of the large-scale disruption at rastatt, germany, in . we define new statistical methods able to detect weak signals in the noisy dataset of recorded punctuality for passenger traffic in switzerland, in the disrupted and undisrupted state, along a period of year. we determine how delay propagation changed, and quantify the heterogeneous, large-scale cascading effects of the rastatt disruption towards the swiss network, hundreds of kilometers away. operational measures of transport performance (i.e. punctuality and delays), while globally being very decreased, had a statistically relevant positive increase (though very geographically heterogeneous) on the swiss passenger traffic during the disruption period. we identify two factors for this: ( ) the reduced delay propagation at an international scale; and ( ) to a minor extent, rerouted railway freight traffic; which show to combine linearly in the observed outcomes. the interconnectivity of transport networks makes them vulnerable when parts of the network experience a reduced performance, or fail . a performance reduction caused by smaller or larger disruptive events has specific geographic and temporal dynamics of reduced transport performance . typically, disruptive events triggered in a specific limited area result in a degradation of performance, which then propagates in the network. the system stabilizes at a less performing state, also considering some management actions, and finally recovers with a certain rapidity towards its original performance. in railway networks, non-performance results in delays, which propagate through the system. the mechanisms of delay propagation directly reflect the complex, concurrent and co-occurring processes characteristics of railway operations. to disentangle them, we study delay propagation (i.e. how railway systems are vulnerable to small reduced performance), through comparison of the same railway system in two operating conditions. we exploit a unique chance in the real world, to observe a systematic change in railway operations based on empirical data, without the typically co-occurring changes in infrastructure or timetable, given by the large-scale railway disruption at rastatt, germany, in . the quantification of delay propagation clarifies how the disruption had a measurable effect on the swiss network. the quantification of the vulnerability of transport networks has been attracting much interest [ ] [ ] [ ] . in topological studies of link or node failures, network-based properties are predominant [ ] [ ] [ ] [ ] . including service level aspects allows quantifying travel time and capacity , . determining the relation between link, node, network characteristics, and risk or exposure of a theoretical small or large disruption, allows a-priori quantification of resilience, and determination of strategic actions such as reducing vulnerability of some links , , [ ] [ ] [ ] . once a disruption happened, management and recovery actions (changing network structure and usage of links scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ disruption, and with no precisely identifiable root cause. we determine how delay propagation changed, and quantify the effects of the rastatt disruption towards delay propagation in the swiss network, more than two hundred kilometers away. we focus on the swiss network, where basel sbb (basel in short) and schaffhausen are the border stations where cross-border trains coming from germany and rastatt have to pass. the rastatt disruption resulted in two explicit changes to the railway traffic in switzerland, see fig. . some freight trains coming from germany and the north, along the rhine alpine corridor, changed route from the boundary point at basel to the boundary point at schaffhausen; up to a quarter of the freight traffic has been canceled or rerouted, not entering anymore switzerland . this is similar to a pressure decreased at one place, and applied in reduced form to a different part of the system. the passenger trains coming from germany and the north have been short turned, and were running via basel between switzerland and offenburg ( km away) rather than via basel between switzerland and hamburg or berlin ( km away). in the swiss network, the planned timetable and infrastructure usage of all passenger trains remained the same, with no significant cancellations or adjustments. nevertheless, the short turning of the long-distance international trains resulted in smaller variability of actual operations, and less entrance delays when entering into the swiss network at basel, which hints at a reduced epidemic spreading of delays in a network , . we empirically verify how the specific propagation dynamics and cascading effects are heterogeneous, with different development, spreading and fade out, in the disrupted and undisrupted state. the rastatt disruption, with large global negative effects, lead to actual measurable improvements of performance for passenger traffic, on part of the swiss railway network, and smaller performance decrease on other parts. a simulation represents well the observed behavior, and shades light on the relative magnitude, and linear interaction, of the two explicitly modelled changes. in the specific test case, the propagation of a lower initial delay has a much larger network impact than a change and rerouting in freight traffic volume. effects around basel. we analyse with a set of proposed metrics (see detailed description in the methods section) the variations in delay propagation of the passenger traffic arriving in basel sbb from germany; and passenger traffic (regardless if they were coming from germany, or originated in switzerland) at their first stop from basel sbb. we cannot consider delay changes for freight traffic, as its volume was strongly affected by the rastatt disruption; and more generally, freight traffic does not stop at stations thus having a ill-defined delay; and scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ despite its buffer times are typically large, its performance is often erratic. passenger traffic instead experienced no significant cancellation in switzerland, and its timetable remained unchanged throughout the disruption. we graphically report the time series of the th, th, th, and th percentile values of the delays of all long-distance passenger trains arriving at basel sbb from germany. we consider a moving average over days, over the course of the timetable years and . throughout the years, almost half of the traffic had more than min of delay. the and percentile are more stable than the and percentile. the highest delays of this observation period of years are reached just before and after the disruption, presumably due to the construction works in southern germany. during the disruption, all percentile time series have distinctly lower delays than before and after the disruption period. the gap between the low and high percentile (i.e. the variation of the delays) is remarkably smaller during the disruption period. a reduced performance has been linked to a reduced variability of performance during a disruption also in other cases . table reports the quantitative evaluation using the novel metrics proposed, described in the methods section, over the timetable year . i diff quantifies the likelihood that the single shocks at beginning and end of the disruption occur elsewhere throughout the data (the higher, the more exceptional the observed behavior was during the disruption). i sum quantifies the likelihood that the combined shocks at beginning, and recovery at the end of the disruption occur elsewhere throughout the data (the higher, the more exceptional the observed behavior was during disruption). both indicators report that the change during this period is rare (half of i diff and i sum are higher than . ), especially those for the highest percentile levels. the ks-test reports the statistical significance of the pattern observed in a months horizon, i.e. quantifying the likelihood that the samples observed within the disruption belong to the same distribution than before and after the disruption (the lower, the more exceptional the observed behavior was during the disruption). the t-test reports the statistical significance of the pattern observed over the entire year, i.e. quantifying the likelihood that the samples observed during the disruption have the same mean as the data observed outside of the disruption, over a period of year (the lower, the more exceptional the observed behavior was during the disruption). both statistical tests report the significance of the variation, with extreme strength in basel and zürich, and smaller strength in rheinfelden and olten, probably due to the different timetable structure and services running. at the . and . percentile, except liestal, both ks-test and t-test metrics reported significance. the mean quantile deviation (mqd) gives the magnitude of the variation observed, in seconds. all reported values are negative, i.e. the delay decreased during the disruption, with the higher percentiles showing a larger decrease. in basel, the decrease of the th percentile is more than min, and % of the traffic had its delay reduced by a minute or more. in zürich, the effect is a reduction of delay by around s, up to almost s for the highest percentile; the strongly delayed trains performed better in the disruption period. this signal is weak compared to the noise, i.e the median delay has been ranging between and s throughout year . the performance change was not uniform, but depends on the percentile level, i.e. the prevailing delay. effects around schaffhausen. due to the disruption, freight trains running on the rhine alpine corridor have been globally rerouted or cancelled. figure reports the absolute difference in number of freight trains actually running in the swiss network, on an average month of the disrupted period compared with an average month in . the different rail corridors have different total infrastructure capacity, and different ratio of passenger/freight trains. the increase in the schaffhausen-zürich corridor (red, more freight trains per month, www.nature.com/scientificreports/ normally - freight trains per month) is relatively much stronger than the decrease in the basel-gotthard corridor (violet, freight trains less per month, normally - freight trains per month). the impacts of this variation in infrastructure utilization (by the freight traffic) on the performance of the swiss passenger railway traffic (sharing the same infrastructure) are analysed with the proposed metrics in table . we consider the passenger traffic arriving in schaffhausen from germany; and passenger traffic (regardless if they were coming from germany, or originated in switzerland) at their first stop from schaffhausen. the number of passenger trains running (originating in germany, or in schaffhausen) remained unchanged throughout the disruption. in schaffhausen, the metrics report small and insignificant (t-test above . ) changes in delay. all other stations report a positive deviation in delays (mqd). the metrics i sum reports a higher value, i.e. more significant variations, than i diff , and this reflects the slower onset of the management actions in the area. rerouting of freight trains from germany via schaffhausen needed some start-up time; in the first days immediately after the disruption, freight companies preferred to wait, and they had permissions and resources (drivers with permission to run over this part of the network, as well as locomotives and train paths) only some time later , . the reversion to a normal state at the end of the disruption, as identified by the cumulative shocks considered by i sum , was instead rather rapid, and identified as significant. in fact, after the disruption the operation www.nature.com/scientificreports/ changed immediately back to the rhine alpine corridor, which offered faster travel times and larger infrastructure capacity. the freight corridor used by the additional freight trains avoids major stations and had smaller and less significant impact to schaffhausen and zürich, than to bülach, where passenger and freight traffic must necessarily interact. in this last station, the effect is strongest with almost half of the traffic facing an additional delay by half a minute or more; both ks-test and t-test show significance, one or more orders of magnitude smaller than α = . . overall, the delay increase identified is remarkably different from the delay reduction seen for the basel area, hinting at a strong heterogeneity between the two areas. within the schaffhausen area alone, the influences are again heterogeneous, with strongest impacts through microscopic delay propagation along the corridor, as experienced in bülach. the quantitative analysis is in good agreement with the observed actions. global validity of the effects. we also investigated if some systematic variations happened at the same time in the swiss network (co-occurrence) but were unrelated to the rastatt disruption (a weak causality analysis ). we performed the same analysis for a swiss train station of comparable size and traffic, far enough away from the disruption, that according to general theories of disruption impact, should have low or even unquantifiable effects. the data (reported as supplementary material) shows no specific evidence or special effect in the disrupted period, with i diff and i sum mostly around . . the t-test identifies some significant changes for half of the stations investigated, while the ks-test identifies only one significant observation. we trace this to a large variability of yearly operations, and the variability encountered during the disruption has been no different than otherwise throughout the year. the mqd has an overall erratic behavior, with a negligibly small increase, and some decrease at different stations. this shows also how a single metric cannot describe the complexity and noise in the data, and a variation with both fast and persistent dynamics. overall, no clear and generalized variation of performance is evident elsewhere in switzerland during the rastatt disruption. the stations near basel and schaffhausen experienced a variation during the disruption, which was not significantly experienced in the rest of the swiss network, around lausanne. this matches well the currently accepted theory of spreading of disturbances throughout networks, which identifies a maximum geographical dimension of the impact, from few boundary points. identifying magnitude of the root causes by simulation. we now discuss how a state-of-the-art simulation model, ontime, based on typical delay propagation theory (the methods section describes its assumptions and functioning, and its calibration for the test case) can partially replicate the degree by which the delay performance of passenger trains improved in the area of basel, and decreased in the area of schaffhausen. moreover, we aim to identify which root cause is responsible for which observed effects, i.e., disentangling the effects of co-occurring actions, business processes, and operations. we compute the delay of passenger trains in the baseline condition (for a typical day, immediately before the disruption) and for a typical day during the figure reports the variation in median delay from undisrupted to disrupted situation (green-red color, mapped between − to + s for all individual stations and measurement points where trains pass and/or stop, green reports a delay decrease during a disruption) of all passenger traffic originated in the focal points at basel or schaffhausen, for the recorded data (left) and simulated traffic (right). visually, a good agreement between the observed and simulated variation of delay propagation is present, especially when filtered by the volume of traffic (i.e. line zürich-chur). the variation along a line might be discontinuous, as the services running are heterogeneous, and have different stopping patterns; this stresses the need for a detailed study. a set of station areas is analysed more in detail, including basel, olten, schaffhausen, bülach, zürich discussed before, as well as bern, in the interior of the country, south of basel; st. gallen and chur towards the east of the country. table shows, per each station, and for the observed and simulated conditions, the variation of the median arrival delays (in s) between the undisrupted and the disrupted situation, plus sample % confidence intervals, for the observed value; the attribution of the delay variation estimated by the simulation to reduced entrance delay or freight rerouting; and the daily amount of trains considered. confidence intervals of the observations are the largest relatively far from the focal node, resulting in less delay observations (bern, chur), as well as at stations with a major change in median delays (basel, schaffhausen). ontime models well the sign and relative size of the variations, less well the absolute values. all stations connected to basel enjoy a reduction of delays, decreasing with the distance. for schaffhausen, the simulations show no variation, while in reality the delay substantially worsened. this mismatch depends on the modelling of the entrance of freight trains in the network, which does not conflict with the arrival delay of trains at schaffhausen, in the model. the stations connected to schaffhausen are correctly identified having an increase in delay (see bülach); in zürich, both effects interact, with a small net increase of delay. the effects for schaffhausen and chur affect much less traffic than busy stations like basel, olten, zürich. in general, ontime underestimates by a factor to a factor the magnitude of effects, even though their relative magnitude is in good agreement with observation. one reason for this is the usage of exponential distributions in ontime, which do not replicate well the long tails that reality showed. moreover, the passenger traffic at all stations includes many local trains, which (depending on the timetable, and the station) might have a larger or smaller degree of interaction with each other at microscopic level, due to their specific platform used at stations, route chosen in the interlocking area, and precise departure/ arrival time. the delay of long distance traffic (discussed in the previous sections) is therefore diluted into the general picture. those effects of delay dilution are weaker in basel, chur and bern. finally, the interaction of special business rules not considered in a purely table . graphical legend for figure , and observed/simulated performance [s]. www.nature.com/scientificreports/ operative perspective (availability of vehicles, drivers, passenger transfer, etc) has been more complicated than what ontime has been able to replicate. the exceptional situation was so special that modelling the same system with some minor changes (as ontime did) might not have been enough to capture the true magnitude of effects. the extra delay observed in real life compared to the simulation gives an idea of the suboptimal planning of freight traffic done in reality, in a hurry, and under strong time pressure, in the area around schaffhausen, which had moreover to cope with exceptional situations for train drivers, passengers, freight companies, schedulers, dispatchers, as typical in exceptional situations , . we simulate separately the individual effects of the two explicit changes modelled during the disruption, keeping the other as in the baseline, to understand their respective role and impact. the total net effect is the sum of two opposite effects: the improvement in entrance punctuality at basel has positive effects on the network about twice as strong than the worsening due to rerouting of freight traffic. the two effects interact at few stations, like zürich and basel, but have a reduced interaction, adding up mostly linearly. in basel, over a daily total amount of around trains, their observed median delay during the disruption is reduced from to s. the simulator estimates the effects of the reduced entrance delay being % of this gap, while the effects of rerouting of freight trains thereby releasing infrastructure capacity, decrease the median delay by the remaining %. in both this detailed view and the general picture of table , the two effects are quantifiable. this shows how in railway networks both nodes (decreased delay propagation due to smaller entrance delay) and links (increased/decreased delay propagation when more/less traffic is running) are crucial in propagating non-performance. moreover, the magnitude of those effects is variable, and depending on prevailing conditions: each additional train running in a congested infrastructure has increasingly larger negative effects, which might not be compensated by running a train less elsewhere. in the specific case, the negative effects of freight train rerouting around schaffhausen are stronger than the positive effects of train rerouting around basel. transport networks are subject to large-scale changes due to sudden disruptions (i.e. hurricanes, earthquakes; infrastructure collapse; terrorist attacks; pandemics or other limitations to mobility). we exploit those changes as they expose mechanisms which are typically co-occurring and mediated by other factors, to understand railway delay propagation dynamics. due to the rastatt railway disruption, the rhine alpine corridor from the netherlands and northern germany to southern germany, switzerland, and italy, was effectively split into two, and this had a measurable effect on delay propagation on a large part of the swiss network. by using the delay of passenger trains as a measure of the network performance, over more than a year worth of operational data, at more than ten stations, the shocks in delay propagation experienced, related to the disruption could be separated from the overall spurious variations and noise in operations performance. as a result of management actions, passenger trains were entering in switzerland from germany with lower potentially accumulated delay, and freight trains were circulating in reduced number and entering the country at a different boundary point. this resulted in an overall positive effect of reduced delay, with large heterogeneity in space , by consistently lower delay propagation of all long-distance passenger traffic in a large area around basel. the area around schaffhausen experienced increased delays in its passenger traffic, due to the additional freight traffic. the part of the swiss networks not directly connected with those two boundary points showed no clear and generalized variation of performance. similar complex effects of functional changes and reorganization within a system due to a shock, resulting in compensatory, or even superior, local behavior ideally match those which have been long postulated, and recently demonstrated in living beings and humans , . the sheer scope of the global effects of the rastatt disruption spanned multiple countries. even restricted to the swiss railway network, this disruption proposes a test case much larger than most comparable studies in the literature discussing effects of disruptions, focusing on a city , or the area around a collapsed single bridge . due to the high variability encountered over this long duration and large geographical extent, typical statistical tests have limited strength in identifying the difference in delay propagation, and isolate the contribution of the disruption. newly designed statistical tests were applied to delay analysis in railway networks, performed at different percentile levels, over multiple time scales, and two specific factors (reduced entrance delay for passenger trains and rerouted freight trains). they could significantly differentiate the variations in passenger train delays from the noise. their respective magnitude has been quantified by simulation, with overall good agreement. the system-wide delay propagation dynamic has been described, with a reduction of delays from basel propagating along the network during the period, with smaller and less identifiable effects farther away, where the magnitude of other spurious effects becomes relatively larger. for high percentile values (i.e. stronger non-performance) the reduction in delays propagation was larger than for low percentile values, meaning particularly the strongly delayed trains performed better in the disruption period, with an overall stabilizing effect of improved performance. the variability of delays in real-life operations is extremely high; no model of first order or second order, or with a time series analysis could explain all the observed variance of the data. the simulation model used could replicate some effects with a high degree, especially at network scale, but a lot of improvised and non standard business rules in the aftermath of a disruption challenge the mathematical power of such models at the level of precise nodes. compared to the state of the art, this work proves that railway disruptions behave distinctively over time, space and processes. delay propagation in railways, differently from other networks, happens at microscopic level along links and nodes, through conflicts for infrastructure capacity at block section level; global effects are instead appreciated only at macroscopic scale , , . further empirical studies on other railway networks, or other transport networks can identify to which extent the identified dynamics on nodes and links occur in other networks. the specific scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ mix of discrete availability of links and nodes, and continuous movements makes the study of railway systems particularly interesting. our study provides evidence supporting the validity of railway delay propagation theories at a network level. in the analysis proposed, we identified how railway networks suffer from local instabilities (i.e. delay propagation is common) even though having global stability (i.e. delay propagation is of limited magnitude, and can be absorbed by buffer times, sufficiently away from the disruption) . even a highly sophisticated simulator routinely used in practice is not able to replicate all magnitudes of delay variation observed, probably due to the choice of underlying probability distributions, and the limited account to specific routes and service stopping pattern running in a real life network. topological studies have limited power to describe real-life microscopic delay propagation, and relation traffic-performance. exponential scaling models or epidemic spreading models could be further extended to include the observed performance-depending vulnerability effects. simulation , or optimization models need to bridge the large gap between detail and complexity of microscopic and macroscopic studies, which we only scratched in this study . we focused on operational performance only, during the disruption, and did not aim to quantify economic or social costs of the disruption , rerouting or mode choice of passengers , , nor to specify mitigation or management actions reducing delay propagation, or disruption impact , . further studies should consider impacts on different stakeholders with different performance measures . agent-based models could help in computing passenger costs, assuming that sufficiently accurate modelling of the non-equilibrium behavior of passengers during disturbed or disrupted operations can be implemented , . moreover, every disruption is one-of-a-kind, and its occurrence triggers often previously unseen dynamics , . in the rastatt disruption, freight traffic experienced very large variations from its planned and usual performance; most probably, passenger demand has also been affected and reduced. it is almost impossible to identify or quantify all those effects, and the reasons why some mitigation actions have been chosen, their objective, and effectiveness , , . the availability of smart decision support , is very relevant for design of proactive railway traffic control , and future contingency schemes against disruptions , . we quantified that the system-wide effect of specific management actions combine linearly, i.e. (higher-order) interaction of delay propagation by management actions is marginal. this allows decentralized, decomposed approaches each optimizing a specific item to solve parts of the problem with limited interaction. our study indicates how the relation between train volume and performance is non linear, with an increasing vulnerability for each extra train running in a congested network. the benefits of relieving a corridor from some traffic are to be traded off with the increased traffic somewhere else, when proposing rerouting as a railway traffic management action , . finally, one important question is how to manage disruptions of this size, i.e. which optimization model can deal with such extreme conditions; and how to include the newly discovered patterns of delay propagation in timetable design , . specifically, it has been observed that a shorter circulation of international trains (travelling between germany and switzerland) contributed to substantially better delay performance in an entire region. how to integrate this finding in timetable design, and balance punctuality against the comfort of the passengers that would need to transfer between two more punctual trains? non parametric identification of shocks in time series. a disruption is assumed to lead to a significant change in a very short term to the time series (i.e. a fast shock), which remains for a certain amount of time corresponding to the entire disruption duration (persistent degradation), and a significant opposite change at its end (i.e. a second shock recovering the first, while returning to full functionality). the pattern of railway delays is generally highly variable; any difference between any day in the disrupted period and any day in the nondisrupted period can also be imputed to many other sources co-occurring. the goal is to quantify the probability that a difference between two consecutive samples in a time series is a spurious product of existing noises, or is related to a fast, persistent, recovered phenomena, happening at the same dates as the disruption. in this latter case, we assume the variation observed is imputable to the disruption. we define three statistical indicators, at different time scales of week, month, year, to identify in time series a fast persistent and recovered disruption as a shock, stabilization to a disrupted level, and return to normality. to avoid biases from the variations of the timetable within the day (peak hours, nights) and week (reduced service at weekends), we focus on percentile levels ( p ∈ p = { , , , } ) of the time series of observed arrival delays collected across seven consecutive days. we ignore extreme values (min, max) as those might be traced to vehicle failures and result in cancelled services, with no relation to the disruption. for any percentile level p ∈ p and any day i ∈ i , let d i,p be the difference between the p-th percentile of the days right before day i, and the days right after day i. the distribution d p describes the probability of the difference between the percentile at weekly scope (d refers to the set of all d p s). specifically in this distribution d p , relating to the beginning/end of the disruption b/e (respectively), d b,p is the difference between the p-th percentile delays of the days before the disruption and the respective value of the first days in the disrupted period; d e,p is analogously the difference between the p-th percentile delays of the days before the end of the disrupted period with the mean value of the days after the disruption end. the hypothesis to test is whether those differences are significant, i.e. if d b,p and d e,p are common values in the distribution d p , the disruption did not have significant effects; if those two values are extreme events, the disruption had a special impact. two metrics i diff and i sum respectively describe how likely (based on the observed samples) the differences computed for the beginning and end shock are to be found in the overall distribution of d p throughout the entire time series. formally, | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ each i diff ,p determines the likelihood that any two periods of two consecutive weeks have a minimum variation in delay, at the percentile level p, as large as the minimum between the one observed at the beginning and end of the disruption. each i sum,p determines the likelihood that any two consecutive periods of week have a cumulative absolute variation in delay, at the percentile level p, as large as the cumulative absolute variation in delay observed at begin and end of the disruption. both metrics can take values between and . the higher the value is, the more infrequent the change observed at the date of begin/end of the disruption is, in terms of absolute variation ( i diff ,p ) or cumulative shock and recovery ( i sum,p ). the usage of percentiles enables understanding if the small delays (low p), or the large delays (high p), observed during the disruption are more unlikely. both metrics are based on the ideas of a rank wilcoxon test. we consider the time series analysis for the first stop only after a reference point, as timetables have built-in time buffer times , to absorb small delays: an arrival delay does not result in a departure delay. buffer times in timetables are specific to public transport and railway systems, not observed in topological studies; private modes; and also not in airline or maritime networks in the same extent, as trains have multiple stops closely spaced. by suitable choice of buffer times in a timetable, delays can disappear or magnify over time, and specific stations might have smaller (respectively larger) delays without any specific event as cause . the effect of buffer times can be approximated as a systematic baseline of delay; plus a non-linear noise, which affects and reduces delay differently for punctual and non-punctual traffic. to avoid considering effects of buffer times and timetable design, only services connecting two stations without any intermediate stop in between are considered in the delay comparison. in other words, only the variation of delays (to remove systematic baseline), at a station served by a service immediately afterwards a focal point (to remove non-linear delay reduction due to buffer time) is studied. due to the different service levels (long-distance intercity traffic ic, interregional ir, regional re, neglecting urban railways), with less/more frequent stops of main/secondary category, the effect at various distances can be estimated (see tables , ) . a second test looks at longer periods, to identify persistent effects of a shock. this test ignores small fast variations which might be considered shocks by the indicators i diff and i sum , but were spurious, such as small holiday periods, or adjusted timetable in case of short events (maintenance, concerts, etc). as the disruption lasted more than months, a time length of weeks is considered, before and after the disruption, to determine a baseline for the hypothetical delay distribution (baseline set) during the disruption period, had the disruption not happened. the delay distribution during the weeks immediately after the beginning of the disruption, and the weeks immediately before the disruption end is taken as description of delay distribution during the disruption (disrupted set). these two distributions are compared by a two-sample kolmogorov-smirnov (ks) test, aiming to reject the null hypothesis h , that any two samples in the baseline and disrupted set, come from the same distribution. when needed, we refer to a significance level α = . . this test is repeated as ks p for all given percentile levels p considered at daily aggregation, as above. a third test looks at even longer time horizon, comparing the observed behavior during the disruption with synthetic data that describes the best estimate of how the system might have looked, over an entire year, had the disruption not taken place . the delays, at given percentile levels p, are modelled as an arima model, with the parameters yielding the highest aic; this is trained on the entire dataset of , while excluding the disruption period. for the disruption period, the delays at any given percentile level are computed by a kalman smoothing on the state space representation of the arima model for filling gaps in time series . this gives a synthetic baseline of the expected value of the percentile levels. we compare the synthetic baseline in the disruption period with the real observed values. under the assumption of equal variances, a t-test aims to reject the null hypothesis h , that there is no difference between the delay of the baseline time series and the observed time series. we repeat this t-test for all given percentile levels p considered at daily aggregation. when needed, we use a significance level α = . . finally, an absolute metric of the deviation magnitude (measured in seconds) is reported as the mean quantile deviation (mqd), for all percentile levels p, between the observed data during the disruption, and the synthetically generated baseline data used also in the t-test. a positive deviation is an increase in the delay during the disruption; a negative deviation is a decrease in the delay during the disruption. stochastic railway operations simulation. we use the mesoscopic stochastic railway simulator ontime, which is based on large-scale monte-carlo analysis of probability distributions for trains departures, running time, stopping time at platforms, as well as for infrastructure-constrained train interactions, transfer connections between trains, possibly track works, and secondary delays. input data include a timetable structure and perturbations in input (primary delays, considering all implicit sources). based on the planned timetable, and business rules including priorities, primary delays are propagated to the running traffic, determining output propagated delay per each train and station. ontime considers all traffic running, including passenger and freight traffic, at mesoscopic level, that is ignoring specific signals, but modelling multiple block sections along the lines, and modelling stops. such a model is much closer to the actual domain processes than general models proposed elsewhere , , . all delay probabilities are assumed expressed as a combination of a negative exponential distribution, plus an additional dirac distribution for punctual trains. in other terms, delays are compactly represented by two parameters, an average delay (including the variance and expected value of the positive delays), and an intensity (i.e. how many events are actually delayed, and how many are not delayed) . we used a calibrated ontime model based on the official timetable , and primary delays replicating reality, as provided by the swiss railways i diff ,p = p[min(|d b,p |, |d e,p |) > min(|d j,p )|, |d k,p |)], ∀j, k ∈ i i sum,p = p[(|d b,p | + |d e,p |) > (|d j,p | + |d k,p |)], ∀j, k ∈ i scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ sbb . the possibilities to use different distributions (for instance, weibull as in ) is left to future research, when a simulator and related calibrated parameters would be available. ontime considers the entire daily timetable over h, and both passenger and freight traffic, respectively passenger trains ( long-distance trains, regional trains), plus freight trains. the model has been further calibrated to match the baseline ( week before the disruption) and disrupted (during the disruption, when the amount and route of freight traffic entering switzerland stabilized), by adjusting the number of freight trains running, their entrance point in the network, and the input punctuality of passenger trains entering switzerland from germany. for both cases, the figure of merit of the calibration was the quantile absolute deviation (qad) between the measured entrance delay in the swiss network (upstream of basel sbb) and the one considered in ontime. we used affine variations for the parameters of the delay distributions (same values for all affected trains), optimized by a line search. the entrance delay of passenger trains has been changed, its volume kept constant. the number of freight trains was changed as in the observed data, over a total of areas of the national railway network. additional freight trains are considered in the corridor schaffhausen-zürich-gotthard, and fewer freight trains are considered in the corridor basel-zürich-gotthard. in this latter, we decreased the train-path usage of 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in microscopic delay management rheintalbahn: sperrung aufgehoben verkehr rollt wieder the unspoken costs of rail disruptions: the consequences of rastatt on the economy and customer confidence assessment of advanced dispatching measures for recovering disrupted railway traffic situations role of timetable, rolling stock rescheduling, and information strategies to passengers in public transport disruptions performance indicators for railway timetables passenger-centered vulnerability assessment of railway networks comparison of ifferent methods for univariate time series imputation in r ensuring timetable stability with train traffic data stability analysis of railway dispatching plans in a stochastic and dynamic environment seeing what is not there: the impact of the rastatt disruption to swiss railway traffic we would like to thank timothy partl, who performed the first analysis ; the entire team at trafit for making available time and resources to enable us to productively the ontime implementation of ; t. graffagnino at sbb for giving insightful comments and making available opentimetable tooling ; and financially mention the support of the project snf dada grant no. . b.b. and f.c. contributed to the conception of the idea. b.b. and t.s. conducted the analyses. all authors reviewed the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to f.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -c c vi authors: basu, saikat; holbrook, landon t.; kudlaty, kathryn; fasanmade, olulade; wu, jihong; burke, alyssa; langworthy, benjamin w.; farzal, zainab; mamdani, mohammed; bennett, william d.; fine, jason p.; senior, brent a.; zanation, adam m.; ebert, charles s.; kimple, adam j.; thorp, brian d.; frank-ito, dennis o.; garcia, guilherme j. m.; kimbell, julia s. title: numerical evaluation of spray position for improved nasal drug delivery date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: c c vi topical intra-nasal sprays are amongst the most commonly prescribed therapeutic options for sinonasal diseases in humans. however, inconsistency and ambiguity in instructions show a lack of definitive knowledge on best spray use techniques. in this study, we have identified a new usage strategy for nasal sprays available over-the-counter, that registers an average -fold improvement in topical delivery of drugs at diseased sites, when compared to prevalent spray techniques. the protocol involves re-orienting the spray axis to harness inertial motion of particulates and has been developed using computational fluid dynamics simulations of respiratory airflow and droplet transport in medical imaging-based digital models. simulated dose in representative models is validated through in vitro spray measurements in d-printed anatomic replicas using the gamma scintigraphy technique. this work breaks new ground in proposing an alternative user-friendly strategy that can significantly enhance topical delivery inside human nose. while these findings can eventually translate into personalized spray usage instructions and hence merit a change in nasal standard-of-care, this study also demonstrates how relatively simple engineering analysis tools can revolutionize everyday healthcare. finally, with respiratory mucosa as the initial coronavirus infection site, our findings are relevant to intra-nasal vaccines that are in-development, to mitigate the covid- pandemic. www.nature.com/scientificreports www.nature.com/scientificreports/ simulated predictions of respiratory flow physics and transport therein; see e.g. [ ] [ ] [ ] . of interest are nasal spray simulation studies on in silico models, re-constructed from medical imaging, to measure drug delivery along the nasal passages , in the sinuses , , and on the effects of surgical alterations of the anatomy on nasal airflow [ ] [ ] [ ] [ ] as well as on topical transport of drugs [ ] [ ] [ ] [ ] . the latter addresses the role of airway channel's shape in the context of airflow-droplet interactions. notably, while using medical devices like sprayers, which are inserted at the nostril, the anterior airway geometry gets altered. to simplify the situation though, computational results suggest that such initial perturbations do not greatly change or adversely affect the eventual drug deposits at the diseased sites. despite the abundance of computational research on nasal drug delivery, there is a distinct lack of articulate instructions for guidance on what could be the "best" way to use the commercially available sprayers. first, numerical studies often do not use a realistic distribution of droplet sizes while simulating topical sprays. focusing on specific droplet diameters is resourceful while studying the detailed nuances of transport characteristics in that size range; however this somewhat limits the applicability of the subsequent findings while predicting the performance of real sprays, which have a wide variability of droplet sizes in each spray shot. secondly, the inter-subject anatomic variations also render it difficult to identify a generic spray orientation that can work for all and ensures maximal delivery of drugs at the diseased locations inside the nose. in this study, we have numerically tracked the transport of therapeutic particulates from over-the-counter nasal sprays via inhaled airflow. the computational fluid dynamics (cfd) models of droplet transport and the in silico prediction of their deposition sites along the nasal airway walls have been compared with in vitro spray experiments in d-printed solid replicas of the same anatomic reconstructions. we have proposed a new strategy of nasal spray usage and the recommendation is supported by a significant improvement in target site particulate deposition (tspd), when compared to the prevalent spray use techniques. the study also expounds [ ] [ ] [ ] on the potential of cfd as a tool in nasal ailment treatment and subject-specific prognosis, and can contribute to the emergence of non-invasive personalized therapeutics and treatment strategies. preliminary results pertaining to this work have featured at the american physical society (aps) -division of fluid dynamics annual meetings , and at the international society for aerosols in medicine (isam) congress [ ] [ ] [ ] . anatomic reconstructions. all methods were performed in accordance with the relevant guidelines and regulations, including use of de-identified computed tomography (ct) data from three pre-surgery chronic rhinosinusitis (crs) patients -collected under approval from the institutional review board (irb) at the university of north carolina at chapel hill. we also obtained informed consent for participation in this study (which includes obtaining and use of ct data) from the test subjects. subject www.nature.com/scientificreports www.nature.com/scientificreports/ medical-grade ct scans of the subjects' nasal airways were used to re-construct digital models through thresholding of the image radiodensity, with a delineation range of − to − hounsfield units for airspace , , complemented by careful manual editing of the selected pixels for anatomic accuracy. as part of that process, the scanned dicom (digital imaging and communications in medicine) files for each subject were imported to the image processing software mimics v . (materialise, plymouth, michigan). for this study, we subsequently considered each side of the nose in the in silico models as a distinct nasal passage model, while studying the droplet transport properties when the spray nozzle was placed on that side: (a) subject 's right side constituted nasal passage model (npm ) and his left side was nasal passage model (npm ); (b) subject 's left side was nasal passage model (npm ); and (c) subject 's right side was nasal passage model (npm ) and her left side was nasal passage model (npm ). note that subject 's right-side anatomy did not exhibit a direct access to the diseased intra-nasal targets from outside of the nostril and was not selected for this study. this had to do with the scope of our study design; for details see the section on target site identification. also refer to the discussion section for follow-up comments. to prepare the in silico anatomic models for numerical simulation of the inhaled airflow and the sprayed droplet transport therein, the airway domain was meshed and spatially segregated into minute volume elements. the meshing was implemented by importing the mimics-output in stereolithography (stl) file format to icem-cfd v (ansys, inc., canonsburg, pennsylvania). following established protocol , , each computational grid comprised approximately million unstructured, graded tetrahedral elements; along with three prism layers of approximately . -mm thickness extruded at the airway-tissue boundary with a height ratio of . inspiratory airflow and sprayed droplet transport simulations. laminar steady-state models work as a reasonable approximation while modeling comfortable resting to moderate breathing , [ ] [ ] [ ] . furthermore, with our simulations focusing on a single cycle of inspiration, steady state flow conditions were adopted as a feasible estimate. based on the principle of mass conservation (continuity), and assuming that the airflow density stays invariant (incompressibility), we have with u representing the velocity field for the inspired air. conservation of momentum under steady state flow conditions leads to the modified navier-stokes equations: here ρ = . kg/m represents the density of air, μ = . × − kg/m.s is air's dynamic viscosity, p is the pressure in the airway, and b stands for accelerations induced by different body forces. to simulate the airflow, eqs. ( ) and ( ) were numerically solved through a finite volume approach, in the inspiratory direction. the computational scheme on ansys fluent v . employed a segregated solver, with simplec pressure-velocity coupling and second-order upwind spatial discretization. solution convergence was obtained by minimizing the flow residuals (viz. mass continuity ~− o( ) , velocity components − Õ ( ) ), and through stabilizing the mass flow rate and the static outlet pressure at the nasopharynx of the digital models. a typical simulation convergence run-time with iterations clocked approximately hours, for -processor based parallel computations executed at . ghz speed. the numerical solutions implemented the following set of boundary conditions: ( ) zero velocity at the airway-tissue interface i.e. the tissue surface lining the sinonasal airspace (commonly called no slip at the walls), along with "trap" boundary conditions for droplets whereby a droplet comes to rest after depositing on the wall; ( ) zero pressure at nostril planes, which were the pressure-inlet zones in the simulations, with "escape" boundary condition for droplets that allowed outgoing trajectories to leave the airspace through the nostril openings; and ( ) a negative pressure at the nasopharyngeal outlet plane, which was a pressure-outlet zone, also with an "escape" boundary condition for droplets. the negative nasopharyngeal pressure was adjusted to generate inhalation www.nature.com/scientificreports www.nature.com/scientificreports/ airflow rates with less than % variation from subject-specific measurements of resting breathing. the physical recordings were collected with lifeshirt vests that tracked chest compression/expansion during breathing, and accordingly quantified the inhalation rates (for additional details, see table ). after simulating the airflow, sprayed droplet dynamics were tracked through discrete phase particle transport simulations in the ambient airflow, and the corresponding lagrangian tracking estimated the localized deposition along the airway walls through numerical integration of the following transport equations : the parameters here are u d , representing the droplet velocity; along with u as the airflow field velocity, ρ and ρ d respectively as the air and droplet densities, g as the gravitational acceleration, f b as any other additional body forces per unit droplet mass (as for example, saffman lift force that is exerted by a typical flow-shear field on small particulates transverse to the airflow direction), and c d u u re( )/ ( ) quantifies the drag force contribution per unit droplet mass. here, c d is the drag coefficient, d is the droplet diameter, and re represents the relative reynolds number. mean time step for droplet tracking was in the order of − sec., with the minimum and maximum limits for the adaptive step-size being − o( ) sec. and − o( ) sec., respectively. also note that the solution scheme posits the particulate droplets to be large enough to ignore brownian motion effects on their dynamics. post-processing of the simulated data laid out the spatial deposition trends, which were then tallied against in vitro observations. d printing and physical experiments. to assess the reliability of numerically predicted topical deposition vis-à-vis physical experiments, d-printed anatomic replicas were generated for subject 's airway and hence included both npm and npm . the posterior parts of the solid models were made from the stereolithography material watershed (dsm somos, elgin, illinois). post-digitization, the printing job of the posterior component was sub-contracted to protolabs (morrisville, north carolina). printing of the anterior soft plastic part on a connex d printer was done by ola harrysson's group at north carolina state university (at the edward p fitts department of industrial and systems engineering), using polymer inkjetting process on tangogray flx material. see fig. (a-c) for representative pictures of a digitized model and the corresponding d replica. recording deposits through gamma scintigraphy. intra-nasal topical delivery was tracked through in vitro examination of mildly radioactive spray deposits in the d-printed anatomic replicas. to ensure that the spray axis orientation and nozzle location aligned with the corresponding simulated spray parameters, we used specially designed nozzle positioning devices (npd) inserted at the nostril. the spray bottle was fitted into the npd, while administering the spray via hand-actuation. for each sample test, a bottle of commercial nasal spray nasacort was labeled with a small amount of radioactive technetium (tc m) in saline. at the time of dispensing the spray shots, a vacuum line controlled by a flow-valve was used to set up inhalation airflow through the model, and the flow rate was commensurate with the subject-specific breathing data (table ). corresponding setup is in fig. (d,e) . four independent replicate runs of each spray experiment were conducted, followed by compilation of the means and standard deviations of the drug deposits along the inner walls of the solid models. the topical deposition was proportional to the radioactive signals emitted from the spray solution traces that deposited inside a solid model and was quantifiable through image-processing of the scintigraphy visuals, collected using a bodyscan (mieamerica, forest hills, il) -mm width by -mm height d gamma camera. the pixel domain was × , with an image acquisition time of minutes; and one pixel equated to a cartesian distance of . mm in the digital and d models. table . this table incorporates the parameters for measured and simulated inhalation airflow, in the study subjects. symbols: σ = standard deviation, μ = mean, *⇒ inhalation rate is considered to be twice the minute ventilation, **⇒ target simulated airflow is . % of the measured rate, to account for influence of the subjects' awareness of recording of the breathing process. note that the tidal volume is a measure of the lung volume representing the volume of air displaced between normal inhalation and exhalation, without application of any extra effort. the minute ventilation (air inhaled per minute) is computed from the inspiratory phase of a breath , . www.nature.com/scientificreports www.nature.com/scientificreports/ model segmentation for comparison with numerical data. to facilitate the comparison between the numerical predictions on droplet deposition and the physical observation of gamma scintigraphy signals in the corresponding solid replica, we segregated npm and npm into virtual segments oriented along three different directions. figure lays out the cartesian coordinate directions for the d space. x was perpendicular to the sagittal plane traversing from left to right sides of the nasal models (with the model head facing forward), y was perpendicular to the axial plane traversing from inferior to superior aspects of the models, and z was perpendicular to the coronal plane traversing from anterior to posterior aspects of the models. the virtual segments were oriented along the xy (coronal), yz (sagittal), and zx (axial) planes. parallel to the xy coronal plane, the models contained segments (named, c -c ⇒ sagittal columns); there were compartments (c -c ⇒ frontal columns) parallel to the yz sagittal plane, and there were compartments (r -r ⇒ sagittal rows) parallel to the zx axial plane (see fig. ). for each compartment, the particulate deposition fraction predicted from the simulation was compared with the deposition fraction measured based on gamma signals of the deposited particulates in the corresponding compartment of the d-printed model. to achieve this, signals emitted from the solution traces, that settled along the airway walls, were subjected to image processing analysis. therein, by superimposing the compartmental grid on the radio-images, the signals were extracted from each compartment. in order to align the grid on the image in a manner consistent with the virtual model, three inset discs were designed as reference points on the outer surface of the virtual and d-printed models. americium sources from commercial in-home smoke detectors were inserted into the insets as reference points on the d-model and a radio-image was recorded. for the analysis, the scintigraphy images were processed using imagej by constructing a region of interest (roi) referenced to the fixed americium sources. care was taken to align the emitted visual signals with similar reference regions within the superimposed grid. this was done via manual visualization to achieve a best fit of signal intensity within reference regions. the grid compartment planes positioned using this visual best-fit technique were designated as "reference planes". given the nature of the radioactive signals and the resolution of the radio-image, some www.nature.com/scientificreports www.nature.com/scientificreports/ signal intensity resided outside of reference regions even while using best-fit practices. a reasonable fit could be obtained by shifting the image by one pixel in either direction (positive shift/negative shift). in order to account for this variation, alternative plane positions (see fig. (d)) were created by shifting the reference planes one pixel along the positive and negative axes for each set of cartesian planes. these three sets of compartment planes were positioned in the in silico modeling software using the measured distances from the reference regions. the corresponding cartesian coordinates of these planes were used to assign droplet deposition locations from the computational simulations to grid compartments, for comparison with the in vitro model. in these comparisons, we left out the deposits in the anterior nose (from the cfd data as well as the physical recordings) in order to negate the bright radiation signal coming from that zone in the experimental deposits; and focused only on measurements from the posterior parts of the respective models. note that the anterior nose in an in silico model is in fact the removable soft pliable anterior part in the corresponding d print (e.g. see fig. ). , and (c) depict the gridline schematic on npm and npm , that is used to extract the deposition fractions from the gamma scintigraphy-based quantification of the sprayed deposits in the solid replicas. the models are respectively segregated into sets of compartments: sagittal columns, frontal columns, and sagittal rows. panel (d) shows the perturbation of the base gridline by pixel. representative technetium signals are in panel (e). note: in regard to the axis system, the circle with solid dot implies out-of-plane direction from this page, the circle with cross signifies into-the-plane of this page. www.nature.com/scientificreports www.nature.com/scientificreports/ identification of target site and spray parameters. effect of airflow on droplet trajectories. inertial motion of a droplet is linearly proportional to its mass, and hence is exponentially proportional to the droplet diameter. consequently, for bigger droplets, the inertial motion persists longer before being taken over by the ambient airflow. figure (a) tracks the trajectory of a representative μ droplet. in there, the tiny red circle marks the location where the inertial motion of the droplet got overwhelmed by the ambient flow, beyond which the droplet trajectory was same as the airflow streamline on which it was embedded at the red circle's location. note the contrasting μ droplet trajectory in fig. (b) , where the inertial motion persisted longer. the phenomenon has a significant impact on drug deposition trends. the bigger droplets (≥ μ) show a greater propensity to hit the anterior walls directly owing to their high initial momentum, while smaller droplet sizes penetrate further into the airspace; see e.g. figure (c,d). to ensure that the bigger droplets also reach the target sites, we argue that it would be conducive to harness their inertial motion and direct those droplets actively toward the target when they exit the spray nozzle. this can be feasibly achieved by orienting the spray axis to pass directly through an intended anatomic target zone. , indicate a lack of definitive knowledge on the best ways to use a nasal spray device. different commercial sprayers often offer somewhat contrasting recommendations. however, there is a common agreement (see fig. (a)) that the patient should incline her/his head slightly forward, while keeping the spray bottle upright , . furthermore, there is a clinical recommendation to avoid pointing the spray directly at the septum (the separating cartilaginous wall between the two sides of the in panel (a), the smaller droplet has weaker inertial momentum and the ambient airflow streamline takes over its motion much earlier than that in case of a heavier droplet like the one in panel (b), where the inertial momentum of the μ droplet persists longer. the small red circle in (a) depicts the point where the inertial momentum gets overwhelmed by the fluid streamline. evidently, owing to smaller inertia, the droplets with smaller diameters get predominated by the airflow streamlines earlier than the bigger droplets. this results in a better penetration and spread of sprayed droplets in the nasal airspace, as shown in panel (c), for a different nasal model. on the contrary, spray shots with exclusive share of bigger droplets (e.g. ≥ μ here) tend to follow their initial inertial trajectories, without much effect of the airflow streamlines on their paths, and deposit along the anterior walls of the nasal airspace, as depicted in panel (d). the red boundaries in panels (c) and (d) highlight the difference in particulate penetration into the model, in the two cases. note: these images were created using fieldview, as provided by intelligent light through its university partners program. www.nature.com/scientificreports www.nature.com/scientificreports/ nose). these suggestions were adopted in our standardization of "current use" (cu) protocol for topical sprays. the digital models were inclined forward by an angle of . °, and the vertically upright spray axis was closer to the lateral nasal wall, at one-third of the distance between the lateral side and septal wall. also, the spray bottle was so placed that it penetrated into the airspace by a distance of -mm, inspired by the package recommendations of commercial sprayers for a "shallow" insertion into the nose. refer to fig. (b,c) for the schematics of the cu protocol used in this study. target site identification and proposing an alternate spray use criteria. all sinuses, except sphenoid, drain into the ostiomeatal complex (omc), it being the main mucociliary drainage pathway and airflow exchange corridor between the nasal airway and the adjoining sinus cavities. to ensure that as many drug particulates reach the sinus chambers and their vicinity as would be possible, we hypothesize that the spray axis should be directed straight toward the omc . this is supported by our observation of the effect of airflow physics on droplet trajectories. if the spray axis hits the omc directly, the likelihood that the larger droplets will deposit there is higher. we refer to this usage protocol as "line of sight" (los). like the cu protocol, the los protocol also had the sprayer inserted at a depth of -mm into the nasal airspace. representative los orientation is shown in fig. . tspd percentage at the omc and the sinuses was evaluated as = × (m target /m spray ); with m target being the spray mass of the particulate droplets deposited at the omc and inside the sinus cavities, and m spray being the mass of one spray shot. to establish the robustness of the tspd predictions for the cu and los protocols, we also tracked droplet transport and deposition when the spray directions were slightly perturbed. such perturbed peripheral directions for cu initiated mm away on the nostril plane and were parallel to the cu's vertically upright true direction. for los, the perturbed peripheral directions were obtained by connecting the base of the true los direction on the nostril plane with points that radially lie mm away from a point on the los; this specific point being mm away along the los from the base of the los direction on the nostril plane (e.g. see bottom panel of fig. for an illustrative example). parameters for the simulated spray shot. over-the-counter nasacort (triamcinolone acetonide), a commonly prescribed and commercially available nasal spray, was selected for this study. four units of nasacort were tested at next breath, llc (baltimore, md, usa) to characterize the in vitro spray performance. corresponding plume geometry was analysed through a sprayview nosp, which is a non-impaction laser sheet-based instrument. averaged spray half-cone angle was estimated at . °, and the droplet sizes in a spray shot followed a log-normal distribution. with the droplet diameter as x, the droplet size distribution can be framed as a probability density function of the form : here, x = . μ is the mass median diameter (alternatively, the geometric mean diameter ) and σ g = . is the geometric standard deviation. the latter quantifies the span of the droplet size data. measurements were www.nature.com/scientificreports www.nature.com/scientificreports/ also made with and without the saline additive in the sprayer, and the tests returned similar droplet size distribution. note that a saline additive was used during the physical recording of the sprayed deposits. also, as per earlier findings in literature , the mean spray exit velocity from the nozzle approximates at . m/s, based on phase doppler anemometry-based measurements. for the test spray units (at next breath), the actuation forces were found to range between . - . kg-force. considering an actuation area of approximately cm , the force measurements agree well with earlier values in literature [ ] [ ] [ ] and hence the resultant pressure exerted on the droplets in our physical experiments was assumed to maintain a similar droplet size distribution, as was determined in the test cases by next breath. the droplets contained in one spray shot in the numerical simulations followed the same size distribution. while simulating the droplet trajectories, we assumed typical solid-cone injections and tracked the transport for -mg spray shot while comparing the tspd trends from the cfd predictions with the corresponding experimental drug delivery patterns. on the other hand, . mg (which is one shot of nasacort, as quantified by next breath, llc) of spray mass transport was simulated while comparing the cfd-based tspd numbers for the los and cu protocols in each model. comparison between cu and los spray usage protocols. los was found to be consistently superior in comparison to the cu spray placement protocol, while targeting the omc and the sinus cavities for drug delivery. table lists the deposition fraction percentages for each spray release condition in the five airway models (npm -npm ). for a graphical interpretation, we have plotted the same information on fig. . overall, the deposition fraction for the los was on an average . -fold higher than the cu deposition fraction, with the corresponding subject-specific improvement range being . - . folds for the five test models. the improvement www.nature.com/scientificreports www.nature.com/scientificreports/ does decay when the perturbed peripheral spray directions are compared, to assess the robustness of the los protocol's advantage over cu. considering the varying peripheral directions around the true los and cu, the los set registered an average . -fold increase in tspd, with the corresponding subject-specific improvement range being . - . folds. statistical tests -on improvements achieved by the revised spray use strategy. los was compared to cu through a paired study design on the data from five test models. table lays out the computed numbers. for each model, the outcome comprised the percentage of deposition in omc and the sinuses for both cu and los spray usage. null hypothesis considered for this statistical test assumed that the tspd would be same for cu and los in an . panel (f) compares the tspd for peripheral directions in a . -mm perturbation (on the left) with respect to a -mm perturbation (on the right) from the true los orientation, both in npm . as expected from the overall findings, the tspd increased for the perturbed spray directions that were closer to the true los. panel (g) depicts the spatial perturbation parameters for the los spray axis orientation in npm . www.nature.com/scientificreports www.nature.com/scientificreports/ airway model. the deposition percentage corresponding to cu and los protocols in the same nostril were treated as paired observations for a paired t-test to check the null hypothesis. owing to a relatively small study cohort, paired wilcoxon signed rank test was also used for robustness check. in order to study how spatial variation might affect the difference between cu and los, three different ways of calculating the percentage of deposition were implemented. the first strategy considered the average deposition from the true los and cu directions. the www.nature.com/scientificreports www.nature.com/scientificreports/ second strategy compared the tspd averaged from the true cu and los directions, along with the deposition data for spray release parameters obtained by perturbing the respective true directions. the third strategy used tspd averaged exclusively from the deposition data corresponding to the perturbed spray release parameters. this allowed us to assess the robustness of any probable improvement from using los, while still accounting for slight spatial variations of the spray direction. the first comparison method demonstrates an average deposition increase of . percentage points for los ( . -% for los vis-à-vis . % for cu). this difference is significant at the . level with a p-value from the paired t-test of . . the paired wilcoxon signed-rank test has a p-value of . , which was the lowest possible p-value for the wilcoxon signed-rank test given only five pairs of data. in the second comparison scheme, los has an increased deposition of . percentage points relative to cu ( . % vis-à-vis . %). the p-value for this difference is . using the paired t-test and . using the wilcoxon signed rank test. finally, for the third comparison method, los registered an increased deposition of . percentage points relative to cu ( . % vis-à-vis . %). the p-value for this difference is . using the paired t-test and . using the wilcoxon signed rank test. this provides a strong evidence that los leads to higher percentage of deposition in the omc and sinuses. the estimated difference is largest when using just the true directions, but the difference is still statistically significant even when using the spray release points obtained by perturbing the true directions. the p-value from the paired t-test is actually lower when the tspd from just the perturbed points are considered, owing to the reduced variance for the estimated difference. for all three ways of estimating the percentage of deposition, the paired wilcoxon signed-rank test returns a p-value of . . with only five pairs of data, this suggests that the use of los does result in statistically significant higher deposition for all five nostril models. comparison of the simulated tspd predictions with physical experiments. figure compares the numerical tspd predictions with corresponding gamma scintigraphy-based experimental recordings in npm and npm . while the compartmental deposits visibly presented a congruous trend in the sagittal columns, sagittal rows, and frontal columns; we conducted additional statistical tests to verify the homogeneity between the two sets of data so as to establish the reliability of the computational findings. table gives the pearson and kendall's correlation between the numerical and experimental models for the average deposition fractions in npm and npm for the los protocol. the confidence intervals are based on bootstrap samples, instead of asymptotic approximations, because of the relatively small sample size. based on the output, we can see that the pearson correlation is consistently very high while the kendall's correlation is somewhat lower. however, while the kendall's correlation is frequently thought to be more robust to outliers, particularly for small sample sizes like this data-set; in this particular instance the pearson correlation is likely more illustrative. this is because the pearson correlation is able to show that, for the most part, the magnitudes of the estimates are similar and comparable between the numerical and experimental models. in general, there is a strong linear relationship between the percent of deposition prediction from the numerical model and the corresponding physical measurements in the experimental model. the lower kendall's correlation (overall mean measure . ) is largely due to regions where both the numerical and experimental models had very low average deposition but the exact rank of these regions changed considerably between the two data-sets. note that this does not necessarily indicate a poor performing numerical model. however, the relatively high pearson correlation (overall mean measure . ) does indicate that the numerical models perform well while predicting the sprayed droplet transport. cfd-guided nasal spray usage defined by the los protocol was found to significantly enhance topical drug delivery at targeted sinonasal sites, when compared to currently used spray administration techniques. with increased sample size, this work can be the catalysis toward prompting personalized instructions and specifications for improved use of topical sprays. the findings, thus, have the potential to substantially upgrade the treatment paradigm for sinonasal ailments through the ability to ascertain los in individual subjects via endoscopic examinations conducted in the clinic, and to help guide treatment decision-making and patient instructions for spray usage. to quantifying the suitability of a person's airway for the los spray protocol, we exploratorily propose a scoring system that is based on how much of the targeted drug delivery sites (omc, sinuses) are visible when inspected clinically from outside of the nostril. the scoring system will also serve to quantify nasal anatomic variability www.nature.com/scientificreports www.nature.com/scientificreports/ among individuals. accordingly, as part of the current study, the los scores (see table ) were first determined observationally, based on the external visibility of the omc site in the in silico sinonasal reconstructions. we fixed a range of scores ∈ [ , ] , with being used when the los direction was easiest to ascertain. subjective as that scoring procedure may be, it is similar to what attending physicians will gauge during a clinic visit to determine if a particular patient has a "line of sight" in her/his nasal anatomy. so, to establish the relevance of the findings from this manuscript toward revisions of the therapeutic protocol for sinonasal care, it is important to assess the comparability of the observational los scores with more objective score determination techniques. this was achieved by calculating the surface area of the nostril plane and the projected area of the omc on the plane of the nostril. we computed the ratio of the projected area to the nostril area, as a percentage. scores of were assigned if the ratio exceeded %, if the ratio exceeded %, if the ratio was more than . %, and if the ratio was greater than %. the two scoring techniques yielded very similar results (as in table ), with the highest and lowest scores respectively going to the same anatomic models. pearson's rank correlation for the two sets of scores was . . while a broader study, involving clinical trials, will be necessary to revise therapeutic protocol for nasal drug delivery, the present results illustrate the easy adaptability of our findings into clinical practice settings. on the comparability of the experimental data with the numerical findings. the computational simulations assumed a laminar framework to mimic steady breathing. however, one may argue that even with resting breathing rates, the airflow often contains transitional features like vortices, emerging from the roll-up of www.nature.com/scientificreports www.nature.com/scientificreports/ shearing fluid layers during flow-structure interactions - at the anatomic bends. some of these nuances are, in fact, difficult to model without proper turbulence simulations , . however, true as that may be, the effect of these flow artifacts on eventual drug delivery in the sinuses has been found to be somewhat nominal while comparing laminar and turbulence simulation results . on the other hand, the in vitro techniques also often pose challenges. for instance, there can be post-deposition run-off as the deposited solution traces undergo translocation along the inner walls of the solid replica. such drip-off dynamics can lead to a flawed estimate of regional deposition. the effect of post-deposition dripping can be conjectured to be most prominent for the signals extracted from the sagittal rows, as the deposited droplets start moving downward along the internal solid walls of the d-printed models, owing to gravitational effects. this is confirmed by the physical and numerically-predicted signals from the sagittal rows demonstrating relatively lower correlation coefficients (when contrasted with the correlations for the signals from the sagittal and front columns) in the two experimental comparisons (e.g. see table ). in the gamma scintigraphy-based method of recording deposits, the radiation signal undergoes some level of scattering and hence in the process of signal extraction from each of the compartments, there is the possibility that signals from one compartment may contaminate the signals at neighboring compartments. to minimize this effect while carrying out the comparisons, the nose (the soft plastic anterior part in the d-printed models), which had a bright radiation signal owing to the relatively large amount of anterior deposits, was excluded from both the experimental and numerical data. finally, while the inhalation airflow rates were same in vitro and in silico, the airflow partitioning on the two sides of the nasal airways was likely affected by the placement of the npd, while administering the spray through hand-actuation. caveats and future implications. readers should note that this was a computational study with validation from spray transport observations in inanimate solid replicas. also, not every patient will have a clear access to the omc, and hence may be without an los. for instance, in the current study, of the six airway sides in the three study subjects, subject 's right-side airway did not exhibit an los. bulk rheology of the spray also affects the droplet size distribution. the spray property measurement tests having been performed in real over-the-counter sprays, we did not separately examine the effect of different droplet viscosities on the spray deposition trends. a different viscosity of the nasal spray can indeed alter the drug deposits, as observed in multiple studies , , . it should however be pointed out that the spray positioning strategies proposed in this study could be conjectured to be generic and should maximize drug delivery to the omc and the sinuses for other sprays as well. it is also critical to note that the flow simulations for evaluating the spray usage strategies were not multiphase; implying that the sprayed droplets were not affected by constituents such as inhaled air moisture and the mucous lining, nor was there any consideration of droplet evaporation. there are, however, earlier findings in literature www.nature.com/scientificreports www.nature.com/scientificreports/ that have looked at some of these nuances; e.g., on the interaction of deposited particulates with mucus and on the phase change of inhaled droplets during their passage through the respiratory tract . the current study simply tracked the motion of inert droplets against the ambient inspiratory airflow and recorded their regional deposition. based on the surfactants in the spray solution, the droplets might also be rendered hydrophilic; but such effects are beyond the scope of this project and the numerical schemes that have been implemented. it is, however, expository to reckon that such hydrophilicity may at times lead to agglomeration of droplet molecules, which can impact the topical drug deposition estimates. this study, its restricted sample size and limitations notwithstanding, is still, to the best of our knowledge, the first-of-its-kind to propose an alternative easy-to-implement strategy that can significantly improve the intra-nasal delivery of topical drugs at the diseased sites. the recommendation for using the "line of sight" is user-friendly, personalized (the physician can instruct the patient on the spray usage technique based on a fast los check in the clinic), and has the potential to be smoothly incorporated into the nasal standard-of-care. for probable revisions to the clinical regimen, we will need a broader study with more subjects, along with a component for clinical trials to track patient response. comparison of the numerical data with in vivo spray performance will also eliminate errors that contaminate the in vitro tspd numbers (e.g. from drip-off of the deposited solution along the inner wall contours of the d-printed models). nevertheless on a larger intriguing perspective, the current study conclusively postulates how relatively simple engineering analysis and mechanistic tools can usher in transformative changes in the prognosis and treatment protocol for ailments such as nasal congestion and respiratory infection. special comments on the significance of the findings in view of the - coronavirus pandemic. with the rapid spread of the novel coronavirus disease (covid- ) worldwide, it is essential that a vaccine or a curative is developed at the earliest. with respiratory mucosa as the initial site in coronavirus infection and transmission; mucosal immunization through targeted intra-nasal vaccine promises to be an effective strategy for prophylaxis, by inducing mucosal and systemic immune responses. as of may , several research groups are working on the possibility of designing intra-nasal vaccines for covid- - , with supporting data from work carried out on earlier strains of coronavirus . in this context, the intra-nasal anatomic targeting strategies (e.g. see fig. ) discussed in the current study can be of significant help to increase the topical delivery. this project has generated both simulated and experimental, quantitative, de-identified data on the regional deposition of aerosolized nasal medication in the form of nasal spray droplets in the sinonasal passages. for readers' convenience, table details the drug delivery numbers, processed from all the numerical runs; and the narrative, included under methods, elucidates the computational software settings for the airflow and droplet transport simulations. the datasets generated during and/or analysed 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healthy humans effect of obesity on ozone-induced changes in airway function, inflammation, and reactivity in adult females the authors declare no competing interests. correspondence and requests for materials should be addressed to s.b. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ige h pd authors: nie, lei; hou, mengjuan; wang, tianwen; sun, meng; hou, ruixia title: nanostructured selenium-doped biphasic calcium phosphate with in situ incorporation of silver for antibacterial applications date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ige h pd selenium-doped nanostructure has been considered as an attractive approach to enhance the antibacterial activity of calcium phosphate (cap) materials in diverse medical applications. in this study, the selenium-doped biphasic calcium phosphate nanoparticles (seb-nps) were first synthesized. then, silver was in situ incorporated into seb-nps to obtain nanostructured composite nanoparticles ((ag)seb-nps). both seb-nps and (ag)seb-nps were characterized by fourier transform infrared spectroscopy (ft-ir), x-ray diffraction (xrd), ultraviolet–visible spectroscopy (uv–vis), x-ray photoelectron spectroscopy (xps), and raman spectra. the results confirmed that the seo( )( −) was doped at the po( )( −) position and silver nanoparticles were deposited on the surface of seb-nps. next, transmission electron microscopy (tem) analysis displayed that the prepared (ag)seb-nps had a needle-cluster-like morphology. cck- analysis revealed seb-nps and (ag)seb-nps had good cytocompatibility with osteoblasts. the antibacterial activity of the prepared (ag)seb-nps was confirmed by using gram-negative e. coli and gram-positive s. aureus. the above results manifested the significance of the final (ag)seb-nps for biomedical applications. www.nature.com/scientificreports/ material, table s ), and ml solution of ca(no ) · h o ( . m) was added, then the ph of mixed solution in the flask was adjusted to . by adding ammonium solution. the mixed solution was stirred for h, and the precipitates were collected by centrifugation ( , rpm) and washed using millipore water five times. the precipitates were dried at °c for h to obtain seb-nps powder. different seb-nps (sb , sb , and sb ) were synthesized by adjusting ca/(p + se) and ca/se mole ratios, as shown in table . at the same time, biphasic calcium phosphate nanoparticles (bcp-nps) and selenium-doped hydroxyapatite nanoparticles (seha-nps) were prepared for comparison (electronic supplementary material). was dispersed in ml millipore water in a three-neck flask, and mg of agno was added into flask slowly. the solution was stirred for h at room temperature (rt). the precipitates were collected by using centrifugation ( , rpm) and washed five times using millipore water to obtain ag seb-nps. different ag seb-nps samples were fabricated by regulating the amount of agno , as shown in table . copy (ft-ir) analysis. ft-ir (thermofisher, nicolelis ) was used to confirm the presence of specific chemical groups in seb-nps and ag seb-nps. the nanoparticles powders were mixed with kbr, ground, and pressed into thin sections, and the kbr was measured as blank control. ft-ir spectra were obtained within the range between , and cm − with a resolution of cm − . x-ray diffraction (xrd) analysis. both seb-nps and ag seb-nps powders were analyzed by x-ray diffraction (xrd). a x-ray diffractometer (rigaku smartlab kw), operating at kv and ma with cu kα radiation (λ = . Å) and a spinning sample holder, was used to collect the x-ray powder diffraction (xrd) patterns. data were acquired in the θ range of °- ° at a step increment of . °. to investigate the diffuse reflectance spectra of samples, ultraviolet-visible spectroscopy (uv-vis, perkinelmer, lambda ) was used. this instrument was equipped with an integrating sphere attachment. the sample was operated in the range of - nm at k for the optical diffuse reflectance (drs) spectra. dynamic light scattering (dls) analysis. the size distribution of nanoparticles was examined by dynamic light scattering (dls, malvern zetasizer e). zeta potential measurements were performed by laser doppler anemometry with a zetasizer nano zs/masterszer e. electrophoretic mobility (converted into ζ-potential by the smoluckowsky approximation) of nanoparticles was tested at °c. morphology analysis. the morphology of seb-nps and ag seb-nps was investigated by transmission electron microscopy (tem, tecnai g f ) with an energy-dispersive detector (eds) and elemental mapping accessories. the obtained nanoparticles were dispersed in ethanol and sonicated for h, and then the copper grid was dipped into a sample solution and dried under an infrared lamp. furthermore, the morphology of seb-nps and ag seb-nps were observed with a cold field emission scanning electron microscope (sem, hitachi, s- ). before sem observation, the samples were coated with a thin pt conductive layer, energy dispersive x-ray spectroscopy (edx) was used for the elemental composition analysis or chemical characterization. raman spectroscopy analysis. raman spectra of both seb-nps and ag seb-nps were recorded with a spex model spectrometer (labram hr) from to , cm − using the nm wavelength excitation from an argon-ion laser. www.nature.com/scientificreports/ x-ray photoelectron spectrometer (xps) analysis. both seb-nps and ag seb-nps were also measured by x-ray photoelectron spectrometer (k-alpha . ev, thermo scientific) to obtain their elements composition, the detailed sample preparation for xps test refers to our previous paper . cell culture and cytocompatibility of seb-nps and ag seb-nps. regarding the potential bone tissue engineering application in the future, hfob . cell (osteoblast type, atcc® crl- ™) was used to evaluate the cytocompatibility of seb-nps and ag seb-nps. according to atcc protocols, hfob cells were cultured using dulbecco's modified eagle's medium (dmem) (sigma-aldrich) supplemented with % fetal bovine serum, u ml − penicillin, and μg ml − streptomycin. cells were grown in the tissue culture flask ( ml) under a humidified atmosphere of % air and % co at °c. the culture medium was changed every days. the cells were passaged by trypsinization, and cells at passage were used for the next experiments. first, a certain amount of obtained seb-nps and ag seb-nps were added into the cell medium, and no agglomeration phenomenon was not observed under optical microscopy. the cell viability cultured with nanoparticles was quantitatively investigated by the cell counting kit- (cck- , abcam) assay, and the cck- assay was operated according to the cck- cell proliferation assay kit protocol. finally, the absorbance at nm was measured by using a microplate reader to indicate cell proliferation after culturing with nanoparticles (electronic supplementary material). antibacterial activity assay. gram-negative e. coli (atcc ) and gram-positive s. aureus (atcc ) were used to evaluate the antibacterial activity of prepared seb-nps and ag seb-nps. the single colony of e. coli and s. aureus on the luria bertani (lb) agar plate were transferred to a liquid lb culture medium by growing at °c overnight to obtain seed culture. μl of nanoparticles dispersed in ultrapure water (the concentrations of nanoparticles used were μg/ml, , μg/ml and μg/ml, respectively) were mixed with ml autoclaved lb medium, then μl of seed cultures of e. coli or s. aureus was inoculated into the medium. after culturing for h at °c, the optical density at nm (od ) was tested by a uv-vis spectrophotometer. e. coli and s. aureus were grown at the same conditions without adding nanoparticles as the control group. on the other hand, the seed culture medium was diluted into fresh lb medium and cultured under °c. when the od of medium reached about . , the broth was diluted to cfu ml − with sterile . % nacl solution. after then, the suspension ( μl) was spread onto a mm-diameter lb agar plate. the wells were created with a hole puncher with a diameter of mm. μl of the prepared nanoparticles solutions ( μg/ml, , μg/ ml and μg/ml) were added into the wells. then, the plates were kept in an incubator at °c for h, the inhibition zones for each sample were recorded. statistics. all data were expressed as means with standard deviation. spss software (spss inc, chicago il) was used for the analysis. statistical analyses were performed by anova or -way repeated-measures anova with tukey's test applied to investigate specific differences. statistical significance was defined at a p value of < . for % confidence. physicochemical characterization. first, the chemical groups were analyzed by ft-ir spectroscopy, and the spectra of seb-nps and ag seb-nps were presented in fig. . the broad peak in the range of , - , cm − was due to h o stretching vibrations in absorbed water. as shown in fig. a , while for seb-nps it decreased and disappeared. at the same time, the band at cm − was attributed to o-p-o vibration mode, and the band decreased as the ca/se ratio increased. notably, the sharpness of cm − bands indicated the well-crystallized bcp-nps, and the peak became rounded, www.nature.com/scientificreports/ indicated the decrease of crystallization for se-nps. however, due to the incorporation of selenium, a band of seb-nps at cm − appeared, which was assigned to seo − stretching. the spectra of ag seb-nps were shown in fig. b . it was clearly noted that the peak at , cm − was ascribed to antisymmetric (v ) p-o stretching vibration mode, which was mainly due to the combination of the silver nanoparticles with oh − or po − groups of seb-nps. the slight difference between ag seb , ag seb , and ag seb demonstrated that the chemical bonding between silver and seb-nps barely happened, and silver was mainly deposited on seb-nps via electrostatic attraction. complementary to the above ft-ir analysis, the raman spectra of both seb-nps and ag seb-nps were obtained. the raman spectra of seb-nps and ag seb-nps were shown in fig. . the oh − vibrational bands in the region of cm − were not clearly observed, which was in good accord with the ft-ir results. in the case of seb-nps, an intense peak appeared at cm − due to the stretching mode (v ) of po − group, and peaks at cm − , cm − , and , cm − were attributed to the stretching mode (v ), bending mode (v ), and stretching mode (v ) of the po − group, respectively. the peak at , cm − corresponded to the symmetrical stretching vibration mode (v ) of co − group. in addition, the band near cm − was attributed to the symmetrical stretching mode (v ) of the seo − group. for ag seb-nps, similar chemical groups from seb-nps appeared. compared with ftir spectra, water vibrational modes should give rise to weak intensity stretching and no bending bands in raman spectra were observed. however, the intensity of the vibration peak at cm − increased with increasing silver content. xrd was used to identify the crystalline phase, and the xrd patterns of all of the synthesized samples were shown in fig. a . the peaks of ha and β-tcp were present in bcp, seb-nps, and ag seb-nps, the peaks at . ° furthermore, the uv-vis drs spectroscopy was used to analyze the obtained ag seb-nps, as shown in fig. b . the uv-vis drs absorption broadened in the region of - nm, and the band at nm corresponds to the surface plasmon resonance of silver nanoparticles. this phenomenon indicated that silver was deposited uniformly on seb-nps. to get a further detailed vision for the ionic species of seb-nps and ag seb-nps, xps measurements were performed, as shown in fig. . the xps spectra of seb-nps (fig. a-c) found all the expected elements during the preparation process, including ca, o, c, p, se. the peak at ev corresponded to seo − [se (iv)] indicated that seo − group was already incorporated in bcp-nps lattice. and the peak at ev belonged to se ( ) was not observed, proving that the redox reaction happened during the preparation process. the p p spectra of all samples exhibited a peak at ev, which was assigned to the phosphate group. the double peaks at ev and ev were attributed to the ca p / and ca p / . for ag seb-nps, except for the peaks appeared in seb-nps, the new high peaks around ev and ev were assigned to ag d / and ag d / binding energies, respectively , . the morphology of the prepared seb-nps and ag seb-nps was analyzed by tem. tem micrographs of seb-nps were given in fig. . bcp-nps exhibited an ellipsoidal morphology ( figure s , supplementary materials). seb-nps showed clear needle granular morphology with a length of less than nm. the results revealed that the doping selenium influenced the morphology of seb-nps. agglomeration was observed in all seb-nps samples. sem was used to further confirm the morphology of seb-nps, and the sem images were shown in figure s a -f (supplementary materials), and the nanoparticles of all samples were aggregated. with increased ca/se mole ratio, more seb-nps nanoparticles aggregated, which was certified via dls analysis ( figure s , supplementary materials). furthermore, the high-angle annular dark-field (haadf) mode image of sample seb was present in fig. d , which was inserted in the top-left corner, and the elemental mapping results were shown in fig. e-g. the presence of calcium (yellow), phosphorus (blue), and selenium (green) indicated that selenium was evenly distributed in the seb-nps. the tem images of ag seb-nps were displayed in fig. . compared with seb-nps, the ag seb-nps displayed irregular morphology, and the aggregation was observed, indicating that the morphology of seb-nps was influenced by the deposition of silver nanoparticles. according to the dls test results ( figure s c , supplementary www.nature.com/scientificreports/ materials), multiple peaks appeared and confirmed that ag seb-nps nanoparticles were further aggregated compared to seb-nps, which was also affirmed by sem ( figure s g-i, supplementary materials) . with the increase of agno used during the preparation procedure, the prepared ag seb-nps tended to flock together, and the snowflake-like morphology was shown (fig. d) . the high-magnification image of ag seb revealed that the silver nanoparticles adhered to the surface of ag seb-nps (fig. c) . interestingly, a rooster-like image (like the map of china) was obtained for ag seb sample, as shown in fig. e , and the haadf mode image was inserted in the bottom-right corner. the elemental mapping analysis showed that silver (red, fig. f) , calcium (yellow, fig. g) , phosphorus (blue, fig. h) , and selenium (green, fig. i) were detected, proved that silver and selenium were evenly distributed in ag seb-nps. in addition, edx analysis was used to further determine the elemental composition of seb-nps and ag seb-nps, as shown in figure s (supplementary materials). according to the edx analysis of seb-nps, the elements oxygen (o), phosphorous (p), calcium (ca), and selenium (se) were presented. with increasing ca/se mole ratio, the weight percent of selenium calculated from edx decreased. www.nature.com/scientificreports/ the weight percentage of silver were . , . , and . for ag seb , ag seb , and ag seb , respectively, which was consistent with the amount of agno added during the preparation of ag seb-nps. in vitro cytocompatibility. the in vitro cytocompatibility test of the prepared seb-nps and ag seb-nps is an essential prerequisite for future bone tissue engineering, and biological experiments were conducted by using hfob . cell as the cell model. figure showed the cck- assay results after the hfob . cells cultured with seb-nps and ag seb-nps for and days. the degree of hfob . cells growth was recorded by the absorbance at nm. for all prepared nanoparticles, the number of cells increased with the number of culture days indicated that the obtained nanoparticles had an excellent cytocompatibility. however, the cell number decreased with increasing the concentration of nanoparticles from µg/ml to µg/ml. the cell morphology on day was investigated using optical microscopy and fluorescent microscopy (fitc-phalloidin/dapi staining) and µg/ml of nanoparticles were used ( figures s and s , supplementary materials). the cells incubated with the prepared nanoparticles showed a well-preserved morphology, which was polygonal and fully spread. moreover, the cells proliferated with ag seb-nps faster, than that with seb-nps. there were no statistically significant differences in cell proliferation culturing with different samples. antibacterial test. the antibacterial activity of the prepared seb-nps and ag seb-nps against gram-positive s. aureus and gram-negative e. coli bacteria was systematically evaluated, and the results were shown in figs. and . s. aureus could cause the formation of biofilm on bone implants, and e. coli strains possess the reduction capability of selenite. thus, both bacteria were used here. the optical density at nm (od ) values of s. aureus and e. coli by adding seb-nps and ag seb-nps using different concentrations were presented in fig. . compared with the control group, the doping selenium into bcp-nps had slight influence on the antibacterial activity, and it had a slight inhibitory effect against bacteria of seb-nps after h of culture. however, the proliferation of both s. aureus and e. coli was completely suppressed by ag seb-nps. the photos and inhibition ratio of e. coli and s. aureus grown in the actual culture tubes after adding seb-nps and ag seb-nps after h were shown in figures s and s (supplementary materials) . furthermore, the od values of s. aureus after seb-nps were lower than that of e. coli, indicated that seb-nps had a better antibacterial effect against s. aureus than e. coli. for ag seb-nps, the inhibition ratio of both s. aureus and e. coli increased with the increase of the agno dosage. there were no differences in ag seb-nps against both bacteria. the antibacterial activity of nanoparticles was also confirmed by bacteriostatic circles investigation as shown in fig. . the disk diffusion was used to confirm the antibacterial effect of prepared seb-nps and ag seb-nps dispersed in ultrapure water against bacterial colonies. photos of bacteriostatic circles for nanoparticles using different concentrations were recorded, as shown in fig. . for seb-nps, the bacteriostatic circles of e. coli were not apparently observed, and such circles of s. aureus appeared, in which the concentration of nanoparticles did not have a significant influence on the diameter of circles. however, compared with seb-nps, ag seb-nps have significant inhibition circles against s. aureus and e. coli. the antibacterial activity of ag seb-nps against e. coli was not influenced by the concentration. but, such activity against s. aureus increased with increasing of ag seb-nps the stated objective of this study was to synthesis new-style biphasic calcium phosphate nanoparticles (bcp-nps) with excellent cytocompatibility and antibacterial activity for further hard-tissue engineering applications. recently, silicon, silver, copper, zinc, selenium, iron, lithium, and titanium dioxide, et al., have been employed to mingle with bone scaffolds to improve their physicochemical properties [ ] [ ] [ ] [ ] [ ] [ ] [ ] . such as, silicon could be doped into hydroxyapatite and composited into gelatine, and then, the three-dimensional ( d) printable composites were obtained. the porous scaffold could be fabricated by rapid prototyping at room temperature (rt) . the bone regeneration of bioactive silicate glass could be improved when the copper was doped at a controlled concentration ( - . wt.% copper oxide), while displayed a promising antibacterial activity . furthermore, compared with pure hydroxyapatite (ha) scaffold, lithium-doped hydroxyapatite scaffold not only showed a higher degradation rate but also benefit the proliferation of osteoblasts . ahmed et al. confirmed that the introduction of selenium www.nature.com/scientificreports/ into carbonated hydroxyapatite (chap) could increase the diffusion and infiltration of human fibroblasts on chap based scaffold . developing a multifunctional scaffold, which combines excellent biocompatibility, osteoinductivity, and antibacterial ability, which is considered to be the next-generation orthopedic implants for hard tissue engineering applications , . selenium, silver, and antibacterial drugs, such as cephalexin and chlorhexidine, were commonly used to impart antibacterial function to the composite scaffolds [ ] [ ] [ ] [ ] . nguyen et al. investigated that selenium nanoparticles could inhibit staphylococcus aureus, with low toxicity to mammalian cells . wang et al. identified that after coating poly(ether ether ketone) (peek) medical devices with selenium nanoparticles, the growth of pseudomonas aeruginosa could be significantly inhibited . however, the uncontrolled release of drugs, the potential systemic toxicity of medicine, and the aggregation of nanoparticles hindered the potential functions of the scaffolds. for these reasons, drugs with selenium, and silver were mainly encapsulated, or mixed, coated into the scaffold , . here, we first fabricated the selenium-doped biphasic calcium phosphate nanoparticles, which were incorporated with silver nanoparticles. the physicochemical properties of the obtained ag seb-nps were deeply characterized by ft-ir, xrd, uv-vis, raman, and xps analysis. in this study, seb-nps were synthesized by co-precipitation and ion-exchange sorption, aka, post-precipitation, occurred simultaneously. by substituting po − on the surface of bcp-nps to adsorb seo − group, then a port of seo − ions entered the lattice (ha or β-tcp) . the addition of selenium dopant could influence the phase composition, and the resulting seb-nps could be tentatively represented as ca (po ) x (seo ) ( -x) (oh) + ca (po ) x (seo ) ( -x) . the ft-ir spectra of seb-nps and bcp-nps showed the v + v phosphate vibration in the region of , - cm − and v phosphate bands in the range of - cm − , as shown in fig. a . the o-se-o asymmetric bond stretching at cm − was detected for seb-nps, and the seo − group at cm − was measured as well. with decreased ca/se mole ratio, the peaks belonging to the seo − group became stronger, which confirmed the presence of seo − group in seb-nps . after xps analysis of seb-nps, there was one peak at ev of the se d scanning, which also proved that only seo − [se (iv)] instead of seo − [se (vi)] was incorporated into seb-nps (fig. ). however, after the deposition of silver nanoparticles on the surface of seb-nps, no chemical bonds were formed between silver and seb-nps (figs. , ) . next, the crystallinity of seb-nps and ag seb-nps was evaluated by xrd, as shown in fig. a . the appearance of ha and β-tcp in all samples was expected. the relative intensity of the diffraction peak at . ° ( ) (d ( ) , miller's plane) was chosen to calculate the crystallite size (scherrer equation) , . the crystallite means size varied for bcp-nps, seb-nps, and ag seb-nps, were calculated by scherrer equation (supplementary materials). compared with bcp-nps, the unit cell dimensions of seb-nps were decreased, which proved the incorporation of seo − group. this inclusion was due to the different shape between seo − group (trigonal pyramids) and po − group (tetrahedra), and phosphate-to-selenite substitution resulted in paired ca + and oh − vacancies to rebalance the charge . as the ca/se mole ratio decreased, the corresponding crystallinity increased. however, the crystallinity of ag seb-nps with different agno concentrations changed slightly, mainly because silver nanoparticles were only deposited on the surface of seb-nps without chemical reaction occurred, which was consistent with the uv-vis drs testing results. rameshbabu et al. identified that silver could influence the crystallinity of ha via heat treatment and further changed the nanosize of ha . furthermore, the morphology of seb-nps showed a typical needle-like bundle shape, which was different from bcp-nps ( fig. and figure s , supplementary materials). unfortunately, the strong tendency to agglomerate made it impossible to evaluate its size distribution. after the deposition of silver, the morphology of ag seb-nps www.nature.com/scientificreports/ tended to be further aggregated, and ag seb-nps changed to snowflake-like agglomerate with increasing agno concentration. element mapping analysis confirmed that selenium was uniformly distributed in seb-nps, and silver was spread on the surface of ag seb-nps (figs. and ). selenium has been widely applied in diverse areas such as the food and pharmaceutical industries. many studies have confirmed that selenium doped ha not only exhibited low cytotoxicity for osteoblastic cells but also reduced the chance of tumor recurrence , . it was necessary to estimate the toxicity of the obtained seb-nps and ag seb-nps for further tissue engineering applications. osteoblasts were commonly used to evaluate the cytocompatibility of nanoparticles and/or scaffold in tissue engineering , . in this study, the cytocompatibility of seb-nps and ag seb-nps was assessed by culturing with hfob . cells. bcp-nps possessed excellent cytocompatibility, and the incorporation of selenium into bcp-nps did not reduce the cellular biocompatibility . according to cck- analysis (fig. ) , osteoblasts proliferated with seb-nps over days, and the growth of cells was not influenced by the ca/se mole ratio. after the deposition of silver, cells cultured with all ag seb-nps samples had similar proliferation profiles compared with seb-nps. not only can the addition of selenium and silver improve the growth of the cells with bcp-nps, but the antibacterial activity was acquired, which has potential for infection-resistant replacement materials , . finally, the typical gram-positive s. aureus and gram-negative e. coli bacteria were used to evaluate the antibacterial activity of synthesized seb-nps and ag seb-nps. s. aureus, the most common virulent pathogen, and due to the biofilm formation and documented antibiotic resistance, can cause bone-implants-associated infections in hospitalized patients. here, s. aureus was used together with e. coli (due to the highly capable of reduction) . selenium could promote the formation of superoxide radicals and, enhance oxidative stress, which resulted in the damage of bacterial cell walls besides, selenium could inhibit the biofilm formation of the s. aureus and the growth of e. coli , . figures and illustrated the antibacterial activity of seb-nps and ag seb-nps against s. aureus and e. coli colonies. it was noted that the introduction of selenium in bcp-nps could not empower the antibacterial performance on seb-nps. besides, the antibacterial activity of the selenium-doped hydroxyapatite nanoparticles (seha-nps) was investigated herein for comparison ( figures s and s , supplementary materials). the results revealed that seha-nps didn't display inhibitory activity against s. aureus and e. coli, which was slightly different from previous reports , . the different results might be due to the varying morphology of seha-nps used in this work, demonstrating that the antibacterial effect of nanoparticles is greatly influenced by the shape and size. however, after deposition of silver on seb-nps, the growth of s. aureus and e. coli were both decreased significantly. assuming that a large number of silver nanoparticles might be released from ag seb-nps, which can inhibit the initial bacterial adhesion and growth, the mechanism of prepared ag seb-nps was displayed in fig. . previous papers reported that the release of silver nanoparticles could be regulated by ph, calcium, and phosphate ion concentrations in the surrounding medium . notably, the acidic environment could generate the release of silver nanoparticles. therefore, because of the growth of oral bacteria under acidic conditions, the future use of ag seb-nps could be expanded to dental applications . besides, selenium-doped bone mineral nanoparticles performed effective bone tumor inhibition. thus, ag seb-nps has potential advantages in the fabrication of a multifunctional bone scaffold for healing related bone tumor diseases. the balance between biocompatibility and antibacterial properties of ag seb-nps, i.e., the excellent cytocompatibility, and productive inhibition activity against bacteria, supported the significance of ag seb-nps in biomedical applications. however, future applications require further in vitro and in vivo investigations, including the release profile of silver nanoparticles and selenium. figure . mechanism of antibacterial activity of prepared ag seb-nps against bacteria. silver ions were released from ag seb-nps to produce free radicals, which resulted in reactive oxygen species (ros), and damaged bacteria until bacterial death. in summary, a well-developed nanostructured seb-nps and ag seb-nps with excellent biocompatibility and suitable antibacterial properties were fabricated herein. fi-ir, raman, xrd and xps analysis certified that the seo − was doped at po − position. the needle-cluster-like morphology of seb-nps was obtained, and the formed ag seb-nps tended to flock together, which was confirmed by tem. after culturing with hfob . cells, both seb-nps and ag seb-nps behaved excellent cytocompatibility. next, the gram-negative e. coli and grampositive s. aureus were used to verify that ag seb-nps had better antibacterial activity than seb-nps. the results obtained support the significance of ag seb-nps in diverse biomedical applications. all data generated or analyzed during this study are included in the published article and its supplementary materials files and are available from the corresponding author on reasonable request. received: november ; accepted: august antimicrobial and biocompatible fluorescent hydroxyapatite-chitosan nanocomposite films for biomedical applications development of chitosan/gelatin hydrogels incorporation of biphasic calcium phosphate nanoparticles for bone tissue engineering amorphous calcium phosphate nanospheres/polylactide composite coated tantalum scaffold: facile preparation, fast biomineralization and subchondral bone defect repair application insight into biological apatite: physiochemical properties and preparation approaches nanocrystalline hydroxyapatite enriched in selenite and 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contribution to acquisition and interpretation of data. all authors critically read and approved the final submitted manuscript the authors declare no conflict of interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to l.n.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -ewvnkdr authors: steeds, kimberley; hall, yper; slack, gillian s.; longet, stephanie; strecker, thomas; fehling, sarah katharina; wright, edward; bore, joseph akoi; koundouno, fara raymond; konde, mandy kader; hewson, roger; hiscox, julian a.; pollakis, georgios; carroll, miles w. title: pseudotyping of vsv with ebola virus glycoprotein is superior to hiv- for the assessment of neutralising antibodies date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ewvnkdr ebola virus (ebov) is an enveloped, single-stranded rna virus that can cause ebola virus disease (evd). it is thought that evd survivors are protected against subsequent infection with ebov and that neutralising antibodies to the viral surface glycoprotein (gp) are potential correlates of protection. serological studies are vital to assess neutralising antibodies targeted to ebov gp; however, handling of ebov is limited to containment level laboratories. pseudotyped viruses can be used as alternatives to live viruses, which require high levels of bio-containment, in serological and viral entry assays. however, neutralisation capacity can differ among pseudotyped virus platforms. we evaluated the suitability of ebov gp pseudotyped human immunodeficiency virus type (hiv- ) and vesicular stomatitis virus (vsv) to measure the neutralising ability of plasma from evd survivors, when compared to results from a live ebov neutralisation assay. the sensitivity, specificity and correlation with live ebov neutralisation were greater for the vsv-based pseudotyped virus system, which is particularly important when evaluating ebov vaccine responses and immuno-therapeutics. therefore, the ebov gp pseudotyped vsv neutralisation assay reported here could be used to provide a better understanding of the putative correlates of protection against ebov. www.nature.com/scientificreports/ and time required for plaque development, which can take approximately nine days, makes it time-consuming and restricts high-throughput sample processing. development of novel serological assays that utilise genetically modified recombinant or chimeric viruses with attenuated pathogenicity have enabled more widespread investigation of neutralising antibodies against highly pathogenic viruses including ebov , . pseudotyped viruses are replication-defective chimeric virions that comprise the structural and enzymatic core of one virus, bearing the envelope protein or glycoprotein of another, and encode a quantifiable reporter gene. retroviruses, including lentiviruses and gammaretroviruses such as human immunodeficiency virus (hiv) and murine leukaemia virus (mlv), respectively, and rhabdoviruses, such as vesicular stomatitis virus (vsv), have been used extensively as cores for pseudotyped viruses , , including for ebov , . a number of ebov gp pseudotyped virus neutralisation assays have been developed to investigate immune responses to ebov infection and vaccination [ ] [ ] [ ] , as well as for evaluation of monoclonal antibody (mab) therapies [ ] [ ] [ ] . there are many factors that need to be considered when developing and optimising pseudotyped virus neutralisation assays, to assess experimental parameters that can affect assay performance and to ensure accuracy and reproducibility. these include, choice of core virus and reporter gene, determination of target cell line and amount of pseudotyped virus input, as well as correlation with live virus neutralisation . the aim of this study was to assess the suitability of ebov gp pseudotyped hiv- and vsv systems to measure neutralisation by evd survivor plasma, in comparison with results from a live ebov neutralisation assay. cell tropism of ebov gp pseudotyped viruses. pseudotyped hiv- and vsv bearing the envelope gp from ebov (mayinga) were generated and quantified by measuring luminescence in a range of target cell lines, in order to determine the optimum cell line to use in neutralisation assays. cells only controls were used to determine background levels of luminescence ( supplementary fig. s ). reporter activity was detected in all cell lines infected with ebov gp pseudotyped hiv- and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed (fig. a,b) . for ebov gp pseudotyped hiv- , highest tcid /ml values were observed in t/ cells, followed by huh- cells (fig. c) . titres generated by infection of t/ cells were approximately , and times greater than those produced by infection of huh- , hela and vero e cells, respectively. for ebov gp pseudotyped vsv, highest titres were obtained in during the initial stages of assay development, it is important to evaluate neutralisation of pseudotyped viruses using well characterised antibodies in order to demonstrate the validity and accuracy of the assay. the ebov gp pseudotyped viruses were assessed for neutralisation by the human anti-ebov gp mab, kz . kz is an antibody isolated from a human survivor of the outbreak in kikwit that neutralises ebov in vitro and recognises a conformational epitope at the base of the gp [ ] [ ] [ ] . human anti-ebov gp mab, kz was unable to neutralise the ebov gp pseudotyped hiv- (fig. a) within the range tested, however it was able to neutralise the ebov gp pseudotyped vsv (fig. b) , suggesting that vsv-based pseudotyped viruses are more sensitive to neutralisation then lentiviral-based, possibly the density of ebov gp on the pseudotyped hiv- may differ from that on the pseudotyped vsv or live ebov. to determine the optimal pseudotyped virus input to use in the hiv-and vsv-based assays, neutralisation of different amounts of the ebov gp pseudotyped viruses by plasma from a guinean evd survivor donor or human anti-ebov gp mab kz was assessed. kz was selected as it is commercially available and there is accompanying information regarding its neutralisation activity against ebov gp pseudotyped vsv expressing luciferase. however, as the ebov gp pseudotyped hiv- was not neutralised by kz (fig. a) in the range tested, plasma from an evd survivor was used to assess the effect of pseudotyped hiv- input on neutralisation instead. survivor plasma sample cs was chosen as it displayed strong neutralising ability against live ebov neutralisation, with a geometric mean titre (gmt) of , . percentage infectivity was determined relative to infectivity of cells by the ebov gp pseudotyped viruses alone (fig. a ,b) and % inhibitory concentration (ic ) of pseudotyped virus neutralisation were estimated by model of nonlinear regression dose-response curves (fig. c,d) . plasma from evd survivor cs displayed neutralising activity against all amounts of ebov gp pseudotyped hiv- tested (fig. a) . lower pseudotyped virus input resulted in larger variability and less curve fitting. therefore, an ebov gp pseudotyped hiv- input of at least . × rlu/well, with a target input of . × rlu/well, was used in subsequent neutralisation assays. kz neutralised all dilutions of ebov gp pseudotyped vsv tested (fig. b) and ic values decreased with decreasing amounts of pseudotyped virus input (fig. d , supplementary table s ). when using . × rlu/well of ebov gp pseudotyped vsv, ic of virus neutralisation ( . µg/ ml) was similar to that expected according to the manufacturer's product data sheet ( . µg/ml). therefore, a target input of approximately . × rlu/well was used in subsequent ebov gp pseudotyped vsv neutralisation assays. table s ). neutralisation of ebov gp pseudotyped hiv- and vsv by positive (evd survivor) and negative (uk donor) control plasma was assessed in several independent assays ( supplementary fig. s ). the background level of neutralisation was determined using plasma from a uk negative control donor. for the hiv- -based assay this was calculated as ic . reciprocal dilution. the negative control plasma displayed no neutralising activity against ebov gp pseudotyped vsv, and therefore the background level of neutralisation was assigned the low- www.nature.com/scientificreports/ est dilution of sample tested in the assay ( / ). in the hiv- -based assay, dose-response curves were unable to be fitted for three of the evd survivor samples and six of the samples were deemed below the background level of neutralisation. in contrast, a dose-response curve was unable to be fitted for only one of the evd survivor samples tested in the vsv-based neutralisation assay. in the hiv- -based assay, three of the negative plasma samples tested were above the background level of neutralisation, whereas only one of the negative samples tested was above the background level of neutralisation in the vsv-based assay. although some differences in the discriminatory power of positive and negative samples between the assays were observed, a statistically significant difference in neutralisation titres was detected between the evd survivors and negative plasma samples in the hiv- -based assay (mann-whitney, p = . ) (fig. a ) and in the vsv-based assay (mann-whitney, p < . ) (fig. b , supplementary table s ). remarkably, this difference was more significant and the separation of the positive and negative plasma was better in the vsv-based assay (fig. b) . the sum of these results clearly show that the vsv-based ebov gp neutralisation assay displayed better reliability, specificity and sensitivity compared to the hiv- -based assay. correlation with live ebov neutralisation. the neutralising capacity of the individual plasma samples against authentic ebov was assessed by a live virus neutralisation assay (supplementary table s , supplementary fig. s ). when ic values of ebov gp pseudotyped hiv- neutralisation of the evd survivor and negative plasma samples were compared with gmt values for the live ebov neutralisation assay, a positive correlation (r s = . ) was determined using the nonparametric spearman correlation coefficient (fig. a) and this was statistically significant (p = . ). remarkably, a stronger statistically significant (p < . ) positive correlation (r s = . ) was observed when ic values of ebov gp pseudotyped vsv neutralisation were compared with gmt values for the live ebov neutralisation assay (fig. b) . the correlation coefficients for ebov gp hiv- and vsv ic compared with live ebov gmt without the negative controls were . (p = . ) and . (p < . ), respectively. therefore, the vsv-based ebov gp pseudotyped virus neutralisation assay correlated better with live ebov neutralisation than the hiv- -based neutralisation assay. pseudotyped viruses can be used as alternatives to infectious virus in serological assays to measure neutralising antibodies to viral envelope glycoproteins . pseudotyped virus assays used to profile neutralising antibody responses against severe acute respiratory syndrome-associated coronavirus (sars-cov) , influenza (h n and h n ) [ ] [ ] [ ] , rabies , and chikungunya virus , for example, found that results correlated well with those from replication-competent or live virus assays. a high degree of correlation has been demonstrated between ebov cl prnt and an ebov pseudotyped vsv cl fluorescence reduction neutralisation test (frnt) . however, pseudotyped virus assays may not always accurately determine neutralisation , . live ebov and ebov gp pseudotyped neutralisation assays have previously been shown to yield variable results , , which could be due to differing experimental conditions and viral systems. it is therefore important to optimise pseudotyped virus neutralisation assays in context of the particular viral gp being studied in order to obtain reliable specificity and sensitivity. the aim of this study was to assess the suitability of ebov gp pseudotyped hiv- and vsv systems to measure the neutralising ability of plasma from evd survivors, when compared to live ebov neutralisation. reporter activity was detected in all cell lines ( t/ , huh- , hela and vero e ) infected with ebov gp (mayinga) pseudotyped hiv- and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed. this may reflect general defects in viral entry in different cells. a relatively lower level of ebov gp pseudotyped hiv- transduction was exhibited by vero e cells, which might be due to an intrinsic restriction factor, trim α, which restricts retroviral infection by specifically recognising the hiv- capsid and promoting its rapid, premature disassembly . highest tcid values were obtained following ebov gp pseudotyped hiv- and vsv infection of t/ and vero e cells, respectively. there seemed to be large variability of the luminescent measurement for the vsv-based platform, which may be caused by the www.nature.com/scientificreports/ sensitive nature of the luciferase signal detection. this highlights the importance of titrating each pseudotyped virus batch before use in neutralisation assays, and the inclusion of multiple replicates. the ebov gp pseudotyped viruses were used to assess the neutralising activity of a human anti-ebov gp mab, kz . kz has been shown previously to neutralise ebov pseudotyped viruses , , . however, within the range tested here, kz did not display neutralisation against ebov gp pseudotyped hiv- , suggesting that the ebov gp on the pseudotyped hiv- might be at higher levels, thereby reducing assay sensitivity, and neutralisation may be observed using a higher concentration of kz . in contrast, kz was able to neutralise the ebov gp pseudotyped vsv. to assess the effects of differing amounts of pseudotyped virus input on neutralisation, plasma from an evd survivor of the - ebov outbreak and kz were screened against different amounts of the ebov gp pseudotyped hiv- and vsv, respectively. decreasing quantities of pseudotyped hiv- led to more variable and unreliable results, and the kz ic of pseudotyped virus neutralisation decreased with decreasing amounts of ebov gp pseudotyped vsv input. the variability in neutralisation observed between different amounts of pseudotyped virus input highlights the importance of including standards or reference material with a known activity or potency when comparing neutralising activity, allowing calibration of results . both pseudotyped virus systems were able to measure neutralising antibodies in plasma from evd convalescent patients, and results correlated positively with a live ebov neutralisation assay. however, the discriminatory power of the hiv- -based assay with regards to differing antibody titres appeared to be low. some of the samples tested, which showed neutralising activity against live ebov, did not display neutralisation against the pseudotyped virus and vice versa, therefore raising questions on the sensitivity and specificity of the pseudotyped hiv- assay. in the current study, human embryonic kidney ( t/ ) cells were used for the pseudotyped hiv- neutralisation assays, whereas african green monkey kidney (vero) cells were used in the vsv-based assay and also the live ebov assay. therefore, this could account for some of the differences in results observed between the two assays and for the better performance of the vsv-based assay in relation to live ebov neutralisation. also, the hiv- -and vsv-based pseudotyped virus systems assessed in the current study utilise different transfection methods, which could have implications on the composition of the pseudotyped viruses, density and/or glycosylation of the viral envelope protein on the surface, and consequently neutralisation results. this highlights the importance of assessing experimental conditions and methodology when developing and optimising pseudotyped virus neutralisation assays. a limitation to this study was that the level of ebov gp incorporation per pseudotyped virus type could not be assessed. also, for the vsv-based pseudotyped virus system, traces of vsv-g from the rvsv-Δg-luc-vsv-g virus could be recycled into newly pseudotyped virions . therefore, the use of anti-vsv-g hybridoma cell culture supernatant could give rise to pseudotyped virions covered by anti-vsv-g antibodies, but are still infectious due to ebola gp. this could potentially induce plasma specific reactivity of virions due to bound anti-vsv-g antibodies more than ebov gp specific reactivity. there are several differences between ebov gp pseudotyped and live ebov neutralisation assays that could affect their results . due to their non-replicating nature, such pseudotype systems do not recapitulate all steps in the viral life cycle that may potentially be targeted by neutralising antibodies . in addition, the round, spherical shape of ebov gp pseudotyped hiv- or bullet shape of ebov gp pseudotyped vsv compared to the filamentous shape of authentic ebov could affect their susceptibility to neutralisation. also, the density of gp on the surface of the pseudotyped virus may not be the same as that found on live ebov and may result in the loss or masking of quaternary epitopes , . furthermore, gp maturation and assembly in live ebov could be different in the generation of an ebov pseudotyped virus and may result in different targets and/or conformational epitopes when using whole live ebov as opposed to ebov gp alone in a pseudotyped virus. the presence of shed gp or secreted gp (sgp) in the live ebov assay compared to absence in the ebov gp pseudotyped virus assays could also have an effect on neutralisation. in the live ebov assay, shed gp and sgp could reduce neutralisation of circulating virus by cross-reactive antibodies to surface gp. however, in the current study, weaker relative neutralisation was observed in the hiv- based pseudotyped virus assay. therefore, it is possible that cell debris or free gp generated during ebov gp pseudotyped hiv- production by polyethylenimine (pei) transfection could be interfering with neutralisation. finally, detection of infected cells via measurement of luminescence in the ebov gp pseudotyped virus neutralisation assay compared to plaque formation in the live ebov neutralisation assay could affect neutralisation readout. ebov gp pseudotyped virus neutralisation assays have value for vaccine evaluation and assessment of convalescent blood products and mabs for use as immunotherapeutics. however, pseudotyped virus assays may not always accurately determine neutralisation when compared with neutralisation against live virus. in this study, both ebov gp pseudotyped hiv- and vsv assays were able to detect neutralisation of plasma from evd survivors and correlated positively with live ebov neutralisation. however, the vsv-based assay performed better than the hiv- -based assay in relation to specificity, sensitivity, and correlation with the live ebov neutralisation assay. this research has highlighted the importance of optimising pseudotyped virus neutralisation assays in context of the particular viral gp being studied, especially when evaluating vaccine responses and therapeutics, and could provide a better understanding of the correlates of protection against ebov. and from negative control blood donors in the uk and guinea, who were not knowingly exposed to persons with evd and did not attend high risk events such as funerals, were heat inactivated at °c for min. the samples were obtained from a pre-existing biobank, for which live ebov neutralisation production of pseudotyped viruses. the generation of hiv- pseudotyped viruses was performed as detailed previously , , . twenty-four hours prior to transfection, approximately × t/ cells were seeded into sterile, -well cell culture plates (corning, ewloe, uk) and incubated at °c, % co and % humidity until - % confluence. the hiv gag-pol plasmid, p . , and the firefly luciferase reporter construct, pcsflw, were transfected simultaneously with the ebov (mayinga) gp expression vector at a ratio of . : . : . µg (core:reporter:envelope) using µl of µg/ml polyethylenimine (pei) (sigma-aldrich) per µg dna in opti-mem medium (gibco). following overnight transfection, the cells were incubated with fresh medium and incubated at °c, % co . pseudotyped virus supernatants were harvested at and h posttransfection, passed through a . µm pore filter (millex, millipore, watford, uk) and stored at − °c. ebov gp pseudotyped vsvs were prepared using recombinant vsv, in which the vsv-g gene had been deleted (rvsv-Δg) and replaced with a luciferase reporter gene (rvsv-Δg-luc) by a method similar to that described previously . twenty-four hours prior to transfection, approximately . × t/ cells were seeded into sterile, mm cell culture dishes (corning) and incubated at °c, % co and % humidity until - % confluence. the cells were transfected with the ebov gp expression vectors using transit-lt transfection reagent (mirus bio, madison, wisconsin (wi), usa) as per the manufacturer's instructions. following overnight transfection, the medium was removed and the cells were infected with rvsv-Δg-luc that was pseudotyped with the vsv glycoprotein (rvsv-Δg-luc-vsv-g) (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a multiplicity of infection (moi) of in opti-mem medium and incubated at °c, % co . after h, the inoculum was removed, cells were washed twice with dulbecco's phosphate buffered saline (dpbs) (gibco) and fresh medium was added. pseudotyped virus supernatants were harvested at - h post-infection, clarified twice by centrifugation at xg for min at °c and stored at − °c. prior to use, the pseudotyped viruses were incubated with anti-vsv-g hybridoma cell culture supernatant (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a : dilution for h at °c to reduce background infection mediated by residual virus possessing vsv-g, which can be carried over during preparation . all experiments involving pseudotyped viruses were performed in a cl facility at public health england (phe), porton down, uk. pseudotyped virus titration and neutralisation assays. titration and neutralisation assays were performed in -well solid white flat bottom polystyrene tc-treated microplates (corning) and were based upon previously described protocols , , . for pseudotyped hiv- titration assays, five-fold serial dilutions of pseudotyped virus at a starting dilution of : were prepared in quadruplicate in opti-mem medium at a final volume of µl/well. µl of approximately × t/ , huh- or vero e cells, or × hela cells were then added to each well and incubated at °c, % co for h. the medium was removed and µl of a : mix of bright-glo luciferase assay reagent (promega, southampton, uk):fresh medium was added to each well and incubated for at least min at room temperature to allow complete cell lysis. luminescence was measured using a glomax-multi + detection system luminometer (promega) and relative luminescence units per ml (rlu/ml) were determined. the negative cut-off was set at . times the average rlus of the cells only control wells. % tissue culture infectious dose (tcid )/ml values were determined using the reed-muench method . for the pseudotyped hiv- neutralisation assay, two or threefold serial dilutions of plasma samples at a starting dilution of : or : , respectively, were prepared in duplicate in opti-mem medium at a final volume of µl/well and incubated with µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for h at °c. µl of approximately × t/ cells were then added to each well and incubated for h at °c, % co , prior to taking a chemiluminescent readout as described above. infectivity was calculated using the formula: percentage (%) infectivity = [(rlu with sample)/(rlu without sample)] × . www.nature.com/scientificreports/ for pseudotyped vsv titration assays, h prior, approximately . × t/ or × huh- , hela cells or vero e cells were seeded in -well microplates and incubated at °c, % co and % humidity. the medium was removed and two-fold serial dilutions of pseudotyped virus in opti-mem medium, starting with neat pseudotyped virus were added to each well in quadruplicate at a final volume of µl/well. after h, a chemiluminescent readout was taken and tcid /ml values were determined as described above. twenty-four hours prior to pseudotyped vsv neutralisation, approximately × vero e cells were seeded and incubated as for titration above. twofold serial dilutions of plasma samples at a starting dilution of : were prepared in duplicate in opti-mem medium at a final volume of µl/well in -well microplates, and incubated with µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for h at °c. the medium was removed from the cells, µl of the plasma-pseudotyped virus mixtures were added to each well in quadruplicate at incubated at °c, % co . after h, µl of fresh medium was added to each well. luminescence was measured after h and infectivity was calculated as described above. statistical analysis. pseudotyped virus neutralisation assay raw data were normalised as percentage (%) infection relative to mean values for pseudotyped virus only controls (equivalent to % infection), then ic of pseudotyped virus neutralisation were estimated by model of nonlinear regression fit with settings for log (inhibitor) vs. normalised response curves using graphpad prism v (san diego, california (ca), usa). statistical comparison between two unpaired groups was performed using the mann-whitney test (graph-pad prism v ). correlation between two variables was quantified using spearman nonparametric correlation (graphpad prism v ). www.nature.com/scientificreports/ open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. ebola haemorrhagic fever emergence of zaire ebola virus disease in guinea world health organization. situation report - biochemical analysis of the secreted and virion glycoproteins of ebola virus a mutation in the ebola virus envelope glycoprotein restricts viral entry in a host species-and cell-type-specific manner characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines antibody-mediated neutralization of ebola virus can occur by two distinct mechanisms systematic analysis of monoclonal antibodies against ebola virus gp defines features that contribute to protection role of antibodies in protection against ebola virus in nonhuman primates immunized with three vaccine platforms the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination current progress with serological assays for exotic emerging/re-emerging viruses construction and use of a human immunodeficiency virus vector for analysis of virus infectivity a system for functional analysis of ebola virus glycoprotein distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps specific neutralizing response in plasma from convalescent patients of ebola virus disease against the west africa makona variant of ebola virus ebola virus neutralizing antibodies detectable in survivors of the yambuku, zaire outbreak years after infection mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb- cocktail antibodies protective monotherapy against lethal ebola virus infection by a potently neutralizing antibody potent neutralizing monoclonal antibodies against ebola virus infection technical considerations for the generation of novel pseudotyped viruses ebola virus can be effectively neutralized by antibody produced in natural human infection pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody structure of the ebola virus glycoprotein bound to an antibody from a human survivor longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to h n influenza viruses characterization of lentiviral pseudotypes with influenza h n hemagglutinin and their performance in neutralization assays safe pseudovirus-based assay for neutralization antibodies against influenza a(h n ) virus a robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in africa development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection high degree of correlation between ebola virus bsl- neutralization assays and pseudotyped vsv bsl- fluorescence reduction neutralization test human immunodeficiency virus type env clones from acute and early subtype b infections for standardized assessments of vaccine-elicited neutralizing antibodies broadly neutralizing human monoclonal antibodies to the hepatitis c virus e glycoprotein comparison of platform technologies for assaying antibody to ebola virus specific recognition and accelerated uncoating of retroviral capsids by the trim α restriction factor a shared structural solution for neutralizing ebola viruses ebola virus: pseudotypes, libraries and standards characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis c virus glycoproteins neutralizing antibodies inhibit chikungunya virus budding at the plasma membrane maturation of west nile virus modulates sensitivity to antibody-mediated neutralization multiply attenuated lentiviral vector achieves efficient gene delivery in vivo investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison phase trials of rvsv ebola vaccine in africa and europe a sensitive retroviral pseudotype assay for influenza h n -neutralizing antibodies lyophilisation of influenza, rabies and marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit generation of vsv pseudotypes using recombinant Δg-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines a simple method of estimating fifty per cent endpoints the authors would like to thank masayuki saijo for providing the rvsv-Δg-luc-vsv-g virus and anti-vsv-g hybridoma cell culture supernatant. we are grateful to eccac for providing vero e and hela cells, and to arvind patel for providing huh- cells. this work was funded by the u.s. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to m.w.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -h mtpcyc authors: mathé-hubert, hugo; colinet, dominique; deleury, emeline; belghazi, maya; ravallec, marc; poulain, julie; dossat, carole; poirié, marylène; gatti, jean-luc title: comparative venomics of psyttalia lounsburyi and p. concolor, two olive fruit fly parasitoids: a hypothetical role for a gh β-glucosidase date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: h mtpcyc venom composition of parasitoid wasps attracts increasing interest – notably molecules ensuring parasitism success on arthropod pests – but its variation within and among taxa is not yet understood. we have identified here the main venom proteins of two braconid wasps, psyttalia lounsburyi (two strains from south africa and kenya) and p. concolor, olive fruit fly parasitoids that differ in host range. among the shared abundant proteins, we found a gh β-glucosidase and a family of leucine-rich repeat (lrr) proteins. olive is extremely rich in glycoside compounds that are hydrolyzed by β-glucosidases into defensive toxic products in response to phytophagous insect attacks. assuming that psyttalia host larvae sequester ingested glycosides, the injected venom gh β-glucosidase could induce the release of toxic compounds, thus participating in parasitism success by weakening the host. venom lrr proteins are similar to truncated toll-like receptors and may possibly scavenge the host immunity. the abundance of one of these lrr proteins in the venom of only one of the two p. lounsburyi strains evidences intraspecific variation in venom composition. altogether, venom intra- and inter-specific variation in psyttalia spp. were much lower than previously reported in the leptopilina genus (figitidae), suggesting it might depend upon the parasitoid taxa. strikingly, a high venom diversity was observed between the closely related drosophila parasitoids leptopilina boulardi and l. heterotoma (hymenoptera, figitidae), with none of the abundant venom proteins in common . to assess whether this variation between two figitid species that differ in their host range similarly exists in other parasitoid taxa, we compared here the venom composition of two braconid wasps, psyttalia lounsburyi and p. concolor (hymenoptera, braconidae, opiinae) that belong to the same complex of species . both psyttalia species are used as biological control agents of the olive fruit fly bactrocera oleae and they differ in their host range. p. lounsburyi is specialized on b. oleae whereas p. concolor successfully develops in b. oleae and at least other fruit fly species . comparison of p. lounsburyi and p. concolor venom was performed using a combined transcriptomic and proteomic approach, and it was extended at the intraspecific level using two geographically distant african strains of p. lounsburyi (south africa and kenya). we also compared data with large-scale venomics results from other braconids, either associated with pdvs, as chelonus inanitus and microplitis demolitor , or devoid of pdvs, as aphidius ervi , since using various parasitism strategies could possibly impact venom evolution and composition. this study will contribute to a better picture of the diversification of venom components at a short evolutionary scale, opening the way to the characterization of underlying mechanisms. structure of the psyttalia venom apparatus. as typically observed for braconidae, the venom apparatus of both psyttalia species is composed of venom gland filaments (which secrete venom), a venom reservoir, and a venom duct that extends into the ovipositor (fig. a) . as was previously described in opiinae , , p. lounsburyi and p. concolor venom gland is multi-lobed, each lobe displaying an external thick layer of tissue and a central lumen filled by a large volume of venom (fig. b,c) . the gland lobes are joined together at the base where the ovoid shaped reservoir is connected. the reservoir is composed of a large muscular layer surrounding a small internal volume of venom, suggesting it may serve as a "pump" for injecting venom at the time of oviposition rather than as a storage organ. the reservoir also shows internal structures that form intricate spirals possibly involved in maintaining the shape of the reservoir, like spiral springs, by passively counteracting the muscular contraction (fig. b,c) . at the base of the venom apparatus of p. concolor, we also observed a "round gland" filled with large vesicles (fig. c) , already described by quicke and called "basal bulb" by wharton . interestingly, the round gland and the intima spirals of the reservoir showed a strong endogenous green fluorescence (fig. d) , suggesting the presence of universal cellular auto-fluorophores such as nad(p)h and flavins, pigments, or cuticular compounds . no vlp or pdv in p. lounsburyi and p. concolor. among the few studies on psyttalia species, two had reported the occurrence of unidentified virus-like particles (vlps) within the venom secretory cells (in p. concolor, previously opius concolor , and the close species o. caricivorae fisher ). we thus performed electron microscopy on p. concolor venom glands to search for vlps, as well as on p. lounsburyi and p. concolor ovaries to ensure the absence of pdvs (polydnaviruses). we did not observe vlps or vesicular material resembling vlps in venom gland cells or venom (fig. a) , nor viral structures or pdv particles in the ovarian cells and fluid close to the eggs (fig. b-d) . accordingly, pl and pc transcriptomes contained no transcript having similarities with genes specific of nudiviruses (from which braconid pdvs derive) or the sister group of baculoviruses. as coronaviruses and cypo-like viruses were also described in p. concolor venom glands , previous observations likely corresponded to small viruses infecting the reproductive tract of the samples, as reported also in other hymenoptera . in the absence of vlps and pdvs, secreted venom proteins are likely the main maternal actors of parasitism success of psyttalia species. comparison of electrophoretic profiles of psyttalia venom proteins. venom samples collected from individuals of p. concolor (pc) and the p. lounsburyi south african (pl sa ) and kenyan (pl k ) strains were the empty space is due to retraction during dehydration. no vesicle is observed in the size range of the vlps described in parasitoids venom ( to nm). bar = nm. (b) picture of whole mounts p. concolor ovaries. oogenesis occurs in egg tubes (et), late oocytes being located in reservoirs (or) and moving down to the calyx (c) where tubes fuse to form the oviduct. bar = μ m. (c,d) tem micrographs of sections through the calyx region of p. lounsbury (c) and p. concolor (d) ovaries, showing the egg chorion (ec), the ovarian fluid (of) and the calyx cells. no pdv particle is observed inside the cells nor in the fluid surrounding the egg chorion. bar = nm. analyzed by d and d gel electrophoresis (figs and ) . on a - % sds-page, the protein content of venom glands was resolved into numerous bands from kda to more than kda (fig. ) . on the silver-stained d gels, to spots were clearly visible, having a to . - isoelectric point, and ranging from to more than kda (fig. ) . some trains of spots were also observed that likely corresponded to post-translational modifications of the same protein. the pl sa and pl k d electrophoretic profiles were very similar with presence/absence or intensity variation detected for only a few bands (fig. ) . the distribution of d spots evidenced more differences, in particular the absence of the pl sa spot number ( kda) in the pl k strain (fig. a,b) . we observed a greater variation at the interspecific level with several profile differences in the - kda range (figs and ) . comparison of transcriptomic and proteomics data between psyttalia wasps. all the major bands and spots on d and d electrophoretic profiles of pl and pc venom, and a number of minor spots ( bands for pl, bands for pc, from at least two d gels per species; spots for pl, for pc, from at least three d gels per species) were excised, and tryptic peptides were analyzed by lc-ms-ms. in parallel, a transcriptomic analysis of pl and pc venom glands was performed, based on illumina sequencing. de novo assembly of the pl transcriptome was improved using additional (full body) and sanger (venom apparatus) sequence data (supplementary figure s ) and it yielded a total of , and , unisequences for pl and pc, respectively (supplementary table s ). data suggested a similar quality of the transcriptomes, based on general features and similarity searches (supplementary table s ), as well as go terms comparison (supplementary figure s ) . the combined transcriptomic and proteomic data resulted in and unisequences for pl and pc, respectively, among which and had a putative function (supplementary tables s and s ) . some of these, such as actin- c or glyceraldehyde- -phosphate dehydrogenase , were typical cellular proteins with no predicted signal peptide (supplementary tables s and s ). whether their identification in venom is due to cellular damage during collection, or these proteins are actually secreted by non-canonical export mechanisms remains unclear. therefore, we only considered as putative venom proteins the unisequences (i) found in venom proteomics, and (ii) either predicted to be secreted or for which the presence of a signal peptide was not tested due to the incompleteness of the sequence. this resulted in a total of and putative venom proteins for pl and pc respectively (tables and ), whose relative abundance was compared using (i) the rpkm normalized number of illumina reads from pl and pc venom apparatus, mapped to the assembled transcriptomes and (ii) the number of peptides matches in mascot searches. interestingly, most of the proteins identified in the proteomics of the reservoir (detection of the most abundant putative venom proteins only, data not shown), such as actin or paramyosin, had a predicted muscular function, as expected from microscopy observations (see above; fig. ). comparison of venomics data from the two psyttalia species evidenced that % and % of the proteins identified in pl and pc were shared with the other species, respectively (tables and ; fig. a ). in comparison, l. boulardi and l. heterotoma shared less than % of the identified venom proteins . when considering only the most abundant venom proteins (rpkm > and mascot matches > ), and out of the pl and pc proteins, respectively, were shared between p. concolor and p. lounsburyi ( fig. b ; tables and ) whereas the two leptopilina species had no protein in common . finally, pl and pc venom proteins ( % and %) had already been identified in the venom of another braconid species ( fig. c ; tables and ) . identified venom proteins. putative venom proteins described below are classified based on their abundance in venom (rpkm values and mascot matches; tables and ) and their occurrence in the venom of (i) both psyttalia species, (ii) p. lounsburyi only and (iii) p. concolor only. venom proteins with a putative function are described in table (with the proposed biochemical function, previous identification in parasitoid venom, and demonstrated or proposed role in parasitism success). several proteins with low rpkm values and lacking n-terminal sequence were not considered since they were typical cellular proteins and the number of proteomic matches was low (tables and ) . proteins identified in the venom of both psyttalia species. proteins of unknown function. several abundant unisequences with no predicted function were characterized by a high number of matches (tables and ) and rather intense protein spots (fig. ) . among these, a family of five related proteins contains a signal peptide and the duf domain of unknown function (supplementary figure s ). they share similarities with venom proteins of the braconids c. inanitus , m. demolitor and m. hyperodae , and of n. vitripennis . leucine-rich repeat protein. two and four unisequences encoding leucine-rich repeat (lrr) proteins were identified in pl and pc, respectively (tables and ; supplementary tables s and s ). one of these (pl_ ) was highly abundant (rank ) in the p. lounsburyi sa strain only, and it corresponded to one of the most intense spot (spot , fig. a ). the complete unisequences contain a n-terminal signal peptide and to canonical lrr motifs similar to the lrr motif in toll-like receptors (tlrs). yet, the majority of tlrs are multidomain table , locus comp _c ). protein not found in the proteomic analysis but with rpkm > and for which a homolog was found in p. lounsburyi. proteins while psyttalia predicted proteins only contain the lrr domain, as already observed for a. ervi venom lrr proteins . as suggested for a. ervi, psyttalia truncated lrr proteins might interfere with the host immune response by targeting the toll pathway. neprilysin-like metalloproteases. three and two unisequences encoded neprilysin-like (nep) zinc-dependent metalloproteases in pl and pc, respectively (tables and ; supplementary tables s and s ) , one of which was in high abundance (rank ; and peptide matches in pl and pc venom, respectively). nep-like proteins occur in the venom of several parasitoid wasps (table ) . annexins are a family of ca + -dependent lipid binding proteins believed to be engaged in membrane transport processes, although recent work suggests a more complex set of functions. annexins normally lack signal sequences for secretion, but some members of the family have been identified extracellularly where they can act as receptors . annexins had never been described so far in the venom of parasitoids. however, some data suggest that different mammalian parasite clades possess annexins with unique properties that can be secreted and are likely involved in host-parasite interactions and host immune-modulation . moreover, some parasitic nematodes secrete an annexin-like effector into host root cells that may mimic plant annexin function during the parasitic interaction . at last, it has been shown that annexins are also involved in the binding and internalization of toxins in eukaryotic cells . arginine kinase plays a crucial role in the energy metabolism of insects and other invertebrates through the use of atp to catalyze the phosphorylation of arginine in phosphoarginine. this enzyme was detected in the venom of pteromalus puparum and leptomastix dactylopii , but its role in the host-parasitoid interaction is unknown. calreticulin is a calcium (ca + )-binding protein with multifunctional properties including chaperone functions . calreticulin was shown to inhibit host cell encapsulation in cotesia rubecula and p. puparum , although the mechanism is still unclear. calreticulin was found in the venom of several phylogenetically distant species and seems thus largely shared among parasitoids. endoplasmin (alternative names: hsp b , gp , grp- ), which belongs to the heat shock protein family, is a molecular chaperone located in the er and involved in the final processing and export of secreted proteins . among parasitoids, endoplasmin has only been detected so far in the venom gland of aphidius ervi . this venom protein was suggested to play a role in the secretion, stabilization, transport and host cell targeting of the different a. ervi venom proteins. enolase is a key enzyme in cell metabolism which is also associated with virulence of several pathogens . an extracellular enolase was recently described in the oviposition injecta from a. ervi and the venom of toxoneuron nigriceps . enolase is also released by teratocytes surrounding the a. ervi embryo . this enzyme was proposed to play an important role in the regulation of the host physiology and the host nutritional exploitation , . esterase/lipase-like esterases and lipases belong to a superfamily of hydrolytic enzymes that act on carboxylic esters . proteins belonging to this functional class were previously found in the venom of several phylogenetically distant species , and appear to be common in parasitoids. the functions of these hydrolase enzymes in host-parasitoid interactions have not been investigated yet. gh β -glucosidases gh β -glucosidases are a family of enzymes found from bacteria to mammals that hydrolyze glycosidic bonds from glycosides and oligosaccharides, and remove non reducing terminal glucosyl residues . among parasitoids, a β -glucosidase enzymatic activity was detected in the venom of pimpla hypochondriaca . a member of this enzyme family was also recently identified, but not abundant, in the venom of microplitis demolitor and, in a low quantity, in a transcriptomic study of the a. ervi venom apparatus . gh β -glucosidases include myrosinases that play a central role in the glucosinolate-myrosinase system, one of the best-studied activated plant defense system . some insects have co-opted this system to defend themselves against enemies, by sequestering plant-derived glucosinolates and producing their own myrosinase-like enzyme [ ] [ ] [ ] . heat shock protein heat shock proteins (hsp ; alternative name: grp- ) are a family of chaperones with distinct sub-cellular localization and function . an hsp protein was identified in the venom of the parasitoid p. puparum whose function remains to be elucidated. leucine-rich repeats (lrrs) are motifs involved in protein-protein interactions . lrrs are generally composed of - amino acid stretches rich in leucine. to our knowledge, lrr domain-containing proteins were only identified in the venom of a. ervi until now . they were suggested to act as scavengers for the pea aphid toll-like receptors, thus impairing the host immune response via the toll pathway. neprilysin-like (nep) proteins are zinc-dependent metalloproteases belonging to the m peptidase family. they are involved in the degradation of a number of regulatory peptides in the nervous or immune system of mammals and insects . although they are typically membrane-bound, ectopeptidases such as nep may also be shed from the membrane through a proteolytic process and found in the surrounding fluid . nep-like proteins were detected in the venom of the braconidae a. ervi , microctonus hyperodae , m. demolitor and t. nigriceps , as well as of the figitidae leptopilina boulardi . they were also found associated with the vlps produced in the ovary of venturia canescens . neplike proteins have been hypothesized to modulate the host immune system by degrading immune-specific peptides . secreted phospholipases a (pla s) are a family of relatively stable enzymes found in venoms. pla has in vitro and in vivo immunomodulatory effects in bee venom , and neurotoxic and myotoxic effects in snake venoms . this enzyme was recently detected in the venom of m. demolitor and t. nigriceps , but its function in the host-parasitoid interaction is unknown. protein disulfide isomerases (pdis) are enzymes involved in the folding and stabilizing of nascent polypeptides in the endoplasmic reticulum (er) through catalysis of disulfide bond formation and isomerization . although this protein is normally recycled back to the er from the golgi via its c-terminal kdel motif, secreted pdis can escape this turnover mechanism . among parasitoids, pdis have only been detected so far in the venom gland of a. ervi . they have a broad substrate specificity and are involved in the folding of toxin peptides in different venomous organisms , . reprolysin-like (rep) proteins are zinc-dependent metalloproteases belonging to the m peptidase family, commonly found as constituents of snake venom. they were previously detected in the venom of the parasitoids pimpla hypochondriaca classical serine carboxypeptidases are enzymes that hydrolyze a peptide bond at the c-terminal end of peptides and proteins. a related enzyme (scpep ) that do not show proteolytic activity but is involved in other functions was described in mice . to our knowledge, serine carboxypeptidase has only been identified so far in the venom of m. demolitor and t. nigriceps . interestingly, serine carboxypeptidases have also been described as candidate virulence factors in several pathogens . serpins (serine protease inhibitors) are a large family of functionally diverse protease inhibitors. they share a conserved structural architecture with an exposed reactive center loop (rcl) of about amino acids, which acts as bait for target serine proteases . the l. boulardi venom serpin lbspny was previously shown to be involved in host immune suppression : it interferes with melanization in the drosophila host through inhibition of the phenol oxidase activation. more recently, serpins were described in the venom of a. ervi and m. demolitor but their role in parasitism success is unknown. another zinc-dependent metalloprotease was identified in each psyttalia wasp, with low inter-species sequence similarity. both proteins are weakly related to venom reprolysin-like proteins of p. hypochondriaca and eulophus pennicornis . however, the sequences were incomplete and the number of matches in the venom was rather low (tables and ; supplementary tables s and s ). gh β -glucosidases. peptides from major d spots at - kda (spots and in pl k and pl sa , respectively, spots , , in pc) matched with two unisequences (one for each psyttalia species) that encoded proteins containing a glycosyl hydrolase family (gh ) domain (pfam ) found in gh β -glucosidases (fig. ) . the high rpkm values and number of peptides matches confirmed that the pl_ and pc_ unisequences, that share % identity (supplementary figure s ) , were among the most abundant in venom (tables and ; supplementary tables s and s ). the pc sequence seems full-length, with a predicted signal peptide of residues, while the pl sequence probably lacks the n-terminal part (supplementary figure s ) . the . kda predicted mw of the mature protein is close to that of the observed spot on d gels, suggesting none or few glycosylation, although several n-glycosylation sites are predicted. yet, several spots were observed, having different isoelectric points, suggesting post-translational modification(s) and thus several isoforms (fig. ) . two pl and one pc additional unisequences, not found in proteomics, shared similarities with gh β -glucosidases, suggesting occurrence of a multigene family (supplementary figure s ) . gh β -glucosidases were previously identified in venomics data from m. demolitor and a. ervi, but they did not correspond to abundant proteins (tables , and ) . finally, the venom of the ichneumonid pimpla hypochondriaca was reported to display β -glucosidase enzymatic activity (table ) . gh β -glucosidases are found from bacteria to mammals. they play an essential role in the metabolism of glycolipids and exogenous glycosides by hydrolyzing glycosidic bonds and removing non-reducing terminal glucosyl residues from saccharides and glycosides. this enzyme family includes for instance the myrosinases, well-known for their role in the "glucosinolate-myrosinase" plant defense system (table ) , and also identified in the cabbage aphid brevicoryne brassicae that feed on crucifers . the alignment of pl and pc venom unisequences with that of well-described plant myrosinase (sinapsis alba) and insect β -glucosidases (b. brassicae, phyllotreta striolata, spodoptera frugiperda) shows conservation of all critical enzymatic site residues , , suggesting that psyttalia enzymes are indeed functional (fig. ) . insect β -glucosidases differ from plant myrosinases in that they have two glutamates in the catalytic site instead of one glutamate and one glutamine. the possible role of psyttalia venom gh β -glucosidase in host-parasitoid interaction is discussed below. heat shock proteins (hsps). molecular chaperones belonging to the heat shock protein (hsp) class, including hsp , calreticulin and protein disulfide isomerase (pdi), were found in variable amounts in the two species (tables and ; supplementary tables s and s ). endoplasmin, also identified in transcriptomic data from both species (rpkm values of . for pl and . for pc) was detected in pl venom only, suggesting a lower abundance in pc venom (tables and ; supplementary tables s and s ). in the endoplasmic reticulum (er), all these hsps cooperate to form multi-chaperone complexes involved in the folding and export of secreted proteins. hsp , pdi and endoplasmin are normally retained in the er through the binding to specific receptors via a k/r/hdel motif. since these proteins are abundant in cells, we cannot totally exclude that their detection in the venom results from tissue contamination during venom collection from the gland. however, one of the venom pdis, identified in both species, contains a substitution that changes the hdel motif into a sdel motif, suggesting a loss of binding and then of retention in the er. moreover, the binding of hsps to er receptors is labile and the retention is not absolute, allowing hsps to be secreted and to play a role in intercellular transport and signaling , . endoplasmin, for example, has been associated as a chaperone with the secretion of pancreatic lipases and their further internalization by intestinal cells in rat . venom hsps may thus play a role in stabilization of other venom proteins and/or their transport and targeting of host cells. among these, endoplasmin is of particular interest because it is a master chaperone for tlrs, a family of lrr domain-containing proteins of which members were found in psyttalia venom (see above). in accordance with a possible role of endoplasmin as a chaperone of venom tlrs, endoplasmin was only detected in the venom of pl that contains higher amounts of lrr proteins than the pc venom (tables and ; supplementary tables s and s ). interestingly these two proteins are also secreted in the venom of the braconid a. ervi . pdis play a central role in the protection of disulfide bounds of secreted proteins . interestingly, the rapid expansion of a large gene family encoding pdis specifically-expressed in the venom glands of cone snails has been recently suggested to facilitate the folding of the numerous conotoxins produced by these marine predators . here, however, phylogenetic analysis shows that psyttalia venomous pdi sequences group with different pdis from braconid wasps (supplementary figure s ) , suggesting that no specific expansion occurred. proteins of low abundance. proteins with similarities with serpin and enolase (tables and ; supplementary tables s and s ) were identified. the pl and pc serpin sequences shared % identity but only the pc sequence was complete, the pl serpin lacking the n-and c-termini (supplementary figure s ) . the pc serpin contains a signal peptide as well as the consensus hinge sequence essential for the conformational change involving the rcl and necessary to inhibit the target protease (supplementary figure s ). an extracellular enolase was already identified in a. ervi injecta and also produced by specialized extra-embryonic cells (the teratocytes) and suggested to be involved in nutritional host exploitation ( toxin-like peptides were previously identified in a. ervi venom but no toxin-like signature was found in pl venom proteins. proteins of low abundance. all the other proteins found in p. lounsburyi venom only were in low abundance (table ; supplementary table s ) . among these, an arginine kinase-like protein was identified, and similarities were found with members of the esterase/lipase-like superfamily, detected in many parasitic wasp species (table ) . proteins detected in p. concolor venom only. proteins of unknown function. two different unisequences (pc_ and pc_ ) were detected ( table ; supplementary table s ) but the sequences were not complete and the presence of a signal peptide could not be assessed. pc_ (rank based on rpkm values) was one of the most intense d spots at less than kda (fig. ) . pc_ was better ranked based on rpkm values, but the corresponding spot was less intense (fig. ) and the number of peptide matches was lower (table ; supplementary table s ) , suggesting a lower abundance in venom. annexin. one unisequence, corresponding to peptide matches, had similarities with annexins ( table ; supplementary table s ). this protein contains a aa n-terminal signal peptide followed by two annexin domains. based on data from their role in mammalian parasites, p. concolor venom annexin might be involved in binding and cell internalization of other venom proteins ( table ). proteins of low abundance. among these, a secreted phospholipase a (pla s) and a retinoid-inducible serine carboxypeptidase (table ; supplementary table s ) were identified (table ). large scale combined "omics" studies have recently increased knowledge of the nature and diversity of the venom content of parasitoid wasps , . yet, very few studies were designed to evaluate how far closely-related parasitoid species differ in venom composition. venom glands of the braconidae m. hyperodae and m. aethiopoides were shown to express a similar set of genes but analyses relied on less than ten genes, and proteomics data were only available for m. hyperodae. more recently, we evidenced striking differences in venom composition of the closely-related figitidae l. boulardi and l. heterotoma , drosophila parasitoids differing in their host range. we observed here a rather similar protein composition of the venom of p. lounsburyi and p. concolor, albeit they also differ in their host specificity level. the host range is determined by both behavioral and physiological traits that include host choice and venom adequacy to the host (i.e. "virulence"). in some taxa, the diversity of venom composition could then be decoupled from the diversity of parasitized hosts in natura if specialization mainly relies on behavioral traits. for instance, although p. lounsburyi is a specialist of the olive fly in the field, it can develop in laboratory conditions on some non-natural hosts such as ceratitis capitata. similarly, although intraspecific variation in p. lounsburyi venom was notably evidenced for a lrr protein, the difference between psyttalia strains from distant geographic origin was much lower than observed between l. boulardi strains . whether the level of diversity of venom differs among taxa (being lower in braconidae than in figitidae), or the extensive venom variation in leptopilina wasps is specific of these genus/species remains an open and stimulating question. unfortunately, a majority of the most abundant unisequences, either common or specific to each psyttalia species, had no predicted function. it is thus difficult to make assumptions about how these wasps counteract the host immune defense and regulate the host physiology, and whether they use similar mechanisms. yet, the identification of a gh β -glucosidase as one of the most abundant venom protein in both species suggests a possible role of this protein in parasitoid wasp's success. in plants, glycoside compounds and hydrolytic enzymes form a classic two-component defense system, with glycosides inducing biological effects after being activated by the enzymes. the vast array of secondary metabolites produced is used as a protection against phytophagous organisms and pathogens , . hydrolysis-products can indeed be repellent or toxic to insects, nematodes, fungi and bacteria, and they also serve as volatile attractants for specialist herbivores and their parasitoids . plant compounds and endogenous glycosidases are usually stored in separated compartments so that activation only occurs upon tissue damage. to reduce toxic effects, some phytophagous insects were shown to downregulate their gut glycosidases while others have even evolved their own glycosylation system to reglycosylate the produced aglycons. the neo-formed glycosides can then possibly be stored, similarly to the ingested plant glycosides that are indeed sequestered by several insect species. by doing so, these insects prevent the production of toxic compounds [ ] [ ] [ ] , and some even use them for their own defense . the main psyttalia hosts, b. olea and c. capitata, oviposit in developing olives and fruits that contain large quantities of various glycoside compounds. olive is particularly rich in phenol-glucosides such as oleuropein, verbascoside and rutin, which accumulate during its development while b. olea is growing inside . oleuropein was shown to be converted into a toxic compound with a glutaraldehyde-like structure -a potent protein crosslinker -by a defense-related olive β -glucosidase . assuming that fly larvae use a sequestering mechanism to survive within fruits during development, the injection of psyttalia venom β -glucosidase inside their body might result in a burst of toxic compounds that could weaken the host and increase the parasitoid probability of success. the release of sugar moieties from glycosides could also increase the amount of energy available for the developing parasitoid larvae. although this is an attractive hypothesis, the fact remains that the alleged role of psyttalia venom β -glucosidase is based on the sequestration of phenolic glycosides by the olive fly larvae, which has not been tested yet. the importance of a tri-trophic understanding of plant-herbivore and herbivore-predator/parasitoid interactions is increasingly recognized. for instance, the role of glucosides/glucosinolates has started to be evaluated not only in insect-plant interactions but also for their cascading effects on the performance of herbivore enemies through metabolic impacts or emission of volatile products . the report of the large production of a β -glucosidase in a parasitoid venom suggests that this enzyme might participate in parasitoid phenotypes such as counter-defense and parasitism success, in addition to its well-described defensive role in plants and herbivore insects. altogether, this study illustrates that parasitoid venom is a complex mixture of proteins whose relative abundance in a given species or group is still hardly explained. the study of a new species often reveals new types of abundant venom molecules, as exemplified here with the β -glucosidase, and it highlights the role of gene duplication in the rapid evolution of venom and acquisition of new features. deciphering the role of major venom proteins in psyttalia parasitism success, especially those with no predicted function, is a key challenge. this will require further exploration using techniques such as rnai, demonstrated as an efficient approach for impairing the production of parasitoid venom proteins . biological material. the south african (sa) and kenyan (k) strains of p. lounsburyi (pl) were previously described . they were reared on a laboratory strain of the fruit fly c. capitata under a : h light/dark cycle at °c. the p. concolor (pc) population was collected in in sicily (italy) and reared for one generation on c. capitata under the same conditions, prior to analysis. c. capitata is a natural host for p. concolor but it is used as a substitute host for p. lounsburyi due to the difficulties of rearing the main natural host, bactrocera oleae (the olive fly). light, fluorescence, and transmission electron microscopy. light and fluorescence microscopies were performed using epifluorescent microscopes fitted with differential interference contrast (dic) optics (imager z , zeiss) fitted with a black and white camera (axiocam mrm, zeiss). images were pseudo-colorized digitally (adobe photoshop). for transmission electron microscopy (tem), blocks were prepared from ovaries or venom glands per sample. dissected samples were pooled into μ l of ringer's saline (kcl mm; nacl mm; cacl mm; tris-hcl mm) in a centrifuge vial on ice. an equivalent volume of fixative ( % glutaraldehyde (sigma) in . m sodium cacodylate buffer, ph . ) was then added and the sample was kept for h at °c. fixed samples were centrifuged ( × g, min) to pellet tissues and remove the fixative prior to post-fixation in % osmium tetroxide in cacodylate buffer. following dehydration in graded series of ethanol solutions, samples were embedded in epon. sample sections were cut with a diamond knife using a lkb ultramicrotome, mounted on copper grids, stained with uranyl acetate and lead citrate, and observed with a zeiss em cr electron microscope at kv. total rna isolation and cdna library construction. the transcriptomic analysis was performed from samples of pl and pc venom glands using illumina rna-seq. to improve de novo assembly for pl, we also generated sanger sequences from samples of venom glands, and sequences from full insect bodies of males and females obtained from six siblings (supplementary fig. s ). pl and pc venom glands were dissected in ringer's saline and stored at − °c. total rna was extracted using trizol reagent (invitrogen) according to manufacturer's instructions, and quality was checked using an agilent bioanalyzer. cdna library construction for illumina rna-seq and sequencing was performed by beckman coulter genomics (usa). cdna library used for sanger sequencing was constructed from μ g of total rna using the creator smart cdna library construction kit (clontech). ligation products were transformed into electromax dh b escherichia coli competent cells (invitrogen). sequencing and assembly. illumina rna-seq sequencing (hiseq , × pb), sequencing ( gs-flx titanium platform) and trimming were performed by beckman coulter genomics. quality of illumina raw reads was controlled using fastqc software and reads were cleaned by removing low quality sequences and reads containing n or adaptor sequences. for sanger sequencing, a total of , clones were analyzed by the genoscope (cea, evry, france) on an abi sequencer using the standard m forward primer and bigdye terminator cycle sequencing kit (applied biosystems, foster city, ca, usa). sanger ests were then trimmed using tigr seqclean software. for each species, we performed de novo transcriptome assembly using velvet/oases assembler (https://www. ebi.ac.uk/~zerbino/oases/) after the filtering process of illumina raw reads. the first assembly step used a multiple kmer approach with kmer size ranging from to (k = , , and coverage = ). a meta-assembly (kmeta = , coverage = ) was then performed using all previously obtained transcripts (> bases long) ( supplementary fig. s ). at both assembly steps, we used cd-hit-est to remove the shorter redundant transcripts entirely covered by other transcripts with more than % identity. finally, a clustering of transcripts was performed using tigr-tgicl. to improve the quality of the assembly for pl, we included the cleaned and sanger sequences as long sequences (minimum size of bases) or otherwise as short sequences, in addition to the short illumina reads. sequence annotation and analysis. to identify similarities with known proteins, the unisequences were compared to ncbi non-redundant protein sequence database, uniprotkb/swiss-prot database, insect predicted proteome databases (drosophila melanogaster v . and nasonia vitripennis v . ) and all braconid venom proteins found in uniprotkb ( venom proteins from different braconid species), using blastx with a cut-off e-value of e- . comparisons with previously published venom gland transcriptomes of a. ervi and leptopilina spp were performed using tblastx with a cut-off e-value of e- . search for nudivirus/baculovirus-related specific genes was performed using tblastn with a cut-off e-value of e- . orf prediction and translation were performed with framedp (https://iant.toulouse.inra.fr/framedp/), signal peptide prediction with signalp (http://www.cbs.dtu.dk/services/signalp/), and prediction of n-glycosylation sites with the netnglyc . server (http://www.cbs.dtu.dk/services/netnglyc/). search for protein domains was achieved using pfamscan (ftp://ftp.sanger.ac.uk/pub/databases/pfam/tools/) and cd-search against conserved domain database (cdd) at ncbi (http://www.ncbi.nlm.nih.gov/structure/ cdd/cdd.shtml). identification of leucine-rich repeats (lrr) in protein sequences was done using lrrfinder (http://www.lrrfinder.com/). search for duf domain-containing, gh β -glucosidase and protein disulfide isomerase sequences was performed using blastp at ncbi (http://www.ncbi.nlm.nih.gov/blast/) and hmmsearch from the hmmer package (http://hmmer.org/). pairwise and multiple amino acid sequence alignments were respectively obtained using the needleman-wunsch algorithm and mafft implemented in geneious software (biomatters). phylogenetic analyses were performed using maximum likelihood (ml) with phyml (http://phylogeny.lirmm.fr/). prottest was used to select the best fit model of amino acid substitution for ml phylogeny (https://github.com/ddarriba/ prottest ). gene functions and go terms were automatically assigned to the predicted proteins based on the identification of domains with pfamscan. only the root domain of the hierarchical domain organization available from ebi was conserved. comparison of go terms between pc and pl unisequences and homogenization of the annotation level were performed using go slim terms. for each species, we used bowtie (http://bowtie-bio.sourceforge.net/) to map back all input trimmed illumina raw reads (minimum size of bases) to the assembled transcriptome with up to nucleotides mismatches allowed. to compare the unisequence expression levels, the number of mapped raw reads for each transcript was normalized with the rpkm (reads per kilobase per million reads), using the r package edger (https://bioconductor.org/packages/release/bioc/html/edger.html). analysis was performed independently on pc and pl wasp venom ( supplementary fig. s ). venom apparatus were dissected from individuals per sample and glands were collected in - μ l of ringer's saline supplemented with a protease inhibitor cocktail (sigma). glands were opened to release the venom and centrifuged for min at × g to remove residual tissues. for d sds-page, samples were mixed with × laemmli buffer containing β -mercaptoethanol (v/v) and boiled for min. proteins were then separated on a - % linear gradient sds-page and the gel was silver stained as previously described , . for isoelectric focusing (ief), samples were prepared by boiling the protein solution for min with % (v:v) of a denaturing solution ( . m dithioerythritol, % sds). after cooling, the samples were mixed with an equal volume of a solution containing . m urea, . m dithioerythritol, and % chaps. ief was performed using slab gel. slab gels were made on glass tubes cm in length ( . mm internal diameter) that were filled with % acrylamide, . m urea, % ampholytes [ % ph - (pharmacia) and % ph - (servalytes)], and % chaps. isoelectric focusing was run in two steps: a first run at ma, . w/tube, v for a total of , v/h, followed by a second run at ma, . w/tube, , v for a total of , v/h. for the second dimension, - % linear gradient sds-page was used. ief gels were incubated with x laemmli buffer containing ß-mercaptoethanol and loaded on top of the d sds-page. after separation, proteins were silver stained as previously described , . identification of proteins by mass spectrometry was performed on d bands and d spots excised from the gels as previously described , . ms/ms data analysis was performed with the mascot software (http://www. matrixscience.com) licensed in house using the combined pc and pl unisequences and non-redundant nr (ncbi). data validation criteria were (i) one peptide with individual ion score above (the mascot significant identity threshold corresponding to p < . is in our case) or (ii) at least two peptides of individual ion score above (corresponding to % probability that a peptide spectrum match is a random event). the mascot score was calculated as − *log (p). the calculated fdr (based on an automatic decoy database search) ranged from to . % depending of the individual gel analysis. mascot analysis was performed with a fragment ion mass tolerance of . da and a parent ion tolerance of . da. carbamidomethyl of cysteine was specified in mascot as a fixed modification, and oxidation of methionine as a variable modification. the maximum missed cleavage allowed was set to . insect immune resistance to parasitoids insights into function and evolution of parasitoid wasp venoms venom proteins from parasitoid wasps and their biological functions diversity of 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morphology of the venom gland and reservoir in opiine and alysiine braconid wasps (insecta, hymenoptera, braconidae) generic relationships of opiine braconidae (hymenoptera) parasitic on fruit-infesting tephritidae (diptera). contributions of the american entomological institute use of confocal laser scanning microscopy in systematics of insects with a comparison of fluorescence from different stains virus-like particles in the poison gland of the parasitic wasp opius concolor venom apparatus of the endoparasitoid wasp opius caricivorae fischer (hymenoptera: braconidae): morphology and ultrastructure presence of a novel small rna-containing virus in a laboratory culture of the endoparasitic wasp venturia canescens (hymenoptera: ichneumonidae) the constituents of microctonus sp. parasitoid venoms insights into the venom composition of the ectoparasitoid wasp nasonia vitripennis from bioinformatic and proteomic studies a venom protein from the endoparasitoid wasp pimpla hypochondriaca is 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action protein disulfide isomerase secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails serine carboxypeptidase scpep and cathepsin a play complementary roles in regulation of vasoconstriction via inactivation of endothelin- the peptidases of trypanosoma cruzi: digestive enzymes, virulence factors, and mediators of autophagy and programmed cell death serpin structure, function and dysfunction a serpin from the parasitoid wasp leptopilina boulardi targets the drosophila phenoloxidase cascade we are grateful to m. thaon for help in insect rearing, n. ris and t. malausa for fruitful discussions, m.c. bon for providing p. lounsburyi samples, and k. varikou and v. caleca for providing p. concolor samples. we also thank a. schmitz, j. villalba, and the microscopy platform of the sophia agrobiotech institute (isa) for technical advices. we are grateful to the anonymous reviewers for their careful reading of our manuscript. this work received support from the genoscope ( accession codes: p. concolor and p. lounsburyi raw illumina sequencing data are available in the genbank sequence read archive (sra) database under the accessions srr , srr , srr and srr , and raw sequencing data for p. lounsburyi under the accession srr . all trimmed ests for p. lounsburyi can be found in the genbank dbest repository under the accessions: jz -jz . the transcriptome shotgun assembly (tsa) projects were deposited in genbank under the accessions gcdx (p. concolor) and gceq (p. lounsburyi). versions in this paper are the first versions gcdx and gceq . key: cord- -ysspkbj authors: milewska, aleksandra; kindler, eveline; vkovski, philip; zeglen, slawomir; ochman, marek; thiel, volker; rajfur, zenon; pyrc, krzysztof title: apobec -mediated restriction of rna virus replication date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ysspkbj apobec family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other dna viruses, such as herpesviruses, parvoviruses and hepatitis b virus. although effects of apobec members on viral dna have been demonstrated, it is not known whether they edit rna genomes through cytidine deamination. here, we investigated apobec -mediated restriction of coronaviridae. in experiments in vitro, three human apobec proteins (a c, a f and a h) inhibited hcov-nl infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. apobec -mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive apobec . moreover, apobec proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of apobec-mediated restriction of rna virus infections. . other apobec proteins have been found to affect a wide variety of viruses, including other retroviruses (e.g., simian immunodeficiency virus (siv), equine infectious anemia virus (eiav), murine leukemia virus (mlv), foamy viruses), and dna viruses (e.g., hepatitis b virus, parvovirus) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as well as the inhibition of viral replication by hypermutation, apobec -induced mutations also cause degradation of viral genomic material, mediated by cellular dna-repair mechanisms . the rna-editing capability of apobec proteins was identified in , since when extensive mutational activity of a a on rna transcripts in monocytes and macrophages has been demonstrated . our current understanding of the apobec proteins suggests that they may also constitute an innate defense system against rna viruses. for example, a g known to be involved in restriction of three rna pathogens (the mumps, measles and respiratory syncytial viruses), but whether this effect is associated with cytidine deamination is not yet known . members of the family coronaviridae are positive-strand rna viruses with large genomes, ranging in size from to kb. six human coronaviruses (hcovs) have been identified, four of which (hcov-oc , hcov- e, hcov-nl and hcov-hku ) circulate continuously in human populations. these four viruses cause the common cold in otherwise healthy adults, and may cause more severe symptoms in young, elderly and immunocompromised individuals [ ] [ ] [ ] [ ] . other coronaviruses that infect humans can cause severe and life-threatening diseases. in , severe acute respiratory syndrome coronavirus (sars-cov) emerged and affected ~ , individuals, causing ~ deaths , . middle east respiratory syndrome coronavirus (mers-cov) is the most recently identified coronaviruse that infects humans. mers-cov was isolated in and is associated with severe pneumonia and renal failure, with a mortality rate of ~ % , . hcov-nl was identified in and is distributed worldwide, with highest prevalence during winter and early spring in temperate climate . hcov-nl accounts for a considerable number of hospitalisations among children < years of age, the elderly and immunocompromised individuals . a notable characteristics of coronaviruses is that their genomes are highly u/a-rich and c/g-poor. for example, hcov-nl has % u and % a content, with only % c and %, g nucleotides . the explanations for this bias is not yet known , . one possibility is that, over an evolutionary timescale, genomes of coronaviruses might have been shaped by cytidine deamination. our aim, therefore, was to determine whether apobec proteins might be responsible for this modification. our results demonstrate that three of the apobec proteins (a c, a f and a h) can inhibit coronaviral infection in vitro. human apobec -family transcript levels in human airway epithelium (hae) cultures during coronavirus infection was evaluated by quantitative rt-pcr (qrt-pcr) with primers and standards developed in our lab. the level of each apobec mrna was analysed in fully differentiated cells from healthy adults, and a high level of expression was observed for six out of seven human apobec genes (fig. a) . the same six genes (apobec a, apobec c, apobec d, apobec f, apobec g and apobec h) were expressed during hcov-nl infection, and these genes were analysed in subsequent experiments. hae cultures were infected with hcov-nl and cultured for days at °c. as a control, hae cultures were infected with influenza a h n virus, which induces considerable upregulation of apobec g . rt-qpcr analysis of total rna extracted from infected cells revealed upregulation of apobec a, apobec c, apobec d and apobec f transcripts in hcov-nl -infected cells relative to mock-infected cells, with an almost -fold increase in apobec a expression (fig. b) . as anticipated, apobec g was upregulated in the influenza-infected cells, and apobec h expression also increased (fig. c) . were prepared and transfected into t cells, which were cultured and analysed for protein expression by western blotting (fig. a, supplementary figure ). for expression in the naturally permissive llc-mk cell line, in which the dna transfection efficiency is very low (data not shown), mrna transcripts encoding apobec proteins or green fluorescent protein (gfp) were prepared by in vitro transcription. transfection of llc-mk cells was efficient, reaching ~ % for the control gfp mrna (fig. b) . expression of ha-tagged apobec proteins in mrna transfected llc-mk cells, measured by flow cytometry, was similar for all proteins (fig. c ). human apobec proteins a c, a f and a h inhibit hcov-nl replication. mrna-transfected llc-mk cells were infected with hcov-nl and cultured for days at °c. no cytopathic effect (cpe) was observed in cells expressing a c, a f and a h proteins, but cpe was observed in untransfected or gfp-transfected cells, and in cells expressing a a, a de and a g (fig. a) . analysis of hcov-nl replication showed a . - log reduction in virus yield in cells expressing a c, a f and a h, relative to the control sample. by contrast, hcov-nl replication was not inhibited in cells expressing gfp, a a, a d or a g (fig. b ). hcov-nl infection resulted in upregulation of expression of a a, a c, a d and a f and a c, a f and a h all inhibited hcov-nl replication, we tested whether this inhibition was the result of the catalytic activity of the apobecs. plasmids encoding variants of a c, a f and a h with glu → gln substitutions in the catalytic site were prepared and mrnas encoding active or inactive proteins were transfected into llc-mk cells, which were infected with hcov-nl and cultured prior to visualisation of viral proteins with specific antibodies. the percentages of hcov-nl -infected cells were measured by flow cytometry. wild-type apobec proteins resulted in pronounced reductions in hcov-nl infection rates, whereas catalytically inactive proteins produced moderate inhibition, with infection rates that were not significantly different from those in controls (fig. a) . western blot analysis showed an even expression of all proteins, showing that the inhibitory effect scientific reports | ( ) : | doi: . /s - - - did not result from unequal mrna translation (fig. b, supplementary figure ). this level of inhibition of hcov-nl replication by catalytically inactive proteins required transfection of µg of mrna, whereas for active proteins, inhibition was achieved with transfection of µg of mrna. next, we tested the activity of apobec proteins in vitro. a dna template containing several possible sites for cytidine deamination (gc-rich domains) was used for in vitro transcription, producing rna that was incubated with lysates from t cells overexpressing a c protein or gfp. the rna was then isolated, reverse transcribed, amplified by pcr and sequenced, revealing several c → c/t changes following incubation with a c (but not gfp) cell lysate (fig. c) . however, hypermutation did not occur, and no pattern was identified in the nucleotide changes. to examine the progressive accumulation of mutations, we performed a series of virus passages on cells expressing a c, a f or a h. notably, after the first passage, the inhibitory effect of the three apobecs diminished, and no decline in virus yield was seen (data not shown). cell-culture supernatants were collected after four passages of hcov-nl , and several regions of the viral rna genome were sequenced. point mutations (g → a or c → t) were identified in the genes encoding the ab polyprotein and spike protein in virus passaged results are presented as mrna copy number per ml. infection was performed at tcid per ml, for days at °c (for hcov-nl ) or for days at °c (for iva). each experiment was performed at least three times with clinical material from different donors, and with two technical replicates. for comparisons by student's t-test, *indicates p < . ; **indicates p < . . in apobec -expressing cells, but not in wild-type cells. these mutations were not specific to virus collected from cells expressing a c, a f or a h, but were also identified in virus passaged in cells expressing a a, ade or a g. to identify whether apobec protein were capable of inducing hypermutation, we used a recombinant gc-rich hcov- e virus (ic e-gc), into which we had introduced silent mutations (either a → g or t → c) in the a/t-rich region of nsp , resulting in a potential 'hot-spot' for cytidine deamination. the virus was propagated and titrated to determine the % tissue-culture infective dose (tcid ) in naturally permissive huh- cells. the effectiveness of mrna transfection in huh- cells was confirmed by detection of green fluorescence in cells transfected with gfp mrna. huh- cells were then transfected with mrna encoding an apobec protein, infected with ic e-gc at tcid per ml and cultured for days. pronounced inhibition of viral infection occurred in cells expressing a c, a f or a h (data not shown). after four passages of ic e-gc, viral rna was sequenced. no g → a or c → t mutations were identified within the gc-rich region in any of the samples. however, ic e-gc passaged in cells expressing a f protein showed considerable sequence heterogeneity, compared with passaged in gfp-positive cells, indicating a possible a f deaminase activity (fig. d ). our results demonstrated that, although cytidine deamination was involved in inhibition of coronavirus by apobec proteins, deaminase mutants retained some anti-coronaviral activity, suggesting that an additional mechanism may be involved in apobec -mediated coronavirus restriction. we therefore analysed the interactions between apobec proteins (a c, a f and a h) and hcov-nl nucleocapsid protein (hcov-nl -n), which is the most abundant viral structural protein. human t cells were transiently transfected with plasmid dna constructs for expression of individual ha-tagged apobec proteins (or their catalytically inactive derivatives) or gfp. cell lysates containing the expressed proteins were incubated with purified hcov-nl -n, and protein complexes were co-immunoprecipitated with anti-ha-tag resin. western-blot analysis demonstrated co-immunoprecipitation of hcov-nl -n with a c, a f and a h, whereas no hcov-nl -n could be detected in samples incubated with gfp lysate. notably, the catalytically inactive derivatives of a c, a f and a h also bound to hcov-nl -n protein (fig. a, supplementary figure ). on the other hand, we could not observe the same effect with other apobec proteins (a a, a d and a g). co-localisation of a c with hcov-nl -n was investigated by co-transfection of llc-mk cells with mrnas encoding a c and hcov-nl -n, followed by immunofluorescence staining and confocal microscopy. the analysis showed several clearly visible foci of co-localisation (fig. b) . calculation of manders' coefficient gave a value of . , indicating the existence of an interaction between the two proteins. apobec -mediated cytidine deamination has been described as an interim antiviral mechanism. although apobec proteins were initially shown to primarily modify dna templates, it has subsequently been demonstrated that they also use rna as a substrate. the finding that apobec deaminases can mutate the hiv rna genome first expanded their possible specificity . thereafter, it was shown that a g impairs the replication of mumps, measles and respiratory syncytial viruses. this antiviral activity is not an effect of apobec following incubation for h at °c in an atmosphere containing % co , cells were infected with human coronavirus (hcov)-nl at tcid per ml. after h incubation at °c in an atmosphere containing % co , cells were fixed and immunostained for hcov-nl nucleoprotein (n) using specific antibodies. the rate of viral infection was analysed by flow cytometry and is presented as the percentage of cells that were infected. for comparisons by student's t-test, *indicates p < . . (b) llc-mk cells were transfected with capped and polyadenylated mrna encoding active or catalytically inactive (m) apobec proteins. following incubation for h at °c in an atmosphere containing % co , cells were lysed in ripa buffer. apobec proteins and control gapdh protein were detected by western blotting using specific antibodies. (c) t cell lysates enriched in the apobec protein a c (lower panel) or green fluorescent protein (gfp) (upper panel) were incubated with in vitro-prepared rna containing a gc-rich sequence for h at °c. the rna was then reverse transcribed, amplified by pcr and sequenced. experiments were performed at least three times, with two technical replicates. (d) huh- cells were transfected with capped and polyadenylated mrna encoding active a f (lower panel) or gfp (upper panel). following h incubation at °c in an atmosphere containing % co , cells were infected with the recombinant gc-rich hcov- e virus (ic e-gc) at tcid per ml. after four cycles of virus passage in a f-positive or gfp-positive cells, viral rna was isolated, reverse transcribed and sequenced across the gc-rich domain. enzymatic activity, but is thought to result from the interaction of a g with viral transcripts . a a has been shown to act as a mrna-editing factor in human monocytes and monocyte-derived macrophages . however, apobec -mediated deamination of viral rna genomes has not previously been demonstrated for viruses that do not replicate through dna intermediates. here, we investigated the antiviral effects of apobec proteins on human coronaviruses. hcov-nl was selected as the model pathogen because of its prominent bias in nucleotide composition (low gc content), suggesting the possible shaping of the genome by cytidine deamination. we studied the interaction between hcov and hae cultures, as an in vitro model of the human airways, the primary entry site for human respiratory viruses. mrna levels of genes encoding a a, a c and a d were upregulated in hcov-nl -infected cultures. upregulation of apobec protein expression was previously shown to be dependent on interferon alpha, and it is associated with non-cytolytic clearance of human hepatitis b virus in primary hepatocytes . cytoplasmic dna triggers production of type i interferon, leading to massive upregulation of apobec a , . here, we investigated the effects of different apobec proteins, and found that expression of a c, a f or a h limits coronavirus infection. notably, some infectious progeny were produced in the presence of these proteins, and serial passaging of virus was possible. surprisingly, if cells overexpressing a c, a f or a h were infected with the progeny virions, no restriction of virus replication occurred. possible explanations for this effect are that the first viral passage results in selection of viral subspecies that are not affected by apobec activities, or that viral passage activates a viral factor or mechanism that can counteract apobec activities. serial passage of hcov in apobec -expressing cells did not result in hypermutation in progeny viruses. it is possible that hcov-nl has reached a maximum level of at content, so that new c → t or g → a changes in the genome result in a drastic drop in viral infectivity. however, incubation of an rna transcript containing potential deaminase hot-spots with a c-enriched cell lysates did not result in hypermutation. the lack of hypermutation might have resulted from limited accessibility of the template or instability of a c in the lysate. we were not able to develop a hcov-nl molecular clone containing gc-rich elements, most likely because of low stability of the reverse genetics system for this virus (data not shown). we therefore developed a hcov- e clone containing a gc-rich region , . replication of this virus was inhibited in cells expressing a c, a f or a h, but no mutations were observed in the modified region. the lack of observed hypermutation may result from proofreading and repair of the coronavirus rna genome , and suggests that inhibition of coronaviral replication by apobec proteins does not result from deamination of viral rna. it was previously shown that, for some rna viruses, apobec-mediated inhibition is deamination-independent. in our system, catalytically inactive a c, a f and a h proteins only had marginal inhibitory effect on virus replication. we suggest that apobec proteins may affect coronavirus replication by two mechanisms: first, by deamination of yet unidentified cellular targets or deamination of single sites in the coronaviral genome; and second, by direct, non-catalytic interaction with viral rna and/or proteins. apobec proteins may interact with the coronaviral n protein, which is an essential structural protein for formation of the viral ribonucleocapsid, and which also has a role in virus replication [ ] [ ] [ ] [ ] [ ] . our results demonstrated that all apobec proteins with inhibitory activity bound to the n protein. the direct, editing-independent mechanism by which apobec proteins inhibit hcov-nl replication is still to be elucidated, but it is possible that interaction with the n protein interferes with virus replication and progeny production. in conclusion, our results show that three apobec proteins (a c, a f and a h) inhibit coronaviral replication. the molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive and may depend on deamination of single sites in the coronaviral genome, or in a cellular target or targets. passage of hcov-nl in cells expressing apobec proteins results in virus that is resistant to apobec-mediated inhibition, possibly indicating the presence of an unidentified factor that counteracts apobec activity. the deaminase-independent component of viral inhibition may result from interaction between viral n protein and apobec proteins. human airway epithelium (hae) cultures. human epithelial cells were isolated from conductive airways resected from transplant patients. the study was approved by the bioethical committee of the medical university of silesia in katowice, poland (approval no: knw/ /kb / / dated on . . ). a written informed consent was obtained from all patients. tissues were procured at the silesian center for heart diseases, zabrze, poland. no organs/tissues were procured from prisoners. cells were dislodged by protease treatment, and later mechanically detached from the connective tissue. resulting primary cells were first cultured in selective media to proliferate. further, cells were trypsinised and transferred onto permeable transwell insert supports (φ = . ). cell differentiation was stimulated by media additives and removal of media from the apical side. in such a manner cells were cultured for - weeks to form well-differentiated, pseudostratified mucociliary epithelium . all experiments were performed in accordance with relevant guidelines and regulations. for the virus infection, hae cultures were washed thrice with μl of × pbs, following inoculation with hcov-nl (isolate amsterdam ) or influenza a virus (iva, strain h n ) or mock (cell lysate). after h incubation at ° (for hcov-nl ) or °c (for iva) unbound virions were removed by washing with μl of × pbs and hae cultures were cultured at an air -liquid interphase until the end of the experiment. virus preparation and titration. the stock of hcov-nl (isolate amsterdam ) was prepared using llc-mk cells. six days post-infection (p.i.) infected cells were lysed by freeze-thawing and the resulting supernatant was stored at − °c. mock sample was prepared in the same manner, from non-infected cells. virus yield was determined according to the method described by reed and muench . briefly, cells infected with serially diluted virus were incubated at °c for days and the appearance of cytopathic effect (cpe) was monitored. poland) was used for nucleic acid isolation from cell culture supernatants, according to the manufacturer's instructions. cellular rna was isolated using fenozol reagent (a&a biotechnology, poland), followed by dnase i treatment (thermo fisher scientific, poland). cdna samples were prepared with a high capacity cdna reverse transcription kit (thermo fisher scientific, poland), according to the manufacturer's instructions. gc-rich region was generated using the vaccinia virus-based reverse genetic system as described previously , . in short, vaccinia virus containing the full-length hcov- e cdna in which an escherichia coli guanine phosphoribosyltransferase (gpt) was inserted between hcov- e and nucleotides was used to recombine with a plasmid containing hcov- e - nucleotides with a number of silent mutations (gc-rich region) . the resulting vaccinia virus was used to rescue the mutant hcov- e, as described previously , . plasmid dna, recombinant vaccinia virus and recombinant hcov- e identities were confirmed by sequencing. used as a household gene control and rox was used as the reference dye. primer sequences are provided in table . reaction was carried out according to the scheme: min at °c and min at °c, followed by cycles of sec at °c, sec at °c and sec at °c. in order to assess the copy number for each gene, dna standards were prepared, as described before . flow cytometry. llc-mk cells cultured in -well plate format (tpp) were washed with sterile × pbs, trypsinised, fixed with % formaldehyde, permeabilised with . % triton x- in × pbs and incubated for h with blocking buffer ( % bsa in × pbs with . % tween ). to visualise ha-tagged apobec proteins after mrna transfection, cells were incubated for h at room temperature with mouse anti-ha-tag antibody ( μg/ml, antibodies-online, germany) in × pbs with % bsa and . % tween , followed by h incubation with alexa fluor -labeled goat anti-mouse antibody ( . μg/ ml, molecular probes, poland). to evaluate hcov-nl infection using flow cytometry, cells were incubated for h at room temperature with mouse anti-hcov-nl -n antibody ( μg/ml, ingenansa, spain) in × pbs with % bsa and . % tween , followed by h incubation with alexa fluor -labeled goat anti-mouse antibody ( . μg/ml, molecular probes, poland). cells were then washed, re-suspended in × pbs and analysed with facs calibur (becton dickinson, usa) using cell quest software. ha-tagged ptracer cmv plasmids (thermo fisher scientific, poland) encoding human a a (genebank accession number (gb) nm_ ), a c (gb: nm_ ), a de (gb: nm_ ), a f (gb: nm_ ), apobec g (gb: nm_ ) and a h (gb: nm_ ) were prepared. catalytic mutants of a c (a c_ e q = ma c), a f (a f_e q = ma f) and a h (a h_e q = ma h) were prepared using quick change mutagenesis with phusion polymerase (thermo fisher scientific, poland) . sequences of oligonucleotides used are listed in table . design of "hot-spot" rna. plasmid construct containing several gc-rich domains was ordered at geneart gene synthesis (thermo fisher scientific, germany). the region was sequenced with primers ′-ctg tgg aaa acc ttt ggc atc- ′ and ′-ctg tgg aaa acc ttt ggc atc- ′. the plasmid was amplified in e. coli top strain and further used as a template in in vitro transcription reaction using t promoter, as described below. preparation of mrna molecules for transfection. apobec plasmids described above were used as templates for amplification of apobec genes with forward primer containing the t promoter region ( ′-tcg gcc tcg tag gcc taa tac gac tca cta tag gga gac tga gag aac cca ctg ctt ac- ′) and specific reverse primer (listed in table , nm each). reaction was carried out with phusion polymerase (thermo fisher scientific, poland), according to the manufacturer's instructions. dna templates were purified using genejet pcr purification kit (thermo fisher scientific, poland) and further used for in vitro transcription with t ribomax express large scale rna production system and ribo m g cap analog (promega, poland). resulting rna was precipitated with licl, purified with % ethanol and polyadenylated using poly(a) antisense ctg tgg aaa acc ttt ggc atc probe atg tta ttc agt gct ttg gtc ctc gtg at a a sense tgg ttc ctt ctt tgc agt tgg acc antisense gca gca ttt gca gtg cct cct tat a b sense agc aca tgg gct ttc tat samples were centrifuged ( min at , × g), pelleted cell debris was discarded and resulting supernatants were stored at − °c. hcov-nl n protein was prepared as described previously . cell lysates were incubated antisense gac tgc ggc cgc cta tca agc gta atc tgg aac atc gta tgg gta gtt tcc ctg att ctg gag aa a c sense acg cga att cca cca tga atc cac aga tca gaa acc cga tg antisense gac tgc ggc cgc cta tca agc gta atc tgg aac atc gta tgg gta ctg gag act ctc ccg tag for h at °c with the purified n protein in × activity buffer, containing mm kcl, mm hepes (ph . ), µm zncl , mm dtt and mm edta . subsequently, µl of agarose spheres coated with anti-ha antibodies (pierce, poland) was added and samples were loaded onto co-immunoprecipitation columns. after overnight incubation at °c on a rotary shaker, proteins were eluted, according to the manufacturer's instructions. analysis of hcov-nl co-localisation with apobec c. llc-mk cells were seeded on -wells plates and cultured for h at °c with % co . . μg of hcov-nl n mrna and . μg of a c mrna was co-transfected using the mrna-in reagent (ams biotechnology, united kingdom). after h incubation at °c with % co , cells were fixed with % formaldehyde, permeabilised with . % triton x- in × pbs and incubated overnight at °c with % bsa in × pbs with . % tween . to visualise hcov-nl n molecules, cells were incubated for h at room temperature with rabbit anti-hcov-nl -n serum ( : dilution, kindly provided by dr lia van der hoek, uva, the netherlands) in × pbs with % bsa and . % tween , followed by h incubation with atto -labeled goat anti-rabbit igg ( μg/ml; sigma-aldrich, poland). to detect ha-tagged a c protein cells were incubated for h at room temperature with mouse alexa fluor -labeled anti-ha-tag antibody ( μg/ml; thermo fisher scientific; poland) in × pbs with % bsa and . % tween . nuclear dna was stained with dapi ( . μg/ml; sigma-aldrich, poland). immunostained cultures were mounted on glass slides with prolong gold antifade medium (thermo fisher scientific; poland). fluorescent images were acquired under a zeiss lsm confocal microscope (carl zeiss microscopy gmbh). images were acquired using zen sp software (carl zeiss microscopy gmbh) and processed using imagej . v (national institutes of health, bethesda, maryland, usa). all the experiments were performed in triplicate and the results are presented as mean ± sd. student's t-test was used to determine significance of the obtained results. p values < . were considered significant. an anthropoid-specific locus of orphan c to u rna-editing enzymes on chromosome dual inhibitory effects of apobec family proteins on retrotransposition of mammalian endogenous retroviruses an endogenous inhibitor of human immunodeficiency virus in human lymphocytes is overcome by the viral vif protein isolation of a human gene that inhibits hiv- infection and is suppressed by the viral vif protein identification of apobec de as another antiretroviral factor from the human apobec family human and rhesus apobec d, apobec f, apobec g, and apobec h demonstrate a conserved capacity to restrict vif-deficient hiv- restriction of equine infectious anemia virus by equine apobec cytidine deaminases cytidine deamination of retroviral dna by diverse apobec proteins apobec g targets specific virus species the antiretroviral activity of apobec is inhibited by the foamy virus accessory bet protein restriction of foamy viruses by apobec cytidine deaminases g to a hypermutation of hepatitis b virus extensive editing of both hepatitis b virus dna strands by apobec cytidine deaminases in vitro and in vivo apobec a is a potent inhibitor of adeno-associated virus and retrotransposons virion-associated uracil dna glycosylase- and apurinic/apyrimidinic endonuclease are involved in the degradation of apobec g-edited nascent hiv- dna apobec-mediated editing of viral rna apobec a cytidine deaminase induces rna editing in monocytes and macrophages the innate antiviral factor apobec g targets replication of measles, mumps and respiratory syncytial viruses croup is associated with the novel coronavirus nl the novel human coronaviruses nl and hku mosaic structure of human coronavirus nl , one thousand years of evolution characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia the severe acute respiratory syndrome sars-beginning to understand a new virus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group isolation of a novel coronavirus from a man with pneumonia in saudi arabia identification of a new human coronavirus genome structure and transcriptional regulation of human coronavirus nl cytosine deamination and selection of cpg suppressed clones are the two major independent biological forces that shape codon usage bias in coronaviruses high level expression of the anti-retroviral protein apobec g is induced by influenza a virus but does not confer antiviral activity catalytic activity of apobec f is required for efficient restriction of vif-deficient human immunodeficiency virus interferon-inducible expression of apobec editing enzymes in human hepatocytes and inhibition of hepatitis b virus replication self-cytoplasmic dna upregulates the mutator enzyme apobec a leading to chromosomal dna damage deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases systematic assembly of a full-length infectious clone of human coronavirus nl infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus coronaviruses as dna wannabes: a new model for the regulation of rna virus replication fidelity the nucleocapsid protein of human coronavirus nl the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein apobec g cytidine deaminase association with coronavirus nucleocapsid protein novel polymeric inhibitors of hcov-nl a simple method of estimating fifty per cent endpoints generation of recombinant coronaviruses using vaccinia virus as the cloning vector and stable cell lines containing coronaviral replicon rnas targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus htcc: broad range inhibitor of coronavirus entry fast and easy method for construction of plasmid vectors using modified quick-change mutagenesis this work was supported by grants from the national science center (umo- / /e/nz / and umo- / /n/nz / (kp and am, respectively) and by the swiss national science foundation (snf; # ) to vt. faculty of biochemistry, biophysics and biotechnology of the jagiellonian university is a partner of the leading national research center supported by the ministry of science and higher education of the republic of poland. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors are grateful to katarzyna owczarek and artur szczepanski for assisting in the confocal studies and image analysis. a.m. and p.v. conducted the experiments. e.k., s.z., m.o. and v.t. provided materials for the study. z.r. participated in confocal imaging. a.m. and k.p. designed the experiments, analysed the data and wrote the manuscript. all authors reviewed the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - pnqpljt authors: munster, vincent j.; adney, danielle r.; van doremalen, neeltje; brown, vienna r.; miazgowicz, kerri l.; milne-price, shauna; bushmaker, trenton; rosenke, rebecca; scott, dana; hawkinson, ann; de wit, emmie; schountz, tony; bowen, richard a. title: replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: pnqpljt the emergence of middle east respiratory syndrome coronavirus (mers-cov) highlights the zoonotic potential of betacoronaviruses. investigations into the origin of mers-cov have focused on two potential reservoirs: bats and camels. here, we investigated the role of bats as a potential reservoir for mers-cov. in vitro, the mers-cov spike glycoprotein interacted with jamaican fruit bat (artibeus jamaicensis) dipeptidyl peptidase (dpp ) receptor and mers-cov replicated efficiently in jamaican fruit bat cells, suggesting there is no restriction at the receptor or cellular level for mers-cov. to shed light on the intrinsic host-virus relationship, we inoculated jamaican fruit bats with mers-cov. although all bats showed evidence of infection, none of the bats showed clinical signs of disease. virus shedding was detected in the respiratory and intestinal tract for up to days. mers-cov replicated transiently in the respiratory and, to a lesser extent, the intestinal tracts and internal organs; with limited histopathological changes observed only in the lungs. analysis of the innate gene expression in the lungs showed a moderate, transient induction of expression. our results indicate that mers-cov maintains the ability to replicate in bats without clinical signs of disease, supporting the general hypothesis of bats as ancestral reservoirs for mers-cov. scientific reports | : | doi: . /srep arab emirates and egypt , [ ] [ ] [ ] . mers-cov sequences and virus isolates obtained from dromedary camels in qatar and the kingdom of saudi arabia showed high sequence identity with those obtained from epidemiologically linked human cases , . together, these data suggest that rather than direct zoonotic transmission from a bat reservoir, dromedary camels are involved as the primary reservoir host for mers-cov. phylogenetic and epidemiological data suggest that rather than a single introduction in the human population, mers-cov appears to continue to be transmitted by multiple independent spillover events from dromedary camels , . after the emergence of severe acute respiratory syndrome coronavirus (sars-cov) in , the targeted focus on reservoir studies in bats has resulted in a vast increase of our knowledge on the genetic diversity of bat coronaviruses. despite the increase in genetic data on coronavirus diversity in their natural reservoirs, only very limited data are available on the impact of these viruses on the reservoir host and controlled infection experiments with coronaviruses in their reservoir hosts have not been performed. to understand the drivers of mers-cov emergence, a more comprehensive understanding of the interaction between the virus and its natural and intermediate reservoir hosts is needed. here we present data on the first experimental infection of bats with mers-cov to model the infection kinetics in a coronavirus host species, the jamaican fruit bat (artibeus jamaicensis). artibeus jamaicensis dpp receptor and cell susceptibility. the mers-cov receptor dpp is the main host restriction factor ; therefore, we first studied the interaction between mers-cov and jamaican fruit bat dpp . the paj-dpp plasmid expressing the dpp coding sequence of jamaican fruit bat under control of a cmv promoter was transfected into bhk cells, which are not permissive to mers-cov . the expression of aj-dpp in transfected cells was confirmed by flow cytometry, showing the presence of bat dpp on the surface of transfected bhk cells by determining the increase over untransfected cells ( figure s ). transient expression of bat dpp in bhk cells supported mers-cov replication, whereas transient expression of hamster dpp in bhk cells did not (fig. a) . subsequently, the replication kinetics of mers-cov were compared in llc-mk cells (macaca mulatta) and jamaican fruit bat primary kidney cells. mers-cov replicated efficiently to high titers in both cell lines (fig. b) , indicating that there is no restriction at the receptor or cellular level for mers-cov replication in jamaican fruit bat cells. clinical signs in bats inoculated with mers-cov. ten adult jamaican fruit bats were inoculated via the intranasal and intraperitoneal routes with tcid of mers-cov strain emc/ ; mock inoculated jamaican fruit bats, housed in a separate cage, were used as controls, the mock inoculated animals were inoculated with tissue culture medium via the same routes and volumes. the bats were observed at least once daily for signs of disease. bodyweight and temperature were measured throughout the experiment for a maximum of days post inoculation (dpi) for bat and (mers-cov inoculated) and bat and (mock inoculated controls). none of the bats showed signs of disease, weight loss or increased body temperature throughout the experiment ( figure s ). to examine mers-cov shedding in inoculated bats, oral and rectal swabs were collected for the duration of the experiment. mers-cov shedding was first detected on dpi, as indicated by the presence of viral rna in throat and rectal swabs and continued for a maximum duration of days. all animals, except bat , shed mers-cov from the respiratory tract ( fig. a) ; all bats except and , shed mers-cov from the intestinal tract (fig. b ). viral loads in swabs collected from the respiratory tract were higher than viral loads in swabs from the intestinal tract. tissues collected at the sequential necropsy dates of , , , and dpi were analyzed for the presence of viral rna, infectious virus, and evaluated by histopathology and immunohistochemistry. mers-cov viral rna was detected in various tissues of all inoculated bats, except bat on dpi and bat on dpi. the highest viral loads were detected in the lower respiratory tract (fig. ) . mers-cov viral rna was detected at dpi in trachea, lung, liver, spleen, bladder and nasal turbinates; at dpi in lung, spleen, duodenum, colon, bladder, turbinates and brain; at dpi in lung, liver, turbinates and brain; at dpi in heart, lung, liver, spleen and duodenum. no mers-cov viral rna was detected at dpi (fig. ) . in additon, mers-cov viral rna was detected in blood on and dpi, in bats - , indicative of viremia ( figure s ). mers-cov mrna was detected in tissues of bats to , confirming mers-cov replication on the transcriptional level (table s ) . infectious mers-cov was isolated from the lungs of bats ( dpi) and ( dpi), the bladder and nasal turbinates of bat ( dpi), and the duodenum of bat ( dpi), indicating active virus replication, mainly in the respiratory tract. only two of ten bats (bat and bat ) exhibited histopathology associated with mers-cov infection, which was mild. all mers-cov associated lesions were detected in the respiratory tract of the infected bats (fig. , table s ). bat and bat ( dpi) displayed a mild acute rhinitis, but mers-cov replication was not detected by immunohistochemistry (table s and s ). bat and displayed a multifocal interstitial pneumonia that was characterized by minimal alveolar interstitial thickening by small numbers of macrophages and neutrophils (fig. , table s ). the adjacent alveolar spaces contained small numbers of alveolar macrophages. mers-cov antigen and rna was detected by immunothroughout the lungs of bat ( dpi), but no associated pulmonary pathology was detected (fig. , table s ). cytokeratin and anti-mers-cov co-staining demonstrated mers-cov antigen in type i pneumocytes of the lungs of bat (fig. ). scientific reports | : | doi: . /srep innate immune response to mers-cov. mers-cov was most consistently detected in the lower respiratory tract of the bats. the mx , isg and rantes gene expression in the lungs of jamaican fruit bats was analyzed as an indicator of the induction of an innate immune response to mers-cov infection. a -fold increase in expression of mx was observed in the lungs of the infected jamaican fruit bats at dpi. a statistically significant upregulation of mx gene expression was detected when comparing the lungs of bats collected on dpi and dpi (two-tailed unpaired t-tests, p < . ). the maximum isg expression of . -fold occurred at dpi. statistically significant differences were observed between the dpi and dpi, dpi and dpi animals (two-tailed unpaired t-tests, p < . , p < . and p < . respectively. in addition significant differences were observed between the dpi and the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ). the rantes expression at its peak at dpi was increased . fold. statistically significant differences were observed between the versus the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ), the versus the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ), and the dpi versus the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ) (fig. ). antibody response to mers-cov. sera were collected prior to inoculation and at the scheduled necropsy dates. each of the bats was seronegative for mers-cov prior to inoculation. only bat developed a mers-cov specific antibody response, both by elisa and virus neutralization assay. the sera obtained from bat had a neutralizing titer of at days post inoculation. the high sequence similarity of mers-cov to coronavirus sequences detected in bats suggests that mers-cov or its immediate ancestor originated in bats . direct contact between bats and humans is uncommon, and a domestic or peridomestic intermediate species often plays a role in the emergence of zoonotic viruses from natural reservoirs to humans [ ] [ ] [ ] [ ] . similar to the emergence of sars-cov in from the masked palm civet (paguma larvata) as an intermediate host , the dromedary camel appears to have initially served as the intermediate host for mers-cov . several aspects of the emergence of mers-cov are currently still unknown, including the role of the natural reservoir and the relationship between the natural and intermediate reservoirs. with their ability to support efficient replication of mers-cov, the availability of an annotated transcriptome , and the relative easy housing and husbandry practices of jamaican fruit bats suggest that this bat species can become an important model system to investigate the relationship between coronaviruses and their bat hosts . although the jamaican fruit bat is not the direct ancestral reservoir for mers-cov, as it is a new world bat species, generalized responses towards viruses of bat-origin rather than a direct host-pathogen relationships can be modelled. the ability of mers-cov to use dpp of multiple species as a receptor, including dpp of human, dromedary camel, and bat origin , , suggests that no prior adaptation was needed on the dpp receptor level for cross-species and zoonotic transmission to occur. with batcov-hku , a closely related coronavirus, it was shown that replication in human cells required two mutations in the spike protein . these amino acid residues, which are conserved in mers-cov, results in the activation of the batcov-hku spike protein by human cellular proteases. this suggests that batcov-hku needs these residues for replication in humans. interestingly, our data show that mers-cov replicates efficiently in jamaican fruit bat cells, suggesting that the mers-cov spike can efficiently be processed by jamaican fruit bat cellular proteases and that there is no host restriction on the post-translational modification level of the mers-cov spike in dromedary camels, humans and bats. bat coronaviruses have been primarily detected in fecal samples in field studies suggesting that these viruses have a intestinal tract tropism , , . mers-cov was able to replicate to higher titers in the respiratory tract in comparison with the intestinal tract of the jamaican fruit bats. the tissue tropism of mers-cov in jamaican fruit bats is comparable to the respiratory tract tropism observed in dromedary camels and humans , . this might suggest that mers-cov, upon cross-species transmission from bats into dromedary camels evolved from a gastrointestinal tract virus into a respiratory tract virus, similar to influenza a viruses . the ability for mers-cov to antagonize the innate immune response appears to correlate with its pathogenic potential in humans. mers-cov and related batcov-hku can inhibit innate immune signaling in a variety of human cell lines in vitro via the orf b-encoded accessory proteins lungs of jamaican fruit bat were stained with α -cytokeratin as an epithelial marker (purple) and with a polyclonal α -coronavirus antibody (brown-red) to demonstrate that viral antigen was located along the basement membrane of alveolar pneumocytes of bat at dpi (indicated by black arrows). original magnification: × . with % fetal calf serum (hyclone, logan), mm l-glutamine (lonza), u/ml penicillin and μ g/ml streptomycin (gibco). vero e , llc-mk , bhk and jamaican fruit bat primary kidney cells were maintained in dulbecco's modified eagle's media (dmem) supplemented with % fetal calf serum, u/ml penicillin and μ g/ml of streptomycin. sequencing and cloning of the jamaican fruit bat dpp sequence. total rna was extracted from primary kidney cells using the rneasy mini kit (qiagen) and cdna was synthesized using random hexamer primers and superscript iii reverse transcriptase (applied biosystems). dpp was then amplified using iproof high-fidelity dna polymerase (biorad) and primers dpp unvf and dpp unvr (primer sequences are available upon request). the obtained dpp gene sequence was synthesized in expression plasmid pcdna . (+ ) cate with mers-cov with a multiplicity of infection (moi) of . (cell lines) or (transfected cell lines) % tissue culture infectious dose (tcid ) per cell. one hour after inoculation, cells were washed once with dmem and culture medium replaced. supernatants were sampled at , , and h after inoculation. mers-cov was titrated by end-point titration in quadruplicate in vero e cells cultured in dmem supplemented with % fetal calf serum, mm l-glutamine (lonza), u/ml penicillin and μ g/ml streptomycin. cells were inoculated with ten-fold serial dilutions of virus, and scored for cytopathic effect days later. the tcid was calculated by the method of spearman-karber. animal experiments. twelve captive-bred jamaican fruit bats were used for this work , . ten bats were inoculated with tcid emc/ via a combination of intranasal ( μ l each nostril) and intraperitoneal ( μ l) routes. two mock inoculated bats were included as controls for histopathology and gene expression analyses. mock inoculated bats were inoculated with standard tissue culture media via the same routes and volumes. bats were injected with an iptt- temperature transponder (bmds) to monitor body temperature daily. animals were weighed daily and observed for signs of disease. oropharyngeal and rectal swabs were obtained on , , , , , , , and dpi and analyzed for the presence of viral rna. on , , , and days post inoculation (dpi), two bats were euthanized and trachea, heart, lung, liver, spleen, kidney, duodenum, colon, bladder, nasal turbinates and brain were collected for virological and histopathological analysis. histopathology. histopathology was performed on bat tissues. after fixation for at least days in % neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (h & e) staining. immunohistochemistry was performed using a mers-cov emc/ polyclonal rabbit antibody at a : dilution and in situ hybridization was performed using probes directed against the mers-cov emc/ n gene as described previously . rna extraction. rna was extracted from swab samples using the qiaamp viral rna kit (qiagen). rna was eluted in μ l. tissues ( mg) were homogenized in rlt buffer and rna was extracted using the rneasy kit (qiagen). rna was eluted in μ l. quantitative pcr. for detection of viral rna in samples, μ l rna was used in a one-step real-time rt-pcr upe assay using the rotor-gene tm probe kit (qiagen) according to the manufacturer instructions. in each run, standard dilutions of a titered mers-cov stock were run in parallel, to calculate tcid equivalents in the samples. for the detection of viral mrna, μ l rna was used in a one-step real-time rt-pcr using the mers-cov m mrna assay in the rotor-gene tm probe kit . artibeus jamaicensis orthomyxovirus resistance gene (mx ) gene expression was determined by qrt-pcr using mx , isg and rantes specific primers (derived from transcriptome sequencing ). the fold-change of each gene was calculated by normalizing the change in ct (cycle threshold) value of mx (Δ ct) to the ct values for hypoxanthine phosphoribosyltransferase (hprt) as an internal reference gene for each sample and comparing this to the ct values of mock inoculated bats and ( ∧ (−Δ Δ ct). mx specific primers: ′ -ccagacctgaccctgataga- ′ , ′ -tggatgtacttcctgaatgagttg- ′ and ′ -fam-atctagtgtccgatgtcagctggc-iabkfq- ′ . isg specific primers: ′ -gctgtctatcgtctgaatggg- ′ , ′ -ttcttgtccgatgtcctgaag- ′ and ′ -hex-cgatgaggc/zen/attttgtctgcaaaccc-iabkfq- ′ . rantes specific primers: ′ -agttgtcctaatcacccgaaag- ′ , ′ -cagagtgttgatgtagtcccg- ′ and ′ -fam-tgtgccga c/zen/ccggagaagaaat-iabkfq- ′ . hprt specific primers: ′ -agatggtgaaggtcgcaag- ′ , ′ -cctgaagtattcattatagtcaaggg- ′ and ′ -fam-actttgttggatttgaaattccag acaagtttg-bhq . virus isolation. tissue samples were homogenized in a tissuelyzer ii (qiagen) after addition of ml dmem. homogenates were centrifuged to pellet cellular debris and subsequently inoculated onto veroe and llc-mk cells. after hr adsorption, cells were washed once with dmem and media was replaced. elisa. antibody responses were measured in an enzyme-linked immunosorbent assay (elisa) using hcov-emc/ as described previously . briefly, emc/ containing cell culture supernatant was used to coat immuno microwell maxisorp plates (nunc) at °c overnight and diluted serum samples were added. bound antibodies were detected using a secondary protein a/g conjugated with horseradish peroxidase (hrp; pierce). sera were considered positive when absorbance was higher than three standard deviations above the mean of negative control sera. sera obtained from rabbits immunized with emc/ were used as a positive control. virus neutralization assay. two-fold serial dilutions of heat-inactivated sera were prepared in a microwell tissue culture plate and tcid of mers-cov was added and incubated for hour at °c. after incubation the virus-sera mixture was transferred to a microwell tissue culture plate with a % confluent scientific reports | : | doi: . /srep monolayer of veroe cells. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, that still inhibited emc/ virus replication. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who. middle east respiratory syndrome coronavirus (mers-cov) full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans a coronavirus detected in the vampire bat desmodus rotundus highly diversified coronaviruses in neotropical bats close relative of human middle east respiratory syndrome coronavirus in bat novel bat coronaviruses, brazil and mexico detection of a virus related to betacoronaviruses in italian greater horseshoe bats circulation of group coronaviruses in a bat species common to urban areas in western europe human betacoronavirus c emc/ -related viruses in bats, ghana and europe coronaviruses in bats from 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viruses related to the sars coronavirus from animals in southern china hendra virus: an emerging paramyxovirus in australia discrepancies in data reporting for rabies a decade after sars: strategies for controlling emerging coronaviruses the emergence of the middle east respiratory syndrome coronavirus walker's bats of the world detection and phylogenetic analysis of group coronaviruses in south american bats neotropical bats from costa rica harbour diverse coronaviruses transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) immunology of bats and their viruses: challenges and opportunities dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc two mutations were critical for bat-to-human transmission of mers coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome practical considerations for high-throughput influenza a virus surveillance studies of wild birds by use of molecular diagnostic tests the orf b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling comparison of serological assays in human middle east respiratory syndrome (mers)-coronavirus infection presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study experimental nipah virus infection in pteropid bats (pteropus poliocephalus) pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission tacaribe virus causes fatal infection of an ostensible reservoir host, the jamaican fruit bat experimental inoculation of plants and animals with ebola virus seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection bats and lyssaviruses experimental rabies virus infection in artibeus jamaicensis bats with cvs- variants middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction. euro surveillance : bulletin européen sur les maladies transmissibles growth and quantification of mers-cov infection the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the authors would like to thank drs. bart key: cord- -sisfxdso authors: banskar, sunil; bhute, shrikant s.; suryavanshi, mangesh v.; punekar, sachin; shouche, yogesh s. title: microbiome analysis reveals the abundance of bacterial pathogens in rousettus leschenaultii guano date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: sisfxdso bats are crucial for proper functioning of an ecosystem. they provide various important services to ecosystem and environment. while, bats are well-known carrier of pathogenic viruses, their possible role as a potential carrier of pathogenic bacteria is under-explored. here, using culture-based approach, employing multiple bacteriological media, over thousand bacteria were cultivated and identified from rousettus leschenaultii (a frugivorous bat species), the majority of which were from the family enterobacteriaceae and putative pathogens. next, pathogenic potential of most frequently cultivated component of microbiome i.e. escherichia coli was assessed to identify its known pathotypes which revealed the presence of virulent factors in many cultivated e. coli isolates. applying in-depth bacterial community analysis using high-throughput s rrna gene sequencing, a high inter-individual variation was observed among the studied guano samples. interestingly, a higher diversity of bacterial communities was observed in decaying guano representative. the search against human pathogenic bacteria database at % identity, a small proportion of sequences were found associated to well-known human pathogens. the present study thus indicates that this bat species may carry potential bacterial pathogens and advice to study the effect of these pathogens on bats itself and the probable mode of transmission to humans and other animals. of previous reports of bacterial identification from bat guano or the bats intestinal content , there remains an immense scope of identifying additional bacterial species from bats. hence, a serious need of detailed and thorough study of bat guano associated bacteria was experienced. such studies are important to catalogue the various bacteria from bats, so that their potential role in zoonosis can be recognized, interpreted and can be comprehended during the zoonosis. metagenomic studies using s rrna gene sequencing have been used to characterize gut microbial communities of different bats species , . a report from philips et al. mentioned that, the herbivory dietary strategy and the reproductively active bats carried more diverse microbiota than carnivore and reproductively inactive individuals . similarly, the carrillo-araujo et al. reported the diet to be a primary factor to define the gut microbiome of bats . further, our previous study reported the considerable similarity of microbial communities of frugivorous and insectivorous bats indicating that there was dietary overlap among the bats of different feeding habits . in addition to the diet, the decaying guano communities were also explored revealing a higher abundance of bacterial communities involved in nitrogen cycling . interestingly, using the same approach, veikkolainen et al. reported and confirmed the presence of important human bacterial pathogen bartonella mayotimonensis . there are more than bats species worldwide which should be explored for their microbial communities to understand a comprehensive 'bats' microbiome' from every aspect including host-microbe relationship as well as their possibility of carrying putative bacterial pathogens. this study deals with the isolation and identification of general bacterial communities and potential bacterial pathogens present in rousettus leschenaultii guano. further, to evaluate the pathogenic potential, virulence genes were positively identified from the most commonly isolated bacteria from the gut microbiome i.e. escherichia coli. additionally, s rrna gene sequencing was performed using ion torrent pgm to identify the bacterial communities and screened for the presence of putative human pathogens. therefore, this study discourages the human interference in the wildlife which otherwise may cause irreparable loss to the ecosystem and severe human health hazards. bat guano collection. fresh bat guano pellets were collected at the robber's cave, mahabaleshwar, maharashtra, india. collection plates were left inside the cave for two hours to capture the guano dropping. this way, a total of fresh guano pellets were collected from which guano of sufficient quantity and free from contaminations were used for the further study. bat identification. sequence analysis of the cytochrome b (cytb) gene amplified from total extracted dna of the guano pellets revealed that the collected fecal pellets were from a single bat species i.e. rousettus leschenaultii, a predominant bat species reported from the robber's cave . this confirmed that the collected guano pellets were from single bat species. total viable counts and identification of bacteria. the total viable count of bat guano was performed, it ranged from . × to . × cfus/gram of the guano among four different bacterial media used. all bacterial isolates were identified by s rrna gene sequencing followed by the blast analysis at ncbi. a total of bacterial isolates from bat guano and bacterial isolates from cave environment samples (total ) were identified (the entire list of bacteria identified is provided in supplementary table s ). all the isolates showed % or above similarity except five isolates. all s rrna gene sequences of isolates were further analyzed for media-wise genus level distribution. maximum numbers of bacterial genera (i.e. ) were obtained using luria hiveg agar (la) media followed by zobell's marine agar (zma) ( genera), arret and kirshbum agar (ak) ( genera) then streptomycetes isolation agar (str) ( genera). similarly, la was found to be the most efficient in capturing unique bacterial genus ( ) followed by zma ( ) then ak ( ) and str ( ) (fig. ) . thus, total species (supplementary figure s ) belonging to different genera (fig. ) and bacterial phyla i.e. proteobacteria, firmicutes, actinobacteria and bacteroidetes were isolated from bat guano. the most dominating bacterial genera obtained was enterobacter ( isolates) followed by enterococcus ( isolates) and escherichia ( isolates). three unique bacterial genera i.e. aquitalea, cedecea and pontoea were obtained from cw (cave stream water) sample but not obtained in any other samples whereas only one bacterial genera i.e. staphylococcus was obtained from bcs (bats' cave ceiling soil) sample (supplementary figure s ). literature survey for bacterial pathogens. as taxonomic affiliation of most of the isolates (~ %) was proteobacteria and majority of pathogens belong to phylum proteobacteria, we speculated that guano could contain a higher abundance of pathogenic bacteria. hence, a detailed literature survey was performed for all bacterial species obtained from r. leschenaultii guano, to identify their pathogenic potential. only bacteria belonging to risk group- or above were considered a pathogen. this survey revealed that only two bacteria belongs to rg (risk group) (table ) , although other bacterial species were also reported to have association with different opportunistic infections. to cause an infection successfully, bacteria must possess some virulence factors. in this study, e. coli was represented by more than % of total isolates. although, it is a common component of a healthy gut microbiome, many virulent strains of e. coli (pathotype) also exist. hence, e. coli isolates were assessed for their pathogenic potential by the identification of eight virulence genes (supplementary table s ). this assessment revealed . % of isolates to be ibea and hlya gene positive (indicative of extraintestinal pathogenic e. coli (expec)) and . % were east gene positive (indicative of enteroaggregative e. coli (eaec)). additionally, one isolate possessed the enterotoxigenic e. coli (etec) heat-stable enterotoxin-b (estii) gene. interestingly, one of the e. coli isolates was entero-hemorrhagic (ehec) scientific reports | : | doi: . /srep bearing shiga toxin-ii (stx ) gene which additionally co-harbored east gene. seven other cultivated isolates were found positive for ibea and east genes ( fig. ) while none of the e. coli isolates belonged to enteroinvasive (eiec) pathotype (ipah negative). on comparison with the previous studies on avian pathogenic strains of e. coli , a higher prevalence of the ibea gene was revealed in this study ( . % vs previous ~ %). nonetheless, e. coli strains isolated from human vagina and neonatal meningitis appears to have similar prevalence of ibea gene , . in present study the prevalence of east gene in e. coli strains was quite similar to the previous study ( . %) in weaned pigs with the diarrheal infections . thus, a comparable frequency of virulent genes was observed. ion torrent sequence analysis. further, to get an in-depth overview of total bacterial communities of r. leschenaultii guano, s rrna gene sequencing was performed using ion torrent pgm. the generated sequences were analyzed using qiime (quantitative insight into microbial ecology) package . the results indicated the presence of different bacterial phyla in bat guano which revealed the huge uncultivated bacterial communities present in the bat guano (fig. ) . samples g and g showed the abundance of proteobacteria (~ %) while g had the abundance of actinobacteria (~ %) and g and g had the abundance of firmicutes ( % and % respectively). the cave ground surface soil (cgs) sample showed the abundance of proteobacteria (~ %) followed by bacteroidetes (~ %) and actinobacteria (~ . %). further, a huge inter-individual variation in bacterial communities was also observed. the various indices of alpha diversity indicated the cgs to be the most diverse sample, whereas, among fresh guano samples g had maximum bacterial diversity (supplementary figures s and s ). further, computation of core microbiome from ion torrent sequences of fresh guano revealed the presence of five bacterial phyla i.e. proteobacteria, tenericutes, candidate division tm , firmicutes and actinobacteria but in different proportions (fig. , supplementary figure s ). bat guano microbiome comparison. two previous bat microbiome studies were compared with the present bat guano microbiome study. to accomplish this, fastq files were retrieved from sra (sequence read archive) ncbi, quality filtered and analyzed using qiime pipeline. the analysis revealed that these three bat microbiome studies are quite different in terms of composition and the relative proportion of bacterial communities. only three phyla i.e. chlamydiae (~ . %), proteobacteria (~ . %) and bacteroidetes (~ %) were represented in veikkolainen et al. study (location: finland; bat species: myotis daubentonii). on contrary, the study by de mandal et al. (location: india; bat species: not available) and present study (location: india; bat species: r. leschenaultii) comprised of and bacterial phyla predominated by actinobacteria (~ . %) and firmicutes (~ . %) respectively (fig. ). in an attempt to identify the core bacterial microbiome, at a broader taxonomic level, two bacterial phyla were identified in all three studies (supplementary figure s ) . as sample cgs of the present study and composite guano samples used in de mandal et al. study are representatives of decaying guano hence a comparison between them was performed. the beta diversity analysis using the jaccard distance revealed that the cgs and the composite guano contained different bacterial communities which in turn were different from the rest of fresh guano samples (fig. ). pathogen identification from ion torrent sequences. the presence of potential bacterial pathogens in culture-based study prompted us to assess ion torrent data for their presence. hence, ion torrent sequences were searched against the available human bacteria pathogen database . upon analysis, at % sequence identity cutoff and ≥ % query coverage (about . %) sequences showed identity to the well-known pathogens belonging bats are crucial and the integral part of healthy ecosystem, serving it in many ways such as chiropterophily, a mode of pollination exclusively carried by the bats. apart from their ecological services, the bio-fertilizers made from the bat guano serve as the rich source of nutrients for better crop production, farming and gardening. bats also has promising prospects for human health. 'draculin' (desmoteplase), a glycoprotein from the vampire bat's saliva, a natural anti-coagulant, is currently in trial to be used as the medication to treat patient of ischaemic stroke. it can reopen the clogged blood vessels so that the damage can be prevented hence, it can prove to be a life saver. despite grandness of bats their every aspect including their gut microbial communities need to be addressed. the present study has attempted to identify the bacterial communities present in rousettus leschenaultii's (a frugivorous bat species) guano using culture-based approach and has led to the identification of about a thousand bacteria belonging to more than a hundred different bacterial species. a majority of them have been isolated for the first time from bats. since all cultivated bacteria were identified using nearly full length s rrna gene sequencing, therefore, identity was ensured. additionally, ion torrent sequencing of s rrna gene provided information about the general bacterial communities with specific emphasis on pathogens among the total communities present in bat guano. our observation of bacterial count for the r. leschenaultii guano is in the range of . × - . × cfus/gram, which is higher than previously reported count i.e. × for the guano of myotis sp. of bats; possibly the use of fresh guano here as against the old and dried pellet used previously could be the reason . the reported bacterial count for the stomach and intestinal contents of different species of bats was in the range of - cfus , , indicating that the main differences, apart from the sample itself, could be because of inter-individual variation due to host species, diet, body size, specificity and geographical location . so far, most studies have used one or two bacteriological media to identify the bacterial communities associated with bats , , hence were confined. few of such studies have reported about , and bacterial species from , and species of bats respectively , , . in contrast, using four different bacteriological media, we were able to acquire four different bacterial phyla belonging to genera and different species, thus, signifying the importance of traditional approach in microbial communities' exploration. this fact is further strengthened by the presence of unique bacterial species not reported earlier from bats. interestingly, only a single genus of bacteria i.e. staphylococcus was found in bcs. therefore, the possibility of bcs contaminating the guano was excluded and assumed that all bacterial species isolated from bat guano are autochthonous community. such rock dwelling staphylococcus has been associated with manganese mobilization in basalt rocks , hence, its high abundance in bcs is explained. while guano shared some bacterial genera with those observed in cw and cgs; the possibilities of these contaminating the guano was taken care during the sampling. the next generation sequencing platforms are rapidly changing the ways of characterizing microbial community from various sources. metagenomic studies using these sequencing technologies have contributed enormously to our understanding of structure and functioning of a given system. accordingly, using ion torrent sequencing, it was observed that two guano samples has more than % sequences representing the proteobacteria, one showed the higher abundance of actinobacteria and another one has the firmicutes in abundance. previous studies have demonstrated the huge variation in gut bacterial communities even in genetically similar human individuals . therefore, the major reason appears to be the host specificity, individual's diet, health and physiological state . recently, bat guano microbiome was reported from the composite guano collected from cave floor , hence, no information was available about the host bats. conversely, in addition to multiple guano microbiomes, we identified the bat to be rousettus leschenaultii, a most dominating frugivorous bat in robber's cave . additionally, alpha diversity and rarefaction analysis indicated the significant microbial enrichment in cgs sample indicating that the decaying process may have led to the increased bacterial diversity . the decaying or the disintegration process is affected by the various physical and environmental factors. additionally, the biogeochemical processes crucial for the recycling of the organic and inorganic matters are primarily carried out by the microorganism which causes the change in ph (due to the release of ammonia) and nutritional contents which further allow the growth of diverse bacteria . hence, the enrichment of bacterial communities in the decaying guano is explained. further, the underlying soil may also cause an increase in the microbial load. the comparison between composite guano and samples from this study revealed that both cgs and composite guano are quite dissimilar in microbial composition from each other as well as rest of the fresh guano samples which further indicates the bacterial communities of decaying guano is rather different from the fresh guano. we speculate that the differences in two different decaying guano could be because of their origin, host bats' species, duration of the decaying process and other physicochemical properties leading to the different trajectories of development of microbial communities. at the broader taxonomic assignments, we were able to detect bacterial phyla as against only phyla in composite guano samples . while this holds true, the core microbiome contained only five different phyla: actinobacteria, candidate division tm , firmicutes, proteobacteria and tenericutes, which indicates probably all fresh guano of r. leschenaultii are characterized by the presence of these five phyla. we believe that the other bacterial phyla that we were able to recover could be derived from the consumed food material, as bats have a huge diet which remains only transiently in the gastrointestinal tract . further, on a comparison between ion torrent sequencing results and culture based finding, it was observed that three major bacterial phyla i.e. actinobacteria, firmicutes and proteobacteria were shared confirming the stable nature of these bacterial phyla in bat gut environment. a higher abundance of actinobacteria was observed in ion torrent sequence data, whereas, the higher relative proportion of firmicutes and proteobacteria was obtained in culture, suggesting fewer actinobacteria were cultivable on the employed media. similarly, due to the stringent growth requirements, tenericutes could not be obtained in culture even though it was one of the members of core communities. furthermore, an absence of bacteroidetes in the core microbiome and its presence in culture indicated the individual variations of guano samples. comparison of the present study with the two previously published microbiome studies revealed that this study reported a higher number of bacterial phyla, probably the fresh guano samples collected for the study appears to be the prime reason. further, in an attempt to find the core microbiome from different microbiome studies indicated that only two phyla i.e. proteobacteria and bacteroidetes are present commonly in three compared studies. the other two recent microbiome studies showed the strong influence of host phylogeny, dietary pattern, physiology and geography on the gut microbial communities of bats , . earlier bats have been suspected as the reservoir of the human bacterial pathogens and their guano was considered important in pathogen dissemination . here, two bacterial species i.e. escherichia coli (risk group-i/ii) and staphylococcus aureus (risk group-ii) were cultivated. additionally, bacterial species obtained from guano have been reported to cause various human infections. except acinetobacter johnsonii (cause fish infection) and staphylococcus lentus (cause mastitis in goats) others were mainly found involved in the sepsis, urinary tract infections (utis) and other infections especially in immune compromised individuals. in the present study, a majority of the cultivated bacteria belong to the family enterobacteriaceae and most of these isolates were identified as escherichia coli ( , ~ . %), a well-known commensal gut inhabitant and opportunistic rg-i/ii pathogen . hence, a high percent of expec and eaggec pathotype in r. leschenaultii guano could be a serious health concern. additionally, shiga toxin producing strain has been linked with the severe outbreak in the germany, leading to more than cases and more than deaths . these results are quite staggering, indicating that the e. coli recovered from r. leschenaultii guano may prove to be pathogenic. few studies have indicated the presence of virulence genes in wild animals , . cabal et al. compared the virulence gene profile of e. coli isolated from cattle, swine and broiler. the results indicated the presence of stx and stx gene (from both o :h and non-o :h e. coli serotypes) in cattle and swine . similarly, post weaning diarrhea in piglets is a common problem in piggery farms and the major reason appears to the epec/etec carrying different virulence genes especially the heat labile/stable toxins (sta and stb) , . recently, even the healthy dairy cows have been reported to carry virulent genes (stx , st and lt) in e. coli isolated from their dung . additionally, the humans are reported to be the prime reservoirs for eaec, epec and eiec , , though they remain healthy. hence, commensals like e. coli may carry virulence genes but often do not cause the infections as the appropriate combination of virulent genes, required to cause the infection, is not available . likewise, merely the presence of rg- bacteria from the host does not indicate it to be the pathogen as all strains of a species may not be the pathogen. nevertheless, a possibility of gradually acquiring the additional virulence genes, required to cause a successful infection cannot be ignored. in the light of presence of various pathotypes of e. coli, it is essential to discuss other potentially pathogenic bacteria as well. in the discussion that follows numbers in the parentheses (in bold) indicate an abundance of the said bacterial species. the other isolated member of genus escherichia is escherichia fergusonii ( ) , a close relative of e. coli which has been isolated from the feces of many warm blooded animals. it has also been recovered from the wound infections, utis, diarrhea etc. and reported to cause the bacteremia in diabetic patient . the other dominating genera cultivated was enterobacter e.g. includes enterobacter asburiae ( ) , enterobacter ludwigii ( ) , enterobacter cloacae ( ) and members of enterococci i.e. enterococcus faecalis ( ) and enterococcus hirae ( ) which causes various infections especially in immunocompromised individuals [ ] [ ] [ ] [ ] . notably, e. faecalis is among the most common species isolated from human clinical samples. both citrobacter koseri ( ) and citrobacter freundii ( ) are the opportunistic pathogens causing neonatal infections but the neonatal meningitis caused due to c. koseri have a high mortality rate . similarly, serratia marcescens ( ) is a common cause of nosocomial, urinary tract and wound infections , . relatively fewer gram-positive bacteria were obtained from guano. the main genus among these is staphylococcus. it included staphylococcus aureus (a rg- organism), staphylococcus nepalensis ( ) and staphylococcus lentus ( ) , which are well-known to cause various infections in humans as well as in other animals , . therefore, it may pose a threat to the herds of sheep and goats grazing nearby bats' roosting sites. similarly, potential bacterial pathogens were also searched in ion torrent data using the human bacteria pathogen database. this database was created from the 'human pathogenic bacteria virulence factor database' and only the bacterial species whose proteins were experimentally validated were considered for its creation . although, the ion torrent generates the sequences of shorter read length (here ~ bp), the use of v region for the identification ensured maximum coverage of diversity (~ - %) . in addition, the only sequence which had high query coverage (≥ %) and high similarity value (> %) during the blast search against the database was considered for the analysis . hence, the possibility of the presence of these pathogens may not be ignored. pathogens identified from ion torrent sequencing includes pseudomonas aeruginosa, and clostridium perfringens etc. p. aeruginosa ( ) is a gram-negative pathogen responsible for several nosocomial infections and opportunistic infections , . the bacterial species salmonella enterica ( ) and shigella flexneri ( ) causes gastroenteritis (food poisoning) and shigellosis , respectively. these can be transmitted by the direct contact, fecal or oral route. hence, the consumption of food contaminated with bat guano should be avoided. the other species found scientific reports | : | doi: . /srep i.e. streptococcus pneumoniae ( ), enterococcus faecalis ( ), staphylococcus aureus ( ) which may cause streptococcal pneumonia, meningitis, bacteremia, infectious lesions, neonatal infections and septicemia [ ] [ ] [ ] . the important observation was the presence of sequences affiliated to mycobacterium tuberculosis ( ) and corynebacterium diphtheriae ( ) . mycobacterium tuberculosis is an airborne disease and spread through the droplet nuclei. similarly, corynebacterium diphtheriae, a causative agent of diphtheria, may cause skin infections and septicemia by the droplets, secretions or direct contact . hence the proper caution should be taken when entering a cave having bats' roosting site. additionally, bartonella henselae ( ) , one of the causative agents of bartonellosis (cat scratch disease), has been suspected earlier from bats which can be transmitted to humans by accidental bite or scratches. additionally, it can also be transmitted by the ectoparasites harbored by the infected animals . some sequences were also found affiliated to brucella melitensis ( ) which is the most pathogenic brucella species to humans . it principally affects the goats and sheep, hence, the grazing goats and sheep which comes in contact with bats' guano may get infected. hence, these may cause zoonotic infections in humans by direct contact, aerosol inhalation or indirectly by consumption of improperly cooked food products from diseased animals. in conclusion, the present study revealed that each gram of r. leschenaultii's guano contains millions of bacteria, belonging to hundreds of different bacterial species, some of which can be potentially pathogenic to humans. additionally, computed core microbiome revealed five bacterial phyla, three of them are found in the culture-based study. further, a fairly large inter-individual variation in microbiome composition of guano was observed indicating the individual specificity. the virulence gene profiling of cultivated e. coli strains from guano revealed the presence of many virulent genes, though it may not be declared pathogens. this study reports the largest culture-based study from r. leschenaultii bat guano to identify the cultivable bacterial diversity and potential human pathogenic bacteria. thus it is strongly advised that the humans should not interfere with the wild life otherwise it may cause huge loss to the environment and health related issues to the humans. sampling site and sample collection. guano samples were collected from the 'robbers cave' ( ° ′ "n and ° ′ "e) situated in the basalt rock structure of western ghats, of maharashtra state, india. collection plates were placed on cave ground surface under the roosting bats' colony and were left for about hours. after hours individual guano pellets were observed and collected separately. thus, a total of guano pallets were collected and kept at °c for transportation to the laboratory for further processing. only guano samples with sufficient quantity were used for the bacterial isolation. other potentially interfering and guano contaminating factors were also included in the study e.g. cave water (cw, from a stream which flows from a side of the cave, cave ceiling soil (bcs, which continuously fall on the ground and hence in guano pellets) and cave ground surface soil (cgs). sample processing. about the half quantity of all selected samples was suspended in ml of water and mixed thoroughly. the supernatant was serially diluted up to − followed by plating of dilution − in different media plates (dilutions − and − were used for la media) and incubation for - hours at °c incubators. post incubation plates were observed and total colony forming units (cfus) were counted. then total viable counts were calculated by considering the number of cfus observed, dilution factor, the volume of supernatant (inoculum) and the weight of guano used. subsequently, about random colonies of guano sample from each media were sub-cultured in their respective medium. post-incubation a loop-full of each colony was suspended in te (tris-ethylenediamine tetra acetate) buffer (ph . ) to use further for dna isolation. the remaining guano sample was used for the total bacterial community dna extraction for ion torrent sequencing. bacterial dna isolation and species identification. for bacterial identification, dna of pure cultures was extracted using purelink ® pro genomic dna purification kit (invitrogen, inc. usa). pcr amplification of s rrna gene was performed in triplicates using x pcr buffer, . mmol/l of dntps, . units of taq polymerase (bangalore genei) and picomols of each primers f ( ′-aga gtttgatcctggctcag - ′) and r ( ′-cggttaccttgttacgactt- ′). pcr parameters include initial denaturation at °c for min followed by min at °c denaturation, °c annealing and extension at °c for cycles followed by final extension at °c for minutes then incubation at °c. pcr amplified products were gel checked for positive products. the positive products were purified using % peg-nacl (polyethylene glycol-nacl). the purified products were processed and sequenced using abi xl dna analyzer (applied biosystems, usa). to obtain nearly full-length sequence ( . kb) of bacterial s rrna gene, internal primers, f ( ′-gtagcggtgaaatgcgtaga- ′) and r ( ′-ccgtcaattcmtttgagttt- ′) were also used. the sequences were concatenated using chromaspro v . (http://www.technelysium.com.au/chromaspro.html), followed by blast analysis at ncbi. a blast hit, with sequence similarity ≥ % and query coverage ≥ is considered as the species of the isolate. identification of bacterial pathogens. only bacterial species belonging to risk group- or above were considered pathogen in present study (nih guidelines; http://osp.od.nih.gov/sites/default/files/nih_guidelines. html#_to c ). table s ) were used to identify the five different pathotype of the e. coli . the dna extracted from the e. coli isolates was used to amplify and check the pathotype of e. coli. the pcr was performed using x pcr buffer, . mmol/l of dntps, . units of taq polymerase (thermo scientific inc. usa) and - picomols of respective primers. pcr parameters include initial denaturation at °c for min followed by min at °c denaturation, different annealing temperatures for seconds and extension at °c for minutes for cycles followed by a final extension at °c for minutes then incubation at °c. bat guano community dna extraction and pcr amplification of s rrna gene. total community dna extracted from bat guano using stool dna isolation kit (qiagen, the netherlands), was used for the ion torrent analysis. the extracted dna was quantified using nanodrop (nanodrop, thermo scientific, usa), followed by pcr amplification of v region of s rrna using hi-fidelity amplitaq gold (invitrogen inc., usa) and eubacterial universal primers f ( ′-cctacgggaggcagcag- ′) and r ( ′-attaccgcggctgctgg- ′). resulting pcr products were purified using agencourt ampure xp dna purification beads (beckman coulter, usa) which were then subjected to end repair. the blunt ended products were used as a substrate for sample-specific barcode and adapter ligation reaction as per the manufacturer's instructions. prior to the sequencing, all amplicons were assessed for size distribution and molar concentrations using bioanalyzer (agilent technologies, usa). the concentration of all the amplicons was adjusted to lowest dna concentration and subsequently, amplicons were pooled in an equimolar ratio and diluted so as to obtain the pooled amplicons of around pm. the pooled amplicons were attached to ion sphere particles (isps) and used for emulsion pcr using ionxpress template- kit using iononetouch system. next, template-positive isps were enriched using iononetouch es system. the enriched isps were then loaded onto chip and sequencing was performed on ion torrent personal genome machine (life technologies, usa) for cycles. ion torrent data analysis. barcode specific fastq files were processed using mothur pipeline to obtain fasta and quality file. these two types of the files were quality filtered using following conditions: size - bp, homo-polymer max. , ambiguity max , and average quality score . this way we were able to obtain good quality sequences. all these reads were pooled into a single fasta file and analyzed using qiime (quantitative insight into microbial ecology) package. briefly, otus picking was done using open reference approach at % sequence similarity cutoff. a representative sequence of each otu was picked up and lowest possible taxonomic rank was assigned to each of them by using rdp classifier (v . ) and silva database (silva_ ) as reference. in order to assess the relationship between sequencing depth and discovery of new otus, rarefaction analysis was performed. further, diversity measurements were made using alpha diversity indices such as shannon, simpson and chao . for computing core otus, cgs sample was excluded and a core otu was defined as an otu present in all ( % of) the samples. microbiome data was retrieved from sra (sequence read archives) ncbi in fastq format to compare present bat guano microbiome studies with the previously published microbiome studies. thus including ours, a total three studies i.e. de mandal et al. , veikkolainen et al. were compared. all the files were quality filtered using mothur pipeline and only good quality sequence having a length more than bases and homopolymers allowed no more than bases and having (zero) ambiguity were used in the comparison. later all the filtered, good quality sequences were compiled into a single fasta file and processed using qiime pipeline. a closed reference approach using silva_ _database as reference was used to pick otus so that different studies which has utilized the different region of s rrna gene can be compared. pathogen identification from ion torrent sequencing data. bacterial pathogen database was used for the search of the bacterial pathogens from ion torrent sequencing reads. all the s rrna gene sequences of bat guano (excluding cgs) were compiled into a single fasta file (containing sequences) and searched against the above mentioned database for the sequence similarity using ncbi-blast- . . + algorithm . thus, every sequence was assigned the closest hit. only blast hit showing query coverage ≥ % and maximum identity with maximum score value and lowest e-value was considered. the number of sequences having blast hit with ≥ % identity were short-listed and calculated the proportion of sequences affiliated to the bacterial pathogens. statistical analysis. statistical analysis was performed in graphpad prism (v . ) and the venn diagram was prepared in the venny . 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the new bacterial taxonomy the silva ribosomal rna gene database project: improved data processing and web-based tools basic local alignment search tool escherichia coli endocarditis: seven new cases in adults and review of the literature virulence factors in escherichia coli urinary tract infection staphylococcus aureus infections s.b. and y.s.s. designed the study. s.b. did the sampling from the field, bacteria isolation and identification, all pre-sequencing work for the ion torrent sequencing and virulence gene profiling. s.s.b. and m.v.s. assisted in ion torrent sequencing. m.v.s. assisted in initial ion torrent data analysis. s.b. and s.s.b. performed the data analysis and manuscript writing. s.p. assisted in the conceptualization of the study. all authors commented on the final draft. key: cord- -oxlv d authors: o’boyle, nicky; berry, catherine c.; davies, robert l. title: differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of mannheimia haemolytica date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: oxlv d mannheimia haemolytica is the primary bacterial species associated with respiratory disease of ruminants. a lack of cost-effective, reproducible models for the study of m. haemolytica pathogenesis has hampered efforts to better understand the molecular interactions governing disease progression. we employed a highly optimised ovine tracheal epithelial cell model to assess the colonisation of various pathogenic and non-pathogenic m. haemolytica isolates of bovine and ovine origin. comparison of single representative pathogenic and non-pathogenic ovine isolates over ten time-points by enumeration of tissue-associated bacteria, histology, immunofluorescence microscopy and scanning electron microscopy revealed temporal differences in adhesion, proliferation, bacterial cell physiology and host cell responses. comparison of eight isolates of bovine and ovine origin at three key time-points ( h, h and h), revealed that colonisation was not strictly pathogen or serotype specific, with isolates of serotype a , a , a and a being capable of colonising the cell layer regardless of host species or disease status of the host. a trend towards increased proliferative capacity by pathogenic ovine isolates was observed. these results indicate that the host-specific nature of m. haemolytica infection may result at least partially from the colonisation-related processes of adhesion, invasion and proliferation at the epithelial interface. heavily colonised by serotype a or a isolates [ ] [ ] [ ] [ ] . these observations have led to the conclusion that serotype a and a strains are pathogenic in cattle whereas serotype a strains are considered non-pathogenic and a commensal of the normal respiratory flora , , . by contrast sheep displaying respiratory disease become heavily colonised by m. haemolytica serotype a , while diverse serotypes such as a , a , a and a are more rarely associated with disease [ ] [ ] [ ] . however, it is important to appreciate that genetically distinct sub-populations of m. haemolytica isolates of serotypes a , a and a are associated with disease and carriage in cattle and sheep and that capsular serotype is not, by itself, an indicator of host-specificity and virulence . nevertheless, these observations indicate interesting host-specific and strain-specific virulence traits in m. haemolytica. serotyping of m. haemolytica is dependent upon variation in the polysaccharide capsule that is thought to play a role in adhesion to host cells and evasion of phagocytosis by macrophages and neutrophils . surface proteins such as ompa and lpp have also been shown to play a role in initial colonisation . transferrin binding proteins tbpa and tbpb and a kda filamentous haemagglutinin have also been proposed to play a role in this process based on surface expression and homology with adhesins in other species, although their roles in m. haemolytica adhesion have not been experimentally confirmed. in addition to colonisation and proliferation, excessive stimulation of the immune system is a hallmark of m. haemolytica pathogenesis. lipopolysaccharide (lps) causes the activation of proinflammatory cytokine secretion from leukocytes , while secretion of leukotoxin (encoded by lkta)-widely regarded as the primary virulence factor of m. haemolytica-leads to degranulation and apoptosis of neutrophils, greatly contributing to the inflammatory necrosis associated with ruminant respiratory disease , . in recent years, there has been a growing interest in the development of cost-effective, biologically relevant models to gain a more complete understanding of the molecular mechanisms underlying these pathogenic processes and the factors determining host-specificity of m. haemolytica. with this in mind, our group have developed highly optimised in vitro tissue engineered models of both cattle and sheep respiratory epithelia [ ] [ ] [ ] [ ] . by optimising medium, growth factor concentrations, substrate pore density, atmospheric gas composition and period of differentiation, these models best reflect the thickness, ciliation level, mucus production and cellular composition of their source of isolation. these models provide an excellent platform for the study of initial interactions between m. haemolytica and the respiratory epithelium. we have shown that differentiated bovine bronchial epithelial cells (bbecs) support the colonisation of pathogenic m. haemolytica . in this study, we aimed to assess whether differentiated ovine tracheal epithelial cells (otecs) displayed similar pathogen-specific colonisation. through the assessment of a broader collection of pathogenic and non-pathogenic isolates of known genetic relatedness and host origin, we also assessed whether the ovine model displayed selectivity towards bovine and ovine isolates. surprisingly, we did not observe a strict pathotype or host-origin selectivity using this model, but the physiology of both tissue-adherent bacteria and the colonised host epithelia were different comparing pathogenic and non-pathogenic m. haemolytica isolates. these findings have implications for future use of the model for the study of commensal respiratory species and agents of the respiratory disease complex. in order to allow the study of m. haemolytica disease progression in vitro, it was important that our model support the colonisation of disease-causing strains. we first assessed the colonisation of differentiated otecs by ph and ph , which represent the major ovine disease-associated serotype (a ) and a serotype rarely associated with disease in sheep (a ), respectively (table ) . strain ph was isolated from the lungs of a sheep with pneumonic pasteurellosis, while ph was isolated from the nasopharynx of a healthy sheep. both isolates were capable of colonising the differentiated otec layer with varying dynamics of growth on the tissue surface. in general, there was a trend of increased initial adhesion and early colonisation ( . , and h) by ph , with higher mean levels of adhesion being observed for ph at all time-points after h. however, it should be noted that a statistically significant difference between the strains was only observed at h (fig. a) . table . properties of bacterial strains. a reference isolates identify et strain groups, and associated characteristics, to which isolates belong as defined in reference davies et al. . b et, electrophoretic type as defined in reference davies et al. www.nature.com/scientificreports/ colonisation levels equating to , % of the inoculum were observed for ph at h indicative of extensive proliferation. this was followed by a reduction to % by h as the excessive bacterial proliferation led to sloughing of the cell layer during washing. by contrast, at h, colonisation by ph reached only % of the inoculum indicating comparatively poor proliferation. there was a reasonably high level of consistency between experiments with overlap being observed in colonisation efficiency between tissues isolated from independent animals at the majority of time-points (fig. s ) , indicating that the model provides a high level of reproducibility. extensive tissue disruption was observed by haematoxylin and eosin (h&e) staining from h with ph and from h with ph (fig. b) . regions of nuclear condensation and cell-sloughing were evident at these time-points. while bacteria were difficult to visualise in h&e-stained sections, they could be readily observed at h by immunohistochemistry using an anti-ompa ph antibody (fig. c ). ompa is a good candidate for detection of multiple strains due to its high conservation in m. haemolytica . strikingly, at h post-infection in some sections ph displayed large regions of bacterial growth greater than μm deep and μm wide (fig. b) , consistent with microcolony formation. ph on the other hand did not form microcolonies and instead appeared to completely disrupt the tissue layer by this time-point, leading to flooding of the apical chamber and growth of bacteria on the cell culture insert, while ph -infected cell layers remained intact. scanning electron microscopy ( fig. ) and immunofluorescence microscopy ( www.nature.com/scientificreports/ amorphous material reflective of exopolysaccharide production in bacterial biofilms ( fig. a) . the composition of this material was not confirmed experimentally. microcolonies of ph were remarkably discrete and well-isolated with some inserts harbouring only two/three microcolonies. as such, in spite of consistently high colonisation levels ( fig. a) , histological sections occasionally appeared negative for ph colonisation, even at later stages of infection. the tissue surrounding ph microcolonies appeared well-ciliated and healthy unlike that surrounding regions of invasion by ph . having observed that differentiated otecs could support the colonisation of both pathogenic and nonpathogenic ovine isolates of m. haemolytica, we next assessed whether isolates associated with disease in cattle could also colonise the tissues. bovine pathogenic serotype a and a isolates (ph and ph , respectively) and bovine non-pathogenic serotype a isolates (ph , ph ) were tested for colonisation of otecs alongside ovine disease-causing serotype a isolates (ph , ph ) and ovine serotype a isolates (ph , ph ) that are rarely associated with disease, in order to robustly assess the strain-specificity of the infection model (table ; fig. ). these isolates represent part of our laboratory collection, obtained through diagnostic screening over several years, and having been investigated to establish the clonal, serotype and host-specific nature of m. haemolytica infections , [ ] [ ] [ ] . in selecting these eight isolates, it was intended that host species and disease status, together with isolate pathogenicity and genetic relatedness/serotype could all be interrogated for a role in otec colonisation. all isolates colonised the differentiated otecs at h, h and h with between . (ph , h) and . % (ph , h) of the inoculum being recovered (fig. a ). colonisation levels were statistically equivalent for all strains at each time-point except for ph and ph at h, which displayed significantly higher initial adherence/colonisation efficiency than the non-pathogenic bovine isolates ph and ph and lower initial adhesion than ph (ph was also found to have significantly higher initial adhesion than ph at h), and ph at h which displayed significantly higher colonisation efficiency than ph , ph , ph and ph (fig. a ). the following trend of decreasing colonisation efficiency at h was observed: ovine pathogenic (ph = %; ph = %) > bovine pathogenic (ph = %; ph = %) > ovine non-pathogenic (ph = %; ph = %). the data obtained for the bovine non-pathogenic isolates (ph = %; ph = %) was more variable and as such, the testing of further bovine non-pathogenic isolates would be www.nature.com/scientificreports/ required in order to discriminate where these isolates fit on this trend. as with the more extensive time-course (fig. s ), a good deal of overlap was observed between colonisation levels on otec cultures derived from independent animals (fig. s ), highlighting reproducibility in the model. immunofluorescence microscopy revealed that bovine pathogenic strains produced invasive foci of infection similar to those induced by ph with localised tissue damage and cellular rounding evidenced by cytoskeletal reorganisation and condensation in the otecs adjacent to these foci (fig. b) . in healthy tissues, β-tubulin staining allows for visualisation of the apical surface-localised cilia, while f-actin staining allows for visualisation of the underlying epithelium . this pattern of cytoskeletal organisation and cellular morphology becomes dramatically disrupted in the regions surrounding invasive foci of pathogenic strains (fig. b ). by contrast, tissues infected with non-pathogenic isolates of bovine and ovine origin showed disperse regions of bacterial immunostaining, predominantly co-localised with cilia (fig. b) . this was surprising given that these isolates colonise efficiently at h, however it should be noted that the invasive foci observed with ph , ph and ph were few in number and as such, the greatest contribution to the cell-associated bacterial numbers may be the discrete aggregates associated with the cilia at these time-points. indeed, ph showed no foci of infection in these experiments despite being of very close genetic relatedness to ph . these results demonstrate that while all m. haemolytica isolates are capable of colonising differentiated otecs at least to some extent, there are differences in the cellular patterns of colonisation and some ovine strains may display enhanced initial adhesion or long-term persistence/proliferation within the model. during gaseous exchange, the mammalian conducting airways are repeatedly exposed to potentially harmful agents. in the case of intensive farming of livestock, this is particularly problematic as infectious respiratory pathogens can circulate rapidly through populations . one of the primary functions of the respiratory epithelium is to function as an innate immune barrier to invading pathogens. the complex cellular architecture of this tissue plays a role in such defences by physically occluding passage of bacteria and viruses , entrapping particles www.nature.com/scientificreports/ in goblet cell-secreted mucus and clearing mucus globules via the coordinated beating of ciliated epithelial cells . submerged monolayer cell cultures fail to recapitulate these fundamental processes , , , and therefore are poorly suited to the study of colonisation by pathogenic species. we recently developed well-differentiated primary otecs for use in modelling interactions of relevant pathogens with the sheep respiratory epithelium in vitro , . the model forms a heavily ciliated, mucus-producing cell layer with similar physiology to that of the native respiratory tissue. in tandem, we have developed a bbec culture model which well represents the respiratory epithelium of cattle , . the sero-specific nature of severe cases of respiratory disease by m. haemolytica , , coupled with a suggested role for the polysaccharide capsule (on which the serotyping classification is based) in cell adhesion , indicates that isolates may be specifically adapted to colonising the airway tissues of discrete hosts. for example, sheep displaying severe respiratory disease are often colonised heavily by m. haemolytica serotype a but rarely by serotype a , . however, capsular polysaccharide type by itself is not likely to be the only factor associated with host-specificity and virulence. indeed, we have previously demonstrated specific associations of lkta , ompa and tbpa and tbpb alleles with m. haemolytica lineages or clones associated with either cattle or sheep. we previously put forward evidence to support the hypothesis that ompa, a major surface-exposed outer membrane protein, is under strong selective pressure from the host species and plays an important role in hostadaptation . it was subsequently shown that ompa plays a role in adherence of m. haemolytica to bovine airway following permeabilisation, bacteria were immunostained with an anti-m. haemolytica whole-cell antibody (green). cilia and cytoskeletal microtubules were stained with anti-β-tubulin (magenta), host cell actin was stained with phalloidin alexa- (red) and nuclei were stained with dapi (blue). samples were mounted and viewed by wide-field microscopy. in healthy regions of tissue, β-tubulin is concentrated in the apical cell surface-localised cilia while f-actin staining allows visualisation of the underlying epithelium. upon formation of invasive foci of infection with pathogenic strains, this localisation and normal cellular morphology becomes disrupted. by comparison, with non-pathogenic strains and ph only sporadic aggregates of cilia-attached bacteria were observed. www.nature.com/scientificreports/ epithelial cells . thus, in vitro differentiated cattle and sheep airway epithelial cell culture models represent an excellent opportunity to study the molecular basis of such host-specificity. on comparison of m. haemolytica ph (a pathogenic bovine serotype a isolate) with ph (a non-pathogenic serotype a isolate) colonisation on differentiated bbecs, it was observed that only ph was capable of proliferation beyond h post-infection, whereas ph was completely cleared from the majority of bbec cultures . we hypothesised that this may have been due to susceptibility of ph to bbec-produced antimicrobial compounds such as defensins and cathelicidins, which are well-defined innate defence mechanisms of the respiratory epithelial cells , . pathogenic isolate ph invaded through the apical surface of the bbec layer, proliferated intracellularly and paracellularly, resulting in a drop in trans-epithelial electrical resistance and a break down in the epithelial barrier. by contrast, in this study both ph (a pathogenic ovine serotype a isolate) and ph (a non-pathogenic ovine serotype a isolate) were capable of colonising differentiated otecs throughout the duration of the time-course, albeit with higher levels being observed for ph at time-points post h (fig. ) . viable counts of ph were recovered from infected otecs at all time-points ( fig. ) indicating that antimicrobial-induced killing of this commensal isolate did not occur. pathogenic isolate ph caused the formation of similar foci of infection in otecs to those observed with ph in bbecs, with invasive proliferation and tissue disruption being observed (figs. , , ) . ph produced a previously unseen phenotype during colonisation, with the formation of large microcolonies within the infected tissue (figs. b, a) . the superficial differences in the pattern of adherence between pathogenic and non-pathogenic isolates observed here may reflect important attributes of the behaviour of these strains in vivo. for example, pathogenic isolates such as ph may engage in unchecked replication, leading to extensive tissue disruption, while non-pathogenic isolates form discrete microcolonies with minimal disruption to the surrounding epithelium. in general, across the eight strains tested, a trend towards increasing colonisation by pathogens, particularly ovine pathogens, was observed (fig. a ). this trend is likely reflective of the enhanced proliferative capacity of pathogenic m. haemolytica isolates. however, there were marked differences in the colonisation efficiencies of the bovine serotype a isolates ph ( %) and ph ( %) at h. although these isolates are representative of the same electrophoretic type (et) and would be expected to perform similarly, there may be subtle differences between them (they do have slightly different omp-profiles-see table ) which could account for these observations. klima et al. suggested the existence of a distinct subset of serotype a strains that are adapted for proliferation in immuno-compromised bovine hosts and the observed differences in colonisation between these two isolates might reflect such strain variation. the analysis of further strains would help resolve this but, importantly and as we have demonstrated, the differentiated airway epithelial cell model described herein has the capability of discriminating between such strains without the need for in vivo challenge experiments. it should be noted that sloughing of tissue from otecs heavily infected with pathogenic m. haemolytica at h could result in loss of enumerable bacterial cells, thereby reducing the difference in colonisation efficiency between pathogenic and non-pathogenic isolates and blurring the significance of differences between these groups. the stark contrast between the colonisation efficiency of the non-pathogenic isolates of bovine (ph ) and ovine (ph ) origin on tissues from their respective animals of isolation is interesting. it may reflect variability in the colonisation factor repertoire of the isolates themselves, or receptor variation in the differentiated bbecs and otecs. we chose to use bronchial tissue of bovine origin due to enhanced differentiation over bovine tracheal tissue in our hands and tracheal tissue of ovine origin due to difficulty encountered in obtaining sufficient bronchial cell numbers for large scale experimentation (without the need for extensive passage, which might compromise differentiation capacity). therefore, it is possible that non-pathogenic isolates are specifically capable of colonising upper airway epithelial cells in vitro. a four-way comparison of upper (tracheal/nasal) and lower (bronchial/bronchiolar) airway tissues would prove useful in dissecting these potential tissue tropisms. the possession of models which allow for the study of commensal interactions as well as pathogenic interactions with m. haemolytica will prove useful in uncovering the mechanisms by which m. haemolytica switches between these two important lifestyles. cattle and sheep display susceptibility to discrete clonal groups of m. haemolytica . we assessed whether interactions with differentiated otecs could reflect this species-specificity by analysing colonisation of pathogenic and non-pathogenic isolates of both bovine and ovine origin. all of the isolates tested were able to colonise and proliferate within the differentiated otec layer (fig. ) . it is therefore apparent that the host-specificity of m. haemolytica does not arise at the level of colonisation (at least within differentiated otecs). as such, there appears to be an inherent lack of specificity associated with colonisation of otecs in direct contrast to the selectivity we have observed for bbecs , highlighting the potential for variation in the outcome of infection experiments performed using tissue culture models of different origins. it has been established that leukotoxin is the primary virulence factor of m. haemolytica contributing greatly to the fibrinous necrotising pneumonia observed in infected animals. as leukotoxin binding is highly species-specific and cattle and sheep isolates tend to encode different lkta alleles , it is possible that host-specificity occurs downstream of colonisation and is mediated by virulence factors that are not involved in attachment. however, the possibility of more species and pathotype specific interactions occurring exclusively with tissues from deeper in the respiratory tract should not be excluded. finally, the experiments conducted herein involve pure culture infections with perhaps the most important pathogen of the bovine respiratory disease complex, m. haemolytica. however, we are currently exploring how further levels of complexity in the modelling of this disease may advance our understanding. for example, three individual viral components of the respiratory disease complex have been shown to replicate efficiently within differentiated bbecs . we are interested in how such infections might affect the outcome of a subsequent infection with m. haemolytica. as mentioned previously, the progression of respiratory disease in ruminants is not exclusively dependent on epithelial interactions but also interactions with immune cells. the addition scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ of neutrophils or macrophages to our infection model would allow for analysis of resistance to phagocytosis and importantly cytolysis and degranulation events stimulated by leukotoxin. overall, we are optimistic that continued study with these models will lead to a significant enhancement of our understanding of ruminant respiratory disease at a molecular level. isolates of bovine and ovine origin and known capsular serotypes were chosen for inclusion in this study. the isolates were selected to represent specific ets which reflected their genetic relatedness . bovine pathogenic isolates were represented by serotype a (ph ) and a (ph ) isolates of et that had been recovered from the lungs of pneumonic cattle; bovine non-pathogenic isolates were represented by serotype a (ph and ph ) isolates of et that had been recovered from the nasopharynxes of healthy animals. ovine pathogenic isolates were represented by serotype a (ph and ph ) isolates of et that had been recovered from the lungs of pneumonic sheep; ovine non-pathogenic isolates were represented by serotype a (ph and ph ) isolates of et , of which ph had been recovered from the nasopharynx of a healthy lamb. most of these strains have been characterised with respect to their outer membrane protein (omp)-and lipopolysaccharide (lps)-types and their lkta, lktcabd, ompa and tbpba allele types [ ] [ ] [ ] . properties of the strains are shown in table . bacteria were routinely cultured on brain heart infusion (bhi) agar containing % (v/v) defibrinated sheep's blood. overnight cultures were prepared by inoculating several colonies in bhi and culturing at °c, rpm for approximately h. all reagents were purchased from sigma-aldrich unless otherwise specified. cultures of differentiated otecs were prepared as previously described , . briefly, otecs were extracted from tracheal epithelia using protease xiv from streptomyces griseus, at °c overnight. digestion was halted by the addition of foetal calf serum (fcs) and loosely bound epithelial cells were removed by vigorous washing. the cells were strained through a μm strainer, centrifuged at × g for min and washed with serum-containing growth medium (sgm), which comprised , μg ml − insulin, ng ml − hydrocortisone, ng ml − epinephrine and μg ml − transferrin) by feeding apically and basally with a : mix of aegm/ali medium approximately half-way through the submerged growth phase. after Ω × cm teer was observed, the cells were washed with phosphate buffered saline (pbs) and fed from the basal compartment only with ali medium. feeding and washing were carried out every - days throughout submerged and ali growth. a total of days growth at ali was allowed before otecs were considered fully differentiated and ready for infection. infection of differentiated otecs. twenty-four hours prior to infection, the otecs were washed apically and basally before feeding with antibiotic-free ali medium basally (to allow the cells to equilibrate and produce mucin prior to infection). bacteria were cultured to exponential phase (approximately h min) before harvesting by centrifugation, washing and resuspending in pbs. the od nm was recorded and the cell suspension was adjusted to . × cells ml − . twenty-five microlitres of this suspension ( . × cfu) were used to infect each differentiated cell culture insert. infection was carried out at °c in % co and % o for the indicated periods of time. at each time-point, tissues were washed three times with pbs and tissues were analysed by lysis and plate counting, histological analysis, immunofluorescence microscopy and sem. assessment of colonisation efficiency. inocula used for infection of differentiated otecs were titered by serial dilution and spot-plating. infected tissues were lysed by incubating in % (v/v) triton x- in pbs for min before scraping from the cell culture insert and homogenising by repeated pipetting. lysates were titered as with inocula. colonisation efficiency was calculated by expressing the numbers of cfu in the lysate at a given time as a percentage of the inoculum. three independent inserts were used for each experiment and three experiments with tissue from independent animals were carried out. histological and immunohistochemical processing and analysis. otec the samples were washed three times with pbst and incubated in primary antibody diluted in blocking buffer for h. rabbit anti-β-tubulin (abcam, #ab ) was used at a : dilution, while bovine anti-m. haemolytica whole-cell antibody was used at a : dilution. the samples were washed three times and incubated in secondary antibody diluted in blocking buffer for h. goat anti-rabbit alexa fluor (thermofisher, #a- ) and goat anti-bovine fitc (thermofisher, #a ), were used at a : dilution. the samples were washed three times and stained with rhodamine phalloidin ( u per sample) and femtomoles dapi for min. following three final washes with pbs, the membranes were cut from the inserts and mounted in vectashield. wide-field images were acquired using a leica dmi microscope. confocal images were acquired using a zeiss lsm . infected otec cultures were fixed with . % (v/v) glutaraldehyde in . m sodium cacodylate at °c for h. all subsequent incubations were carried out at room temperature. the samples were then washed three times with . m sodium cacodylate and incubated in % (w/v) osmium tetraoxide for h. following three washes with distilled water, the tissues were incubated for h in . % (w/v) uranyl acetate while protected from light. after two additional washes, the tissues were dehydrated via a series of increasing ethanol concentrations and incubated in hexamethyldisilizane for h before drying overnight in a desiccator. the tissue samples were mounted on aluminium sem stubs using carbon tape and sputter-coated with gold. images were acquired using a jeol electron microscope. bacteria were manually false-coloured yellow using photoshop elements. statistical analysis. data were analysed using graphpad prism . each experiment was performed using tissues isolated from independent animals, with three inserts being employed for quantitation of colonisation efficiency at each time-point. triplicate experimental means were used for statistical analysis. for each independent time-point, strains were compared by two-tailed student's t test or welch's unequal variance t test with multiple comparisons as indicated. significance values of *, ** and *** represent p < . , . and . , respectively. scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. mannheimia haemolytica: bacterial-host interactions in bovine pneumonia failure of respiratory defenses in the pathogenesis of bacterial pneumonia of cattle estimation of nasal shedding and seroprevalence of organisms known to be associated with bovine respiratory disease in australian live export cattle bovine tonsils as reservoirs for pasteurella haemolytica: colonisation, immune response, and infection of the nasopharynx bacterial pathogens of the bovine respiratory disease complex economic effect of pneumonia and pleurisy in lambs in new zealand pasteurellosis of cattle prevalence of pasteurella haemolytica in transported calves specificity of bovine serum antibody to capsular carbohydrate antigens from pasteurella haemolytica characterization of mannheimia haemolytica isolated from feedlot cattle that were healthy or treated for bovine respiratory disease molecular genetic analysis of virulence in mannheimia (pasteurella) haemolytica genetic characterization and antimicrobial susceptibility of mannheimia haemolytica isolated from the nasopharynx of feedlot cattle serotypes of pasteurella haemolytica in ovine pasteurellosis pasteurellosis of sheep mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia evolutionary genetics of pasteurella haemolytica isolates recovered from cattle and sheep electron microscopic examination of cells of pasteurella haemolytica-a in experimentally infected cattle resistance to host immune defense mechanisms afforded by capsular material of pasteurella haemolytica, serotype identification of mannheimia haemolytica adhesins involved in binding to bovine bronchial epithelial cells discrimination between apo and iron-loaded forms of transferrin by transferrin binding protein b and its n-terminal subfragment lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to pasteurella (mannheimia) haemolytica leukotoxin mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (bl- ) via a caspase- -dependent mitochondrial pathway effect of leukotoxin of mannheimia haemolytica and lps of e. coli on secretory response of bovine neutrophils in vitro temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface pathogenic mannheimia haemolytica invades differentiated bovine airway epithelial cells sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompa) of mannheimia(pasteurella) haemolytica, mannheimia glucosida, and pasteurella trehalosi mosaic structure and molecular evolution of the leukotoxin operon (lktcabd) in mannheimia (pasteurella) haemolytica, mannheimia glucosida, and pasteurella trehalosi evidence for a common gene pool and frequent recombinational exchange of the tbpba operon in mannheimia haemolytica, mannheimia glucosida and bibersteinia trehalosi bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors innate immunity in the lung: how epithelial cells fight against respiratory pathogens the airway goblet cell mucociliary clearance in the airways inhibition of ciliated cell differentiation by fluid submersion air-liquid interface culture of serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies investigations on the species specificity of mannheimia (pasteurella) haemolytica serotyping sequence diversity and molecular evolution of the leukotoxin (lkta) gene in bovine and ovine strains of mannheimia (pasteurella) haemolytica inducible innate resistance of lung epithelium to infection innate barriers against skin infection and associated disorders monomeric expression of bovine β -integrin subunits reveals their role in mannheimia haemolytica leukotoxin-induced biological effects three viruses of the bovine respiratory disease complex apply different strategies to initiate infection intra-specific diversity and host specificity within pasteurella haemolytica based on variation of capsular polysaccharide, lipopolysaccharide and outer-membrane proteins outer membrane protein a of bovine and ovine isolates of mannheimia haemolytica is surface exposed and contains host species-specific epitopes we thank ms. margaret mullin and ms lynn stevenson (university of glasgow, united kingdom) for their assistance with sem and histological sample preparation. this work was supported by the biotechnology and biological sciences research council (grant reference: bb/l / ; url:https ://www.bbsrc .ac.uk) and by msd animal health (url: https ://www.msd-anima l-healt h.com/). this study was part funded by msd animal health. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to r.l.d.reprints and permissions information is available at www.nature.com/reprints. key: cord- -cu nx authors: luo, lingfei; gu, yiqin; wang, xiaoguang; zhang, yinghua; zhan, longwen; liu, jiqian; yan, hongjing; liu, yun; zhen, shanshan; chen, xiuhua; tong, rui; song, chiping; he, yingying title: epidemiological and clinical differences between sexes and pathogens in a three-year surveillance of acute infectious gastroenteritis in shanghai date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: cu nx acute infectious gastroenteritis cases in shanghai, reported over three years, were analyzed. pathogens were identified in patients; of these, and were bacterial and viral cases, respectively. vibrio parahemolyticus and salmonella were the dominant bacteria, and caliciviridae and reoviridae were the dominant viral families in the local area. the acute gastroenteritis epidemic peaks appeared in august and january, which represented the active peak periods of bacteria and viruses, respectively. logistic regression analyses with sex stratification showed that abdominal pain, fever and ingestion of unsafe food at restaurants were independent factors more frequently associated with bacterial gastroenteritis irrespective of sex; red cell-positive fecal matter was associated with bacterial gastroenteritis with an odds ratio (or) of . only in males; and white blood cell count was associated with bacterial gastroenteritis with an or of . only in females. pathogen stratification showed that age, vomiting and red cell-positive fecal matter were associated with males with ors of . , . and . , respectively, in bacterial gastroenteritis; and the migrant ratio was higher in males with an or of . only in viral gastroenteritis. in conclusion, although bacterial and viral gastroenteritis shared many features, epidemiological and clinical factors differed between sexes and pathogens. isolation and identification of bacterial pathogens. bacterial detection is a vital part of the acute gastroenteritis control and prevention strategy established by the local health administration department. bacterial identification is the systematic work of a multisectoral collaboration in china's centers for disease control and prevention, and it is difficult to detail all processes in this report. briefly, detection was carried out via classic bacterial isolation, culture, and identification in combination with molecular diagnostic strategies . in addition, bacterial samples were subjected to vitek ® compact (biomérieux, france) for simultaneous microbial identification. the above technologies covered the most common diarrhea-related bacteria, including vibrio cholerae, salmonella, shigella, vibrio parahemolyticus, yersinia enterocolitica, campylobacter jejuni, enterotoxigenic escherichia coli (etec), enteropathogenic e. coli (epec) and others . to learn the pathotypes of diarrheagenic e. coli, suspected e. coli colonies on smac or macconkey plates were further analyzed by pcr using primers targeting the genes eae, bfp, stx , stx , ipah, pcdv, elta and esta. a first pcr detection was performed with stx / stx and eae primers focusing on the identification of stec or epec. positive eae and negative stx /stx samples were then examined by pcr with bfp primers to differentiate tepec from aepec. negative eae and stx /stx samples were further screened by pcr using pcvd primers for plasmidic eaec sequences, ipah primers for detecting invasion plasmid antigen genes of eiec, and detection of the elta and esta genes of etec labile and stable enterotoxins , . the diagnosis of epidemic dysentery and cholera was performed according to the laboratory methods for the diagnosis of epidemic dysentery and cholera recommended by the u.s. centers for disease control and prevention and the world health organization . laboratory standard operating procedures for detection were performed according to the practical guidance for clinical microbiology-laboratory diagnosis of bacterial gastroenteritis and the final report and executive summaries from the aoac international presidential task force on best practices in microbiological methodology . detection of viral pathogens. viral detection is another vital part of the acute gastroenteritis control and prevention strategy established by the local health administration department. gastroenteritis virus detection was performed according to methods reported by other peers , , , , . briefly, fecal specimens were prepared as % (w/v) suspensions in distilled water and then centrifuged for min at g in a . ml collection tube www.nature.com/scientificreports www.nature.com/scientificreports/ (biovisualab, shanghai, china) to remove debris. viral dna and viral rna were extracted from the suspensions using a qiaamp dna mini kit and a qiaamp viral rna mini kit (qiagen, the netherlands), according to the manufacturer's instructions. the virus panel was established with routine diarrhea surveillance data that were accumulated by local centers for disease control and prevention and by reference to information from peers [ ] [ ] [ ] [ ] . rotavirus, norovirus, enteric adenovirus, astrovirus, sapovirus, mimiviruses, aichivirus, bocavirus, parechovirus, cytomegalovirus, hepatitis a, coronaviruses, picornaviruses, toroviruses, and other enteroviruses were detected by polymerase chain reaction (pcr) or reverse transcription pcr using primer sets, as reported previously , , , - . statistical analysis. continuous variables are presented as the means ± standard deviations (sds); differences between groups were evaluated using the mann-whitney u-test for independent samples. categorical variables are presented as frequencies (percentage); differences in frequencies were evaluated using the chi-square test or fisher's exact probability test. to identify independent factors and to distinguish factors associated with pathogen or sex, logistic regression analyses were applied in subjects with known pathogens. to identify factors that tended to be associated with pathogens, the dependent variables were defined as viral case = and bacterial case = , and the multivariable logistic regression analyses were performed by sex. to identify factors that tended to be associated with sex, the dependent variables were defined as female = and male = , and the multivariable logistic regression analyses were performed by viral and bacterial gastroenteritis. potential independent variables were selected by univariate analyses; factors with p < . were introduced into the starting model of the multivariable logistic regression analyses and then eliminated manually using the backward step-by-step approach, depending on the largest p-value. all analyses were performed using spss software for windows (ver. . ; spss inc., chicago, il, usa), and the significance level (alpha) was set at . . general information from three-year-round surveillance. this report included annual cases from three full years of acute infectious gastroenteritis that occurred in two sentinel hospitals of shanghai from to . in total, subjects, males and females, were involved. as shown in table , the average age of patients was older among females, and the ratio of local residents was significantly higher in the female group. regarding the symptoms associated with acute gastroenteritis, the percentages of subjects with nausea, vomiting, watery stools and abdominal pain were significantly higher in the female group than in the male group. the rate of patients with fever was significantly higher in males ( . %) than in females ( . %). no significant difference in vomiting frequency, duration of vomiting, diarrhea frequency, duration of diarrhea, average body temperature or rate of dehydration existed between the sexes. since the number of patients with dehydration was low in both sexes, differences in blood pressure may be an inherent sex difference rather than a pathological feature associated with acute gastroenteritis. the epidemiological questionnaire showed that . % and . % of male and female patients, respectively, had a history of the possible ingestion of unsafe foods. the foodborne source of males more frequently tended to be from restaurants and delivery foods, while the foodborne source of females more frequently tended to be from home ( table ). the rates of drinking unsafe water, exposure to patients with diarrhea within days and travel within one week were very low in both sexes (table ) . clinical laboratory tests showed that the fecal red cell-positive rate was significantly lower in females ( . %) than in males ( . %) ( table ). in conclusion, age, symptoms, epidemiological factors and clinical laboratory tests differed between sexes in patients with acute gastroenteritis. annual dynamic characteristics of the acute gastroenteritis epidemic. to understand the seasonal epidemic characteristics of acute infectious gastroenteritis, all cases were aligned by month. as shown in fig. a , all annual acute gastroenteritis epidemics, including , , and merged data, displayed two peaks in the summer and winter. to learn if the epidemic peaks were associated with pathogen differences, all identified viral and bacterial cases were aligned again by month. as shown in fig. b , cases infected by bacteria peaked in august, and cases infected by viruses peaked in january. pathogenic spectrum. pathogens were successfully identified in patients, and and subjects were infected by bacteria and viruses, respectively. of the identified cases of bacterial acute gastroenteritis, . %, . %, . % and . % were infected by vibrio parahemolyticus, salmonella, epec and etec alone, respectively; . %, . % and . % were coinfected by vibrio parahemolyticus and salmonella, epec and vibrio parahemolyticus and epec and salmonella, respectively (fig. ) . of differences between viral and bacterial gastroenteritis by sex. the difference between the sexes shown in table does not taken into account the possible role of pathogens. therefore, the clinical and epidemiological characteristics of bacterial and viral cases were further compared by sex. of males, as shown in table , and subjects were identified as having bacterial and viral gastroenteritis, respectively. the rates of nausea, watery stools, abdominal pain, fever, ingesting possible unsafe foods, ingesting unsafe foods at restaurants, leukocyte-and red-cell-positive fecal matter and the average vomiting frequency, body temperature and white blood cell count variables were significantly higher in the bacterial group than in the www.nature.com/scientificreports www.nature.com/scientificreports/ viral group; conversely, the average vomiting duration, heart rate and diastolic pressure and the rate of ingesting unsafe foods at home variables were significantly higher in the virus group than in the bacterial group. no significant difference in the age, percentage of local residents, vomiting rate, diarrhea frequency, duration of diarrhea, dehydration rate, systolic blood pressure, or rate of ingesting delivery food variables was observed between the viral and bacterial gastroenteritis groups ( table , left panel) . of females, and subjects were identified as having bacterial and viral gastroenteritis, respectively. the rates of nausea, vomiting, abdominal pain, fever, ingesting unsafe foods at restaurants, leukocyte-and red-cell-positive fecal matter and the white blood cell count variables were significantly higher in the bacterial group than in the viral group; conversely, the average vomiting duration was significantly longer and the rate of ingesting unsafe foods at home was significantly higher in the viral group than in the bacterial group. no significant differences in the age, percentage of local residents, frequency of vomiting, frequency of watery stools, frequency of diarrhea, duration of diarrhea, rate dehydration, heart rate, blood pressure, or rate of ingesting possible unsafe food variables was observed between the viral and bacterial gastroenteritis groups ( independent factors differentially associated with pathogen by sex. bacterial gastroenteritis shares many clinical manifestations and epidemiological features with viral gastroenteritis [ ] [ ] [ ] . although the above stratified analyses showed differences between sexes and pathogens, such analyses could neither identify www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ independent factors nor quantify the associations. thus, logistic regression analyses were adopted to distinguish the associations. among males, univariate analyses showed that nausea, vomiting frequency, watery stools, abdominal pain, fever, ingesting unsafe food at restaurants, fecal leukocyte-positive, fecal red cell-positive and white blood cell count were potential independent factors that were differentially associated with viral and bacterial gastroenteritis. multivariable logistic regression analyses revealed that only abdominal pain, fever, fecal red cell-positive and ingesting unsafe food at restaurants were independent factors that more frequently occurred in bacterial gastroenteritis than viral gastroenteritis with ors of . , . , . and . , respectively (table ) . among females, nausea, vomiting, duration of vomiting, abdominal pain, fever, ingesting unsafe food at restaurants, fecal leukocyte-positive, fecal red cell-positive and white blood cell count were potential independent variables that were differentially associated with viral and bacterial gastroenteritis. further multivariable logistic regression analyses revealed that abdominal pain, fever, and ingesting unsafe food at restaurants were independent factors that more frequently occurred in bacterial gastroenteritis with ors of . , . and . , respectively (table ) . white blood cell count was also an independent factor and higher in bacterial gastroenteritis than in viral cases (table ) . table . clinical and epidemiological differences between viral and bacterial gastroenteritis by sex. continuous variables are presented as the means ± standard deviations (sds); difference between groups were evaluated using the mann-whitney u-test for independent samples. categorical variables are presented as frequencies (percentages); differences in frequencies were evaluated using the chi-square test or fisher's exact probability test. www.nature.com/scientificreports www.nature.com/scientificreports/ independent factors differentially associated with sex by pathogen. to identify the independent factors of acute infectious gastroenteritis that were differentially associated with sex by pathogen, logistic regression analyses were applied to bacterial and viral cases. initial univariate analyses showed that age, domiciliary register and vomiting frequency and age, domiciliary register, nausea, abdominal pain, vomiting and fecal red cell-positive were potential independent variables in associated with sex in the viral and bacterial groups, respectively. further multivariable logistic regression analyses using the above candidate variables revealed that, in bacterial gastroenteritis, an increase of year old resulted in a decreased male/female (ratio) with an or of . (table ) , vomiting occurred less frequently in males than in females with an or of . , and red cell-positive fecal matter occurred more frequently in males than in females with an or of . (table ). in the viral group, only domiciliary register remained an independent factor associated with males with an or of . (migrant vs. local resident) (table ). in this report, we collected data across a -year period from two sentinel hospitals located in the minhang district of shanghai. our analysis strategy consisted of three steps: first a pooled analysis with male and female patients irrespective of pathogens to understand sex differences; second, sex and pathogen stratification analyses with patients whose pathogens were identified to determine epidemiological and clinical differences between sexes and pathogens; and third, logistic regression analyses to distinguish the factors associated with sex and pathogens. in the pooled analysis, the percentage of local residents and the rate of ingesting possible unsafe food at home variables were lower in males, and the rate of ingesting possible unsafe food at restaurants was higher in males, which is consistent with the characteristics of male social behavior. the rates of nausea, vomiting, watery stools and abdominal pain were higher, and the rates of fever and red cell-positive fecal matter were lower in females, suggesting a sex difference in symptoms associated with acute gastroenteritis. although the pooled analysis did not implement pathogen classification, it reflected the features of acute gastroenteritis that clinicians face every day. next, stratification analyses by sex and pathogen showed that the distributions of clinical and epidemiological factors differed not only between viral and bacterial groups but also between sexes. the results obtained by foregoing two steps guided us to implement sex and pathogen stratification in subsequent logistic regression analyses. independent factors differentially associated with pathogens were evaluated by sex stratification; logistic regression analyses revealed that abdominal pain and fever were two common independent symptom factors that more frequently occurred in bacterial gastroenteritis than in viral gastroenteritis, regardless of sex. clinically, acute infectious gastroenteritis is classified into two pathophysiologic types: noninflammatory and inflammatory. the noninflammatory gastroenteritis is mostly caused by viral infection with milder disease; while, inflammatory gastroenteritis is more severe and always resulted from infection of invasive or with toxin-producing bacteria , - . in addition, fever and abdominal pain are two common symptoms in acute gastroenteritis caused by salmonella infection ; our data showed that . % identified bacterial cases were salmonella infection (fig. ) . thus, higher prevalence of fever and abdominal pain in acute bacterial gastroenteritis were observed. ingesting unsafe food at restaurants was a common transmission route for both sexes and was more frequently associated with bacterial gastroenteritis; which is consistent with that bacterial infections are more often associated with foodborne transmission [ ] [ ] [ ] and acquired easily at places with high population mobility , . white blood cell count was higher in bacterial cases only in females with low or of . , suggesting the relative pathology of www.nature.com/scientificreports www.nature.com/scientificreports/ infectious acute gastroenteritis differed between sexes. however, testing stool for leukocytes to screen for inflammatory diarrhea has fallen out of favor due to a wide variability in sensitivity and specificity , . independent factors differentially associated with sex were further evaluated in logistic regression analyses with pathogen stratification. in bacterial gastroenteritis, age and vomiting were associated with males with ors of . and . , respectively; for ease of understanding, these associations were translated to females, and age and vomiting were associated with females with ors of . and . , respectively, meaning that with a -year old increase, the female/male ratio will increase . times, and vomiting is more frequently associated with female patients. the age quartiles of female and male bacterial cases are . , . and . ; and . , . and . years old respectively; the nd and particularly the rd quartiles of female age were older; which is why female/male ratio will increase . times with a -year old increase. red cell-positive fecal matter remained an independent factor that was more frequently observed in males with or of . (table ); in addition, the above sex stratification analysis showed that red cell-positive fecal matter was an independent factor that was more frequently observed in bacterial cases only in males with or of . (table ) . these two analysis methods mutually confirmed that red cell-positive fecal matter is common in males with bacterial infections. bacteria, such as vibrio parahemolyticus and salmonella, predisposing to cause inflammatory infections with bloody stool [ ] [ ] [ ] , which could be used to explain why bloody stool is more frequently observed in bacterial infections, but could not interpret gender difference. since the proportions of vibrio parahemolyticus and salmonella infections were similar between males ( . % and . %) and females ( . % and . %), higher bloody stool rate in male cases should not be caused by differences in rates of bacterial infection. although we could not give a reasonable explanation to partial gender differences; these evidences have clinical significance and will guide translational study to interpret a pathological mechanism. in fact, gender differences exist widely in clinical medicine and have been paid more and more attention in recent years . in viral cases, only domiciliary register remained an independent factor associated with males; the migrant/local resident ratio was . times higher in males than in females, suggesting the prevalence of viral gastroenteritis is relatively higher in migrant workers. some parameters, such as body temperature, heart rate and blood pressure, were not potential independent variables in univariate analyses; this finding is because body temperature was only recorded in subjects with fever, and heart rate and blood pressure were only recorded in patients with dehydration. as to why some variables did not remain independent factors in the final equation, this is partially explained by the high colinearity between variables, for example, the pearson correlation coefficient between fecal leukocyte-positive and fecal red cell-positive was . (p < . ), and the narrow differences between two groups, for example, the percentage of nausea between bacterial and viral cases ( . % vs. . %) in males and the vomiting frequency between bacterial and viral cases ( . ± . vs. . ± . days) in males. however, most of the above differences involved complex pathophysiological principles, and we cannot offer a perfect explanation. regardless, logistic regression analysis can effectively assess confounders and select independent variables associated with sex and pathogen. acute gastroenteritis caused by viral infections was dominated by rotavirus and norovirus , . rotavirus always causes severe gastroenteritis in young children , , while norovirus causes most outbreaks of nonbacterial acute gastroenteritis in all age groups . recently, some scholars reported that the rotavirus predominance of acute gastroenteritis has been replaced by norovirus due to the wide implementation of rotavirus vaccination . our data showed that caliciviridae (norovirus gii and gi) and reoviridae (rotavirus a) constituted more than % of viral infections. since a norovirus vaccine is still under development , rotavirus vaccination needs to be implemented in local areas. bacteria are the second leading cause of acute gastroenteritis; shigella, salmonella, campylobacter, diarrhoeagenic escherichia coli, pathogenic vibrio, yersinia, and clostridium difficile are the most commonly reported bacteria correlated with acute gastroenteritis , . our data showed that vibrio parahemolyticus and salmonella were the dominant bacteria identified in the local area, and these findings are consistent with those of a previous study with a small sample size . this report included the following limitations: first, this report only focused on the outpatients of two sentinel hospitals, and all subjects were sporadic adult cases; thus, this study does not represent the infectious gastroenteritis epidemic in schools, although it is known that acute gastroenteritis is highly prevalent in schools and always causes outbreaks , . second, based on our sampling rates, pathogens were only identified in . % ( / ) of patients, and more effort should be made to improve the representative accuracy of our results. third, in the stratified and logistic regression analyses, we only considered mixed viral cases and mixed bacterial cases and did not subgroup the subjects by bacterial classification, virus classification or by multiple infections due to the restrictions of sample size. since all of the above factors might influence the symptoms and clinical examinations of acute gastroenteritis, our analyses may be influenced by related bias. acute gastroenteritis aetiological characteristics of adult acute diarrhoea in a general hospital of shanghai acute gastroenteritis outbreak caused by a gii. norovirus laboratory methods for the diagnosis of epidemic dysentery and cholera acg clinical guideline: diagnosis, treatment, and prevention of acute diarrheal infections in adults viral gastroenteritis laboratory diagnosis of bacterial gastroenteritis burden of acute gastroenteritis caused by norovirus in china: a systematic review molecular epidemiology of genogroup ii norovirus infections in acute gastroenteritis patients during - 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ovbto authors: yu, xiaojuan; zhang, senyan; jiang, liwei; cui, ye; li, dongxia; wang, dongli; wang, nianshuang; fu, lili; shi, xuanlin; li, ziqiang; zhang, linqi; wang, xinquan title: structural basis for the neutralization of mers-cov by a human monoclonal antibody mers- date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ovbto the recently reported middle east respiratory syndrome coronavirus (mers-cov) causes severe respiratory illness in humans with an approximately % mortality rate. the envelope spike glycoprotein on the surface of mers-cov mediates receptor binding, membrane fusion, and viral entry. we previously reported two human monoclonal antibodies that target the receptor binding domain (rbd) of the spike and exhibit strong neutralization activity against live and pesudotyped mers-cov infection. here we determined the crystal structure of mers-cov rbd bound to the fab fragment of mers- antibody at . Å resolution. the mers- epitope in the rbd overlaps with the binding site of the mers-cov receptor dpp . further biochemical, viral entry, and neutralization analyses identified two critical residues in the rbd for both mers- recognition and dpp binding. one of the residues, trp , was found to function as an anchor residue at the binding interface with mers- . upon receptor binding, trp interacts with the n-linked carbohydrate moiety of dpp . thus, mers- inhibits mers-cov infection by directly blocking both protein-protein and protein-carbohydrate interactions between mers-cov rbd and dpp . these results shed light on the molecular basis of mers- neutralization and will assist in the optimization of mers- as a tool to combat mers-cov infection. route of transmission to humans could result from direct or indirect contact with this reservoir or with another intermediate. the prevalence of mers-cov-neutralizing antibodies in camels and the recent isolation of mers-cov from dromedary camels suggest that camels could be one of the intermediate hosts [ ] [ ] [ ] . unlike the severe acute respiratory syndrome coronavirus (sars-cov) outbreak in [ ] [ ] , which resulted in ~ infections with a case-fatality rate of ~ % , mers-cov has limited human-to-human transmission , . transmission potential analyses have indicated that mers-cov has a lower reproduction number (r ) than sars-cov ( . vs . ) and has not yet achieved pandemic potential , . however, the sustained epidemic in animal reservoirs would be expected to lead to spill-over into humans . the current case-fatality rate of mers-cov infection is estimated to be as high as % among hospitalized patients, and no approved pathogen-specific vaccines or antivirals against mers are available. therefore, the development of prophylactic and therapeutic reagents is urgently needed to combat mers-cov infection. the envelop spike (s) glycoprotein of coronaviruses, a class i transmembrane protein, is responsible for receptor binding, membrane fusion, and viral entry . the s protein exists as a trimer, with each monomer consisting of a globular membrane-distal s domain, a membrane-proximal s domain, and a transmembrane domain. the functional cellular receptor for mers-cov has been identified as dpp (dipeptidyl peptidase , also known as cd ) . the receptor binding domain (rbd) is located in the s domain of mers-cov spike and is responsible for binding to dpp , . both our laboratory and another research group recently determined the crystal structure of the rbd in complex with dpp and identified rbd residues that are critical for dpp binding and viral entry , . furthermore, neutralizing monoclonal antibodies capable of disrupting the interaction between rbd and dpp were identified and characterized [ ] [ ] [ ] [ ] . specifically, we utilized rbd as an antigen bait to screen a non-immune human single-chain variable domain fragment (scfv) library displayed on yeast and identified two neutralizing antibodies mers- and mers- . these two antibodies inhibited in vitro infection of mers-cov live and pseudoviruses with ic values at nanomolar concentrations , indicating their potential for prophylactic and therapeutic development. here we report the crystal structure of mers-cov rbd bound to the mers- fab. the mers- fab interacts with the receptor binding subdomain of rbd. its binding epitope on the rbd overlaps with the dpp binding site in a small area consisting of four residues . we utilized biochemical, pseudovirus-entry, and neutralization assays to examine the roles of these four residues in mers- recognition and dpp binding. we found trp as an anchor residue in the rbd for mers- recognition, and its interaction with n-linked carbohydrates of dpp is important for the binding to dpp and for viral entry of mers-cov. overall structure of mers-cov rbd bound to mers- fab. we generated the rbd of the mers-cov spike glycoprotein (residues - ) by baculovirus expression in insect cells ( figure s ). this fragment was previously utilized as an antigen bait to isolate the human neutralizing antibodies mers- and mers- from a yeast-displayed single-chain variable region (scfv) fragment library . recombinant igg mers- was expressed in hek t cells, and the fab was generated by endoproteinase lys-c digestion and further purified by size-exclusion chromatography ( figure s ). the mers-cov rbd and mers- fab were mixed in vitro to allow the formation of the complex ( figure s ). the complex structure was determined by molecular replacement to a resolution of . Å (table ). in the crystallographic asymmetric unit, the rbd and fab form two : binding complexes that are structurally similar with a root-mean-square deviation (rmsd) of . Å for all cα pairs ( figure s ). the model consists of residues val to lys of mers-cov rbd, ala to arg (light chain) and val to lys (heavy chain) of mers- fab, and two n-acetyl-d-glucosamine (nag) molecules attached to asn and asn of rbd, respectively (fig. a ). it has been previously shown that the mers-cov rbd comprises the core subdomain and receptor-binding subdomain (fig. a) . both the heavy and light chain of mers- interacts with the receptor-binding subdomain in the complex (fig. a) . the rbd core subdomain comprises a five-stranded anti-parallel sheet (β , β , β , β , and β ) in the center, with five short helices (α , α , α , α , and α ) decorating the central β -sheet on both sides (fig. b) . the fold is maintained by three disulfide bonds that connect c to c , c to c , and c to c in the core subdomain (fig. b) . the rbd receptor-binding subdomain is a four-stranded anti-parallel sheet (β , β , β , and β ) located between β and β of the core-subdomain (fig. b) . a long loop containing a short helix (α ) connects β to β in the receptor-binding subdomain (fig. b) . the loop crosses the β -sheet perpendicularly on one side, and a disulfide bond between c and c further links strand β to helix α (fig. b) . the other side of the β -sheet lacks this structural decoration, leaving a relatively flat surface in the receptor-binding subdomain, thereby facilitating interaction with human receptor dpp . the mers-cov rbd structure was previously determined alone and in complex with receptor dpp , , . the rbd structure was shown to be maintained in the unbound, dpp -bound, and mers- -bound states with three pairwise structural superimpositions showing rmsd for cα atoms of . , . , and . Å ( figure s ). four disulfide bonds maintain the fold in the rbd structure (fig. b) . the α -β loop and the α helix in the core subdomain can be seen interacting with the β -sheet and the n-terminal segment of the linking loop in the receptor-binding subdomain (fig. b) . these features, especially the rigid orientation between the two subdomains, may account for the structural conservation of rbd observed in the different states ( figure s ). of approximately Å of surface area, including roughly Å of the heavy chain, Å of the light chain, and Å of the rbd. residues from the complementary determining regions (cdrs) h (tyr ), h (tyr , ser , tyr , and thr ), and h (phe and trp ) of the heavy chain and l (asn and phe ) and l (tyr , asp , lys , and pro ) of the light chain comprise the interacting residues of mers- ( fig. a) . the mers- fab interacts with the c-terminal segment of the linking loop and β strand of the receptor-binding subdomain. the epitope on the receptor-binding subdomain consists of val , ser , ile , val , pro , ser , trp , glu , and asp in the long linking loop and tyr , tyr , and arg in the β strand (fig. b) . a cavity is formed by the h , h , h , and l cdrs between the mers- heavy and light chains (fig. a) . the rbd trp inserts into this cavity and interacts with the surrounding mers- residues tyr , tyr , and trp of the heavy chain and tyr , lys , and pro of the light chain (fig. a) . outside the cavity, phe and trp of the heavy chain show hydrophobic interactions with tyr , tyr , and arg of rbd (fig. b ). heavy chain residues tyr and tyr form hydrogen bonds with asp and glu of rbd, respectively (fig. b) . light chain residue phe shows hydrophobic table . crystallographic data collection and refinement statistics. r work and r free are defined by r = Σ hkl ||f obs | − |f calc ||/Σ hkl |f obs |, where h, k, and l are the indices of the reflections (used in refinement for r work ; %, not used in refinement for r free ) and f obs and f calc are the structure factors, deduced from intensities and calculated from the model, respectively. interactions with val and pro of rbd, while light chain residues asn and asp form hydrogen bonds with main chain atoms of rbd residues val and ser , respectively (fig. c ). dpp show steric clashes between the variable domain of the heavy chain and the propeller domain of dpp when they simultaneously bound to rbd (fig. a ). they bind to the receptor-binding subdomain from different directions (fig. a) , resulting in an overlap on an epitope of the receptor-binding subdomain that includes trp , glu , asp , and tyr and that is relatively small compared with the large binding surfaces of mers- and dpp on the rbd (fig. b ). we previously found that pseudoviruses bearing a triple glu ala/asp ala/asp ala mutant of the mers-cov spike glycoprotein had significantly reduced efficiency of viral entry . we performed a more thorough mutagenesis study by introducing single mutations of trp , glu , asp , and tyr into the spike glycoprotein. we first measured the efficiency of entry of pseudoviruses bearing these single-site mutations. we found that trp ala, asp ala, and glu ala mutations reduced the efficiency of viral entry, with the first two mutations (trp ala and asp ala) showing more profound effects (> % reduction) than the last one (glu ala, ~ % reduction) (fig. c) . in contrast, a mutation at tyr had negligible effects on the efficiency of viral entry (fig. c ). we then further examined the effects of these mutations on resistance to mers- neutralization. pseudoviruses bearing spike glycoproteins with trp ala, glu ala, or asp ala mutations failed to be neutralized by the mers- antibody, whereas cell entry by pseudoviruses with the tyr ala mutation was still inhibited by mers- (fig. d ). finally, we examined effects of these rbd mutations on the binding of mers- fab by using surface plasmon resonance (spr). wild type rbd bound the mers- fab with an affinity of nm (fig. e ). the glu ala mutation reduced the binding affinity by -fold to . μ m, and the tyr ala mutant did not affect the binding, showing an affinity of nm (fig. e ). the individual trp ala and asp ala mutations reduced binding to a level that was undetectable by spr (fig. e ). in sum, results of viral entry, neutralization, and binding assays consistently indicated that trp and asp are critical for both mers-cov spike glycoprotein recognition by mers- and binding with receptor dpp . in this study, we determined that the two rbd residues trp and asp are critical for both mers- recognition and dpp binding (fig. ) . asp forms a hydrogen bond with heavy chain residue tyr upon mers- recognition and it forms a salt-bridge with lys of dpp upon receptor binding ( figure s ) . therefore, the binding of mers- antibody prevents the formation of the asp -lys salt bridge that is important for the receptor binding and viral entry (fig. c) . rbd residue trp inserts its side chain into the cavity of mers- (fig. a) , forming an anchor at the interface of mers- recognition. in contrast to asp and other rbd residues involved in dpp binding, trp does not directly interacts with dpp residues. instead, trp has close contacts with the carbohydrate moiety linked to residue asn of dpp (fig. a) . taken together, these results indicate that the protein-carbohydrate interaction might be important for dpp binding and the viral entry of mers-cov. to further confirm this possibility, we generated a human dpp asn gln mutant and measured its ability to bind to rbd and to mediate the cell entry of pseudoviruses. we found that the efficiency of viral entry into cos cells expressing the dpp asn gln mutant was significantly reduced (~ % reduction) compared with cos cells expressing wild-type dpp (fig. b) . similarly, the rbd binding affinity was also reduced by approximately fold from the . nm for wild-type dpp to nm for the dpp asn gln mutant (fig. c) . therefore, although the mers- binding epitope overlaps with a small area of the dpp -binding site on rbd, mers- lodges rbd trp in the cavity between its heavy and light chains (fig. a) and forms hydrogen bond with rbd asp (fig. b) . in this way, mers- disrupts both the protein-protein and mers-cov is the second reported example of a zoonotic coronavirus that results in severe respiratory infection with high mortality rate in humans after sars-cov. therefore, mers-cov has attracted significant attention in basic research and clinical studies since its discovery in , . after revealing the structural basis for the specific binding of mers-cov spike glycoprotein to the cellular receptor dpp and identifying two human neutralizing monoclonal antibodies , , we report here the structural, biochemical, and cellular mechanism of mers-cov inhibition by one antibody mers- . notably, our study revealed that mers- inhibits both protein-protein and protein-carbohydrate interactions between mers-cov rbd and dpp . although the close contact of trp with the dpp asn -linked carbohydrate moiety has been previously observed , the role of the interaction in receptor binding and viral entry of mers-cov has remained unclear. we showed that the recognition of rbd by mers- is anchored at rbd residue trp through structural and biochemical studies. the interaction between trp of rbd and the dpp carbohydrate is also important for receptor binding and viral entry of mers-cov. the critical rbd residues considered to be involved in receptor binding and viral entry should include trp , which was not defined in our previous study . notably, recent studies showed that the isolated rbd of the hku spike was able to bind dpp and pseudoviruses bearing hku spike infected cells through dpp , . structural determination also found that the dpp asn -linked carbohydrate moiety also interacts with the rbd of hku . these results further underscore that the asn -linked carbohydrate moiety is important for facilitating the binding of dpp to the rbd of mers-cov and its close relatives. in addition to mers- , the scfv library screen against mers-cov rbd identified another neutralizing antibody, mers- . mers- and mers- exhibited a synergistic inhibitory effect on the entry of pseudoviruses . previously, we generated a series of mutants of the mers-cov spike glycoprotein with residues involved in dpp binding and analyzed the escape of these mutant-bearing pseudoviruses from antibody neutralization. the mers- epitope largely overlapped with dpp binding site . however, none of the mutants that efficiently escaped mers- neutralization showed similar effects with mers- , indicating that mers- and mers- recognize different epitopes . the dpp binding site in the rbd has been separated into patch , which includes tyr , glu , asp , and asp , and patch , which includes the hydrophobic leu , trp , val and the surrounding hydrophilic asp , arg , and glu . in this study, we showed that mers- epitope mainly overlaps with the patch dpp -binding site. upon mers- binding, more patch dpp -binding rbd residues are still exposed. structural data for mers- with rbd have not yet been made available, though we hypothesize that mers- binding targets and blocks the rbd residues in patch , which are separated from the mers- epitope in patch and could explain the synergistic effect of mers- and mers- . other groups also reported the identification of ten human and one mouse neutralizing monoclonal antibodies targeting the rbd of mers-cov spike glycoprotein [ ] [ ] [ ] . the structural information of these antibodies with rbd has not yet made available. epitope mapping showed that the mouse antibody mersmab recognizes leu , asp , arg , glu , and trp in patch . mers-cov spike mutations escaped neutralization by seven human neutralizing antibodies, as reported by tang et al. . nearly all of the mutated residues mapped to patch , with the exception of one mutation in the s domain of spike . ying et al. reported that asp and trp of patch are important for the binding of rbd to all three of the identified human neutralizing antibodies . the binding epitope of one antibody, m , includes glu and asp , which are also present in the epitope of mers- . however, trp , which has been shown to be important for mers- binding, was not included in the binding epitope of m . mers- seems to differ from other identified mers-cov neutralizing antibodies in that trp is used as an anchor recognition site, blocking protein-carbohydrate as well as protein-protein interactions. in summary, our study reported here revealed the structural basis for the neutralization of mers-cov by mers- . the structural analysis of mers- with mers-cov rbd further led us to delineate the importance of the protein-carbohydrate interaction in the binding of rbd to the mers-cov cellular receptor dpp . our study therefore provides important information required for a complete understanding of mers-cov neutralization by different antibodies, which will in turn assist the optimization of these antibodies to ultimately combat mers-cov infection. protein expression and purification. the mers-cov receptor-binding domain (rbd) (residues to of the spike glycoprotein) and the ectodomain of human dpp (residues to ) were expressed in spodoptera frugiperda sf insect cells using the bac-to-bac baculovirus expression system (invitrogen). sf insect cells were maintained in insect-xpress protein-free medium (lonza) without serum at °c. the cdnas encoding the mers-cov rbd and dpp ectodomain were cloned into pfastbac-dual vector with an n-terminal gp signal peptide to facilitate secretion and a c-terminal histag to facilitate purification. the plasmid was transformed into bacterial dh bac competent cells, and the extracted bacmid was transfected into sf cells in the presence of cellfectin ii reagent (invitrogen). after incubation of the transfected cells at °c for days, the low-titer baculoviruses were harvested by collecting the cell-culture supernatant with centrifugation at , rpm for minutes. after two rounds of amplification, the high-titer viruses were used to infect sf cells at a density of × cells per milliliter. the cell-culture supernatant containing the mers-cov rbd or dpp ectodomain were harvested hours after infection, concentrated, and buffer-exchanged to hbs buffer ( mm hepes, ph . , and mm nacl). the mers-cov rbd or dpp ectodomain was captured with nickel beads, eluted with mm imidazole in hbs buffer, and purified by gel-filtration chromatography using a superdex high performance column (ge healthcare) with the hbs running buffer. the purified mers-cov rbd was digested with endoglycosidase f and f at room temperature overnight and was then further purified by gel-filtration chromatography. the crystallization and data collection. the purified mers-cov rbd and mers- fab were mixed at a molar ratio of : , incubated for hours at °c, and then purified by gel-filtration chromatography using the superdex high performance column (ge healthcare). the complex was collected and concentrated to ~ mg/ml in hbs buffer for crystallization. crystals were successfully grown at °c using the sitting drop vapor diffusion method, which involved mixing equal volumes of protein and reservoir solution containing . m mes monohydrate (ph . ) and % (w/v) polyethylene glycol , . all crystals were cryoprotected by soaking in reservoir solution supplemented with % glycerol for several seconds and then were cooled in liquid nitrogen. diffraction data were collected on the bl u beamline at the shanghai synchrotron research facility (ssrf) and processed with hkl . all data collection and processing statistics are listed in table . structural determination and refinement. the structure was determined by molecular replacement with the crystallographic software phaser . the search models are the mers-cov rbd structure (pdb id: l ) and the structures of the variable and constant domain of the heavy and light chains available in the pdb with the highest sequence identities. iterative refinement with the program phenix and model building with the program coot were performed to complete the structure refinement , . structure validation was performed with the program procheck , and all structural figures were generated with pymol . all structure refinement statistics are listed in table . surface plasmon resonance analysis. real-time binding and analysis by surface plasmon resonance (spr) were conducted on a biacore t instrument (ge healthcare) at °c. the mers- fab was immobilized on a research-grade cm sensor chip by the amine-coupling method. flow cell was left blank as a reference. mers- fab ( μ g/ml) in mm sodium acetate ph . was immobilized to response units on the flow cell . for the collection of data, mers-cov rbd and its mutants were injected in a buffer of mm hepes, ph . , mm nacl, and . % (vol/vol) tween- over the flow cells at various concentrations with a μ l/min flow rate. the mers-cov rbd and mers- fab complex was allowed to associate for seconds and dissociate for seconds. data were analyzed with the biacore t evaluation software by fitting to a : langmuir binding model. pseudoviruses were generated in hek t cells by co-transfection of the human immunodeficiency virus backbone expressing firefly luciferase (pnl r-e-luciferase) and the mers-cov spike glycoprotein expression vector (pcdna . + , invitrogen) into hek t cells. the mutant spike glycoprotein expression vectors were generated using site-directed mutagenesis, and mutations were confirmed by sequencing. the viral supernatant was harvested hours after transfection and was then normalized using a p elisa kit (beijing quantobio biotechnology co., ltd, china). mers-cov pseudoviruses bearing wild-type or mutant spike glycoprotein were used to infect huh target cells endogenously expressing hdpp . the infected huh cells were lysed hours after infection and viral entry efficiency was quantified by comparing the luciferase activities of the pseudoviruses bearing the mutant and the wild-type mers-cov spike glycoproteins. for viral infection into cos cells, the cells were transiently transfected with pcdna . + vector encoding wild-type or mutant hdpp . the transfected cos cells were then incubated with goat anti-hdpp polyclonal antibody (r&d) followed by incubation with fluorescein phycoerythrin (pe)-labeled rabbit anti-goat igg antibody (santa cruz). the expression levels of wild-type and mutant hdpp were measured by flow cytometry (bd aria ii), and the mean fluorescence intensity (mfi) was analyzed. the cos cells infected by mers-cov pseudoviruses were lysed hours after infection and viral entry efficiency was quantified by comparing the luciferase activities of pseudovirus-infected cos cells expressing wide-type and those expressing mutant hdpp . neutralization assays were performed by incubating tcid (median tissue culture infectious dose) of pseudoviruses with serial : dilutions of purified antibody at °c for hour. huh 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camels: an outbreak investigation severe acute respiratory syndrome middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk the molecular biology of coronaviruses dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc a predicted receptor-binding and critical neutralizing domain in s protein of the novel human coronavirus hcov-emc the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a -residue region in the spike protein that efficiently elicits neutralizing antibodies structure of mers-cov spike receptor-binding domain complexed with human receptor dpp molecular basis of binding between novel human coronavirus mers-cov and its receptor cd potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus bat origins of mers-cov supported by bat coronavirus hku usage of human receptor cd processing of x-ray diffraction data collected in oscillation mode method phaser crystallographic software phenix: building new software for automated crystallographic structure determination coot: model-building tools for molecular graphics procheck: a program to check the stereochemical quality of protein structures we thank professor jianhua. he and scientists at ssrf bl u beam line for assistance in diffraction data collection. we also thank dr. shilong fan and the china national center for protein science (beijing) for providing the facility support. this work was supported by the ministry of science and technology ( cb , cb , and cb - ), the national science and technology major projects ( zx - ), and the national natural science foundation of china ( and u ). x.y., s.z., y.c. and d.l. expressed, purified and crystallized the protein. l.j. and l.f. carried out the pseudovirus entry and antibody neutralization analyses. x.y. and d.w. carried out the spr analysis. x.y. and s.z. collected and processed the diffraction data. x.w. carried out the structural determination and refinement. x.y., l.z. and x.w. wrote the manuscript. x.w., l.z., n.w., x.s. and z.l. supervised the project and/or commented on the manuscript. accession codes: the coordinates and structural factors have been deposited into the rcsb protein data bank. the psb accession code is zs . key: cord- -l dm pp authors: santhakumar, diwakar; rohaim, mohammed abdel mohsen shahaat; hussein, hussein a.; hawes, pippa; ferreira, helena lage; behboudi, shahriar; iqbal, munir; nair, venugopal; arns, clarice w.; munir, muhammad title: chicken interferon-induced protein with tetratricopeptide repeats antagonizes replication of rna viruses date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: l dm pp the intracellular actions of interferon (ifn)-regulated proteins, including ifn-induced proteins with tetratricopeptide repeats (ifits), attribute a major component of the protective antiviral host defense. here we applied genomics approaches to annotate the chicken ifit locus and currently identified a single ifit (chifit ) gene. the profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. this highly virus- and ifn-responsive chifit protein interacted with negative sense viral rna structures that carried a triphosphate group on its ′ terminus (ppp-rna). this interaction reduced the replication of rna viruses in lentivirus-mediated ifit -stable chicken fibroblasts whereas crispr/cas -edited chifit gene knockout fibroblasts supported the replication of rna viruses. finally, we generated mosaic transgenic chicken embryos stably expressing chifit protein or knocked-down for endogenous chifit gene. replication kinetics of rna viruses in these transgenic chicken embryos demonstrated the antiviral potential of chifit in ovo. taken together, these findings propose that ifit specifically antagonize rna viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance. innate immune responses, primarily triggered by interferons (ifns) and their antiviral effectors, can establish an extremely potent antiviral state to efficiently restrict virus replication and virus-induced pathologies in the susceptible host . to initiate these innate responses, viruses are detected by the host pathogen recognition receptors (prrs) through recognition of viral molecular signatures known as pathogen associated molecular patterns (pamps) . pamps associated with most viruses include viral double-stranded rna (dsrna), which is produced as replicative intermediate and single-stranded adenosine/uridine (au)-rich regions . these pamps effectively activate cascades of signalling events that culminate in the production of ifns in virus-infected cells. the released ifns transcriptionally activate hundreds of ifn-stimulated genes (isgs) in uninfected neighbouring cells that instigate direct or indirect antiviral activities , . well-studied examples of these antiviral effectors are dsrna-activated protein kinase r and ′- ′ oligoadenylate synthetase which bind to dsrna, and interferon induced proteins with tetratricopeptide repeats (ifit)- and - that bind to ′-triphosphate containing rna , . ifit genes are evolutionary conserved and are originated possibly by gene duplication . all members of the ifit family consist of multiple tetratricopeptide repeats (tpr) throughout the length of the protein that are mainly responsible for the protein-protein interaction and assembly of larger protein complexes , . most mammals encode several ifit genes including ifit /isg , ifit /isg , ifit /isg and ifit /isg , however, mice and rats lack ifit and horses lack ifit . amongst all known members of the family, ifit and ifit are highly responsive both at the transcription and translation levels to diverse cellular stresses including those induced by dsrna, virus infections and lipopolysaccharides , . the ifit proteins family is responsible for diverse array of cellular activities including nucleic acid sensing and direct antiviral effects . specifically, ifit is proposed to be a negative-feedback regulator of virus-triggered induction of type i ifns and cellular antiviral responses whereas ifit potentiate antiviral signalling . additionally, mammalian ifit can sense and bind to numerous short cellular rnas such as initiator trna, and these interactions are mapped across the protein surface . the most prominent features of ifit proteins are attributed to their involvements in the inhibition of virus replication through nucleic acid sensing and leading to possible inhibition of translation , . ifit protein, in addition to ifit , discriminates the cellular and viral mrna for initiating downstream antiviral activities by recognition of discrete features at the ′ termini , . most eukaryotic cellular ribosomal and transfer rnas (rrnas and trnas) carry monophosphate at ′-termini whereas messenger rnas (mrnas) bear n -methlguanosine cap (cap ) attached to the first base through ′- ′ triphosphate bridge that recruits cellular factors involved in rna processing and translation initiation , . additionally, in most higher eukaryotes methylation occurs at the ′-o position of the first or second base yielding the cap (m gpppnmn) or cap (m gpppnmnm) structures, respectively . although cap and cap are not crucial for mrna translation, human and murine ifit protein can inhibit translation of cap -lacking mrna , . these features of cellular rnas are also mimicked by several viruses as countermeasure strategies . however, viral genomic and subgenomic rna of negative sense single-stranded viruses such as influenza a viruses (iav) and newcastle disease viruses (ndv) bear triphosphate at the ′-termini . these pamps are sensed by cellular prrs and initiate innate immune responses, which ultimately restrict virus growth , . significant genetic, functional and structural features have been recently attributed to the mammalian ifit genes and proteins [ ] [ ] [ ] [ ] . there is limited information available on the repertoires of cellular proteins that recognizes different populations of viral nucleic acid in avian species especially in chickens which differ significantly in mounting innate immune responses and are infected by pathogens that continuously pose zoonotic threats to public health . here, we have genetically characterized chicken ifit and revealed that chickens, in contrast to other vertebrates, encode only one ifit gene (chifit ). the chifit gene was transcriptionally highly responsive to both type i ifns and rna viruses (iav and ndv) and interestingly this responsiveness was mapped to isre motif in the cis-acting elements. we also demonstrated that chifit specifically interacts with ssrna carrying ′-ppp moiety. through this interaction, chifit applies strong antiviral activities in lentivirus-transduced stable cell lines whereas crispr/cas -mediated chifit knockout promoted virus replication. finally, employing the rcas-based retroviral gene transfer vector system , we generated transgenic chicken embryos expressing chi-fit and demonstrated its antiviral potential in ovo. these findings were further evaluated by rcas-mediated gene silencing in developing transgenic chicken embryos by assessing the replication kinetics of rna viruses. these analyses provide evidence of the presence of a functional homologue of ifit and expand our understanding on the breaths and dynamics of nucleic acid sensing in chicken. genomic annotation of ifit locus revealed that chicken genome encodes ifit gene. the genome of all major mammalian species (including human, mouse, dog and horse) encodes for ifit genes, however, these genes are only genetically and functionally characterized in a limited number of species , . the human genome encodes four ifit genes (ifit , ifit , ifit and ifit ) whereas rats and mice lack ifit and horses lack ifit (fig. a ). in addition, several pseudogenes have been identified in different animal species including ifit b (in human, mice and rabbits), ifit c (in mice), ifit -like (in dogs and mice) and ifit -like gene (in dogs and rabbits). to identify corresponding ifit homologues in chicken, initially several ifit genes from human and mouse were used in the blast algorithm in the ensembl database. based on high genetic similarity with huifit , only a single gene (ensgalt ) was identified in the chicken genome (fig. a) . the ifit locus, encoding all ifit genes, is primarily mapped in between the lysosomal acid lipase/cholesteryl ester hydrolase (lipa) and pantothenate kinase (pank ) genes. since only a single ifit gene (ifit ) was identified in the chicken genome, we next examined an approximately . kb genomic sequence spanning aadn . contigs in the chicken chromosome (chromosome : , , - , , ). immediately adjacent to the lipa gene, a sequence gap was identified whose estimated length was bps in the ensembl chicken genome build (ensembl release -july ) ( supplementary fig. a ). long primers (supplementary table ) flanking each end of the gap were used to amplify genomic fragments and subsequent sequencing of the products was used to cover the genomic gap. we used the sequenced fragments as input in webaugustus and predicted any possible genes in the entire ifit locus. only one gene (ifit ) was predicted in any strand of the input dna using chicken as species parameters ( supplementary fig. a ). the genomic gap has now been filled with the latest ensembl chicken genome (ensembl release -dec ) and no gene other than ifit has been identified (until april ). to confirm the sequence of the cdna and to identify the genomic structure of the identified ifit gene, the transcript was amplified from rna extracted from ndv-infected cefs. complete sequence analysis of the gene revealed an open reading frame (orf) of bps ( amino acids excluding stop codon) with high sequence identity ( % and %) with the human and duck ifit proteins, respectively ( supplementary fig. b) . the identified gene showed a characteristic exon/intron organization where two exons were separated by a few kilobases (kb) long intron (fig. b) . the first exon encodes barely a kozak sequence (catg) and ′ untranslated region (utr). the second exon codes the rest of the orf ( bps) and ′ untranslated regions (utr) for chicken ifit gene. based on the transcriptomic data from marek's disease virus (mdv, strain rb b)-infected cefs (fig. b) and cdna sequencing data, a complete ifit gene was annotated. phylogenetic analysis of characterized chicken (ch) ifit gene with all known human, mouse and duck ifit genes clustered the chicken gene with scientific reports | ( ) : | doi: . /s - - -y duck and human ifit (fig. c) . one of the structural hallmarks of all ifit proteins is the presence of multiple trp motifs dispersed throughout the length of the protein , . the consensus chicken, duck and human ifit sequences were used to predict trps using ncbi's conserved domain database. both duck and human ifit proteins structurally carried ten trp motifs and two multi-domains whereas chicken ifit encoded eight predicted and ten structure-based tprs (fig. d) . taken together, the gene synteny, genetic similarity, genomic architecture and annotation indicate that chickens encode only one ifit gene compared to at least four in mammals. based on genetic clustering, it is highly similar to ifit genes of other avian (chickens and ducks) and mammalian species. moreover, no ortholog for ifit , ifit , ifit and no pseudogenes were identified in the current ensembl chicken genome build (ensembl release -dec ). mouse ifit protein is predominantly expressed in the cytoplasm and upon stimulation with the ifn, it accumulates in the cytoplasm (> × molecules per cell), and this abundance is identified to be crucial for its antiviral function . beside the distribution pattern of mouse ifit , no other ifit proteins have been investigated for their subcellular distributions. to delineate the expression dynamics and sub-cellular distribution, df- cells were transfected with v -tagged chifit and transient expression of chifit was compared in ndv-stimulated or mock-treated cells. confocal microscopy figure . genomic architecture along with relative loci around ifit genes in human, mouse, dog, horse and chicken, and gene annotation in chicken. (a) the ifit locus in compared species is flanked upstream with lipa gene and downstream with pank gene. direct syntenic analysis identified a single ifit gene in chicken compared to four in other species aslong with other pseudogenes. (b) transcriptomics profiling of chicken primary fibroblasts which were mock-infected or infected with rb b strain of mdv. blue bar represents the transcript in the current chicken genome assembly whereas the red bar represents the mappability of the transcript to the chicken genome. a final transcript from mdv-infected data and gene characterization is shown at the bottom. (c) phylogenetic analysis of ifit genes in different species. based on the clustering patterns and sequence homologies, the single identified gene clustered closer to ifit of duck and human. (d) putative tetratricopeptide repeats (tpr) showing characteristic features of ifit proteins. (e) expression and subcellular distribution of chifit in chicken embryo fibroblasts. chicken cells were transfected with ng of mammalian expression vectors encoding v -tagged chifit for hours and were left untreated (ndv-) or were treated with moi of ndv-gfp (ndv+) for another hours before fixation, staining for nucleus (blue), chifit (red) and gfp marker (ndv). scientific reports | ( ) : | doi: . /s - - -y using anti-v antibodies showed that chifit was exclusively cytoplasmic and was expressed throughout the cytoplasm; however, this expression was concentrated at the cell surface (fig. e, upper panel) . we were unable to demonstrate the distribution pattern of chifit under ndv infection (fig. e , lower panel) probably due to the profound antiviral state induced by chifit so that infection of ndv in the chifit -expressing cells could not be achieved. it has previously been demonstrated that human ifit enhances the innate immune responses by interacting with rig-i (not identified in chicken) and mavs and this interaction occurs at the mitochondria . since mavs localizes on both mitochondria and endoplasmic reticulum (er)-derived membranes (mem), we labelled mitochondria and er in presence of chifit to assess the distribution and localization of chifit with mavs. in df- cells, chifit localized in close proximity to the mitochondria and no co-localization was observed between er and chifit (data not shown). owing to lack of cross-reactivity of human ifit antibodies and specificity of anti-sera which we have raised against chifit , these experiments were performed using tagged-chifit . therefore, future studies are required to assess the expression patterns of the endogenous chi-fit under virus or non-virus stimuli. transcriptional profiling of chifit in vitro and ex vivo. we next assessed the nature of ligands that can transcriptionally induce the expression of chifit . different ligands that can either directly induce the transcription of ifit such as ifn-β or stimuli that result in the expression of ifns, which in turn induce the ifit transcription, were assessed ( fig. a) . tlr (lipopolysaccharide, lps) and tlr (poly i:c, synthetic dsrna) ligands significantly induced the expression of chifit gene by and folds, respectively (fig. b) . the induction is likely through the activation of ifns since the chifn-β stimulated cells profoundly increased the transcription of chifit by folds compared to untreated or mock-treated cells. there are evidences that negative sense single stranded rna viruses (e.g. influenza and ndv) produce dsrna as intermediate by-product during the virus replication cycle , - . therefore, it is plausible that induction of chifit expression in chicken cells infected with ndv is mediated by virus-generated dsrna (fig. b ). to further assess the temporal effect of ndv infection on the induction of chifit , a time course profiling ifn and ifn-regulated genes was evaluated. the virus-induced expression of chifit was profoundly observed as early as hours post-infection (fig. c ) and the expression was maintained for hours. a slight reduction in ifit expression was observed at hours post-infection (hpi) was reconstituted at hpi (fig. c ). this biphasic expression of ifit after virus infection was repeatedly observed in chicken cells. however, the pattern of expression of myxovirus-resistance protein (mx) gene, which is another well-characterized isg , shows a steady up-regulation and peak expression was observed at the latest time post-infection (fig. e ). the levels of chifn-β gene induction was proportional to the ndv replication (fig. d) , therefore, it can be inferred that the virus-induced expression of chicken ifit is ifn-dependent and that chifit is an early-isg with capacity to modulate initial steps of virus life cycle. to investigate transcriptional activation of ift following virus infection in chicken, we analysed a panel of rna transcripts derived from selected tissues (liver, kidney, spleen, beak, trachea, lungs and duodenum) of chickens ( -weeks-old rhode island red) which were mock-infected or infected with h n avian influenza virus strain a/chicken/pakistan/udl / (udl /h n ). all tissues from infected birds contained relatively higher levels of chifit compared to the corresponding tissue samples derived from mock-infected chickens (fig. g) . these results conclude that virus infection positively regulates the transcriptional dynamics of chifit in chickens. promoter structures contributing to the ifn, dsrna or virus-mediated transcriptional activation of chifit gene. most studied-ifit genes respond to ifn or ifn stimuli through one to four ifn-stimulated response elements (isre), which are mainly present within base pair (bp) of the transcriptional start site in the orientation of the encoded gene or in the reverse complement order . on the other hand, several ifit genes lack isre motifs in their promoters including ifit from chimpanzee, ifit b from human, chimpanzee and dog, and ifit from horse, chimpanzee and dog . since our data and previously published transcriptomics studies support the profound expression of ifit gene against a wide range of stimuli, we analysed the ′ flanking region of chifit gene for motifs that regulate the expression of chifit . in addition to the gene-encoding sequences, approximately . kb sequence including the putative promoter (supplementary fig. a ) was isolated and sequenced. inspection of the promoter region revealed the presence of two consecutive isre motifs within bps from the transcriptional start site ( fig. a and supplementary fig. b ). these elements were preceded by tata element, and the binding motif for specificity protein (sp ) transcription factor. a putative and weak ifn-gamma-activated site (gas) was identified at the distal end of the promoter and six consecutive gaaann elements were predicted between gas and sp motifs (fig. a) . gas is essential for ifn-gamma-mediated transcription of the genes whereas gaaann elements have been demonstrated to regulate the virus-induced and type i ifn-regulated genes via the binding of interferon regulatory factors , . to determine the role of these cis-acting regulatory elements in the responsiveness of different ifn ligands and stimuli, the entire promoter region ( bp) was cloned upstream to the luciferase gene in a promoter-less pgl . -basic vector (fig. a) . additionally, five reporter constructs were generated each containing either all of the gaaann elements, sp , tata box, double or single isre promoter sequences (fig. a,b) . luciferase reporter assays were performed to assess whether the upstream region of the chifit gene can mediate responses to stimuli from chifn-β, dsrna or virus infection. df- cells were transiently transfected with these reporter plasmids and subsequent luciferase activity was monitored after stimulation with either chifn-β (fig. c) , dsrna ( fig. d ) or with ndv (fig. e ). similar to ifn-induced promoter activation of chmx gene , the chifn-β induced -fold higher luciferase activities in full-length promoter (fig. c ). however, further deletion of gas, gaaann, sp and tata box resulted in an approximately -fold reduction in basal transcription activity. additional deletion of the distal isre reduced the ifn-responsiveness of the promoter. however, repetitive luciferase assays demonstrated that isre motif just proximal to the transcriptional start site was exceedingly responsive to the ifn-treatment with a highest of -fold induction in luciferase activity as compared to the control vector without a promoter sequence (fig. c ). in the dsrna-dependent promoter induction (fig. e ), results demonstrated that the above elements and motifs (gaaann, spi and tata box) are least important in controlling the transcriptional activation of luciferase gene and a non-significant difference was observed in the absence or presence of these motifs. however, promoter sequence containing dual isre motifs severely compromised the responsiveness of dsrna on the ifit promoter and the dsrna-dependent promoter induction was restored when distal-isre was deleted, further suggesting the importance of this isre motif (fig. e ). similar to dsrna induction, the construct containing only one isre motif was highly inducible by ndv compared to the construct carrying both isre motifs (fig. e) . as was observed in the ndv-induced chmx promoter activation, all other constructs with variable lengths and motifs were also responsive to virus infections. comparison of these three stimuli indicated lower luciferase activities in ndv and dsrna-treated chicken cells compared to chifn-β induced promoter activation, presumably due to indirect activation of ifit promoter by inducing endogenous ifns. following the demonstration of robust reporter function of the chifit promoter-containing proximal isre motif by the chifn-β, dsrna and ndv, we compared the sequence of cis-acting regulatory elements with the previously characterized chmx promoter . in silico analysis of highly responsive bp sequence indicated that the isre motif ( ′-gctttcactttct- ′ at position − to − in pchifit − /+ construct) was identical to the consensus isre (g/a/t)g/ctttcn - tttc(a/t/c) found in most of the ifn-inducible genes including chmx promoter sequence (fig. a ). for further absolute delineation of importance of this motif, we mutated both triplet thymidines (ttt), the core residues for the ifn-inducible activation of genes ( fig. b and supplementary fig. c ). responsiveness of the wild type (wt) and mutated isre motifs was assessed in chicken df- cells with or without stimulation by chifn-β, dsrna or ndv using luciferase assays. while both pchifit - /+ and pchifit - /+ -mut promoters were not auto-stimulatory, stimulation with either of the stimuli positively regulated the luciferase genes in pchifit - /+ . interestingly, stimulation of mutated promoter with chifn-β ( . . chicken and human ifit restricts the replication of rna virus in stably expressing chicken fibroblasts. to assess the antiviral potential of chifit protein, we generated a lentivirus construct which bicistronically expresses chifit and a red fluorescent protein tagrfp under the control of emcv (encephalomyocarditis virus) internal ribosome entry sequence (ires) . cef cells were transduced with the appropriate vsv-g pseudotyped lentiviral particles and infected with gfp expressing ndv. after hours, the virus infectivity (gfp+) was quantified in lentivirus transduced (rfp+) cell population using fluorescence-activated cell sorting (facs) (fig. a ). lentivirus expressing firefly luciferase (ffluc), not expected to affect the virus replication, was used as a negative control. human irf (huirf ), a potent virus restriction factor , was used as a positive control ( fig. b and supplementary fig. a ). compared to ffluc control, both huirf and chifit significantly inhibited ndv replication (fig. b,c) , while the antiviral effect of chifit was greater than that of huirf in cefs. since two populations of gfp+ cells were observed, further gating of low gfp+ and high gfp+ indicated a profound and cumulative antiviral effect compared to ffluc control ( supplementary fig. b ). these results showed that the chifit -mediated antiviral effect is not profound at individual cell levels but it attenuates replication of the virus after initial infection. to compare anti-viral effects of chifit (only ifit gene identified in chickens so far) with its orthologous and homologous human proteins, huifit , huifit , huifit and huifit were expressed in chicken cells and antiviral affect was monitored (fig. a ). similar to chifit , both huirf and huifit showed significant inhibition of ndv replication whereas no antiviral activities were observed by other huifit proteins in chickens (fig. d ). to further demonstrate the effect of ifit -induced inhibition of ndv replication, we observed an exclusive replication of either ndv (gfp+) or transduction of lentivirus expressing ifit (rfp+) in % cells compared to ffluc expressing cells (n = ) (fig. e, upper panel) . it is possible that transduction of lentivirus particles may interfere with the virus entry and/or ndv replication. to examine the lentivirus-mediated restriction of ndv replication, we used ffluc-transduced cells in the control group. the analysis of several microscopic fields showed simultaneous expression of lentiviruses-transduced ffluc protein and ndv infection (fig. e , lower panel and fig. b ), demonstrating that the transduction of lentivirus particles doesn't interfere with the ndv replication. collectively, these results demonstrate that ifit proteins of both human and chicken are potent cellular restriction factors against rna virus infection in chicken primary cells. to further evaluate the antiviral effect of chifit on virus replication, we applied a loss-of-function approach using crispr/cas genome editing technology. using synthetic grna and cas expression plasmid (fig. a) , the chifit was targeted for editing. df- cells were co-transfected with hybridized crrna and tracr-rna as well as a vector expressing hspcas and puromycin resistance marker (puro r ) gene. for fast and efficient enrichment of genetic modification, a population of stably-integrated cells was selected with puromycin, and was further enriched using facs (fig. a) . the relative frequency of gene editing in the puromycin-resistant and facs-enriched cell was estimated from a dna mismatch detection assay using t endonuclease i (t e ) (fig. b) . t e assay showed a mutation frequency of % within the chifit gene, however, t e assays are likely to underestimate the fold enrichment . we subsequently sequenced the mutated sites and confirmed the in-frame or out-of-frame gene editing (fig. c) . results of the t e assay and sequencing showed that sgrna sufficiently edited the gene and puromycin selection greatly improved the enrichment of the cells. additionally, most of the mutations in the chifit gene appeared to be deletions, which introduce a pre-mature stop codon to the beginning of exon of the chifit gene. the chifit deletion-confirmed df- cells (chifit ko df- ) were isolated, expanded and assessed for virus replication. both wt df- and chifit ko df- were infected with ndv and vsv, and replication of viruses was monitored for hours. both ndv (fig. d) and vsv (fig. e ) replicated efficiently in wt df- cells over the time course of infection ( to hours). however, deletion of chifit by crispr/cas further supported the replication of ndv (fig. d) and vsv (fig. e) , confirming that chifit is a crucial antiviral effector and elimination of such factors weakens the hostile barriers of the host. these results also highlight the possible exploitation of innate immune genes in promoting virus replication for vaccine production. additionally, we applied facs to quantify the percentage infectivity of the wt df- and chifit ko df- cells for gfp-expressing recombinant ndv. data demonstrated that the wtdf- cells infectivity by ndv ( . %) could further be enhanced ( . %) in the absence of chifit (supplementary fig. ) . cumulative quantitative measurement of ndv-infectivity was significantly enhanced in chifit ko df- cells compared to wt df- cells (fig. f ). next, in order to counter-confirm the antiviral potential of chifit , the level of vsv replication was assessed in conventional plague assay in df cells either overexpressing or knocked out with chifit (fig. g) . the vsv replicated effectively in wt df- cells and overexpression of chifit suppressed the vsv whereas crispr/cas knockout df- cells substantially supported the virus replication (fig. g) . together, these results confirm the potential of chifit as an important host restriction factor, at least against evaluated rna viruses. chicken ifit interacts with ′ppp-containing rna. different human and mouse ifit proteins (ifit , ifit , ifit and ifit ) interact with rna carrying multiple genetic modifications at their ′ ends , , - . since ifit was the only identified ifit protein in chickens, we aimed to explore whether chifit interacts with rna in a similar mechanism reported for huifit or chifit -rna interaction is redundant to other members of ifit family. in order to explore the molecular mechanisms of chifit recognition of rna, we used bp rna without any known similarity to viral rna sequences and structure, and generated (i) rna bearing terminal ′ hydroxyl group ( ′ohrna), and (ii) rna bearing ′ triphosphate ( ′ppprna) (fig. a) . these populations of rna, mimicking viral rna ends, were biotinylated and coupled with agarose beads. the beads were then incubated with chicken df- cells expressing v -tagged chifit , the ribonucleoproteins were purified (fig. b ) and the interaction of chifit was determined by staining chifit . while neither huifit nor chifit interacted with the ′ohrna, both proteins recognized rna carrying ppp at the ′ end (fig. c) . overexpression of chicken chifit in transgenic chicken embryos restricts the replication of rna virus. we next asked whether the in vitro demonstrated antiviral activities of chifit are translatable in ovo in developing chicken embryos. for this purpose, we constructed rcasbp(a)-chifit recombinant viruses to generate mosaic-transgenic chicken embryos that are constitutively expressing chifit , and monitored its impact on the replication of ndv (fig. a) . in addition, a reporter virus, rcasbp(a)-egfp, was generated to monitor the rescue of the virus and to investigate the effect of retroviruses on host responses. rcasbp(a)-chifn-β, expressing chifn-β which is a known antiviral cytokine , was generated as a positive control (fig. a) . expression of this transgene did not compromise the replication of retroviruses, and the induction of innate immune responses was not significant (data not shown), confirming previous reports that rcasbp is a safe in ovo gene transferring system , . stable cells lines expressing retrovirus-mediated chifit and chifn-β were used to monitor virus replication. while mock-infected or rcasa(bp)-wt infected df- cells did not interfere with the replication of ndv (fig. b) and vsv (fig. c) , stable expression of chifit and chifn-β established a profound antiviral state against both viruses over the time course of infection. these functionally validated infectious df- cells were used to generate transgenic chicken embryos. three-day-old embryonated chicken eggs were inoculated with wt retroviruses or viruses that were expressing chifit or chifn-β to generate mosaic transgenic chicken embryos (fig. d) . additionally, embryos were either mock-inoculated with pbs or with non-infectious df- cells to exclude the possibility of antiviral effects by the retrovirus infected df- cells. these five groups of embryonated eggs were either mock-challenged with pbs or virus-challenged with ndv, and were incubated for another five days (fig. d ). compared to controls, retroviral expression of ifit negatively affected the development of healthy embryos until day post-embryonation before challenge with ndv (fig. e) . quantitative analysis of ndv in allantoic fluids showed that the wt retroviruses or non-infectious df- cells alone were unable to interfere with the replication of ndv, however, transgenic embryos stably expressing chifit or chifn-β significantly restricted the virus replication compared to the mock-treated control (fig. f) , indicating that chifit can restrict virus replication in developing transgenic chicken embryos. collectively, these results further confirm the antiviral activity of chifit in ovo. ablation of chicken chifit in transgenic chicken embryos ameliorates the replication of rna viruses. next, we asked whether silencing of endogenous chifit in developing embryos would facilitate the replication of ndv. for this purpose, we streamlined two individual gene delivery protocols for specific and optimal gene silencing in chicken cells and embryos. first, a total of three short hairpin rnas (shrna) targeting the chifit and a scrambled shrna with a highly confident predicted score were cloned in the prf-prnaic as described before (fig. a) . transfection of chicken df- cells with these vectors expressing shrna under a chicken u promoter showed a reliable level of expression of the rfp marker gene indicating the functional integrity of the system (fig. b) . although all shrna were effective in silencing, quantitative analysis of ndv-induced chifit mrna showed that shrna# was the most effective (> %) (fig. d) . next, the validated shrna cassette that included chicken u promoter, shrna, leader and trailer sequences was transferred to the compatible rcasbp(a) vectors for silencing of chifit in ovo (fig. c) . additionally, we used shrna silencing-resistant rcasbp(a)-chifit retroviruses ( supplementary fig. ) to stably express the chifit in developing embryos in the presence of shrna against endogenous chifit gene. the replication competency of shrna expressing retroviruses was compared to the wt retroviruses (fig. e ) before using them to generate transgenic chicken embryos. using the experimental strategy depicted in fig. f , we monitored the replication of ndv in transgenic embryos that were either stably expressing chifit or were expressing chifit in the endogenously silenced ifit gene. the retroviral overexpression of chifit or silencing of chifit were normalized to non-infectious df- cells or wt rcaspb(a) that did not express the transgene. quantitative analysis of ndv showed an expected reduction in replication of the virus in ifit -overexpressing embryos, whereas silencing of endogenous chifit favoured the replication of ndv compared to the control df- cells (fig. g) . a moderate reduction in the virus replication was observed in transgenic embryos that were simultaneously silenced for endogenous chifit and were stably expressing silence-resistant chifit (fig. g) . taken together, these results not only confirm the antiviral activities of chifit in different experimental settings but also highlight the potential use of rcaspb(a) in enhancing the replication of avian viruses in embryonated eggs. innate immune responses are key to the success of host resistance to virus infection and isgs provide a front-line role in these defences by acting at multiple stages of the virus replication cycle [ ] [ ] [ ] [ ] . among the myriad of isgs, members of the ifit family have attracted recent attention for both immunological as well as virological reasons wild type and chifit ko -confirmed df- cells were infected with ndv or were left uninfected for hours before processing them for facs. cumulative mfi of gfp positivity in wild type and chifit ko df- cells based on at least independent experiments. (g) df- cells were either mock transfected (wt df- cells) or were transfected with ug of chifit expression plasmid (chifit oe df- cells). these cells and crispr/cas knockout df (chifit ko ) were either left un-infected or were infected with vsv for hours before staining with crystal violet. the plagues developments were imaged using hand-held camera. scientific reports | ( ) : | doi: . /s - - -y due to their abundant and profound transcription and translation responses to diverse stimuli such as ifns and viruses , . significant advances have been made during studies of the mechanisms of both human and mouse ifit proteins . however, the knowledge on the breadth and plasticity of the antiviral properties of ifit proteins are currently inadequate for non-mammalian hosts. characterizing the repertoire of ifit proteins and investigating their functions in chicken are of special interest because of the unique features of the chicken immune system including the absence of essential components of innate immune induction and signalling such as rig-i, irf , and irf in chickens. intriguingly, while chickens are lacking these components, they still mount profound innate responses against virus infections. understanding the alternative means of innate immune regulation and antiviral defences in chicken could establish the foundation to control chicken-mediated emergence of zoonotic infections such as influenza viruses . in order to explore the ifit genes in chicken, we began with genetics and functional genomic approaches and revealed that chicken encodes only a single ifit gene compared to genes in mammals (human and mice). based on its sequence and structure similarities, and phylogenetic associations, this single chicken ifit gene is classified as ifit (chifit ). currently the chicken genome is about % annotated and all chromosomes are correctly characterized , however, the possibility of orthlogous and pseudogenes in the non-annotated part ( %) of the chicken genome cannot be excluded at this stage. with regard to the complex ifn pathways and a number of major missing genes in chicken, the innate immune genes, as general sensors of self and non-self, are under continues evolutionary selection pressure compared to the pathogen-specific adaptive immune responses. therefore, it is plausible that ifit proteins are generated by paralogue expansions and/or gene deletions in chicken . ifit is the only gene that is missing in the mice and rat genomes whereas it is the only gene identified in chicken, as this study shows. it remains to be determined in future if chifit also plays additional antiviral functions of the redundant ifit and ifit . we observed a profound transcription of chifit gene by diverse stimuli that acted upon the ifn induction or ifn signalling pathways, which was further confirmed by the transcriptomics profiling of the mdv-infected chicken fibroblasts. interestingly, structural and functional characterization of the chifit promoter, required for effective transcriptional regulation of chifit , was mapped within bp of the transcriptional start site and carried a single isre motif. this minimum essential requirement of the promoter justifies several folds induction of chifit gene as early as hours of post ndv-infection. although transcription of ifit was generally proportional to the virus replication and ifn induction, an ifn-independent regulation of chifit is also possible, especially when the chifit gene up-regulation was observed in earlier time points of virus-infection when the ifn gene was barely detectable. it is also plausible that transcriptional activation of chifit is directly regulated by irf or related transcription factors and thus warrants future investigations. nevertheless, these transcriptional patterns augment essential roles of the chifit protein during the early stages of virus replication such as interaction with viral genetic material (viral rna/dna) and viral protein translation. previous rna-protein analysis revealed that all ifit proteins assemble into multimeric complexes (except ifit ) and that these interactions are crucial for co-functionalities of these proteins . while all ifit proteins can make multimers, ifit exists as a poorly characterized monomer . probably due to these structural flexibilities, reports on ifit functionalities are inconsistent , - . however, ifit protein can arguably sense a broad range of rna structures including single stranded rna with mono-(p) and tri-phosphates (ppp) at the ′ ends, double stranded dna and rna with cap modifications , , . in order to understand the binding potential of chifit to modified rna that either interacts specifically with human ifit or with human ifit , we coupled modified rna-coated beads with quantitative binding assays for chifit . our results indicated that chifit specifically interacted with rna that carried ′-ppp modifications and failed to interact with rna in which ′-ppp was replaced with oh. these rna-protein interaction studies highlighted the principal roles of chifit for direct recognition of foreign ppp-rna and to subsequently exert downstream antiviral activities. since ppp-rna is found within the genome of most viruses carrying negative sense single stranded rna genomes such as influenza, ndv, vsv , and ppp-rna is produced as an intermediate product during the replication of viruses with positive sense rna genomes such as coronaviruses , it is plausible that chifit sense foreign rna (bearing ppp-rna) while ignoring self rna (bearing cap in the case of mrna and monophosphate in the case of rrna and trna). compared to four essential ssrna cellular sensors including rig-i, tlr , tlr and ifit in mammals , , rig- is missing and tlr is disrupted due to insertion of retroviruses in the tlr locus in chickens , , . because of these fundamental differences in innate immune genes between avian (e.g. chicken) and mammals, future studies are needed to investigate if the interaction of ifit with ′-ppp-ssrna can induce downstream ifn signalling , and if so, does this interaction compensate for the deficiency of rig-i and tlr in chickens. this specific interaction with ppp-rna could lead to attenuation of virus replication by sequestering viral rna for transcription and translation. to investigate this possibility, we applied both gain-in-function and loss-in-function methodologies and evaluated the antiviral potential of ifit against negative sense single stranded rna viruses, including a poultry specific (ndv) and model rna virus (vsv). lentivirus-delivered stable expression of chifit or huifit compromised the replication of viruses, whereas crispr/cas mediated knocking-out of the chifit gene supported virus replication in chicken fibroblasts. intriguingly, overexpression of human ifit , ifit and ifit failed to establish an antiviral state in chicken fibroblasts, suggesting that chickens have opted exclusively for the antiviral activities of ifit . our attempts to monitor virus replication in mosaic transgenic chickens overexpressing chifit , or silenced for endogenous chifit yielded strong evidence that this cytokine possesses antiviral activities in developing embryos. thus rcas-mediated gene delivering and silencing approaches can be exploited to study gene functionalities in ovo at the early embryonic developmental stage and may establish the basis for evaluation of genetic resistance against pathogens. taken together, we characterized the ifit locus in chicken and systemically analysed the functional rationale for antiviral activities of chifit against rna viruses using both functional genomics and molecular biological approaches. the foundations built in this study warrant future investigations to assess the potential of chifit in sensing the nucleic acid of many diverse viruses and bacteria (which also generate ppprna), and the impact of these interactions on host innate immunity. data mining and sequence analysis. chicken genome (ensembl) and expressed sequence tags (est) databases were screened for the homologues of ifit family gene members using the basic local alignment search tool (blast) algorithm. a single transcript showing high sequence-similarity to human ifit was identified in the putative ifit locus. using sequences from public databases and transcriptomics data from marek's disease virus (mdv)-infected chicken embryo fibroblasts (cef), an open reading frame (orf) was revealed and extracted. the chicken ifit (chifit ) coding region was amplified from ndv-infected primary cefs, whereas sequence-covering gap in the ifit locus was amplified from genomic dna using primers mentioned in supplementary table . the genomic nucleotide sequence of chifit promoter region was amplified using primers provided in supplementary table . the orf and homology searches for chifit were carried out in the orf finder programme (http://www.ncbi.nlm.nih.gov/projects/gorf) and blast tool (http://www.ncbi.nlm.nih.gov/ blast/) integrated in the ncbi database. possible gene transcription start sites were identified using promoter predictor programme (http://www.fruitfly.org/seq_tools/promoter.html) whereas potential transcription factor binding sites were identified using the matinspector server (http://www.genomatix.de). gene synteny and tetratricopeptide repeats (tpr) were predicted using ensemble as well as conserved domain databases, respectively. the ifit sequences from aves and non-aves were acquired from ncbi and aligned using clustalw programme. the phylogenetic analysis was performed using neighbour-joining method with bootstrap value of n = , in the mega programme version . cells culture, media and antibodies. cefs were prepared from -day-old embryonated eggs at the pirbright institute as described previously . immortalized chicken fibroblasts (df- ), human embryonic kidney cells t (hek- t) and madin-darby canine kidney (mdck) cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with % foetal bovine serum (fbs), % penicillin and streptomycin (p/s) at °c in % co incubator. amv- c -s (gag) antibodies were purchased from hybridoma bank of iowa, university of iowa. α-v and j antibodies for the detection of v tag and dsrna were from genetex, and scicons, respectively. alexa-flour secondary antibodies were purchased from invitrogen carlsbad, ca, usa and irdye cw α-mouse secondary antibodies were acquired from li-cor, nebraska usa. poly i:c scientific reports | ( ) : | doi: . /s - - -y (a synthetic analogoue of dsrna), dimethyl sulfoxide (dmso) and lipopolysaccharide (lps), were purchased from invivogen and sigma whereas chicken ifn-β was produced in hek- t cells . (ndv-gfp) was generated using reverse genetics system as described before and rescued virus particles were propagated in embryonated chicken eggs . the ndv-gfp strain was quantified using immunostaining and expressed as focus-forming units (ffu). vesicular stomatitis virus (vsv) expressing gfp (vsv-gfp) was kindly provided by dennis rubbenstroth (institute for virology, medical centre -university of freiburg, germany). vsv-gfp was propagated and quantified in df- cells and was represented in ffu or images showing plaques. allantoic fluids and infectious viruses from cell culture supernatants were titrated by plaque assays on mdck cells. briefly, mdck cells were inoculated with -fold serially diluted samples and overlaid with . % agarose (oxoid, hampshire, uk) in overlay dmem ( × mem, . % bsa v, mm l-glutamate, . % sodium bicarbonate, mm hepes, × penicillin/streptomycin (gibco, carlsbad, ca, usa) and . % dextran deae, with µg ml − tpck trypsin (sigma-aldrich, dorset, uk). plates were incubated at °c for h and plaques were developed using crystal violet stain containing methanol. plasmids construction and mutagenesis. the full-length chicken and human ifit was pcr-amplified using primers that were tailed with ′ bamhi site, ′ ecori/spei site and a consensus kozak translation sequence (ccaccatg) (supplementary table ). the bamhi and ecori/spei digested amplicons were sub-cloned in the mammalian expression vector, pefplink-v (kindly provided by steve goodbourn, st. george's university of london), which contains an n-terminal v tag. for identification of cis-acting elements in the chifit promoter, a . kb genomic sequence was amplified and cloned into the kpni and xhol sites in the promoter-less vector, pgl . basic (promega) and named as pchifit - /+ . subsequent five truncated versions of the promoter were amplified from full-length pchifit - /+ using primers mentioned in the supplementary table and cloned between kpni and xhol sites in the pgl . basic vector. for production of the chifn-β, the orf for chifn-β (accession number, nm_ ) was cloned in pcdna . + and final constructs were labelled as pcdna . -chifn-β . the reporter plasmid pgl -p-chmx-luc was kindly provided by nicolas ruggli, switzerland and renilla luciferase plasmid (phrl-sv ) was purchased from promega, madison, wi, usa. mitochondrial (dsred -mito- , # )) and er (mcherry-er- , # ) markers were obtained from addgene. triple thymidine duplex (tttnnnttt) in pchifit - /+ -wt construct was mutated into tatnnntat using quikchange lightning site-directed mutagenesis kit (agilent technologies) and was named as pchifit - /+ -mut. all mutagenesis oligonucleotides were designed in the quikchange primer design tool and these primers are provided in the supplementary table . all clones were sequenced from both ends for correct frame and orientation or were digested with unique cleavage sites to confirm the gene inserts. confocal microscopy. chicken cells were transfected with individual or combined plasmids for indicated time points using lipofectamine (invitrogen) at a ratio of : or were infected with lentiviruses, retroviruses or ndv-gfp for indicated time points. these cells were then fixed for h in % paraformaldehyde and permeabilized using . % triton-x before incubation with primary antibodies raised against v tag, dsrna (j ), or gag protein of retroviruses. additionally, depending upon the experimental needs, different fluorescent markers (rfp, gfp) were used. afterwards, cells were incubated with corresponding secondary antibodies at °c for h. after brief staining with ′, -diamidino- -phenylindole (dap ) (nuclear), slides were visualized using a leica sp confocal laser scanning microscope. western blotting. all the transfections for subsequent western blot analysis were performed following the same protocol as described for immunofluorescence unless otherwise indicated. cells were lysed in a hypotonic buffer and protease inhibitor cocktail (sigma). proteins were separated by sds-page under reducing conditions and analysed by western blotting using anti-v (gentek) and irdye-labelled secondary antibodies (li-cor biosciences). the signals were acquired and quantified using the odyssey infrared imaging system (li-cor biosciences). ifn bioassay. ifn-induced protection against vsv-gfp was used to identify ifn-producing stable clones and to quantify ifn preparations, as described before . briefly, df- cells were seeded in -well plates until they are % confluent and treated with serial dilutions of supernatants containing interferons for hours. these interferon stimulated cells were inoculated with vsv-gfp (moi of ). at hours post-infection, vsv-gfp replication was correlated with the change in gfp fluorescence signal intensities using luminometer (promega, madison, wi, usa). the percentage antiviral activity of ifns were determined by comparing the percentage reduction of corrected gfp signal intensity (gfp signal intensity of ifn treated and virus infected wells minus background fluorescence signal intensity of uninfected control) with the mock treated and vsv-gfp-infected control wells. one unit (u) of ifn in the tested ifn preparations was defined as the volume containing % inhibitory activity against vsv-gfp. a total of us of ifns were used for stimulation of cef or df- cells. tronic expression and gateway-compatible destination vector (ptrip.cmv.ivsb.gene.ires.tagrfp) for lentiviruses was kindly provided by charles rice, the rockefeller university, usa. to generate chifit entry clone, gene encoding chifit was pcr amplified with oligonucleotides (supplementary table ) containing attb sites flanking gene-specific sequences. pcr products were purified over qiagen columns (qiagen) and cloned into pdonr (invitrogen) with bp clonase. bp clonase reactions were transformed into escherichia coli (invitrogen), and colonies were screened by restriction digestion and sequencing. the gene sequences from pentr clones scientific reports | ( ) : | doi: . /s - - -y were moved into ptrip.cmv.ivsb.gene.ires.tagrfp using lr clonase ii (invitrogen) according to the manufacturer's instructions. after lr reaction products transformation, one or two colonies for each construct were grown in ml luria-bertani (lb) broth with ampicillin, and transfection-quality plasmid dna was purified over anion-exchange columns (qiagen). lentivirus constructs expressing human ifit , ifit , ifit , ifit , irf (positive control) and ffluc (negative control) were kindly provided by charles rice, the rockefeller university, usa. all constructs were sequenced using primers provided in supplementary table to confirm the gene insertion before rescuing lentiviruses. poly-lysine pre-coated plates with seeding density of × per well of -wells plates and were co-transfected with gene expressing proviral dna (huifit , huifit , huifit , huifit , chifit , huirf , or ffluc), hiv-i gag-pol and vsv-g in a ratio of : . : . using lipofectamine (invitrogen). supernatants collected at h and h post-transfection were cleared by centrifugation ( rpm for min) and were pooled and supplemented with mm hepes and μg/ul polybrene (sigma). for titration of lentiviral pseudoparticles, cef cells ( × ) were transduced with serial dilutions of individually rescued pseudoparticles for hours. trypsinized cells were fixed with % paraformaldehyde and processed for facs for quantification of percentage rfp+ cells. the volume of the lentiviral pseudoparticles that infected % of cef cells was used to transduce cefs. titrated lentiviral pseudoparticles were stored at − °c before use and the same stock of the virus was used for all experimentation. duced with moi of lentivirus-expressing specific gene in dmem media containing % fbs, mm hepes and μg/ml polybrene. transduction was facilitated by centrifugation ( g for h at °c) and cells were incubated at °c. a day later, cells were infected with gfp-tagged virus (ndv-gfp) at . moi and the infection was stopped after hr and replaced with fresh dmem containing % fbs. after hours of infections, cells were trypsinised and the cell suspensions were incubated with live dead marker (near ir cat no: l ) according to the manufacturer's protocol. the cells were then fixed with % paraformaldehyde (pfa) for min before analysing the cells by flow cytometry. as described before , live and singlet cells were gated based on forward and side scatter, and four-quadrant plots were generated using the untransduced and uninfected (rfp negative and gfp negative), uninfected (rfp positive and gfp negative), and untransduced (rfp negative and gfp positive) cells. analysis was carried out using flowjo software applying the same gating and analysis strategies for all samples. construction of ifit knockout cell line using crispr/cas . a synthetic gene-targeting approach was applied to specifically knockout the chicken ifit from the chicken embryo fibroblasts. for this purpose, two components (crrna and tracrrna) of single guide rna (sgrna) which are crucial for targeting specificity and scaffolding/binding ability for crispr associated protein (cas ) nuclease were synthesized by dharmacon. targeting the beginning of the second exon of chicken ifit , two individual crispr rna (crrna) were designed with the highest-predicted score and lowest off-target affects; sgrna acaggagaagtctcgttacc and sgrna gcttggatctactaccacat. using dharmafect duo transfection reagents, df- cells were co-transfected with individual crrna and a common trans-activating crrna (tracrrna) as well as plasmid expressing cas nuclease and puromycin resistance gene (puro r ) separated by self-cleavage t a sequence. cells were split after hours transfection and selected for puromycin antibiotics ( μg/ml) for one week or until complete eradication of non-transfected control cells. for fast and efficient enrichment of genetic modification, a population of cells with stable integration was enriched using facs. for this purpose, puromycin-selected cells were transfected with gfp-expression plasmid and individual cells were sorted by facs before being seeded in -well plates. at least clones were expanded and the relative frequency of gene editing in the puromycin-resistant and facs-enriched cells was estimated from a dna mismatch detection assay using t endonuclease i (t e ) (neb). the dna fragments flanking the target editing sites were amplified from genomic dna extracted by dneasy kits (qiagen) using primers mentioned in supplementary table . a total of ng of the pcr products were denatured at °c and allowed to anneal gradually at room temperature to form heteroduplex dna. the re-hybridized dna was digested with t ei and resolved in a . % agarose gel to determine the gene editing efficiency. additionally, the pcr products were sequenced using pcr-amplification primers and aligned with the corresponding wild-type genomic sequence to identify mutations, deletions and insertions. t ei and sequence verified clones were used to monitor virus replication. rna was extracted from ifn-β ( u), lps ( μg/ml), dsrna ( μg/ml), or ndv-stimulated (moi of ) df or cefs using trizol reagents (invitrogen, carlsbad, ca, usa). additionally, organs were collected from specific pathogen free (spf) chickens, which were infected or mock-treated (intranasally) for days with pfu of a/ chicken/pakistan/udl- / (h n ). a total of ng of rna was used in pcr reactions using superscript ® iii platinum ® sybr ® green one-step qrt-pcr kit (invitrogen, carlsbad, ca, usa). the abundance of specific mrna was compared to the s rrna (supplementary table ) in the applied biosystems prism system. the reaction was carried out in abi light cycler using the following thermo profile; °c for minutes hold, °c for minutes hold, followed by cycles of °c for seconds and °c for seconds. melting curve was determined at °c for seconds, °c for minute, °c for seconds and °c for seconds. primers for isgs including chifit are provided in supplementary table . primers specific for a conserved region of the influenza a and ndv matrix genes were used as described previously , . short hairpin design and expression systems. to silence endogenous chifit gene in developing embryos, a total of three -nucleotides-long rna interference (rnai) short hairpin rnas (shrna) were designed using the genscript rnai target finder (https://www.genscript.com/ssl-bin/app/rnai). double stranded dna products for each of three chifit specific and a control scrambled target were generated by pcr using random and gene-specific oligonucleotides together with hp-l and hp-r (supplementary table ) as described before . the amplified pcr products were cloned between nhei and mlui into microrna (mirna) cloning sites of prfprnaic (kind gift of stuart wilson, the university of sheffield, uk). all shrna coding plasmids were sequenced to confirm the inserts and orientations. to evaluate the silencing potential of individual shrna, df- cells were transfected with ng plasmid using lipofectamine according to the manufacturer's protocol and the knockdown effects on the chicken ifit was monitored and compared with the scrambled rna transfected control. next, the validated shrna cassette that included chicken u promoter, individual shrna, leader and trailer sequences was transferred to the rcasbp(a) retrovirus vector between noti and clai sites. rescue of rcasbp(a)-sh ifit retroviruses and generation of mosaic transgenic chickens are detailed below. to determine responsiveness of chicken ifit promoter to chicken ifn-β, dsrna, and virus-stimulation, chicken fibroblasts were grown in -well plate format at × to × cells in addition pgl -p-chmx-luc was used as a positive control whereas pgl . basic vector was used as a negative control. correspondingly, df- cells were co-transfected with phrl-sv and pchifit - /+ -wt or pchifit - /+ -mut constructs. all transfections were performed using lipofectamine sk-as) was linearized with unique bamhi restriction digestion and the purified dna was used for in vitro transcription in the presence of bioin- -utp using ribomax ™ large scale rna production system-sp (promega, cat# p ) as recommended by the manufacturer and reported previously with a few modifications. briefly, a reaction of μl was established containing μl × sp buffer, μl ntp-bioutp mixtures, μg linearized plasmid, and μl enzyme mix. the reaction mixture was first incubated at °c for hours followed by digestion of the dna remnant with u rnase-free dnase (thermo scientific) for another minutes at °c. the biotinylated uracil triphosphate (bioin- -utp) was incorporated during in vitro transcription for purification of ribonucleoproteins and due to nascent nature of polymerase a ′-ppprna was over-hanged as a signature for ifit protein interaction. after the completion of in vitro transcription, the quality of in vitro transcription rna was assessed by agarose gel electrophoresis and rna was purified with rneasy minelute cleanup kit (qiagen) according to the manufacturer's recommendations. a total of μg of purified in vitro transcribed and biotinylated ppp-rna was either mock treated or rna samples were purified with rneasy minelute cleanup kit and eluted with μl nuclease-free water for use in the rna-protein interactions to prepare chifit protein, chicken df- cells ( × ) were transfected with μg v -tagged chifit plasmid for hours and lysed with tap buffer in the presence of protease and rnase inhibitors. the rna-coated beads were incubated with mg chifit protein lysate on a rotary wheel for hours at °c, and washed three times to remove unbound proteins the gfp/gag expression-confirmed cell cultures were split into cm flasks and were passaged again into cm flasks after days. finally cells were expanded into cm flasks until the desired number ( cells/egg) was achieved. mosaic-transgenic chicken embryos were generated by inoculation of one million rcasbp(a)-chifn-β, rcasbp(a)-chifit , rcasbp(a)-shrna and rcasbp(a)-wt infected df- cells at day post-embryonation or were left un-infected. at day post-embryonation ( days post-infection), each egg was either left unchallenged or was challenged with pfu ndv-gfp. embryo mortality was monitored daily and allantoic fluids were harvested at days of embryonation and were subjected to the plaque assay for virus quantification. statistical analysis. pairwise comparisons of treated and control groups were performed using student's t-test interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures cytosolic sensing of viruses interferon-stimulated genes and their antiviral effector functions interferons and their actions lineage-specific expansion of ifit gene family: an insight into coevolution with ifn gene family ifit is an antiviral protein that recognizes ′-triphosphate rna structural basis for viral ′-ppp-rna recognition by human ifit proteins interferon-induced ifit proteins: their role in viral pathogenesis isg is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response ifit potentiates anti-viral response through enhancing innate immune signaling pathways trna binding, structure, and localization of the human interferon-induced protein ifit broad and adaptable rna structure recognition by the human interferon-induced tetratricopeptide repeat protein ifit conventional and unconventional mechanisms for capping viral mrna eukaryotic ribonuclease p: a plurality of ribonucleoprotein enzymes when your cap matters: structural insights into self vs non-self recognition of ′ rna by immunomodulatory host proteins sequestration by ifit impairs translation of ′ o-unmethylated capped rna inhibition of translation by ifit family members is determined by their ability to interact selectively with the ′-terminal regions of cap -, cap -and ′ ppp-mrnas the broad-spectrum antiviral functions of ifit and ifitm proteins the rcas vector system the glucocorticoid attenuated response genes garg- , garg- , and garg- /irg encode inducible proteins containing multiple tetratricopeptide repeat domains when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna double-stranded rna is detected by immunofluorescence analysis in rna and dna virus infections, including those by negative-stranded rna viruses activation of the pkr/eif α signaling cascade inhibits replication of newcastle disease virus the chicken mx-promoter contains an isre motif and confers interferon inducibility to a reporter gene in chick and monkey cells differential expression profile of chicken embryo fibroblast df- cells infected with cell-adapted infectious bursal disease virus triggering the innate antiviral response through irf- activation the rna polymerase ii core promoter genotyping with crispr-cas-derived rna-guided endonucleases distinct induction patterns and functions of two closely related interferoninducible human genes, isg and isg crystal structure and nucleotide selectivity of human ifit /isg coordinated regulation and widespread cellular expression of interferon-stimulated genes (isg) isg- , isg- , and isg- in the central nervous system after infection with distinct viruses prolonged effect of baff on chicken b cell development revealed by rcas retroviral gene transfer in vivo antiviral activity of lambda interferon in chickens a robust system for rna interference in the chicken using a modified microrna operon association of rig-i with innate immunity of ducks to influenza rig-i in rna virus recognition a virological view of innate immune recognition identification and characterization of a functional, alternatively spliced toll-like receptor (tlr ) and genomic disruption of tlr in chickens molecular evolutionary genetics analysis version . cell-culture methods a novel cytokine with antiviral activities tissue tropism in the chicken embryo of non-virulent and virulent newcastle diseases strains that express green fluorescence protein biological characterization and phylogenetic analysis of a novel genetic group of newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in pakistan a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h and h hemagglutinin subtypes construction and rescue of ifit expressing rcas system, and generation of transgenic embryos. the orf of chicken ifit and coding regions (signal and mature peptide sequence) of the chifn-β were amplified from rna extracted from the ndv-infected primary cefs. the amplified products were sub-cloned into an improved version of rcasbp(a)-Δf (kindly provided by stephen h. hughes, national cancer institute, md, usa) via the clai and muli restriction sites which replace the src gene while maintaining the splice accepter signals. the resultant constructs were named as rcasbp(a)-chifit and rcasbp(a)-chifn-β. similarly, a gfp encoding rcasbp(a), referred as rcasbp(a)-egfp, was generated by introducing the coding sequence of the gfp in between the clai and muli sites. additionally, a codon optimized and shrna-silencing resistant chifit gene was chemically synthesized (geneart, invitrogen) and cloned in the corresponding sites and labelled as rcasbp(a)-shrna . the inserted gene orientation and sequence validity were confirmed by dna sequencing.to rescue recombinant rcasbp(a) viruses, a total of . × df- cells were seeded in cm flasks and maintained at °c, % (vol/vol) co for hours (~ % confluent). cells were washed with pbs and transfected ethics statement. all animal studies and procedures were carried out in strict accordance with the guidance and regulations of european and united kingdom home office regulations under animal risk assessment numbers ar and ar . as part of this process the work has undergone scrutiny and approval by the ethics committee at the pirbright institute. supplementary information accompanies this paper at https://doi.org/ . /s - - -y. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -jkzxjk authors: papineau, amber; berhane, yohannes; wylie, todd n.; wylie, kristine m.; sharpe, samuel; lung, oliver title: genome organization of canada goose coronavirus, a novel species identified in a mass die-off of canada geese date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: jkzxjk the complete genome of a novel coronavirus was sequenced directly from the cloacal swab of a canada goose that perished in a die-off of canada and snow geese in cambridge bay, nunavut, canada. comparative genomics and phylogenetic analysis indicate it is a new species of gammacoronavirus, as it falls below the threshold of % amino acid similarity in the protein domains used to demarcate coronaviridae. additional features that distinguish the genome of canada goose coronavirus include novel orfs, a partial duplication of the gene and a presumptive change in the proteolytic processing of polyproteins a and ab. due to the remote location of the die off, samples from the dead birds were not collected immediately and sent to a diagnostic laboratory until severe predation and decomposition had occurred. the poor sample quality, in addition to the difficulty of coronavirus isolation, led to the failure to isolate infectious virus using standard methods. however, the complete genome of a novel gammacoronavirus was assembled from high throughput sequencing reads derived from the cloacal swab of a single canada goose. the assembled genome of the novel canada goose coronavirus (cgcov) is , nts in length (excluding the poly(a) tail) and has . % gc-content. the genome of cgcov is approximately nts longer than the reference genomes for acov available in genbank. the genome organization of cgcov is presented in fig. . the ′ utr of cgcov is nt in length and contains a higher gc content ( . %) relative to the genome as a whole. the ′ utr of cgcov shares only % pairwise identity with that of duck coronavirus (dcov) and . % pairwise identity to that of sw . like all coronavirus genomes reported to date, cgcov's genome is dominated by the coding regions for the large polyproteins a and ab, followed by the structural and accessory genes. the heptanucleotide slippery sequence uuuaaac, associated with the ribosomal slippage that produces polyprotein ab, was present at nt positon , . cgcov's genome contains genes for all four structural proteins common to coronaviruses; spike (s), envelope (e), membrane (m) and nucleocapsid (n). in addition, cgcov contains open reading frames (orfs) predicted to encode accessory proteins. the order of the structural and accessory protein-coding orfs in cgcov resembles that of acov, but there are notable differences. the general genome organization of acov is ab-s- a- b-e-m- b- c- a- b-n- b . however, there is some variance in the genome organization within the acov species. for example, australian ibv strains lack orfs a, b and b . overall, cgcov contains a larger number (n = ) of orfs coding for predicted accessory and structural proteins downstream of the polyprotein ab coding region. two additional orfs ( a and b) are found between the cgcov m and n orfs. there are also two additional orfs ( and ) following the n gene. while some acovs do have orfs following the n gene, orfs and in cgcov do not share obvious homology to those of ibv and tcov. the ′ utr of cgcov is nucleotides in length and contains the stem loop-like motif bp upstream from the poly(a) tail. this stem loop-like motif was first identified in astroviruses but is also present in acovs and sars-cov . further downstream in the ′ utr, the octanucleotide motif (ggaagagc) is found bp upstream of the poly(a) tail. the ′ utr of cgcov shares % pairwise identity to the partially sequenced gcov and % pairwise identity to ibv. a trait suggesting common ancestry between cgcov and acov is the canonical acov transcription regulatory sequence (trs) found at the end of the leader sequence in cgcov. the trs of cgcov is identical to that identified by cao et al. ( ) as the trs of tcov (cttaacaaa). body trs's regulate viral gene expression by forming a complex with the leader trs, causing discontinuous transcription of mrna . ten putative body trss were found in the ′ end of the cgcov genome (fig. ) . four of the ten putative trss ( , , , ) were exact matches to the canonical leader trs. three trss ( , , ) contained one mismatch and the remaining three trss ( , , ) contained two mismatches to the leader trs. the functionality of these trss would need to be experimentally determined; however, previous studies have shown that trss of acovs are subject to some variation , . cgcov contains twice the number of trs's as acovs and a similar number compared to the nine contained in sw . table demonstrates the nucleotide distances between the trs and the start codon of orfs found in cgcov's, which are comparable to those of tcov . the start codon of cgcov's polyprotein ab is located nucleotides downstream of the leader trs. the coronavirus polyprotein ab is cleaved into - non-structural proteins (nsps) by two viral proteases . putative cleavage sites for these proteases are present in cgcov's a and ab polyproteins, with the exception of the nsp / (polyprotein a) and nsp / (polyprotein ab) cleavage sites. the missing cleavage site would be located near the end of polyprotein a, producing the nsps and , and also in the alternatively transcribed polyprotein ab, producing nsps and . the absence of the nsp / and / protease recognition site was confirmed with sanger sequencing. with the exception of the missing cleavage sites, the putative cleavage sites would produce nsps of sizes congruent with other gammacoronavirus species (table ) . no gammacoronavirus species to date, including cgcov, have a papain-like protease cleavage site between nsp - . while the genome structure of cgcov resembles that of acov, there are some notable differences. for example, there are no homologues to acov's a or b accessory proteins in cgcov, a trait shared with sw . furthermore, cgcov has a number of orfs that do not appear to have homologues in other sequenced gammacoronavirus species, such as the orfs for putative proteins and a (fig. ) . these two orfs are found in cgcov in the corresponding location of acov's a and b orfs (between the s and e orfs) and are also similar in size to acov's a and b proteins. however, they share no obvious sequence similarity with any a or b gene, or any other entry in ncbi (table ) . acov's a and b proteins have been shown to be unnecessary for replication , however knock-out mutants for these accessory genes are attenuated . the ibv's gene is functionally tricistronic, meaning the a, b and e proteins are under the control of a single trs , . this is not the case www.nature.com/scientificreports www.nature.com/scientificreports/ in cgcov, as the e orf of cgcov shares a trs with only the a orf in cgcov and orf is preceded by a separate trs (fig. ). an additional trs is also found in between cgcov's m and n orfs, preceding the proteins a and b (fig. ) . commonly acov's have two orfs between the m and genes, coding for the b and c accessory proteins. cgcov contains orfs between the m and gene (acov gene homologue). two of these orfs ( b and a) are acov b homologues, likely the result of gene duplication. this area in ibv has been identified as a hotspot for recombination . the region between the acov m and gene was formally called the intergenic region because of the lack of a trs. however, it was later shown that gene is expressed using an alternative trs in ibv . notably, one of the b homologs (i.e. b) in cgcov does have a trs (fig. ) . the use of template switching at trss is thought to lend to recombination in coronaviruses . the two cgcov b homologs are not identical to each other (table ) . amino acid sequence identity to other b proteins is low for both cgcov b homologues, % to ibv and % to dcov respectively. the gene duplication was also confirmed by sanger sequencing of the genomic region between the m orf to the gene. the acov a and b accessory proteins ( a and b in cgcov) appear to be the only accessory proteins conserved in all gammacoronavirus species, although gene order differs. orfs encoding putitive proteins a and b belong to the bicistronic gene of acovs and are also unnecessary for replication . to date, all publically available sequence information suggest that gammacoronavirus species have lost the nsp cleavage site. the function of nsp in alphacoronaviruses and betacoronaviruses is the inhibition of host protein production. accessory protein a is shown to have adopted this function in place of nsp in ibv . the majority of structural proteins of cgcov also share low amino acid sequence identity ( - %) with ibv and dcov. phylogenetic analysis of the spike gene show that the cgcov spike gene clusters with the ibv spike gene, separate from the tcov cluster (fig. a) . figure b also demonstrates the nucleocapsid gene of cgcov is distantly related to those of acovs. however the cgcov nucleocapid protein does share % amino acid sequence identity with the nucleocapsid protein encoded in the partially sequenced graylag gcov genome . in addition, orfs and , which are preceded by the nucleocapsid gene, also share high amino acid identity with graylag gcov proteins, % and % respectively. it should be noted that, among full and partial genomes of gammacoronaviruses sequenced to date, orfs and seem to be unique to cgcov and gcov and are both preceded by a trs, suggesting that these orfs are very likely expressed. the fact that some cgcov proteins share higher amino acid sequence similarity with the partial gcov sequences available suggest these two viruses are more closely related to each other than to other gammacoronaviruses known to date. the phylogenetic tree built using the coding regions for the conserved replicase and helicase domains demonstrates that cgcov clusters with gammacoronaviruses and shares a more recent common ancestor with acov than with the cetacean gammacoronaviruses (fig. ) . further comparisons suggest that cgcov is a separate species from acov. current taxonomy of coronaviridae is determined using pairwise comparisons of the amino acid sequence of seven conserved domains in the ab polyprotein. members of the same species share over % amino acid identity in these seven conserved domains . percent identity of cgcov falls well below the % threshold set by ictv with acov and sw , suggesting cgcov is a separate species (table ) . within coronaviridae, cgcov shares the highest homology ( %) in the conserved domains to the gammacoronaviruses tcov and dcov. as the full genome was sequenced from only the cloacal swab of a single canada goose, a screening pcr was designed based on the b duplication region unique to cgcov and performed on all samples. the sanger sequencing primers of the region between the m and gene were used, as this area of the genome is specific to cgcov. all samples were found to be positive, with the exception of the pharyngeal swab of the snow goose and the lung tissue of the second canada goose which could not be tested as the sample was exhausted. amplicons were sanger sequenced and confirmed to match the cgcov genome. high throughput sequencing conducted on rna extracted from cloacal swabs from the second canada goose and the snow goose also resulted in partial ( and %) genomes of the cgcov. while this does not confirm the virus's presence in all animals that perished in the die off, this shows cgcov was present in all birds that were available for testing. further studies will require the availability of an infectious virus to determine the pathogenicity of cgcov and its ability to cause mortality in canada geese and snow geese. to summarize, the complete genome of cgcov, a novel gammacoronavirus species was sequenced directly from the cloacal swab of a canada goose associated with a mass die-off. the cgcov genome was also detected in samples derived from a second canada goose and a snow goose that perished in the die-off, using pcr, sanger and high throughput sequencing. comparative genomics and phylogenetic analysis indicate cgcov clusters with acov but is a distinct gammacoronavirus species. interesting features of this new species include the cloacal and pharygenal swabs were collected from all three birds, lung tissue was collected from one canada goose. other organs were not present or were in extremely poor condition. detection of both common avian pathogens, such as avian influenza and avian paramyxovirus by the national reference laboratory, by routine laboratory testing gave negative results. virus isolation was performed by two serial passages in spf chicken eggs using protocols prescribed by the world organization for animal health (oie) for the most closely related gammacoronavirus, infectious bronchitis virus (ibv). samples were then subjected to targeted sequence enrichment and next-generation sequencing on an illumina miseq platform. sample pre-treatment. tissues were homogenized using a precellys evolution homogenizer (bertin instruments) according to the manufacturer's instructions. following a clarification by centrifugation at rpm for minutes, nucleic acids were extracted using the magmax pathogen rna/dna kit (ambion) according to the manufacturer's instructions. cdna synthesis was then performed using superscript ™ iv first-strand synthesis system (ssiv) (thermofisher) according to the manufacturer's recommendation. a total of ul of extracted total nucleic acid was mixed with dntps ( mm) and a tagged random nonamer primer ( um) (gtt tcc cag tca cga tan nnn nnn nn). samples were incubated at °c for minutes, and then placed on ice for minute. a reagent mixture of x ssiv buffer, ribonuclease inhibitor ( u/μl), dtt ( mm) and superscript ™ iv reverse transcriptase was then added. the samples were incubated for minutes at °c, minutes at °c and minutes at °c. second strand synthesis was performed using sequenase version . dna polymerase (thermofisher) according to the manufacturer's recommendation. the first strand synthesis product was incubated with ul of sequenase version . dna polymerase diluted in x reaction buffer and nuclease free water. samples were then heated to °c over five minutes and incubated at °c for minutes, followed by minutes at °c. samples were then cooled to °c and . ul of sequenase dna polymerase in dilution buffer was added. samples were again ramped to °c over five minutes and incubated at °c for minutes, followed by minutes at °c. a total of ul of the second strand synthesis product was then used as template for amplification. accuprime ™ taq dna polymerase (thermofisher) was mixed with x accuprime ™ pcr buffer i, nuclease free water and a primer for the nonomer's tag ( um). cycles of pcr were then performed with the following parameters: seconds at °c, seconds at °c, seconds at °c and minute at °c. cdna/dna mixtures were then cleaned with genomic dna clean & concentrator columns (zymo research) and eluted in mm tris (thermofisher). library preparation and sequencing. sequence libraries were prepared with the kapa hyperplus library kit (roche). sequence library construction and capture were carried out according to nimblegen's seqcap ez hypercap workflow user's guide v . samples were pooled in equal amounts by weight prior to capture. sequencing was performed on an illumina miseq instrument in the national centre for foreign animal disease biocontainment level sequencing facility. a v flow cell was used with a cycle reagent cartridge (illumina). ′ race and sanger sequencing. ′ race was used to obtain the missing leader sequence ( bp). the smarter ′ race and ′ race kit (takarabio) was used according to the kit instructions. the gene specific primer used for ′ race was tcagctacagtagagggagatgtcataggtgc. for sanger sequencing, amplicons was performed using kapa hifi hotstart readymixpcr kit (kapabiosystems). the primers ctaaagagaaggtggacactggt and ctaagaatgcgaacttcacagagc were used to amplify the gene b homologue region. the primers gttgttgtgttacaaggcaaggg and ggattatgatcaaaccatgaacctgg were used to amplify the nsp / region. cycling conditions table . comparison of the amino acid pairwise identity of conserved coronavirus domains in the poly ab protein of canada goose coronavirus to other gammacoronaviruses. a cluster of cases of severe acute respiratory syndrome in hong kong an apparently new syndrome of porcine epidemic diarrhoea discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group c coronavirus revision of the family coronaviridae. taxonomic proposal to the ictv executive committee the early history of infectious bronchitis global distributions and strain diversity of avian infectious bronchitis virus: a review characterization of turkey coronavirus from turkey poults with acute enteritis identification of a novel coronavirus from a beluga whale by using a panviral microarray discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus viral respiratory diseases (ilt, ampv infections, ib): are they ever under control? molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) complete nucleotide sequence of polyprotein gene and genome organization of turkey coronavirus infectious bronchitis viruses with a novel genomic organization a common rna motif in the ′ end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous rna synthesis identification of a noncanonically transcribed subgenomic mrna of infectious bronchitis virus and other gammacoronaviruses virus-encoded proteinases and proteolytic processing in the nidovirales infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein b neither the rna nor the proteins of open reading frames a and b of the coronavirus infectious bronchitis virus are essential for replication deletion of accessory genes a, b, a or b from avian coronavirus infectious bronchitis virus induces an attenuated phenotype both in vitro and in vivo a polycistronic mrna specified by the coronavirus infectious bronchitis virus comparisons of envelope through b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes infectious bronchitis viruses with naturally occurring genomic rearrangement and gene deletion why do rna viruses recombine? enhanced virome sequencing using targeted sequence capture trimmomatic: a flexible trimmer for illumina sequence data rapid and sensitive removal of background sequences from next generation sequencing data spades: a new genome assembly algorithm and its applications to single-cell sequencing the pfam protein families database in clustal w and clustal x version . molecular evolutionary genetics analysis across computing platforms the authors acknowledge funding from canadian food inspection agency (cfia) project win-a- and canadian safety and security program project ti- for the student stipend of a.p. the authors would also like to acknowledge michelle nebroski and mathew fisher for review of the manuscript and technical assistance. a.p. and o.l. designed the experiment and wrote the main manuscript text. y.b. and s.s. performed sample collection and routine testing. t.w. and k.w. designed the targeted sequence capture method used for enrichment of viral sequences. a.p. performed the experimental work and performed the analysis. all authors reviewed and approved the manuscript. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -dejh q authors: konishi, tomokazu title: re-evaluation of the evolution of influenza h viruses using direct pca date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: dejh q the history of influenza h virus was re-evaluated by applying a new methodology to sequencing data; this objective method enables comparisons among viral types. the approach led to the segregation of all segments of swine and human viruses into three distinct groups: two of them included the pandemic and human viruses, and the remaining group may be new in humans. these three groups might have originated from avian viruses and drifted out independently. genome shifts occurred occasionally among swine viruses; however, distances between avian and swine/human viruses negated the existence of direct shifts from avian viruses. in humans, only one or two viruses appeared each year, which suggests the presence of competition among viruses that migrated freely. all segments drifted continuously under certain rules and constant velocity. viruses that had caused an outbreak did not appear again over subsequent decades, which may mean populations had become immune to such viruses. in contrast, the viruses in livestock were rather conserved and maintained unique strains in small, separate areas. such collections of swine strains included human segments, which could become an epidemic in the future. russia viruses, respectively. black, human; green, swine, and blue, avian. the orange-coloured "h" in the m group is one of the strains classified as the "triple reassortant" . the h in the u group is the human virus found in switzerland (ncbi taxonomy id: ). ( fig. s e ). differences within a type appeared in low-level pcs; however, swine can accept the wide variations that appeared in pc and (fig. c) . the tendency to segregate into the three groups was also observed for the other genome segments (fig. s ) ; the r, m, and u groups appeared from the top, clockwise. the groups that include the segments of the two pandemic viruses, russia and mexico , are termed r and m, respectively, with u being the remaining group. as the distances among the groups are similar in all segments, they were placed similarly on the plots (fig. s ) . variations in the rotation angle of the triangle were caused by differences in the number of samples among the groups. fig. s includes several very old strains: the and human pandemic strains (green) and the swine strains (grey); they showed a tendency to be located closer to the origin of the plot. the magnitudes of pcs were similar among the segments, showing that the groups were separated by similar distances. this suggests a constant mutation rate among the segments, which might be branched from the ancestral strain coincidentally as a new set of viruses. the complete separations observed negate the possibility of homologous recombination between viruses, which would yield some intermediates among the groups. those of the swine strain may be an exception among the intermediates, which will be discussed below. the combinations of segments varied in swine strains (table : pb, rna polymerase basic protein; pa, rna polymerase acidic protein; np, nucleoprotein; nep, nuclear export protein and non-structural protein). in contrast, in human strains, the combinations were all m or all r. it should be noted that avian viruses showed lower pc values and appeared around the centre of the pca, while swine and human viruses exhibited extreme values (figs. c and s ). thus, avian viruses had sequences that were similar to the average among samples at amino acids that were characteristic to the three groups of human and swine viruses: r, m, and u. avian samples also showed characteristic motifs at other positions, which may appear in lower pcs. the relationships observed among the strains presented here are different from the classic ones , [ ] [ ] [ ] [ ] [ ] in several elements. for example, the number of swine classes was conspicuous. previously, "triple reassortant", "eurasian avian-like, " and "classic" viruses were estimated . however, the triple-reassortant group was absorbed into the two groups, u and m, and the sorting differed among segments in the strains presented here (fig. s , blue, table ). the frequency of shifts detected was another of the differences detected; i.e., only a few shifts were estimated , , , . these may be derived from differences in the classification methods used, which will be discussed below. only limited positions of a virus can be changed. although amino acid sequences varied among the types, the three-dimensional ( d) structures of the viruses were rather conserved regarding ha (fig. s a) , which suggests that at least some positions were conserved. in fact, several positions were common to type a viruses; e.g., they were located at the core of the protein, the rod of the alpha helix (fig. s b) . these positions seem to be important for the maintenance of the protein functions. from to , many residues in the type r viruses changed (fig. s c , asterisks). they are mainly located on the surface of the protein, where they may form epitopes. therefore, the changes that occurred at those positions would be beneficial for escaping the immune system of humans. conversely, we identified characteristic residues of the r, m, and u groups, which were indicated by the hexagonal directions of pc and pc (fig. s e , green). they exhibited higher scores because they appeared unchanged in only one of the groups (fig. s c ). these residues might play important roles in the function of the respective protein. let us consider the annual changes of the group r in humans (figs. a-c and s a). in any given year, only a few variations of the h subtypes were found. they were changed slightly by drifting and were detected in another area in the subsequent year (figs. d and s ). as the lives of humans span several decades, they might be immune to viruses that appeared in the past. this may represent a selective pressure to viruses. moreover, humans may travel to other countries by air while carrying viruses. hence, the viruses spread worldwide, unifying the outbreaking strains. although changes in one to several residues may be sufficient to escape specific antibody defenses , humans have various antibodies that target different epitopes. the drifts that occur within a year would be insufficient for viral spread in the same district; hence, h outbreaks occurring in two consecutive years were rare (figs. d and s ). thus, the areas that record an outbreak change yearly (fig. s ) . the axis of pc exhibited contributions from bases that changed once, whereas pc was characterized by bases that changed back and forth (fig. s a ). this oscillation might have been caused by sequential random walk mutation . the random walk model was also supported by the rapid attenuation of the contributions and the accordance with the tangent rule ( fig. s ) . however, a constant pattern of oscillation did not appear in lower-level pcs, suggesting that the changes were not fully random; rather, they seemed to obey some unidentified rules. as the number of samples is much smaller than the number of bases, the dimension of the pca is determined by the samples. in particular, as the samples do not differ much within a year, the actual dimension is determined by the total number of years, forming the drops at the end of the contributions; this also bends the tangent regression at its lowest part (fig. s ). in addition to the annual continuous drifts, several leaps were also observed ( fig. c , arrows between different colours). for example, variants coloured in blue started in , kept drifting, and were recorded through to , going around the world several times. those coloured in black were found in and were recorded through to . those in grey did not change in pc , but they went back toward the pc axis (other directions also appeared in lower pcs, with contributions from positions that changed several times; fig. s a ). the leaps would refresh the character of the epitopes; e.g., the black leap might allow the parallel evolution of the blue and black variants detected in pc (fig. b ). the leaps seemed to occur during the years in which the viruses were not detected in the areas that reported the surveillance viral counts and sequences (figs. d and s ). leaps were also observed in other segments; although the distance of jumps may have exhibited a certain variation, their timing was similar, and the relationships among the segments that appeared in pc and pc were all lambda shaped, pointing to the three terminals (fig. s ). the magnitude of the differences was very similar among the segments, which suggests the presence of a constant mutation rate among the segments. as was the case for the h viruses, the changes seemed to be a choice between two amino acids, each of which showed extreme positive or negative values in pc for bases; therefore, the directions of pcs for residues ran through the origin, showing a symmetrical hexagonal shape in ha (fig. s f) . the variation of permissive amino acids should be limited at each position. livestock viruses. let us consider the annual changes among the viruses of livestock (swine h : fig. s b ; chicken h : fig. s c ). many variations were present coincidently in any given year, especially in recent years, when the sequences became available from various areas. such a variety may hide the effects of drifts from higher axes of the pca. in fact, the pc values formed horizontal rows in the plots. however, the variations detected in an area did not change much, even in lower pc axes (fig. s b,c) . the small changes may result from differences in the districts that were surveyed in specific years, rather than from those caused by drifts. moreover, strains that may be transmitted from humans to swine were detected several years after the human epidemics; this may have occurred if the strains were kept unchanged among swine (fig. a,b) . these observations suggest that livestock viruses are rather conserved. each district may keep its own set of a virus library and we may be observing only a limited portion of the variation. the conserved character of these viruses suggests the presence of a weaker scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports www.nature.com/scientificreports/ selective pressure for newer epitopes. the variety suggests an absence of competition among viruses; i.e., they might have a limited ability to migrate. there were a few exceptions to this conservative endemic rule; e.g., many types of h n chicken viruses were detected in east asia (fig. ) . viruses might be carried by imported livestock. such migration has also been reported for viruses from swine that were transported from europe to mexico , . the group u swine viruses were further separated into european and asian subgroups and one of the european viruses was found in mexico ( fig. s b ; u, pc , indicated as " "). origin of pdm . the pdm virus appeared soon after the last outbreak of type r, which occurred in , and spread worldwide within year (fig. s ) , , , . the origin of pdm is thought to be swine viruses from mexico , , ; however, swine strains were not recorded before the pandemic in that country. a later study disclosed that several viral types existed , which included groups that were mainly found in asia or europe (fig. s b) , as well as human m and r viruses (fig. a,b) . although group m viruses were reported sporadically in humans before , the human pdm virus was characteristic as a small cluster in the plot (fig. c) . such characteristics were also observed in the other segments (fig. s ) . the analysis of data for scientific purposes requires objectivity. this cannot be achieved without considering the falsifiability of models , . here, we presented views that are very different from those of previous studies. the discrepancies were generated by differences in the methodologies, because many of the data analysed were common among studies. here, we wish to show that our study has better falsifiability . a sequence matrix is essentially composed of multivariate data with a large number of dimensions . previous studies used tree-based models (fig. ) which can handle only a single dimension ; such tree models have to ignore the real data structure. the understanding of clusters requires advanced judgement calls; hence, it may fail easily and the comparison of the results of different segments or types is difficult. in contrast, pca does not have limitations regarding the shape of presentations or the number of dimensions; thus, it has better objectivity . for instance, the attributions of clusters presented in figs. c and s should not cause confusion. moreover, the assumption that "the relationship among samples should be a tree-like structure" cannot be verified. our analyses are free from such assumptions. www.nature.com/scientificreports www.nature.com/scientificreports/ one of the major differences observed was the direct shift from avian to swine or human viruses. although these shifts were thought to explain the , , and , , pandemic viruses, the segments of avian viruses were separated from any swine or human segments, even those that were thought to be close to the source (fig. s , a, orange) . therefore, it is difficult to estimate such direct shifts. in fact, the cluster called "triple reassortant" disappeared (fig. s) ; this class was detected as a pandemic swine h n virus and also presented some clusters of h n , , . although avian viruses might be the origin of the h viruses, they might have a limited capacity to infect swine or humans, and vice versa. among the samples studied here, there was only one example of such transmittance, which may have occurred from humans to turkeys (ncbi taxonomy id: ) in contrast to the occasional transmittance observed between humans and swine ( figs. c and a,b) . the three groups, r, m, and u, may have branched out from the origin to achieve better infectivity in swine or humans (fig. ) . the human strain may represent an earlier process of branching out (fig. s ). swine may be the origin of the pandemic virus, i.e., the swine strain may be a derivative of this group of viruses. drifts and spreading: the genomes of the r group of human viruses have been changing yearly (figs. a-c and s a). the changed residues covered almost the whole surface of the ha protein (fig. s c) , showing the trajectory of the years of battle against the human immune system. in the last few years, drifting was not observed in pc , which records positions that changed only once ( fig. a, grey) . it seems that few positions that were amenable to variation were left unchanged, and that spontaneous mutation failed to hit those positions (fig. s c , positions located on the surface of ha and not marked). even the viruses that almost exhausted the number of unchanged positions kept drifting in the lower pcs, where repeated mutations were recorded (fig. s a) . the leaps of drifting observed here (fig. c ) may not denote punctuated evolution; rather, they may be the result of accumulating mutations that occurred in areas that do not report sequences. for instance, very few records are available from the african continent. the history of the r type also included some bifurcations ( fig. a,c) ; i.e., viruses located on different branches appeared in different districts in parallel. if these viruses competed within a small area, only the strain with the highest infectivity would remain. if some drifts occurred coincidently in areas located far apart, such branching might have occurred and might have been maintained for a while. in contrast to the frequent drifts observed in the human strains, those of livestock seemed to be conserved. the livestock strains differed among districts and remained constant for years. the conservative nature of these viral sequences may be attributed to the short life-span of livestock. as they rarely live for a year, they are not immune to viruses that spread over the previous year. hence, these viruses are not under a selective pressure toward the formation of newer epitopes. likely, a conservative character was also found in our preliminary studies of human infection with filoviruses and coronaviruses (not shown). moreover, because livestock rarely move around, the area of viral transmission is limited. for example, even the very infectious pandemic avian virus of was unable to spread worldwide. instead, it infected a limited number of farms . this limitation precludes a competition among strains, which enabling the maintenance of unique strains in small, separate areas. some of the strains that are retained may include segments that spread among humans in the past. it is even possible that several human viruses are kept intact among swine. such collections may be the source of the pandemic virus, which is thought to be a frozen laboratory strain that was released accidentally , . the migration of livestock viruses could be mainly caused by trading , although migrant birds might be able to transport avian viruses to chickens in some cases . however, if such transfers occur frequently, the characteristics of specific areas would disappear, and viruses would be better distributed worldwide; in particular, europe www.nature.com/scientificreports www.nature.com/scientificreports/ should have viruses that originated in other areas (fig. s ) . therefore, viruses of livestock would be transmitted rarely by migrant birds. we should be aware of the risk of keeping groups of swine that have different influenza strains in adjacent farms because new viruses may be produced by genome shifting. to prevent this, the segregation of imported swine from domestic ones is critical. districts that report several viral types should prevent farm-to-farm contamination. moreover, the exchange of genomes between human and swine viruses may give rise to a new strain with new sets of surface proteins that is able to infect humans. the switzerland virus (ncbi taxonomy id: ) proved that the group u strain is also capable of infecting humans; therefore, a new pandemic may occur in the case of an outbreak of this type of virus. such viruses of human origin are maintained among swine, maybe latently (fig. a,b) . origins of the segments: the pdm virus may have originated via shifts among swine viruses , , . the american classic virus and the reassortant virus could be defined using the segments (r, r (table ) . replacing the u groups of na and mp with those of the european type (u, u, u, u, u, m, m, u) can complete all m segments of pdm . such shifts may occur occasionally (table ) . it should be noted that assortment of the m segments was insufficient to produce the pdm virus. although the reassortant virus was repeatedly detected in human samples, it was transmitted from swine and did not spread among humans. the segments showed pcs located nearer the average compared with pdm (figs. c and s ). if a human-to-human transition requires the specific characteristics of a virus, those characteristics should be detected in some of the pcs when enough samples are included in the calculation and the extreme values observed in pc and pc may reflect such characteristics. the archiving of the characteristics requires further shifts in all the segments of the assorted m virus. the origin of m type polymerases in the reassortant and pdm viruses may not be the result of a direct shift from avian viruses (fig. s ) . instead, they might be maintained among swine and may have drifted away from the avian type (fig. ) . the same would be true for the r type segments. although most of the sequencing data were obtained from humans, the r type may have been overlooked among swine. if these viruses do not cause severe symptoms, they may not attract the attention of farmers or researchers. in fact, focused approaches detected the r type in swine (fig. b) . such endemic r types may show lower values in pc closer to the origin; e.g., those found in england and dötlingen in might satisfy this condition ( fig. s ; ha) , and other segments also exhibited such endemic candidates. unfortunately, it is difficult to estimate the history of old viruses because of the shortage of data. some of the segments of the strain exhibited interesting positions on the branches: i.e., some were r type, while others were located between the r and (m or u) types (fig. s ) . the segments would be those close to the branching point of the groups. however, this does not necessarily mean that the branching occurred recently; i.e., it could have been preserved among swine at least until . whether the and/or human strains are lineal ancestors of the pandemic virus remains uncertain. it should be noted that these viruses were included in different epidemic series on different memories of immunity among humans. these immune memories are changing because of the change of human generations, i.e., the return of the r group observed over recent years (fig. c , grey) might have been enabled by such changes. estimation of future viruses and vaccination: patterns of change were observed for the human r and m types in each of the pc axes ( figs. a,b, a, and s a ). although pca produces sin curves for the records of random walk mutations , the real patterns may reflect some rules in the drift of genomes. as patterns of m groups become apparent, we can compare them between m and r viruses to estimate the hidden rules. although ha is thought to be the primary target of the human adaptive immune response , other segments that include proteins located inside the virus particle also drifted with a magnitude similar to that of ha, which suggests a common mutation frequency (fig. s c ). this observation was unexpected because mutations may lead to malfunction; if they are not the target of immunity, they should be conserved, similar to those of livestock viruses. as the segments are displayed on the surface of infected cells, they will activate specific immune cells, which may play important roles in the human immune system. it is obvious that any strain that has caused an outbreak will not appear again for decades among humans. in contrast to the case of the h n viruses, h n outbreaks rarely occur in two consecutive seasons (figs. d and s ). this shows that most people exposed to the virus are infected asymptomatically during the viral outbreak and become immune; otherwise, the virus could infect a portion of receptive people that were not infected in the previous year. also, the drift occurring in a single year is insufficient for escaping the immune system. the pdm virus appeared after the flu season and spread worldwide from april to march (fig. s ) . the extraordinary infectivity of this virus may have been caused by epitopes that were new to humans. therefore, the present system of vaccine production to control influenza infections, which uses eggs, has a critical defect: i.e., none of the stock strains that are used for viral production are the one that will cause an outbreak in the following season. the population might have become immune to the stock viruses and the vaccine just helps to recall the memory of these strains. sequence data analysis. all of the sequence data used here were obtained from the ncbi database (https:// www.ncbi.nlm.nih.gov/nuccore). data pertaining to amino acid sequences were used to observe multiple subtypes or hosts (figs. and ) , while nucleotide sequences were used for specific subtypes (figs. - , s and s ). data alignment was performed using muscle . all the aligned sequences are presented in figs s , s and s . the sequence matrix was casted to direct principal component analysis (pca) , which analyses sequence matrix without losing any of original information in the matrix , using r . the direct pca is resistant to differences caused by the alignment: differences in the conditions or the alignment methods may not alter the results practically . the sequence matrix were translated into a boolean vector x; for n samples of l bases, x is a matrix of n × l, which correspond to the nucleotides and inserted space "-" for alignment. a mean vector → m was estimated as → = ∑ = n x m / i n i j , , where j = , ,,,, l. the boolean vector was then catered to estimate differences to the mean vector, , where k = , ,,,, n. then d was subjected for singular value decomposition as Σ = ⁎ d l r . scaled values of the principal components (spc) were found by placing back the singular values Σ to the unitary matrixes l or r; those for the samples were estimated as Σ = l l spc / s and the nucleotide bases were estimated as Σ = n r spc / n . they were scaled for comparisons that enables to identify which base contributed for the differences of samples . therefore, each spc presents directions and distances of the differences, which were recorded by the unitary matrix and the singular value, respectively, as sets of orthogonal vectors executed as the columns of the spc matrixes. scripts of the r used for the calculation is presented in fig. s . pc signs were derived in a random manner; thus, the results were adjusted to align different presentations for comparison. a starter kit for the direct pca is available in the accompanying article . the d structures. the pdb files were obtained from the rcsb protein data bank (https://www.rcsb.org/). position data were found using atom header: i.e., positions at the alpha carbon of amino acids were connected to draw the structure and were then aligned using pca. the html output was generated using the writewebgl function of the rgl package of r (https://cran.r-project.org/web/packages/rgl/vignettes/rgl.html). the evolution of seasonal influenza viruses avian influenza: assessing the pandemic threat influenza virus evolution, host adaptation, and pandemic formation historical perspective-emergence of influenza a (h n ) viruses the pig as a mixing vessel for influenza viruses: human and veterinary implications principal component analysis applied directly to sequence matrix molecular phylogenetics: principles and practice origins and evolutionary genomics of the swine-origin h n influenza a epidemic antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans influenza a viruses of human origin in swine, brazil. emerging infectious disease journal origins of the h n influenza pandemic in swine in mexico global migration of influenza a viruses in swine the persistent legacy of the influenza virus the stanford encyclopedia of philosophy concerns regarding the deterioration of objectivity in molecular biology characterization of the influenza virus polymerase genes genetic reassortment of avian, swine, and human influenza a viruses in american pigs the re-emergence of h n influenza virus in : a cautionary tale for estimating divergence times using biologically unrealistic sampling dates & related influenza, v. role for migratory wild birds in the global spread of avian influenza h n muscle: multiple sequence alignment with high accuracy and high throughput r_core_team. r: a language and environment for statistical computing. (r foundation for statistical computing principal component analysis for designed experiments the author declares no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to t.k. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - cxyogor authors: widagdo, w.; begeman, lineke; schipper, debby; run, peter r. van; cunningham, andrew a.; kley, nils; reusken, chantal b.; haagmans, bart l.; van den brand, judith m. a. title: tissue distribution of the mers-coronavirus receptor in bats date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: cxyogor middle east respiratory syndrome coronavirus (mers-cov) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase- (dpp ) as its receptor. the distribution of dpp in the respiratory tract tissues of humans and camels reflects mers-cov tropism. apart from dromedary camels, insectivorous bats are suggested as another natural reservoir for mers-like-covs. in order to gain insight on the tropism of these viruses in bats, we studied the dpp distribution in the respiratory and extra-respiratory tissues of two frugivorous bat species (epomophorus gambianus and rousettus aegyptiacus) and two insectivorous bat species (pipistrellus pipistrellus and eptesicus serotinus). in the frugivorous bats, dpp was present in epithelial cells of both the respiratory and the intestinal tract, similar to what has been reported for camels and humans. in the insectivorous bats, however, dpp expression in epithelial cells of the respiratory tract was almost absent. the preferential expression of dpp in the intestinal tract of insectivorous bats, suggests that transmission of mers-like-covs mainly occurs via the fecal-oral route. our results highlight differences in the distribution of dpp expression among mers-cov susceptible species, which might influence variability in virus tropism, pathogenesis and transmission route. immunohistochemistry to detect dpp was performed on nose, lung, intestine, kidney, salivary gland, and liver tissues of different bat species: common pipistrelle bat, serotine bat, gambian epauletted fruit bat (further referred as gambian fruit bat), and egyptian fruit bat. the assay was replicated two-three times for each tissue. we have used the same technique to map dpp localization in the respiratory tract tissues of human, dromedary camel, sheep, horse, pig, and llama , . the antibody used in this study recognizes bat dpp as was demonstrated in transfection experiments using cloned pipistrelle bat dpp . hematoxylin and eosin staining on subsequent slides of the same tissues from the bats did not show significant histological changes. dpp was not found in the nasal olfactory epithelial cells of common pipistrelle bat, serotine bat, gambian fruit bat, or egyptian fruit bat (fig. ). in the nasal tissues of common pipistrelle bat, dpp was not detected in the respiratory epithelial cells lining the nasal cavity, but was detected in the epithelial cells lining the ducts of the submucosal glands in this species. in the serotine bat and gambian fruit bat, multifocal dpp expression was detected in a limited number of nasal respiratory epithelial cells. in contrast, in the nasal tissues of the egyptian fruit bat, dpp was prominently detected at the apical surface of the respiratory epithelial cells lining the nasal cavity as well as in glandular and ductular epithelial cells of the submucosal glands. in the lungs of the common pipistrelle and serotine bat, dpp was found in the endothelial cells of the capillaries but not in the bronchial, bronchiolar or alveolar epithelial cells. in the gambian and egyptian fruit bat, dpp was detected in the bronchial, bronchiolar and alveolar epithelial cells as well as in endothelial cells of small blood vessels (fig. ). in the intestinal tissues of all four bat species, dpp was prominently expressed on the apical surface of both small and large intestinal epithelial cells (fig. ). in the kidney of all four bat species, dpp was found in glomerular cells, parietal squamous epithelial cells of the bowman's capsule, and in the proximal tubular epithelial cells. in the salivary gland of common pipistrelle bat, dpp was only detected in the ductular epithelial cells, while in the serotine bat, it was detected in a limited number of glandular epithelial cells. in the gambian and egyptian fruit bat, it was detected in both the glandular and ductular epithelial cells of the salivary gland. in the liver of the common pipistrelle bat and serotine bat, dpp was present in a limited number of endothelial cells lining the sinusoids. in contrast, in the liver of the gambian and egyptian fruit bat, dpp was detected in the bile duct epithelial cells, in the endothelial cells of the hepatic arteries, and in the endothelial cells of the sinusoids (fig. ) . variation in dpp signal and localization were occasionally observed between animals within the same species. in one common pipistrelle bat, the paranasal sinus and pharynx were examined and showed a limited number of dpp positive epithelial cells. the results of the dpp immunohistochemistry staining were scored qualitatively and summarized in table . in general, our results showed that dpp was prominently expressed in the intestine and the respiratory tract tissues of the frugivorous bats, i.e. the gambian and the egyptian fruit bat. however, it is limitedly expressed in the respiratory tract tissues of the insectivorous bats, i.e. the common pipistrelle bat and the serotine bat. in the common pipistrelle bat, dpp was not detected in the nasal respiratory, nasal olfactory, bronchiolar, or alveolar epithelium, but was abundant on the apical surface of the epithelium lining the small and large intestine. we compared these findings to our previous results on dromedary camel and human tissues . in dromedary camels, dpp is strongly detected in the nasal respiratory, tracheal, and bronchial epithelium, while there is limited expression in the alveolar epithelium (fig. ). in humans, it is not found in the nasal epithelium and is present mainly in the alveolar epithelium. additionally, we performed dpp staining on intestinal tissues of dromedary camels obtained from a previous study . we found that dpp was expressed mainly on the apical surface of the small intestinal epithelium (data not shown), similar to what has been reported for humans [ ] [ ] [ ] [ ] (fig. ). the tissue distribution of the mers-cov receptor, dpp , has previously been studied in humans, dromedary camels, and other livestock animals , . here, we show that dpp is differentially expressed among bat species, especially between insectivorous and frugivorous bats. it is strongly detected in the intestine of the common pipistrelle bat, the serotine bat, the gambian fruit bat and the egyptian fruit bat. it is also prominent in the respiratory tract epithelium of the gambian and egyptian fruit bat, but expression is limited in that of the common pipistrelle and serotine bat. given the essential role of dpp in the entry of mers-cov into cells, these results suggest that mers-like-covs are not likely able to replicate in the respiratory tract in these two insectivorous bats. this is in line with our previous report on mers-cov infection experiment in sheep, showing that the lack of dpp in the respiratory tract of the sheep was associated with restricted mers-cov replication in these animals upon intranasal inoculation . rather, in these two insectivorous bats, mers-like-covs may preferentially replicate in the gastrointestinal tract. this is partly supported by the fact that viral genomes of mers-like-covs were mainly obtained from faecal and intestinal tissue samples of insectivorous bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this intestinal tropism indicates that these viruses transmit mainly through the fecal-oral route. therefore, future screening of mers-like-covs from insectivorous bats, particularly the common pipistrelle bat, might focus on fecal material, rectal swabs, or intestinal tissues, rather than throat or nasal swabs. prominent dpp expression in both respiratory tract and intestinal epithelium of the gambian fruit bat and the egyptian fruit bat suggests that mers-cov is able to replicate in both the respiratory tract and intestine of the fruit bats. these results are in line with the fact that mers-cov was able to replicate in the lungs of jamaican fruit bat (artibeus jamaicensis) upon intranasal and intraperitoneal inoculation be detected in the rectal swabs of these animals up to day p.i. and infectious virus was also isolated in the duodenum of one of the bats at day p.i. . these data suggest that mers-cov infects and replicates in the intestine of these bats, not only in the respiratory tract. mers-cov infection in these bats is likely mediated by dpp , since hamster bhk cells, a non-susceptible cell line, could be infected by mers-cov when modified to express jamaican fruit bat's dpp . dpp expression in the intestine and respiratory tract of these jamaican fruit bats, however, was not investigated. since the jamaican fruit bat is a new world fruit bat, unlike the gambian fruit bat and the egyptian fruit bat, which are old world fruit bats, their genetic difference might influence the variation in dpp expression among these species. in contrast to the fruit bats, where dpp is expressed throughout the respiratory tract, dpp is rarely detected in the respiratory tract tissues of insectivorous bats. this limited dpp expression in insectivorous bats might significantly restrict the replication of mers-like-covs in these tissues and minimize the possibility of transmission of these viruses from the respiratory tract. the limited dpp expression in the respiratory tract of the two insectivorous bat species, particularly the common pipistrelle bat, is different from what has been reported for dromedary camels and humans. in humans, dpp is merely expressed in the lower respiratory tract, while in the dromedary camels, it is detected in the upper respiratory tract epithelium . this renders humans to develop pneumonia upon mers-cov infection, while camels develop upper respiratory tract infection , , . in the intestine of both dromedary camels and humans, dpp is mainly present in the apical surface of the small intestine epithelium [ ] [ ] [ ] [ ] . mers-cov has been isolated from faecal samples of a naturally infected dromedary camel, which suggests that this virus is able to replicate in the intestinal tract of this species . however, in dromedary camels, the chance of detecting mers-cov rna in faecal samples is much lower than from nasal swabs . we also observed that low amounts of viral rna are detectable in rectal swabs taken from mers-cov-inoculated dromedary camels . while mers-cov has not yet been isolated from human faecal samples, low amounts of viral rna could be detected in stool samples of mers patients , and several mers patients have also been reported to suffer from diarrhoea [ ] [ ] [ ] . these observations suggest that mers-cov replicates in the intestine of both dromedary camels and humans although only to a limited extent. it is currently unclear what factors restrain mers-cov replication in the intestinal tract of dromedary camels and humans. the human intestinal tract is protected by a mucus layer, commensal microorganisms, multiple innate and adaptive immune cells . also, adenosine deaminase (ada), a natural antagonist of dpp that can inhibit mers-cov infection in vitro , has also been found in the human intestine. the amount of ada in the human intestine is four times higher compared to that in the lung . the presence of dpp in the intestinal tract of bats suggests an intestinal tropism of mers-like-covs. we also detected dpp in the salivary glands and kidneys in all of the bats. in vitro, mers-cov has also been shown to replicate in primary kidney cell culture derived from common pipistrelle bat . however, there has been no further evidence supporting the susceptibility of these two tissues in vivo, nor have there been any reports of mers-like-covs isolated from these two tissues or from bat urine samples. whether these viruses are transmitted through bat saliva or urine, therefore, is currently unclear. in general, our study describes the variation in dpp distribution among four bat species, with notable differences between insectivorous and frugivorous bats. more importantly, the tissue distribution of dpp in insectivorous bats, believed to be one of the natural hosts for mers-like-covs, is different to that in dromedary camels and humans. our results indicate intestinal tropism of mers-like-covs in the insectivorous bats we examined. the existence of a co-receptor that might influence mers-like-covs tropism and replication in these bats, however, could not be disregarded. ceacam is recently reported as an attachment factor that facilitates entry of mers-cov in-vitro . whether ceacam plays an important role in-vivo, particularly in bats, remains to be investigated. in-vivo infection experiments are necessary to confirm our findings, but such studies are ethically and technically challenging. nevertheless, our data are relevant for future monitoring and surveillance of mers-like-covs in insectivorous bats, particularly in the common pipistrelle bat , , as well as for future efforts to better understand the pathogenesis and transmission of mers-like-covs in their natural host. common pipistrelle and serotine bats were found stranded and severely wounded on different occasions, and admitted to an official local bat shelter in the netherlands. the animals were euthanized by veterinarians due to ethical reasons using officially approved methods. the gambian fruit bats and three of four egyptian fruit bats used in this study originated from free-ranging populations in ghana. the bats were sampled for an unrelated study and this study was approved by the ethics committee of the zoological society of london (ref. wle ) and the council for scientific and industrial research in accra, ghana. one of the egyptian fruit bats was obtained from the captive colony at the friederich loeffler institute, riems, germany. it had been euthanized due to reasons not related to this study. all methods were performed in accordance with the relevant guidelines and regulations. after euthanasia, the bats were necropsied and tissues were collected. parts of the lung, intestine, salivary gland, liver, and kidney were obtained from nine common pipistrelle bats, seven serotine bats, three gambian fruit bats, and four egyptian fruit bats. parts of the noses were obtained from five common pipistrelle bats, six serotine bats, three gambian fruit bats, and three egyptian fruit bats. these tissues were all fixed in % formalin and embedded in paraffin. the noses were decalcified in % edta for days before being embedded in paraffin. the formalin fixed paraffin embedded tissues were sectioned ( μm), deparaffinized, and subsequently hydrated. citric acid buffer mm ph was used to retrieve antigens. blocking with normal rabbit serum % was performed prior to staining with polyclonal goat igg anti-dpp (r&d systems, abingdon, uk) in µg/ml concentration. normal goat serum (mp biomedicals, santa ana, ca, usa) in equal concentration was used as negative control. dpp staining was performed at °c overnight. secondary antibody rabbit anti-goat igg labeled with peroxidase were applied subsequently in : dilution for hour at room temperature (dako, glostrup, denmark). the red signal was revealed with -amino- -ethyl-carbazole (sigma-aldrich, st. louis, missouri, usa) before counterstaining with hematoxylin. dromedary camel intestinal tissues were obtained from three different animals sacrificed at 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analysis of a case of middle east respiratory syndrome coronavirus infection a case of imported middle east respiratory syndrome coronavirus infection and public health response epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission intestinal epithelial cells: regulators of barrier function and immune homeostasis adenosine deaminase isozymes in human tissues carcinoembryonic antigen-related cell adhesion molecule is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus this study is supported by a top project grant ( ) and by the zoonoses in the night project ( o- - - ) both funded by zonmw. the e. gambianus and three of four r. aegyptiacus used in this study originated from a study in ghana in collaboration with richard suu-ire, from the forestry commission, accra. we would like to thank anne buschmann-balkema for her assistance in preparing the r. aegyptiacus tissues that come from the friederich loeffler institute, riems, germany. we would like to thank stichting vleermuisopvang oss for providing the tissues of p. pipistrellus. we thank brigitta m laksono for her advice on the schematic figure. competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -htx ul o authors: chothe, shubhada k.; bhushan, gitanjali; nissly, ruth h.; yeh, yin-ting; brown, justin; turner, gregory; fisher, jenny; sewall, brent j.; reeder, deeann m.; terrones, mauricio; jayarao, bhushan m.; kuchipudi, suresh v. title: avian and human influenza virus compatible sialic acid receptors in little brown bats date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: htx ul o influenza a viruses (iavs) continue to threaten animal and human health globally. bats are asymptomatic reservoirs for many zoonotic viruses. recent reports of two novel iavs in fruit bats and serological evidence of avian influenza virus (aiv) h infection in frugivorous bats raise questions about the role of bats in iav epidemiology. iavs bind to sialic acid (sa) receptors on host cells, and it is widely believed that hosts expressing both sa α , -gal and sa α , -gal receptors could facilitate genetic reassortment of avian and human iavs. we found abundant co-expression of both avian (sa α , -gal) and human (sa α , -gal) type sa receptors in little brown bats (lbbs) that were compatible with avian and human iav binding. this first ever study of iav receptors in a bat species suggest that lbbs, a widely-distributed bat species in north america, could potentially be co-infected with avian and human iavs, facilitating the emergence of zoonotic strains. with a wide host range, ability to undergo genetic recombination and cross species barrier, influenza a viruses (iavs) continue to spread globally, causing huge economic losses to the poultry industry and threatening public health. unlike the low pathogenic avian influenza viruses (lpaivs), highly pathogenic avian influenza viruses (hpaivs) cause very severe disease in gallinaceous poultry often leading to % mortality within - days . hpaivs of h n subtype are of particular concern as certain contemporary eurasian lineage h n viruses can carry an alarming case fatality rate of up to % in humans . negative strand segmented rna genomes contribute to the genetic variability of iavs. in addition, iavs can infect a wide range of avian and mammalian host species resulting in the emergence of novel subtypes with altered species tropism and/or virulence. it is widely known that aquatic birds such as ducks, gulls, and shorebirds serve as a natural reservoir of most known iavs . iavs are known to infect a wide range of avian and mammalian hosts, and it is highly likely that their host range could be broader than currently known, with more reservoirs to be revealed. for example, northwest atlantic gray seals have recently been suggested to be an endemically infected wild reservoir population for diverse iavs . from these natural reservoirs, iavs can evolve into novel variants which can potentially lead to human pandemics. influenza pandemics occur time to time with the most recent pandemic being in . it is undisputed that the next influenza pandemic will happen, but the only question is when it will happen. despite several reports investigating the basis of genetic variability of iavs, the precise mechanism of pandemic iav generation still remains an unresolved mystery. it is possible that additional iav reservoirs are yet to be identified which will help to see the full picture of iav ecology and evolution. bats (order: chiroptera) are one of the ancient mammals, and their speciation occurred long before the development of most modern mammals. bats are globally distributed, relatively long lived and represent approximately % of all known mammalian species. further, bats are one of the most diverse families of mammals found in nearly every habitat/continent around the world except antarctica. more importantly, certain old world bat species are known to be natural reservoirs of zoonotic viruses that cause some of the deadliest diseases in humans including filoviruses (such as ebola and marburg viruses), lyssaviruses, severe acute respiratory syndrome (sars)-related coronaviruses and henipaviruses (e.g. hendra and nipah viruses) , . in addition, bats also act as a major natural reservoir for hepaciviruses and pegiviruses (hepatitis c virus and gb virus b) . notably, all the zoonotic viruses of bat-origin identified to date are rna viruses . however, it is believed that the actual diversity of viruses in bats could be much more than what is currently known, as most of the investigations have focused on searching for specific viruses of interest and many additional viruses must have been overlooked . the prospects for bats contributing to iav epidemiology came to light after the identification of two novel influenza-like viruses in fruit bats by next generation sequencing . these two viruses are genetically distinct from all previously known iavs and hence are designated as novel subtypes, namely h n and h n . these novel iavs have been recently recovered in cell culture using synthetic dna . however, these ha and na subtypes have not been identified in birds serologically or virologically. consequently, the reservoir(s) of these novel iav subtypes is still undefined. phylogenetic studies raised a possibility that bats have the capacity to harbor more influenza virus genetic diversity than all the other mammalian and avian species combined . in addition, it was demonstrated that little yellow-shouldered bats in central america could constitute a potential sylvatic mammalian reservoir of influenza . susceptibility of bats to iavs has been confirmed by recent serological evidence of aiv h subtype in about % of frugivorous bats from africa . it is worth noting that detection of antibodies against one aiv subtype in % of the bats tested is very significant. influenza viruses bind to sialic acid (sa) residues that are bound to glycans through α , or α , linkage on the host cells . the expression of the appropriate host cell receptor to which viral haemagglutinin (ha) can bind is the key determinant of the ability of iavs to infect a host . avian influenza viruses (aivs) preferentially bind to sa receptors that are linked to galactose by an α , linkage (sa α , -gal), while human and classical swine influenza viruses show preference to α , linked sas (sa α , -gal). a key source of iav genetic diversity could be from the replication of iavs in a non-native host species that initiate evolution of new virus variants . hosts that co-express both sa α , -gal and sa α , -gal receptors such as chickens, ducks and pigs have been hypothesized to potentially support re-assortment of iavs and hence play a major role in the evolution of iavs , . while the role of bats in iav evolution is not yet known, recent evidence raises a primary question, "can bats support co-infection of avian and human iavs?". consequently a logical first unknown which needs to be addressed is whether bats express appropriate sa receptors compatible with avian and human iav binding. to resolve this enigma, we investigated for the first time the distribution of sa receptors in little brown bats (lbbs) (myotis lucifugus), a widely-distributed bat species in north america and their compatibility to support avian and human iav binding. tissues sections from a total of juvenile and adult lbbs were subjected to lectin histochemistry for the detection of influenza virus receptors. no differences in sa receptor distribution between the juvenile and adult lbbs were found. co-expression of both sa α , -gal and sa α , -gal receptors in lbb respiratory tract. abundant co-expression of avian (sa α , -gal) and human (sa α , -gal) type influenza receptors was found throughout the lbb respiratory tract (fig. ) . the relative abundance of avian and human type influenza receptors were distinctly different in the upper and lower airway of lbb. while sa α , -gal receptors were predominant on the mucosal lining of the trachea (fig. a) , predominance of sa α , -gal receptors was found in the alveolar epithelium, alveolar duct and visceral pleura of the lung (fig. b) . further, abundant expression of sa α , -gal receptors was found in the lamina propria and submucosa of the tracheal tissue (fig. a ). expression of sa α , -gal receptors was found in the stomach and intestines of lbb (fig. ) . cells lining the mucosa of stomach exhibited co-expression of both avian and human receptors ( fig. a) . the luminal epithelium of intestines primarily expressed sa α , -gal receptors whereas considerable expression of sa α , -gal receptors were found on the goblet cells, lamina propria, muscularis and serosa of the intestine (fig. b ). ing assays were performed on lbb trachea and lung sections using an lpaiv h n (a/h n /chicken/ pennsylvania/ / ) virus. extensive binding of avian h n virus to lbb trachea, lung and intestines was observed (fig. ) . h n virus binding pattern was in accordance with the relative abundance of sa α , -gal receptors in tissues such that greater virus binding to the tracheal ( in addition to the virus binding assay using antibody-based detection, we also visualized virus binding using scanning electron microscopy (sem) which also confirmed abundant binding of avian h n virus (fig. a ) and human h n virus (fig. b ) to lbb trachea (fig. ) . it has been shown that lectins from different suppliers may show different binding specificities; in particular the source of maa has been shown to significantly affect specificity. lectins (sna and maaii) used in this study were purchased from vector laboratories which were shown to bind to the appropriate sialic acid linkages with a high degree of specificity by glycan microarray screening (http://www.functionalglycomics.org). we treated sections with sialidase a (n-acetylneuraminate glycohydrolase; prozyme, san leandro, ca), for h at °c, which cleaves all non-reducing terminal sialic acid residues in the order α( , ) > α( , ) > α( , ) > α ( , ) . sialidase a treatment completely abrogated lectin binding (supplementary figure s ) and h n influenza virus binding (supplementary figure s ) which further confirmed the specificity of the lectins used in this study to appropriate sialic acid linkages. in summary, we found that the sa receptors on lbb tissues supported binding of avian h n and human h n influenza viruses. further, the virus binding pattern was in accordance with the relative distribution of sa α , -gal and sa α , -gal receptors in tissues. with the continued recognition of the role of various wild animals in influenza virus evolution, the interest in understanding the tissue sa distribution in wild animals has intensified . among the various factors, the ability to bind to sa receptors on host cells is considered a key feature of iav pathogenicity. there is serological evidence of avian influenza virus infection in certain frugivorous bats and pteropus alecto bat cells were found to be susceptible to iav infection and reassortment . in addition bats have been shown to harbor novel influenza-like viruses . however, to date, the sa receptor distribution in any bat species is completely unknown. for the first in contrast, sa α , -gal receptors gradually increases towards the lower respiratory tract. tissue sections were stained with biotinylated maaii (red -specific for avian type receptor, sa α , -gal) and fitc labelled sna (green -specific for human type receptor, sa α , -gal) lectins, and dapi nuclear stain (blue). a": haematoxylin and eosin (h and e) stained tracheal tissue section. . respiratory epithelium, . lamina propria, . submucosa, . hyaline cartilage b": h and e stained lung tissue section. . alveolar duct, . visceral pleura, . pulmonary blood capillary. time, this study demonstrated that little brown bats (lbbs), a widely-distributed bat species in north america, co-express both avian and human type influenza receptors in their respiratory and gastrointestinal systems. co-expression of avian and human type influenza receptors was found in lbb trachea and lung. however avian type (sa α , -gal) receptors were predominant in tracheal mucosa similar to ducks and some species of passeriformes and charadriiformes . in contrast sa α , -gal receptors are predominant in the tracheal mucosal lining in chickens, pigs and humans , . we found co-expression of both avian and human type receptors in the mucosal lining of stomach and to a lesser degree in intestines. sa α , -gal receptors were predominant throughout the digestive tract much like chickens and ducks , , . in contrast, other mammalian species such as humans and pigs predominantly express sa α , -gal receptors in the gi tract . unlike chickens and ducks, low levels of sa α , -gal receptor expression was also observed in lbb digestive tract which decreased progressively from stomach to intestines. we did not find any difference in the receptor distribution among various locations in intestines as it is difficult to distinguish large and small intestine in most bat species . similarly, no major differences in the receptor distribution pattern between large and small intestine were found in most other species including chickens, ducks and pigs , . a fair amount of sa α , -gal receptor expression was found in the intestinal crypts, lamina propria, muscularis and serosa of lbb intestine. it is worth noting that a similar co-expression pattern of influenza receptors is observed in various wild birds of the families columbiformes, anseriformes and gruiformes , which constitute the natural influenza virus reservoir, and also in pigs . virus binding assays with an avian h n and a human h n virus confirmed that the sa receptors found in lbb tissues are compatible with avian and human iav binding. bats act as natural reservoirs for a variety of zoonotic viruses and they coexist with viruses through several mechanisms including elevated metabolism and body temperature . it is believed that this unique feature of bats leads to the selection of viruses that adapt better at higher body temperature, and hence are more virulent to humans . bats carry a number of rna and dna viruses asymptomatically, and the detection rate of new viruses or virus sequences seems to be much higher in bats than any other mammals . the perfect equilibrium between various zoonotic viruses and bats have been studied extensively in the past two decades , . iavs have been isolated from more than different bird species belonging to different families . although anseriforms (ducks, geese, and swans) and charadriiforms (gulls, terns, and shorebirds) are considered to be the major influenza reservoirs, iavs have also been isolated from gaviiformes (loons), podicepediformes (grebes), procellariiformes (shearwaters and petrels), pelecaniformes (pelicans and cormorants), ciconiiformes (ibis and herons), and gruiformes (moorhen and coots) . many of these species may indeed be reservoirs of iavs, but no systematic surveys have been conducted, leaving a gap in our current understanding of the natural reservoirs of iavs. we showed for the first time that lbbs co-express both the avian (sa α , -gal) and human (sa α , -gal) influenza receptors in their respiratory and gastrointestinal system that are compatible with avian h n and human h n virus binding. in addition, there is strong evidence that cell lines from a range of bat species support productive iav replication . the sum of this evidence suggests that bats could play an important role in iav epidemiology and zoonotic iav emergence. as the novel bat influenza viruses are different from other iavs, it was proposed that these viruses would therefore require significant changes before they can infect and spread among humans . however, a recent study rescued these viruses using reverse genetics in cell culture and found that the novel bat influenza viruses can infect a range of mammalian cells including canine cells . all the existing data suggests that bats could be susceptible to many different iav subtypes and even support co-infection of avian and human iavs. it is believed that the novel bat influenza viruses found in fruit bats are probably the ancient influenza viruses from which the modern world iavs have been derived over time . evidence of high seroprevalence of avian influenza in frugivorous bats together with the evidence of abundant sa receptors in lbbs found in this study, raises a strong possibility that bats could be a major influenza virus reservoir. despite many rigorous scientific pursuits, we have been unable to understand the mechanism by which new pandemic influenza viruses emerge. consequently, we do not yet have sufficient scientific understanding needed to accurately predict which iav strains may cause the next pandemic. the extensive diversity of bat species globally and the limited understanding of the role of bats in iav biology raises an urgent need for comprehensive epidemiological surveillance of iavs across different bat species. experimental animals. little brown bat respiratory and gastrointestinal tissues were collected and provided by pa game commission from an ongoing collaborative study with bucknell university, on little brown bats from the state of wisconsin and new york. the study (dmr- ) was approved by the bucknell university institutional animal care and use committee and all methods were carried out in accordance with relevant guidelines and regulations. a total of adult and juvenile male little brown bat tissues were included in the study. lectin histochemistry. lectin histochemistry on paraffin embedded tissues was performed as previously described . briefly, µm thick tissue sections were deparaffinized in xylene and rehydrated in ethanol. following min presoaking of the rehydrated sections in tris-buffered saline (tbs), sections were incubated with biotinylated maackia amurensis (maaii) and fitc labelled sambucus nigra (sna) lectins specific to sa α , -gal and sa α , -gal receptor respectively each at a concentration of μg/ml, overnight at °c. both lectins were purchased from vector laboratories (burlingame, ca, usa). following three washes with tbs, sections were incubated with streptavidin alexafluor conjugate for h at °c. the sections were washed three times with tbs and mounted in prolonggold antifade reagent with nuclear stain ′, -diamino- -phenylindole, dihydro-chloride (dapi). negative controls were performed omitting the primary reagents. following h of curing at room temperature (rt), sections were imaged using olympus fluoview ™ fv confocal microscope. settings of the confocal microscope were determined using negative controls to avoid any background fluorescence and the same settings were used to scan all the other sections for consistency. to rule out nonspecific binding of the lectins and iavs, control tissue sections were treated, prior to lectin staining or virus binding, with sialidase a (n-acetylneuraminate glycohydrolase; prozyme, san leandro, ca), for h at °c, which cleaves all non-reducing terminal sialic acid residues in the order α( , ) > α( , ) > α( , ) > α ( , ) . sialidase treated and control sections were further subjected to lectin staining or virus binding. virus binding assay. virus binding assays with a low pathogenic aiv (lpaiv) h n (a/h n /chicken/ pennsylvania/ / ) and human pandemic h n virus (a/h n /virginia/ ) were performed as previously described . virus binding assays were performed following strict biosafety level- (bsl- ) practices. briefly deparaffinised tissue sections were incubated with a μl of lpai h n virus ( tcid /ml) or human h n virus ( tcid /ml) for h at rt. tissues incubated with pbs served as mock treated controls. sections were washed with tbs before blocking with inactivated goat serum for min and immunostained with primary mouse monoclonal antibody to influenza hemagglutinin h (ab , abcam) or influenza nucleoprotein (ab , abcam). following min of incubation with the primary antibody, sections were washed and incubated with a secondary goat anti mouse igg-cy antibody (ab , abcam). after min of incubation with secondary antibody at rt, sections were washed with tbs and mounted in prolonggold antifade reagent with dapi. following h of curing, the sections were imaged using olympus fluoview ™ fv confocal microscope. scanning electron microscopy (sem). virus binding was also visualized using scanning electron microscopy. deparaffinized sections were incubated with lpai h n virus or human h n virus for h as described above. subsequently the sections were washed with distilled water and fixed using increasing ethanol concentrations ( %, %, % and %). the sections were then dried by a critical point dryer (leica em cpd ). gold was coated over the dried sections by applying sputter coating for seconds (cressington, auto sputter coater) and sem images were taken by a field emission sem (fe-sem, merlin zeiss) under ev. image processing and pseudo coloring of virus particles bound to tissues was performed using adobe photoshop cc. experimental infection of chickens, ducks and quails with the highly pathogenic h n avian influenza virus mammalian innate resistance to highly pathogenic avian influenza h n virus infection is mediated through reduced proinflammation and infectious virus release highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses prevalence of influenza a virus in live-captured north atlantic gray seals: a possible wild reservoir. emerging microbes & infections bats and their virome: an important source of emerging viruses capable of infecting humans. 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accompanies this paper at doi: . /s - - - competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -qwl nzkr authors: omori, ryosuke; matsuyama, ryota; nakata, yukihiko title: the age distribution of mortality from novel coronavirus disease (covid- ) suggests no large difference of susceptibility by age date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: qwl nzkr among italy, spain, and japan, the age distributions of covid- mortality show only small variation even though the number of deaths per country shows large variation. to understand the determinant for this situation, we constructed a mathematical model describing the transmission dynamics and natural history of covid- and analyzed the dataset of mortality in italy, spain, and japan. we estimated the parameter which describes the age-dependency of susceptibility by fitting the model to reported data, including the effect of change in contact patterns during the epidemics of covid- , and the fraction of symptomatic infections. our study revealed that if the mortality rate or the fraction of symptomatic infections among all covid- cases does not depend on age, then unrealistically different age-dependencies of susceptibilities against covid- infections between italy, japan, and spain are required to explain the similar age distribution of mortality but different basic reproduction numbers (r( )). variation of susceptibility by age itself cannot explain the robust age distribution in mortality by covid- infections in those three countries, however it does suggest that the age-dependencies of (i) the mortality rate and (ii) the fraction of symptomatic infections among all covid- cases determine the age distribution of mortality by covid- . since its emergence, coronavirus disease (covid- ) has resulted in a pandemic and has produced a huge number of cases worldwide . as of may , , the number of confirmed cases in italy was . (per , population), with . in spain, and . in japan . of those infected, it has been reported that elderly individuals account for a large portion of fatal cases inducing a large heterogeneity in the age distribution of mortality [ ] [ ] [ ] . the expected value of mortality (the number of deaths, hereafter referred to as mortality) is calculated as the product of the number of cases and the mortality rate among cases (hereafter referred to as morality rate). as the background mechanism of the heterogeneity of mortality by age, the association of two epidemiological factors with mortality can be considered: (i) the age-dependency of susceptibility to infection, which is related to the heterogeneity in the number of cases, and (ii) the age-dependency of severity, which is related to the heterogeneity in the mortality rate, e.g. the rate of becoming a symptomatic, severe, or fatal case among infected individuals. for the first factor, a high susceptibility for infection will generate a larger number of infections and result in an increase in fatal cases. the possibility of heterogeneity in susceptibility by age was pointed out by the analysis of epidemiological data reported from wuhan, china - and from iceland . for the second factor, an increase in severity will result in a higher mortality rate and subsequently a rise in the number of fatal cases. this assumption is also reasonable because elder age as well as the existence of comorbidities, which are likely with aging, have been reported as risk factors for severe covid- infections [ ] [ ] [ ] [ ] [ ] [ ] . although not yet shown in relation to severe acute respiratory syndrome coronavirus (sars cov- ), which is the causal agent of covid- , the presence of age-dependent enhancement of severity has been suggested in sars coronavirus by the analysis of the innate immune responses in the balb/c mouse model [ ] [ ] [ ] . additionally, it has been suggested that antibody-dependent enhancement (ade) can contribute to the formation of the observed age-dependency of severity, as suggested in sars and middle east respiratory syndrome (mers) cases [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, the age distribution of mortality by covid- (the distribution of the proportion of deaths per age group among all deaths), is similar between italy, japan, and spain, even though the number of deaths are quite different among them [ ] [ ] [ ] (fig. ) . the reported number of deaths was in - years old (yo), in - yo, in - yo, in - yo, in - yo, , in - yo, , in - yo, and , in + yo in italy as of may , . in japan, that was in - yo, in - yo, in - yo, in - yo, in - yo, in - yo, in - yo, and in over + yo as of may , . in spain, that was in age - yo, in - yo, in - yo, in - yo, in - yo, in - yo, in - yo, and , in over + yo as of may , . according to projections by the united nations , the population size for per , , was . in - yo, . in - yo, . in - yo, . in - yo, . - yo, . in - yo, . in - yo, and . in + yo. the large difference in the number of deaths between the countries suggests a large difference in their basic reproduction numbers, r s. an independency between age distribution of mortality by covid- and r is suggested. from this independency of age distributions of mortality from r , it can be expected that the contribution of heterogeneity in susceptibility by age to forming the age distribution of mortality is small. that is because, as we will show in this paper, though the age-dependency of severity will naturally produce a proportional effect on the distribution of mortality and result in the formation of robust distributions, when the age-dependency of susceptibility forms the age distribution of mortality, the age distribution of mortality highly depends on r and shows variability. to understand the background of robust age distribution of mortality with varied r , we constructed a mathematical model describing the transmission dynamics of covid- and analyzed the impact of age-dependent susceptibility on the age distribution of mortality. the heterogeneity in social contacts by age may also contribute to the age distribution of mortality. our model took into account the heterogeneity in social contacts by age and country, and the effect of behavioral change outside of the household during the outbreak. we also estimated and compared the age-dependent susceptibility in japan, italy, and spain to argue the existence of heterogeneity in susceptibility among age groups. our result shows variation of susceptibility among age groups measured by the exponent parameter φ can explain the age distribution of mortality by covid- (fig. a) . however, the age distribution of mortality formed by the age-dependency of susceptibility is influenced by the value of r (fig. b) , which cannot explain the similarity in age distributions of mortality among italy, japan, and spain. on the other hand, if susceptibility is constant among age groups, the impact of r is quite small on the age distribution of mortality (fig. ) . assuming that the age-dependency of mortality by covid- is determined by only age-dependent susceptibility (model ), i.e., the mortality rate does not depend on age, the exponent parameter, φ, describing the variation of susceptibility among age groups for each country, italy, japan, and spain, was estimated as shown in fig. . from the difference of the r value and country, the estimated value of φ is largely varied. the impact of reductions in contacts outside of the household on the estimated value of φ was small. the estimate of φ in italy, assuming a range of r = . - . , was . ( % ci . - . ), . ( % ci . - . ), and . ( % ci . - . ) for %, %, and no reduction in contacts outside of the household. for japan, the estimate of φ assuming r = . was . ( % ci . - . ), . ( % ci . - . ), and . ( % ci . - . ) for %, %, and no reduction in contacts outside of the household. when it comes to spain, the estimate of φ assuming an r = . was . ( % ci . - . ), . ( % ci . - . ), and . ( % ci . - . ) for %, %, and no reduction in contacts outside of the household. www.nature.com/scientificreports/ the estimates of φ, assuming that the mortality by covid- infections depends on age but the fraction of infections becoming symptomatic does not depend on age (model ), were also varied by the value of r and by country (fig. , s and s ). employing the same assumptions of r value, the estimate of φ in italy was . ( % ci . - . ), . ( % ci . - . ), and . ( % ci . - . ) for %, %, and no reduction in contacts outside of the household. for japan, the estimate of φ was . ( % ci . - . ), . ( % ci . - . ), and . ( % ci . - . ) for %, %, and no reduction in contacts outside of the household. for spain, the estimate of φ was . ( % ci . - . ), . ( % ci . - . ), and . ( % ci . - . ) for %, %, and no reduction in contacts outside of the household. in the present study, we explored the role of susceptibility to covid- in explaining the age distribution of mortality by covid- . interestingly, the age distributions of mortality from covid- are quite similar between italy, japan, and spain ( fig. ) . when comparing the age distributions of mortality, only the comparison between italy and spain is significant (p < . in wilcoxon rank sum test with bonferroni correction). on the other hand, the numbers of deaths are quite different ( , for italy, for japan, , for spain). indeed, r values are largely different: . - . for italy , , . for japan , and . for spain . if the variation of mortality by age is determined by only the age-dependency of susceptibility, the age distribution of mortality is affected by r as shown in fig. b . however, we observed a similarity in age distributions of mortalities between italy, japan, figure . the sensitivity of transmission coefficient β against age distribution of mortality when it was assumed that age-dependent mortality was proportional to ccfr per age group. all parameters were fixed and parameterized as the setting for spain except the transmission coefficient β. www.nature.com/scientificreports/ and spain where their r s are quite different. indeed, unrealistically different φs among these three countries are required to explain their age distribution of mortality for both settings, (i) age-independent mortality, and, (ii) the fraction of infections that becomes symptomatic among all covid- cases, f s , does not depend on age. although we cannot fully reject the existence of age-dependency in susceptibility, our results suggest that it does not largely depend on age, but rather that age-dependency in severity highly contributes to the formation of the observed age distribution in mortality. the estimates of φs assuming age independency in symptomatic infections were smaller than those that assumed age independency in mortality. this suggests that the age-dependency of the confirmed case fatality rate (ccfr), which can be biased by the age-dependent difference of the fraction of symptomatic infections among all cases, partially explains the age distribution in mortality. indeed, when we assumed that the fraction of symptomatic infections was not dependent on age, the estimate of φ in japan was close to zero in all scenarios regarding the fraction of symptomatic infections, meaning that susceptibility is constant among age groups (fig. ) . although we observed φs not close to zero in italy and spain, this does not mean straightforwardly that susceptibility is age dependent because there is room for an alternative explanation: not susceptibility, but an age-dependent fraction of symptomatic infections can explain this age-dependency. unfortunately, as we do not yet have detailed data regarding the age-dependent fraction of symptomatic infections and the rate of diagnosis in covid- , we cannot conclude which factors (i.e., susceptibility or the fraction of symptomatic infection among all cases) contributed to the observed age-dependency. wu et al. showed variation of susceptibility to symptomatic infection by age. this susceptibility can be expressed as the product of the susceptibility and the fraction of symptomatic infection among all cases. to accurately understand susceptibility (i.e., without the constraint of the symptom onset), estimates of the agedependent fraction of symptomatic infections is required. to understand the mechanism of age-dependency of mortality by covid- , an accurate age-dependent mortality rate is required. the data of mortality by covid- infections used in this study might not cover all mortalities by covid- infections. to estimate the age-dependent mortality rate, an accurate estimate of the case fatality rate is required. however, the number of cases, which is the denominator of the case fatality rate, is www.nature.com/scientificreports/ difficult to estimate for covid- due to changes in the testing rate [ ] [ ] [ ] , the change of case definition , selection biases , and the delay between the onset of symptoms and death , - as were the cases we experienced in the surveillance of other emerging diseases , . to address this problem, implementation of active epidemiological surveillances, such as a large-scale cohort study including real-time detection of infections, should be considered. from the modelling perspective on mortality by covid- , age-dependency of severity should be carefully taken into consideration. in particular, in the mathematical models of ade, previous models employed three types of assumptions , the assumption of: increasing susceptibility to infection , , increasing transmissibility once infection occurred , , , and increasing severity and/or mortality associated with infection . based on our results and from the biological/epidemiological observations of past sars and mers cases, the "increasing severity" assumption should be taken into account when analyzing sars cov- epidemics. we modelled the age-specific susceptibility as a power law function based on the monotonic increase of mortality by covid- over age as seen in fig. . the power law function is widely used to model heterogeneity, e.g., the heterogeneity in risks of sexually transmitted infections . although our model for age-specific susceptibility covers a wide variation of monotonic changes, our results might be biased by this formulation if the susceptibility changes over age in non-monotonic fashion. the increase in width of the confidence interval for the estimate of φ by increasing r values were observed in fig. . to explain with the "left-skewed" age-distribution of mortality with high r , a large φ is required since the higher r value decreased the heterogeneity of mortality by age (fig. b ) and the large φ increased the heterogeneity of mortality (fig. a) . the sensitivity of φ to the age-distribution of mortality becomes smaller when φ is larger (fig. a) , the large widths of the confidence intervals for the estimate of φ is necessary to explain the age-distribution of mortality when r is high. in conclusion, the contribution of age-dependency to susceptibility is difficult to use to explain the robust age distribution in mortalities by covid- , and it suggests that the age-dependencies of the mortality rate and the fraction of symptomatic infections among all covid- cases determine the age distribution in mortality from www.nature.com/scientificreports/ covid- . further investigations regarding age-dependency on the fraction of infections becoming symptomatic is required to understand the mechanism behind the mortality by covid- infections. data. we analyzed the number of mortalities caused by covid- in italy reported on th may , japan reported on th may , and spain reported on th may . the data were collected from public data sources in each country - . model. a simple seird model taking into account mixing between age groups (model ). to understand the background of robust age distribution of mortality with varied r , we employed a mathematical model describing transmissions of covid- . clinical observations suggest that both asymptomatic and symptomatic cases are infectious after the latent period , , we used a simple age-structured seird (susceptible-exposedinfectious-recovered-dead) model, which can be written as; where s n , e n , i n , r n and d n represent the proportion of susceptible, latent, infectious, recovered and dead among the entire population, and the subscript index n denotes age group. we stratified the entire population by into eight groups, n = , , , , , , , and for < yo, - yo, - yo, - yo, - yo, - yo, - yo, and + yo. β, k n,m , ε, γ and δ represent a transmission coefficient, an element of the contact matrix between age group n and m, the progression rate from latent to infectious, recovery rate and mortality rate by covid- infections, respectively. σ n denotes the susceptibility of age group n. for the sake of simplicity, based on the short study duration of covid- epidemics compared to the length of a human lifespan, births and deaths from causes other than covid- were ignored. to take into account the effect of behavioral changes outside of the household during the outbreak, k n,m is decomposed by a matrix for contacts within household k in,n,m and that for contacts outside the household k out,n,m ; where α denotes the reduced fraction of contacts outside of the household. we modelled age specific susceptibility as where c is susceptibility among age group and a constant among all age groups, φ denotes the exponent parameter describing the variation of susceptibility among age groups. an increase in φ means an increase in the variation of susceptibility among age groups, and φ = means that susceptibility is equal among all age groups. seird model taking into account mixing between age groups, asymptomatic/symptomatic, and age-dependency of mortality by . model does not classify the cases into asymptomatic and symptomatic cases explicitly. if the progression of disease is largely different between asymptomatic and symptomatic cases, the estimates using model can be biased. in addition, the age-dependency of mortality by covid- infections is not taken into account. model takes into account both the different progression of disease between asymptomatic and symptomatic cases and the age-dependency of mortality by covid- infections; ( ) s ′ n = −βσ n s n m k n,m i m , www.nature.com/scientificreports/ where i s,n and i a,n represent the proportion of symptomatic and asymptomatic cases among age group n. other compartments are the same as model . f s represents the fraction of symptomatic infections among all covid- cases and δ n represents the mortality rate by covid- infection among age group n. γ s and γ a denote the recovery rates among symptomatic and asymptomatic cases. other parameters are the same as model . we parameterized ε and γ using values from a previous modelling study of , . the average length of the latent period (i.e., /ε) was set to . days , , assuming that the latent period is equal to the incubation period, and the average length of the infectious period (i.e., /γ) was days , for model . in model , to take into account the different infectious period between symptomatic and asymptomatic infections, we set an average length of infectious period among asymptomatic cases (i.e., /γ a ) as days and an average length of infectious period among symptomatic cases (i.e., /γ s ) as days. we referred to the contact matrices for italy, japan, and spain from prem et al. . β and c were controlled such that the basic reproduction number, r , becomes arbitral values. r was calculated by constructing a next generation matrix , using each country's demographic data obtained from a public data source . in terms of parameterization for mortality rate by covid- infection, a reliable estimate of δ n for covid- is difficult to obtain. due to the uncertainty of the fraction of symptomatic infections per age group, δ n is difficult to estimate from observed data, i.e., the confirmed case fatality rate among age group n (ccfr n ). since an estimate of δ n is difficult to obtain, we employed two different settings (i) δ n is assumed to be a constant among all age groups as assumed in the model , i.e., δ n = δ for any age group n, or, (ii) δ n is calculated from ccfr n assuming that the fraction of symptomatic infections among all covid- cases (f s ) is not dependent on age as assumed in model . in the setting for model , the value of δ is not required to estimate d n once the value of r is given. we calculated d n by calculating the proportions of recovered persons per age group among all recovered persons r n (∞)/ n r n (∞) instead of d n (∞)/ n d n (∞) . in our model, shown in eq. ( - ), r n (∞)/ n r n (∞) is determined by the value of r completely when all parameter values other than β and δ are fixed, and d n (∞)/ n d n (∞) = r n (∞)/ n r n (∞) if δ n = . the proof can be found in the supplemental text. the assumption in model , δ n is constant among all age groups, may be too strong for the covid- epidemic. to take into account the age-dependency of mortality by covid- , δ n was calculated from the ccfr n assuming that f s is not dependent with age. for the setting in model , assuming three scenarios; f s = . , . , and . , δ n for each country were calculated using ccfr n in each country. we obtained δ n by solving ccfr n = δ n / (δ n + γ s ). fitting. we calculated the proportions of deaths in the age group n among all deaths, d n ( = d n (∞)/ n d n (∞) ), and fitted them to the observed data in each country. we solved model shown in eqs. ( ) ( ) ( ) ( ) ( ) and model shown in eqs. ( ) ( ) ( ) ( ) ( ) ( ) numerically, and d n was calculated after sufficient time was given to finish the epidemics. we estimated φ using a log likelihood function describing the multinomial sampling process of deaths per age group; maximum likelihood estimates of φ with given r were obtained by maximizing eq. 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- - .correspondence and requests for materials should be addressed to r.o.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -xgtqfwyn authors: liu, bing; han, junyan; cheng, xiaohuan; yu, long; zhang, li; wang, wei; ni, lan; wei, chaojie; huang, yafei; cheng, zhenshun title: reduced numbers of t cells and b cells correlates with persistent sars-cov- presence in non-severe covid- patients date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xgtqfwyn covid- has been widely spreading. we aimed to examine adaptive immune cells in non-severe patients with persistent sars-cov- shedding. non-severe patients with persistent sars-cov- presence that were transferred to zhongnan hospital of wuhan university were retrospectively recruited to the pp (persistently positive) group, which was further allocated to ppp group (n = ) and ppn group (n = ), according to their testing results after days (n = negative). epidemiological, demographic, clinical and laboratory data were collected and analyzed. data from age- and sex-matched non-severe patients at disease onset (pa [positive on admission] patients, n = ), and lymphocyte subpopulation measurements from matched healthy subjects were extracted for comparison (hc). compared with pa patients, pp patients had much improved laboratory findings. the absolute numbers of cd (+) t cells, cd (+) t cells, and nk cells were significantly higher in pp group than that in pa group, and were comparable to that in healthy controls. ppp subgroup had markedly reduced b cells and t cells compared to ppn group and healthy subjects. finally, paired results of these lymphocyte subpopulations from ppn patients demonstrated that the number of t cells and b cells significantly increased when the sars-cov- tests turned negative. persistent sars-cov- presence in non-severe covid- patients is associated with reduced numbers of adaptive immune cells. monitoring lymphocyte subpopulations could be clinically meaningful in identifying fully recovered covid- patients. data collection. demographic information, clinical characteristics (including medical history, exposure history, comorbidities, surgery history, signs, and symptoms), chest computed tomographic (ct) scan or x-ray results, and laboratory findings of each patient were obtained from the electronic medical record system of zhwu and analyzed by three independent researchers. laboratory testing. patient nasopharyngeal swab specimens were collected for the sars-cov- viral nucleic acid detection using real-time reverse transcriptase-polymerase chain reaction (rt-pcr) assay. the viral nucleic acid testing for all patients was performed by the clinical laboratory from zhongnan hospital of wuhan university in wuhan. detailed protocol was described previously . lymphocyte subpopulations were examined by facs aria iii cytometer (bd bioscience, usa) and analyzed using flowjo software v. . (bd bioscience, usa). other laboratory indicators, including blood routine, c-reactive protein (crp), serum amyloid a (saa), and il- , were collected for each patients. statistical analysis. data analysis was performed using spss (statistical package for the social sciences, version ). categorical variables were reported as absolute (relative frequencies) and compared by χ tests or fisher's exact tests. continuous variables were expressed as mean (sd) if they are normally distributed or median (interquartile range, iqr) if they are not and compared by independent group t tests or mann-whitney u tests, respectively. p < . was considered as statistically significant. baseline characteristics. after initial screen, non-severe covid- patients that were tested positive for sars-cov- more than days were recruited to the pp group. the median age for these patients was years (iqr - ; table ), and ( . %) patients were men. since no patients had direct exposure history of huanan seafood market, we presumed all patients in this study were community-infected cases. the most common symptoms at onset of illness were fever ( . %) and dry cough ( . %), followed by dyspnea ( . %), expectoration ( . %), and diarrhea ( . %). the less common symptoms included pharyngalgia ( . %), hemoptysis ( . %) and weep tears ( . %). common complications included cvd ( . %), followed by diabetes ( . %) and hepatitis ( . %). there were current smokers. the baseline characteristics were summarized in table . table presented the laboratory testing results of these patients (pp group) on admission to our hospital. unfortunately, the results of the same patients at disease onset were not available since these patients were first admitted to mobile cabin hospitals and then transferred to our hospital, we therefore randomly selected another age-and sex-matched covid- patients confirmed with non-severe disease (pa group), who had lymphocyte subsets in peripheral blood. it has been reported that dysregulated immune response were correlated with the severity of covid- . however, changes in adaptive immune cells in non-severe covid- patients with persistent sars-cov- shedding has yet to be examined. for this purpose, peripheral blood samples from patients in the pa and pp group were collected, the absolute numbers and relative frequencies of each lymphocyte subpopulations were compared between these two groups. in addition, age-and sex-matched healthy subjects were randomly selected as healthy control (the hc group). as shown in table , we failed to find any differences between the pp group and the hc group, but patients from both groups had increased numbers of cd + t cells, cd + t cells, and nk cells compared to those from the pa group. in addition, pa patients had significantly lower frequency of b cells compared with healthy subjects (table ). these results indicated that non-severe covid- patients (pa group) have already dysregulated immune system at disease onset, and those with persistent sars-cov- shedding could restore this abnormality to certain level. upon admission, pp patients received the same standard treatment in our hospital. after at least days, of them that were tested negative for sars-cov- in two consecutive examinations were retrospectively allocated we did not find any differences in symptoms and laboratory findings for these two groups (supplementary tables and ). however, when lymphocyte subpopulations were examined, ppp patients were found to have significantly lower numbers of cd + t cells (p = . ), cd + t cells (p = . ), cd + t cells (p = . ), and b cells (p = . ), but higher proportion of nk cells (p = . ) than ppn patients (fig. a,b) . next, we determined the abnormalities for each parameters by using reference ranges published elsewhere (table , fig. c ,d) . similar trends were found in cd + t cells (p = . ), cd + t cells (p = . ), cd + t cells (p = . ), and b cells (p < . ). since the reference ranges of lymphocyte subpopulations were established based on all chinese han population, we therefore selected age-and sex-matched healthy subjects from wuhan for comparison. again, ppp patients exhibited less numbers of cd + t cells (p = . ), cd + t cells (p = . ), and b cells (p = . ) than healthy subjects (fig. a,b ). finally, we were able to extract paired results of lymphocyte subpopulations for patients on admission (last positive), and on the first day they tested negative for viral rna (first negative). these patients demonstrated markedly increased cd + t cells (p = . ), cd + t cells (p = . ), cd + t cells (p = . ), and b cells (p = . ) after turned negative for sars-cov- (fig. ) . together, these results indicated that the abnormalities in adaptive immune cells, but not symptoms and laboratory indicators, were associated with sars-cov- viral rna detection in non-severe covid- patients. this retrospective investigation was designed to examine immunological characteristics of non-severe covid- patients with persistent viral presence. we reported here that despite their alleviated symptoms and much improved laboratory findings, these patients demonstrated significantly lower numbers of t cells and b cells than healthy controls, and than those turned negative for viral rna. non-severe covid- patients with persistent viral presence were included in this study and were allocated to the pp group. multiple symptoms, including fever, dry cough, dyspnea, expectoration, diarrhea, pharyngalgia, hemoptysis and weep tears were recorded at disease onset (table ) , and most of these patients were abnormal in radiographic examination (data not shown). upon treatment in mobile cabin hospitals and transferred to our hospital, they turned almost asymptomatic with much improved laboratory findings, as showed in table and compared with those in the pa group. however, persistent sars-cov- presence were evident in all these patients. the presence of sars-cov- has been the golden standard for both diagnosis and disease management of covid- . in fact, two consecutively negative results for viral rna is required for patients to be discharged from hospitals . nasopharyngeal swabs were frequently used for detecting viral rna by rt-pcr because these samples are easily accessible. however, some limitations were noticed. first, the kinetics of sars-cov- shedding was different from that of sars-cov and mers-cov. rna copies of sars-cov- were very high in nasopharyngeal swab during the first week of symptoms, with peak on day post-onset, whereas the peak figure . absolute numbers (a) and relative frequencies of lymphocyte subpopulations (b) in peripheral blood of pp patients were tested positive again at least days after they were admitted to our hospital (ppp), and pp patients were tested negative in days after they were admitted to our hospital (ppn). fifty six ageand sex-matched healthy subjects were used as control (hc). the proportion of abnormalities of lymphocyte subpopulations in terms of absolute numbers (c) and relative frequencies of (d) in peripheral blood of ppp and ppn patients were also indicated. *p < . ; **p < . ; ***p < . . | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ value appeared until - days post-onset with much lower rna copies during sars-cov and mers-cov infection , [ ] [ ] [ ] . second, the presence of virus rna in lower respiratory tract (sputum or bals), stool, and blood samples were reported, and the kinetics of virus shedding in these sites were distinct from that in throat [ ] [ ] [ ] [ ] . third, sampling error and the technical limitations of rt-pcr sometimes led to a false testing result . with these limitations, it is not surprising that some patients who tested negative in two consecutively rt-pcr tests and were discharged from hospital had positive results - days later , . in together, these notions posed a great challenge to discharge management for covid- patients, especially for non-severe cases having obtained clinical cure. since the presence of viral rna might come from fragments of dead virus, isolating live sars-cov- is therefore useful in determining viral infectivity . however, this method is required to be performed in a biological safety level (bsl- ) laboratory, which limited its application in clinical practice for discharge management. indicators from the immune system are promising candidates in this regard. detection for virus-specific igm and igg has been widely used in hepatitis and other virus infectious diseases for helping the diagnosis of viral infection, as well as for evaluating disease status and prognosis . it was reported that sars-cov-specific igm and igg were generated - days and - days post infection, respectively . in fact, detection for virus-specific igm and igg were recently included in the latest version of the guideline of sars-cov- (trial version of the chinese national health commission), for assisting the diagnosis of sars-cov- infection . however, antigen selection and assay sensitivity may cause both false positive and false negative results . thus, its efficacy in diagnosis and discharge management is yet to be tested by large clinical investigations. the production of both antibody isotypes requires the cooperation between virus-specific t cells and b cells. therefore, alterations of these adaptive immune cells might precede the changes of antibodies and could be useful for discharge management. lymphopenia was observed at illness onset in . % of non-severe covid- patients (the pa group) in our study, which is similar to those reported by zhang et al. ( . %), mo et al. ( . %), wang et al. ( . %), and guan et al. ( . %), suggesting the involvement of lymphocytes in the early phase of sars-cov- infection. furthermore, lymphocyte count was reported to be correlated with disease severity. significant higher numbers of lymphocytes were found in survivors versus non-survivors , as well as critically ill versus severe , , and severe versus non-severe cases , . we focused on non-severe patients with persistent viral presence, and found that the pp group had markedly higher lymphocyte count ( . [ . - . ] vs . [ . - . ]; p < . ) than the pa group, and were comparable to healthy subjects. this finding, together with alleviated symptoms and improvements of other laboratory findings, indicated that pp patients might be in the process of recovery, albeit their viral rna were still tested positive. however, other parameters are required to determine if they were fully recovered. we therefore examined lymphocyte subsets and found that ppp patients had significantly lower numbers of cd + t cells (p = . ), cd + t cells (p = . ), cd + t cells (p = . ), and b cells (p = . ) than ppn patients (fig. a,b) . when compared with healthy subjects, ppp patients again exhibited much less cd + t cells (p = . ), cd + t cells (p = . ), and b cells (p = . ) (fig. a,b) . most strikingly, ppn patients www.nature.com/scientificreports/ showed markedly increased cd + t cells (p = . ), cd + t cells (p = . ), cd + t cells (p = . ), and b cells (p = . ) after they turned negative for sars-cov- (fig. ) . together, these results suggest that measurement of these lymphocyte subpopulations could be used to distinguish non-severe patients with persistent viral presence from healthy subjects and those turned negative, and thus have clinical relevance for discharge management. t cells and b cells are the two most important lymphocytes in fighting against viral infection. cd + t cells are particularly efficient in clearing virus-infected cells, after receiving help from cd + t cells . the latter can induce the activation and differentiation of cognate b cells, and subsequently promote the production of virusspecific antibodies, including neutralizing antibodies . in turn, neutralizing antibodies are able to mediate antibody-dependent cell-mediated cytotoxicity to kill virus-infected cells, and to block the entrance of extracellular virus . therefore, it's not surprising that changes in these cells could reflect the viral presence. accordingly, t cell subsets were reported to be profoundly affected in severe cases with sars-cov- infection . however, we could not determine from our data and the current knowledge whether sars-cov- can directly infect these lymphocytes, or indirectly caused these alterations. we did not find any difference in nk cells between the ppp group and healthy subjects, in terms of both absolute numbers and relative frequency (fig. ) . instead, the relative frequency of nk cells were even higher in the ppp group than in the ppn group (p = . , fig. b) , although the absolute numbers of nk cells between these two groups were comparable. this discrepancy is likely due to the relative increase of t cells and b cells in the ppn group. as an innate immune cells, nk cells are among the first cell types to combat virus infection . however, pp patients in our study were likely to be in the late phase of sars-cov- infection, during which the role of nk cells remained to be defined. several limitations to the present study warrant mention. first, this retrospective study was conducted in a single hospital, which may result in selection bias. our conclusion could be further strengthened by a multicenter, prospective study in a randomized setting. second, only non-severe covid- patients with persistent viral presence were included in this investigation, interpretation of our findings might be limited by the sample size. nevertheless, our study cohort included patients that were transferred from two large mobile cabin hospitals that have treated hundreds of thousands patients during the pandemic, in addition to those directly from zhongnan hospital. in fact, that's all the patients that were persistently positive for sars-cov- rna we can successfully recruit for this study. despite the small sample size of this study, similar phenomena have been observed by other groups while this manuscript was under consideration. using viral clearance days of days as the cutoff to divide patient population into long viral persistence group (n = ) and short viral persistence group (n = ), chang et al. found that the frequencies of lower than normal cd + t cell and cd + t cell counts in the long viral persistence group were significantly higher than that in the short viral persistence group. similarly, ling et al. found that the cd + t lymphocyte count may help predict the duration of viral rna detection in patients' stools. third, these patients were transferred to our hospital, we do not have their laboratory results and lymphocyte measurements at disease onset, we therefore randomly selected age-and sex-matched pa patients for comparison. fourth, quantitative viral rna detection and isolation of live virus were not performed due to limited resources in our hospital, which prevent us from building connections between lymphocyte subpopulations and these parameters. despite these limitations, the present study, to the best of our knowledge, is the first investigation to examine changes of lymphocyte subpopulations in non-severe covid- patients with persistent viral presence. we found that cd + t cells, cd + t cells, and b cells were markedly decreased in these patients. our findings suggest that monitoring lymphocyte subpopulations could be clinical meaningful in discharge management for non-severe covid- patients with persistent viral presence. www.nature.com/scientificreports/ open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. coronavirus disease (covid- ) situation report- clinical characteristics of coronavirus disease in china novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) clinical course and risk factors for mortality of adult in patients with covid- in wuhan, china: a retrospective cohort study virological assessment of hospitalized patients with covid- evaluation of sars-cov- rna shedding in clinical specimens and clinical characteristics of patients with covid- in macau clinical features of patients infected with novel coronavirus in wuhan human kidney is a target for novel severe acute respiratory syndrome coronavirus (sars-cov- ) infection sars-cov- viral load in upperrespiratory specimens of 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positive rt-pcr test results in patients recovered from covid- post-discharge surveillance and positive virus detection in two medical staff recovered from coronavirus disease (covid- ), china role of humoral immunity against hepatitis b virus core antigen in the pathogenesis of acute liver failure production of specific antibodies against sars-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses e and oc national health commission of the people's republic of china development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection t cell-mediated immune response to respiratory coronaviruses recent progress in broadly neutralizing antibodies to hiv regulation and function of nk and t cells during dengue virus infection and vaccination persistent viral presence determines the clinical course of the disease in covid- persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients we thank all patients and their families involved in the study, and all the healthcare professionals from zhongnan hospital of wuhan university. y.h. and z.c. conceived the original idea of the work and performed, analyzed and interpreted of data and reviewed the manuscript. y.h. and z.c. take responsibility for the integrity of the data and the accuracy of the data analysis. b.l., y.h., z.c., and j.h. drafted the manuscript. b.l. and x.c. collected the data. b.l., x.c., l.y., w.w., l.n., and c.w. did the analysis, and all authors critically revised the manuscript for important intellectual content and gave final approval for the version to be published. all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to y.h. or z.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - az h h authors: schiller, bastian; kleinert, tobias; teige-mocigemba, sarah; klauer, karl christoph; heinrichs, markus title: temporal dynamics of resting eeg networks are associated with prosociality date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: az h h as prosociality is key to facing many of our societies’ global challenges (such as fighting a global pandemic), we need to better understand why some individuals are more prosocial than others. the present study takes a neural trait approach, examining whether the temporal dynamics of resting eeg networks are associated with inter-individual differences in prosociality. in two experimental sessions, we collected healthy males’ resting eeg, their self-reported prosocial concern and values, and their incentivized prosocial behavior across different reward domains (money, time) and social contexts (collective, individual). by means of eeg microstate analysis we identified the temporal coverage of four canonical resting networks (microstates a, b, c, and d) and their mutual communication in order to examine their association with an aggregated index of prosociality. participants with a higher coverage of microstate a and more transitions from microstate c to a were more prosocial. our study demonstrates that temporal dynamics of intrinsic brain networks can be linked to complex social behavior. on the basis of previous findings on links of microstate a with sensory processing, our findings suggest that participants with a tendency to engage in bottom-up processing during rest behave more prosocially than others. more specifically, we used a spatio-temporal analysis approach to cluster the resting eeg signal into a circumscribed number of scalp electrical potential topographies that remain stable for certain time periods (ca. - ms) before dynamically changing into a different topography that remains stable again [ ] [ ] [ ] [ ] [ ] . one has referred to these periods with stable topographies as "microstates" and one has interpreted transitions between microstates to represent sequential coordinated activity of different, distributed neural networks. remarkably, almost % of the variance in the resting eeg data can be explained by just four archetypal microstates a-d, i.e., resting networks which may result from evolutionarily determined, brain-intrinsic biases toward particular patterns of co-activation particularly suited to representing environmentally relevant information . these networks' temporal dynamics have been proven to be highly reliable, specific, and reproducible across multiple independent studies , , ideally qualifying them as neural trait markers. several studies have attempted to identify the neural sources and functions related to these four resting eeg networks , , . microstates a and b have been associated with bottom-up sensory processing (microstate a has been linked to activity in temporal areas involved in phonological processing and microstate b has been linked to activity in extrastriate areas involved in visuo-spatial processing [ ] [ ] [ ] [ ] . microstate d, on the other hand, has been associated with top-down cognitive processing (microstate d has been linked to activity in fronto-parietal areas involved in attention and control , , ). the function of microstate c has remained more controversial, as it was originally linked to activity in fronto-insular areas considered to be involved in salience processing , but recent research associates it with activity in the default mode network and stimulus-independent processing , . being the first study of its kind, our general research question was to examine whether there is any association among the four canonical resting eeg networks' temporal dynamics with prosociality. for that purpose, we generated a domain-general index of prosociality that we aggregated across self-reported prosocial concern (using the interpersonal reactivity index scale empathic concern), self-reported prosocial values (using the portrait value questionnaire scale benevolence), collective prosocial behavior (using the public goods game in a monetary reward domain), and individual prosocial behavior (using the social value orientation task in a non-monetary reward domain). on the basis of resting eeg networks' significance for non-social behavior, personality, and psychiatric conditions , , we expected that revealing an individual's tendency to engage these networks at rest would help explain inter-individual differences in prosociality and illuminate the potential psychological processes that underlie these differences. for example, our findings might contribute to the debate on the role of bottom-up and top-down processing in driving inter-individual differences in prosocial behavior [ ] [ ] [ ] [ ] . due to our study's exploratory nature, we applied bonferroni-correction for multiple tests to our findings. prosociality. we did observe considerable variability in prosociality (m = . , s.d. = . , range: − . - . ), aggregated across self-reported prosocial concern (non-standardized values in the - prosociality and resting eeg networks. in accordance with previous findings, the applied cluster analysis identified the four canonical microstates a-d (see fig. ) which explained % of the variance in our whole sample and at least % of the variance in every single individual (m = . , s.d. = . , range: . - . ). to assess reliability, we correlated microstate parameters that were identified separately for the first and second halves of artifact-free data available in each individual (for details, see "methods"). we detected correlations ranging from . - . (all p < . ) with regard to microstate coverage, duration, and occurrence, and correlations ranging from . to . (all p < . ) concerning microstate transitions (for details, see table s ). these findings show the potential of microstate parameters to serve as neural trait markers that might be associated with inter-individual differences in prosociality. we first tested for associations of microstate coverage and prosociality. correlative analyses revealed that participants with a higher coverage of microstate a were more prosocial [r s ( ) = . , p = . , significant after bonferroni-correction for multiple testing; prosocial concern: r s ( ) = . , p = . ; prosocial values: table s ). furthermore, we found the significant association of microstate a's coverage and prosociality in both the first and the second half of data (first half: r( ) = . , p = . ; second half: r( ) = . , p = . ), demonstrating the robustness of this finding. in sum, participants with a higher temporal stability (= mean duration) and, in turn, coverage of microstate a tended to show more prosocial concern, values, and behavior. next we tested for associations of microstate transitions and prosociality. correlative analyses revealed that participants with more transitions from microstate c to a were more prosocial [r( ) = . , p = . , scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ significant after bonferroni-correction for multiple testing; prosocial concern: r s ( ) = . , p = . ; prosocial values: r s ( ) = . , p = . ; collective prosocial behavior: r s ( ) = . , p > . ; individual prosocial behavior: r s ( ) = . , p = . , see fig. ]. there were no other significant associations of microstate transitions and prosociality after bonferroni-correction for multiple testing (see table s ). meng's z-tests indicated that the correlation of prosociality with transitions from microstate c to a was significantly higher than in sum, participants with more transitions from microstate c to a tended to show more prosocial concern, values, and behavior. finally, to enable better comparability of our findings with the literature, we repeated all of our analysis sorting individual microstate maps according to normative grand mean templates . these analyses yielded highly similar results confirming the robustness of our study's findings [correlation of coverage of microstate a and prosociality: r( ) = . , p = . ; correlation of transitions from microstate c to a and prosociality: r( ) = . , p = . ; for details see table s ]. can someone's task-free neurophysiological processing reveal information on someone's prosociality? by analyzing the spatio-temporal dynamics of resting eeg recordings the present study demonstrates that an individual's propensity of how to engage the four canonical eeg resting networks (i.e., microstates a, b, c, and d) is associated with an index of an individual's domain-general prosociality. this index was aggregated across self-reported prosocial concern and values, as well as incentivized behavior collected in different reward domains (time and money) and social contexts (individual and collective). more specifically, we found that participants with a higher coverage of microstate a and more transitions from microstate c to a were more prosocial. how can we interpret the association of microstate a's coverage with prosociality based on previous research examining the functional significance of the four canonical eeg resting networks (for reviews, see , )? in healthy participants, microstate a's coverage has been associated with activity in the phonological fmri resting network and been shown to increase during hypnosis-a state characterized by a sense of automaticity and effortlessness , . one could thus deduce from these findings that a higher coverage of microstate a indicates an individual's tendency to engage in sensory, bottom-up processing during rest which in turn predisposes towards prosociality. notably, microstate b, associated with visual sensory processing , but also shown to decrease during hypnosis , was not associated with prosociality, indicating a complex relationship between different kinds of sensory, bottom-up processing and prosociality. furthermore, we found indications of a negative association of attention-related microstate d , , with prosociality (this correlation was no longer significant after correction for multiple comparisons across microstate classes), tentatively suggesting an antagonistic relationship of specific kinds of top-down compared to bottom-up processing with prosociality which may be corroborated in future research. www.nature.com/scientificreports/ our study also demonstrates that inter-individual differences in prosociality are associated with differences in the communication between eeg resting networks. more specifically, the networks underlying microstates a and c seem to act as a crucial gateway, as demonstrated by positive associations of transitions from microstate c to a with prosociality. so far, no consensus has been reached about the function of microstate c, but recent research has proposed that this state is associated with stimulus-independent processing , , . one could thus speculate that individuals with more transitions from microstate c to a tend to shift more often from stimulusindependent processing to stimulus-dependent sensory-related, bottom-up processing which in turn seems to predispose them to prosociality. we encourage future research to zoom in on the transitions between resting eeg microstates in order to better understand the functions underlying the communication between neural resting networks on a millisecond scale , . the discussed findings and interpretations might also contribute to our understanding of the interactive role of bottom-up and top-down processing in driving inter-individual differences in prosocial behavior [ ] [ ] [ ] . it has been reported that individuals who self-report a bottom-up processing style tend to maintain more successful interpersonal relationships, exhibit greater prosocial concerns, and tend to behave more prosocially [ ] [ ] [ ] . other research both examining and manipulating the response times underlying social behavior showed that individuals who took or possessed less time for their decisions behaved more prosocially , (but see also , ). these findings have been interpreted to indicate that people are "intuitively cooperative" and behave prosocially if they do not take or have the time to engage in time-consuming top-down processing during the (decision) task (see also recent research for specification under which conditions these findings hold [ ] [ ] [ ] ). here, we complement these findings by demonstrating correlative evidence that a tendency to engage in bottom-up rather than top-down, task-free neural processing predisposes towards prosociality. while prosociality is clearly determined by situational factors as well , , recent research has demonstrated that there is a cross-situational and temporally stable individual tendency for prosociality , . the present study proposes potential neural traits affecting this tendency by uniquely revealing the temporal dynamics of resting eeg networks with millisecond resolution, thereby extending findings from research on the role of resting fmri networks in prosociality , . it shows that an individual's propensity of how to engage the four resting eeg networks is associated with an individual's level of prosociality. future studies should test the generalizability of these findings across situations, as the aforementioned associations seemed to differ in their significance and strength across distinct measurement approaches, social contexts, and reward domains. furthermore, given that males and females differ in their prosociality in several dimensions [ ] [ ] [ ] , it is unclear how our findings would apply to females. we hope that our study inspires future research aiming to better understand the nature of the relationship between resting eeg networks' temporal dynamics and prosociality in participants of both genders, www.nature.com/scientificreports/ for example by experimentally modifying the environment of resting eeg recordings , analyzing the relationship between task-independent and task-dependent neural activity , , , analyzing brain-to-brain synchronization affecting prosociality , and linking the four resting eeg networks to psychological constructs known to affect prosociality (e.g. empathy, perspective-taking [ ] [ ] [ ] . due to potential confounds associated with hormonal variation in the menstrual cycle and the complexities associated with controlling for this variation in the experimental design , , only male participants were enrolled in this initial project. all participants were right-handed, and free of current or previous history of physical and psychiatric disorders, and alcohol or drug abuse. the ethics committee of the university of freiburg approved this study, which was conducted according to the principles expressed in the declaration of helsinki. procedure. there were two experimental sessions. at the first session, participants received detailed information on the experiment and gave informed consent. then, participants were comfortably seated in a darkened, electrically shielded cabin for the recording of -channel resting eeg. our measurement protocol consisted of -s eyes open periods followed by -s eyes closed periods, repeated five times. this resting state paradigm has been routinely used in resting eeg research , , , in order to minimize fluctuations in participants' vigilance state. participants can become drowsy already after min of recording resting state brain activity, if there is no alternation of eyes-open/eyes-closed periods . we gave the instructions about eye opening/closing via intercom. to exclude the possibility that instruction delivery confounds the resting state during the eyes-closed periods, instructions were delivered at the beginning and end of the eyes-open periods. after the resting eeg measurement participants completed three reaction time paradigms that are unrelated to the purpose of the current study and will be analyzed elsewhere. finally, participants completed self-report measures related to prosociality. the first session lasted approximately . h. the second session was conducted in groups of six participants in a group-laboratory specifically designed for computerized interaction experiments several weeks after the first appointment. after receiving detailed instructions and answering comprehension questions, participants played two social-decision making paradigms. finally, participants had to complete reaction time tasks which they had to repeat several times dependent on the time units earned in one of the social decision-making paradigms; we did not analyze this task further. the second session lasted approximately . eeg recording. the eeg was recorded with a -channel recording system (brainamp with acticap, brain products gmbh, munich) according to the extended - system montage . scalp impedance was kept below kΩ. fcz served as the reference electrode, afz as the ground electrode. horizontal and vertical electrooculographic signals were recorded with two additional electrodes at the left and right outer canthi and one electrode at the left infraorbital. the eeg was online band-pass filtered between . and hz, and the data digitized with a sampling rate of hz. eeg pre-processing. we used the brain vision analyzer program (version . . . ; brain products gmbh, munich) to pre-process eeg data. only the s eyes-closed periods were used for the analysis, because the influence of external visual stimulus processing and confounding eye blinks is minimized , . next, we band-pass filtered eeg data (high-pass hz, low-pass hz and re-derived them to average reference. ocular correction was conducted via a semi-automatic independent component analysis based correction process. eeg signals with excessive noise were replaced by using a linear interpolation of neighboring electrodes. after an automatic artifact rejection (maximum amplitude: ± μv), data were visually examined to eliminate residual artifacts. eeg microstate analysis. microstate analysis was conducted using the microstate-plugin for the matlab toolbox eeglab . first, in line with the standard procedure , , the maps at the momentary peaks of the global field power (i.e., maximum voltage values at all electrodes that represent time points of optimal signalto-noise ratio were extracted and submitted to a modified spatial cluster analysis using the atomize-agglomerate hierarchical clustering method (aahc , ). this clustering approach identified the four most dominant cluster maps in every single participant. the individual cluster maps were submitted to a second cluster analysis yielding grand mean maps which were then sorted according to the standard labeling of the four canonical eeg resting networks , (see fig. ). next, the maps of each individual were sorted according to these grand mean maps on the basis of spatial correlations. to enable comparability of our findings with the literature, we repeated this sorting procedure using the grand mean template maps as identified in normative eeg data collected from multiple sites (n = ; electrodes . finally, the gfp peaks of individual eeg data were assigned to the individually identified cluster maps to which they best fitted. this assignment was linearly interpolated to the time periods between the gfp peaks, yielding a continuous temporal stream of microstates occurring in each individual. from this last step, we extracted several microstate parameters. we focused on the temporal coverage a given microstate is dominant, representing the total presence of the underlying network. we also www.nature.com/scientificreports/ investigated underlying associations with average microstate duration (i.e., an index of the temporal stability of the underlying network; unit: milliseconds) and microstate occurrence (i.e., an index of the relative usage of the underlying network; unit: occurrences/second), both of which together determine a microstate's temporal coverage. to reveal communication between the underlying networks, we finally studied microstate transitions, which were operationalized as the percentage of observed transitions from one microstate class to another relative to expected transitions [transitions = (observed transitions per second -expected transitions per second) / expected transitions per second * ; i.e., a value of indicates that transitions from one microstate class to another occurred % more frequently than expected from each microstate's occurrence]. finally, to assess the reliability of microstate parameters, we performed the fitting procedure (i.e., assigning the gfp peaks to the four individually identified maps, which were sorted according to the grand mean maps or grand mean templates maps) separately for the first and second halves of each participant's artifact-free eeg data. self-report measures of prosocial concern and prosocial values. we measured self-reported prosocial concern using the interpersonal reactivity index (iri , german version by ). the iri is a questionnaire including items for the assessment of empathic abilities on four different scales with seven items each (empathic concern, perspective taking, fantasy, and emotional distress). per item, participants have to report how well these items describe them as a person from "not at all" to "very strong" ( -point likert scale). for the purpose of this study, we focused on the scale empathic concern which assesses feelings of prosocial concern for others (cronbach's alpha = . - . ). additionally, we measured self-reported prosocial values using the portrait value questionnaire (pvq , german version by ). we used the -item version of the pvq measuring personal values on different scales with - items each (benevolence, power, achievement, hedonism, stimulation, self-direction, universalism, tradition, conformity, and security). the pvq measures values indirectly by obtaining judgments of the similarity of another person, who is portrayed in terms of her or his goals, aspirations, and wishes, to oneself, on a scale ranging from "not like me at all" to "very much like me" ( -point likert scale). for the purpose of this study, we focused on the scale benevolence, which assesses prosocial values, i.e., attaching importance to the preservation and enhancement of the welfare of other people (cronbach's alpha = . ). decision-making paradigms to assess prosocial behavior. we measured prosocial behavior involving actual consequences to interaction partners across two distinct reward domains (money and time) and social contexts (individual and collective). to control for strategic considerations (e.g., reputation, reciprocity), we kept the decisions anonymous and non-reciprocal. because we were interested in explaining inter-individual differences, we used a fixed order of the two decision-making paradigms , . participants first played the public goods game, a well-established behavioral measure of collective prosocial behavior . each participant was randomly assigned to a group of three people and endowed with points (exchange rate: points = euro) which he could either keep for himself or contribute to the public good (possible contributions ranged from to in steps of points). the sum of all contributions to the public good was multiplied by the factor . and then equally divided among all players of the assigned group regardless of individual contributions. this game induces a conflict between self and group interests, because participants can earn the maximal personal profit by contributing nothing and profiting from other participants' contributions, whereas the group can earn the maximal profit by all participants contributing all their resources. the amount contributed to the public good is entered into the calculation of domain-general prosociality. second, participants played the social value orientation task , which assesses individual prosocial behavior and has previously been linked to prosocial behavior such as helping or pro-environmental intentions , . in our study, two participants who were explicitly not aligned to the same group in the public goods game were randomly paired for the svo. both participants had to decide how to distribute time units between themselves and another participant among a series of six preset choices which affected the total duration of the experiment for each participant (see fig. s for an exemplary item of the svo; points = min; for another experiment using time units, see ). after the decisions it was randomly determined which of the two paired participant's decisions were implemented into actual consequences. this task induces a conflict between self and another person's interests, because participants can earn the maximal personal profit in terms of time units by selecting a distribution which does not yield the maximal profit for both paired participants and vice versa. from all these decisions an svo angle is calculated which represents a continuous measure of how much weight someone attaches to the welfare of others in relation to their own. the svo angle is also entered into the calculation of domain-general prosociality (see statistical analysis,see fig. s for details on the calculation of the svo angle). to obtain a domain-general measure of prosociality we averaged z-standardized scores of self-reported prosocial concern and prosocial values, and of pgg contributions and svo angles (cronbach's alpha = . ). to test for any association between specific microstates' coverages and prosociality and for the reliabilities of microstates parameters (first vs. second half), pearson-coefficients were calculated for normally distributed variables, and spearman-coefficients otherwise ( -sided tests, alpha level = . , 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multidimensional approach to individual differences in empathy a proposal for measuring value orientations across nations predicting voting behavior with implicit attitude measures. the german parliamentary election cooperation and punishment in public goods experiments social value orientation and helping behavior moderating effects of social value orientation on determinants of proenvironmental behavior intention relationship orientation as a moderator of the effects of social power comparing correlated correlation coefficients this work was supported by the wissenschaftliche gesellschaft freiburg (grant "the role of self-control and individual values in social decisions" to bastian schiller). the article processing charge was funded by the university of freiburg in the funding programme open access publishing. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to b.s. or m.h. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -s vdazq authors: huang, yan-jang s.; vanlandingham, dana l.; bilyeu, ashley n.; sharp, haelea m.; hettenbach, susan m.; higgs, stephen title: sars-cov- failure to infect or replicate in mosquitoes: an extreme challenge date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: s vdazq this research addresses public speculation that sars-cov- might be transmitted by mosquitoes. the world health organization has stated “to date there has been no information nor evidence to suggest that the new coronavirus could be transmitted by mosquitoes”. here we provide the first experimental data to investigate the capacity of sars-cov- to infect and be transmitted by mosquitoes. three widely distributed species of mosquito; aedes aegypti, ae. albopictus and culex quinquefasciatus, representing the two most significant genera of arbovirus vectors that infect people, were tested. we demonstrate that even under extreme conditions, sars-cov- virus is unable to replicate in these mosquitoes and therefore cannot be transmitted to people even in the unlikely event that a mosquito fed upon a viremic host. www.nature.com/scientificreports/ from various arthropods , , no virus in the family has been isolated from mosquitoes. to date, only one report related to epidemic coronaviruses and mosquitoes has been published . this study that evaluated the potential use of mosquitoes for surveillance, included feeding of mers virus to anopheles gambiae mosquitoes. residual viral rna, probably in the remains of the bloodmeal in the midgut, was detected up to -day post-feeding. similarly, positive pcr detection was observed for bacillus anthracis, trypanosoma brucei gambiensis, and zika virus, none of which infect or are transmitted by an. gambiae. levels of detected rna were equal to or below the input level, indicating a lack of replication. by analyzing samples using in vitro cultivation, rather than using molecular approaches, we focused specifically on detection of infectious virus rather than on rna. as illustrated by, for example, the use of inactivation techniques specifically developed to enable safe handling and shipping of viral material, the mere presence of rna does not mean that any infectious virus is actually present. it is well known that viral rna can be detected in mosquitoes simply because they have fed on a viremic host, and so rna detection should never be interpreted as proof of mosquito susceptibility to infection and competence to transmit the virus. in this study, the susceptibility of three mosquito species, ae. aegypti, ae. albopictus and cx. quinquefasciatus, were determined through the intrathoracic inoculation with sars-cov- . infectious viruses were recovered from / mosquitoes collected within two hours of inoculation. it is possible, that in the two negative mosquitoes, the inoculated virus lost infectivity during the holding period. no virus was detected in the inoculated mosquitoes collected and titrated at time points beyond h, suggesting a rapid loss of infectivity and the lack of replication after injection. from a total of mosquitoes analyzed, infectious viruses were only recovered from one ae. albopictus collected at h post-inoculation. the quantity of infectious virus in this mosquito corresponded to the amount of inocula, producing infectious titers at approximately . logtcid /ml. no virus was detected in control l- medium inoculated mosquitoes. collectively, our findings suggest that mosquitoes in the aedes and culex genera are refractory to sars-cov- and unlikely to contribute to viral maintenance and transmission in nature (table . ). the most extreme approach for viral challenge of mosquitoes, namely intrathoracic inoculation, was used as an ultimate test of the capacity of sars-cov- to infect and replicate in mosquitoes. the hypothesis was that if the virus did not replicate in mosquitoes after intrathoracic inoculation, then even if mosquitoes did feed on viremic people, and the virus disseminated from the midgut, the lack of replication would preclude the possibility of biological transmission. three widely distributed species of mosquito, representing the two most significant genera of arbovirus vectors that infect people, were tested. all three of the species: aedes aegypti, ae. albopictus, and culex quinquefasciatus are present in china, the country of origin of sars-cov- . samples collected within two hours of inoculation confirmed efficient delivery of infectious viruses to mosquitoes. based upon the lack of detectable infectious virus in any of the samples collected at all time points beyond h post-inoculation, we conclude that sars-cov- is unable to replicate in mosquitoes and that even if a mosquito fed on a person with virus in the blood, that the mosquito would not be a vector if feeding on a naïve host. virus: sars-cov- virus wa / strain was obtained from bei resources (catalog # nr- ). virus was propagated in vero cells at the approximate multiplicity of infection of . . using serial tenfold dilutions in -well plates , infectious titers of viral stocks used for intrathoracic injection were approximately . logtcid /ml. mosquitoes: the colonized aedes aegypti strain rex d, higgs white eye was originally obtained from puerto rico , ae. albopictus generation f originated from new jersey, and culex quinquefasciatus f were from florida , . all mosquitoes were reared at °c, relative humidity of % and a h light: h dark photoperiod. these colonized mosquitoes have proven to be susceptible to several arboviruses , [ ] [ ] [ ] [ ] [ ] [ ] . viral challenge of mosquitoes: for intrathoracic inoculation , mosquitoes were cold-anaesthetized on ice, transferred to a secure glove box, and then inoculated with approximately . µl of viral stock. it was anticipated that each mosquito received approximately . logtcid /ml of infectious viruses. l- medium was inoculated table . recovery rates of sars-cov- in mosquitoes receiving intrathoracic injection. * mosquitoes obtained at day post infection were collected within h from the time of intrathoracic injection. ** all mock control groups of mosquitoes received leibovitz's l- media. www.nature.com/scientificreports/ as a negative control. the results were compiled from two experiments using ae. aegypti and ae. albopictus and one experiment using cx. quinquefasciatus. experimentally challenged mosquitoes were maintained and sampled under conditions as described above. mosquitoes were individually triturated in ml of medium using a tissuelyser ii platform (qiagen, valencia, ca), and titrated on vero cells as previously described. world health organization. coronavirus disease (covid- ) advice for the public: myth busters sars-cov- infection protects against rechallenge in rhesus macaques clinical features of patients infected with novel coronavirus in wuhan, china potential for stable flies and house flies (diptera: muscidae) to transmit rift valley fever virus nonviremic transmission of west nile virus nonviremic transmission of west nile virus: evaluation of the effects of space, time, and mosquito species is nonviremic transmission of west nile virus by culex mosquitoes (diptera: culicidae) nonviremic? the use of toxorhynchites mosquitoes to detect and propagate dengue and other arboviruses the use of mosquitoes to detect and propagate dengue viruses pink bollworm larvae infection with a double subgenomic sindbis (dssin) virus to express genes of interest ectopic gene expression and homeotic transformations in arthropods using recombinant sindbis viruses brugia malayi microfilariae (nematoda: filaridae) enhance the infectivity of venezuelan equine encephalitis virus to aedes mosquitoes (diptera: culicidae) centers for disease control and prevention. international catalog of arboviruses runde" virus, a coronavirus-like agent associated with seabirds and ticks mechanical transmission of turkey coronavirus by domestic houseflies (musca domestica linnaeaus) the use of xenosurveillance to detect human bacteria, parasites, and viruses in mosquito bloodmeals growth characteristics of chimerivax-den vaccine viruses in aedes aegypti and aedes albopictus from thailand chemical and gamma-ray mutagenesis of the white gene in aedes aegypti application of a nonpaper based matrix to preserve chikungunya virus infectivity at ambient temperature. vector borne zoo culex species mosquitoes and zika virus. vector borne zoo differential outcomes of zika virus infection in aedes aegypti orally challenged with infectious blood meals and infectious protein meals culex tarsalis is a competent vector species for cache valley virus infection and transmission of cache valley virus by aedes albopictus and aedes aegypti mosquitoes a single mutation in chikungunya virus affects vector specificity and epidemic potential evaluation of simultaneous transmission of chikungunya virus and dengue virus type in infected aedes aegypti and aedes albopictus (diptera: culicidae) north american culex pipiens and culex quinquefasciatus are competent vectors for usutu virus /scientificreports/ reprints and permissions information is available at www.nature.com/reprints this work was performed in the acl- insectary at kansas state university's biosecurity research institute. the research was in part supported by the state of kansas national bio and agro-defense facility (nbaf) transition fund. the authors declare no competing interests. correspondence and requests for materials should be addressed to s.h.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -ddpfox authors: mindikoglu, ayse l.; abdulsada, mustafa m.; jain, antrix; jalal, prasun k.; devaraj, sridevi; wilhelm, zoe r.; opekun, antone r.; jung, sung yun title: intermittent fasting from dawn to sunset for four consecutive weeks induces anticancer serum proteome response and improves metabolic syndrome date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: ddpfox metabolic syndrome is characterized by central obesity, insulin resistance, elevated blood pressure, and dyslipidemia. metabolic syndrome is a significant risk factor for several common cancers (e.g., liver, colorectal, breast, pancreas). pharmacologic treatments used for the components of the metabolic syndrome appear to be insufficient to control cancer development in subjects with metabolic syndrome. murine models showed that cancer has the slowest progression when there is no food consumption during the daily activity phase. intermittent fasting from dawn to sunset is a form of fasting practiced during human activity hours. to test the anticancer effect of intermittent fasting from dawn to sunset in metabolic syndrome, we conducted a pilot study in subjects with metabolic syndrome who fasted (no eating or drinking) from dawn to sunset for more than h daily for four consecutive weeks. we collected serum samples before -week intermittent fasting, at the end of th week during -week intermittent fasting and week after -week intermittent fasting. we performed serum proteomic analysis using nano ultra-high performance liquid chromatography-tandem mass spectrometry. we found a significant fold increase in the levels of several tumor suppressor and dna repair gene protein products (gp)s at the end of th week during -week intermittent fasting (calu, ints , kit, crocc, pigr), and week after -week intermittent fasting (calu, calr, igfbp , sema b) compared with the levels before -week intermittent fasting. we also found a significant reduction in the levels of tumor promoter gps at the end of th week during -week intermittent fasting (polk, cd , camp, nifk, srgn), and week after -week intermittent fasting (camp, plac ) compared with the levels before -week intermittent fasting. fasting from dawn to sunset for four weeks also induced an anti-diabetes proteome response by upregulating the key regulatory proteins of insulin signaling at the end of th week during -week intermittent fasting (vps , polrmt, igfbp- ) and week after -week intermittent fasting (prkcsh), and an anti-aging proteome response by upregulating h b histone proteins week after -week intermittent fasting. subjects had a significant reduction in body mass index, waist circumference, and improvement in blood pressure that co-occurred with the anticancer, anti-diabetes, and anti-aging serum proteome response. these findings suggest that intermittent fasting from dawn to sunset actively modulates the respective genes and can be an adjunct treatment in metabolic syndrome. further studies are needed to test the intermittent fasting from dawn to sunset in the prevention and treatment of metabolic syndrome-induced cancers. | ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports/ one of the grand challenges of our times is the rising prevalence of metabolic syndrome . metabolic syndrome is characterized by central obesity, insulin resistance, elevated blood pressure, high triglyceride, and low highdensity lipoprotein levels . metabolic syndrome has adversely impacted many aspects of society . importantly, metabolic syndrome is a significant risk factor for several common cancers (e.g., liver, colorectal, breast, endometrium, pancreas) , . disrupted circadian clock rhythm has been recognized as one of the causes of metabolic syndrome and metabolic syndrome-induced cancers [ ] [ ] [ ] . pharmacologic treatments used for the components of the metabolic syndrome appear to be insufficient to reduce the risk of developing metabolic syndrome-induced cancers . moreover, pharmacologic treatments cannot reset the circadian clock rhythm; thus, there is an urgent need for an effective intervention to reset the circadian clock and prevent metabolic syndrome and metabolic syndromeinduced cancers. animal studies showed that resetting the circadian clock by time-restricted feeding improves metabolic syndrome and inhibits the development of cancer , . therefore, resetting the disrupted circadian clock in humans by consecutive daily intermittent fasting could provide a primary strategy to improve metabolic syndrome and reduce the incidence of metabolic syndrome-induced cancer [ ] [ ] [ ] . fasting during activity hours in contrast to the inactivity hours of the day appears to be important for the optimization of anticancer effect and gene expression. mice with no access to food during the activity phase (dark phase) of a -h light/ -h dark cycle had a significantly slower tumor progression and higher survival compared with mice that had no access to food during the inactivity phase (light phase), and mice that had access to food ad libitum . consistent with these findings, an earlier murine study showed that the uncoupling of the peripheral clocks from the control of the central clock only occurred when mice had no access to food during the active phase . in contrast, a minimal change in the phase of gene expression was observed when mice had no access to food during the inactive phase , corresponding to night time in humans. the findings of murine studies should be interpreted in the context of the fact that humans are diurnal (activity occurs during daytime), and mice are nocturnal (activity occurs during nighttime). the findings of these murine studies are in accord with the findings of our preliminary studies conducted in healthy subjects . our results showed that -day intermittent fasting from dawn to sunset, the human activity phase, was associated with an anticancer serum proteome response and upregulated several key regulatory proteins that play a key role in tumor suppression, dna repair, insulin signaling, glucose, and lipid metabolism, circadian clock, cytoskeletal remodeling, immune system, and cognitive function . importantly, the increase in the levels of these critical regulatory proteins occurred in the absence of any significant weight loss and calorie restriction . several human studies showed beneficial effects of intermittent fasting (e.g., ramadan fasting [ ] [ ] [ ] , and time-restricted eating in subjects with metabolic syndrome. however, in none of these studies, proteomic profiling was performed to understand the mechanism behind the anticancer effect of intermittent fasting and time-restricted eating in subjects with metabolic syndrome. to this end, we hypothesized that intermittent fasting from dawn to sunset practiced exclusively during the human activity hours for four weeks would be associated with an anticancer serum proteome response, upregulate anticancer proteins and regulatory proteins of dna repair and insulin signaling, and downregulate pro-cancer proteins. study subjects. this study was approved by the institutional review board of the baylor college of medicine biomedical research and assurance information network (brain) under protocol number h- . all research was conducted in accordance with relevant guidelines and regulations after written informed consent was obtained from all subjects. the study did not qualify for prospective registration as a clinical trial because there was no study directed intervention. the religious fast was habitual personal conduct, not study directed, and as such, the study was observational in study design. inclusion criteria were as follows: ( ) subjects who are years old or older; ( ) subjects who plan to fast during the religious month of ramadan ; ( ) subjects should meet any three of the following five criteria for metabolic syndrome as described by grundy et al. (a) central obesity assessed by waist circumference equal to or greater than cm ( inches) in men, and equal or greater than cm ( inches) in women; (b) fasting serum triglyceride level equal to or greater than mg/dl or on drug therapy for hypertriglyceridemia (e.g., fibrates, nicotinic acid); (c) low high-density lipoprotein level less than mg/dl in men and less than mg/dl in women or on drug therapy for low high-density lipoprotein level (fibrates, nicotinic acid); (d) elevated systolic blood pressure equal to or greater than or elevated diastolic blood pressure equal to or greater than or on drug therapy for hypertension, (e) elevated fasting glucose level equal to or greater than mg/dl or on drug therapy for hyperglycemia/diabetes); ( ) subjects who agreed to undergo fibroscan testing for evaluation of hepatic steatosis and fibrosis . subjects were excluded if they had any of the following: ( ) inability to provide informed consent; ( ) women who are pregnant or breastfeeding; ( ) active cancer; ( ) active infection requiring antibiotic use; ( ) seizure disorder; ( ) cardiovascular event during the last months; ( ) use of alcohol or recreational substances. the primary outcome of this pilot study was the induction of an anticancer proteome response at the end of th week during -week intermittent fasting and week after -week intermittent fasting. the secondary outcomes were improvement in the components of metabolic syndrome, lipid panel (total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein), hepatic panel (albumin, total protein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase), and adiposity, oxidative stress, and inflammation biomarkers. study procedures. subjects www.nature.com/scientificreports/ criteria, and written informed consent was taken. medical history and physical examination were performed. blood pressure measurements were performed at rest and sitting position. a urine pregnancy test for the female subjects at childbearing age was performed. hepatic steatosis and fibrosis were assessed using fibroscan with a controlled attenuation parameter (cap) . subjects started fasting at dawn after a pre-dawn breakfast and ended fasting at sunset (dusk) with a dinner for consecutive days. strict fasting occurred without eating or drinking between dawn and sunset (dusk), which are symmetrical transition time zones of the day. there was no interventional calorie or energy restriction otherwise. subjects had their main meals at the transition time zones of the day, including pre-dawn breakfast (at the first transition time of the day) and dinner at sunset (at the second transition time of the day) and were allowed to eat (e.g., snacks) or drink if they needed between sunset and dawn in addition to the pre-dawn breakfast and dinner at sunset. data on weight, waist circumference and blood pressure and blood specimens were collected within three weeks before the initiation of -week intermittent fasting to assess the effect of ad libitum eating, at the end of th week during -week intermittent fasting to assess the effect of intermittent fasting and week after -week intermittent fasting to assess the carryover effect of intermittent fasting on serum proteome, components of metabolic syndrome, lipid and hepatic panels, and adiposity, oxidative stress, and inflammation biomarkers. at each time point, data on weight, waist circumference, and blood pressure and blood specimens were collected after at least h of fasting. the compliance with fasting was monitored by a c-isotopic breath enrichment test, as previously described , . serum proteomics. we previously described a robust, streamlined proteomic approach to perform quantitative analysis of human serum samples using nano ultra-highperformance liquid chromatography-tandem mass spectrometry (nano uhplc-ms/ms) . briefly, μl of serum was incubated with the top abundant serum protein depletion kit (thermo scientific pierce, cat# ) and digested with trypsin on s-trap column (protifi, ny). the digested peptide was eluted, vacuum dried, and fractionated using high ph stage (stopand-go extraction) method into two pools, then subjected to nano-hplc-ms/ms analysis. the parameters for mass spectrometry analysis and the process for mass analysis is maintained the same as previous publication . we also explained the details of the gene protein product (gp)s quantification in our previous publication . briefly, we quantified gps using the label-free, intensity-based absolute quantification (ibaq) method and then normalized to final quantificational value (fot) defined as the ibaq value of an individual protein divided by the total ibaq values of all identified proteins within one experiment. the fot represents the relative abundance of each gp. the fot values of a particular gp in different conditions (e.g., fot value of a gp before -week intermittent fasting and at the end of th week during -week intermittent fasting) can be divided to get a fold change. fot value also provides the relative quantification of different gps in the same condition (e.g., fot value of two different gps at the end of th week during -week intermittent fasting). for statistical analysis of serum proteomics, we used excel application (microsoft, redmond, wa, usa). to determine statistically significantly regulated protein levels at the end of th week during -week intermittent fasting and week after -week intermittent fasting, we performed paired two-tailed student's t-test using log converted ifot values . we considered protein levels that showed an equal to or greater than fourfold average paired change and a p value of < . as significant . we performed a volcano plot analysis to display the gps that had an equal to or greater than fourfold significant change at the end of -week intermittent fasting during -week intermittent fasting and week after -week intermittent fasting compared with the levels before -week intermittent fasting . components of metabolic syndrome, lipid and hepatic panels, adiposity, oxidative stress and inflammation biomarkers. we measured the components of metabolic syndrome, lipid panel, hepatic panel and adiposity, oxidative stress, and inflammation biomarkers within three weeks before -week intermittent fasting, at the end of -week intermittent fasting during -week intermittent fasting, and week after -week intermittent fasting. we estimated the insulin resistance by using homeostatic model assessment for insulin resistance (homa-ir) equation as described by matthews et al. we calculated the mean arterial blood pressure using the following formula: (diastolic blood pressure) + [(systolic blood pressure-diastolic blood pressure)/ ] . statistical analysis. we used sas version . ts level m x _ pro platform (sas, cary, nc, usa) to perform statistical analysis of the components of metabolic syndrome, lipid panel, hepatic panel, and adiposity, oxidative stress, and inflammation biomarkers. we performed a student's paired t-test to determine statistically significant changes in the levels of the components of metabolic syndrome, lipid panel, hepatic panel, and adiposity, oxidative stress, and inflammation biomarkers measured at the end of th week during -week intermittent fasting, and week after -week intermittent fasting. we calculated pearson's correlation coefficient to assess correlations between significant gps (i.e., gps that showed significant fold changes at the end of th week during -week intermittent fasting and week after -week intermittent fasting) and the components of metabolic syndrome, lipid panel, hepatic panel, and adiposity, oxidative stress, and inflammation biomarkers. in these analyses, we considered a two-tailed p value of ˂ . statistically significant. subjects. we enrolled subjects with metabolic syndrome ( males: females) with a mean age of years (sd = ). all subjects fasted for more than h daily for days beginning from may , , until june , ( fig. ). mean fibroscan cap was (sd = ) db/m, and the mean elastic modulus was . (sd = . ) kpa. ten subjects had moderate to severe hepatic steatosis (s -s ), two had mild hepatic steatosis (s ), and two had no hepatic steatosis (s ). two subjects had f hepatic fibrosis, two had f hepatic fibrosis, one had f hepatic fibrosis, and nine had f -f hepatic fibrosis. nine subjects were on anti-hypertensive medications; seven subjects were on antidiabetic medications, and six subjects were on statins. the minimum required duration of daily fasting from dawn to sunset (dusk) was h, min for the shortest day (may , ), and h, min for the longest day (june , ). all subjects tolerated intermittent fasting well without any complications. the components of metabolic syndrome, lipid and hepatic panels, and adiposity, oxidative stress and inflammation biomarkers. table shows the mean levels of the components of metabolic syndrome, lipid panel, hepatic panel, and adiposity, oxidative stress, and inflammation biomarkers before -week intermittent fasting and their mean paired changes at the end of th week during -week intermittent fasting and week after -week intermittent fasting. there was a significant reduction in weight (p < . ), body mass index (p < . ), waist circumference (p = . ), systolic (p = . ), diastolic (p = . ) and mean (p = . ) arterial blood pressures at the end of th week during -week intermittent fasting and a significant reduction in weight (p < . ), body mass index (p < . ), waist circumference (p = . ) and homa-ir (p = . ) week after -week intermittent fasting compared with the levels before -week intermittent fasting. we observed a reduction in insulin, glucose, homa-ir, triglyceride, leptin, and several oxidative stress and inflammation biomarker levels and an increase in high-density lipoprotein and adiponectin levels at the end of th week during -week intermittent fasting, however, these parameters did not reach statistical significance. and has several major unique features: ( ) fasting is exclusively practiced during the human activity hours from dawn to sunset and is for both eating and drinking, which differentiates the dawn to sunset intermittent fasting from the other forms of intermittent fasting where eating light meals and/or drinking are allowed during the fasting window; ( ) although the main meals are at transition time zones of the day (pre-dawn breakfast and dinner at sunset), eating and drinking outside these transition time zones is allowed as long as it is within the non-fasting window; ( ) there is no interventional calorie or energy restriction; ( ) daily fasting window is in synchrony with circadian rhythm and earth's rotation on its axis because the daily fast starts at dawn (the first transition time zone of the day) after a pre-dawn breakfast and ends at sunset (dusk) (the second transition time zone of the day) with dinner; ( ) monthly fasting window is in synchrony with the moon's rotation around the earth and lunar phases because the monthly fasting starts and ends when the new moon is sighted (with permission from baylor college of medicine). | ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports/ table s ). figure a ,b, and table show the selected ones from these gps associated with tumor suppression, carcinogenesis, dna repair, and insulin signaling. there was an average -fold increase in adaptor related protein complex subunit zeta (ap z ) (log fold = . www.nature.com/scientificreports/ p = . ), nucleolar protein interacting with the fha domain of mki (nifk) (log fold = − . , p = . ) and serglycin (srgn) (log fold = − . , p = . ) gp levels compared with the levels before -week intermittent fasting. the proteome coverage and its dynamic order from samples collected week after -week intermittent fasting are shown in fig. c . there were gps recovered with over eight orders of magnitude of dynamic range. there was a significant average paired fold change in the levels of several gps week after -week intermittent fasting compared with the levels before -week intermittent fasting (supplementary table s ). figure c ,d, and table show selected ones from these gps associated with tumor suppression, carcinogenesis, insulin signaling, and prolonged lifespan. there was an average fold increase in prkcsh protein kinase c substrate k-h (prkcsh) (log fold = . , p = . ), fold increase calu (log fold = . , p = . ), fold increase in calreticulin (calr) (log fold = . , p = . ), fold increase in insulin-like growth factor-binding protein (igfbp ) (log fold = . , p = . ), tenfold increase in semaphorin b (sema b) (log fold = . , p = . ), sixfold increase in h b clustered histone (hist h bb) (log fold = . , p = . ), h b clustered histone (hist h bd) (log fold = . , p = . ), and h b clustered histone (hist h ba) (log fold = . , p = . ). we found a significant reduction in cathelicidin antimicrobial peptide (camp) (log fold = − . , p = . ) and placenta enriched (plac ) (log fold = − . , p = . ) gp levels week after -week intermittent fasting compared with the levels before -week intermittent fasting. there was a significant average paired fold change in the levels of several gps week after -week intermittent fasting compared with the levels at the end of th week during -week intermittent fasting (supplementary table s ). supplementary fig. and table show the average paired fold changes in the selected gps week after -week intermittent fasting compared with the levels at the end of th week during -week intermittent fasting. several gps that showed significant average paired fold changes at the end of th week during -week intermittent fasting and week after -week intermittent fasting were correlated with the components of metabolic syndrome, lipid and hepatic panels and adiposity, oxidative stress and inflammation biomarkers at the end of th week during -week intermittent fasting (supplementary table ) and week after -week intermittent fasting (supplementary table ). there was no significant correlation between log fold changes in the selected proteins (gene names are displayed in table ) and changes in weight, waist circumference and body mass index at the end of th week during -week intermittent fasting and week after -week intermittent fasting compared with baseline. we reported the results of the first human study of serum proteomics of -week intermittent fasting from dawn to sunset conducted in subjects with metabolic syndrome. the results showed that intermittent fasting from dawn to sunset for more than h daily for four consecutive weeks induced a unique anticancer, anti-diabetes and anti-aging proteomic response ( table , figs. and ) , upregulated several regulatory proteins that play a key role in tumor suppression, dna repair, humoral defense, insulin signaling, and downregulated several tumor promotor proteins. these changes in protein expression are likely related to the reset of the circadian clock rhythm by intermittent fasting from dawn to sunset and in line with the results of previous murine studies , . intermittent fasting from dawn to sunset for weeks is associated with an anticancer serum proteomic signature. this pilot study has important clinical implications, specifically from the standpoint of type of intermittent fasting on cancer prevention in subjects with metabolic syndrome. there are two major forms of daily intermittent fasting based on the time to start and end fasting: ( ) fasting that starts at dawn and ends at sunset (dusk). the fasting window is between two symmetrical transition time zones of the day (dawn and dusk) which is the human activity period (fig. ); ( ) fasting that starts at a self-determined time of the day and lasts for a fixed number of hours consisting of both human activity (daytime) and inactivity (nighttime) periods (e.g., : intermittent fasting that starts at pm and ends at noon). ramadan fasting is a unique form of intermittent fasting from dawn to sunset (dusk) without eating or drinking during the month of ramadan based on the lunar calendar and has several major unique features: ( ) fasting is exclusively practiced during the human activity hours from dawn to sunset (dusk) and is for both eating and drinking, which differentiates the dawn to sunset intermittent fasting from the other forms of intermittent fasting where drinking and/or eating light meals are allowed during the fasting window; ( ) although the main meals are at the transition time zones of the day (pre-dawn breakfast and dinner at sunset), eating (e.g., snacks) and drinking outside these transition time zones is allowed as long as it is within the non-fasting window; ( ) there is no interventional calorie or energy restriction; ( ) daily fasting window is in synchrony with circadian rhythm and earth's rotation on its axis because the daily fast starts at dawn (the first transition time zone of the day) after a pre-dawn breakfast and ends at sunset (dusk) (the second transition time zone of the day) with dinner; ( ) monthly fasting window is in synchrony with the moon's rotation around the earth and lunar phases because the monthly fasting starts and ends when the new moon is sighted. it is surmised that this fasting pattern results in energy reserves being accessed without leading to micronutrient deficiencies due to replenishment after sunset. fasting during activity hours of the day appears to be of paramount importance in cancer prevention and treatment. a study showed that mice with no access to food during the activity phase of a -h light/ -h dark cycle had a significantly slower tumor progression and higher survival compared with mice that had no access to food during the inactivity phase, and mice that had access to food ad libitum . the highest anticancer response occurred in the mice with no access to food during the activity phase, and the worse outcome was in the mice that scientific reports | ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports/ had access to food ad libitum . there was a minor anticancer effect and no survival benefit in the mice with no access to food during the inactivity phase . in regards to humans, a study conducted among women with breast cancer without diabetes mellitus showed that fasting equal to or longer than h at night (the combination of inactivity and activity hours) was associated with a reduced risk of breast cancer recurrence . of note, this study did not have control subjects who fasted exclusively during the activity hours (daytime) . altogether, these animal and human studies show that intermittent fasting either during daily activity or inactivity hours (with or without extending to activity hours) have an anticancer effect compared with ad libitum eating; however, the most robust anticancer response appears to occur when prolonged fasting is practiced exclusively during the activity hours . in accord with the findings of these murine and human studies , , we found a significant fold increase in the levels of specific tumor suppressor/anticancer proteins at the end of th week during -week intermittent fasting from dawn to sunset and/or week after -week intermittent fasting from dawn to sunset, including calr, calu, ints , kit, crocc, pigr, igfbp , and sema b that are downregulated in several cancers resulting in cancer metastasis and poor prognosis (table , fig. ). the calr gene encodes for calreticulin, which is a calcium-binding protein located in the endoplasmic reticulum and nucleus . cancer cells tagged by calreticulin on their surface stimulate an immunogenic cancer cell death by enabling their phagocytosis by the dendritic cells of the immune system which in turn triggers t-cell-mediated immune response [ ] [ ] [ ] . obeid et al. showed that anthracyclines translocate calreticulin to the tumor cell surface, and trigger immunogenic tumor cell death ("eat me" signal). calu gene encodes for calumenin, a calcium-binding protein that plays a significant role in the endoplasmic reticulum functions, including folding and sorting of proteins . calumenin is an inhibitor of cell migration and metastasis in several cancers , e.g., hepatocellular , , pancreatic , head and neck squamous cell , and lung squamous cell carcinomas. ints gene encodes for a dead box rna helicase that has a motif of asp-glu-ala-asp . it is a tumor suppressor gene that plays a significant tumor-suppressive role in hepatocellular carcinoma and prostate ca , . the induction of ints gene was shown to suppress castration-resistant prostate cancer . mutations in kit are linked to several cancers , and the loss of proto-oncogene c-kit expression has been associated with poor prognosis in breast cancer , . several other anticancer gps require elaboration. crocc, also known as tax bp , is a tumor-suppressor gene that was shown to suppress hepatocellular carcinoma via p /p /p pathway activation . the downregulation of crocc was associated with poor survival in patients with hepatocellular carcinoma after surgical resection . pigr, that encodes for polymeric immunoglobulin receptor , was found to be downregulated in pancreatic and periampullary adenocarcinoma and lung cancer . the upregulation of igfbp (encodes for a protein that binds insulin-like growth factors i and ii ) , was shown to function as a potent tumor suppressor in hepatocellular carcinoma and delay tumor formation in prostate cancer cells , . sema b is involved in protein-coding and encodes for a protein that inhibits tumor growth in non-small cell lung cancer . a significant fold-reduction in the levels of several tumor promoter/pro-cancer gps was observed at the end of th week during -week intermittent fasting and/or week after completion of -week intermittent fasting. these include polk, nifk, srgn, camp, cd , and plac that are upregulated in several cancers resulting in metastasis and poor prognosis ( table , fig. ). polk gene (polq) encodes for a specialized dna polymerase . specialized dna polymerases are ectopically overexpressed in several cancers, can function as an oncogene and enhance mutations induced by dna damage [ ] [ ] [ ] . overexpression of polk has been reported in lung cancer , . polk overexpression contributes to cancer development by inactivating wild-type p , and this was shown in lung cancer . polk gene also plays a role in breast cancer . a case-control study showed a higher risk of developing breast cancer in women with two specific single nucleotide polymorphisms in the polk gene compared with controls . nifk that encodes for a protein that functions in mitosis and progression of cell cycle was found to be upregulated and associated with poor prognosis in lung cancer . srgn encodes for a proteoglycan in hematopoietic cells and its overexpression is associated with poor prognosis in hepatocellular , colorectal cancer , non-small cell lung cancer , and nasopharyngeal carcinoma . camp that is also known as ll and cap , encodes for cathelicidin antimicrobial peptide . camp was shown to increase the growth of colon cancer via wnt/β-catenin signaling pathway activation and function as a tumor promoter for lung and ovarian cancers. cd encodes for a glycosyl phosphatidylinositol-linked glycoprotein that was found to be overexpressed in several tumors, e.g., malignant melanoma , squamous cell carcinoma of the lung and oral cavity . plac , which has a biased expression in placenta , was shown to be expressed in hepatocellular carcinoma , and overexpressed in breast cancer , non-small cell lung cancer , and pancreatic ductal adenocarcinoma . intermittent fasting from dawn to sunset for weeks can play an important role in humoral defense against severe acute respiratory syndrome-associated coronavirus (sars-cov). calreticulin was shown to enhance igg-mediated immune response when it is fused with spike (s) protein of sars-cov . recombinant fusion protein that combines calreticulin and sars-cov s protein - fragment had much higher immunogenicity when compared with sars-cov s protein alone . we found an average -fold increase in the calr gp level week after completion of -week intermittent fasting compared with the level before -week intermittent fasting. our findings, combined with the prior report from qiu et al. www.nature.com/scientificreports/ mittent fasting and prkcsh week after -week intermittent fasting. the induction of vps , polrmt, and igfbp gps at the end of th week during -week intermittent fasting preceded the significant reduction in insulin resistance estimated by homa-ir that occurred week after -week intermittent fasting. vps , a subunit of corvet complex , plays a critical role in integrin requiring cell adhesion and migration, and recycling of beta- integrins . integrins are transmembrane receptors connecting the extracellular matrix to the actin cytoskeleton of the cells and thereby acting as a sensor for cell adhesion . an impaired integrin signaling in the extracellular matrix of skeletal muscle, adipose tissue and liver can lead to insulin resistance . an intact skeletal muscle and beta-cell mitochondrial function is vital for insulin synthesis. type diabetes mellitus is associated with skeletal muscle mitochondrial dysfunction and reduced oxidative capacity . polrmt encodes for the mitochondrial rna polymerase that plays a critical role in transcription in pancreatic beta-cells and insulin secretion in the pancreas . as the loss of polrmt results in severe mitochondrial dysfunction in cardiac muscle , a similar mitochondrial dysfunction should occur in beta-cells with the loss or dysfunction of polrmt, resulting in insulin resistance and diabetes mellitus. igfbp plays an active role in myoblast differentiation by binding to insulin growth factor ii and upregulating its expression . prkcsh encodes for a protein called hepatocystin or k-h, which is a beta subunit of glucosidase ii association of -week intermittent fasting from dawn to sunset with autophagy and oxidative stress parameters. autophagy appears to be one of the mechanisms of intermittent fasting in cancer prevention . downregulation of the hepatocystin encoded by prkcsh results in dysfunctional glucosidase ii, and thereby increase in autophagy via mtor dependent pathway . the fact that we found reduction at the end of th week during -week intermittent fasting and then -fold increase in prkcsh gp level week after -week intermittent fasting is suggestive of increased autophagy during -week intermittent fasting and decreased autophagy with the subjects' return to ad libitum eating ( table ) . although it did not reach statistical significance, we found a reduction in multiple oxidative stress and inflammation biomarkers as well as gammaglutamyl transferase levels, suggesting glutathione repletion at the end of th week during -week intermittent fasting (table ) . intermittent fasting from dawn to sunset for weeks upregulates proteins associated with prolonged longevity and dna repair. we observed an average sixfold increase in h b histone gp levels week after -week intermittent fasting compared with the levels before -week intermittent fasting. feser et al. demonstrated similar findings in yeast cells. they reported that histone proteins are lost in aging yeast cells, whereas histone protein overexpression and supplying extra histone proteins prolonged longevity . authors suggested that extra histone protein supply extends lifespan by providing a tighter chromatin packaging, and thereby restoring the transcriptional silencing that is lost in aging . we also observed a significant positive correlation between h b histone gp levels and high-density lipoprotein levels week after -week intermittent fasting (supplementary table ). the association between high-density lipoprotein levels and longevity was previously reported . our findings of a significant positive correlation between h b histone gp and high-density lipoprotein levels week after -week intermittent fasting shed light on the mechanistic understanding of the association between high-density lipoprotein levels and longevity. besides the extended life span, the overexpression of histone proteins may also be associated with increased dna expression and repair because irradiation-induced dna damage was shown to downregulate histone gene transcription via the g checkpoint pathway . additionally, we observed an average -fold increase in the ap z gp level at the end of th week during -week intermittent fasting compared with the level before -week intermittent fasting. ap z is a helicase that likely plays a role in the repair of homologous recombination dna double-strand break . a variant in the ap z gene was associated with extreme longevity in a genome-wide association study conducted among , participants of the uk biobank . the strengths of our study are as follows: ( ) we performed a robust, streamlined proteomics method to quantify proteins in the human serum using nano uhplc-ms/ms . with this unique proteomics method, we were able to recover more than gps at the end of the th week during -week intermittent fasting ( fig. a ) and week after -week intermittent fasting (fig. c) ; ( ) we evaluated serum proteome simultaneously with the components of metabolic syndrome, lipid, and hepatic panels, and adiposity, oxidative stress, and inflammation biomarkers; ( ) we assessed the carryover effect of -week intermittent fasting after switching to ad libitum eating. for this, we compared the gp levels measured week after -week intermittent fasting with the levels measured at the end of th week during -week intermittent fasting. several gps that had a significant fold change at the end of th week during -week intermittent fasting compared with the levels before -week intermittent fasting, did not have any significant fold change week after -week intermittent fasting compared with the levels at the end of th week during -week intermittent fasting (e.g., calu, camp) ( table ). these findings suggest that intermittent fasting has a carryover effect on these proteins. on the other hand, several gps that had no significant fold change at the end of th week during -week intermittent fasting compared with the levels before -week intermittent fasting, had a significant fold change week after -week intermittent fasting compared with the levels at the end of th week during -week intermittent fasting (e.g., prkcsh, hist h ba) ( table ). these findings may suggest either decreased or scientific reports | ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports/ delayed carryover effect of intermittent fasting on the proteome. there is also a possibility that intermittent fasting might have triggered a cascade of proteomic changes in a continuum that may not be explained by the assessment of the serum proteome at a single time point alone after intermittent fasting is stopped. several gps that showed significant fold changes in subjects with metabolic syndrome had also shown similar changes (e.g., increase or decrease) at the end of th week during -day intermittent fasting (kit, crocc, dntt, polk, srgn, clstn ) and week after -day intermittent fasting (prkcsh, calu, specc l, igfbp , myh , cdh , h b histone, pkp , lrrc , fgb, enpp ) in our previous study conducted in healthy subjects although they had not reached statistical significance . the lack of caloric measurement by dietary assessment is one of the limitations of our study. the improvement in the components of metabolic syndrome could partially be explained by the significant weight reduction. nonetheless, we did not find any significant correlation between log fold changes in the selected proteins (names are displayed in table ) and changes in weight, waist circumference and body mass index at the end of th week during -week intermittent fasting and week after -week intermittent fasting compared with baseline suggesting that the effect of -week intermittent fasting from dawn to sunset on the proteomic changes was independent of weight reduction. furthermore, our previous study conducted in healthy volunteers who fasted from dawn to sunset for days showed the induction of an anticancer proteome in the absence of a significant weight change . we estimated insulin resistance by homa-ir equation instead of performing an oral glucose tolerance test. a non-significant reduction in homa-ir at the end of th week during -week intermittent fasting might be related to insufficient accuracy of the homa-ir equation in our study population. homa-ir equation was found to have limited accuracy in adults between the ages and and subjects on insulin treatment (unless the glucose and insulin are in steady-state levels in subjects on insulin) , . given the fact that out of study subjects were years old or older and one subject was on insulin treatment, homa-ir might have underestimated the reduction in insulin resistance that occurred at the end of th week during -week intermittent fasting. nonetheless, there was a reduction in glucose, insulin, and homa-ir levels at the end of th week during -week intermittent fasting. although these findings did not reach statistical significance, they provide additional evidence for the antidiabetic effect of the -week intermittent fasting from dawn to sunset. serum proteomic signature of -week intermittent fasting from dawn to sunset for more than h a day contributed to the mechanistic understanding of the effect of intermittent fasting from dawn to sunset on anticarcinogenesis, dna repair, insulin signaling, humoral immunity and increased longevity in subjects with metabolic syndrome. our findings suggest that intermittent fasting from dawn to sunset for four consecutive weeks, which is in synchrony with circadian rhythm and earth's rotation can be an adjunct treatment in metabolic syndrome and should be tested in the prevention and treatment of metabolic syndrome-induced cancers. altogether, our findings are in line with the results 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calreticulin exposure dictates the immunogenicity of cancer cell death calreticulin exposure increases cancer immunogenicity extracellular calumenin suppresses erk / signaling and cell migration by protecting fibulin- from mmp- -mediated proteolysis proteome analysis of hepatocellular carcinoma cell strains, mhcc -h and mhcc -l, with different metastasis potentials identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry molecular cloning, identification and analysis of lung squamous cell carcinoma-related genes pseudogene ints p regulates its cognate gene ints through competitive binding of mir- - p in hepatocellular carcinoma small rna-induced ints gene up-regulation suppresses castration-resistant prostate cancer cells by regulating Β-catenin signaling breast cancer is associated with loss of the c-kit oncogene product loss of c-kit expression in breast cancer correlates with malignant 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links between dna polymerases, mutagenesis and human disease elevated expression of dna polymerase kappa in human lung cancer is associated with p inactivation: negative regulation of polk promoter activity by p association between single nucleotide polymorphisms in dna polymerase kappa gene and breast cancer risk in chinese han population: a strobe-compliant observational study. medicine (baltimore) , e the nucleolar protein nifk promotes cancer progression via ck α/β-catenin in metastasis and ki- -dependent cell proliferation serglycin (srgn) overexpression predicts poor prognosis in hepatocellular carcinoma patients srgn promotes colorectal cancer metastasis as a critical downstream target of hif- alpha serglycin in tumor microenvironment promotes non-small cell lung cancer aggressiveness in a cd -dependent manner serglycin is a theranostic target in nasopharyngeal carcinoma that promotes metastasis cathelicidin, an antimicrobial peptide produced by macrophages, promotes colon 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adenocarcinoma. tumor biol. , calreticulin as a hydrophilic chimeric molecular adjuvant enhances igg responses to the spike protein of severe acute respiratory syndrome coronavirus vps and vps control integrin trafficking from early to recycling endosomes and regulate integrin-dependent functions integrins: bidirectional, allosteric signaling machines the extracellular matrix and insulin resistance type diabetes mellitus and skeletal muscle metabolic function transcribing Β-cell mitochondria in health and disease polrmt regulates the switch between replication primer formation and gene expression of mammalian mtdna igfbp- regulates muscle cell differentiation by binding to igf-ii and switching on the igf-ii autoregulation loop identification of k-h as a protein involved in glut vesicle trafficking autophagy and intermittent fasting: the connection for cancer therapy? deficiency of hepatocystin induces autophagy through an mtor-dependent pathway elevated histone expression promotes life span extension phenotypes and genotypes of high density lipoprotein cholesterol in exceptional longevity dna damage induces downregulation of histone gene expression through the g checkpoint pathway human longevity is influenced by many genetic variants: evidence from , uk biobank participants limitation of the homeostasis model assessment to predict insulin resistance and beta-cell dysfunction in older people use and abuse of homa modeling authors thank scott c holmes, c.m.i., a member of the michael e. debakey department of surgery research core at baylor college of medicine, for his assistance during the preparation of fig. . a.l.m. formulated the study hypothesis and study concept, designed the study, drafted the manuscript, contributed with conducting the study, analyzing data, and critically reviewing and finalizing the manuscript. m.m.a. contributed with conducting the study, and critically reviewing the manuscript. a.j. contributed with performing serum proteomic analysis and critically reviewing the manuscript. p.k.j. contributed with conducting the study, and critically reviewing the manuscript. s.d. contributed with performing the analysis of components of metabolic syndrome, lipid and hepatic panels, and adiposity, oxidative stress and inflammation biomarkers and critically reviewing the manuscript. z.r.w. contributed with conducting the study and critically reviewing the manuscript. a.r.o. contributed with conducting the study, and critically reviewing the manuscript. s.y.j. contributed with performing serum proteomic analysis, analyzing data, and critically reviewing the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - -w.correspondence and requests for materials should be addressed to a.l.m.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - bwdb o authors: liu, xiaofan; zhou, hong; zhou, yilu; wu, xiaojun; zhao, yang; lu, yang; tan, weijun; yuan, mingli; ding, xuhong; zou, jinjing; li, ruiyun; liu, hailing; ewing, rob m.; hu, yi; nie, hanxiang; wang, yihua title: temporal radiographic changes in covid- patients: relationship to disease severity and viral clearance date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: bwdb o covid- is “public enemy number one” and has placed an enormous burden on health authorities across the world. given the wide clinical spectrum of covid- , understanding the factors that can predict disease severity will be essential since this will help frontline clinical staff to stratify patients with increased confidence. to investigate the diagnostic value of the temporal radiographic changes, and the relationship to disease severity and viral clearance in covid- patients. in this retrospective cohort study, we included patients admitted to the renmin hospital of wuhan university, with laboratory confirmed moderate or severe covid- . temporal radiographic changes and viral clearance were explored using appropriate statistical methods. radiographic features from hrct scans included ground-glass opacity, consolidation, air bronchogram, nodular opacities and pleural effusion. the hrct scores (peak) during disease course in covid- patients with severe pneumonia (median: . ) were higher compared to those with pneumonia (median: ) (p = . × (− )), with more frequency of consolidation (p = . ) and air bronchogram (p = . × (− )). the median values of days when the peak hrct scores were reached in pneumonia or severe pneumonia patients were vs. , respectively (p = . ). log-rank test and spearman’s rank-order correlation suggested temporal radiographic changes as a valuable predictor for viral clearance. in addition, follow up ct scans from pneumonia patients showed full recovery. given the values of hrct scores for both disease severity and viral clearance, a standardised hrct score system for covid- is highly demanded. www.nature.com/scientificreports www.nature.com/scientificreports/ our knowledge, at present, there is limited standardised method to predict which infected patient will remain moderately symptomatic and which will progress to more severe disease as well as viral clearance. here, we present details of patients admitted to the renmin hospital of wuhan university (hubei, china), with laboratory confirmed moderate or severe covid- . we aim to describe the temporal radiographic changes, and the relationship to disease severity and viral clearance in covid- patients. study design and participants. all methods were carried out in accordance with relevant guidelines and regulations. this retrospective study was approved by the ethics committee of renmin hospital of wuhan university, hubei, china (no. wdry -k ), and the requirement for informed consent was waived by the ethics committee due to a public health outbreak investigation. we identified consecutive patients with moderate or severe covid- discharged from the general wards of renmin hospital of wuhan university between february to march . all patients had been diagnosed with covid- according to who interim guidance , , . on admission, patients are classified into the following severity stages: mild, moderate, severe and critical, as defined by the covid- chinese guidelines document (version ). mild cases are defined by mild symptoms and no evidence of pneumonia while moderate cases are defined by fever, respiratory tract and other symptoms, and radiological evidence of pneumonia. severe cases meet any of the following criteria: respiratory rate ≥ breaths/min; oxygen saturation ≤ % at a rest state; arterial partial pressure of oxygen (pao )/oxygen concentration (fio ) ≤ mmhg; patients with > % progression of lesions on lung imaging within to hours should be treated as severe cases. critical cases meet any of the following criteria: occurrence of respiratory failure requiring mechanical ventilation; presence of shock; other organ failure that requires monitoring and treatment in the icu. in the who interim guidance (version . ), moderate cases are known as "pneumonia" while severe cases as "severe pneumonia". critical cases were not available in this cohort. in this cohort, all the patients were discharged. the criteria for patient discharge was the absence of fever for at least days, substantial improvement in both lungs on chest ct, clinical remission of respiratory symptoms, and two throat-swab samples negative for sars-cov- rna obtained at least hours apart . the same cohort of patients was used to investigate non-radiographic risk factors associated with disease severity and length of hospital stay in covid- . laboratory procedures. sars-cov- infection in patients was confirmed using real-time rt-pcr with a standard protocol recommended by china center for disease control and prevention (cdc). throat-swab specimens were obtained for examination every other day after admission, but only qualitative data were available. the specimens were considered positive if the ct (cycle threshold) value was ≤ , and negative if the results were undetermined. specimens with a ct higher than were repeated. the specimen was considered positive if the repeat results were the same as the initial result and between and . if the repeat ct was undetectable, the specimen was considered negative. routine blood examinations were performed in the same hospital and results were retrieved from electronic medical records. high-resolution computed tomography (hrct) scans. all patients underwent chest non-contrast enhanced ct examinations in the supine position and with breath-holding following inspiration. the technical parameters included a -section scanner with mm collimation at mm intervals. images were obtained with both mediastinal (width hu; level hu) and parenchymal (width hu; level − hu) window settings. frequency of examinations was determined by the treating physician. pneumonia patients were followed up after being discharged, imaging evaluation. for imaging evaluation, experienced chest radiologists reviewed the images independently in a consistent manner, with a final finding reached by consensus when there was a discrepancy. they were blinded to the clinical information or clinical progress of the patients, except for the knowledge that these were cases of covid- patients. the ct features included ground glass opacity, consolidation, air bronchogram, nodular opacities and pleural effusion. the ct scans were scored on the axial images referring to a method described previously . the extent of involvement of each abnormality was assessed independently for each of zones: upper (above the carina), middle (below the carina and above the inferior pulmonary vein), and lower (below the inferior pulmonary vein). the ct findings were graded on a -point scale: normal attenuation ( ), ground-glass attenuation ( ) , and consolidation ( ). each lung zone, with a total of lung zones in each patient, was assigned a following scale according to distribution of the affected lung parenchyma: normal ( ), < % abnormality ( ), - % abnormality ( ), - % abnormality ( ), and > % abnormality ( ). the -point scale of the lung parenchyma distribution was then multiplied by the radiologic scale described above. points from all zones were added for a final total cumulative score (hrct score), with value ranging from to . data collection and sharing. demographic, clinical, laboratory and radiographic findings were extracted from electronic medical records , . the data that support the findings of this study are available from the corresponding author upon reasonable request and with permission of renmin hospital of wuhan university, hubei, china. mann-whitney u test or wilcoxon test if appropriate; categorical variables were compared by χ test or fisher's exact test if appropriate. continuous and categorical variables were expressed as median (interquartile range, www.nature.com/scientificreports www.nature.com/scientificreports/ iqr) and number (n) (%), respectively. kaplan-meier plot was used to present viral clearance in covid- patients. the viral clearance day was determined as the median of the last day of viral rna positive and the first day of viral rna negative. p-values were calculated by log-rank test. correlations between variables were accessed using spearman's rank-order correlation. p-values less than . were considered statistically significant. all data analyses and graphs were done in rstudio (v . . ) or graphpad prism (v . . ). patients ( pneumonia and severe pneumonia) with key information in their medical records were included in this study. main features in covid- patients with pneumonia or severe pneumonia. the median age of covid- patients with severe pneumonia was years (iqr ~ , range to ), compared with years (iqr ~ , range from to ) in patients with pneumonia (p = . ). the proportion of male patients with severe pneumonia was higher than in the pneumonia patients, although statistical significance was not reached (p = . ). comorbidities were present more frequently in severe pneumonia cases compared to pneumonia www.nature.com/scientificreports www.nature.com/scientificreports/ ( % vs. %, p = . ), with hypertension being the most common comorbidity, followed by diabetes and cardiovascular disease. the frequency of covid- patients with hypertension was % in cases with severe pneumonia compared with % in the pneumonia cases (p = . ) ( table ). www.nature.com/scientificreports www.nature.com/scientificreports/ the most common symptoms on admission were fever, cough and dyspnea, followed by fatigue, anorexia and/ or lethargy, myalgia and/or arthralgia and diarrhoea. symptoms such as dyspnea, fatigue, and anorexia and/or lethargy were observed more often in severe pneumonia patients (p < . ) ( table ) . www.nature.com/scientificreports www.nature.com/scientificreports/ in laboratory findings, lymphocyte counts were significantly decreased whereas neutrophil counts, the neutrophil to lymphocyte ratio (nlr), d-dimer, lactate dehydrogenase and c-reactive protein levels were all increased in severe pneumonia cases (p < . ), while there was no significant difference in white blood cell counts (p = . ) ( table ) . radiographic features from hrct scans included ground glass opacity, consolidation, air bronchogram, nodular opacities and pleural effusion ( fig. ; table ). temporal radiographic changes in covid- patients were shown in figs. and a. we managed to follow up covid- patients with pneumonia after being discharged, and found their ct scans were all back to normal (fig. b ). the hrct scores (peak) during disease course in covid- patients with severe pneumonia (median: . ; iqr range: ~ . ) were higher compared to those with pneumonia (median: ; iqr range: ~ ) (p = . × − ) (table ; fig. c ), with more frequency of consolidation ( . % vs. . %, p = . ) and air bronchogram ( . % vs. . %, p = . × − ) ( table ). the median values of days when the peak hrct scores were reached in pneumonia or severe pneumonia patients were (iqr range: ~ ) vs. (iqr range: ~ ), respectively (p = . ) (table ; fig. d ). to check viral clearance in covid- patients, throat-swab specimens were collected to detect sars-cov- www.nature.com/scientificreports www.nature.com/scientificreports/ rna (positive or negative) using real-time rt-pcr routinely. interestingly, there was no difference in viral clearance in pneumonia vs. severe pneumonia patients (fig. a, p = . ) . the median viral clearance day for pneumonia patients was , ranging from to ; while in severe pneumonia patients, it was , ranging from . to . (fig. b, p > . ) . the viral clearance day was determined as the median of the last day of viral rna positive and the first day of viral rna negative. we next asked whether there were any demographic, laboratory and radiographic findings associated with viral clearance (summarised in table ). we found in covid- patients with pneumonia, hrct scores (peak) were positively correlated with days to viral clearance using either the log-rank test (fig. a , p = . ) or spearman's rank-order correlation (fig. b , r = . , p = . × − ). for the kaplan-meier plot of viral clearance, the median hrct score ( ) from all covid- patients in this cohort was used to separate the groups (fig. a ). in addition, while the hrct score peak day was positively correlated with days to viral clearance in all covid- patients (fig. c , r = . , p = . × − ) and severe pneumonia patients (fig. e , r = . , p = . ), this failed to reach significance in the pneumonia patients (fig. d , r = . , p = . ). covid- is "public enemy number one", says the director general of the who. at the time of writing, more than . million cases have been recorded, with over k associated deaths. it is estimated that the case-fatality risk for covid- is in a broad range of . ~ . % . high mortality rate has been observed in several areas due to over stretched medical resources amongst other things. as a result, understanding the prognostic factors that can predict disease severity will be essential, since this will help frontline clinical staff to stratify patients with www.nature.com/scientificreports www.nature.com/scientificreports/ increased confidence. in addition, to battle against a highly contagious disease, like covid- , elucidating factors that can predict viral clearance is important. in order to provide insight into these questions, we designed a retrospective study, with covid- patients ( pneumonia and severe pneumonia) with key information from their medical records. here we report temporal changes in hrct scores as a valuable predictor for both disease severity and viral clearance. the features and importance of hrct scans in the diagnosis of covid- patients have been reported , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . similarly, in our cohort, radiographic features included ground glass opacity, consolidation, air bronchogram, nodular opacities and pleural effusion, with more frequency of consolidation and air bronchogram in severe cases, indicating a more severe clinical course for these abnormalities can be pathologically correlated with diffuse alveolar damage . as caused by members within the same virus family, covid- shared common radiographic characteristics with middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) [ ] [ ] [ ] [ ] . interestingly, with various statistical tools, we were able to show that a higher hrct score (peak) is strongly correlated with days to viral clearance in covid- patients with pneumonia, while hrct peak day correlates with days to viral clearance in severe cases. the length of days to viral clearance may be determined by virus load, immune response, treatment and others; while the chest hrct changes represent the underlying pathophysiology of the disease process. these findings suggest that in covid- patients with pneumonia, damages to the lungs, reflected by hrct score (peak), are positively correlated to the viral clearance. on the other hand, in severe cases, it is the time taken to develop maximal damage (reflected by hrct peak day) that positively correlates to the viral clearance. in addition, with follow up ct scans in pneumonia patients after being discharged, we found that pathological changes in the lungs can be completely healed, although more investigation is required in severe cases. nevertheless, given the values of hrct scores for both disease severity and viral clearance, a standardised hrct score system for covid- is thus highly recommended. there are several limitations in this study. ( ) quantitative viral rna detection (ct values) was not available. the estimated duration of the presence of viral rna is limited by the frequency of respiratory specimen collection. ( ) some patients may have received medicine before admission, which may affect chest ct findings. ( ) interpretation of our findings might be limited by the sample size. despite these limitations, with appropriate statistical tools, we are able to identify the values of monitoring temporal radiographic changes in covid- patients. a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding coronaviridae study group of the international committee on taxonomy of, v. the species severe acute respiratory syndromerelated coronavirus: classifying -ncov and naming it sars-cov- a new coronavirus associated with human respiratory disease in china clinical features of patients infected with novel coronavirus in wuhan clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a singlecentered, retrospective, observational study. the lancet. respiratory medicine risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease association of radiologic findings with mortality of patients infected with novel coronavirus in wuhan, china risk factors associated with disease severity and length of hospital stay in covid- patients case-fatality risk estimates for covid- calculated by using a lag time for fatality ct imaging of the novel coronavirus ( -ncov) pneumonia. radiology prediction of prognosis for acute respiratory distress syndrome with thin-section ct: validation in cases acute interstitial pneumonia: comparison of high-resolution computed tomography findings between survivors and nonsurvivors clinical characteristics of coronavirus disease in china novel coronavirus ( -ncov) pneumonia ct imaging features of novel coronavirus ( -ncov) chest ct findings in coronavirus disease- (covid- ): relationship to duration of infection correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases fleischner society: glossary of terms for thoracic imaging severe acute respiratory syndrome: temporal lung changes at thin-section ct in patients severe acute respiratory syndrome: radiographic appearances and pattern of progression in patients coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers. current opinion in infectious diseases middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings x.l., h.z., y.l., y.w., h.n. and y.h. had the idea for and designed the study. x.w., y.z. and h.n. had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. y.w., h.n., y.h. and r.e. drafted the paper. y.z., x.l., h.z. and y.w. did the analysis, and all authors critically revised the manuscript for important intellectual content and gave final approval for the version to be published. x.w., y.z., x.l., h.z., y.l., w.t., m.y., x.d., j.z., r.l. and h.l. collected the data. all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. the authors declare no competing interests. correspondence and requests for materials should be addressed to y.h., h.n. or y.w.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -iaxkfszq authors: nachman, dean; constantini, keren; poris, gal; wagnert-avraham, linn; gertz, s. david; littman, romi; kabakov, eli; eisenkraft, arik; gepner, yftach title: wireless, non-invasive, wearable device for continuous remote monitoring of hemodynamic parameters in a swine model of controlled hemorrhagic shock date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: iaxkfszq accurate and continuous monitoring of critically ill patients is frequently achieved using invasive catheters, which is technically complex. our purpose was to evaluate the validity and accuracy of a photoplethysmography (ppg)-based remote monitoring device compared to invasive methods of arterial line (al) and swan-ganz (sg) catheters in a swine model of controlled hemorrhagic shock. following a baseline phase, hemorrhagic shock was induced in pigs by bleeding % of their blood volume, followed by a post-bleeding follow-up phase. animals were monitored concomitantly by the ppg device, an al and a sg catheter, for a median period of min. heart rate (hr), systolic and diastolic blood pressure (sbp and dbp, respectively), and cardiac output (co) were recorded continuously. the complete data set consisted of paired observations. correlations between the ppg-based technique and the invasive methods were significant (p < . ) during baseline, bleeding and follow-up phases for hr (r = . – . ), sbp (r = . – . ), dbp (r = . – . ), and co (r = . – . ). intraclass correlations for all phases combined were . , . , . and . for hr, sbp, dbp and co, respectively. correlations for changes in co, sbp and dbp were significant (p < . ) and strong (r > . ), with concordance rates (determined by quadrant plots) of %, % and %, respectively. the novel ppg-based device was accurate and valid compared to existing invasive techniques and might be used for continuous monitoring in several clinical settings following further studies. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ demonstrated no improvement in the mortality of patients following the use of these invasive methods compared to patients without invasive monitoring [ ] [ ] [ ] . yet, some studies have shown that use of a pre-emptive strategy of hemodynamic monitoring including hr, bp and co reduces morbidity and surgical mortality with the advantages and precision of the invasive measurement methods considered to outweigh their complications , , . on the other hand, although any medical intervention has associated risks, it has been noted in several clinical studies that the number of life-threatening complications associated with the sg catheter is relatively high . in this study, we evaluated the monitoring capability of a new device (biobeat technologies ltd, petah-tikva, israel), which measures cardiovascular parameters using a reflective photoplethysmography (ppg) sensor. this sensor allows non-invasive, cuff-less, and wireless measurements of hr, systolic blood pressure (sbp), diastolic blood pressure (dbp), and co. the accuracy and validity of the device were determined by head-to-head comparison with readings from an al and a sg catheter in a swine model of controlled hemorrhagic shock, an accepted model to study trauma care as described later. during all study phases, sg and al, as well as the wearable device, provided continuous assessment of physiological parameters. two animals did not undergo a bleeding phase due to early systemic decompensation, and therefore only baseline (i.e., pre-bleeding) data is reported for those animals. details about the blood volume removed from each animal, and clinical data recorded during the study are presented in table . in general, we did not see any major clinical event in any of the animals during the study. the number of observations (total of ) and mean ± sd values for each variable per stage are presented in table . degree of agreement. pearson correlations and bland-altman plots with % limits of agreement (loa) for each variable are presented in fig. . hr values obtained by the al ( ± bpm) and the ppg-based device ( ± bpm) were significantly (p < . ) and strongly correlated during all phase (pre-bleeding: r = . ; table . clinical data of animals included in the study. www.nature.com/scientificreports/ r = . ; bleeding: r = . ; post-bleeding: r = . ). intraclass correlation coefficients (icc) and standard error of the mean (sem) per phase between each pair of measurements have been tested and presented in fig. . for all phases combined, icc (± sem) was . ( . ), . ( . ), . ( . ), and . ( . ) for hr, sbp, dbo, and co, respectively. correlations between al/sg and the ppg-based device for changes in sbp, dbp and co were significant (p < . ) and strong (r > . for all; fig. ). the concordance rates, determined by four quadrant plots, were %, % and % for sbp, dbp and co, respectively. invasive monitoring methods provide continuous measurement of numerous cardio-pulmonary parameters, yet they are prone to complications that could endanger patients' lives , . thus, advanced hemodynamic monitoring by non-invasive means might enable thoughtful diagnosis and treatment of unstable patients while minimizing morbidity and mortality. in this work, we have shown that the ppg-based device provides accurate and valid readings with marginal bias and narrow loas for sbp, dbp, hr, and co when compared to the invasive, gold-standard measurements obtained from al and sg catheters. our findings demonstrated a high level of correlation between the techniques during all stages of the study, including during dynamic and substantial fluctuations that occurred throughout the bleeding and post-bleeding phases. these phases are characterized by high hemodynamic variability and low reading inputs, suggesting that the wearable device is valid for use under unstable physiological conditions. in the past, there have been several commercial and academic efforts to develop non-invasive, advanced hemodynamic monitoring technologies [ ] [ ] [ ] . as of today, none of these techniques have gained widespread acceptance or use due to conflicting validation data and cumbersome implementation of the instrumentation , . ppg is commonly-used for non-invasive monitoring of arterial oxyhemoglobin saturation and hr in research and applied settings; however, several studies have shown that the waveform produced is affected by systemic circulation changes , , and the accuracy and reliability of data obtained from ppg-based devices remain questionable, particularly under dynamic conditions such as in hemodynamic shock. nevertheless, results from this study show that the higher temporal resolution of the ppg sensor we used allows accurate detection of ppg www.nature.com/scientificreports/ waves even during periods of hemodynamic instability. the advanced algorithms applied to the pulse contour readings by the device allow for enhanced accuracy and reliability of hr, sbp, dbp, and co monitoring (fig. ) . although in many situations bp is measured non-invasively (i.e. without the insertion of an al) and by automated devices, the necessity of using a cuff makes this measurement cumbersome and inconvenient for both patients and care providers, particularly in circumstances where beat-to-beat measurements are required or in isolated patients. while criteria exist for assessing the validity of automatic bp devices and acquiring approval by the european society of hypertension (esh), american heart association, food and drug administration and other governing bodies, criteria for cuff-less monitors such as the sensor used in this study have yet to reach widespread, general acceptance. based on the esh's international protocol for the validation of bp measuring devices , post-hoc analysis revealed as many as % of the sbp values obtained from the wearable device were within ± mmhg of those recorded by al. a similar analysis for dbp yielded even more remarkable results with , , . a recent study investigating the agreement between electrocardiogram and ppg-based hr measurements reported an icc of . and mean difference between devices of . beat/min − . while these findings may appear promising, it should be mentioned that the overall icc for hr in this study was . , the mean difference between techniques was < beat/min (figs. , ) , and % of non-invasive measurements were within beat/min of al-derived hr . identifying accurate hr and bp readings, particularly under unstable hemodynamic conditions, is fundamental in providing optimal medical treatment and improving patient outcomes and prognosis. therefore, the high level of accuracy and strong agreement between the wearable device and the al measurements for hr and bp under dynamic physiological conditions suggests the need for testing this device in humans. of the hemodynamic variables measured in this study that are relevant and important for clinically unstable patients, co is the most challenging one to assess accurately using non-invasive methods. as mentioned above, although various attempts have been made to develop techniques for minimal or non-invasive measurement of co, the accuracy and reliability of these methods have been inconsistent and therefore unsatisfactory for clinical use at this time , , , , . for example, while the clinically acceptable percentage error (i.e. loa as a proportion of mean co) for co monitors is as high as % , , most devices have an error of ~ % , , making them unacceptable for clinical use. to the best of our knowledge this is the first scientific study to compare a high-resolution ppg technique with gold standard invasive measurements of co. we have shown that the percentage error of the wearable device was as low as %, with a small bias ( . l/min − ) and relatively narrow loa (fig. ) compared to sg in a swine model of controlled hemorrhagic shock. when analyses were performed per phase, intraclass and pearson correlations between the sg and the ppgbased device for the pre-bleeding and bleeding phases were relatively high, while post-bleeding correlation analyses were not as strong (fig. ) . it should be emphasized that due to workflow considerations, the thermodilution co monitor was calibrated once prior to the beginning of data collection for each animal, which may have contributed to the slight discrepancies between devices during the post bleeding phase. nevertheless, given the high level of accuracy between the wearable device and the sg measurements, further studies should investigate whether these findings are also translatable to humans particularly under conditions of hemodynamic instability. from a clinical standpoint, an important advantage of the wearable device is its reliance on ppg technology, which is widely used in medical care. this technology allows easy patient monitoring without the need for extensive training of care providers. on the basis of our results, it appears that implementation of this wearable device in humans would allow reliable, yet safer, monitoring of hospitalized patients who may be in critical, unstable conditions similar to the hemorrhagic shock tested in our study or other life-threatening situations such as sepsis, anaphylaxis or cardiogenic shock. alongside in-hospital intensive care, such a device might enable better monitoring, triage, and treatment in prehospital scenarios, including prolonged field care and natural disaster zones with limited access. moreover, the current sars-cov- pandemic illustrated that frequent, non-invasive and wireless means for advanced hemodynamic monitoring is important for proper management of large numbers of isolated patients without compromising the health of the medical staff or quality of treatment . thus, although the results from this study are promising, future studies should test the capabilities of the ppg-based technology in humans under these specific indications. in conclusion, there is a clear need for reliable non-invasive technology capable of advanced hemodynamic monitoring. in this animal model of hemorrhagic shock, we report that measurements obtained from a novel ppg-based device offer a high level of reliability compared to existing standard invasive techniques, even in circumstances of moderate to severe and unstable hemorrhagic shock. pursuant to further trials, such a device could offer reliable and straightforward monitoring capabilities in a variety of clinical settings, from various hospital departments to pre-hospital and ambulatory settings. most importantly, this novel ppg-based technology enables continuous non-invasive monitoring and timely focused care, which may minimize morbidity and mortality without compromising measurement accuracy. animals. eleven white domestic female pigs (laboratory animals farm, lahav, israel; table ) were housed in the institutional animal facility accredited by the association for assessment and accreditation of laboratory animal care international (aaalac). water and normal appropriate diet were available ad libitum. the experimental procedures were performed days after acclimatization. food was withheld starting the night before the procedure. study design. animals were sedated and connected to various monitoring devices as described below. the animals were monitored for ~ min ("pre-bleeding" phase) before % of their total blood volume was withdrawn ("bleeding phase"; table ). animals were then monitored ("post-bleeding phase") for up to h (median of ± min) or until death if occurred earlier. no additional intervention was performed. hemodynamic parameters were documented every min until the end of bleeding (both pre-and bleeding phases) and every min during the post-bleeding phase using the al and the sg catheter. at the same time, measurements were recorded every min using the ppg-based wearable device, attached either to the animals' skin, tongue, or tail in order to achieve the best ppg signal readings. despite the capability of the wearable device to allow a high rate surgical procedures. the following vessels were cannulated using the over-the-wire (seldinger) technique with introducers (cordis, fremont, ca) inserted into: the left common carotid artery for invasive blood pressure monitoring and heart rate; the right internal jugular vein for venous blood sampling; pulmonary artery catheter placement (swan-ganz ccombo, edwards lifesciences, irvine, ca); right femoral artery cannulation for arterial blood sampling and induction of bleeding. a urinary catheter (tiemann catheter f) was placed for collection and output monitoring. body temperature was monitored rectally. pulse oximeter was placed on the tail lingual or buccal surface to measure arterial oxyhemoglobin saturation. continuous three-lead electrocardiogram monitoring was obtained using electrodes placed on the animal's right forelimb, left forelimb, and left hind limb. at the end of the observation period, surviving animals were euthanized by an intravenous injection of kcl solution (fagron group bv, rotterdam, netherlands). hemorrhagic shock. hemorrhage was induced by the controlled bleeding of % of the animal's calculated blood volume . blood was withdrawn manually from the right femoral artery by using a syringe in ml aliquots. the rate of bleeding was controlled to keep mean arterial pressure from dropping below mmhg. when necessary, bleeding was stopped, and the animal was allowed to recover prior to the resumption of bleeding. the total bleeding time was kept between and min. hemodynamic measurements. sbp, dbp, hr, and co were monitored and recorded every min up to the end of bleeding and every min thereafter. pulmonary artery pressures were monitored using a datex-ohmeda cardiocap (datex-ohmeda inc, madison, wi). co was monitored using a vigilance ii monitor (edwards lifesciences, irvine, ca). photoplethysmography-based monitoring device. ppg is commonly applied for pulse oximetry, usually transmitting light in specific red and infrared wavelengths which is absorbed by a detector on the other side of the tissue (hence attached to relatively thin body parts such as fingers, ear lobes, etc.). while passing through the tissue, and upon interaction with oxy-and deoxyhemoglobin, these wavelengths show a unique absorbance pattern. the detector can measure the changing absorbance at each of the wavelengths, and by that it can determine the absorbance resulting from the pulsating arterial blood. the currently used device is based on reflective ppg (fig. ) , in which the transmitted light is partially reflected from the tissue and detected by a www.nature.com/scientificreports/ photodiode detector positioned near the light source transmitter. the high resolution of the ppg wave combined with advanced algorithms allows the sensor to capture minute changes as well as tracking numerous vital signs derived from the pulse contours, including advanced hemodynamic parameters such as bp, hr, co, and more. tracking the changes of blood pressure is achieved after a pre-set baseline calibration process as described below, and is based on pulse wave transit time (pwtt) technology combined with pulse wave analysis (pwa). the algorithm used was not trained or adapted using the data acquired in this study, rather it was trained using data from the public mimic critical care database source (https ://mimic .physi onet.org/; https ://doi.org/ . / sdata . . ), and from numerous clinical studies conducted earlier by the company. the baseline calibration measurement is by a simple offset using either an approved non-invasive, cuff-based device or an invasive (al) device as in the case of this study, with the average value of three consecutive measurements entered into the device's management application, and from that moment on it tracks changes in blood pressure. calibration is needed once every months, which increases its clinical usability. within the context of this study, calibration was conducted only once after the animals were sedated, ventilated, and the instrumentations (including the al and sg catheters) were inserted. data processing and analysis. data obtained from the al and sg catheter for sbp, dbp, hr, and co were screened for outliers according to the following criteria: initially, percentage differences (%∆) between each value and the preceding and proceeding values were calculated. for each variable separately, the average standard deviation (sd) for the sum of preceding and proceeding %∆ was then obtained. next, the individual data points (%∆) were screened, and any value for which the percentage difference from both the previous and next values was > ± sd was eliminated. in total, out of (< . %; hr: n = ; sbp: n = ; dbp: n = ; co: n = ) outlier values were excluded from the final analysis. statistics. statistical analyses were performed separately for each of the three phases (i.e., pre-bleeding, bleeding, post-bleeding) and for all phases combined. the kolmogorov-smirnov and shapiro-wilk tests were used to assess normality, as these tests are sensitive to outliers. to define the degree of correlation between the two methods (i.e. invasive vs. ppg-based), the linear regression formula was defined, pearson's correlation coefficient was calculated, and the hypothesis that the slope and intercept are equal to zero was tested. the level of absolute agreement between al/sg measurements and those obtained from the wearable device for sbp, dbp, hr, and co was evaluated using intraclass correlation coefficients (icc) and standard error of the mean (sem) as well as bland-altman plots. results of the bland-altman analyses are reported as mean biases ± % limits of agreement (loa). all other results are presented as means ± sd. due to the unstable hemodynamic state imposed on the animals and to better assess the ppg device's ability to track changes (between successive measurement points) or trends in bp and co, four quadrant plots analyses were performed. concordance rate was calculated as the percentage of data points lying in the upper right and lower left quadrants relative to all points. to reduce statistical noise, we implemented an exclusion zone of ± mmhg and . l/min for bp and co measurements, respectively, as suggested by critchley et al. and saugel et al. . statistical analyses were considered significant if p < . . data were analyzed using spss . 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considerations on the -quadrant plot and the polar plot methodology none. d.n. and a.e. conceived and designed the study. d.n., g.p., l.w.a. and a.e. conducted the study. d.n., k.c., g.p., l.w.a., s.d.g., r.l., e.k., a.e. and y.g. analyzed the data. d.n., k.c., g.p., l.w.a., s.d.g., r.l., e.k., a.e. and y.g. wrote the manuscript. all authors read and approved the manuscript. a. eisenkraft and r. littman are employees at biobeat technologies ltd, they have no additional financial interests. all other authors declare no competing interests exists. correspondence and requests for materials should be addressed to a.e.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -mmcu c authors: sahu, amit ranjan; wani, sajad ahmad; saxena, shikha; rajak, kaushal kishor; chaudhary, dheeraj; sahoo, aditya prasad; khanduri, alok; pandey, aruna; mondal, piyali; malla, waseem akram; khan, raja ishaq nabi; tiwari, ashok kumar; mishra, bina; muthuchelvan, d.; mishra, bishnu prasad; singh, raj kumar; gandham, ravi kumar title: selection and validation of suitable reference genes for qpcr gene expression analysis in goats and sheep under peste des petits ruminants virus (pprv), lineage iv infection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: mmcu c identification of suitable candidate reference genes is an important prerequisite for validating the gene expression data obtained from downstream analysis of rna sequencing using quantitative real time pcr (qrt-pcr). though existence of a universal reference gene is myth, commonly used reference genes can be assessed for expression stability to confer their suitability to be used as candidate reference genes in gene expression studies. in this study, we evaluated the expression stability of ten most commonly used reference genes (gapdh, actb, hsp , hmbs, s rrna, b m, polr a, hprt , acac, ywhaz) in fourteen different peste des petits ruminants virus (pprv) infected tissues of goats and sheep. reffinder and rankaggreg software were used to deduce comprehensive ranking of reference genes. our results suggested hmbs and b m in goats and hmbs and hprt in sheep can be used as suitable endogenous controls in gene expression studies of pprv infection irrespective of tissues and condition as a whole, thus eliminating the use of tissue specific/ condition specific endogenous controls. we report for the first time suitable reference genes for gene expression studies in pprv infected tissues. the reference genes determined here can be useful for future studies on gene expression in sheep and goat infected with pprv, thus saving extra efforts and time of repeating the reference gene determination and validation. based studies where both quality control and validation are essential criteria . the major concern in qrt-pcr is a suitable endogenous control/reference gene to nullify variations that arise in the due course of experiment . the variation can be introduced at any step starting from rna extraction to quantification of qrt-pcr in terms of quality and quantity , . endogenous control genes are assumed to be constitutively and uniformly expressed within the samples to be compared, irrespective of experimental conditions or treatments and tissue differentiation , . housekeeping genes are the most commonly used endogenous control genes. these genes are used as reference control genes to normalize the variations in the qrt-pcr experiment , . however, varying expression of housekeeping genes under different experimental conditions has been reported in viral infections , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , cancer research , and heat stress response in sheep . the use of an invalidated reference gene in normalization leads to unreliable conclusions especially when used with tissue samples , , . this warrants for a need to identify suitable reference gene(s) for normalization for every gene expression experiment to do away with the hurdles in qrt-pcr . sometimes, validated endogenous controls for the desired experimental conditions can be derived from the literature describing the similar type of experiment. peste des petits ruminants (ppr) is one of the most economically important diseases of goats and sheep, characterized by acute febrile condition, erosive stomatitis, diarrhea and pneumonia [ ] [ ] [ ] . eradication of rinderpest (rp) has put ppr in spotlight to be the next eradicable disease due to similar nature of the causative agent , . ppr caused by peste des petits ruminants virus (pprv) belongs to genus morbillivirus of family paramyxoviridae. it is known that virus infection (e.g. sars corona virus, yellow fever virus, human herpes virus, cytomegalovirus etc.) often results in modified or fluctuating gene transcription patterns of conventionally used housekeeping genes , . recently, gapdh was identified to be the most suitable reference gene for evaluating the gene expression in pprv infected goats pbmcs in vitro . no published data is currently available on the use of specific reference gene(s) in goats and sheep infected with pprv in vivo. rna-seq experiments are being carried out in our laboratory to identify specific host gene expressions signatures in goats and sheep under pprv infection. the indiscriminate use of any endogenous control to validate the rna-seq experiment may lead to erroneous conclusions. therefore, in our study we used a panel of ten reference genes viz. gapdh (glyceraldehyde- -phosphate dehydrogenase), s rrna ( s ribosomal rna), b m (β microglobulin), hsp (heat shock protein ), acac-alpha (acetyl coenzyme carboxylase alpha), hmbs (hydroxymethylbilane synthase), ywhaz (tyrosine -monooxygenase activation protein zeta polypeptide), polr a (polymerase ii (dna directed) polypeptide a), actb (beta actin) and hprt (hypoxanthin phosphoribosyl transferase ) in fourteen different tissues obtained from healthy and pprv infected goats and sheep to identify the best possible reference gene(s) for qrt-pcr normalization. we recommend different sets of reference genes based on the experimental condition. performance of qrt-pcr primers. gene specific amplification was confirmed by a single peak in the melting-curve analysis for all the genes (supplementary figs s and s ). the linear regression equation, correlation coefficient, pcr efficiency and standard curve for each gene are shown in supplementary figs s and s . the efficiency of all the primers for the genes ranged from - %. ct value of candidate reference genes. the mean ct value of the genes in control, infected and combined groups is given in supplementary table s . the mean ct value of the reference genes ranged from . ± . (b m) to . ± . (actb) in goats and . ± . (actb) to . ± . (polr a) in goats. expression profile of all the reference genes in both the species is represented by box whisker plots ( supplementary fig. s ). stability of candidate reference genes under specific experimental conditions. the lower the m-value coefficient, higher is the stability ranking in genorm and normfinder. in control goats, infected goats and goats combined, b m and hsp ; gapdh and hmbs; and acac and hmbs, respectively, were the most stable candidate genes by genorm analysis. similarly, hmbs and hprt were co-ranked as most stable genes in control sheep, infected sheep and sheep combined group by genorm analysis ( table s ). normfinder and comparative delta ct method analysis ranked hmbs as the stable gene for control goats, infected goats, goats combined, control sheep and sheep combined groups, and hsp for infected sheep group (table and supplementary tables s and s ). the stability of a gene is inversely proportional to the standard deviation value in the bestkeeper algorithm. hmbs was found to be most stable reference gene in all groups of goats as well as for infected sheep and sheep combined groups and b m for control sheep group (table and supplementary table s ). comprehensive ranking of reference genes. reffinder is a comprehensive program that integrates all four above mentioned software tools to rank the candidate reference genes based on their stability. the overall ranking suggested hmbs (fig. a -f and table and supplementary table s ) to be the most stable among all groups of goats, control sheep and sheep combined groups while hsp ( fig. e ) was found to be the most stable reference genes in infected sheep group. tissue specific studies among various goat tissues revealed hmbs as the most stable gene in spleen ( fig. a) , caecum ( n ). rankaggreg provides the consensus ranking by bruteaggreg function of the package. rankaggreg suggested hmbs to be the most stable endogenous control gene in all groups of goats and sheep (table and supplementary table s ). similarly, for tissues specific studies, rankaggreg suggested hmbs for caecum, large intestine, lower lip, small intestine, spleen and trachea; gapdh for abomasum, mesenteric lymph node, prescapular lymph node and rectum; polr a for liver and lung; acac for tongue and b m for upper lip as the most stable candidate reference genes in goats (supplementary table s ). in sheep, rankaggreg suggested hmbs for caecum, lower lip and trachea; hprt for abomasum and mesenteric lymph node; gapdh for large intestine and small intestine; actb for liver and spleen; b m for lung and rectum; acac for prescapular lymph node; ywhaz for tongue and hsp for upper lip, as the most stable genes (supplementary table s ). final consensus ranking was obtained for each condition and tissue from rankaggreg results. the consensus ranking was obtained for each condition and tissue by considering the results of both reffinder and rankaggreg. a detailed list of recommended endogenous control genes for individual tissues is given in table . due to its high specificity and sensitivity, qrt-pcr dominated the world of gene expression studies among all other contemporary techniques. it is extremely useful in gene expression studies to document host cell responses to virus infection , , , - . elucidation of molecular pathogenesis from global gene expression profile by high-throughput omics study ultimately ends up in a number of candidate genes. qrt-pcr provides the simplest platform for its validation. in spite of these facts, qrt-pcr requires a robust normalization of the data to overcome the variability introduced at any of the steps in an experiment , . an ideal reference gene should be stably expressed in tissues under varied experimental conditions. however, this constant expression of any reference gene only refers to a specific condition under certain environment and the expression level in different cell types and tissues significantly varies under different experimental systems , [ ] [ ] [ ] . thus identification and validation of reference genes for expression studies in an experiment is widely supported and practiced , . the data in the study was analyzed using genorm, normfinder, bestkeeper and comparative Δct method. genorm calculates the standard deviation of the expression ratio of two candidate reference genes, which are not co-regulated as a pairwise variation . the stability value (m) is calculated as the average pairwise variation of a specific gene compared with all other reference genes. genes with the highest m values have the least stable expression. genorm also identifies the number of reference genes required for the normalization of a particular experiment . the normfinder allows comparison of intra-and inter-group variation and calculates expression stability value (m) , . bestkeeper uses repeated pairwise correlation analysis to determine the optimal reference genes . the comparative delta ct method evaluates the average of standard deviation values derived from comparison of relative expression between a reference gene with other reference genes. the difference in ranking results obtained from different software programs in our study may be attributed to the use of different algorithms by different softwares to determine gene expression stability , , . most of the reports recommend consensus comprehensive ranking for use as best endogenous control , , . therefore, we recommend the candidate reference genes obtained through comprehensive ranking method in all the three different experimental conditions i.e. control, infected and combined. in control goats and control sheep, hmbs and hsp , and hmbs and b m are recommended as the most stable endogenous controls. these genes can be used as suitable reference genes in studies where basal expression of target genes across healthy tissues is compared in goats and sheep. in pprv infected tissue studies for comparing across tissues, we recommend the use of hmbs and acac in goats and hmbs and hsp in sheep. in comparative studies of pprv infected with uninfected tissues as a whole, we recommend the use of hmbs and b m in goats and hmbs and hprt in sheep, thus eliminating the use of multiple tissue specific endogenous controls. the purpose of the combined analysis was to demonstrate the stability of reference genes with respect to different conditions and tissues. we recommend to use that reference gene which shows highest stability in the combined groups for studies under pprv infection. isg plays a key role in the innate immune response to viral infection either via its conjugation to a target protein (isgylation) or via its action as a free or unconjugated protein. isgylation involves a cascade of enzymatic reactions to alter host immune system. it exhibits antiviral activity towards both dna and rna viruses, including influenza a, hiv- and ebola virus , - . irf , a key innate immune modulator controlling the induction of type i interferons during viral infections , . upon activation, phosphorylated irf induce expression of genes responsible for type i interferon production inside the nucleus in virus infection . isg and irf were chosen as the target gene of interest as these genes have been identified and predicted as important antiviral molecules by rna-sequencing data analysis of pprv infection studies in our lab (data not shown). the significant difference in expression of isg and irf on use of the most stable reference genes in goats and sheep corroborated with the findings of the rna-seq experiment conducted in the laboratory. the reference genes determined herein can be the protocols were approved vide letter no /cpcsea. apparently healthy, non-descriptive hill goats (local rohilkhand breed) and sheep (muzaffarnagri breed) between months to year of age were used in the present study. virulent pprv (izatnagar/ -lineage iv, accession number kr . ) isolate was used as a challenge virus for infection. the tissue samples -upper lip, lower lip, tongue, trachea, lung, pre-scapular lymph node, mesenteric lymph node, spleen, liver, small intestine, large intestine, abomasum, caecum and rectum were collected from pprv infected sheep and goats (n = for each of the species). the counterpart healthy tissues were collected from apparently healthy animals (negative for pprv antibody by competitive elisa and serum neutralization test) housed separately. the apparently healthy animals are referred as control. these animals were handled in a humane manner and euthanized as per the cpcsea guidelines. the graphical abstract of this study is represented in supplementary fig. s . a total of ten candidate reference genes were selected based on, their use as reference genes in diverse studies on gene expression in goats and sheep, availability of their sequences in databases and their function in the cell ( table ) . the ten reference genes used were gapdh, actb, b m, hsp , acac-α, hmbs, ywhaz, polr a, hprt and s rrna , , , [ ] [ ] [ ] [ ] , , . primers for ywhaz, polr a, actb and hprt were designed based on the sequence obtained from ncbi with the help of software primer plus . the quality parameters for these primers were checked in oligo analyzer and ncbi primer blast . the primers for rest of the genes were obtained from already published literature , , . samples. ppr sandwich-elisa kit for pprv antigen detection was obtained from national morbilivirus referral laboratory, division of virology, ivri, mukteshwar . selisa for the tissue samples was carried out as per the instructions provided with the kit. absolute quantification of n gene in all tissues. absolute quantification of n gene for viral load by qrt-pcr for infected tissues was performed using primers specific to pprv n gene. primers -pprv n-fp: atctgcaggaaaggtcagct- ′ and pprv n-rp: tccctctcctgttttgtgct- ′ were designed using primer plus. the standard curve was generated using a series of -fold dilutions of gel purified pcr product of n gene. the amplification efficiency was calculated from the slope of the standard curve using the formula e = (− /slope) . copy number was calculated from the standard curve ( supplementary fig. s ) . ct values greater than were considered negative. . isg expression in control and infected lung tissues of goats with the two most stable reference genes (a) and two least stable reference genes (b). isg expression in control and infected lung tissues of sheep with the two most stable reference genes (c) and two least stable reference genes (d). isg expression in control and infected spleen tissues of goats with two most stable reference gene (e) and two least stable reference genes (f). isg expression in control and infected spleen tissues of sheep with two most stable reference genes (g) and two least stable reference genes (h). the expression was calculated as delta ct value (ct (isg ) − ct (geometric mean of ct of the best endogenous control genes) or ct (geometric mean of the least stable endogenous control genes) ). significance (p < . ) of difference in expression between the control and infected groups was tested using t-test. levels not connected by the same superscript are significantly (p < . ) different. scientific reports | ( ) : | doi: . /s - - - figure . expression of irf in lung and spleen tissues of both goats and sheep with two most stable reference genes (hmbs and b m in goats; hmbs and hprt in sheep) and two least stable reference genes (actb and ywhaz in goats; actb and polr a in sheep). irf expression in control and infected lung tissues of goats with the two most stable reference genes (a) and two least stable reference genes (b). irf expression in control and infected lung tissues of sheep with the two most stable reference genes (c) and two least stable reference genes (d). irf expression in control and infected spleen tissues of goats with two most stable reference gene (e) and two least stable reference genes (f). irf expression in control and infected spleen tissues of sheep with two most stable reference genes (g) and two least stable reference genes (h). the expression was calculated as delta ct value (ct (irf ) − ct (geometric mean of ct of the best endogenous control genes) or ct (geometric mean of the least stable endogenous control genes) ). significance (p < . ) of difference in expression between the control and infected groups was tested using t-test. levels not connected by the same superscript are significantly (p < . ) different. reverse transcriptase-quantitative polymerase chain reaction (qrt-pcr) of reference genes. gene specific primers ( data analysis. the ct values for the control (uninfected), and pprv infected samples were initially analyzed for each of the species to determine the best possible endogenous control(s) for healthy and pprv infected conditions separately. then, the data (ct values) from the control and infected were combined for each of the species to identify the best endogenous control for the case where pprv infected samples were compared with control healthy samples. the data was analyzed for six groups: goats -control goats, infected goats (pprv infected) and goats combined (combining both the control and infected ct values); sheep -control sheep, infected sheep and sheep combined. to determine tissue specific endogenous controls, both infected and control tissue ct values were taken into consideration for each species. stability of the candidate reference genes were evaluated by algorithms genorm , normfinder , bestkeeper , and the comparative Δ delta ct method in reffinder (http://leonxie.esy.es/reffinder/). a comprehensive overall ranking of the stability by integrating all four algorithms was provided in the reffinder. the final consensus ranking was obtained with rankaggreg package by considering the results obtained from all the above analysis. the rankaggreg package of r software combines the stability measurements obtained from softwares (genorm, normfinder, bestkeeper, comparative delta ct method and reffinder) and establishes a consensus rank of reference genes . a weighted rank aggregation was applied by using bruteaggreg function of the package. this function performs rank aggregation using the brute force approach. the aim of rank aggregation is to find an aggregated ranking that minimizes the distance to each of the ranked lists in the input set. validation of reference genes identified. the stability of the identified best reference genes was validated by evaluating the expression of isg and irf as target genes in the pprv infected lung and spleen tissues with respect to the control tissues in both the species. isg and irf were chosen as the target genes of interest as these genes have been identified and predicted as important antiviral molecules by rna-sequencing data analysis of pprv infection studies in our lab. forward primer ′-cagttcatcgcccagaagat- ′ and reverse primer ′-gtcgttcctcaccagg atgt- ′ were used for isg . similarly, for irf ′-gacacgcccatctttgactt- ′ and ′-actgtccagggaggacacac- ′ were used as primers. the amplification efficiency was calculated from the standard curve generated by point, fold serial dilutions. the ct values for control and infected tissue samples with two most stable endogenous control genes (hmbs and b m in goats, and hmbs and hprt in sheep) and two least stable endogenous control gene (actb and ywhaz for goats and actb and polr a for sheep) were estimated. expression for control and infected groups was represented by delta ct value (ct (target genes) − ct (geometric mean of ct of the two best endogenous control genes) or ct (geometric mean of ct of two least stable endogenous control genes) ). t-test in graphpad prism was used to compare the expression of isg and irf in infected relative to control. miqe guidelines. this enables the researcher to evaluate the technical quality of the qrt-pcr experiments , . all the experiments were carried out as per the miqe guidelines. a summary sheet of miqe guideline of this experiment is provided in supplementary table s . design and validation issues in rna-seq experiments rna-seq: an assessment of technical reproducibility and comparison with gene expression arrays understanding mechanisms underlying human gene expression variation with rna sequencing mapping and quantifying mammalian transcriptomes by rna-seq rna-seq: a revolutionary tool for transcriptomics experimental validation of methods for differential gene expression analysis and sample pooling in rna-seq validation' in genome-scale research infection of bovine dendritic cells by rinderpest or measles viruses induces different changes in host transcription identification of suitable reference gene in goat peripheral blood mononuclear cells (pbmcs) infected with peste des petits ruminants virus (pprv) absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays the need for transparency and good practices in the qpcr literature quantification of mrna using real-time rt-pcr validation of reference genes for gene expression studies in virus-infected nicotiana benthamiana using quantitative real-time pcr evaluation and validation of candidate endogenous control genes for real-time quantitative pcr studies of breast cancer real-time rt-pcr normalisation; strategies and considerations selection of reliable reference genes for gene expression studies using real-time pcr in tung tree during seed development apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation normalization of qrt-pcr data: the necessity of adopting a systematic, experimental conditions-specific, validation of references evaluation of suitable reference genes for gene expression studies in porcine pbmcs in response to lps and lta s rrna is a reliable normalisation gene for real time pcr based on influenza virus infected cells careful selection of reference genes is required for reliable performance of rt-qpcr in human normal and cancer cell lines use of maximum likelihood-mixed models to select stable reference genes: a case of heat stress response in sheep accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-pcr in somatic cells from goat milk global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control peste des petits ruminants expression kinetics of isg , irf , ifngamma, il , il and il genes vis-a-vis virus shedding, tissue tropism and antibody dynamics in pprv vaccinated, challenged, infected sheep and goats peste des petits ruminants virus supporting livelihoods and supporting livelihoods and peste des petits ruminants (ppr) and small ruminant diseases control reference gene selection for quantitative real-time pcr analysis in virus infected cells: sars corona virus, yellow fever virus, human herpesvirus- , camelpox virus and cytomegalovirus infections using host s ribosomal rna as a housekeeping gene for quantitative real-time reverse transcription-pcr (qrt-pcr) in virus-infected animal cells selection of reference genes for quantitative real-time pcr normalisation in adipose tissue, muscle, liver and mammary gland from ruminants early changes in cytokine expression in peste des petits ruminants disease toll-like receptor responses to peste des petits ruminants virus in goats and water buffalo the validation of housekeeping genes as a reference in quantitative real time pcr analysis: application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus real-time pcr in virology cytokines expression profile and kinetics of peste des petits ruminants virus antigen and antibody in infected and vaccinated goats selection of reference genes for quantitative real-time pcr analysis in chicken embryo fibroblasts infected with avian leukosis virus subgroup systematic selection of housekeeping genes for gene expression normalization in chicken embryo fibroblasts infected with newcastle disease virus reference gene selection for normalization of pcr analysis in chicken embryo fibroblast infected with h n aiv reference gene screening for analyzing gene expression across goat tissue refgenes: identification of reliable and condition specific reference genes for rt-qpcr data normalization selection of reference genes for gene expression studies related to intramuscular fat deposition in capra hircus skeletal muscle the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments quantitative pcr pitfalls: the case of the human placenta guideline to reference gene selection for quantitative real-time pcr reference gene validation for rt-qpcr, a note on different available software packages selection of reliable reference genes for rt-qpcr analysis normalization of real-time quantitative reverse transcription-pcr data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets validation of suitable reference genes for assessing gene expression of micrornas in lonicera japonica determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper-excel-based tool using pair-wise correlations selection of housekeeping genes for gene expression studies in human reticulocytes using real-time pcr reference genes selection for quantitative real-time pcr using rankaggreg method in different tissues of capra hircus antiviral activity of innate immune protein isg the antiviral activities of isg emerging role of isg in antiviral immunity irf- is the master regulator of type-i interferon-dependent immune responses role of interferon regulatory factor in t cell responses during acute lymphocytic choriomeningitis virus infection genome sequencing of an indian peste des petits ruminants virus isolate, izatnagar/ , and its implications for virus diversity, divergence and phylogeography design and evaluation of a unique sybr green real-time rt-pcr assay for quantification of five major cytokines in cattle, sheep and goats primer plus, an enhanced web interface to primer primer-blast: a tool to design target-specific primers for polymerase chain reaction a sandwich-elisa for the diagnosis of peste des petits ruminants (ppr) infection in small ruminants using anti-nucleocapsid protein monoclonal antibody rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay rankaggreg, an r package for weighted rank aggregation why the need for qpcr publication guidelines?-the case for miqe a practical approach to rt-qpcr-publishing data that conform to the miqe guidelines r.k.s., b.p.m., a.k.t. and r.k.g. conceived and designed the research. a.r.s., s.a.w., s.s., d.c. and a.k. conducted the wet lab work. a.r.s., a.p. and r.k.g. analyzed the data. a.r.s. and r.k.g. wrote the manuscript. a.r.s., p.m., w.a.m., r.i.n.k., r.k.g., a.p.s., k.k.r. and d.m. helped in manuscript drafting and editing. r.k.s., b.p.m., a.k.t. and r.k.g. proof read the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -hmsidrc authors: grunwell, jocelyn r.; stephenson, susan t.; mohammad, ahmad f.; jones, kaitlin; mason, carrie; opolka, cydney; fitzpatrick, anne m. title: differential type i interferon response and primary airway neutrophil extracellular trap release in children with acute respiratory distress syndrome date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: hmsidrc acute respiratory distress syndrome (ards) is a heterogeneous condition characterized by the recruitment of large numbers of neutrophils into the lungs. neutrophils isolated from the blood of adults with ards have elevated expression of interferon (ifn) stimulated genes (isgs) associated with decreased capacity of neutrophils to kill staphylococcus aureus and worse clinical outcomes. neutrophil extracellular traps (nets) are elevated in adults with ards. whether pediatric ards (pards) is similarly associated with altered neutrophil expression of isgs and neutrophil extracellular trap release is not known. tracheal aspirate fluid and cells were collected within h from seventy-seven intubated children. primary airway neutrophils were analyzed for differential isg expression by pcr, stat phosphorylation and markers of degranulation and activation by flow cytometry. airway fluid was analyzed for the release of nets by myeloperoxidase-dna complexes using an elisa. higher stat phosphorylation, markers of neutrophil degranulation, activation and net release were found in children with versus without pards. higher nets were detected in the airways of children with ventilator-free days less than days. increased airway cell ifn signaling, neutrophil activation, and net production is associated with pards. higher levels of airway nets are associated with fewer ventilator-free days. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ we have previously demonstrated that neutrophils exposed to airway fluid from mechanically ventilated children with virally-induced respiratory failure and bacterial coinfection have decreased respiratory burst and bacterial killing capacity . however, the mechanisms underlying these observations are still unclear. respiratory viruses have been shown to induce type i interferon (ifn) (ifnα/β) responses that then upregulate expression of interferon-stimulated genes (isgs) such as mx , isg , and ifit , , which influence neutrophil behavior and function , . since viral lower respiratory tract infections are a primary trigger for pards, we questioned whether differential isg expression was associated with neutrophil responses in intubated children at risk or with pards. we hypothesized that children with pards would have a greater type i ifn response resulting in more neutrophil extracellular trap (net) release. this prospective, observational study was performed in the thirty-six-bed academic medical/surgical pediatric intensive care unit (picu) at emory university/children's healthcare of atlanta at egleston from january through february . the study was approved by the institutional review board at emory university (irb and irb ) and all methods were carried out in accordance with relevant guidelines and regulations in the declaration of helsinki. informed consent was obtained from the parents of all subjects prior to collection and use of their samples. all patients admitted to the picu who were greater than days, with a corrected gestational age of at least weeks, and were younger than years old that met criteria for being at risk or having pards, as defined by the pediatric acute lung injury consensus conference (palicc), were screened for eligibility . children had to have lung injury within days of a known clinical insult, new infiltrate(s) consistent with acute pulmonary parenchymal disease on chest imaging and be receiving oxygen delivered either noninvasively or invasively to maintain an oxygen saturation between and %. children were excluded if they had any peri-natal related lung disease, respiratory failure fully explained by cardiac failure or fluid overload, chronic respiratory failure with mechanical ventilation via a tracheostomy or ram cannula, confirmed immunodeficiency disorder, immunosuppression from chemotherapy for an oncologic process, chronic immunosuppression in a bone marrow transplant or solid organ transplant recipient, no parent or legal guardian present to provide written informed consent, or the attending physician did not wish the patient to participate in the study. children were enrolled as controls if they were endotracheally intubated for airway protection and without lung pathology or signs of systemic infection or inflammation. for example, children selected as controls were electively intubated to facilitate radiologic imaging, for non-airway or cardiothoracic surgeries, and for airway protection following an acute ingestion or altered mental status without suspicion for infection or systemic inflammation. clinical data were abstracted from the medical record onto a standardized form. variables included demographics; fraction inspired oxygen, mean airway pressure, arterial oxygen saturation or arterial oxygen pressure used to calculate an oxygen saturation index (osi) or oxygenation index (oi), respectively; laboratory and microbiology results; length of mechanical ventilation and need for reintubation; length of picu stay, use of high frequency oscillatory ventilation (hfov) or extracorporeal life support (ecls), and vital status. severity of illness was determined by the pediatric risk of mortality (prism)-iii and pediatric logistic organ dysfunction (pelod) scores were calculated within h of intubation , . need for mechanical ventilation to -days was monitored to calculate ventilator-free days . lung injury severity was categorized according to palicc criteria . tracheal aspirate collection. tracheal aspirates were obtained from patients on conventional mechanical ventilation by instilling - ml of sterile saline through the inline ballard suction catheter and into a sterile luken's trap as part of routine suctioning per published protocols . children who were mechanically ventilated with hfov were suctioned only if clinically indicated and approved by the attending physician. tracheal aspirate samples were immediately placed on ice for transport to the laboratory for processing. airway sample processing. tracheal aspirate was gently dissociated using repeated passage through an g needle after the addition of ml of pbs-edta. dissociated tracheal aspirate was then centrifuged at ×g to generate a cell pellet and a fluid fraction. the fluid fraction was spun at ×g to generate cell-free airway supernatant (asn), aliquoted, and stored at − °c . airway cells were resuspended in pbs-edta and cell density was quantified using a countess hemocytometer using trypan blue exclusion to determine cell viability. cell purity was also assessed by cytospin preparations and diff-quik staining of airway cells. cell viability was also assessed using a live/dead aqua stain in the surface staining flow cytometry panel. gene expression assays. rna was isolated from airway cells stored at − c in rna later using the nucleospin rna ii kit with on-column genomic dna digestion according to the manufacturer's protocol (takara, mountain view, ca). rna was quantified using a nanodrop fluorospectrometer (therma scientific). rna integrity (rin) was measured at the emory integrated genomics core on an agilent bioanalyzer (see supplementary table s statistical analysis. statistical analyses were performed using jmp pro (sas institute, cary, nc) and graphpad prism for windows. unless otherwise stated, comparisons between samples of children with versus without pards were made using a two-tailed mann-whitney u test for nonparametric data. for pcr studies, a rout outlier test using a % false discovery rate was performed prior to a two-tailed mann-whitney u test. statistical significance was defined as a p value less than . . subject characteristics. seventy-seven patients were enrolled into the study within h of intubation and mechanical ventilation. of these children, ( . %) had pards and ( . %) did not have pards. table shows the demographics and clinical characteristics of the enrolled patients. the respiratory viral pcr panel and respiratory culture results from the clinical microbiology lab are reported in supplementary table s online. children with pards were more likely to be supported by extracorporeal life support (ecls), had a longer duration of mechanical ventilation, and spent more days in the hospital and the picu (table ) . there were no significant differences in severity of illness (prism iii) or organ dysfunction (pelod) scores, the absolute number or percentage of alive neutrophils from children based on pards status (table ) . this study was a survey of the activation status of neutrophils is pards. we assessed markers of airway neutrophil activation by flow cytometry. the gating strategy for selecting airway neutrophils is shown (fig. a-c) . there was no difference in total number (fig. d ) or percent cd b + neutrophils (fig. e ) in intubated children with or without pards. children with pards also had increased neutrophil surface expression of cd , a marker of primary granule exocytosis (fig. f) , and sphingosine -phosphate receptor (s pr ), a protein that forms a heterodimer with the neutrophil il -induced chemotaxis receptor, cxcl , and is detected with increased abundance in blood neutrophils from adults with bacterial pneumonia (fig. g ) . there was no significant difference in surface expression of the type iii fcγ receptor, cd or arginase i (arg ), an enzyme stored in the primary and tertiary granules of human neutro- www.nature.com/scientificreports/ phils ( fig. h-i) . examples of cytospin preparations stained with diff-quik from four unique patient tracheal aspirates show the predominance of neutrophils in the airway samples (fig. j) . we next determined whether the type i interferon (ifnα/β) signaling pathway was activated by measuring the phosphorylation of signal transducer and activator of transcription (stat ) by intracellular staining of fixed airway samples. airway cells from children with pards had increased expression of phosphorylated-y stat (p-stat ) and stat compared with children who did not meet pards criteria (fig. ) . cells were gated on forward and side scatter and representative histograms for both p-stat and stat are shown ( fig. a) . quantification of both the mean fluorescence intensity (mfi) and percent positive cells are summarized in fig. b -e. children with pards have higher levels of stat and p-stat compared with children without pards. activation of the type i interferon signaling pathway results in increased expression of many www.nature.com/scientificreports/ www.nature.com/scientificreports/ ifn-stimulated genes (isgs) . three isgs, ifit , isg , and mx , were used to identify activation the type i ifn signaling pathway , . isg expression of children with versus without pards were compared. isg expression was variable; however, children with pards did not show a significant difference in expression level of isg , ifit or mx compared to children without pards (fig. ) . our results show that markers of neutrophil degranulation and activation are elevated in children with versus without pards. in addition, activation of the type i ifn signaling pathway, as indicated by increases in p-stat , and isg expression occurs in some children with pards. (fig. a) . higher airway net levels were associated with fewer ventilator-free days (f = . , p value < . ), a lower proportion of children with ventilator-free days over days (fig. b) acute lrti are the trigger for the majority of pards . viral and bacterial infections activate type i ifn signaling pathways resulting in an increase in antiviral isg expression and proinflammatory responses critical for host defense , . a finely tuned type i ifn response is crucial to a host as an excessive or prolonged type i ifn response may lead to impaired gas exchange in the lung due to cellular and tissue destruction . ifns prime mature neutrophils for net release upon stimulation with a second stimulatory signal . although antimicrobial proteins expressed on nets can inactivate pathogens and prevent viral spread to neighboring cells, the lung is www.nature.com/scientificreports/ vulnerable to the damage that histones and proteolytic enzymes contained within nets can inflict to host tissue. therefore, excessive and prolonged type i ifn signaling may contribute to the production of nets which fill the alveolar spaces, lead to increased inflammation, and result in impaired lung function, which are the hallmarks of pards. our study assessed neutrophil activation, the differential expression of isgs and activation of the stat signaling pathway, and netosis in the airways of intubated children with acute respiratory failure due to lower respiratory tract infections. we found increased neutrophil degranulation markers, phosphorylation of stat (y ), and nets, as measured by mpo-dna complexes, in the airways of children with pards compared with children without pards. higher levels of airway nets were associated with fewer ventilator-free days. our findings are summarized in fig. . the timing and magnitude of the type i ifn response are important in modulating the immune response to viral infection , . at higher levels, isgs contribute to dysregulated lung inflammation, disease progression, and are linked to worse clinical outcomes . for example, in a mouse model of sars-cov infection, the delayed expression of high levels of type i ifn in the presence of high viral titers resulted in lethal pneumonia ; however, early treatment with type i ifn within the first six hours of sars-cov infection was protective. in influenza a virus, disease severity and progression are associated with overshooting the ifn-driven inflammatory response whereby exogenous supplementation with type i ifn correlated with increased morbidity and mortality , . rsv also induces high levels of pro-inflammatory cytokines directly related to type i ifn, and mice that lack ifnar have less proinflammatory cytokine release resulting in a less severe disease course following rsv infection . by contrast, there was no effect of ifnar deletion on pathogenicity of mice infected with sars-cov; however, stat deficient mice showed increased susceptibility, prolonged viral shedding and mortality . differential type i ifn and isg expression are associated with neutrophil dysfunction and worse clinical outcomes , . for example, high expression of a panel of isgs (mx , ifit , isg ) from circulating neutrophils from a subgroup of adults with ards compared with normal isg expressing neutrophils had reduced migration toward the neutrophil chemokine interleukin- (il- ), decreased p map kinase phosphorylation, superoxide anion release, il- release, and a shift from necrotic to apoptotic cell death that was associated with a diminished capacity to kill staphylococcus aureus, but not pseudomonas . subsequent hierarchal clustering analysis of the aforementioned isg panel from circulating neutrophils of ards patients within h of initiation of mechanical surface expression of the primary granule exocytosis marker, cd , and the lipid signaling g protein-coupled receptor, sphingosine- -phosphate receptor (s pr ), are higher in children with versus without pards. the type i interferon (ifn) signaling pathway transcription factor, stat , is upregulated and phosphorylated, and transcript levels of isg is increased in children with versus without pards. neutrophil extracellular trap (net) release is regulated by a nadph oxidase (nox) respiratory burst (reactive oxygen species (ros) triggered mechanism and an intracellular calcium-dependent trigger. it is not known which trigger dominates in the airways of children with pards. children with pards have elevated levels of nets in their airways as detected by myeloperoxidase (mpo)-dna complexes in our study. elevated net levels are associated with a higher number of ventilator-free days (vfd) over days in a -day period (i.e. if the child survived, then they were more likely to spend ≥ days endotracheally intubated and mechanically ventilated). created with biorender.com using the web version, which may be accessed at https ://biore nder.com/, with a paid individual subscription granting permission to publish in journals. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ ventilation showed that both high-and low-range expression had fewer -day ventilator-free and icu-free days and higher -day mortality compared with mid-range isg expressing patients . these data suggest that a targeted middle ground for ifn levels exists to benefit the host. an ifn signal below a lower threshold would result in an ineffective antiviral host response, while an ifn signal above an upper threshold would trigger a detrimental inflammatory host response. ifn signaling outside the target zone would increase lung inflammation and ards severity , , . by contrast, our findings are from airway cells with a neutrophil predominance rather than from circulating neutrophils for adults with ards , . additionally, we assessed each isg, ifit , isg , and mx , individually rather than in aggregate, and we note that isg expression is highly variable in children with versus without pards. finally, the release of nets was not assessed in the aforementioned studies. excessive net formation has been reported in the serum and bronchoalveolar lavage fluid from adults with ards; however conclusions with respect to clinically relevant outcomes are difficult to draw due to the heterogeneity in study design , , . nets play prominent roles in bacterial pneumonia [ ] [ ] [ ] , rsv bronchiolitis , , influenza pneumonia , , , sepsis [ ] [ ] [ ] [ ] , sars-cov , small-vessel vasculitis , systemic lupus erythematosus , , and transfusion-related acute lung injury . nets induce dose-dependent cytotoxic effects on human alveolar epithelial cells due to histone and myeloperoxidase induced damage to alveolar epithelial and endothelial cells , . nets also influence the macrophage function , t cell proliferation , and amplify ifn production by plasmacytoid dendritic cells . conversely, interferons influence the process of netosis in mature neutrophils , . high ifn/isg expression polarizes neutrophils to an activated "n " phenotype that can lead to increased net production, degranulation, and influence neutrophil interactions with other immune and airway epithelial cells . interferonopathies, autoimmune diseases, and tumor-associated neutrophils are all examples where high ifn/isg levels influence neutrophil activation and function , . priming of neutrophils with ifn-α and subsequent stimulation with c a resulted in increased stat phosphorylation at tyrosine and net production in mature neutrophils . activation of neutrophils by type i ifns led to increased netosis that triggered biofilm formation by pseudomonas aeruginosa and persistence in the lung . net production, like ifn signaling, exists in a balance. excessive net release can lead to the systemic spread of inflammation through platelet interactions and result in multiple organ dysfunction , , ; however, depletion or defective net production can lead to the spread of infection early in the course of disease . several mechanisms regulating the formation of nets are known and include nadph oxidase (nox ) dependent and calcium channel nox-independent netosis - . netosis is driven by net-specific kinases that regulate transcription initiation in neutrophils. for example, erk was shown to differentially regulate noxdependent netosis . interestingly, khan and colleagues also noted that stat was a transcription factor that was upregulated in the nox-dependent netosis pathway . we were not able to study the relative importance of nox-dependent versus calcium influx (nox-independent) mechanisms on net formation given that netosis has already occurred by the time of tracheal aspirate sampling in our patients. additionally, we did not quantify the relative amounts of neutrophils undergoing netosis versus apoptosis. quantifying neutrophil apoptosis should be part of future studies as nox-dependent netosis is dependent on the activation of the kinase akt to suppress apoptosis and switch to netosis . while we detected higher type i ifn signaling and net production in the airways of children with versus without pards, due to the clinical nature of our study, we are not able to attribute causation of neutrophil dysfunction to high isg expression in study participants. there are several additional limitations to our study. first, this is a single-center study with a limited sample size; however, despite the limited number of children studied, we were able to detect differences in neutrophil type i ifn signaling pathway phosphorylation and function in intubated children with versus without pards. the majority of children in this study were at risk for developing pards, with the remaining children being equally distributed amongst mild, moderate and severe pards categories; however, the degree of pards severity was not associated with neutrophil dysfunction due to heterogeneity in neutrophil function and a limited sample size. we also excluded immunocompromised patients from our study which is a major risk factor associated with pards-related mortality [ ] [ ] [ ] . second, there is heterogeneity in the identity of viral and bacterial pathogens infecting study participants precluding any conclusions regarding the influence of organism on study results. we did not stratify the patients based on bacterial growth in an endotracheal respiratory culture as there is controversy regarding whether this is a true bacterial coinfection versus merely codetection of bacterial and viral organisms . thirdly, we are not powered to detect differences in mortality associated with higher isg gene expression as seen in the adult ards cohort; however, we did detect a significant difference in ventilator-free days in children with a higher airway net burden . we are limited in the ability to study the mechanisms regulating airway net release as netosis has already occurred in patients at the time of sample collection' however, network analysis of transcription factor signaling pathways from recovered airway neutrophils could be performed as previously described . additionally, future experiments using blood-derived neutrophils from healthy donors could be incubated in patient airway fluid to simulate the airway environment with pma and calcium-ionophore stimulation, and appropriate nox inhibition, to study mechanistic regulation of net formation. finally, we did not explore the role of other signaling pathways, such as that initiated through the sphingolipid receptor or other kinase signaling cascades, as mechanisms involved in the pathogenesis of nets or pards severity. in summary, we describe the differential type i ifn signaling based on the presence or absence of pards and show that airway levels of nets are higher in children with versus without pards. higher levels of airway nets are associated with fewer ventilator-free days. future work will explore the influence of type i ifn signaling, nets and the pards airway environment on macrophage and t-cell activation and function. defining underlying mechanistic differences in children with acute respiratory failure due to lower respiratory tract infections is needed to move beyond supportive care and toward targeted personalized therapies for pediatric patients with moderate/severe pards. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. received: july ; accepted: october paediatric acute respiratory distress syndrome incidence and epidemiology (pardie): an international, observational study the acute respiratory distress syndrome acute respiratory distress syndrome a role for neutrophils in viral respiratory disease bacteria-specific neutrophil dysfunction associated with interferon-stimulated gene expression in the acute respiratory distress syndrome extremes of interferon-stimulated gene expression associate with worse outcomes in the acute 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macrophage polarization t lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses the essential role of type i interferons in differentiation and activation of tumor-associated neutrophils detrimental effect of type i ifns during acute lung infection with pseudomonas aeruginosa is mediated through the stimulation of neutrophil netosis a distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type i ifns neutrophil extracellular traps promote hypercoagulability in patients with sepsis sk channel and mitochondrial ros mediate nadph oxidaseindependent netosis induced by calcium influx transcriptional firing helps to drive hypertonic saline suppresses nadph oxidase-dependent neutrophil extracellular trap formation and promotes apoptosis alkaline ph promotes nadph oxidase-independent neutrophil extracellular trap formation: a matter of mitochondrial reactive oxygen species generation and citrullination and cleavage of histone. front. immunol. , akt is essential to induce nadph-dependent netosis and to switch the neutrophil death to apoptosis a simple and robust bedside model for mortality risk in pediatric patients with acute respiratory distress syndrome predicting mortality in children with pediatric acute respiratory distress syndrome: a pediatric acute respiratory distress syndrome incidence and epidemiology study subtypes of pediatric acute respiratory distress syndrome have different predictors of mortality pediatric ventilator-associated infections: the ventilator-associated infection study we acknowledge the emory + children's flow cytometry core for flow cytometry instrumentation. this study was supported in part by the emory integrated genomics core (eigc), which is subsidized by the emory university school of medicine and is one of the emory integrated core facilities. additional support was provided by the georgia clinical and translational science alliance of the national institutes of health under award number ul tr . the content is solely the responsibility of the authors and does not necessarily reflect the official views of the national institutes of health. the authors thank the bedside caregivers of the patients involved in this study for their skilled and compassionate care. j.g. and a.f. conceived and developed the study, supervised the acquisition of the biological data, analyzed and interpreted the data. j.g. drafted and edited the manuscript. a.f. assisted with drafting and editing the manuscript. s.s. and a.m. helped with patient sample processing, performed experiments and helped to interpret the data. k.j. and c.o. assisted in identifying, consenting, acquiring patient samples. k.j., c.o., and c.m. assisted in collecting clinical information about the patients. all authors edited and approved the final version of this manuscript. drs. grunwell and fitzpatrick received support for research from the national institutes of health. funding was provided by nih grants k hd (atlanta pediatric scholars program), k hl - , and an emory university pediatrics research alliance junior faculty focused pilot award to jg. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.r.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -gzuug p authors: kwiyolecha, elizabeth; groendahl, britta; okamo, bernard; kayange, neema; manyama, festo; kidenya, benson r.; mahamba, dina c.; msanga, delfina r.; gehring, stephan; majigo, mtebe; mshana, stephen e.; mirambo, mariam m. title: patterns of viral pathogens causing upper respiratory tract infections among symptomatic children in mwanza, tanzania date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: gzuug p upper-respiratory tract infections (urti) are the leading causes of childhood morbidities. this study investigated etiologies and patterns of urti among children in mwanza, tanzania. a cross-sectional study involving children was conducted between october- and february- . children with features suggestive of urti such as nasal congestion, dry cough, painful swallowing and nasal discharge with/without fever were enrolled. pathogens were detected from nasopharyngeal and ear-swabs by multiplex-pcr and culture respectively. full blood count and c-reactive protein analysis were also done. the median age was (iqr: – ) months. majority ( . %) had fever and nasal-congestion ( . %). rhinitis ( . %) was the commonest diagnosis followed by pharyngitis ( . %). viruses were isolated in % of children, the commonest being rhinoviruses ( . %). nineteen percent of children had more than viruses; rhinovirus and enterovirus being the commonest combination. the commonest bacteria isolated from ears were staphylococcus aureus and pseudomonas aeruginosa. children with viral pathogens had significantly right shift of lymphocytes ( %—sensitivity). majority ( / ) of children were symptoms free on eighth day. viruses are the commonest cause of urti with rhinitis being the common diagnosis. rapid diagnostic assays for urti pathogens are urgently needed in low-income countries to reduce unnecessary antibiotic prescriptions which is associated with antibiotic resistance. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ in many low-and middle-income countries. in tanzania, there is only one study that has documented the viral etiologies of rti . therefore, there is a paramount need to establish information on the common etiologies of rtis in tanzania, the information that can stimulate further studies and possible control interventions including introduction of cheap and reliable methods to detect these pathogens in clinical settings. in addition due to increased use of antibiotic without a support of a diagnostic test in the treatment of urti as observed in number of previous studies [ ] [ ] [ ] , make the availability of epidemiological data on the patterns of etiology of urti of paramount important. overuse of antibiotics without prescriptions for urti is widespread in developing countries , this is partially contributed by lacking of data on the etiologies of urtis. therefore, these data are relevant to clinicians in developing countries and policy makers in order to invest on the improved diagnostic facilities and reduce antibiotic prescriptions for urtis. study area, design and study population. a cross sectional hospital based study involving children aged - months presenting with rti symptoms was conducted from october to february in the city of mwanza, tanzania. the study was conducted in two health facilities namely: buzuruga health center (bhc), and nyamagana district hospital (ndh). these are public health facilities providing free services to children below years of age. the study included all children presented with nasal congestion or runny nose, hoarseness of voice with dry cough, painful swallowing with tender cervical lymph nodes and enlarged tonsils on examination, ear pain or ear discharge, nasal discharge with or without fever (axilla body temp of . °c and above). in this study we defined: pharyngitis as painful swallowing dry cough, plus or minus hoarseness of the voice (sore throat), tonsillitis as painful swallowing with tender cervical lymph nodes and enlarged tonsils(primarily tonsillar inflammation) and rhinitis as presence of one or more symptoms including sneezing, itching, nasal congestion, and rhinorrhea. sample size estimation and sampling procedures. sample size was calculated using yamane taro ( ) with precision level of %. the minimum sample size estimated was children. however, a total of children were enrolled. all children who met the inclusion criteria were serially enrolled until the desired sample size was attained. data collection and sample collection. sociodemographic and clinical information were collected using pretested structured data collection tool. nasopharyngeal swabs (copan diagnostics inc. usa, canada) were obtained as previously described by inserting the swab into one nostril straight back along the floor of the nasal passage until reaching the nasopharynx. the swabs were rotated gently for - s to loosen the epithelial cells and collect the sample. the swabs were then inserted into viral transport medium and stored at − °c until processing. in children presenting with ear discharge; ear swabs were collected using flexible shaft swab via an auditory speculum in case of inner ear while a sample from outer ear was obtained by firmly rotating swab in outer canal. swabs were immediately taken to the laboratory for bacterial culture and sensitivity. for each consenting participant, about mls of blood was also collected for blood cell counts and quantitative c reactive protein analysis. a thorough general and physical examination was performed to all enrolled children to establish clinical features. all children were managed as per local hospitals protocol. patients were followed two times; the first follow up was on the th day, where these patients were fully examined and their full blood count, crp and ear swab culture results were revealed. in case of positive ear swab culture treatment was changed based on susceptibility patterns. the second follow up was done on the th day as clinical review to evaluate for disease progression. laboratory procedures. ear swab specimens were inoculated onto chocolate agar, blood agar (ba) and macconkey agar (mca) plates and incubated aerobically at °c for - h. in-house biochemical identification tests were used to identify isolated bacteria to their species level . the identified isolates were tested for antimicrobial susceptibility following clsi guidelines, the tested antibiotic discs included: amikacin ( µg), gentamicin ( µg), erythromycin ( µg), vancomycin ( µg), clindamycin ( µg), ciprofloxacin ( µg) which were used for gram positive bacteria and ampicillin ( µg), ceftazidime ( µg), meropenem ( µg), amikacin ( µg), piperacillin-tazobactam ( / µg) and ciprofloxacin ( µg) for gram negative bacteria . bacterial isolates obtained were inoculated into brain heart infusion broth with % glycerine and stored at - °c freezer. e. coli atcc and staphylococcus aureus atcc were used as control strains. nasopharyngeal swabs were transported to mainz university germany and were tested to detect enterovirus (ev), influenza virus type a (iva), influenza virus type b (ivb), respiratory syncytial virus (rsv), parainfluenza virus type (piv ), parainfluenza virus type (piv ), parainfluenza virus type (piv ), parainfluenza virus type (piv ), adenovirus (av), rhinovirus (rv), human metapneumovirus (mpv), coronavirus (cv), bocavirus, mycoplasma pneumoniae (mpn), chlamydophila pneumoniae (cpn), bordetella pertussis (bp), bordetella parapertussis (bpp) and legionella pneumophila (lpn) using multiplex pcr as previously described . blood samples were quantitatively tested for c-reactive protein following manufacturer instructions (medical instruments co., ltd, shanghai, china). blood in edta container (bd vacutainer, nairobi, kenya) was used to estimate complete blood count (fbc) using hematological analyzer (beckman coulter (uk) ltd) . www.nature.com/scientificreports/ statistical data analysis. data entry was done using microsoft excel then exported to stata version for analysis. continuous variables (age and temperature) were summarized using median with interquartile ranges. categorical variables (sex and level of education) were summarized using frequency and proportions. to determine the utility of fbc, and crp in the determination of causative agents among children below years of age, a by table and receiver operating curve (roc) characteristic analysis were used to determine the sensitivity, specificity, positive and negative predictive values. children presenting with symptoms for more than days were classified as having chronic illness. a child who had no any symptom on day eight of follow was declared cure. ethical approval and consent to participate. the approval for conducting the research was sought from the joint cuhas/bmc research ethics and review with ethical clearance number: crec/ / . permission to conduct the study was also sought from the pediatrics departments at bhc and ndh. the aim and importance of the study was explained to parents/caretakers before enrollment of children to the study, followed by a signed informed consent by the parent/caretaker. all information regarding the patient remained confidential. patient's records were kept such that the identity of the patient was not disclosed. for those who refused to participate, were provided with services similar to the participants and had equal chance to treatment regardless of their inclusion status. all methods were carried out in accordance with relevant guidelines and regulations. socio-demographic characteristics of study participants. the www.nature.com/scientificreports/ ing from to days and were classified as acute illness in this study ( table ). the slightly majority ( . %) of children presented with rhinitis ( fig. ) . lymphocytes, neutrophils and crp were used to predict the possible causative agents. children with viral pathogens had significantly elevated lymphocytes, with normal or elevated crp. the sensitivity of elevated lymphocytes in detecting viral pathogens was . % (fig. a,b ). etiologies of urti among under five children in mwanza city. this is the first study to establish etiologies of rtis in the lake victoria zone tanzania. findings from this study shows that, number of viruses are responsible for rtis among children below years of age attending outpatient clinics in the city of mwanza. the prevalence of . % reported in the current study is low compared to a previous study conducted in ifakara and dar es salaam, which reported prevalence of . % . the possible explanation for these differences could be criteria used in the enrollment of the study participants. in the previous study fever was the main inclusion criterion which was not the case in the current study. in addition, this study was conducted in different season of the year (october through february ) compared to the previous which was conducted from april to august and from june to december at two different sites, respectively. viral infections have been found to be influenced by season variations [ ] [ ] [ ] [ ] [ ] . further studies to establish seasonality of these viruses are warranted in developing countries. the findings in this study are comparable to the previous study in kenya among children below years of age, whereby viruses were isolated in % of children with rtis . in comparison to a previous study in refugee www.nature.com/scientificreports/ camp in kenya the prevalence of viral infection reported in this study is low ( % vs. . %) . the possible explanation could be overcrowding conditions in refugee camp which has been found to facilitate transmission of rtis viruses , . regarding distribution of viruses; rhinovirus, adenovirus and parainfluenza were the commonest viral pathogens in the current study which is contrary to a previous study in kenya whereby influenza a virus, respiratory syncytial virus and influenza b were the commonest. the predominance of rhinovirus, influenza viruses was also reported in a previous study in tanzania . the distribution of respiratory viruses is associated with climatic changes; the peak of infection usually occurs in winter period in temperate regions which is equivalent to wet season in hot climates like tanzania , . as documented earlier, a significant proportion of children who had viral infection presented with acute illness . however, it should be noted that some studies have shown that some viral diseases may last for several weeks , which may account for the few children in the present study who presented with chronicity. regarding ear bacterial infections, staphylococcus aureus, pseudomonas aeruginosa, serratia marcescens, klebsiella pneumoniae, providencia spp. and streptococcus spp. were isolated. this is contrary to the previous study whereby streptococcus pneumoniae was the commonest isolate . the possible explanation could be wide coverage of pneumococcal vaccines. decrease in streptococcus pneumoniae infections has been observed after introduction of the vaccine. a previous study by mushi et al. in similar settings established that staphylococcus aureus and pseudomonas aeruginosa were the commonest pathogens causing csom among adult patients. in the current study, only one nasopharyngeal specimens yielded bacterial isolate (bordetella parapertussis) which is contrary to a previous observation by ndossa et al. that detected streptococcus pneumoniae to be colonizing the nasopharynx of children in the city of mwanza. moreover, the bacterial isolates in the current study were highly resistant to the readily available and the over counter antibiotics like amoxicillin, trimethoprim/ sulphamethoxazole and ampicillin with majority being susceptible to ciprofloxacin ear drops. this could be explained by the fact that, ciprofloxacin is not readily used in children below years of age. in the current study, rhinitis was the commonest presenting disease, followed by pharyngitis and pneumonia. this is further supported by findings in the current study which reported rhinovirus to be the commonest pathogen. a previous study , documented rhinovirus to cause up to - % of the upper respiratory tract infections. systemic responses of th for rhinovirus results into stimulation of specific clones of cd t cells and secretions of large amount of granulocytes macrophage colony stimulating factor (gm-csf) which are also responsible for the urti symptoms . moreover, rhinovirus infection has also been associated with lower respiratory tract disease, asthma exacerbations and fatal pneumonia [ ] [ ] [ ] . on the other hand, the spectrum of diseases in this study could be also explained by the commonly isolated viruses; influenza viruses, rsv, parainfluenza viruses and adenoviruses which are the common viruses responsible for most of the upper respiratory diseases as previously reported , . in the current study, children with viral pathogens had a right shift of lymphocytes with an increased sensitivity of . %, with left deviation of the neutrophils or slightly raised crp (not more than mg/dl). these findings are important and could be used to predict these infections in resource limited setting and assist in decision making on the management of the patients which eventually might reduce unnecessary prescriptions of antibiotics. the observation is further supported by the fact that the majority of children were free from the initial symptoms on day eighth, underscoring that viral caused urti have mild symptoms which tend to disappear with time , . study limitations. diagnosis of patterns of urti was done clinically with no imaging to support the diagnosis, this might have caused misclassification. however, efforts were made to minimize this by consulting senior pediatricians whenever overlap of symptoms occurred. another potential limitation was failure to perform multiplex pcr for all ear swabs therefore viral pathogens might have been missed in these samples. urti are common in children below years of age and are predominantly caused by viruses. elevated lymphocytes, normal neutrophils with elevation or normal crp levels can predict viral causes of urti while the raise in neutrophils and crp are more likely to predict bacterial infections. clinicians should suspect urti caused by viral infections whenever the children present with runny nose with congestion, fever and dry cough. the use of antibiotics should be minimized in children with urti symptoms since most of the symptoms disappears within a week. further studies to determine etiologies of lower respiratory tract infections are warranted in this setting. all data are included in the manuscript. raw data is available upon request and the request should be made to the director of research and innovation, catholic university of health and allied sciences. scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ human bocavirus symptomatic treatment of upper respiratory tract symptoms in children beyond malaria-causes of fever in outpatient tanzanian children estimates of world-wide distribution of child deaths from acute respiratory infections antibiotic use in acute upper respiratory tract infections pneumonia case management trials group: effect of pneumonia case management on mortality in neonates, infants, and preschool children: a meta-analysis of community-based trials global estimate of the incidence of clinical pneumonia among children under 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flocked swabs risk factors for acute respiratory tract infections in under-five children in enugu southeast nigeria respiratory tract infections in the tropics seasonal variations of respiratory viruses detected from children with respiratory tract infections in riyadh, saudi arabia epidemiology and seasonality of respiratory tract virus infections in the tropics predictors of disease complications and treatment outcome among patients with chronic suppurative otitis media attending a tertiary hospital factors associated with colonization of streptococcus pneumoniae among under-fives attending clinic in mwanza city rhinovirus: more than just a common cold virus rhinovirus infections in the upper airway an update on the pathophysiology of rhinovirus upper respiratory tract infections adenoviral infections in children: the impact of rapid diagnosis epidemiological analysis of respiratory viral etiology for influenza-like illness during in zhuhai incubation periods of acute respiratory viral infections: a systematic review estimating incubation period distributions with coarse data the authors acknowledge the assistance provided by department of pediatrics mainz university, the department of microbiology and immunology-cuhas-bugando, and the department of pediatrics and child health-cuhas-bugando. this study was supported by the departments of pediatrics-university medical center of the johannes gutenberg university mainz, mainz, germany, department of microbiology and immunology cuhas-bugando and the department of pediatrics and child health of the cuas-bugando. the authors declare no competing interests. correspondence and requests for materials should be addressed to m.m.m.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - zan yf authors: counoupas, claudio; pinto, rachel; nagalingam, gayathri; britton, warwick j.; petrovsky, nikolai; triccas, james a. title: delta inulin-based adjuvants promote the generation of polyfunctional cd (+) t cell responses and protection against mycobacterium tuberculosis infection date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: zan yf there is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as mycobacterium tuberculosis. advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. in this report we determined if advax displays broad applicability against important human pathogens by assessing protective immunity against infection with m. tuberculosis. the fusion protein cysvac , comprising the m. tuberculosis antigens ag b (rv c) and cysd (rv ) formulated with advax provided significant protection in the lungs of m. tuberculosis-infected mice. protection was associated with the generation of cysvac -specific multifunctional cd (+) t cells (ifn-γ(+)tnf(+)il- (+)). addition to advax of the tlr agonist, cpg oligonucleotide (advax(cpg)), improved both the immunogenicity and protective efficacy of cysvac . immunisation with cysvac /advax(cpg) resulted in heightened release of the chemoattractants, cxcl , ccl , and tnf, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of cysvac -specific cd (+) t cells. as delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, cysvac /advax(cpg) is a strong candidate for further preclinical evaluation for progression to human trials. a limited number of adjuvant formulations have been tested in clinical trials of tb subunit vaccine candidates. these adjuvants are typically complex, multicomponent formulations that have been selected for their ability to induce a th response (usually measured as ifn-γ production by antigen-specific t cells) and are similar in their proposed mode of action. for example the immunomodulatory molecule mpl (monophosphoryl lipid a) in as adjuvant or the mpl synthetic analogue glucopyranosyl lipid adjuvant (gla) and the mycobacterial cell wall component trehalose dimycolate (tdm) as part of the caf adjuvant are inflammatory adjuvants that initiate responses through pattern recognition receptors such as toll-like receptor (tlr)- (mpl and derivatives) or macrophage inducible ca + -dependent lectin (mincle) for tdm. oligonucleotide as a component of ic adjuvant acts through tlr to similarly activate nf-kb and resultant pro-inflammatory cytokine release . in the current study the adjuvanticity of a novel polysaccharide adjuvant called advax ™ was assessed in a murine model of virulent m. tuberculosis infection. advax is made up of semicrystalline, delta inulin polysaccharide particles approximately - microns in diameter . advax has been shown in a wide range of animal models to enhance vaccine immunogenicity and protection against pathogens including japanese encephalitis virus , , west nile virus , hepatitis b virus , influenza , hiv , sars coronavirus , listeria monocytogenes , and bacillus anthracis , as well as non-infectious diseases such as alzheimer's disease . in mice advax generates protective immunity with a reduced localized inflammation compared to alum formulations . importantly, advax adjuvant has been shown to be safe and effective in human trials of influenza , hepatitis b and allergy vaccines thereby significantly de-risking use for tb vaccine development. in this study formulations of advax adjuvant alone or combined with cpg oligonucleotide was combined with cysvac fusion protein and assessed for ability to protect against virulent m. tuberculosis infection, together with mechanistic studies on the impact of the advax-containing vaccines on innate and adaptive immune responses. advax-formulated vaccines induce polyfunctional cd + t cells. cysvac , a fusion protein comprising the m. tuberculosis ag b and cysd antigens, was previously found to provide protective immunity against pulmonary m. tuberculosis challenge in mice when formulated with mpl combined with dimethyldioctadecylammonium (dda) . because of its high adjuvanticity, mpl is reported to be highly reactogenic and does not adequately stimulate long-term t cell memory . as concerns of mpl-dda toxicity make the combination unsuitable for human use, there was a necessity to identify an alternative safe and effective human adjuvant for cysvac to advance to human trials. advax adjuvant has proved safe and well tolerated in human influenza vaccines and hence we sought to test its ability to enhance cysvac -induced protective immunity. mice were vaccinated intramuscularly (i.m.) with doses of cysvac /advax or cysvac /advax incorporating cpg (advax cpg ) and immunogenicity was assessed by examining cysvac -specific responses in the peripheral blood mononuclear cells (pbmcs) weeks after the last vaccination. the cysvac /advax cpg group showed a high frequency of triple positive ifn-γ + il- + tnf + and double positive ifn-γ + il- + producing cd + t cells in antigen-stimulated pbmcs (fig. a,b) . by contrast, the levels of these poly-functional t cells induced by cysvac /advax were much lower (fig. b) . examination of the frequency of ifn-γ-secreting cells by elispot demonstrated that both cysvac /advax and cysvac /advax cpg induced comparable ifn-γ responses (fig. c) . no il- a production was detectable from pbmcs from mice vaccinated with either cysvac /advax formulation (data not shown). together, these results show that vaccination with cysvac /advax cpg , and to a lesser extent cysvac /advax, induces a vaccine-specific th -like response. protection afforded by cysvac /advax against aerosol m. tuberculosis infection. considering the th response elicited by the advax-adjuvanted vaccines, we next determined if they could afford protection against low dose aerosol challenge with virulent m. tuberculosis. vaccination of c bl/ mice with bcg, cysvac /advax or cysvac /advax cpg resulted in an approximate log reduction in lung m. tuberculosis cfu counts compared to unvaccinated mice, and this difference was statistically significant ( fig. a ). cysvac / advax cpg induced the greatest level of protection, although this was not significantly greater than that seen in cysvac /advax-vaccinated mice. the protection afforded by cysvac /advax cpg was equivalent to that observed with bcg (fig. ) . the lungs of unvaccinated mice were characterised by large unorganised areas of inflammatory infiltrate mostly composed of cells with large amounts of cytoplasm, most likely macrophages (see supplementary fig. s ). in the lungs of bcg-vaccinated mice there was generally less tissue involvement with smaller lesions of macrophage-like cells and high numbers of lymphocytes. lungs of both cysvac /advax-and cysvac / advac cpg -vaccinated animals demonstrated reduced cellular infiltration with more organisation and were characterised by the presence of higher number of lymphocytes compared to the unvaccinated group. these results indicate that cysvac when formulated with advax adjuvant formulations can reduce pulmonary bacterial load and infection-induced pathology in mice challenged with aerosolised m. tuberculosis. post-challenge immune response induced by advax-formulated vaccines. we next looked for the pattern of post-challenge immune responses that correlated with protection in advax-vaccinated mice, initially by the determination of cytokine-release by cells taken from mice days post-challenge and stimulated ex vivo with cysvac or its individual protein components (ag b or cysd). the highest level of ifn-γ production was observed upon cysvac or ag b re-stimulation of cells from cysvac /advax cpg -vaccinated animals (fig. a) . ifn-γ responses were next highest in cells from cysvac /advax-vaccinated animals, which were higher than the levels for unvaccinated mice (fig. a) , correlating with the general pattern observed in the pre-challenge cytokine profiles (fig. ) . re-stimulation with cysd protein induced lower levels of ifn-γ release, with the highest response measured in the cysvac /advax vaccinated group (fig. a) . while tnf release was low (pg/ml range) in all groups, nevertheless it was increased for cysvac /advax and cysvac /advax cpg -immunised mice (fig. b) . no increase in il- a production was detected for any group (fig. c ). scientific reports | : | doi: . /s - - -y to further characterise immunity post-challenge, the frequencies of antigen-specific, multi-cytokine-secreting cd + t cells were compared between groups. cysvac /advax cpg -vaccinated mice had the highest frequency of multifunctional ifn-γ + il- + tnf + and il + tnf + cd + t cells (fig. d) . a similar profile was observed for cysvac /advax-vaccinated mice, albeit at a lower frequency. this expansion of multi-functional cells was the number of ifn-γ producing cells after cysvac re-stimulation was determined by elispot (c). data (average ± sem) is representative of two independent experiments. statistical significance between the groups was determined by anova (*p < . , **p < . ; **p < . ). advax-formulated vaccines provide protection against aerosol m. tuberculosis infection. c bl/ mice (n = ) were vaccinated with bcg (s.c. × cfu) or times i.m. with μg cysvac formulated in either advax or advax cpg . control mice were left unvaccinated (unv), advax or advax cpg alone. twelve weeks after the first vaccination, the mice were challenged with approximately cfu of m. tuberculosis by aerosol route and the bacterial load was assessed weeks later in the lung. the data is representative of two independent experiments and are presented as log cfu ± sem. statistical significance between the groups was determined by anova (**p < . ; **p < . ). statistically different from that observed in unvaccinated and bcg-vaccinated mice, where the frequency of single-cytokine-secreting cd + t cells, particularly those secreting ifn-γ, was most pronounced (fig. d ). these results demonstrate that cysvac /advax cpg and, to a lesser extent, cysvac /advax elicit the generation of fig. and weeks after the first vaccination mice were challenged with approximately cfu of m. tuberculosis. four weeks after infection splenocytes were re-stimulated with cysvac or its singular components (ag b and cysd) and the levels of ifn-γ (a), tnf (c) or il- (c) production in the supernatants were measured by elisa. splenocytes were re-stimulated in vitro with cysvac in the presence of brefeldin a and the frequency of cysvac -specific cytokine secreting cd + t cells determined by flow cytometry (d). data (average ± sem) is representative of two independent experiments. statistical significance between the groups was determined by anova (*p < . , **p < . ; **p < . ). multifunctional cd + t cells, the frequency of which correlates with the level of protection afforded against m. tuberculosis challenge. we next investigated the possibility that advax adjuvants may potentiate vaccine protection by enhancing the recruitment of immune cells to the site of immunisation. mice were injected i.d. in the ear with advax formulations or a pbs control without antigen to allow quantification of cell recruitment to the site of injection . this route of vaccination was shown to induce similar immunogenicity post-challenge with m. tuberculosis and equivalent level of protection to that previously observed with the i.m. route (see supplementary fig. a ). the leukocyte composition in the skin at the site of injection was determined after days using the gating strategy shown in supplementary fig. s . local accumulation of neutrophils (cd + cd b + ly g + cells, fig. a ,d) and cd + macrophages/monocytes (cd + cd + cd b + ly g − , fig. b ,e) was observed days after injection of advax or advax cpg . advax cpg induced the greatest chemotaxis with ~ -fold higher frequency of neutrophils and macrophages/monocytes compared to advax alone (fig. d,e) . interestingly, most of the increase observed within the cd + population was within the ly c hi subset, which is the inflammatory subset that may differentiate into inflammatory dcs . however, no apparent difference in the frequencies of conventional dcs between groups was observed at the time point examined (cd + ly g − cd c + mhcii hi , fig. c,f) . to investigate what were the possible immune mediators of this immune cells recruitment, we measured the levels of cytokines/chemokines in ear cells cultures. cells from mice vaccinated with advax or advax cpg exhibited higher levels of the neutrophil attractant cxcl compared to pbs-injected mice (fig. g ). cellular recruitment also correlated with higher levels of ccl (fig. h) , tnf (fig. i) , and il- (data not shown), which were more pronounced in the advax cpg -injected mice. by contrast no ccl , ccl , ccl , il- β or il- p were detected. considering the potent ability of advax cpg to recruit immune cell subsets to the injection site, the ability of cysvac /advax cpg to drive the priming of antigen-specific t cells was next assessed. cfse-labelled splenocytes from p -tgtcr mice, whose t cells recognise m. tuberculosis ag b protein, were adoptively transferred into naïve c bl/ mice, which were then vaccinated i.d. with cysvac alone or with advax cpg . at days post-immunisation, cfse-labelled p -tgtcr cd + t cells had started to proliferate in the auricular lymph nodes (aln) in both cysvac and cysvac /advax cpg groups, with proliferation more pronounced in the mice immunised with cysvac /advax cpg (fig. a ). at this early timepoint, the total number of p -tgtcr cd + t cells was significantly greater in cysvac /advax cpg compared to pbs-vaccinated mice (fig. b ). by days post-immunisation, division of cfse-labelled p -tgtcr cd + t cells in the aln started to be seen in the cysvac alone group (fig. a ), but with ~ -fold higher numbers of proliferating p -tgtcr cells in the cysvac / advax cpg group (fig. c) . furthermore, analysis of cytokine release by cd + t cells after ag b - peptide re-stimulation revealed that a high proportion of p -tgtcr cd + t cells from cysvac + advax cpg -vaccinated animals displayed a triple positive phenotype (ifn-γ + il- + tnf + ), followed by double-positive cells producing either ifn-γ + tnf + or il- + tnf + (fig. d ). by contrast, mice vaccinated with cysvac alone, advax cpg alone or pbs exhibited mainly tnf + cd + t cells, although the cysvac alone group also exhibited a significant proportion of double positive il- + tnf + -secreting cd + t cells (fig. d) . thus, advax cpg is a potent chemotactic agent that stimulates the early priming and expansion of antigen-specific cd + t cells at the immunisation site with promotion of multi-potent cd + t cells subsets. the limited number of adjuvants currently licensed for use in human vaccines (e.g. aluminum-based salts and squalene-based emulsions) are relatively poor inducers of th type responses , . consequently, the identification of adjuvants for use in tb vaccines for humans is a major unresolved challenge critical for progression of vaccine candidates . this study assessed the capacity of advax delta inulin-based adjuvants to induce protective cellular immunity against m. tuberculosis infection. advax adjuvants have been used to induce protective immunity against a wide range of pathogens across multiple animal species and, most importantly, have already been shown to be well-tolerated and immunogenic in human subjects [ ] [ ] [ ] . this report demonstrates for the first time that advax adjuvants, when formulated with cysvac fusion protein, confer protection against aerosol challenge with m. tuberculosis (fig. ) . the level of protection induced by advax-adjuvanted vaccines (ranging from . to log cfu reduction compared to unvaccinated mice) is similar to that seen in preclinical studies of other tb vaccine candidates that have entered clinical trials, for example h /caf , id /gla-se or m /as . notably, advax alone, as a single component adjuvant, could provide significant protection against m. tuberculosis, while the adjuvants used above are more complex and require multiple components to achieve a protective effect. the identification of advax as a novel antigen that protects against a broad array of pathogens, including tb, complements the recent report demonstrating that different adjuvants display distinct immunological signatures that is independent of the vaccine antigen . the addition of cpg to advax (advax cpg ) further improved protection, similar to results when cpg had been added to other tb candidates such as id /gla-se . enhanced protection through addition of cpg to advax has previously been seen in vaccines against sars in mice , japanese encephalitis and west nile virus in mice and horses and pandemic avian influenza in ferrets . this suggests the synergistic protection observed in the current study with these two adjuvant components is a widely generalizable phenomenon. based on these beneficial effects, human phase i vaccine trials involving advax cpg adjuvant are currently underway (petrovsky et al., unpublished observations). the mechanism whereby cpg synergises with the delta inulin component of advax is currently under investigation. as shown here, use of advax cpg was particularly associated with an increased frequency of triple-positive ifn-γ + il- + tnf + cells and greater chemotaxis of immune cells to the injection site than advax alone. this suggests that the addition of cpg to advax helps drive greater chemotaxis and a stronger effector memory t cell response. analysis of both the pre and post-infection immune response revealed that cysvac combined with either advax formulations elicited a vaccine-specific th response greater than the one elicited by bcg vaccine (figs and ) . the ability of advax to induce a strong ifn-γ recall response by antigen-specific t cells was also seen in influenza and hepatitis b immunisation models, amongst others. analysis of cytokine secretion showed that advax formulations successfully induced poly-functional cd + t cells; cysvac /advax cpg induced an appreciable level of triple-positive ifn-γ + il- + tnf + cells both pre-and post-challenge, higher than that induced by cysvac /advax, bcg or in unvaccinated mice . levels of poly-functional cd + t cells have been shown to correlate with better protection in numerous models of infection including tb , . in particular, it has been suggested that optimal tb protection can be achieved by the generation of a pool of triple-positive multifunctional t cells that can mediate rapid effector functions . in cysvac /advax cpg vaccinated mice a double positive il- + tnf + cd + t cell subset was observed, which are characteristic of central memory t cells with high ) . the next day mice were injected i.d. in each ear with pbs, advax cpg , cysvac protein alone, or cysvac / advax cpg . p -tgtcr cd + t cells cfse dilution profiles and proliferation index (±sem) were calculated (a). total cell numbers were evaluated by flow cytometry at day (b) or day (c) in the auricular lymph node (aln). cells isolated from the aln were re-stimulated over night in the presence of ag b - /brefeldin a and the frequency of p -tgtcr cd + t cells producing ifn-γ, il- or tnf was determined by intracellular staining and flow cytometry (d). data (average ± sem) is representative of two independent experiments. statistical significance between groups was determined by anova (**p < . ). scientific reports | : | doi: . /s - - -y proliferative capacity that correlate with protective efficacy of tb vaccine candidates in mice . however, in other studies, the presence of polyfunctional t cells in either mva a-vaccinated adults or bcg-vaccinated infants did not correlate with protection against tb in humans, highlighting the incomplete understanding of immune correlates of vaccine-induced tb protection. th responses are thought to contribute to the protection against mycobacterial infection by triggering the expression of chemokines in the lung, which in turn may mediate the recruitment of protective t cells to the airways , , however, advax-adjuvanted vaccine formulations afforded significant protection against infection without inducing detectable levels of il- (fig. ) . notably, il- may be a double-edged sword as excessive il- is associated with heightened inflammation and tissue damage during mycobacterial infection . hence, adjuvants such as advax that primarily induce multifunctional th responses, rather than il- dominated responses, may represent safer candidates for human tb vaccine use. the local response induced by adjuvants at the site of injection represents the first series of events that leads to a protective immune response, and has been characterised for a number of experimental adjuvants including mf , mpl/dda and caf . in this study we showed that injection of advax formulations induced chemoattractants/cytokines, such as cxcl , ccl , il- and tnf, which may be responsible for the observed rapid influx of neutrophils and monocytes/macrophages to the site of vaccination (fig. ) . innate cell populations interact in a very complex microenvironment and as such the distinct roles of each of these populations and their individual contribution to effective vaccine-induced immunity is not completely defined . for example, neutrophils rapidly internalise mycobacteria and can participate in the initiation of adaptive immunity and supress the release of inflammatory cytokines by th cells , . the suppression of neutrophil apoptosis appears to be a strategy used by m. tuberculosis to delay the activation of cd + t cells . adjuvant-induced neutrophils have been shown to regulate the level of antigen presentation by dcs to apcs , , and hence neutrophils attracted to the site of immunization by advax adjuvants may assist in enhancing antigen presentation and local t-cell activation. however, the role of neutrophils in protective immunity to tb is complex as excessive neutrophil infiltration during m. tuberculosis infection of the mouse is associated with increased lung pathology, which is more pronounced in susceptible hosts , . we also observed recruitment of large numbers of monocytes/macrophage (fig. ) , in particular cd + ly c hi monocytes/macrophages, a subset shown to be able to differentiate into dcs and migrate to the draining ln to facilitate antigen presentation . indeed, we observed that a single dose of cysvac when formulated with advax cpg induced significant recruitment and local priming of vaccine-specific cd + t cells, resulting in generation of polyfunctional cd + t cells secreting multiple th effector cytokines in the draining ln (fig. ). this supports a role for advax adjuvants in inducing local injection site chemotactic signals, that recruit antigen presenting cells to the site of immunisation, leading to enhanced antigen presentation and activation and expansion of memory t cells. upon injection advax particles are rapidly endocytosed by dendritic cells (dc), although the specific receptor(s) mediating this process is still unknown (petrovsky et al., unpublished observations) . delta inulin retains its adjuvant action in myd /trif double knockout mice indicating that it does not require tlr signalling for its action (petrovsky et al., unpublished observations) . however, further studies are required to determine if these cell subsets are directly involved in advax uptake and t cell priming; antigen-loaded apcs are refractory to t cell stimulation during mycobacterial infection , while the timing of antigen and adjuvant delivery to apcs is critical for the induction of cd + t cell responses . like neutrophils, monocyte/macrophage accumulation can have a deleterious effect during mycobacterial infection and therefore the level of myeloid cell recruitment induced by vaccine recall responses may be critical for the balance between effective immunity and immunopathology. reassuringly, there was no evidence of any local or systemic toxicity either pre-or post-challenge in mice that received cysvac /advax cpg immunisation, which supports the safety and tolerability data on advax adjuvants seen in recent human trials . in conclusion, the results presented here demonstrate that the advax adjuvant can be incorporated into tb subunit vaccines to confer strong immunogenicity and protection against m. tuberculosis in the mouse model. its effects are further enhanced by the addition of a cpg component which potentiates immune cell recruitment and the subsequent multifunctional effector t cell response. considering the acceptable safety and tolerability profile of advax in humans , the cysvac /advax cpg combination is a strong candidate for further preclinical evaluation and progression to human trials. h rv and m. bovis bcg pasteur were grown at °c in middlebrook h medium (bd) supplemented with . % glycerol, . % tyloxapol, and % albumin-dextrose-catalase (adc) or on solid middlebrook h medium (bd) supplemented with oleic acid-adc. antigens and adjuvants. protein antigens ag b (rv c), cysd (rv ), cysvac were produced in recombinant form from eschericia coli as described previously . antigen purity was > % as assessed by sds page analysis. the absence of contaminant e. coli proteins in the cysvac vaccine was further demonstrated by the lack of cytokine release by cells from cysvac -vaccinated mice after re-stimulation with an irrelevant recombinant mycobacterial protein produced by the same methodology (see supplementary fig. s ). ag b - peptide was synthetised by genescript. advax and advax cpg were provided by vaxine pty ltd (adelaide, south australia). vaccination and infection of mice. female c bl/ ( - weeks of age) were purchased from the animal resources centre (perth, australia). mice were maintained in specific pathogen-free condition and experiments were performed with the approval of the sydney local health district animal welfare committee (approval number / c) in accordance with relevant guidelines and regulations. animals were randomly assigned to experimental groups. for protection experiments, mice were vaccinated subcutaneously (s.c.) at the base of the tail either once with × cfu of bcg pasteur ( µl in pbs), or i.m. times at weeks interval with µg of recombinant protein formulated in advax or advax cpg ( mg delta inulin per dose with or without μg of mer type b oligonucleotide containing cpg-motif, μl in each thigh). for intradermal (i.d) experiments, mice were anaesthetised by intraperiteneal injection with ketamine/ xylazine ( / μg/kg). four microliters of protein and/or adjuvants ( μg and/or μg, respectively), adjuvant alone or pbs were injected i.d. into each ear under a surgical leica m microscope (leica, wetzlar, germany) using an ultrafine syringe ( g, bd biosciences) as described by lin et al. . for m. tuberculosis challenge experiments, six weeks after the final vaccination mice were infected with m. tuberculosis h rv via the aerosol route using a middlebrook airborne infection apparatus (glas-col) with an infective dose of approximately viable bacilli. four weeks later the lung and spleen were harvested, homogenized and plated after serial dilution on supplemented middlebrook h agar plates. colonies forming units (cfu) were determined approximately weeks later and expressed as log cfu. histology. for histological analysis, the middle right lobe of each infected mouse was perfused with a % buffered formalin solution. tissue samples were embedded in paraffin, and μm thickness tissue sections were cut and stained with hematoxylin and eosin (h&e). slides were observed with leicadm microscope (leica microsystems, north ryde, australia) with a magnification of x or x and acquired as a mosaic. assays of cytokine production. pbmcs were isolated by gradient centrifugation of approximately μl of blood per mouse on histopaque (sigma) according to manufacturer's instructions. splenocytes and auricular lymph nodes (mln) were prepared from vaccinated or infected mice by passage through a cell strainer (bd). dorsal and ventral pinnae were separated using tweezers, and cells from the ears were dissociated with collagenase i ( . mg ml − ; worthington, lakewood, nj) and dnase ( u ml − ; worthington) and then passaged through a cell strainer. cells were resuspended in buffered ammonium sulfate (ack buffer; . mm edta (sigma), mm khco (sigma), mm nh cl (sigma) to lyse erythrocytes and then washed and resuspended in rpmi (life technologies) supplemented with % heat-inactivated fetal bovine serum (scientifix, cheltenham, australia), μm -mercaptoethanol (sigma), and u ml − penicillin/streptomycin (sigma). antigen specific ifn-γ producing cells were detected by elispot assay as described previously . all antigens were used at a concentration of µg ml − . for cytokine elisas, cells were stimulated with antigens and supernatants collected after hours, and ifn-γ, tnf and il- were detected as described previously . for cytokine assessment after i.d. vaccination, ear cell suspensions were cultured - hours in complete rpmi at × cells ml − . supernatants were frozen at − until use, and cytokine concentrations (cxcl , ccl , ccl , ccl , il- b, il- , il- p , tnf) were determined using cytokine bead array (bd) following manufacturer's instructions. the data was acquired on a bd lsr-fortessa flow cytometer (bd) and then analyzed using the fcap array software (bd, usa). for adoptive transfer studies, p transgenic tcr (p -tgtcr) mice (expressing the tcr specific for residues - of the m. tuberculosis ag b protein) were bred in house under specific pathogen free conditions. splenocytes were prepared and labelled with cfse as described . c bl/ mice (cd . ) received i.v. × cfse labelled p -tgtcr splenocytes (cd . ) and the next day were immunized i.d. as described previously. at selected timepoints ears or lymph nodes were harvested and single cell suspensions prepared and stained for flow cytometry (see below). intracellular cytokine staining and flow cytometry. for intracellular cytokine staining, cells were stimulated for - hours in the presence of the cysvac fusion protein ( µg ml − ) and then for up to hours with brefeldin a ( µg ml − ). two million cells were incubated with . μg ml − anti-cd /cd (ebioscience, san diego, ca) in facs wash buffer (pbs/ % fcs/ . %) for min to block fc receptors, then washed and incubated for min with either anti-cd -percpcy . (clone - c ), anti-cd -alexafluor (clone rm - ), anti-cd a-allophycocyanin (apc)-cy (clone - . ), or anti-cd -fluorescein isothiocyanate (fitc) (clone im , bd). fixable blue dead cell stain (life technologies) was added to allow dead cell discrimination. cells were then fixed and permeabilized using the bd cytofix/cytoperm tm kit according to the manufacturer's protocol. intracellular staining was performed using the following antibodies: anti-ifn-γ-phycoerythrin (pe)-cy (clone xmg . all samples were acquired on a bd lsr-fortessa flow cytometer (bd), and analyzed using flowjo tm analysis software (treestar, macintosh version . , ashland, or). a boolean combination of gates was used to calculate the frequency of single-, double-and triple-positive cd + cd + cell subsets. the pbmc gating strategy for intracellular cytokine staining is described in ref. . statistical analysis. the significance of differences between experimental groups was evaluated by one-or two-way analysis of variance (anova), with pairwise comparison of multi-grouped data sets achieved using tukey or dunnet post hoc test. global tuberculosis report progress in tuberculosis vaccine development and host-directed therapies-a state of the art review. the lancet towards an understanding of the adjuvant action of aluminium adjuvants and antibody production: dispelling the myths associated with freund's complete and other adjuvants safety and immunogenicity of the m /as candidate tuberculosis vaccine in hiv-infected adults on combination antiretroviral therapy: a phase i/ii, randomized trial the importance of adjuvant formulation in the development of a tuberculosis vaccine cationic liposomes formulated with synthetic mycobacterial cordfactor (caf ): a versatile adjuvant for vaccines with different immunological requirements ag b-esat- adjuvanted with ic promotes strong and long-lived mycobacterium tuberculosis specific t cell responses in naive human volunteers delta inulin: a novel, immunologically 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differential priming of cd t cells in response to the bacille calmette guerin vaccine or mycobacterium tuberculosis infection this work was supported by a national health and medical research council (nhmrc) project grant (app ) and the nhmrc centre of research excellence in tuberculosis control (app ). we acknowledge the support of the european h grant tbvac . np is supported by national institutes of health contract hhsn c and development of advax adjuvant was supported by nih contracts ai and hhsn c. this publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of the national institutes of health, national institute of allergy and infectious diseases. c.c., n.p., and j.t. conceived and designed the study. c.c., r.p., g. n. performed the experiments. all authors processed and analysed the data. c.c., n.p., and j.t. wrote the manuscript. all authors read and approved the final manuscript. supplementary information accompanies this paper at doi: . /s - - -y competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - gvay i authors: hsieh, yu-chia; tsao, kuo-chien; huang, ching-tai; chang, kuang-yi; huang, yhu-chering; gong, yu-nong title: clinical characteristics of patients with laboratory-confirmed influenza a(h n )pdm during the / and / clade b/ b. / b. -predominant outbreaks date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: gvay i a novel pandemic influenza a(h n )pdm virus emerged in globally, and it continues to circulate in humans. the national influenza surveillance network in taiwan identified five a(h n )pdm -predominant seasons, representing the / , / , / , / , and / outbreaks from to . independently, a retrospective cohort study (which enrolled infected patients during the five seasons) was conducted at chang gung memorial hospital to explore the risk factors associated with influenza a(h n )pdm -related complications. a phylogenetic analysis of hemagglutinin (ha) sequences showed that the circulating a(h n )pdm virus belonged to clades , , and in / ; clades , , , and in / ; clades and c in / ; clades b in / ; and b/ b. / b. in / . compared to individuals infected in non- b/ b. / b. seasons ( / , / , and / ), those infected in b/ b. / b. seasons ( / and / ) were at higher risk for influenza-related complications (adjusted odds ratio [aor]: . , % confidence interval [ci]: . – . ), pneumonia (aor: . , % ci: . – . ), mechanical ventilation (aor: . , % ci: . – . ), and acute respiratory distress syndrome (aor: . , % ci: . – . ). for the increased severity of infection during the influenza a(h n )pdm clade b/ b. / b. seasons, aspects related to the antigenic change of a(h n )pdm virus, immune response of the host, and environmental factors required further investigation. scientific reports | ( ) : | doi: . /s - - - number of severe cases and outcomes in the groups at risk and healthy young adults; these events were associated with a(h n )pdm clade b. infection , . since , the taiwan centers for disease control (cdc) has established a nationwide surveillance system requiring contract virologic laboratories to perform continuous virologic surveillance for respiratory viruses, particularly influenza and enteroviruses; this system was established after an epidemic of enterovirus in . the long-term national influenza surveillance network described the epidemiologic pattern of circulating viruses, and it has successfully identified the outbreaks of severe acute respiratory syndrome (sars)-associated coronavirus and adenovirus , . moreover, it also identified the novel h n and h n influenza viruses were significantly different between each season (table ) . a phylogenetic analysis of hemagglutinin (ha) sequences recovered in these epidemics, along with geographically diverse global influenza a(h n ) pdm viral sequences, has revealed that the sequences are members of clades , seasons versus non-clades b/ b. / b. seasons, the patients were classified into two groups. the median (interquartile range, iqr) age of patients in the b/ b. / b. season was older than that in the non b/ b. / b. seasons ( table ). the number of infected individuals aged - years was higher in b/ b. / b. seasons than that in the non-clade b/ b. / b. seasons ( table ). the rate of underlying conditions; complications, including pneumonia and acute respiratory distress syndrome (ards); icu admission; respiratory failure with mechanical ventilation; -day mortality; and in-hospital mortality in b/ b. / b. seasons were significantly higher than that in non-clade b/ b. / b. seasons ( table ). the rate of underlying conditions; complications, such as ards; icu admission; and respiratory failure with mechanical ventilation in b/ b. / b. seasons was significantly higher than that in non-clade b/ b. / b. seasons (table ) . the results of the logistic regression analysis on the risk factors associated with influenza a(h n )pdm -related complications and pneumonia are shown in table , and respiratory failure with mechanical ventilation and ards are also presented in table . in the univariate analysis, b/ b. / b. season, age ( - years), onset to presentation, underlying conditions, obesity, smoking, alcoholism, and antiviral therapy were significant risk factors of complications, pneumonia, mechanical ventilation, and ards (tables and ). in the multivariate logistic regression analysis, b/ b. / b. season, age ( - years and ≥ years), underlying conditions, and antiviral therapy were significant independent risk factors of complications, pneumonia, and mechanical ventilation (tables and ). only b/ b. / b. season and obesity were considered as significant independent risk factors of ards ( table ). the effect of b/ b. / b. season on the total number of influenza-related complications was not significant in children aged ≤ years. however, it was significantly stronger among individuals aged ≥ years (table s ). among the hospitalized patients with laboratory-confirmed influenza a(h n )pdm infection, male patients and those with underlying conditions were significantly at risk for -day mortality (overall death within the first days after hospital admission) and all-cause in-hospital mortality (overall death during hospital admission) as assessed using the multivariable cox proportional hazard model ( table ). the same analysis showed that season was not associated with an increased risk for -day and all-cause in-hospital mortality (table ). the during the / season, an unusually high hospitalization rate in adults aged - years was observed in the united states and mexico , . the increased morbidity in middle-aged adults during the / season had been attributed to the low vaccination rate in this age group . however, that hypothesis cannot explain the unusual number of severe cases because the vaccination rate had already been low during the previous years , . an interesting study has shown that up to % of middle-aged adults born between and , who had been exposed to seasonal h n viruses circulating in , had reduced serologic reactivity with the / a(h n )pdm ; notably, the / virus harbors the distinctive k q ha antigenic mutation . in the cohort of individuals born between and , ha-specific antibodies with activity against a(h n )pdm must have been produced and shaped by exposure to prior-season h n viruses (the so called "original antigenic sin") . nonetheless, the ha-specific antibodies in this cohort failed to recognize the / a(h n ) . compared to the a/california/ / vaccine virus, viruses of clade b harbor d n, k q, s t, k e, and a t substitutions in ha . viruses of subclade b. harbor further amino acid substitutions s n, s n, and i t and b. and carry amino acid substitutions v t and v i . more extensive studies must be conducted to identify the potential antigenic differences between clade b and subclade b. / b. ; such studies are expected to improve our understanding of how a(h n )pdm evolved (and continues to evolve) and how it affects and interacts with the human immune system. in the / season, the who has selected a new vaccine virus, which is the a/michigan/ / (h n )pdm -like virus (a member of the b. subclade), as the influenza vaccine virus component for the northern hemisphere. the national influenza surveillance network coordinated by the taiwan cdc was established more than years ago. policies favoring government funding for vaccines and antiviral agents have been consistent during the subsequent intervals. between and , government-funded vaccines have been administered primarily to those aged months to years, elderly individuals aged ≥ years, healthcare workers, and individuals with underlying diseases. individuals aged - years were not included in the government-funded vaccination program. elementary school children aged - years had the highest influenza vaccination rate, with coverage reaching - % annually . the present study is limited by its observational nature and the incorporation of a retrospective investigation. a potential bias may exist due to the exclusion of all cases with a(h n )pdm infection for years. nonetheless, no change was observed in terms of admission or management procedures during these outbreaks. the surveillance and reporting system in taiwan has long been established. taken together, the increased frequency of complications in / and / is unlikely due to detection bias. in addition, the major drawback of this study was the lack of documentation about the history of influenza vaccination in the records used to generate this study. however, a study by taiwan cdc has reported that % of patients with complications in the / season had not received the influenza vaccine . the vaccine coverage rate in non-elderly adults and elderly individuals would have been low during each of the outbreaks, particularly during the first wave, given that no a(h n )pdm vaccine was available in / . thus, the increased severity of influenza during the / and / seasons is unlikely to reflect a decreased rate of vaccination. the study has shown that taiwan experienced the greatest burden of influenza-related complications due to a(h n )pdm clades b/ b. / b. in the sixth year of its circulation. the reasons for the increased impact of influenza-related complications remain uncertain. aspects related to the antigenic change of a(h n )pdm virus, immune response of the host, and environmental factors required further investigation. this report shows the importance of influenza disease surveillance and requires that the influenza a(h n )pdm virus should always be considered. national influenza surveillance network. the network consists of eight regional commissioned laboratories located in the northern (n = ), central (n = ), southern (n = ), and eastern (n = ) parts of taiwan. these laboratories have steadily collected more than respiratory specimens for surveillance per year, including more than influenza virus specimens annually, all of which are sent to the taiwan cdc for the monitoring of influenza viral activity. the taiwan influenza express, a weekly online influenza surveillance report, has been published by the taiwan cdc from july to may of each year since (http://www.cdc.gov.tw/english/ submenu.aspx?treeid = ed d c bb &nowtreeid = f e d fd a) , . this report includes the total number of respiratory specimens; isolate number of influenza a(h n ), influenza a(h n ), and influenza b; and case number of laboratory-confirmed influenza cases in intensive care units (icus), a class of events that is considered a category nationally notifiable disease. however, data on weeks - are not available annually. a confirmed case involved a patient who had acute influenza-like illness (temperature ≥ °c with either cough or sore throat) and nasopharyngeal/throat or bronchoalveolar lavage samples harboring influenza a(h n )pdm virus as detected using real-time (rt) reverse-transcription polymerase chain reaction (pcr) assay or via viral culture , . for the purposes of the present study, each season was defined as extending from july of the same year to may of the following year. the annual population figures provided by the department of household registration affairs of the interior ministry were used for the calculation of the incidence of laboratory-confirmed influenza a(h n )pdm cases in the icu. , a -bed, university-affiliated teaching hospital that is located in northern taiwan and provides both primary and tertiary care. in addition, cgmh is one of the regional commissioned laboratories of the taiwan cdc. patients who had acute influenza-like illness (temperature ≥ °c with either cough or sore throat) and had influenza a(h n )pdm virus as detected using rt-pcr assay or via viral culture using respiratory specimens were included in the study. patients whose data are not available were excluded. the institutional review board of cgmht approved the study, and it was carried out in accordance with the relevant guidelines and regulations. informed consent was waived due to the study's retrospective nature. all medical records of the enrolled patients were reviewed. demographic characteristics, underlying medical conditions, clinical course, antiviral treatment (oseltamivir or zanamivir), mechanical ventilation, admission to an icu, and death were recorded using a structured questionnaire. body mass index (bmi), a measure of obesity, was calculated for patients whose height and weight data were available. obesity was defined as follows: ) body weight ≥ th percentile in children < years of age; ) bmi ≥ kg/m in patients aged between and years; and ) bmi > kg/m (chinese criteria) in patients > years . medical conditions associated with a high risk for influenza complications were defined based on those listed by the united states advisory committee on immunization practices . patients with confirmed pneumonia on radiography, acute respiratory distress syndrome (ards), acute onset of cardiovascular, neurologic condition, respiratory failure with mechanical ventilation; those who were admitted in the icu; and those who died were considered to have influenza-related complications. pneumonia on radiography was diagnosed based on the presence of a consolidation, infiltrate, or opacity . ards was defined according to the standard criteria . the primary study outcome was the occurrence of (any) influenza-related complications. the secondary study outcomes were pneumonia, mechanical ventilation, ards, -day mortality, and in-hospital mortality. genetic characterization of the virus. a total of isolated influenza a(h n )pdm virus were randomly selected for the analysis of viral hemagglutinin (ha) and neuraminidase (na) genes across the five seasons. the rna was extracted using the qiaamp viral rna mini kit (qigen, germany) according to the manufacturer's instructions. rt-pcr and primer pairs used for sequencing ha and na genes were performed, as previously described . sanger sequencing of the viral ha and na genes was performed to establish clade designation and to detect differences in amino acid . the obtained amplicons were assembled into a full-length , -bp span for ha and -bp for na using dnastar lasergene (dnastar, madison, wi). newly reported sequences in this study were deposited at the genbank database under the accession numbers shown in fig. s for ha and na genes. the evolution history was inferred by the maximum likelihood method based on the hasegawa-kishino-yano model . the percentages of replicate trees ( , replicates) are shown next to the branches in which the associated taxa clustered together in the bootstrap test. phylogenetic analysis in this study was conducted using mega . statistical analysis. continuous variables were presented as medians and interquartile ranges (iqrs); categorical variables were presented as numbers and percentages. all analyses were performed using the statistical package for the social sciences software package version . (spss inc., chicago, il, the usa). the incidence rate ratio (irr) was generated using poisson regression with % confidence intervals to compare the rates of laboratory-confirmed influenza a(h n )pdm cases in the icu per , populations across different seasons; % confidence intervals for which the upper and lower bounds did not include were considered as statistically significant. differences in categorical variables were compared using the chi-square test or a fisher's exact test. continuous variables were compared using the kruskal-wallis one-way analysis of variance test. multivariate logistic regression analysis and multivariate cox proportional hazards model were used for outcome analysis. the variables included sex, season, age group, onset to presentation, underlying condition, obesity, smoking, alcoholism, and antiviral therapy. variables with a p value < . in the univariate analysis were included in the multivariate model. the hosmer-lemeshow goodness-of-fit test was performed 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definitions, mechanisms, relevant outcomes, and clinical trial coordination amino acids transitioning of h n pdm in taiwan from dating of the human-ape splitting by a molecular clock of mitochondrial dna molecular evolutionary genetics analysis version . for bigger datasets this work was supported by a grant from national science council, taiwan, and three grants (grant cmrpg f and cmrpg f to yc hsieh, most - -b- a- -my ,nmrpg g to kc tsao) from the chang gung memorial hospital. y.c.h., c.t.h., y.c.h., and t.y.l. designed the study. k.c.t. and y.n.g. conducted the virologic characterization. y.c.h. and k.y.c. performed the statistical analysis. h.y.l. collected data. y.c.h. wrote the first draft of the manuscript, and all authors contributed to the final draft. all authors contributed to data interpretation and critically reviewed the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -n iewmx authors: li, chunmei; teng, xin; qi, yifei; tang, bo; shi, hailing; ma, xiaomin; lai, luhua title: conformational flexibility of a short loop near the active site of the sars- clpro is essential to maintain catalytic activity date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: n iewmx the sars c-like proteinase (sars- clpro), which is the main proteinase of the sars coronavirus, is essential to the virus life cycle. this enzyme has been shown to be active as a dimer in which only one protomer is active. however, it remains unknown how the dimer structure maintains an active monomer conformation. it has been observed that the ser -leu loop forms a short ( )-helix that disrupts the catalytic machinery in the inactive monomer structure. we have tried to disrupt this helical conformation by mutating l to t in the stable inactive monomer g a/r a/q a. the resulting tetra-mutant g a/l t/r a/q a is indeed enzymatically active as a monomer. molecular dynamics simulations revealed that the l t mutation disrupts the ( )-helix and helps to stabilize the active conformation. the coil- ( )-helix conformational transition of the ser -leu loop serves as an enzyme activity switch. our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations. the first outbreak of severe acute respiratory syndrome (sars) occurred over years ago in . this outbreak has led to significant attention and intensive study of the treatment and prevention of coronaviruses over the past decade. recently, a new coronavirus called the middle east respiratory syndrome coronavirus (mers-cov) has emerged. mers-cov first appeared in saudi arabia in and quickly spread into europe. to date, approximately - out of every patients who have been diagnosed with mers have died . although significant advances in the understanding of coronaviruses have been made over the past decade through the study of sars-cov, more research is needed to develop effective countermeasures, such as drugs or vaccines, to control the new pathogen. the c-like proteinase ( clpro) has received much attention as a potential key anti-cov target . like other known cov- clpro structures, such as tgev, hcov- e, hcov-hku , and ibv - , sars- clpro has a highly conserved three-dimensional structure, dimer interface, catalysis dyad, and substrate binding site, but an extremely low homology with cellular proteases. sars- clpro forms a homodimer in crystal structures , that consists of two protomers oriented almost perpendicularly. each protomer of the sars- clpro contains three domains. domains i (residues - ) and ii (residues - ) have trypsin-like folds and control catalysis. domain iii (residues - ) is an extra domain with five α -helices that is related to enzyme dimerization and activity regulation . the substrate binding site is located in the cleft between domains i and ii, which is composed of six subsites (s -s ) corresponding to the p -p peptides of the substrate peptide. s is the most important subsite, because residue gln-p of this subsite is conserved in all coronaviruses. sars- clpro exists in a monomer-dimer equilibrium in solution, but only the dimer is active . the n-finger α -helix a' of domain i (residues - ) and helix domain iii have been recognized as the main components of the dimer formation. changes in these components are known to disrupt the monomer-dimer equilibrium. for example, the proteinase loses its activity and remains monomeric in solution when the n-finger is deleted . mutations of either gly or arg to ala also lead to inactive monomers , . the g a mutation may shorten the α -helix a' of domain i, which in turn may disrupt the n-finger conformation, preventing the n-finger from correctly squeezing into the pocket of another monomer during dimerization and leading to the monomer structure . similarly, the r a mutation may trigger the switch from a dimer to a monomer by releasing the constraints between the n-finger and c terminus and destroying their precise positioning and orientation . ser and phe are two key residues that not only contribute to interactions between the two protomers in the parent dimer but also maintain the correct conformation of the s subsite in the substrate-binding pocket. although ser and phe are adjacent, their mutations cause different conformational changes in the crystal structures . the s a mutation forms monomers in the crystal structure but maintains partial activity in solution due to the existence of a small fraction of dimers in solution. in contrast, the f a mutation forms dimers with a highly collapsed substrate-binding pocket and is totally inactive. the active site mutation, c a, loses hydrolysis activity completely but remains a dimer . other residue mutations, which are neither on the dimer interface nor key to catalysis, can also influence enzyme activity and dimer association-dissociation of sars- clpro via long-range interactions , . all current experimental and computational evidence supports the hypothesis that the dimer structure is necessary for sars- clpro activity . meanwhile, all mutants that disrupt the dimer structure are inactive [ ] [ ] [ ] [ ] . however, the conformational characteristics in the active protomer that are essential to catalysis remain to be determined. to the best of our knowledge, no active monomer mutant has been reported previously for sars- clpro or the main proteinases of other covs. in the present study, we successfully designed an active monomer of sars- clpro by comparing the active and inactive structures of the proteinase. we studied the mechanism that regulates this active monomer using mutational and enzymatic studies, as well as molecular dynamics simulations. active monomer design. by comparing the active protomer (protomer a) of the wild-type sars- clpro with both the inactive protomer (protomer b) of the wild-type sars- clpro (pdb code uk ) and the inactive monomer r a (pdb code qcy) , we found that the most distinguishable differences between the active and inactive structures were in the conformational changes of residues ser -phe -leu . ser -phe -leu is the part of the oxyanion loop that participates in maintaining the correct conformation of the s subsite in the active enzyme. in contrast, in inactive enzymes, this short peptide forms a short -helix that twists the oxyanion hole of the s subsite ( fig. ). this same conformational change is also observed in the collapsed clpro of avian infectious bronchitis virus (ibv- clpro) . formation of a -helix in inactive structures implies that disruption of this -helix will make the structure more flexible, and may produce an active monomer. among the three residues in the -helix, leu is a strong helix former , . we hypothesized that changing leu to another residue with a lower helical tendency and strong β -structure tendency may destroy the -helix. in the present work, we mutated leu to thr , a residue with strong β -structure and low helix tendency. simultaneously, we mutated gly , arg , and gln , which are residues at the dimer interface, to ala , , to generate a stable monomeric protein. the triple-mutant g a/r a/q a (grq) was confirmed to be a monomer, and the tetra-mutant g a/l t/r a/ q a (glrq) was a monomer with enzyme activity as designed. enzyme activity of glrq. both grq and glrq were incubated with a pna substrate (thr-ser-ala-val-leu-gln-pna) at a concentration of μ m at °c. grq exhibited no enzyme activity. after introducing l t, however, glrq was shown to have regained hydrolytic activity (fig. ) . to determine the amount of activity recovered in glrq, the reaction rates of equal amounts of glrq and wild-type clpro (as a control) were measured (fig. s ). compared with clpro, glrq regained % of proteolysis activity. we derived k cat and k m of glrq using the lineweaver-burk plot with a fixed enzyme concentration and a series of substrate concentrations. k m of glrq was . ± . mm and k cat was . ± . min − . in comparison, k m is . ± . mm and k cat is . ± . min − in the wild-type enzyme . thus, glrq and clpro have similar binding affinity, whereas catalytic activity is times lower and k cat /k m is times lower in glrq compared with the wild-type enzyme. this finding demonstrates that introducing thr at position to disrupt the -helix can indeed recover proteolysis activity in glrq. oligomeric state of glrq. analytical gel filtration and sedimentation velocity methods were used to check whether both the grq and glrq mutants maintain monomer structure. the same concentration of wild-type clpro served as a control. the superdex hr / gl column (ge healthcare, pittsburgh, pa, usa) was used for gel filtration analysis. a standard calibration curve was fitted according to the retention volume of known markers (supplemental table s , fig. s ). grq and glrq appeared as a single peak, and the maximum retention volume did not change with the concentration, indicating that only one species was present in solution. for clpro, the peak shifted to the high molecular weight side as the concentration increased, indicating the existence of subunit exchange (fig. ) . the retention volumes for grq and glrq were all approximately ml at both high and low concentrations, corresponding to the molecular weight of a monomer (table s ) . a sedimentation velocity experiment was conducted at °c in pbs with mg/ml concentrations for clpro, grq, and glrq to further verify the oligomeric state of the glrq mutant. a single peak was observed for both grq and glrq at approximately . s (fig. ) , which corresponds to a monomer . two peaks were observed at the monomer and dimer positions, respectively, consistent with previous reports , . previous studies have shown that dimer formation can be induced or enhanced by either substrate or substrate-analogue ( f) binding , . f has a similar binding pattern to the enzyme as the substrate, and was used as a probe to test whether the active pocket of the sars- clpro adopts the correct conformation. under our experimental condition, . μ m glrq was totally inhibited by μ m f (supplemental fig. s ), which implies that the substrate binding pocket of the glrq mutant maintains the active conformation. in addition, when μ m f was incubated with μ m glrq in pbs and analysed using the sedimentation velocity experiment described above, no peaks were induced except for the monomer peak (supplemental fig. s ). this finding further supports that the glrq mutant is monomeric in solution and does not dimerize in the presence of the substrate or its analogue. exists as a monomer-dimer equilibration in solution, and the dimer is the active form of the proteinase. its enzymatic-specific activity, k cat /k m , increases with an increase in enzyme concentration , , , which provides evidence that further dimerization can be induced by the substrate during hydrolysis. furthermore, a previous study has shown upward curvature in the reaction rate versus enzyme concentration plots for clpro . we . clpro exhibits both a monomer and dimer peak at mg/ml, but grq and glrq exhibit only a monomer peak at the same concentration. measured the enzyme activity of glrq at different enzyme concentrations, and found that the velocity increases in a linear manner with an increase in enzyme concentration, which is in contrast to the activity of the wild-type enzyme (fig. a) . although glrq hydrolyses the colorimetric pna substrate with a lower efficiency than the wild-type proteinase, the reaction rate increases proportionally with a positive slope that goes through the origin; i.e., k cat /k m did not change with enzyme concentration (fig. b) . this result indicates that the enzyme is active as a monomer with no occurrence of substrate-induced dimerization. to understand why glrq retains enzymatic activity with a monomeric structure, we carried out molecular dynamics simulations and analysed the structural changes of the catalytic machinery. four simulation systems were constructed (table ) : the wild type (wt, pdb id uk ), the inactive monomer r a (monomer, pdb id qcy), the glrq mutant structure built from the inactive monomer (mutantm), and the glrq mutant structure built from the active monomer of the wild-type structure (mutantw). we used several criteria to quantify the catalytic machinery of the enzyme, as in previous studies on clpro , . the catalytic dyad. his and cys are two residues of clpro that are directly involved in catalysis . the distance between his and cys is critical for the maintenance of their hydrogen bond, which is one stabilizing factor of the catalytic site. we monitored the formation of hydrogen bonds between these two residues in the trajectories ( table ). the percentages of hydrogen bond formation in the mutantm and mutantw systems were . % and . %, respectively. these percentages were larger than that observed in the monomer system ( . %) but smaller than those observed in the wt system ( . % and . % for the first and second chains, respectively). the y-x-h motif. the y-x-h motif contains a hydrogen bond between the oh atom of tyr and the nd atom of his . this motif stabilizes the p substrate binding site and is conserved in the main protease of coronaviruses . the percentages of hydrogen bond formation in the mutantm ( . %) and mutantw ( . %) systems fall between those observed for the wt ( . % and . % for the first and second chains, respectively) and the monomer ( . %) systems (table ) . hydrophobic packing of his and phe . the hydrophobic packing of his and phe is another stabilizing factor of the substrate p binding site of clpro. we analysed the packing between these two residues using two measurements: the distance and the cosine of the dihedral angle between the imidazole ring of his and the phenyl ring of phe (fig. ). in the monomer system, the distance between his and phe is approximately Å. in the first chain of the wt system (wt chain a), the side chains of his and phe are well packed. the distance is approximately Å and the cosine is close to − , which means that the rings adopt a face-to-face conformation. these two conformations observed in the monomer system and wt chain a represent the inactive and active state of the catalytic site, respectively. in the second chain of the wt system (wt chain b), the distance is approximately Å and the cosine is shifted to . the distribution in mutantw is between the distributions in wt chains a and b, with two minima at cosine values of − and , respectively. although the mutantm system originates from the monomer system, the mutantm system has a local minimum at a distance of Å and a cosine of . this minimum may represent an intermediate state of the inactive-active transition. these results indicate that the conformations of his and phe in the mutants fall between the active and inactive state. secondary structure of ser -phe -leu/thr . in the inactive structure of the enzyme, the catalytic site collapses due to the formation of a -helix at the ser -phe -leu residues . to determine whether the l t mutation disrupts this helix, we compared the backbone dihedrals (φ , ψ ) of these residues (fig. ) . in the monomer system, the -helix was well preserved. in wt chain a, however, all residues had non-helical conformations. in wt chain b, l had a stable conformation in the helical region. the conformational distributions of wt chain a and wt chain b confirm results from previous studies, which have reported that only one protomer in the dimer is active. in the mutantm system, the helical conformations of s and t were partially disrupted, showing a small population in the β -region around (φ , ψ ) = (− , ). in the mutantw system, which originated from the non-helical conformation, a sub-stable helical conformation of residue t was observed. these results suggest that the l t mutation partially disrupts the -helix. taken together, the simulation results show that in glrq, the mutation of leu to thr partially restores the correct catalytic conformation as designed. previous studies using molecular dynamics simulations and hybrid protein experiments have shown that only one protomer of the clpro dimer is active . the reason why nature uses this dimer-monomer combination of activity regulation is unknown but has received much attention. in the present study, we successfully turned sars- clpro into an active monomeric enzyme with four mutations: g a, l t, r a, and q a. three of the mutations, g a, r a, and q a, were used to maintain a stable monomeric structure. the fourth mutation, l t, was used to disrupt the -helix structure that is formed by the ser -leu tri-peptide in the inactive monomer and restore the active conformation. the active monomer of the glrq mutant maintains a stable monomer structure as shown by analytical gel filtration and analytical ultracentrifugation analysis. glrq maintains its monomer structure in the presence of a substrate analogue. in contrast to the wild-type enzyme, k cat /k m of glrq did not change with changes in enzyme concentration. these experimental results verify that glrq is an active monomer. these results also confirm that the flexibility of the ser -leu loop is essential for clpro enzyme activity. the short -helix formed by ser -leu , which twisted the oxyanion hole in the monomer structure, has also been found in the inactive c protease ( cpro) mutant of the hepatitis a virus (hav), which belongs to the picornavirus family. the wild-type hav- cpro is active as a monomer . residues - of hav- cpro, which normally form an oxyanion pocket, adopt a -helical conformation in the crystal structure of the inactive c a mutant. results from the mutant c a of hav- cpro demonstrate that the enzyme is active when the loop that forms the oxyanion hole is in the correct conformation, regardless of whether the enzyme molecules form a stable dimer or remain monomeric. our molecular dynamics simulations showed that ser -leu maintains a stable -helix conformation in the inactive monomer structure and a well-defined loop conformation in the active protomer of the dimer structure. for the mutant glrq, the loop conformation and active site structure transitioned between inactive and active structures regardless of whether the simulation started from an inactive conformation or an active conformation. because the three mutations, g a, r a, and q a, stabilize the monomer structure, the only mutation in the ser -leu loop, l t, was deduced to be the major driving force that destabilizes the -helix towards the active loop conformation. this single mutation may not be sufficient to achieve a highly active enzyme, however. more mutations may be necessary to increase the activity of glrq. the successful design of an active monomer of sars- clpro nevertheless demonstrates the importance of the ser -leu loop conformation, as the loop -helix transition serves as a switch for enzyme activity. although sars- clpro uses the dimer structure to maintain its enzyme activity, our study shows that the monomer can also be evolved into an active enzyme via mutations. why does nature select the dimer structure as the active form for sars- clpro when multiple strategies for enzyme activity regulation are available? our current study provides more evidence that the virus needs a main proteinase to process its viral polyprotein at the appropriate time. a previous study has shown that the substrate can enhance sars- clpro dimerization . during the viral replication process, the enzyme modulates its activity to control the digestion process as the amount of polyproteins change via dimer association and dissociation . in addition to maintaining enzyme activity, sars- clpro also needs to tune its activity according to the substrate concentration. the sars virus may use this strategy to simultaneously prepare proteins that are necessary for assembly. in conclusion, by tuning the conformation flexibility of an active site loop, we have successfully designed an active monomer mutant of sars- clpro. the present work not only serves as a model system to study the regulatory mechanism of enzyme activity, but also facilitates the discovery of anti-viral treatments and the response to emerging coronaviruses such as mers-cov . site-directed mutagenesis, expression, purification, and activity detection. the mutants g a/ l t/r a (grq) and g a/l t/r a/q a (glrq) of sars- clpro were generated with the quikchange site-directed mutagenesis kit (stratagene, la jolla, ca, usa) using pet clp- h or its derivatives as templates. the primers for all mutations are listed in supplemental table s . the mutation was verified by dna sequencing (invitrogen, beijing, china). all resulting plasmids were transformed into e. coli bl (de ) cells for expression. the recombinant sars- clpro and its mutants were prepared using ammonium sulfate fractional precipitation and chromatography, as previously described . enzyme activity was determined using a colorimetric substrate, thr-ser-ala-val-leu-gln-pna, as previously reported . substrate ( μ m) was mixed with μ m grq, . μ m glrq, or . μ m clpro at °c to compare their reaction rates. k m and k cat of glrq were studied by adding the preheated substrate to a reaction mixture to obtain a final concentration of . μ m glrq and - μ m substrate. values of k m and k cat were calculated by the lineweaver-burk plot. enzyme activity dependence of glrq at different concentrations was measured by fixing peptide-pna substrate at μ m and varying the enzyme concentration from . to . μ m. inhibition assay. the isatin derivative, -( -naphthlmethyl) isatin- -carboxamide ( f) , is known to inhibit sars- clpro. f was dissolved in dimethyl sulfoxide (dmso) to test inhibition efficiency towards glrq. glrq ( . μ m) and f ( μ m ) or control % dmso were pre-incubated in mm of phosphate buffer (ph . ) at °c for min, and then μ m of the substrate (thr-ser-ala-val-leu-gln-pna) was added to the mixture and allowed to react for s. beckman optima xla analytical ultracentrifuge using a previously reported procedure . one mg/ml clpro, grq, or glrq was loaded into the double-sector centrepieces. absorbance scans were collected at nm with a speed of , rpm. in addition, μ m glrq in mm of phosphate buffer (ph . ) with or without μ m of the substrate-like inhibitor f were also loaded and analysed at nm with the same speed. three μ m clpro with or μ m f was also scanned at nm with the same speed and temperature as the controls . analytic gel filtration analysis. the oligomeric states of grq and glrq were tested on the superdex hr / gl column using previously reported procedures . the purified protein was loaded onto the column at two different concentrations: mg/ml and . mg/ml for grq and mg/ml and . mg/ml for glrq. four gel filtration molecular weight markers, albumin from bovine serum ( mg/ml), ovalbumin ( . mg/ml), carbonic anhydrase ( . mg/ml), and cytochrome c ( mg/ml), were also loaded onto the column and calibrated to determine a standard calibration curve based on the retention volumes (supplemental fig. s and table s ). two concentrations of clpro ( mg/ml and . mg/ml) were also analysed on the superdex hr as controls. simulations were conducted with the gromacs package using the opls-aa force field . the simple point charge (spc) model of water was used to solvate the protein in a periodic dodecahedron box extending Å from the nearest protein atom. the solvated system then was neutralized with na + and cl − ions, minimized by the steepest descent method ( steps), and equilibrated with a -ps constant volume (nvt) simulation. the production runs were conducted in the constant pressure ensemble (npt). the temperature was set to k and controlled with a modified berendsen thermostat . long-range electrostatic interactions were treated with the particle-mesh ewald method . the pressure was coupled to bar with the parrinello-rahman method . all bond lengths were constrained with the linear constraint solver (lincs) algorithm . a cut-off of Å was used to calculate short-range van der waals and electrostatic interactions. the time step was fs and the trajectories were saved every ps. information about middle east respiratory syndrome (mers) coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain structure of the main protease from a global infectious human coronavirus, hcov-hku structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor production of authentic sars-cov m-pro with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction the catalysis of the sars c-like protease is under extensive regulation by its extra domain biosynthesis, purification and substrate specificity of severe acute respiratory syndrome coronavirus c-like proteinase the n-terminal octapeptide acts as a dimerization inhibitor of sars coronavirus c-like proteinase mutation of gly- on the dimer interface results in the complete crystallographic dimer dissociation of severe acute respiratory syndrome coronavirus c-like protease: crystal structure with molecular dynamics simulations mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus c-like protease two adjacent mutations on the dimer interface of sars coronavirus c-like protease cause different conformational changes in crystal structure mechanism of the maturation process of sars-cov cl protease mutation of asn disrupts the dimerization and enzymatic activity of sars cl(pro) dynamically-driven inactivation of the catalytic machinery of the sars c-like protease by the n a mutation on the extra domain only one protomer is active in the dimer of sars c-like proteinase prediction of the secondary structure of proteins from their amino acid sequence c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism substrate binding and homo-dimerization of sars cl proteinase are mutual allosteric effectors maturation mechanism of severe acute respiratory syndrome (sars) coronavirus c-like proteinase cov main proteinase: the monomer-dimer equilibrium dissociation constant picornaviral c cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases sars veterans tackle coronavirus isatin compounds as noncovalent sars coronavirus c-like protease inhibitors algorithms for highly efficient, load-balanced and scalable molecular simulation development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids canonical sampling through velocity rescaling the effect of long-range electrostatic interactions in simulations of macromolecular crystals -a comparison of the ewald and truncated list methods polymorphic transitions in single-crystals -a new molecular-dynamics method we thank professor jianguo chen for providing clones of the sars-cov cl proteinase. this research was supported, in part, by the national natural science foundation of china (grant numbers and ) and the ministry of science and technology of china. l.l., c.l., x.t. and y.q. conceived the project and analysed the data. c.l. and x.t. performed the experimental study. y.q. performed the molecular dynamics simulations. b.t., h.s., and x.m. participated in conducting the experimental study. y.l. contributed reagents. c.l., y.q. and l.l. wrote the manuscript. all authors reviewed the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord- -b nc ay authors: tsai, andrew; diawara, oumou; nahass, ronald g.; brunetti, luigi title: impact of tocilizumab administration on mortality in severe covid- date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: b nc ay the novel coronavirus disease (covid- ) worldwide pandemic has placed a significant burden on hospitals and healthcare providers. the immune response to this disease is thought to lead to an aberrant inflammatory response or cytokine storm, which contributes to the severity of illness. there is an urgent need to confirm whether the use of tocilizumab provides a benefit in individuals with covid- . a single-center propensity-score matched cohort study, including all consecutive covid- patients, admitted to the medical center who were either discharged from the medical center or expired between march , , and may , , was performed. patients were stratified according to the receipt of tocilizumab for cytokine storm and matched to controls using propensity scores. the primary outcome was in-hospital mortality. a total of patients meeting inclusion and exclusion criteria were identified and patients were included in the matched dataset (tocilizumab = ; no tocilizumab = ). approximately % of the patients were male. hypertension ( %), diabetes mellitus ( %), and chronic pulmonary disease ( %) were the most common comorbidities present. there were deaths ( . %) in the tocilizumab group and deaths ( . %) in the no tocilizumab group (odds ratio, . ; % confidence interval, . – . ; p = . ). advanced age, history of myocardial infarction, dementia, chronic pulmonary disease, heart failure, and malignancy were significantly more common in patients who died. the current analysis does not support the use of tocilizumab for the management of cytokine storm in patients with covid- . use of this therapeutic agent should be limited to the context of a clinical trial until more evidence is available. | ( ) : | https://doi.org/ . /s - - -y www.nature.com/scientificreports/ immunomodulators early on in the treatment algorithm against sars-cov- may pose more harm than benefit. administering these pharmacologic agents too late may be in vain as the inflammatory process may have already contributed to irreparable damage to lung tissue. accordingly, general management with tocilizumab in our institution targeted severe patients prior to the progression of respiratory failure. the objective of this analysis was to evaluate the clinical outcome of in-hospital mortality in patients with covid- treated with tocilizumab in a single medical center. study design and participants. a single-center propensity-score matched cohort study, including all consecutive covid- patients, admitted to the medical center who were either discharged from the medical center or expired between march , , and may , , was performed. patients were stratified according to the receipt of tocilizumab. demographic, clinical, and laboratory data were extracted from the electronic medical records from each of the patients included in the analysis. the baseline (or first available) laboratory values were collected for each patient. at least one ferritin concentration was required for entry into the cohort study, given the relationship with this parameter and cytokine storm , . further, ferritin was used to justify the use of tocilizumab at our institution. in addition, patients were required to exhibit severe disease at time of administration. severe disease was defined as an spo < % on room air, requiring supplemental oxygen, or requiring invasive or non-invasive mechanical ventilation. the timing of tocilizumab administration was dependent on the individual clinician; however, the general approach at the institution was to administer tocilizumab early on before the progression of respiratory failure. the robert wood johnson university hospital somerset institutional review board granted this study expedited approval (irb - ) with waived consent. the reporting of this study conforms to the strengthening the reporting of observational studies in epidemiology (strobe) statement . all methods were performed in accordance with approved guidelines. procedures. data were extracted from the electronic medical record and entered into an electronic dataset for analysis. at least two independent investigators adjudicated study data before analysis. outcomes. the primary outcome was defined as all-cause in-hospital death. patients were followed for the duration of their hospitalization. statistical analysis. propensity scores were generated using ps match in spss v . (ibm corporation). subsequently, propensity score matching was performed to account for treatment strategy influenced by confounding by indication (the tendency of clinicians to prescribe tocilizumab in patients perceived to have cytokine storm and worsening trajectory). propensity scores were calculated using a multivariable logistic regression model where tocilizumab was the dependent variable. covariates included in propensity score included age, sex, body mass index, select baseline laboratory values (lactic acid, ferritin, lactate dehydrogenase (ldh), procalcitonin, serum creatinine), hypertension, and comorbidity score. variables were selected based on clinical significance related to the disease state as well as those that were utilized by prescribers to guide treatment decisions. although il- was not readily available, crp was used as a surrogate marker due to its strong association with the cytokine. il- is a known modulator of acute phase protein synthesis and stimulates the production of crp from the liver and immune cells . controls were matched : using the nearest neighbor approach without replacement using a caliper width of . . standardized mean biases were tested to ensure balance after propensity score matching between groups. categorical data were analyzed using chi-square or fisher's exact test as appropriate. the normality of data was assessed through visual inspection of histograms, and mann-whitney u. continuous data were analyzed using the independent samples t-test or wilcoxon-rank sum as appropriate. the odds ratio and % confidence interval were calculated using logistic regression. we identified a total of patients who received tocilizumab and patients who were treated without the use of tocilizumab as part of covid- management in our hospital. after the propensity score matching process, each group included patients. the matching process yielded well-balanced groups, and no significant differences between any of the covariates were observed. patient demographics and relevant baseline laboratory values are presented in table . the mean age of the study population was ± . years, and patients ( . %) were females. the most prevalent comorbidities included hypertension ( . %), diabetes ( . %), and chronic pulmonary disease ( . %). at baseline, markers of inflammation including ferritin, crp, and ldh, were found to be elevated in almost all patients. the mean ferritin was . ± . ng/ml, mean crp was . ± . mg/dl, and mean ldh was . ± . u/l. there was no significant difference in the number of patients on a ventilator at baseline between groups ( . % versus . %; p = . , tocilizumab versus control group, respectively). furthermore, there were patients who required intubation after tocilizumab administration. hydroxychloroquine and azithromycin were commonly used and a similar proportion of patients in each group received both therapies. bloodstream infections were present in % of patients in each of the groups after matching. of the patients who received tocilizumab, patients ( . %) received mg of tocilizumab, patients ( . %) received mg of tocilizumab, and patients ( . %) received mg. four patients received a second dose of tocilizumab· in terms of mortality, there were deaths ( . %) in the tocilizumab group and deaths ( . %) in the control group (odds ratio, . ; % confidence interval, . - . ; p = . ). a secondary analysis using the entire dataset (unmatched), including the propensity score as a predictor in multivariable logistic regression yielded similar results (odds ratio, . ; % confidence interval, . - . ; p = . ). in an exploratory analysis of the entire patient population (prior to propensity score matching), characteristics of www.nature.com/scientificreports/ patients surviving were compared to those who succumbed to the disease process. table provides a comparison of these covariates between groups. there were significant differences between age, baseline lactic acid, baseline c-reactive protein, baseline oxygen saturation, ventilator use, myocardial infection, heart failure, dementia, chronic pulmonary disease, and any malignancy. given our current knowledge of the pathophysiology of covid- , researchers have proposed repurposing il- receptor antagonists to help curb the cytokine storm. currently food and drug administration approved for use in the management of rheumatoid conditions and cytokine release storm-related to chimeric antigen receptor (car)-t cell therapy, tocilizumab has gained momentum as a potentially effective option in reducing il- associated fevers and preventing clinical deterioration in covid- . contrary to other observational studies that have been reported, we did not find a benefit with tocilizumab treatment , . there was no difference in mortality in patients treated with tocilizumab versus those receiving supportive care. although the inflammatory response induced by cytokines may contribute to the severity of illness with sars-cov- given the essential role that interleukins have in our innate immunity against infectious diseases, our findings suggest that interfering blood stream infection (n, %) ( . ) ( . ) . ( . ) ( . ) . table . patient demographic and clinical characteristics before and after propensity score matching. a ventilator use is before administration of tocilizumab. b third and fourth generation cephalosporins. www.nature.com/scientificreports/ with that response may not be beneficial. moreover, il- is only one proinflammatory cytokine on a list of many that are released in the downstream pathway of crs. important biologic functions of il- include promoting differentiation of b cells into igm and igg and stimulating the development and function of th cells . specifically, in covid- , there is not enough evidence to conclude that inhibiting the signaling of il- alone is enough to quell the ensuing cytokine storm. we identified several demographic and clinical characteristics that were more common in patients with covid- who did not survive. consistent with other studies, non-survivors in our hospital were older and had more underlying comorbidities, especially cardiovascular and respiratory diseases , . elderly patients undergo age-related changes that affect their ability to mount an appropriate immune response to emerging infections. production of new naïve t cells drops significantly between the ages of to , threatening the ability of older adults to fight off new viruses like sars-cov- . as our population advances in age, it is also much more common to see individuals with two or more comorbidities. while cardiac disease, bone disease, and malignancy have been determined to increase the risk for poor clinical outcomes in sars-cov infections, further studies are needed to explore the mechanisms behind the associations found particularly in covid- . small observational studies have investigated il- blockade; however, focus on clinical outcomes has been scant , . these studies focused on improvements in laboratory data (i.e., reductions in il- , crp) with the presumption that improvement in these values leads to clinical benefit . surrogate endpoints, while essential to establish or support proposed hypotheses, do not always translate to improvements in clinical endpoints. for example, il- is another proinflammatory cytokine that has been linked to increased survival in mice infected with the influenza virus. but, in addition to suppressing viral replication, il- is involved in the priming of adaptive t and b cells during infection response , . both early and late phases of virus immunity are influenced by these proinflammatory cytokines. therefore, extreme caution should be exercised with immunomodulatory therapies that blunt our natural protective mechanisms against infectious diseases. these are critical concerns that must be considered as healthcare providers reach for therapeutic strategies in the absence of clear benefit. while there may be a place in therapy for tocilizumab, it should not be given in the routine management of covid- until more studies are conducted. there are several limitations to this study. patients primarily received a single flat mg dose of tocilizumab intravenously, with some receiving a second dose if an additional benefit was considered likely by the treating clinician. the current practice utilizes weight-based dosing for t-cell-induced crs, and elevations in crp have been noted to be inversely related to tocilizumab clearance from the body. therefore, our patients may have been at risk of being underdosed. however, the fixed dosing strategy was based mainly on positive results recently published by xu and colleagues . additionally, serum crp concentration was included in the propensity score matching. furthermore, population pharmacokinetic analysis of tocilizumab supports a flat dosing approach due to reductions in the variability of drug exposure among patient weight categories . other limitations of our study include the retrospective design, as it may have increased the risk of selection bias toward more severe patients in the tocilizumab arm. while propensity score matching was utilized and resulted in well-matched groups, residual confounding cannot be excluded. finally, given the small sample size, a type ii error is possible. remarkably, the mortality was identical in both treatment and control groups, suggesting that even if there is a difference in mortality, it may be modest at best. while the results of the current study were unexpected, recent preliminary reports of randomized controlled trials have dampened enthusiasm for il- antagonizing agents. a clinical trial evaluating sarilumab in covid- patients was discontinued after failing to meet its primary and key secondary endpoints, and instead revealed negative trends in clinical status of patients who were not mechanically ventilated as baseline . preliminary analysis of a french tocilizumab study (nct ) suggested benefit as the study reached its primary endpoint (composite of death or required ventilation). final data analyses are needed to make any conclusive statements of benefits in terms of overall survival. clinicians also await completion of the industry sponsored covacta trial investigating tocilizumab (nct ). recently, results from the tocivid- phase study reported a -day mortality rate of . %, which is comparable to the incidence reported in our results. although the investigators suggest a potential -day mortality benefit with tocilizumab use, the lack of a control group prevents proper evaluation of the benefits for this intervention . in addition, preliminary results from a randomized controlled trial from italy of early-stage covid- pneumonia casts further doubt on the efficacy of tocilizumab as it was stopped early due to a lack of efficacy identified at an interim analysis . our analysis provides a reason for pause and reset. while there are some data that tocilizumab may be beneficial in a subset of patients, the benefits of this agent are nebulous. until more data from randomized clinical trials are available, the use of il- directed therapies should occur only in the context of a clinical trial as outlined in the national institutes of health treatment guidelines . treatment with tocilizumab in patients with severe covid- was not associated with a reduction in mortality. due to the observational nature of our study, these observations require further evaluation in clinical trials to determine the effectiveness of this treatment. available upon request. received: july ; accepted: october covid- : what has been learned and to be learned about the novel coronavirus disease the pathogenesis and treatment of the 'cytokine storm' in covid- cytokine release syndrome: current perspectives covid- : consider cytokine storm syndromes and immunosuppression clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study current concepts in the diagnosis and management of cytokine release syndrome pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology hyperferritinemia and inflammation strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies elevated levels of il- and crp predict the need for mechanical ventilation in covid- interleukin- blockade for severe covid- effective treatment of severe covid- patients with tocilizumab the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area comorbidity and its impact on patients with covid- in china: a nationwide analysis sars-cov- and covid- in older adults: what we may expect regarding pathogenesis, immune responses, and outcomes interleukin- in covid- : a systematic review and meta-analysis use of siltuximab in patients with covid- pneumonia requiring ventilatory support the urgency of care during the covid- pandemic-learning as we go interleukin- is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection the role of neutrophils in the upper and lower respiratory tract during influenza virus infection of mice fixed dosing of intravenous tocilizumab in rheumatoid arthritis sanofi and regeneron provide update on kevzara (sarilumab) phase u.s. trial in covid- patients tocilizumab for patients with covid- pneumonia. the tocivid- phase trial roche rheumatoid arthritis drug fails to help covid- patients in italian study covid- ) treatment guidelines we acknowledge all the members of the covid- response team at rwjs for their contributions during the response to the pandemic. all authors take responsibility for the integrity of the data and the data analysis. a.t., o.d., r.n., and l.b. were responsible for the study concept and design. a.t., o.d., and l.b. were responsible for data acquisition and adjudication. a.t. and l.b. drafted the initial manuscript. r.n. and l.b. provided critical review and expert context. l.b. performed the statistical analysis. all authors reviewed and provided input at each step. there was no funding received for the completion of this study. the authors declare no competing interests. correspondence and requests for materials should be addressed to l.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -dabjcvno authors: poli, piero; boaga, jacopo; molinari, irene; cascone, valeria; boschi, lapo title: the coronavirus lockdown and seismic monitoring of anthropic activities in northern italy date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dabjcvno in march/april the italian government drastically reduced vehicle traffic and interrupted all non-essential industrial activities over the entire national territory. italy thus became the first country in the world, with the exception of hubei, to enact lockdown measures as a consequence of the covid- outbreak and the need to contain it. italy is also a seismically active area, and as such is monitored by a dense permanent network of seismic stations. we analyse continuous seismic data from many stations in northern and central italy, and quantify the impact of the lockdown on seismic ambient noise, as a function of time and location. we find that the lockdown reduces ambient noise significantly in the – hz frequency range; because natural sources of seismic noise are not affected by the lockdown, the seismic signature of anthropic noise can be characterised with unprecedented clarity, by simply comparing the signal recorded before and after the lockdown. our results correlate well with independent evaluations of the impact of the lockdown (e.g., cell phone displacements), and we submit that ambient-noise seismology is a useful tool to monitor containment measures such as the coronavirus lockdowns. noise is of interest to geoscientists, as it can be used at relatively small scales, for instance in mapping and monitoring efforts , . while earlier studies have attempted to characterise high-frequency seismic noise , [ ] [ ] [ ] [ ] , the current lockdown of industrial activities and reduction in road and train traffic in italy is an unprecedented opportunity to discriminate it from ambient noise of natural origin. italy is a highly industrialized and urbanized country, densely covered with non-stationary noise sources , such as traffic and industry-induced vibration . this is particularly true in its northern regions, which account for % of the country's entire industrial output, and where lockdown measures have been enacted earlier than everywhere else in europe. we analyse continuous data from an array of broadband seismic stations, located in the vicinity of known industrial districts in lombardy, emilia-romagna and tuscany (fig. ) ; we identify the spectral signature of the march lockdown, and take advantage of the lockdown to quantify and evaluate the spectral signature of anthropic activities. importantly, measuring the overall reduction in seismic energy associated with the lockdown is also a way to quantify its effects; this is relevant to governmental entities, wishing to monitor the effectiveness of the measures being taken. we downloaded publicly available, continuous, three-component seismic recordings from a set of permanent broadband stations, part of the italian national seismic network operated by the istituto nazionale di geofisica e vulcanologia . all instruments have a flat response at frequencies between ~ . and ~ hz, or broader; we remove ("deconvolve") instrument response from the data prior to our analysis. the locations of stations employed in most of our study are shown in fig. . stations were selected based on their proximity to industrial districts; in particular, miln is located near the city of milano, with a particularly high concentration of vehicle traffic and industrial activities. the seismic signature of the containment measures in italy is apparent from a relatively simple analysis of continuous recordings at station miln, located within the city limits of milano, in a busy area near the university of milano campus and the lambrate train station. we compute spectrograms (fig. ) by fourier-transforming -hour-long segments of continuous signal, with a -minute overlap between subsequent segments; for each calendar day, all segments are then averaged, and the squared modulus of the resulting average fourier transform is computed: this way, a single "power-spectral density" (psd) function is obtained, for each station, component (east-west, north-south, vertical) and calendar day. figure shows clearly that the lockdown has a relevant impact on recorded seismic noise over a broad frequency range; its effect disappears at frequencies below hz, where anthropic noise is weaker. the energy drop associated with the lockdown is comparable with that occurring every weekend and during the winter break, both in / and / . interestingly, loss of energy is gradual over time, starting with the first lockdown measures on february , and increasing with time until a plateau is reached around march (interruption of non-essential industrial activities). a trend similar to that seen in fig. has also been found through the analysis of cell phone displacements . this suggests that vehicle traffic, which was significantly reduced (particularly in and around milano) already with the february measures, contributes significantly to the entire spectrum of anthropic noise; there is also episodical evidence from the press that a number of factories were closed based on the unilateral decision of their owners, before the government-imposed lockdown. the analysis applied to station miln is repeated for all seismic stations of fig. , and the results are illustrated in figs www.nature.com/scientificreports www.nature.com/scientificreports/ lockdown measures apparently impact all stations under consideration, but the character of their effects changes in various ways with station location. in the case of fir, located in the city of florence, the signature of the winter break is almost negligible, while the february/march lockdown still has a prominent effect; it might be possible to interpret this observation through the analysis of anthropic activities usually taking place in the area (e.g., tourism, which is presumably not reduced by the holiday). the drop is gradual at all stations, with no specific governmental decree standing out with respect to the others. at station prma, a slight increase in ambient noise occurs after february and before march . we next characterise anthropic noise by evaluating variations in the spectra of seismic ambient noise before and after the implementation of lockdown measures. we compute the ratios of the psd measured (as described in sec. ) on tuesday march , to that measured at the same station on tuesday december . we carry out this calculation separately for each component, and for all stations analysed thus far; the results of this exercise are shown in fig. . the energy associated with ambient signal is clearly reduced for all stations, at all frequencies in the range of interest. at each station, psd ratios change with frequency almost exactly in the same way for all components. near hz, all stations show a more or less rapid decline in the psd ratio, with ambient noise being more effectively reduced as frequency grows. this trend continues all the way to hz at stations euct and prma, while other stations show a more complex behaviour. above hz station miln stands out, its psd ratio growing quickly with frequency. anthropic noise is known to be relevant at frequencies above hz, and to consist of a range of different excitation mechanisms , , , . natural sources such as rain, wind and sea/ocean waves are typically characterized by frequencies below hz, and are obviously not affected by the lockdown. we infer that, by taking the ratio of noise spectra before and after the lockdown, an estimate of the spectral character of anthropic noise is obtained, and the spectra in fig. can help us estimate the nature of anthropic noise in the region of interest, independent of the lockdown; the frequencies where the psd of ambient signal is most reduced by the lockdown are those where, in normal times, the contribution of anthropic activities to seismic ambient noise is most important. the fact that most energy loss associated with the lockdown is at frequencies between - hz is coherent with what is known of the typical signature of industrial activity and vehicle traffic . seismic data recorded during the lockdown might be particularly useful in identifying sources of anthropic noise, which could be employed by geophysicists, after the lockdown, e.g. to characterise the upper subsoil by cross correlation of ambient signal , . we further analysed the relationship between ambient noise recorded on different components, finding the "h/v" ratio between the psds of horizontal-component and vertical-component signals: first, the psd of each component of signal recorded on a given day is averaged in the frequency range - hz; then the arithmetical average of the resulting east-west-component and north-south-component values is taken; finally, the ratio of the resulting horizontal psd to the vertical one is computed. the procedure is iterated for each station and for each day between december and march , and the results are shown in fig. . in general, the value of h/v is related to how seismic energy in the ambient-noise field is distributed in the form of compressional, shear and surface waves , , ; changes in h/v after vs. before the lockdown would reveal whether the reduction in anthropic noise affects one of these seismic phases/components more or less importantly than the others; in other words, whether traffic and industry-induced vibration can be associated to one particular constituent of the seismic field. figure shows that the lockdown measures have no effect on h/v, and we infer that, while anthropic noise is reduced significantly by the lockdown (fig. ) , the relative contributions of compressional, shear and surface waves remain approximately constant: the noise wave field is stable in the fig. are plotted here on a single graph, for comparison; for each station, the average value of the psd observed in the time interval of interest is subtracted from the corresponding curve, prior to plotting, as this can change significantly from station to station, but is not relevant to our analysis. each colour corresponds to one station, as specified. again, the dates of mentioned governmental decrees are highlighted as in fig. . the italian territory is densely covered by seismic instruments, and by repeating our analysis on the entire network of available stations we are able to quantify the spatial dependence of anthropic noise reduction. for each station, for each day, the psd of signal recorded am to pm is computed, and averaged over different frequency bands. in practice, we employ the direct fourier method , as implemented in the obspy package , : this is standard procedure to identify artefacts related to station operation, episodic cultural noise, overall station quality and level of earth noise at each site. to emphasize the change in ambient noise with the lockdown, we plot the difference between the values so obtained on three dates in , and reference values obtained conducting the same calculation on data recorded for five months until the lockdown, and averaging. we include as supplementary material s an animated version of fig. , showing the psd at the same stations, october , through april , ; through this time-dependent visualization, the drastic effects of the lockdown are further emphasized. our main result, that noise be strongly reduced after the lockdown in the "cultural" frequency range, is confirmed by fig. , and extended to most of northern italy. between - hz, the lockdown effects are more pronounced in the lombardy and veneto regions than in central italy and along the apennine range. the most important reductions in ambient noise are recorded by stations along the alpine arc, near torino, milano and verona, and in the city of florence. we have analysed continuous data from northern italy, and quantified the effects of the march coronavirus lockdown on the seismic ambient noise field. we confirm that this effect is significant, and easily observed in our data: see in particular figs. and . the italian government first imposed a reduction of people (and therefore vehicle) movement, on march ; we find that this date marks the beginning of a gradual loss in ambient-noise energy at all frequencies, which we attribute to the reduction of road and railroad traffic in the region of interest. depending on the station, the energy curve flattens out, or starts to decline more slowly towards the beginning of april, despite the more stringent measures imposed at that time (interruption of all non-essential industrial activities). a similar trend has been found from cell-phone displacement data . one implication of our observations is that seismic data could be useful for governmental institutions to monitor the effectiveness of measures involving a reduction or interruption of human activity in a given area. it is understood that the lockdown only reduces noise of anthropic origin; it follows that by comparing the fourier spectrum of seismic ambient noise before and after the lockdown (fig. ) , one can attempt to characterise anthropic noise. we find that, confirming earlier estimates , , anthropic noise becomes dominant at frequencies coronavirus lockdowns have changed the way earth moves seismic imaging and monitoring with ambient noise correlations stationary-phase integrals in the cross-correlation of ambient noise a theory of the origin of microseisms seismic noise in fennoscandia, with emphasis on high frequencies variations in broadband seismic noise at iris/ida stations in the ussr with implications for event detection the nature of noise wavefield and its applications for site effects studies: a literature review emergence of broadband rayleigh waves from correlations of the ambient seismic noise faster, better: shear-wave velocity to meters depth from refraction microtremor arrays rain and small earthquakes maintain a slow-moving landslide in a persistent critical state characterization of and correction for cultural noise h/v ratio: a tool for site effects evaluation. results from -d noise simulations on the stability and reproducibility of the horizontal to vertical spectral ratio on ambient noise: case study of cavola, northern italy cultural noise and the night-day asymmetry of the seismic activity recorded at the bunker-east (bke) vesuvian station maninduced low frequency seismic events in italy a catalogue of non-tectonic earthquakes in central-eastern italy italian seismological instrumental and parametric database (iside) the reduction of social mixing in italy following the lockdown observations and modeling of seismic background noise recent advances in seismology spectral analysis of seismic noise induced by rivers: a new tool to monitor spatiotemporal changes in stream hydrodynamics sources of long range anthropogenic noise in southern california and implications for tectonic tremor detection shear wave structural models of venice plain, italy, from time cross correlation of seismic noise train traffic as a powerful noise source for monitoring active faults with seismic interferometry observation of equipartition of seismic waves seismic velocity change patterns along the san jacinto fault zone following the m . el mayor-cucapah and m . collins valley earthquakes an algorithm for the machine calculation of complex fourier series obspy: a bridge for seismology into the scientific python ecosystem seismic noise analysis system, power spectral density probability density function: stand-alone software package. united states geological survey open file report seismic noise level variation in south korea global oceanic microseism sources as seen by seismic arrays and predicted by wave action models rete sismica nazionale (rsn) mediterranean very broadband seismographic network (mednet) north-east italy seismic network. international federation of digital seismograph networks regional seismic network of north western italy. international federation of digital seismograph networks the generic mapping tools version we downloaded and analysed continuous seismic data provided by the istituto nazionale di geofisica e vulcanologia, the osservatorio geofisico sperimentale, the university of genova. the generic mapping tools were used to generate the map in figure . piero poli was supported by the european union horizon research and innovation programme (grant agreements, -monifaults). the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to p.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - vucm s authors: franzo, giovanni; tucciarone, claudia maria; moreno, ana; legnardi, matteo; massi, paola; tosi, giovanni; trogu, tiziana; ceruti, raffaella; pesente, patrizia; ortali, giovanni; gavazzi, luigi; cecchinato, mattia title: phylodynamic analysis and evaluation of the balance between anthropic and environmental factors affecting ibv spreading among italian poultry farms date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: vucm s infectious bronchitis virus (ibv) control is mainly based on wide vaccine administration. although effective, its efficacy is not absolute, the viral circulation is not prevented and some side effects cannot be denied. despite this, the determinants of ibv epidemiology and the factors affecting its circulation are still largely unknown and poorly investigated. in the present study, ibv qx (the most relevant field genotype in italy) sequences were obtained between and from the two main italian integrated poultry companies. several biostatistical and bioinformatics approaches were used to reconstruct the history of the qx genotype in italy and to assess the effect of different environmental, climatic and social factors on its spreading patterns. moreover, two structured coalescent models were considered in order to investigate if an actual compartmentalization occurs between the two integrated poultry companies and the role of a third “ghost” deme, representative of minor industrial poultry companies and the rural sector. the obtained results suggest that the integration of the poultry companies is an effective barrier against ibv spreading, since the strains sampled from the two companies formed two essentially-independent clades. remarkably, the only exceptions were represented by farms located in the high densely populated poultry area of northern italy. the inclusion of a third deme in the model revealed the likely role of other poultry companies and rural farms (particularly concentrated in northern italy) as sources of strain introduction into one of the major poultry companies, whose farms are mainly located in the high densely populated poultry area of northern italy. accordingly, when the effect of different environmental and urban parameters on ibv geographic spreading was investigated, no factor seems to contribute to ibv dispersal velocity, being poultry population density the only exception. finally, the different viral population pattern observed in the two companies over the same time period supports the pivotal role of management and control strategies on ibv epidemiology. overall, the present study results stress the crucial relevance of human action rather than environmental factors, highlighting the direct benefits that could derive from improved management and organization of the poultry sector on a larger scale. a total of qx sequences were included in the final dataset. of those, belonged to "company a" and to "company b". the sampled farm location is reported in fig. . overall, farms were mainly located in the "pianura padana" region (central area of northern italy) and, to a lesser extent, in north-eastern, north-western, central and southern italy. although the two companies tend to operate in different italian regions, a clear overlapping was present in the high densely populated poultry area of northern italy (fig. ). qx-population genetics parameter estimation. all considered field sequences formed a monophyletic group including only italian strains ( supplementary fig. ). tempest investigation revealed that the positive correlation between genetic divergence and sampling time (i.e. r = . ) was suitable for phylogenetic molecular clock analysis . the tmrca of the overall qx population in italy (i.e. qx genotype introduction) was estimated in . [ hpd: hpd: . - . ] using the structured coalescent approach. almost identical results were obtained including a third "ghost" deme (i.e. an estimated deme for which no sequences were available, representative of other unsampled companies and farms) in the analysis or using the "traditional" coalescent approach. when strains collected from integrated poultry companies were considered independently, the tmrca was predicted in . [ hpd: [ hpd: . - for company a and in . [ . - . ] for company b. the viral population dynamics evidenced a substantially constant ne*t (effective population size * generation time, or relative genetic diversity) with the remarkable exception of the period between mid- and mid- , when a sudden fluctuation was observed. however, a quite different scenario was demonstrated between the two integrated poultry companies. in fact, company a was featured by a substantially constant population size, with a minor decrease affecting particularly the period - . however, the ne*t hpd were relatively broad and at odds with the significance of the observed variations. on the contrary, a much more changeable pattern was observed in company b (fig. ) . migration among companies. the structured coalescent model fitted with the two company, evidenced the presence of separate clades (fig. a) for the companies, with only exceptions, represented by strains sampled in company b but clustering in the company a clade, thus suggesting the migration of strains from company a to company b. accordingly, the migration rate from company b to company a was . * − [ hpd: . * − - . * − ], while the one from company a to company b was . * − [ hpd: . * − - . ]. the figure . location of farms from which samples have been obtained. different companies have been color coded. samples collected in company b but clustering with company a clade have been colored in red (herein named "imported"). farm location has been jittered using an internal routine of ggplot library to guarantee anonymity. the map was generated in r (version . . ), using the library ggmap . phylogeographic analysis. all the samples phylogenetically belonging to company a but collected from company b originated from farms located in the high densely populated area of northern italy (fig. ) . the continuous phylogeographic analysis reconstructed a spreading pattern originating from a single introduction in emilia romagna region (company a), followed by a progressive expansion and persistence at high level in the pianura padana region. more rarely, spreading episodes toward other italian regions were observed (fig. ) . after qx introduction, the infection wave front increased slowly approximatively until , when a rapid expansion led to the final distribution range by the middle of (fig. ) . accordingly, the dispersal velocity progressively increased in the first years after qx genotype introduction, peaking in the period - and then remaining essentially constant, despite some fluctuations (fig. ) . the presence of a high dispersal velocity after , when no further increase in wave front was observed, suggests that ibv continued to circulate at high rate after its first establishment in a region. the analysis of the effect of different environmental factors on qx genotype dispersal velocity led essentially to negative results (i.e. absence of significant correlation). the only exception was represented by the poultry density www.nature.com/scientificreports www.nature.com/scientificreports/ sl model, which was positively and significantly correlated to viral dispersal velocity: d = . , percentage of d with p-value < . = %. despite the economic relevance, the epidemiology of ibv and the factors affecting its behavior have been only partially investigated. even if a huge amount of knowledge and literature has accumulated over time, most of the reports are anecdotal or based on the analysis of single clinical outbreaks , , . although relevant pieces of information could be obtained, the risk of being biased by personal believes or the particular condition under www.nature.com/scientificreports www.nature.com/scientificreports/ investigation is high. a certain caution is thus required when inferring and extending the same conclusion on a broader/general scale. moreover, most of the available studies are focused on avian influenza and, to a lesser extent, newcastle disease and infectious laryngotracheitis , , . the aim of the present study was to construct an objective and statistically sound framework to understand ibv field strains behavior, the effect of control measures and the factors conditioning their epidemiology. the field of phylodynamics, and all related extensions, provides an invaluable tool for the study of viruses and particularly of rapidly evolving ones, whose evolution can be measured in "real time", over the course of an epidemic . ibv qx genotype is the most relevant field strain in italy , , and despite a relatively long circulation and the efforts devoted to its control, it still remains one of the main menaces for poultry industry profitability. therefore, the understanding of the forces shaping its epidemiology would be of remarkable relevance in order to prevent the induced damages, rather than try to control them. remarkably, the italian ibv strains appear to originate from one introduction events only, as previously reported . therefore, it was possible to reconstruct ibv italian strain evolution and epidemiological pattern without the biasing effect of strains recently introduced from other counties. the implemented approach allowed to reconstruct the migration history of the qx genotype over time. the estimated introduction, in emilia romagna region, shortly predates the first detection, posing in favor of the effectiveness of the italian monitoring and early detection systems. all the analyses, independently of the underlying statistical model, support that company a was the first introduction site (fig. ) . thereafter, the virus circulation was limited to farms belonging to this company for years, until approximatively , when company b became involved. contextually, a progressive increase in diffusion speed was noticed (fig. ) , not unexpectedly considering the rising number of involved farms (especially at the border between veneto and lombardy regions, where most farms are located) and thus the increase in spreading potential and opportunity. the high farm density of this area has been described as a risk factor for different infectious diseases , and ibv seems to be no exception. interestingly, the viral population size remained relatively constant in this time period, evidencing that, even if qx strains were able to effectively spread from farm to farm, their replication was adequately controlled, likely by effective vaccination strategies. actually, a certain slowdown in dispersal velocity was noticed in - , potentially because of a progressive decrease in naive populations availability. a dramatic change was observed in , when a new spreading wave (fig. ) and an increase in diffusion rate (fig. ) and population size (fig. ) were detected. a more detailed analysis demonstrated that this variation affected company b only (fig. ) . a previous study has ascribed this episode to a change in the vaccination scheme adopted by this company, which moved from a heterologous mass+ b based vaccination to a mass only vaccination leading to an increased viral circulation and clinical outbreaks number . moreover, experimental studies demonstrated a significant reduction in r in vaccinated groups compared to unvaccinated ones . it can therefore be speculated that the increase in infectious pressure within-farm and the higher flock susceptibility to infection could have enhanced the risk of ibv spreading to other farms and regions. in support of this hypothesis, the geographical spreading affected mainly northern italian farms (where company b is located). moreover, when a new double vaccination was implemented, the decrease in viral population size was mirrored by a reduction of dispersal velocity. www.nature.com/scientificreports www.nature.com/scientificreports/ continuous phylogeography showed that the areas interested by a more intensive viral circulation were those featured by a higher poultry density, and this evidence was confirmed by a statistically significant correlation between poultry density and dispersal velocity. the association between spatial proximity and farm infection is probably the most consistently reported risk factor for poultry infectious diseases , , . although an airborne transmission has been proposed for ibv, its occurrence has rarely been demonstrated experimentally . however, the spatial proximity likely increases the likelihood of a greater number of horizontal contacts between farms, including the movement of people, vehicles and fomites between farms, as well as sharing similar risk factors (e.g. environmental conditions, climate, presence of wild animals, etc.) , . based on these premises, the presence of segregated poultry companies should represent an effective obstacle to viral shedding and the obtained results partially confirm these evidence. the strains from different poultry companies formed two independent clusters, which suggests the effectiveness of independent production flow/chain in protecting farms from exogenous introductions. additionally, the application of adequate biosecurity measures, enforced also by the italian legislation, likely contributed in limiting new strain introduction. the exceptions to this general rule were farms located in the high densely populated poultry area of northern italy, where an overlap between the two companies occurs. the unidirectionality of the viral flux from company a to company b implies that other factors, besides spatial proximity, must be in place. a detailed survey could shed some insights into relevant factors like different biosecurity measures, structural factors, vaccination strategy etc.. the mediation of other "actors" cannot also be excluded. in fact, the analysis of just two companies, however predominant they are on the italian poultry sector, cannot be considered an accurate depiction of the italian situation. remarkably, the inclusion of a third deme (representative of other unsampled companies and farms) in the analysis model highlighted that several transmission events could be mediated by smaller entities operating in the same region. actually, the high migration rate estimated between company b and this ghost deme poses in favor of its pivotal role in maintaining an active ibv circulation. even if the idea of modeling demes for which no sequences are available could seem counterintuitive, previous studies showed that the structured coalescent can provide meaningful estimates even in absence of samples from one population and this approach has already been applied and proven effective for other diseases, including ebola . since also company a was evaluated in the same analysis run, the absence of relevant links between this company and the ghost deme further supports the analysis reliability, posing in favor of an actual interaction between company b and the ghost deme rather than a mere low specificity of the method. a less effective control of ibv infection could be speculated for small companies, whose management capability and resources are limited compared to big-integrated companies. in fact, all italian farms have to follow national legislation dictating the minimum biosecurity measures to be applied. however, integrated poultry farms, part of major companies, enforce additional managerial practices to increase biosecurity levels. personnel and veterinarian formation, internal audits and periodic controls guarantee a higher level of application of the required standards, compared to most of small non-integrated farms. the higher spatial overlap and the likely sharing of some infrastructures (e.g. streets, accessory personnel, services and infrastructures) could nevertheless have a negative indirect effect on the major companies, especially in northern italy where company b is located. however, differences between company a and company b in the application of biosecurity measures and production flow management could also explain the different ibv epidemiology, as demonstrated by the dissimilar patterns in viral population fluctuations in the two companies (fig. ) . a further risk factor that would deserve further investigation is the presence of the rural sector, which is highly concentrated in the densely populated poultry area of northern italy. this sector is characterized by a complex mix of growers, dealers and backyards flocks, often applying poor biosecurity measures and linked together by a poorly traceable contact network . although interactions with industrial poultry farming is hardly discouraged, illegal/indirect interactions have been documented and multiple epidemiological connections could result in a bidirectional transmission between the two sectors, as demonstrated in the italian low pathogenicity avian influenza (ai) outbreaks occurred in - . after these episodes, a stricter legislation has been developed, imposing limits to animal movements and more active surveillance in the rural sector. nevertheless, no measures were taken for the monitoring and control of ibv in these enterprises, and therefore their role as sources of encroachment in intensive farming cannot be excluded. other environmental factors do not seem to play a relevant role in affecting viral dispersal. while climatic conditions like temperature, humidity and wind could actually affect viral viability and spreading, their effect could be circumvented by a transmission mediated by "fast-moving" vectors like trucks, personnel and, potentially, wild species , . more surprising could be the non-significant role of road density. however, it must be stressed that the available raster reported the overall density of roads, which could significantly differ from those preferentially used for live animal or their byproduct transportation, hindering the detection of an otherwise plausible risk factor. therefore, the mapping of the live animal transportation pathways could provide remarkable benefits in ibv (and other infectious diseases) epidemiology understanding and control. the present study demonstrates that ibv spreading potential is mainly affected by farm and poultry density overall, which can be reasonably claimed as a major risk factor. other environmental/climatic variables do not seem to affect ibv epidemiology, stressing the pivotal role of human action and thus highlighting the direct benefits that could derive from an improved management and organization of the poultry sector on a larger scale. actually, the integration of poultry production seems to provide a relevant constrain to ibv circulation, even though some differences were noted between the two considered companies. in fact, despite differences in management and applied control strategies likely playing a role, the presence in the same area of other minor poultry companies seems to represent a major issue, probably due to the less effective infection control ascribable to the sometimes lower organization capability and resources of small enterprises. the present study results emphasize the need of an active sharing of sequences and related molecular epidemiology data originating from all the actors in poultry production, allowing a proper depiction of the viral exchange dynamics, based on actual data rather www.nature.com/scientificreports www.nature.com/scientificreports/ than estimations. the obtained information would represent a fundamental substrate for the implementation of effective and shared efforts for the infection control on a broad regional scale. ibv strain sampling, diagnosis and sequencing. samples were collected for routine diagnostic purpose in the period - from poultry flocks belonging to the two main poultry companies (here named company a and company b) operating in italy, which account together for about % of italian poultry production. samples were obtained mainly from outbreaks of respiratory disease, following a standard protocol that enforced the collection of a pool of tracheal swabs from randomly selected birds. for each sampling, collection date and farm localization were recorded. all considered samples had been performed in the context of routine diagnostic activity and no experimental treatments or additional assays were implemented during the study. therefore, no ethical approval was required to use specimens collected for diagnostic purpose. additionally, several samples from company a were already sequenced using the same protocol and published in franzo et al. . when detailed information on sampling farm and time could be traced back, these samples were included in the study. the permission to use the collected samples for research purpose was obtained from each company. swab pools were resuspended in ml of pbs and vortexed. thereafter, rna was extracted from µl of the obtained eluate using the high pure viral rna kit (roche diagnostics, monza, italy) kit. diagnosis was performed by amplification and sanger sequencing of the hypervariable region of the s region using the primer pair described by cavanagh et al. . obtained chromatograms quality was evaluated using finchtv (http://www. geospiza.com) and consensus sequences were generated using cromaspro (cromasproversion . ). sequence dataset preparation. all obtained sequences plus the reference dataset provided by valastro et al. ( ) were aligned using mafft and a phylogenetic tree was reconstructed using iq-tree selecting as the best substitution model the one with the lowest akaike's information criterion, calculated using jmodeltest . the strains clustering with the gi- lineage (previously known as qx genotype) were selected and further evaluated for the presence of recombination in the considered region using rdp and gard : to limit the computational burden the sequences were clustered using a % identity threshold using cd-hit and a single representative sequence for each cluster was selected. these sequences plus the valastro et al. ( ) references were re-aligned and recombination analysis was performed. recombinant sequences, including the ones belonging to the same cluster, were removed from the dataset. finally, the dataset was re-expanded to the original size and sequences identical or closely related (p-distance < . ) to the qx-based vaccines administered in italy were also excluded. to evaluate the distribution of italian gi- strains in the international scenario, an extensive dataset of s ibv sequences was downloaded from genbank and a phylogenetic tree was reconstructed as previously described. to reduce computational complexity and increase interpretation easiness (without losing information), only one sequence representative of all identical ones was selected using cd-hit and included in the analysis. the presence of an adequate phylogenetic signal was assessed by a likelihood mapping analysis performed with iq-tree. tempest was used to preliminarily evaluate the temporal signal of the italian qx phylogeny and therefore the applicability of molecular clock-based methods . strain migration among integrated poultry companies. ibv qx strain migration among companies was evaluated using the structured coalescent-based approach implemented in the multitypetree extension of beast . according to this model, the considered population is divided in a series of demes, which can be imagined as different islands, featured by their own populations size and interconnected by a certain migration rate among them. in the particular italian qx scenario, the serially sampled (i.e. with known collection date) strains were used to infer the migration rate and history between the two integrated poultry companies (i.e. considered as different demes) over time. additionally, the bayesian approach implemented in beast allowed to contextually estimate other population parameters, including the time to most recent common ancestor (tmrca), evolutionary rate and population size. accounting for the presence of other farms and companies operating in the italian poultry sector, which could take part in or mediate the viral transmission among the investigated major companies, a third "ghost" deme (a deme for which no sequences were available) was added to the model . the priori of the ghost deme size was set to one tenth of the other demes, according to the estimated poultry population distribution. however, broad priori distribution (i.e. relatively uninformative priori) was chosen to avoid constrains or biases in the parameter posterior estimation. for all analyses, the best substitution model (tn + g ) was selected based on the bayesian information criterion, calculated using jmodeltest , while the relaxed lognormal molecular clock model was selected based on marginal likelihood calculation and comparison using the path sampling and stepping stone method . the final estimations were obtained performing a million generation markov chain monte carlo run, sampling parameters and trees every twenty thousand generations. results were visually inspected using tracer . and accepted only if mixing and convergence were adequate and the estimated sample size was greater than for all parameters. parameter estimation was summarized in terms of mean and % highest posterior density (hpd) after the exclusion of a burn-in equal to % of the run length. maximum clade credibility (mcc) trees were constructed and annotated using treeannotator (beast package). results consistency was also evaluated performing a "traditional" serial coalescent analysis in beast . . . the same substitution and clock model of the structured coalescent analysis were selected, while a nonparametric skyline population model was chosen to reconstruct the viral population dynamic over time . independent ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ analysis for each integrated company were also performed using the same approach but generating two new datasets including only the sequences collected from a specific company. however, sequences introduced from one company to the other were excluded from the company-specific analysis since they did not share a common evolution history. continuous phylogeography and determinants of ibv spreading. the history of qx dispersal was reconstructed over time using the continuous phyogeographic approach described by lemey et al., using beast . . . substitution and clock models were selected as previously described. similarly, the gamma relaxed random walk was preferred over the other phylogeographic continuous diffusion models based on the marginal likelihood calculation and comparison using the path sampling and stepping stone method , . the final estimations were obtained performing a million generation markov chain monte carlo run, sampling parameters and trees every twenty thousand generations. results were visually inspected using tracer . and accepted only if mixing and convergence were adequate and the estimated sample size was greater than for all parameters. the reconstruction of qx movements over time within italian borders was obtained using spread , summarizing and visualizing the full posterior distribution of trees obtained in continuous phylogeographic analyses . pattern and determinants of viral spreading were evaluated as described by (dellicour et al.) , using the seraphim r library . the history of lineage dispersal was recovered from the posterior trees generated using beast and annotated with ancestral longitude and latitude reconstruction. particularly, the distance, duration and velocity of spatial dispersal were recoded as vectors and used to generate different summary statistics of viral spreading, including dispersal velocity and maximal wave front distances (measured from the location of the tree root). several environmental/social variables were considered to determine if they were associated with the dispersal rate of ibv lineages. the environmental rasters describing the variables of are shown in supplementary fig. . more in detail, the values in the raster (i.e. altitude, population density, poultry density, temperature, etc.) were used to associate a weight to the abovementioned vector. two models of spatial movements were considered: ( ) "straight line (sl) path" model, assuming a straight movement between the starting and ending locations of each branch (i.e. the branch weight is computed as the sum of raster cells through which the straight line passes); ( ) "least cost (lc) path" model, using a least cost algorithm (i.e. the branch weight is computed as the sum of the values of cells transition values between adjacent cells along the least-cost path). in this model, the analyzed environmental variable can be considered both as a conductance (i.e. enhancing viral dispersal through the cells with higher values) or resistance factor (i.e. allowing an easier dispersal through cells with lower values). both instances were evaluated for each considered factor. the obtained "environmental" weights were used to calculate a regression with the branch duration and the corresponding coefficient of determination (r env ) was obtained. a null coefficient of determination (r null ) was also calculated assuming the null raster (i.e. when only the spatial distance of each movement is assumed to affect branch duration). the statistic d = r env -r null was selected as final outcome, and describes how much the regression is strengthened when the spatial variation in the environmental variable is included. to account for the phylogenetic uncertainness, the d statistic was calculated for each tree of the posterior distribution. however, for computational constraints, the number of posterior trees was down-sampled to after discharging a % burn-in. only the environmental variables with more than % of d statistics > were considered for further analysis. particularly, the significance of d statistic of those variables was assessed against a d null distribution obtained by randomizing times the phylogenetic nodes location under the constraint that branch length remained equal. a p-value was generated for each initial tree, therefore a percentage of the trees with p-value < . could be calculated, which can be interpreted as a posterior probability of observing a significant correlation between lineage movements and considered environmental variable. according to , a percentage of p-value < . greater than % was considered a strong evidence that the environmental variable is associated to viral movement speed . molecular evolution and emergence of avian gammacoronaviruses s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification review of infectious bronchitis virus around the world effect of different vaccination strategies on ibv qx population dynamics and clinical outbreaks sjaak) & cook, j. k. a. factors influencing the outcome of infectious bronchitis vaccination and challenge experiments infectious bronchitis virus variants: a review of the history, current situation and control measures age-dependent immune responses and immune protection after avian coronavirus vaccination a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain porcine circovirus type (pcv ) evolution before and after the vaccination introduction: a large scale epidemiological study genetic data provide evidence for wind-mediated transmission of highly pathogenic avian influenza modelling the wind-borne spread of highly pathogenic avian influenza virus between farms avian influenza virus infections. ii. experimental epizootiology of influenza a-turkey-wisconsin- virus in turkeys characterisation of influenza a viruses isolated from turkeys in england during avian influenza in caged laying chickens isolation of avian influenza virus in texas assessing the probability of introduction and spread of avian influenza (ai) virus in commercial australian poultry operations using an expert opinion elicitation transmission parameters of highly pathogenic avian influenza (h n ) among industrial poultry farms in northern italy in - risk maps for the spread of highly pathogenic avian influenza in poultry explaining the geographic spread of emerging epidemics: a framework for comparing viral phylogenies and environmental landscape data exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) risk factors for the introduction of high pathogenicity avian influenza virus into poultry farms during the epidemic in the netherlands in risk factors for highly pathogenic h n avian influenza virus infection in poultry during the - epidemic in italy a cross-sectional survey of australian chicken farms to identify risk factors associated with seropositivity to newcastle-disease virus wind-borne transmission of infectious laryngotracheitis between commercial poultry operations phylogenetic and epidemic modeling of rapidly evolving infectious diseases continued use of ibv b vaccine needs reassessment after its withdrawal led to the genotype's disappearance think globally, act locally: phylodynamic reconstruction of infectious bronchitis virus (ibv) qx genotype (gi- lineage) reveals different population dynamics and spreading patterns when evaluated on different epidemiological scales transmission of infectious bronchitis virus within vaccinated and unvaccinated groups of chickens control of avian influenza in poultry studies on australian infectious bronchitis virus. iv. apparent farm-to-farm airborne transmission of infectious bronchitis virus estimating population parameters using the structured serial coalescent with bayesian mcmc inference when some demes are hidden new routes to phylogeography: a bayesian structured coalescent approximation proroga e modifica dell' ordinanza agosto e successive modificazioni, concernente: «misure di polizia veterinaria in materia di malattie infettive e diffusive dei volatili da cortile epidemiology and control of low pathogenicity avian influenza infections in rural poultry in italy coronaviruses in avian species -review with focus on epidemiology and diagnosis in wild birds coronaviruses in poultry and other birds longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions mafft multiple sequence alignment software version : improvements in performance and usability w-iq-tree: a fast online phylogenetic tool for maximum likelihood analysis jmodeltest : more models, new heuristics and parallel computing rdp : detection and analysis of recombination patterns in virus genomes gard: a genetic algorithm for recombination detection cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences efficient bayesian inference under the structured coalescent improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty bayesian coalescent inference of past population dynamics from molecular sequences phylogeography takes a relaxed random walk in continuous space and time spread : interactive visualization of spatiotemporal history and trait evolutionary processes seraphim: studying environmental rasters and phylogenetically informed movements spatial visualization with ggplot this research was partially founded by the grant (bird / ) from the department of animal medicine, production and health, university of padua. g.f., a.m. and m.c. planned the study, g.f., c.m.t., m.l., t.t., r.c., p.p. performed laboratory work and generated the sequences obtained in the present study, c.m.t. and m.l. curated the sequences dataset, g.f. analyzed the data, m.c., a.m., p.m., g.t., g.o., l.g. supervised the respective research groups, g.f. wrote the manuscript, c.m.t., m.l., m.c. revised and improved the manuscript. all authors reviewed and agreed on the current version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to g.f.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -gl i s authors: tang, qin; song, yulong; shi, mijuan; cheng, yingyin; zhang, wanting; xia, xiao-qin title: inferring the hosts of coronavirus using dual statistical models based on nucleotide composition date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: gl i s many coronaviruses are capable of interspecies transmission. some of them have caused worldwide panic as emerging human pathogens in recent years, e.g., severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). in order to assess their threat to humans, we explored to infer the potential hosts of coronaviruses using a dual-model approach based on nineteen parameters computed from spike genes of coronaviruses. both the support vector machine (svm) model and the mahalanobis distance (md) discriminant model achieved high accuracies in leave-one-out cross-validation of training data consisting of representative coronaviruses ( . % and . % respectively). predictions on additional coronaviruses precisely conformed to conclusions or speculations by other researchers. our approach is implemented as a web server that can be accessed at http://bioinfo.ihb.ac.cn/seq hosts. scientific reports | : | doi: . /srep invasion, its sequence must encode the information related to specific hosts; therefore, it is especially useful in identifying hosts of given coronaviruses. as the result of natural selection and evolution, different genomes are characterized with different preferences for nucleotides. according to probability principles, a shorter nucleotide fragment has a lower chance of variation due to evolution, and the copies of this fragment in a genome tend not to change significantly. this phenomenon is helpful for evolutionary analysis. dinucleotides are the most stable of these fragments because they are the shortest, and their bias values are usually diverse among species and they are highly invariant for a given individual genome . dinucleotide abundance has been proven to be reliable in the identification and classification of sequences from viral genomes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . support vector machines (svms) are a group of supervised machine learning methods that were originally introduced by vapnik as a linear classifier . their current standard incarnation (soft margin) comprises associated learning algorithms for classification and regression analysis . the basic principle of class separation for a svm is mapping vectors into a high-dimensional feature space and finding an optimal separating hyperplane between the two classes in this space by maximizing the margin between the classes' closest points. the points on the boundaries are referred to as support vectors, and the middle of the margin is the optimal separating hyperplane, which forms the largest gap between two sets of data . based on this gap, the points of different attributes fall into different classes. several types of algorithms exist for a svm to address classification problems for multiple classes and high-dimension data. svms perform well in multiple areas of biological analysis, including the evaluation of microarray expression data, the detection of remote protein homologies, and the recognition of translation initiation sites . instances in which the established classification is questionable or wrong can be identified if an svm is used for prediction of training samples. mahalanobis distance (md) discrimination is a classical and accurate method that is extensively applied in cluster analysis and classification techniques . md measures the distance between a point and a population and considers the variance of the population distribution; the points are sorted to the closest population in distance. another method-fisher's linear discriminant analysis-has been applied to infer hosts for three novel picorna-like viruses . as it requires data that have a normal distribution, which is not the case for our data, md is adopted in this study. previous studies of coronaviruses were primarily focused on the evolution of genomes or specific genes, serum-neutralization assays for identification of receptors, and crystal structure analysis of spike protein and receptor binding domains. in this study, we analysed the compositions of mononucleotides and dinucleotides in coronavirus spike genes. based on the data matrix of nucleotide composition, the md and svm were applied to predict hosts of coronaviruses. the results of this technique may provide hints regarding natural hosts or potential hosts of the virus and can be used to guide the selection of the cells for virus isolation or to explore the probability of interspecies transmission of coronaviruses. nucleotide composition analysis. nineteen parameters, including three mononucleotide frequencies (g, c and t) and dinucleotide biases, were computed from spike gene sequences (see supplementary table s ). all parameters show significant differences across the host groups (kruskal-wallis tests, p < . e- ); therefore, they were subsequently employed as factors in statistical models for discriminant analyses. empirically, a dinucleotide relative abundance or dinucleotide bias (e.g., ρ xy ) is significantly high if ρ ≥ . . among the dinucleotides in this study, the cpa and tpg show an average abundance that is significantly higher than the expected values (ρ ca = . , ρ tg = . ), whereas the average bias of cpg is extremely low (ρ cg = . ). this result indicates that the observed abundances of cpa and tpg are significantly higher than their expected values, and the observed abundance of cpg is significantly lower than the expected value. the g+ c content is minimal ( - %) . this finding indicates that coronaviruses exhibit a low density of nucleotide sequences and may be sensitive to heat or alkali. the low g+ c content also indicates a preference for codons ending with a or t and a higher mutability. training and validation of statistical models. the data matrix with factors as columns and samples as rows was fitted to svm and md models, all predictions in leave-one-out cross-validations were listed in supplementary table s and summarized in table according to host species. the validations indicate that both models achieved high accuracies on the training data set: . % for the svm and . % for the md. all incorrect cases in unsupervised predictions are listed in table . the only incorrect prediction by the svm is sample nc_ . , which is isolated from an avian species but was predicted to infect humans. among all incorrect predictions by md, bats are the common predicted hosts. no sample was incorrectly predicted by both models. the trained models were applied to additional samples, and the predictions unveiled clues regarding potential interspecies transmission (see table ). sequences - comprise spike genes of coronaviruses that were primarily isolated from palm civets from restaurants, animal markets, or farms in southern china when sars wreaked havoc in . the sequences of these coronaviruses (civet-covs) are similar not only to each other but also to sars-cov. cross-host evolution research of sars-cov in palm civet and humans indicated that the variations in spike genes seemed to be essential for the transition of coronavirus from animal-to-human transmission to human-to-human transmission . in addition to cross-neutralization with sars-cov, these sars-like civet-covs can use human ace as an entry receptor . bats are the reservoir hosts of a number of coronaviruses, and a recent study also suggests that bats are natural reservoirs of these sars-like coronaviruses, whereas palm civets and humans are intermediate hosts . all hosts predicted by the svm are humans, which supports the previously mentioned research. the md identified both bats and humans as hosts of these samples, but bats are the preferable hosts for samples - and the second choice for samples - . this finding is also expected as bats are considered to be natural hosts of these viruses. sequences - comprise spike genes of mers-covs from dromedaries after the outbreak in the middle east in . mers-covs are similar to the bat coronaviruses hku and hku in their amino acid sequences , and they can use human dpp as an entry receptor . mers-covs was assumed to originate from hku in pipistrelle, which is a type of japanese bat . in our study, these mers-covs isolated from camels were predicted to be capable of infecting humans; and bats are also likely hosts next to humans in predictions by md. this result is obviously consistent with above speculations, and also supports the who advices about avoiding close contact with camels (http://www.who.int/csr/ don/ _ _ _mers/en/). the st sample was a sars-associated coronavirus that was transmitted from human to pig , and both svm and md detected its threat to humans. bat and avian might be potential hosts since both models suggest that they are more vulnerable than porcine. samples - (rsshc , rs and sl-cov-wiv ) consist of three sars-like coronaviruses from bats . analyses based on the sequence similarities and cultures in the cell lines suggest that rs and sl-cov-wiv are capable of using a sars-cov receptor for cell entry and pose a threat to humans, whereas rsshc cannot . our study provides a precise support to these conclusions. the md correctly predicts bats as the natural hosts of the three viruses, and the svm indicates that rs and sl-cov-wiv are harmful to humans. the th sample was isolated from an alpaca by jin et al. in with a serotype of bovine; the phylogenetic analysis suggests that it shares the same ancestor with bovine-coronaviruses . our analysis supports the finding that this coronavirus is capable of infecting bovine. these analyses imply that this strain is capable of interspecies transmission between bovines and alpacas. samples and are enteric coronaviruses from bovines and humans; they have been identified as the same strain named "human enteric coronavirus " in the ncbi database due to the similarity between their spike protein sequences of . %. although they are similar to the human coronavirus oc and the bovine coronavirus, evidences from morphological, immunological, and genomic studies indicate that they are closer to bovine coronavirus than to human coronavirus (unpublished research, from personal communication). this finding is consistent with our analysis. in addition, avian and bat are worthy of attentions as potential hosts due to the small md values. two groups of two-dimensional data are plotted in n( , ) , and the red points represent a "tight" population with a smaller sd of n( . , . ). the red line separates the two groups classified by the md, and the groups predicted by the svm are delimited by the blue line. in this figure, two individuals (the red triangles between the two lines) from the "tight" population were classified into the "loose" group by the md, whereas the svm accidentally excluded four points (the blue reversed triangles between the two lines) from the "loose" population. this example shows that md and svm have inverse tendencies in some cases, i.e., when a "loose" population is close to a "tight" population, md intends to classify outliers of the "tight" population into the former. the opposite situation is valid for the svm. nucleotide composition analysis revealed the overrepresentation of cpa and tpg dinucleotides and the suppression of cpg dinucleotides (see supplementary table s ), which indicates that coronaviruses generally prefer motifs that contain cpas and tpgs and avoid cpgs in sequences. these dinucleotide biases are common characteristics of rna viruses in vertebrates , , , . as most vertebrates exhibit a very low cpg representation in genomes, rna viruses may gradually adapt to the accumulation of host mutations and mimic the host gene's dinucleotide patterns for survival . for dna viruses, the most-accepted mechanisms for the suppression of cpg dinucleotides are the methylation of cpg nucleotides and the subsequent deamination of -methylcytosine, which renders cpg a mutational hotspot . for rna viruses, a different hypothesis is that the rna viruses encounter different selection pressures when they switch to a new host, and viral rna genes mimic host mrnas to avoid immune detection . similar to other human ssrna viruses, coronaviruses show a strong correlation between cpg pressure and c+ g content (pearson's correlation coefficient, r = . , p < . e- , our data). a lower c+ g content usually indicates that the nucleotide sequence of the virus is unstable or is highly variable under evolutionary selection pressure. considering that the mutation rates for rna viruses are significantly higher than the mutation rates for dna viruses , mutational pressure may be the most important determinant of the bias in codon usage in human rna viruses, such as coronaviruses . the capabilities to bind with receptors and to replicate in host cells are essential for any virus to infect hosts. different genes contribute to these biological processes. variations on these genes may enable a table . the isolate sources and predicted hosts of coronaviruses. predictions consist of hosts with minimal md or p values, those with md < = or p < = . for svm, and those with md or p values no greater than corresponding values of isolate sources if the isolate sources are among the six categories of hosts. all predictions are listed in ascending of md or p values. * p < = . or md < = . ** p < = . or md < = . scientific reports | : | doi: . /srep virus to transmit cross-species. one famous example would be the polymerase (pb ) of influenza a virus, in which amino acid change from e to k at its th position would render the virus to replicate in mammalian cells [ ] [ ] [ ] . in coronaviruses, the spike protein is functionally associated with recognition of hosts and the rna-dependent rna polymerase (rdrp) is related to proliferation of virus. however, there are two obstacles limiting the use of rdrp gene: ( ) the similarities among nucleotide sequences is too high to train md model, i.e., the variation rate of rdrp sequence is slower and cannot provide enough resolution to discriminate different coronaviruses; ( ) even worse, available full-length cdss in public databases are very limited -only or so. on the contrary, the spike gene perfectly satisfied the requirements for variation rate and availability, therefore was adopted as markers in this study. md and svm show opposite tendencies in judging outliers (see fig. ) , which reflects the different principles of the two classification approaches. unlike the euclidian distance (ed), which measures the absolute distance between points or mass centres in space, the mahalanobis distance considers the variances within a population and the covariance between variables. in some cases, especially when a population with individuals who are scattered across a wide range is located close to a "tight" population with smaller internal variations, the md may classify marginal individuals from the latter into the "loose" population even if they are "close" to a "tight" population according to the ed. the md enables "loose" populations to have a greater number of points. the svm has a different philosophy. svm separates populations by finding a hyperplane that maximizes the distances between populations. when a "loose" population is close to the boundary of a "tight" population, svm is more likely to find this hyperplane within the former. this finding explains svm's tendency to exclude outliers from a "loose" population. bats are the reservoir hosts of a number of coronaviruses that can survive in bats and accumulate variations in the long evolutionary process , , . thus, coronaviruses in bats constitute a "loose" population with larger internal gaps. we assume that some strains of viruses in bats gain sufficient variation to enable them to infect other organisms; these viruses form a new "tight" population at the edge of the original group. in this case, the md emphasizes the connection of a virus with the original source, whereas the svm may be more sensitive to the possibility of infecting new hosts. therefore, the incorporation of analyses using the md and svm can be especially helpful for revealing the profile of interspecies transmission. according to the predictions by md, bats are not only the hosts in all incorrect cases from training data set (see table ), but also in the host list of each coronaviruses for testing (see table ). furthermore, bats were predicted to host of . % training samples isolated from other hosts (see table ). these facts convincingly support the notion that these viruses originated from bats and shifted to other hosts. next to bats, avians could be infected by . % samples from other hosts. if bats are the only reservoir hosts and coronaviruses spread from bats to avians and other animals, according to the stochastic event model, the probability of co-infectivity to both bat and avian can be the product of the infectivity probabilities to each of them, i.e., . ( . × . , see table ), then ( . × ) samples are expected to be of co-infectivity. however, only samples were predicted to be of co-infectivity to bats and avians. so avians might be the second independent source of coronavirus in parallel to bats. if this speculation is true, people will have to maintain vigilance to avian coronaviruses apart from avian influenza viruses. especially, due to the high accuracy of the svm in cross-validation, we should seriously consider its only "wrong" prediction: perhaps it is sensible to investigate whether the nc_ . virus from avian is capable of infecting humans. for the viruses that are capable of spreading across a host species barrier, the combination of the md and the svm is valuable for assessing their potential threat. the origin and interspecies transmission of coronaviruses have been extensively discussed in the past ten years, and the coronaviruses of most mammals are believed to originate from their ancestors in bats , , . our analysis with dual statistical models support the finding that sars-covs and mers-covs spread from bats to humans and other animals. in most cases, our approach provided convincing predictions. the dual-model approach can be expected to become a useful tool in future studies. typically, when a novel coronavirus is isolated, the combination of the md and the svm may provide meaningful hints regarding its origin and potential threat to humans or other animals. as soon as more virus genomes are sequenced, this approach can be applied to investigate the interspecies transmission route of other threatening viruses, including the recent ebola outbreak in west africa. data preparation. all genome sequences and complete coding sequences (cdss) of spike genes were downloaded from the national centre for biotechnology information (ncbi) database (http://www. ncbi.nlm.nih.gov/) on july , . sequences of spike genes were extracted from the coronavirus genomes and pooled with downloaded cdss. then, we removed replicate sequences and sequences that contained non-standard bases or were incapable of coding complete products. the length of each sequence is longer than , bases. among all valid nucleotide sequences that are listed in supplementary data s , sequences fall into six categories according to different hosts: for humans, for porcines, for bovines, for bats, for murines and for avians. the majority of the remaining viruses were isolated from the two epidemic diseases caused by the coronavirus in the past years. although we only listed the hosts from which they were isolated, these viruses have been verified or suspected to have the ability to infect different hosts; thus, all sequences were employed scientific reports | : | doi: . /srep to explore interspecies transmission of coronaviruses. viruses from other mammals, including canines, felines, rabbits, equines, alpacas and whales, were excluded from the data set as the number of spike sequences for each host is insufficient for establishing a separate group. spike sequences were computed using our original python scripts. dinucleotide bias is the ratio of the observed value to the expected frequency of each of the dinucleotides: where ρ xy is the dinucleotide bias, f xy is the frequency of dinucleotide xy, f x and f y are the frequencies of nucleotide x and nucleotide y , respectively. in this study, we considered factors, including three mononucleotide frequencies (g, c and t) and dinucleotide biases. as none of the frequencies has a normal distribution, the nonparametric "kruskal-wallis test" was employed to investigate the difference in each factor among six categories. as a result, significant differences across categories were detected for each factor; thus, all factors were employed for modelling. modelling, validation and prediction. as a classifier, the svm can efficiently perform a nonlinear classification using a kernel technique that is rooted in structural risk minimization. in this study, the r package e (version: . - ) was employed for the svm analysis. "c-classification" was adopted as the model type and "radial" was adopted as the svm kernel in our analysis. the md is a measure of the distance from a point to the centre of a distribution; the principle of this discriminant is that individuals belong to the closest group in the distance. the md is defined as , where x denotes the population, x denotes the individual, μ is the mean value of the population, t denotes the matrix transpose, and ∑ denotes the covariance matrix of population . the r program "distinguish.distance.r" was employed in the md analysis. leave-one-out cross-validation was employed for both svm and md analyses. when the trained models are applied to a sequence for testing, each of the six categories of hosts will obtain a p value from svm and a md value. based on p values and md values, three steps will be taken to determine candidate hosts. first, the host of minimal p value or md value is reasonably regarded as the preferable host. then, two adjustable empirical thresholds can be used for each model to pick out other potential hosts. in this study, we adopted . and . for p value, and for md value; i.e., likely hosts were determined if p < = . or md < = , and very likely hosts were defined by p < = . or md < = . the two steps are unsupervised prediction. in case that the isolate source is among the six host groups for modelling, a supervised prediction can be applied as the third step, i.e., all host species with p values or md values no more than those of the observed host will be listed as potential hosts, which can be practical references for researchers to evaluate a virus's threats to human or other animals. compare the tendencies of md and svm in predictions. two groups of two-dimensional vectors were generated in silico as two populations. the number of vectors in the first population are randomly generated from the normal distribution n( , ), and the number of vectors in the second population are randomly generated from n( . , . ). as the first population has a larger standard deviation (sd), we refer to it as the "loose" population and refer to the second population as the "tight" population. the two groups of data are employed for the leave-one-out cross-validations of md and svm. all python and r scripts employed in this study are available from the authors upon request. the prediction can be performed using the spike gene sequences of the coronaviruses on our web server, which is available to the public at no cost at http://bioinfo.ihb.ac.cn/seq hosts. interspecies transmission and emergence of novel viruses: lessons from bats and birds virus taxonomy, the ninth report of the international committee on taxonomy of viruses genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus a decade after sars: 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and codon usage bias in the genus flavivirus dinucleotide and stop codon frequencies in single-stranded rna viruses on a class of pattern-recognition learning algorithms support-vector networks support vector machines-the interface to libsvm in package e support vector machine classification and validation of cancer tissue samples using microarray expression data the mahalanobis distance use of nucleotide composition analysis to infer hosts for three novel picorna-like viruses compositional differences within and between eukaryotic genomes cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human natural mutations in the receptor binding domain of spike glycoprotein determine the reactivity of crossneutralization between palm civet coronavirus and severe acute respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans structure of mers-cov spike receptor-binding domain complexed with human receptor dpp sars-associated coronavirus transmitted from human to pig isolation and characterization of a bat sars-like coronavirus that uses the ace receptor analysis of the genome sequence of an alpaca coronavirus mutation rates among rna viruses genomic signatures of human versus avian influenza a viruses adaptive mutations in nep compensate for defective h n rna replication in cultured human cells virus-specific factors associated with zoonotic and pandemic potential discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event over-representation and under-representation of short oligonucleotides in dnasequences mahalanobis distance. resonance statistical modeling and r software this work was supported by the -talent program grant from the chinese academy of sciences. q.t. designed the research, collected and analyzed data, and wrote the paper. y.s., m.s., y.c. and w.z. analyzed data, x.-q.x. designed the research, re-analyzed data, wrote the paper, and created the web server. all authors reviewed the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord- -ggo wsrm authors: huang, stephen s. h.; lin, zhen; banner, david; león, alberto j.; paquette, stéphane g.; rubin, barry; rubino, salvatore; guan, yi; kelvin, david j.; kelvin, alyson a. title: immunity toward h n influenza hemagglutinin of historical and contemporary strains suggests protection and vaccine failure date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ggo wsrm evolution of h n influenza a outbreaks of the past years is interesting and significantly complex and details of h n genetic drift remains unknown. here we investigated the clinical characteristics and immune cross-reactivity of significant historical h n strains. we infected ferrets with h n strains from , , , , , and and showed each produced a unique clinical signature. we found significant cross-reactivity between viruses with similar ha sequences. interestingly, a/fortmonmouth/ / antisera cross-reacted with a/ussr/ / virus, thought to be a resurfaced virus. importantly, our immunological data that didn't show cross-reactivity can be extrapolated to failure of past h n influenza vaccines, ie. , and . together, our results help to elucidate h n immuno-genetic alterations that occurred in the past years and immune responses caused by h n evolution. this work will facilitate development of future influenza therapeutics and prophylactics such as influenza vaccines. t he major challenge in regard to influenza surveillance and management is the propensity of the influenza virus to mutate, altering its immunogenic properties thereby allowing it to evade immune recognition and cause disease. influenza a is classified according to the specific combination of its two surface molecules, hemagglutinin (ha) (h -h ) and neuraminidase (na) (n -n ) isotypes - and its diversity is attributed by two mechanisms: genetic mutation or by gene reassortment , . typically genetic mutation is responsible for seasonal influenza outbreaks and the emergence of influenza pandemics is a consequence of gene reassortment among different strains and subtypes [ ] [ ] [ ] . since the extensively documented influenza pandemic in - , there have been a total of five influenza pandemics that have resulted in millions of deaths worldwide, where the fatality rate has reached more than . % as in the case of pandemic , . in , h n surfaced and replaced h n in the human population until when h n resurfaced, leading to co-circulation of the influenza subtypes. compared to h n , the h n subtype is believed to have a lower rate of antigenic drift which is associated with a smaller amount of mutations leading to amino acid changes . importantly the evolutionary behaviours of h n and h n are divergent leading to a dynamic and ever changing influenza climate in the human population where one virus subtype typically dominates over the other , . throughout the year history of h n , the specific clinical parameters and immunogenic response to the genetic drift of h n remains to be clarified. subsequent to the h n pandemic were several h n epidemics occurring from the 's to the late 's [ ] [ ] [ ] which was followed by a year disappearance and a re-emergence in the 's . in a significant antigenic change transpired in the h n virus creating strain distinct from previous viruses . the new h n virus termed ''a-prime'' was relatively mild although widespread (a pseudopandemic) and hypothesized to be a reassortant from two distinct h n strains , , . an unusual h n virus emerged in that was associated with severe disease: also thought to be a reassortant virus with genes from novel viruses and older h n segments from the 's . succeeding a year h n disappearance, two h n epidemics of interest came about, including the children's pandemic and a swine flu epidemic in that was feared to have pandemic potential and led to a massive public vaccination strategy . the epidemic had limited infectivity to the immunologically naïve younger population (persons , years of age) which is thought to be due to the similar circulating h n viruses of the 's . in a substantial change in the h n virus occurred, unlike previous modifications, which allowed the virus to spread rapidly throughout the globe and prompted the who to declare a pandemic on june . genomic analysis determined the virus was of swine origin and contained a triple reassortant of swine, human and avian influenza a genes . unlike previous contemporary seasonal influenza outbreaks, the h n pdm had age-related disparities in the frequency and severity of infection where the older age groups were less susceptible to the disease , . furthermore, these differences in age-related severity are hypothesized to be due to previous exposure to older h n viruses with similar antigenic epitopes , . after influenza exposure, the body generates antibodies against the specific influenza strain it has encountered. anti-ha production is often associated with immunity to the same or homologous influenza strains and some antibodies have virus neutralizing ability blocking viral entry to the host cell , . importantly, previous exposure to influenza viruses influences how the body will respond to a subsequent influenza infection of the same or different genetic subtype . to date, little is known about the immune and clinical response to h n influenza viruses of the past years and how the h n subtype genetic drift and shift affected immune crossreactivity. previously nelson et al., conducted a large scale genetic analysis of h n sequences to determine the evolutionary history of this virus since . in our present study, we investigated the clinical characteristics and immune cross-reactivity of significant h n influenza strains in the past years in ferrets to determine the immunogenicity of important h n viruses. we infected ferrets with historical h n strains from , , , , , and and monitored them for a -day time course to determine the clinical picture of each influenza strain infection and immune crossreactivity. our findings clarify the influenza clinical picture of historical h n strains and the help to elucidate the antigenic alterations of h n that occurred in the past years. the work here will facilitate the understanding of the immune response toward h n and with future work may aid the development of future influenza therapeutics and anti-viral treatments. ferrets infected with historical h n strains show mild clinical symptoms. the ferret, mustela putorius furo, is a superlative animal model for respiratory infections and the influenza ferret infectome has recently been published [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ferrets show respiratory illness similar to humans and clinical features of disease are easily observed where fevers can persist days following infection of viruses such as h n pdm influenza , , . as well as fever, nasal discharge and sneezing can also be observed in animals infected with influenza viruses these influenza a viruses were chosen due to their emergence and influence in h n genetic history (fig. , strains used in this study are marked with an asterisks) as covered in the introduction. following infection, ferrets were monitored for body temperature, weight, inactivity level, sneezing and nasal discharge from each group were observed daily until day post-infection (pi). infection by all strains produced an increase in temperature; the normal range for ferret temperature is indicated by the shaded area of each graph ( supplementary fig. s a ). the pandemic h n strain ny/ induced the greatest fever on day to a temperature of % (of baseline). ncal/ and marton/ infection also caused a high temperature of % from baseline, which peaked on day and day pi, respectively ( table ) . ussr/ and taiwan/ had moderate fevers and fm/ had the smallest increase in temperature reaching only % above baseline ( table ) . analysis of weight loss showed that animals infected with of all viruses except pandemic ny/ were able to recover to original weight or greater following infection (supplementary fig. s b and table ). ny/ infected ferrets had the most significant weight loss compared to normal weight fluctuations (shaded area) which peaked at % of baseline weight on day and day . ussr/ and ncal/ reached less than % and % of baseline weight, respectively, on day pi ( table ) . infection with taiwan/ produced the smallest amount of weight loss and animals infected with fm/ did not lose any weight at all ( table ) . secondary clinical signs were also measured and analysed for all infections, including nasal discharge, sneezing, and inactivity level (supplementary table s ). ussr/ infected ferrets had the highest amount of nasal discharge and ny/ ferrets had the greatest amount of sneezing and lethargy. taken together, analysis of the complete clinical signs for each h n strain infection suggested a unique clinical picture for each strain: ny/ infection had the most severe with significant increase in temperature and an unrecovered weight loss compared to mildest strain, fm/ , which did not produce any weight loss and only a slight increase in temperature. analysis of ha immunogenicity in historical h n strains. the ha protein, a homotrimeric protein that functions in viral entry into host cells , and antibodies reactive toward ha have been associated with host resistance and a decrease in disease severity [ ] [ ] [ ] . it has been shown that % of antibodies produced during an influenza infection are reactive toward the ha protein . after determining the clinical signatures of ferrets infected with historical and contemporary h n viruses, we went on to analyze the aspects of h n ha immunogenicity. the hemagglutination inhibition (hi) assay, is a common assay used to determine the reactivity and/or amount of antibody to the ha protein produced during a viral infection and the cross-reactivity of antibodies raised toward these specific historically important h n viruses has not been investigated. to determine the crossreactivity produced by infection among the historical and contemporary influenza strains, we used the harvested antisera from each ferret strain infection and perform hi assays with our panel of table s ) . interestingly, ferret anti-sera toward the marton/ virus showed hi with its own virus and with the older pr/ but not with its nearest chronological neighbour fm/ (fig. a) . fm/ and ussr/ antisera recognized its respective virus and each other ( fig. b and c) . taiwan/ and h n pdm (ny/ or cal/ ) antisera both only reacted with their respective viruses ( fig. d and h). ncal/ antisera only showed hi with its own ncal/ virus (fig. e) . unexpectedly, the si/ and bris/ antisera were able to inhibit the ncal/ virus ( fig. f and g) . furthermore, si/ antisera also cross-reacted with the bris/ virus and the bris/ antisera recognized the si/ virus. taken together, these results showed antigenic cross-reactivity to infection among historical and contemporary h n influenza strains. this data suggested similarities of the host immune response between marton table s ). importantly, no detection was observed for the tawain/ virus and earlier viruses with the who h n seasonal control. taken together, this work suggested that the who seasonal h n antisera is only capable of detecting infection from recent seasonal h n strains. we investigated the phylogenetic relationship and sequence homology of the ha gene among the h n viruses used in this study and included other strains implicated in important historical h n outbreaks. the temporal structure of phylogenetic tree was comprised of groups separated by long braches (fig. a) . the pandemic strains and pandemic isolates each formed their own distinct cluster. the middle cluster contained epidemic isolates. within this cluster, fm/ was closer to ussr/ than marton/ which was more similar to pr/ . the latest strains of seasonal h n , ncal/ , si/ and bris/ , were positioned as a clade which was separated from the early stains by a long branch and clustal alignment of these three viruses ( supplementary fig. s ) showed several amino acid changes from ncal/ and conserved between si/ and bris/ : a v, t k, y h, k e, v a, r k, w r, t n, and n d. we calculated the protein homology by using the most variable region of the ha gene (ha - ) which covers most of the viral surface and sequence change in this area has a strong impact in immunogenicity . we found that % and higher homology scores led to hi cross-reactivity in all the cases. on the other hand, crossreactivity with lower homologies of - % may or may not take place (fig. b) . taken together, our results suggested an important relationship and cross-reactivity between fm/ and ussr/ as well as the ncal/ , si/ and bris/ group that can be related to the genetic ha sequence. further analysis of other influenza genes may also be important to the understanding of h n evolution. the h n influenza subtype is a significant viral agent affecting public health that has been responsible for pandemics and several influenza epidemics globally. here we investigated the clinical response and subsequent immunogenicity following infection with historically and contemporarily relevant h n strains in ferrets. importantly, we showed that infection with each strain produced a unique clinical signature which may be indicative of disease that affected humans. we also found significant cross-reactivity between viruses with similar ha aa sequences which can be extrapolated to protection or no protection following a subsequent infection with another h n strain. these results are the first to compare the these results add to the understanding of how the immune system responds to an evolving virus. in the current study we analyzed the clinical parameters of evolutionary h n strain infection in ferrets which has not been previously compared experimentally. our results of the evolutionary h n strains showed that infection with the fm/ resulted in the . these records support our findings, where animals infected with fm/ had the lowest percent nasal discharge, mildest fever and was the only virus infection which did not lead to weight loss. furthermore, ussr/ infection produced a moderate disease comparable to the reports of a mild clinical disease . our analysis of the clinical course of taiwan/ infection in ferrets showed a moderate disease consisting of a mild biphasic temperature increase and minimal weight loss. these observations were in agreement with clinical disease reported during the naval base outbreak of influenza which there was no deaths but patients described fever, non-productive cough, sore throat and myalgia . furthermore, in accordance with our ny/ data which had the most significant temperature fluctuations and weight loss, analysis by shrestha and colleagues demonstrated that h n pdm was more severe than the previous circulating seasonal influenza viruses . evolutionary relationships among historical influenza h n strains were conducted in mega using the maximum likelihood method and bootstrap replications (a). antibody cross-reactivity (determined by hi) among influenza strains is indicated by using the same colors. the numerical values displayed next to the branches indicate the percentage of bootstrap replicates in which the associated sequences clustered together. hemagglutinin protein sequence homology among several influenza strains (b). influenza strains were grouped according to the hi cross-reactivity observed in the serum from previously-infected ferrets (groups a through f). alignments of protein sequences were performed using the highly variable ha - region, which belongs to the ha-rbd area. the influenza virus was first identified in and since then the epidemiology and phylogentic analysis of subsequent influenza epidemics and pandemics have been extensively investigated , , . importantly, our results build on the previous investigations as we report the immunogenic evolution of the ha protein of significant pandemic and epidemic strains. the epidemic isolates examined by our phylogenetic analysis showed the and h n pdm strains to comprise each their own cluster and the seasonal strains ranging from to to cluster together. as expected by chronological analysis, our results showed marton/ to cross-react with its predecessor pr/ which was in agreement with the phylogenetic analysis that placed the ha of marton/ close to pr/ in the tree. extensive phylogenetic analysis preformed by nelson mi and colleagues suggested the strain to be a reassortant from two previous h n viruses combining the pb , na and m segements from a virus with the pb , pa, ha, np and ns segments from an unknown h n virus . furthermore, they showed that the ha region of the ha was significantly different from the ha of the earlier 's strains . in accordance with this analysis, our results showed that the fm/ antisera did not inhibit hemagglutination with the earlier pr/ and marton/ viruses and the marton/ antisera did not cross-react with the fm/ virus. the inability for immunity induced by marton/ virus infection to target the fm/ virus compliments the influenza vaccine performance in the human population of the 's as well as previously published studies . specifically, a related h n virus strain (a/weiss/ ), as well as a/pr/ / and b/lee/ were used in the influenza vaccine preparation which was completely ineffective during the ' influenza outbreak , . although fm/ antisera did not recognize the earlier h n strains, it did cross-react with the ha molecule of the ussr/ virus as did the ussr/ antisera with the fm/ virus. in support of these findings, the epidemic was thought to be the resurfacing of the fm/ strain . the ussr/ outbreak was known as 'the children's pandemic' since only persons younger than years old were affected . during this time, older individuals were considered primed for the outbreak strain by previous infections from the 's and 's which is supported by our immunological evidence. together, our results, the phylogenetic analysis and the epidemiological records suggest the triggering of a unique immune response following infection with marton/ and fm/ and a similar response from infection with the related strains fm/ and ussr/ . in the fall of a significant outbreak of influenza occurred on a us naval base which was found to be caused by strains that had been circulating in asia since the spring of that year , [ ] [ ] [ ] . taiwan/ was isolated from this outbreak and genomic analysis of its ha have suggested that the taiwan/ virus had evolved from viruses circulating in the early 's in hong kong . furthermore, it has also been suggested that this virus was the product of a reassortment event that had occurred between two h n viruses . our results showed that the taiwan/ strain shared lower homology to , and viruses seen on our phylogenetic tree. the immunogenic findings showed antisera produced from ferret infection with taiwan/ was not able to inhibit hemagglutination with the other viruses on our virus panel, which compliments the literature describing the strain. the influenza vaccine in included the a/chile/ / influenza strain . interestingly, during the us naval base outbreak, the soldiers who were vaccinated were poorly protected from infection with taiwan/ . furthermore, it was also shown that antibodies produced by vaccination with the a/chile/ / vaccine preparation were not able to provide hemagglutination inhibition toward the newly arisen taiwan/ and taiwan/ -like viruses . from this evidence, it was then recommended that the taiwan/ strain be included in the subsequent influenza monovalent vaccine preparation , . our findings together with the previous reports on the taiwan/ outbreak suggest that the viruses were an antigenically unique group of strains that induced a specific immune response important when considering the evolution of h n immunity. it can be argued that the h n virus underwent minor genetic drift in the year span from to which can be evidenced by the consistent inclusion of the ncal/ in the influenza vaccine preparation , . our genetic analysis showed the three contemporary h n seasonal viruses, ncal/ , bris/ and si/ , to be proximal to each other on the phylogenetic tree with a long separation from the previous taiwan/ virus and in a distinct cluster from the h n pandemic viruses. moreover, bris/ and si/ were closer to each other than they were to ncal/ . interestingly, the antisera generated by infection with ncal/ did not cross-react with the other two contemporary seasonal h n viruses although the si/ and bris/ antisera recognized the ncal/ virus. these results suggested there to be an amino acid change in bris/ and si/ from ncal/ that may have resulted in a new antigenic site that was not originally present in ncal/ strain. to investigate this contention we generated a clustal alignment of these viruses which showed numerous amino acid changes between bris/ and si/ sequences with ncal/ . these results may indicate the gradual yet still present drift of the h n virus in this time period. as well, the immunogenic evolution seen in our analysis may question the consistent usage of the ncal/ virus in influenza vaccine preparations from - to - , . the most significant emergent in h n history since ensued in late spring and was declared a pandemic shortly after . the ha from newly emerged h n virus was identified to be similar to the ha of viruses isolated from north american swine which had recent and historical precedence . this virus was designated h n pandemic (h n pdm) and was shown to be a triple reassortant virus containing genes from swine, human and avian influenza a viruses . in our phylogenetic analysis it was clearly evident that the triple reassortant viruses clustered far from the previously circulating seasonal h n viruses in agreement with similar alignments. furthermore, hi assays performed using ferret antisera produced raised against the ny/ h n pdm antisera was not able to recognize the ha of any other viruses. these results are supported by previous published findings that determined the cross-protection initiated following infection with h n pdm which was preceded by infection with contemporary seasonal h n did not occur through an ha targeted antibody response . interestingly, although an h n component had been included in the seasonal influenza vaccine preparations since , antigen directed pre-existing immunity to h n pdm was only detected in the elderly suggesting the majority of the current human population was naïve to the this novel virus , . cross-reactive antibodies found in the elderly population suggested that protective immunity was induced from exposure to an older h n virus with antigenic similarities to h n pdm , - . our study directly tested the ability of infection with older h n strains from the years to to bind the ha molecule of the h n pdm virus. against current hypotheses, we found that the immune production of antisera from infection with viruses originating from the 's were not able to recognize the ha of the novel pandemic viruses. likewise, the h n pdm antisera did not detect the panel of viruses ranging from to . recently, the effect of priming ferrets with historical h n viruses on the immune response of secondary infection the h n pdm was described , . these studies addressed distinct immunological questions as our study utilizing a different set of h n viruses. these priming studies complimented our study as the historical h n strains used were not the same as ours. the purpose of the previous studies was to determine protection from priming infection/infections whereas we queried immune detection, cross-reactivity and clinical analysis. importantly, a significant issue that remains is the affect of age on the outcome of infection/ www.nature.com/scientificreports scientific reports | : | doi: . /srep sequential infection with the historical and contemporary viruses. historical accounts mention clinical pathology among age groups but do not have a comprehensive analysis of the comparative severity , , , . as well, it is not know how the aging immune system responds to the evolving influenza virus. although a protective response was seen in priming studies in adult animals, it is not know if protection of the same magnitude would remain during age related immunosenescence or if the clinical response in the aged would be similar as we have shown in this study. influenza a h n and influenza a h n have varying patterns of antigenic and genetic evolution where specifically h n has a lower rate of acquired mutations , . these evolutionary behaviours are divergent leading to a dynamic and ever changing influenza climate in the human population where one influenza a subtype typically dominates over the other. importantly, understanding the interplay between the h n and h n infection evolution remains a significant hurdle in influenza reasearch , . nelson and colleagues investigated the genetic evolution of the h n virus by analyzing the evolution of the whole virus as well as the individual viral segments from strains since . in this study the authors were able to identify instances where divergent clades co-circulated as well as events of intra-h n subtype reassortment . previously, smith and colleagues compared the antigenic and genetic evolution of the h n virus from strains originating in to and found that antigenic and genetic evolution showed differing patterns . antigenic evolution was punctuated where the strains formed clusters and genetic change was more even and gradual as time increased . here we sought to elucidate the antigenic evolution of the h n virus. as with the h n virus , we found the antigenic change did not vary evenly by year but instead corresponded to genetic changes in the ha molecule. these studies are significant for elucidating the intricacies of influenza evolution as the continuous change of the influenza virus is the greatest challenge to the management of disease and containment of further outbreaks. a better understanding of the evolution of the influenza virus and the interplay between the h n and h n strains will improve the design of prophylactics such as by guiding vaccine strain selection for yearly trivalent influenza vaccine or may even give insight to the development of a universal influenza vaccine. more work is needed to understand the true nature of influenza virus dynamics and evolution. animal models remain the most appropriate mode for the methodical scientific investigation of human influenza virus pathogenesis and the testing of influenza prophylactics and therapeutics. currently, the ferret is thought to be the most suitable animal model for respiratory influenza virus infection since ferrets are physiologically susceptible to wild type influenza viruses due to a similar respiratory tract and are able to transmit the virus once infected. as mentioned previously, ferrets display similar clinical disease as humans following influenza infection which include fever, weight loss, and sneezing and these positive features have been well discussed previously , , , , . as well, the ferret has been shown to have similar features as human in regard to ifn pathway function and is potential model for the study of human ifn-gamma signalling , . although the ferret is an appropriate model, it is imperative to be mindful of the disparities between animal models and the nature human infection to have an accurate assessment of research findings especially from ferret-influenza studies. during experimental influenza infections in animal models, a specific and known amount of virus is administered to the animal in a controlled manor, usually by intranasal inoculation. modeling the infectious behaviour of a virus is more predictable when the infection route and dose are controlled. this is in contrast to natural infection where humans are infected with an unknown amount of virus in an unknown route, which must be considered when extrapolating results from animal model studies. most significantly, ferrets as well as other animal models used in influenza studies are typically 'specific pathogen free' (spf). in this case, the ferrets are determined to be influenza free prior to study initiation. unlike humans who have been previously exposed to various subtypes of influenza viruses, ferrets used in influenza studies are completely naïve to the virus. the nuances of the immune response subsequent to multiple influenza infections, such as the phenomenon of original antigenic sin , has only started to be elucidated in the ferret model and much work is still required to have a full understanding of the effect of sequential influenza infection on the specific immune response mounted. furthermore, since the influenza virus is subject to both genetic shift and genetic drift , the clinical manifestations of sequential virus infections by a virus that undergone multiple genetic drifts would differ from a response of a virus that had undergone a significant genetic shift as that of the h n pandemic variant. influenza virus infection has been thought to plague the human population for hundreds of years, where symptoms of influenza can be traced back in the writings of the early greek cultures to bc . as many questions still exist in relation to evolution and emergence of influenza strains, we analyzed the immunological evolution of past and contemporary influenza viruses. our results shed light on the cross-protective immune response toward the ha molecule and suggest that unique immunity is induced dependent on the ha sequence. further work is needed to determine the evolutionary placement of the and virus immunogenicity, as well as the placement of newly emerging influenza strains. our results describing the antigenic evolution of the h n influenza subtype together with previous work will add to the understanding of the transcendence of influenza viruses and have implications on the design of future influenza vaccines. ethics statement. all animal work was conducted in strict accordance with the canadian council of animal care (ccac) guidelines. the university health network (uhn) has certification with the animals for research act (permit number: # and # of the ontario ministry of agriculture, food and rural affairs) and follows nih guidelines (olaw #a - ). the animal use protocol was approved by the animal care committee (acc) of the uhn. infections and subsequent sample collection were performed under % isofluorane anesthesia in an effort to minimize suffering. infection and ferret monitoring. maintenance and monitoring of infected ferrets has been previously described . briefly, male ferrets - months old were bred in an on-site spf ferret colony (university health network, toronto, on, canada). prior to infection, all ferrets were screened for influenza and shown to be seronegative by hi assay against circulating influenza a and b strains ( - who influenza reagent kit for identification of influenza isolates (who collaborating center for surveillance, epidemiology and control for influenza infection division)). the kits contain the circulating influenza strains for the particular year. prior to infection, ferrets were randomly selected and pair-housed in cages contained in bioclean portable laminar-flow clean-room enclosures (lab products, seaford, de) in the bsl- animal holding area. baseline body temperature and weight were measured on day for each animal. temperatures were measured by using a subcutaneous implantable temperature transponder (biomedic data systems, inc., seaford, de). upon infection, ferrets were anesthetized and infected ml of virus preparation for each ferret ( . ml in each nostril). ferrets were infected with marton/ (n ), fm/ (n ), ussr/ (n ), taiwan/ (n ), ncal/ (n ) , and ny/ (n ). all viruses were used at eid except for fm/ which was infected at eid since it was reported to be a milder virus . clinical signs (body temperature, body weight, level of activity, nasal discharge, and sneezing) were observed daily for days pi. we examined animals at the same time each day for consistency. nasal discharge includes crusty nose, mucous, and transparent exudates/ fluids. the scores were calculated from the total animals displaying any nasal discharge symptom over the total number of animals. the sneezing scores were calculated from the total animals found sneezing over the total number of animals. scores were calculated daily for days and only the peak values for each infection are www.nature.com/scientificreports scientific reports | : | doi: . /srep summarized. the inactivity scoring system is based on the reference reuman et al., to assess the inactivity level: , alert and playful; . , alert but playful only when stimulated; , alert but not playful when stimulated; , neither alert nor playful when stimulated. a relative inactivity index was calculated as follows: s (day to day ) [score ] n /s (day to day ) n, where n equals the total number of observations. a value of was added to each observation unit score so that a score of could be divided by a denominator, resulting in an index value of . as the minimum value. determination of influenza specific antibody responses. influenza specific antibody responses from the uninfected or infected ferrets were measured by hi as previously described . briefly, receptor destroying enzyme ([rde], accurate chemical & scientific corp., westbury, ny, usa) treated ferret anti-sera was serially diluted and hi titers were determined by the highest dilution that completely inhibited influenza hemagglutination ( hau) of turkey erythrocytes. comparative analysis of hemagglutinin sequences. phylogenetic analysis of influenza hemagglutinin dna sequences were conducted in mega using the maximum likelihood method based on the tamura-nei model , bootstrap repetitions were performed. to analyze the protein similarity of influenza hemagglutinin from different h n strains, the aminoacid sequences from the protein region ha - were aligned using clustalw . this region of the ha protein is part of the hemagglutinin receptor binding domain (ha-rbd), which is located in the external surface of the virus and it concentrates most of the antigenic potential of the ha protein . amino acid ha genbank accession numbers for clustal alignment (supplementary fig. s ): a/newcaledonia/ / (h n ) amino acid ha genbank accession numbers for homology table (fig. b): a/brevigmission/ / (aad ) a/puertorico/ / (caa ) a/taiwan/ / (abf ) a/ newcaledonia/ / (abf ) genbank accession numbers for the phylogenetic analysis of ha dna sequence (fig. a): a/pr/ / (v ) a/brevigmission/ / (af ) a/south carolina/ / (af ) a/fortmonmouth/ / (cy ) a/ussr/ / (cy ) seasonal, avian, and novel h n influenza: prevention and treatment modalities characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls a distinct lineage of influenza a virus from bats rna virus genomics: a world of possibilities a (h n ) influenza virus pandemic: a review world health organization (who) pathogenesis of the pandemic influenza virus mapping the antigenic and genetic evolution of influenza virus multiple reassortment events in the evolutionary history of h n influenza a virus since development of influenza epidemics trends and epidemics of influenza and pneumonia: - on the origin of the human influenza virus subtypes h n and h n importance of antigenic composition of influenza virus vaccine in protecting against the natural disease; observations during the winter of - influenza pandemics of the th century world health organization (who) new influenza a(h n ) virus: global epidemiological situation triple-reassortant swine influenza a (h ) in humans in the united states protection of mice against lethal challenge with h n influenza a virus by -like and classical swine h n based vaccines response to a monovalent influenza a (h n ) vaccine who manual on animal influenzadiagnosis and surveillance seasonal h n influenza virus infection induces cross-protective pandemic h n virus immunity through a cd -independent, b cell-dependent mechanism modeling host responses in ferrets during a/california/ / influenza infection differential pathological and immune responses in newly weaned ferrets are associated with mild clinical outcome of pandemic h n infection comparative analyses of pandemic h n and seasonal h n , h n , and influenza b infections depict distinct clinical pictures in ferrets ferret tnf-a and ifn-g immunoassays in trends in immunolabelled and related techniques transmission and pathogenesis of swine-origin a(h n ) influenza viruses in ferrets and mice the current state of h n vaccines and the use of the ferret model for influenza therapeutic and prophylactic development lack of innate interferon responses during sars coronavirus infection in a vaccination and reinfection ferret model sequencing, annotation, and characterization of the influenza ferret infectome early gene expression events in ferrets in response to sars coronavirus infection versus direct interferon-alpha b stimulation pathogenesis of avian influenza a (h n ) viruses in ferrets influenza virus assembly and budding inactivated influenza vaccines. . laboratory indices of protection antibody response in man to influenza virus neuraminidase following influenza determinants of immunity to influenza infection in man influenza virus antigenic variation, host antibody production and new approach to control epidemics an influenza a/h n / hemagglutinin vaccine produced in escherichia coli age distribution of patients with medically-attended illnesses caused by sequential variants of influenza a/h n : comparison to age-specific infection rates, - an outbreak of influenza a/taiwan/ / (h n ) infections at a naval base and its association with airplane travel estimating the burden of pandemic influenza a (h n ) in the united states a history of influenza the total influenza vaccine failure of revisited: major intrasubtypic antigenic change can explain failure of vaccine in a post-world war ii epidemic cdc update: influenza activity-worldwide. mmwr cdc antigenic variation of recent influenza a(h n ) viruses. mmwr cdc recommendation of the immunization practices advisory committee monovalent influenza a(h n ) vaccine sequence analysis of the haemagglutinin of a/taiwan/ / , a new variant of human influenza a(h n ) virus the evolution of human influenza viruses cdc update: influenza activity -united states and worldwide, - and composition of the - influenza vaccine antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans prior infections with seasonal influenza a/h n virus reduced the illness severity and epidemic intensity of pandemic h n influenza in healthy adults cross-reactive antibody responses to the pandemic h n influenza virus incidence of pandemic influenza a h n infection in england: a cross-sectional serological study in vitro and in vivo characterization of new swine-origin h n influenza viruses sequential seasonal h n influenza virus infections protect ferrets against novel h n influenza the effect of priming with h n influenza viruses of variable antigenic distance on challenge with pandemic h n virus failure of influenza vaccine to prevent two successive outbreaks of influenza a h n in a school community the ferret as a model organism to study influenza a virus infection cloning, expression and characterization of ferret cxcl understanding original antigenic sin in influenza with a dynamical system assessment of signs of influenza illness in the ferret model mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees clustal w and clustal x version . we thank the li ka-shing foundation of canada, immune diagnostics & research, and shantou university medical college for the support of conducting this study. a/mexico/ / virus was obtained through the influenza reagent resource, influenza division, who collaborating center for surveillance, epidemiology and control of influenza key: cord- -q kgzuob authors: choi, jeongan; kang, miran; jung, jae hee title: integrated micro-optofluidic platform for real-time detection of airborne microorganisms date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: q kgzuob we demonstrate an integrated micro-optofluidic platform for real-time, continuous detection and quantification of airborne microorganisms. measurements of the fluorescence and light scattering from single particles in a microfluidic channel are used to determine the total particle number concentration and the microorganism number concentration in real-time. the system performance is examined by evaluating standard particle measurements with various sample flow rates and the ratios of fluorescent to non-fluorescent particles. to apply this method to real-time detection of airborne microorganisms, airborne escherichia coli, bacillus subtilis, and staphylococcus epidermidis cells were introduced into the micro-optofluidic platform via bioaerosol generation, and a liquid-type particle collection setup was used. we demonstrate successful discrimination of syto -dyed fluorescent bacterial cells from other residue particles in a continuous and real-time manner. in comparison with traditional microscopy cell counting and colony culture methods, this micro-optofluidic platform is not only more accurate in terms of the detection efficiency for airborne microorganisms but it also provides additional information on the total particle number concentration. may not satisfy the specific growth requirements of all species of interest, and also requires prolonged incubation (i.e., > hours) . recently, detection and enumeration of airborne microorganisms has been accomplished using techniques based on polymerase chain reaction (pcr) and enzyme-linked immunosorbent assays (elisa), which are highly sensitive and quantitative techniques [ ] [ ] [ ] [ ] [ ] . however, additional pretreatment processes (such as particle condensation/purification), and elaborate sample handling by well-trained operators in a clean environment are required . although these pcr and elisa techniques have been applied to microfluidic chips to simplify the treatment processes, [ ] [ ] [ ] most such microfluidic systems have been developed as disposable chips for point-of-care diagnosis of target microorganisms, which often leads to a limited number of samples or poor accuracy or rates of detection . novel real-time methods for the rapid detection and continuous monitoring of airborne microorganisms have the potential to address the public health problems associated with bioaerosols. one of the most frequently used real-time detection techniques for detection of airborne microorganisms exploits auto-fluorescence following illumination with ultraviolet (uv) light , . auto-fluorescence is caused by metabolites and structural components of living cells . although this technique allows continuous real-time monitoring and detection of bioaerosols directly in the air stream, problems remain due to low fluorescence intensity, which leads to poor limits of detection, and requires precise optical systems for the measurements. for these reasons, such devices are typically not portable, and cannot provide an integrated "micro-total analysis system". to overcome the limitations discussed above, cell and particle detection methods based on microfluidic flow-cytometry have been reported [ ] [ ] [ ] [ ] . microfluidics benefits from miniaturization, low power consumption and high sensitivity. microfluidic methods of counting water-borne microorganisms include tracking the population of marine algae and quantification of bacterial cells using pre-staining [ ] [ ] [ ] . chung et al. ( ) developed a real-time single-cell detection system using target aptamer-conjugated fluorescent nanoparticles in a microfluidic flow-cytometry platform for microbial diagnostic applications . however, there have been no reports of micro-optofluidic platforms for real-time continuous detection and monitoring of airborne microorganisms. here, we demonstrate a micro-optofluidic platform for real-time detection and quantitative analysis of airborne microorganisms. our optofluidic system involves the following steps: ( ) sampling of airborne microorganisms; ( ) mixing and reacting in a microchannel for staining; and ( ) real-time detection and analysis of the particle by means of light scattering (sc) and bacterial fluorescence (fl). from these optical signals, we may discriminate and quantify airborne microorganisms, enabling simultaneous measurement of the total particle concentration. the performance in terms of particle detection is evaluated using standard particles and three bacterial taxa, and is compared with that of conventional microscopy cell counting and colony counting methods. design and operation of the micro-optofluidic platform. figure shows a schematic diagram of the micro-optofluidic platform. the -μ m-deep polydimethylsiloxane (pdms) microfluidic channel was fabricated using conventional soft-lithography processes . the micro-optofluidic platform is based on single-layer pdms channel and has four inlets (one sample inlet, one dye inlet, and two hydraulic focusing sheath inlets), and one outlet for waste. it consists of four main components: sample and dye inlets, a mixing region, a hydraulic focusing zone, and a sample optofluidic detection component (see fig. s ). we used syto dye (molecular probes inc., eugene, or, usa) with an excitation wavelength of nm and an emission wavelength of nm for nuclear staining of live cells in the microfluidic mixing zone. syto provides cell-permeant nucleic acid staining and exhibits low intrinsic fluorescence in cell-free systems, with marked enhancement upon binding to deoxyribonucleic acid (dna) or ribonucleic acid (rna) , . following injection of the sample and dye using a syringe pump (kds ; kd scientific inc., holliston, ma, usa), the two fluids mixed rapidly at the entrance to the mixing zone. numerical analysis was carried out using the commercial computational fluid dynamics (cfd) software package cfd-ace (esi us r&d inc., huntsville, al, usa) to identify the optimal design and operating conditions. the navier-stokes equations were solved with fick's law of diffusion to describe the dynamics and homogenization of the mixing process in the channel. figure (b- ,b- ) show the results of the cfd simulations and optical micrographs of the mixing of the sample and dye in the micro-mixer part. the numerical simulations show that the mixing of the two fluids was complete approximately . - . mm from the entrance of mixing zone with flow rate conditions for each fluid in the range . - μ l/h (simulation duration, ~ - ms) (fig. s ). the mixing zone was . mm long and μ m wide. the residence time for mixing (and hence for the reaction between the sample and dye) was in the range - s for total flow rates in the range - μ l/h. after passing through the mixing zone, the mixed sample fluid was focused horizontally using a dual sheath flow with sterilized distilled water (sdw) in the hydraulic focusing component (see fig. (a) ). the focused core flow was . μ m wide, which is one-twentieth the width of the channel. this minimized variation in the optical signals among locations in the particle detection zone . figure (c- ) shows a schematic diagram of real-time detection of target particles in the core flow, as well as the optical setup for continuous detection and signal quantification. a diode-pumped solid-state (dpss) laser with a wavelength of nm and an output power of . mw (cni laser, changchun, china) was coupled with a single-mode optical fiber (core diameter: . μ m, cladding diameter: μ m). the laser beam was focused on the core fluid using an objective lens (rms x-pf; x/ . ; thorlabs) located beneath the microfluidic channel (~ μ m beam spot size at full-width half-maximum). a highly sensitive multi-pixel photon counter (mppc) (c - - u; hamamatsu photonics, hamamatsu city, japan) module was used to detect the sc and fl signals from the target particles. the particle fl was collected using the objective lens, and entered the mppc via the optical long-pass filter and focusing lens. to bring the sc light from the particles, multi-mode optical fiber with a core diameter of μ m and a cladding diameter of μ m (thorlabs) was carefully inserted into the channel with the axis of the fiber at ° to the direction of flow, and linked to the mppc. a data acquisition (daq) unit (ni usb- ; national instruments, austin, tx, usa) was employed to collect the analog data from the mppc modules, which were recorded using a desktop computer. an oscilloscope (dpo b; tektronix, beaverton, usa) was used to monitor the signal in real-time, as shown in fig. performance evaluation of the micro-optofluidic platform using standard psl particles. the particle detection performance of the optofluidic platform was evaluated under various microfluidic and sample conditions, such that the sample flow rate and the mixing ratios of fl and non-fl particles were varied. we used standard spherical polystyrene latex (psl) and fluorescence psl (flpsl) particles that were μ m in diameter. figure shows the particle fl and sc signals with flow velocities in the range . - . mm/s. the flpsl particle suspension (~ . × /μ l) was injected into sample inlet. the ratio of the flow rate of the flpsl particle suspension to that of the sdw was : in the narrow core region (which had dimensions of . × μ m). as the flow velocity increased from . to . mm/s, the frequency of the particle sc and fl signals increased in proportion to the particle flow velocity, and the average intensity of the measured sc and fl signals (sc/fl) decreased from ~ arbitrary unit (a.u.)/~ a.u. to ~ a.u./~ a.u., respectively ( fig. (a) ). this is attributed to the decreased photon integration time of the photodetector with decreasing residence time of the particles in the detection zone of microchannel . figure (b) shows that the event rate (i.e., number of counts per second) of the particle sc and fl signals followed a linear relationship with the particle flow velocity; for fl we have y = . x + . , with r = . , and for sc we have y = . x + . , with r = . . the index of coincidence between the total frequencies of particle sc and fl signals was ~ . % over the entire range of particle flow velocities. mcclain et al. reported that only ~ % of the particle sc signals were unaccompanied by coincident fl signals, which was attributed to unresolved fl peaks due to the relatively low signal intensity . when the number of counts of the particle sc and fl signals are plotted as histograms as a function of the signal intensity, both sets of signals form gaussian-like distributions, as shown in fig. s . the variation in the intensity results from variations in the particle size and particle position in the detection zone. as expected, the particle sc signal was significantly stronger than the fl signal. we evaluated the particle detection performance of the micro-optofluidic platform with various mixing ratios of flpsl and psl particles. the total particle concentration was fixed at ~ . × /μ l, and the total flow rate of the mixed suspension was μ l/h, giving a flow velocity of ~ . mm/s. the mixing ratio of flpsl and psl particle suspensions was varied in the range - %. as shown in fig. (a) , the ratio of the count rate of fl signals to sc signals increased in proportion to the mixing ratio of flpsl particles. figure (b) shows fluorescence microscopy images of the flpsl particles in the mixing suspension. when the mixing ratio of flpsl particles was %, the flpsl particle number concentration in the micrographs was /μ l; at a mixing ratio of %, the flpsl particle number concentration was /μ l (~ % of the initial flpsl particle concentration); and at a mixing ratio of %, the flpsl particle number concentration was /μ l (~ % of the initial flpsl particle concentration). psl particles could not be enumerated using normal bright-field microscopy because of the limit of resolution and small field of view. from this performance evaluation (figs and ) , we may conclude that the micro-optofluidic platform demonstrated not only real-time and continuous particle detection, with suitable performance for standard psl particles, but also quantitative and accurate discrimination between fl and non-fl particles. real-time detection of airborne microorganisms. to simulate a hazardous airborne bacteria-contaminated environment, we prepared a × × m test chamber containing live, airborne escherichia coli, bacillus subtilis and staphylococcus epidermidis bioaerosols separately. details of the experimental setup can be found in the methods and supplemental information (fig. s ) . figure shows the size and morphology of the bacterial bioaerosols in the test chamber. real-time particle size distribution data of the bacterial bioaerosols were obtained using an aerodynamic particle sizer (aps; ; tsi inc., shoreview, mn, usa). as shown in fig. (a) , the bacterial bioaerosols exhibited mono-modal curves with a specific geometric mean diameter (gmd), peak diameter, and geometric standard deviation (gsd). table lists these data for the three bioaerosols; the gmds of e. coli and b. subtilis were similar at ~ μ m, and that of s. epidermidis was somewhat smaller, at ~ . μ m. airborne bacterial bioaerosols in the test chamber were collected using a biosampler (skc inc., eighty four, pa, usa), which is a highly efficient collection device that traps airborne microorganisms in a swirling liquid for subsequent analysis . the airborne particle collection efficiency of the biosampler was ~ . % for the -μ m-diameter psl particles (fig. s ) . figure samples containing the bacterial suspension were injected into the micro-optofluidic platform using a syringe pump to evaluate the device performance for the real-time detection of airborne bacterial particles. figure (a) shows the acquired sc and fl signals of the test bacterial bioaerosols. the average sc/fl signal intensity ratios were ~ / for e. coli, ~ / for b. subtilis, and ~ / for s. epidermidis. because of the size of these bacteria (i.e., . - . μ m), they may be considered to lie within the mie scattering regime, and the intensity of the sc signals may be assumed to be proportional to the cross-sectional area of particles . in the same manner, the fl signal intensity increased proportionally with the size of the bacteria because larger particles have a higher concentration of nucleic-acid fluorescent dye , . the particle number concentration was determined based on the frequency data for the sc and fl signals. table lists the sc signal concentration, which corresponds to the total particle number concentration. this was higher than the fl signal concentration, which corresponds to the total concentration of microorganisms. the difference between the total particle count (sc signals) and the total bacterial concentration (fl signals) was ~ % for e. coli, ~ % for b. subtilis, and ~ % for s. epidermidis. it follows that the sample included other particles; i.e., non-microorganism particles or impurities, such as non-dyed particles or small debris. to evaluate the particle detection efficiency for airborne microorganisms, the total concentration of bacteria obtained from the fl signal of the micro-optofluidic platform was compared with the conventional fluorescence microscopy cell counting and colony counting methods. figure (b) shows a comparison of the concentration of bacteria measured using fluorescence microscopy and colony counting, both of which were normalized to that measured using the micro-optofluidic platform. for e. coli the normalized cell concentration by microscopic cell counting was ± . %, and was ± . % using colony counting; for b. subtilis, the normalized cell concentration using microscopic cell counting was ± . %, and was ± . % using colony counting; and for s. epidermidis the normalized cell concentration using microscopic cell counting was ± . %, and was ± . % using colony counting. the concentration of bacteria measured using both conventional cell-counting methods was lower than that using the micro-optofluidic platform. the micro-optofluidic platform exhibited the highest cell counts and the lowest standard deviation, which is indicative of superior performance. photobleaching of fluorophores may occur during cell counting via conventional fluorescence microscopy, which results in a reduction in the fluorescence intensity. furthermore, the low signal-to-noise ratio in the imaging process may lead to underestimation of the number of cells. an inappropriate image threshold intensity setting is required to discriminate fluorescence particles from the background, and this may exclude particles that either have a low fluorescence intensity or are slightly out of focus . furthermore, quantitative analysis of the total concentration of particles with sizes of less than μ m is limited by the resolution and the field of view of bright-field microscopy. as shown in figure (b), the colony counting method resulted in the lowest cell concentration of the three methods. the colony counting method measures only culturable cells in the medium. therefore, it is difficult to apply the colony counting method for the analysis of viable but non-culturable (vbnc) microorganisms, or microorganisms that require a specific growth environment and/or specific nutrients . the micro-optofluidic platform with integrated sample preparation and detection simplifies the system, can significantly reduce the measurement time, and can yield more accurate quantitative results . the number concentration of the particles in air was recorded for each aps channel size, divided by the logarithmic interval of the corresponding particle size range and plotted as a function of the aerodynamic diameter table . size characteristics of test airborne bacterial particles. the geometric standard deviation (gsd) is defined as exp( where d j is the diameter of an individual particle, n j is the number of particles in the j th group, n is the total number of particles, and ln d g is the natural logarithm of the geometric mean diameter (gmd) of the particles, defined as ∑ / n d n ln compared with the abovementioned conventional methods. furthermore, our system provides additional information on the total particle number concentration of aerosols, which is difficult to obtain using conventional methods. we have demonstrated continuous, rapid and real-time detection of bioaerosols using a micro-optofluidic platform. the performance of our device was investigated using standard psl particles with various flow speeds and mixing ratios with flpsl particles. accurate quantitative flpsl particle discrimination with high efficiency was achieved. this first application of this integrated micro-optofluidic platform for the analysis of airborne microorganisms showed that our device could reduce the time for sample preparation and manual analysis compared with conventional microorganism-counting methods. the micro-optofluidic platform has potential applications in aerosol analysis, and can enable portable, highly sensitive, continuous real-time detection of airborne particles and microorganisms. in future, we plan to include a d focusing stream to optimize the optical signals, which is expected to enhance the resolution and efficiency of the measurements, and to reduce the noise due to spatial deviations of the particles. accurate particle sizing using the current micro-optofluidic platform is possible ; furthermore, a micro-pump between the biosampler and microfluidic channel could be included for continuous real-time analysis of airborne microorganisms. various species of dye or marker for specific target materials, such as target-specific antibodies or aptamers, could be used to create an analysis system that provides data on the physical, chemical and biological properties of the airborne particles . note that the purpose of this work was to quantitate airborne microorganisms rapidly and in real-time using a fully integrated micro-optofluidic platform. sample preparation. two types of standard uniform particles and three types of bacteria were used in this study. fluorescence polystyrene latex (flpsl) particles (f ; fluorescent microsphere; . -μ m diameter; orange fluorescent ( / ); invitrogen) and polystyrene latex (psl) particles ( a; monosized microsphere; . -μ m diameter; refractive index of . ; density of . g/cm ; duke scientific corporation) were used as standard particles for the performance evaluation of particle detection and quantification. as test airborne microorganisms, gram-negative escherichia coli (korean collection of type cultures (kctc) , biological resource center, republic of korea), gram-positive bacillus subtilis (kctc ), and gram-positive staphylococcus epidermidis (atcc ), were used . the bacteria were incubated in nutrient broth (becton dickinson, franklin lakes, usa) at °c, and harvested using a centrifuge (mini, gyrozen, south korea) at rpm for minutes. bacterial pellets were washed three times using sdw with a centrifuge to remove the residual medium. to create the bacteria suspensions, -ml aliquots were placed in a six-jet collision nebulizer (bgi corp., usa) to aerosolize the bacteria. microchannel design and fabrication. the microchannel was a single-layer pdms channel fabricated using conventional soft lithography. su- ( ; microchem corp.), which is a negative photoresist, was spin-coated onto a . -inch si wafer. the photoresist was soft baked on a hotplate at °c for min, followed by °c for min. a chrome photomask pattern was installed using a uv aligner, and the photoresist was exposed to uv irradiation. following this exposure, the resist was baked at °c for min and then at °c for min. the su- photoresist pattern was developed using -methoxy- -propyl acetate (microchem corp.) for min. after completion of the developing process, the pattern was rinsed using isopropyl alcohol (ipa) and deionized (di) water to yield a master mold. pdms was poured over the developed wafer with the pattern, and cured in an oven at °c. the patterned pdms channel was then removed from the wafer, and bonded onto glass using o plasma to seal the microchannel. fluorescence microscopy cell counting. to enumerate flpsl particles and bacterial cells using the fluorescence microscopy method, - μ l of sample (following enrichment via centrifugation in the case of low particle concentrations) was loaded into the sample injection area of a disposable hemocytometer (dhc-n ; incyto, republic of korea), and visualized using a fluorescence microscope (b x ; olympus, tokyo, japan) with a u-mwg filter set; the excitation wavelength was in the range - nm, and the emission wavelength was > nm. for each sample, images of at least microscopic fields were captured using a ccd array camera. microscopy cell counting was carried out using the imagej software package (http://imagej.nih.gov/ij/). colony counting. bacterial suspensions were serially diluted and spread onto the surface of nutrient agar (becton dickinson) in a petri dish, followed by incubation at °c for hours. the resulting colonies were counted manually. scanning electron microscopy. the morphology of airborne bacterial particles was investigated using sem (nova nano sem ; fei co., hillsboro, or, usa). the modified karnovsky's fixation protocol was used , , and bacterial samples were fixed using % paraformaldehyde ( ; electron scientific reports | : | doi: . /srep microscopy sciences (ems), hatfield, pa, usa) and % glutaraldehyde ( ; ems) in a . m sodium cacodylate buffer (scb; ph . ) ( ; ems) at °c for hours, followed by washing with . m scb at °c for hours. post-fixation, the samples were treated with % osmium tetroxide ( ; ems) in . m scb at °c for hours, and then washed with distilled water at room temperature. the samples were dehydrated at room temperature in a - % ethanol series with -minute exposure to each concentration. the ethanol was then replaced with hexamethyldisilazane ( ; ems). following dehydration, each sample was spread onto polycarbonate membrane filters with a pore size of . μ m (isopore membrane filters http ; millipore, billerica, ma, usa) and coated with osmium by chemical vapor deposition (hpc- sw; vacuum device inc., ibaraki, japan). real-time detection of an airborne microorganism using inertial impaction and mini-fluorescent microscopy the role of particle size in aerosolised pathogen transmission: a review bioaerosol health effects and exposure assessment: progress and prospects measurement of airborne influenza virus in a hospital emergency department rapid quantification of bioaerosols containing l. pneumophila by coriolis ® μ air sampler and chemiluminescence antibody microarrays microbiological quality of indoor air in university rooms exposure to organic dusts, endotoxins, and microorganisms in the municipal waste 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microfluidics for sheath-less highthroughput flow cytometry multiplexed detection of bacteria and toxins using a microflow cytometer three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows disposable flow cytometer with high efficiency in particle counting and sizing using an optofluidic lens rapid, semiautomated quantification of bacterial cells in freshwater by using a microfluidic device for on-chip staining and counting optofluidic characterization of marine algae using a microflow cytometer flow cytometry of escherichia coli on microfluidic devices fast and continuous microorganism detection using aptamerconjugated fluorescent nanoparticles on an optofluidic platform soft lithography analysis of apoptosis by laser scanning cytometry syto probes in the cytometry of tumor cell death single molecule fluorescence under conditions of fast flow improved aerosol collection by combined impaction and centrifugal motion optimization of an automatic counting system for the quantification of staphylococcus epidermidis cells in biofilms accuracy of plate counts the complete genome sequence of the gram-positive bacterium bacillus subtilis optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy adhesion forces between e. coli bacteria and biomaterial surfaces aerosol technology: properties, behavior, and measurement of airborne particles this research was supported by the kist institutional program ( e ). supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord- - x f authors: kim, sung-il; song, jong tae; jeong, jin-yong; seo, hak soo title: niclosamide inhibits leaf blight caused by xanthomonas oryzae in rice date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: x f rice leaf blight, which is caused by the bacterial pathogen xanthomonas oryzae pv. oryzae (xoo), results in huge losses in grain yield. here, we show that xoo-induced rice leaf blight is effectively controlled by niclosamide, an oral antihelminthic drug and molluscicide, which also functions as an anti-tumor agent. niclosamide directly inhibited the growth of the three xoo strains pxo , and k a. niclosamide moved long distances from the site of local application to distant rice tissues. niclosamide also increased the levels of salicylate and induced the expression of defense-related genes such as ospr and oswrky , which suppressed xoo-induced leaf wilting. niclosamide had no detrimental effects on vegetative/reproductive growth and yield. these combined results indicate that niclosamide can be used to block bacterial leaf blight in rice with no negative side effects. effective and economical chemical treatment for rice leaf blight has not been established, although research and development are ongoing. enhancing plant genetic resistance is an effective method for controlling bacterial leaf blight disease. a number of studies have identified plant genes that confer resistance against xanthomonas bacteria. at least bacterial leaf blight resistance gene (r genes), designated in series from xa to xa , have been identified , . six of these genes have been cloned (xa , xa , xa , xa , xa /xa , and xa ), and six additional genes have been physically mapped (xa , xa , xa , xa , xa , and xa ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . two major classes of r genes, receptor kinase (rlk) and nucleotide-binding site leucine rice repeat (nbs)-lrr, are involved in disease resistance in rice. xa is the first r gene of the rlk class to be cloned with a broad spectrum of resistance, and xa is an r gene of the nbs-lrr class , which is the largest r gene class conferring resistance against bacteria, fungi, and viruses . other r genes, including xa and xa , encode proteins such as a small subunit of the transcription factor iia (tfiiaγ ) and a plasma membrane protein, respectively , . these data have been used to develop a breeding program for bacterial leaf blight-resistant rice. the introduction of resistance genes xa , xa , and xa into new cultivars conferred a high level of resistance against xoo , indicating that germplasm screening against bacterial leaf blight and breeding-mediated transfer of resistant genes to target cultivars can generate new rice cultivars with increased resistance to bacterial leaf blight disease. large-scale experiments, including microarrays, were used to isolate genes related to bacterial leaf blight; a large number of candidate genes was selected, although their functions were not clearly identified , . a transgenic approach was employed to protect rice from bacterial leaf blight and fungal blast. for example, transgenic rice overexpressing cecropin b (an antibacterial peptide from bombyx mori) showed increased resistance to bacterial leaf blight . the introduction of multiple bacterial blight resistance genes (xa , xa , xa , and xa ) into rice conferred resistance to six xoo bacteria . rice transformants overexpressing wrky are resistant to xoo and the fungal pathogen magnaporthe oryzae , and those overexpressing wrky displayed markedly enhanced resistance to bacterial leaf blight and fungal blast disease [ ] [ ] [ ] . these results suggest that transgenic approaches can be effectively utilized to develop disease-resistant rice. although several biological, chemical, and genetic control approaches have yielded improvements in rice protection from bacterial leaf blight, there was still a need for an effective approach that would provide large-scale protection. therefore, we focused our current efforts on screening known chemicals for their effects on rice leaf blight. we are interested in the discovery of a master regulator that effectively functions to protect animals and plants from infectious diseases mediated by bacteria, fungi, and/or parasites. therefore, we first considered infectious diseases of humans. we selected two drugs that cure infectious diseases in humans, auranofin [ , , -triacetyloxy- -(acetyloxymethyl)oxane- -thiolate] and niclosamide [ -chloro-n-( -chloro- nitrophenyl)- -hydroxybenzamide], for further analysis. however, because auranofin is much more expensive than niclosamide, we focused our initial screen on niclosamide. niclosamide has been widely used since to treat gastrointestinal tapeworm infections in both humans and animals. recent studies show that niclosamide has antiviral activity against the severe acute respiratory syndrome virus , anti-anthrax toxin properties , and anti-neoplastic activity . niclosamide strongly induces lc -positive autophagosomes , inhibits the wnt/ frizzled pathway , suppresses the autonomous notch-signaling pathway , and inhibits mtor signaling . niclosamide uncouples mitochondrial oxidative phosphorylation and thereby slows cell growth. these combined results indicate that niclosamide has various curative effects on humans and animals. we reasoned that niclosamide might affect plant disease responses by modulating disease signaling pathways as a broad spectrum regulator. therefore, we investigated the effect of niclosamide on bacterial leaf blight in rice. here, we show that niclosamide blocks rice leaf wilting mediated by xoo bacteria, both locally and systemically, without negatively affecting plant growth. the results suggest that niclosamide may be used to prevent rice leaf blight caused by bacterial pathogen attack. niclosamide inhibits xoo bacterial growth. to evaluate the functional effect of niclosamide on bacterial blight, we first examined its effect on the growth of xoo bacteria using three different strains, pxo , , and k a, and xanthomonas axonopodis pv. glycines (xag). the results showed that growth of the xoo strains was completely inhibited by μ g/ml niclosamide, whereas xag growth was inhibited by μ g/ml niclosamide (fig. a) . xag is a pathogen that causes bacterial leaf pustule disease in soybean . we also examined the effect of niclosamide on the growth of the fungal pathogen magnaporthe oryzae. niclosamide did exert an inhibitory effect on the growth of m. oryzae strains, although the inhibition was considerably weaker than that against xoo (fig. b) . we also tested the effects of parthenolide, a sesquiterpene lactone with anti-tumor activity, on the growth of the xoo strains pxo and . parthenolide failed to inhibit the growth of either xoo strain at a concentration of μ g/ml, although it slightly inhibited bacterial growth at μ g/ml (fig. c) . we also tested the effect of niclosamide on the growth of three e. coli strains, top , rosseta , and dh b, which served as gram-negative control bacteria. at lower concentrations, niclosamide had no effect on the growth of these e. coli strains, although it slightly inhibited growth at μ g/ml (fig. d) . to determine the minimum inhibitory concentration (mic) of niclosamide on pxo and growth, we tested concentrations ranging from - μ g/ml. both xoo strains could grow in the presence of μ g/ml niclosamide, but not μ g/ml niclosamide ( fig. e; left panel) . we further narrowed the mic of niclosamide from - μ g/ml. as shown in fig. e , the growth of both pxo and was completely inhibited by . μ g/ml niclosamide ( fig. e ; right panel), indicating that . μ g/ml niclosamide is the mic for both xoo strains. rice bacterial blight is blocked by niclosamide. we examined whether niclosamide blocks bacterial blight in rice. for this experiment, we grew the rice cultivar nipponbare in a growth chamber for weeks (before bolting), and subjected the plants to pathogen and niclosamide treatment. first, to determine the minimum concentration of niclosamide that would block bacterial blight, we examined the niclosamide dosage effect on disease responses to the representative xoo strain pxo . we inoculated pxo onto nipponbare using the leaf-clipping method, sprayed the plants with different niclosamide concentrations, and examined the phenotypes of plants treated with pathogen only or pathogen plus niclosamide. leaf wilting did not develop in plants treated with ≥ μ g/ml niclosamide (fig. a) . we also estimated lesion development by measuring lesion length. lesion development also was significantly inhibited by μ g/ml niclosamide, although a small lesion was still detected at this concentration (fig. b) . lesion development gradually declined with increasing niclosamide concentrations (fig. b) . we also examined the levels of bacterial growth, and found that population levels of xoo bacteria declined with increasing niclosamide concentrations (fig. c) . we next examined the effect of niclosamide on rice disease responses to xoo bacteria after treatment with pxo . first, nipponbare rice was inoculated with pxo by the leaf-clipping method. after incubation for four days until leaf blight lesions with a length of cm developed, the plants were sprayed with μ g/ml niclosamide (fig. ) . leaf blight was completely blocked in niclosamide-treated leaves (fig. a) . lesion development also was completely blocked in niclosamide-treated leaves (fig. b) , and pxo growth was inhibited (fig. c ). niclosamide has a systemic effect on rice disease response. we next investigated whether niclosamide could move from the site of local application to distant tissues and subsequently inhibit pxo -mediated leaf wilting. we inoculated half of the leaves of nipponbare plants with pxo and covered them with polythene bags. then, we sprayed the non-inoculated leaves with niclosamide and examined leaf blight in both local and systemic leaves. leaf wilting and lesion development were completely inhibited in pxo -inoculated leaves that had not been treated with niclosamide ( fig. a,b ). pxo growth also was significantly inhibited in the inoculated leaves (fig. c) . next, we examined the long-distance movement of niclosamide by extracting niclosamide from niclosamide-treated leaves and untreated systemic leaves. niclosamide levels gradually increased in systemic leaves ( fig. a,b) , indicating that niclosamide can systemically move from niclosamide-treated local leaves to untreated distal leaves. niclosamide induces the expression of defense-related genes. we next examined whether niclosamide protects pxo -infected plants through the induction of pathogen-related gene expression. we treated the rice cultivar nipponbare with different niclosamide concentrations for h, and then extracted total rna from niclosamide-treated and untreated control plants. we performed qrt-pcr analysis to examine the transcript levels of seven defense-related genes, including ospr , ospr , oswrky , oswrky , oswrky , oshi-lox, and osacs . the results showed that ospr , ospr , oswrky , oswrky , oswrky , and oshi-lox were induced by niclosamide treatment, and that transcript expression levels increased with increasing niclosamide levels (fig. ). however, osacs expression was only slightly increased min after treatment with μ g/ml niclosamide, which suggests that its expression was not affected by niclosamide (fig. ). the pathogenesis-related genes ospr , ospr , oswrky , oswrky , and oswrky are induced by high salicylate (sa) levels [ ] [ ] [ ] [ ] ; therefore, we measured free sa and glucosyl-sa levels in the leaves of niclosamide-treated rice plants. sa levels increased in response to niclosamide treatment (fig. a,b) , indicating that niclosamide induces the expression of pathogenesis-related genes by increasing the levels of sa and sa-conjugates. niclosamide has no effect on rice growth and development. we examined the effect of niclosamide on rice growth and development from the vegetative stage to seed maturation. three-week-old rice plants were treated with μ g/ml niclosamide at -day intervals and the phenotypic characteristics of niclosamide-treated and -untreated plants were examined at days after planting. plant height and leaf characteristics were not altered by niclosamide treatment, although the contents of chlorophyll and other compounds (spad values) were slightly reduced in niclosamide-treated plants ( fig. a-g) . seed characteristics such as color, number, and weight were not affected by niclosamide treatment (fig. h-l) , indicating that niclosamide does not have any detrimental effects on rice growth, development, and grain yield. xoo-mediated leaf blight is one of the most devastating rice diseases worldwide. for the past two decades, substantial efforts have been made to identify and isolate bacterial blight-resistance genes. in this study, we used an alternative chemical approach to protect rice from xoo infection. more than drugs, including antibiotics, have been utilized to protect crops from pathogen attack. some of these have broad-spectrum bactericidal and fungicidal activity, whereas others specifically target bacteria or fungi. oxytetracycline and streptomycin are commonly used antibiotics in humans and plants , suggesting that some types of human drugs can positively control diseases in plants. therefore, we screened human drugs for their ability to prevent rice leaf blight disease. our long-term goal is to identify a master regulator that inhibits pathogenic disease in both plants and humans. niclosamide was initially characterized as an oral antihelminthic drug and molluscicide . several recent studies report that niclosamide is active against cancer cells , , , which indicates that niclosamide has broad-spectrum disease control activity. therefore, we examined whether niclosamide had inhibitory activity against rice leaf blight, and found that the leaf blight symptoms were suppressed by niclosamide through inhibition of xoo growth and lesion development (figs and ) . niclosamide moved systemically from the local application site to xoo-inoculated distal leaves, and inhibited lesion development and leaf wilting by inhibiting xoo growth in distal leaves (fig. ) . we examined the effect of niclosamide on the growth of three xoo strains, and found that it inhibited their growth (fig. a,e) . next, we evaluated whether niclosamide induced the expression of the defense-related genes ospr , ospr , oswrky , oswrky , oswrky , and oshi-lox, and detected higher transcript levels for these genes (fig. ) . these results clearly indicate that niclosamide blocks the development of rice leaf blight by directly inhibiting xoo bacterial growth (figs c and c) and/or by inducing defense-related gene expression (fig. ) . niclosamide also inhibited xag growth, which causes bacterial leaf pustule disease in soybean , although its inhibitory effect against xag was weaker than that against xoo strains (fig. a) . this suggests that niclosamide may inhibit the growth of select bacterial pathogens of other crops and thereby protect them from disease. jasmonate (ja) and sa function in plant defense pathways by inducing the expression of numerous genes . treatment of rice leaves with benzothiadiazole or probenazole induced sa accumulation and increased resistance to bacterial blight and fungal blast caused by xoo and m. grisea, respectively , , . our qrt-pcr analysis showed that niclosamide induced the expression of sa-dependent genes in rice leaves (fig. ) . we investigated whether niclosamide induced defense mechanisms directly by functioning like a phytohormone, or indirectly via sa-mediated signaling pathways. we measured the levels of free sa and its conjugate glucosyl-sa, and found that their levels were higher in niclosamide-treated leaves than in control leaves (fig. ) . this indicates that increase in sa level can contribute to the blockage of xoo lesion development by niclosamide. wrky proteins are involved in ja response pathways in rice. oswrky triggers the expression of ja-responsive genes and ja accumulation, and provides rice with resistance to fungal pathogens rhizoctonia solani and m. grisea . oswrky expression regulates ja accumulation and resistance to the rice blast fungus m. grisea . our results showed that the expression of oshi-lox, a ja-responsive gene, was induced in niclosamide-treated leaves (fig. ). this indicates that ja-dependent defense signaling pathways can also contribute to the blockage of xoo lesion development by niclosamide. pathogens are sensitive to the activities of certain classes of drugs. the antibiotic streptomycin, which is commonly used in animals and humans, protects crops from some bacterial and fungal pathogens . niclosamide serves as an oral antihelminthic drug, molluscicide, and anti-tumor agent in human disease; therefore, we reasoned that niclosamide might have beneficial effects on bacterial and fungal diseases of rice. niclosamide inhibited growth of the fungal pathogen m. oryzae, although this effect was much weaker than that against pathogenic xoo bacteria (fig. b) . this suggests that niclosamide may inhibit the growth of other fungal pathogens and thereby protect plants from fungal diseases. benzothiadiazole and probenazole induce sa pathway-mediated defense responses in plants by acting as chemical inducers , , , - , leading to strong resistance against bacterial and fungal pathogens. niclosamide induces the expression of both sa-and ja-responsive genes in rice (fig. ). this indicates that niclosamide may also be effective against fungi due to ja. further study of the effects of niclosamide against other fungal pathogens will indicate whether the compound can protect plants from diseases caused by fungal pathogens. the biochemical function and physiological action of niclosamide in human health and disease have been elucidated. niclosamide inhibits glucose uptake, mitochondrial oxidative phosphorylation, and anaerobic metabolism in parasitic helminths . it also inhibits transcription and dna binding of the nfκ Β pathway, and increases ros levels to induce apoptosis in acute myelogenous leukemia cells . recent work reports that niclosamide inhibits pseudomonas aeruginosa quorum sensing . these results suggest that niclosamide controls signaling networks of prokaryotes and eukaryotes by modulating metabolic and signaling pathways. currently, we do not know the mechanism underlying niclosamide-mediated inhibition of xoo growth and induction of sa-and ja-dependent plant defense responses. future work will examine the effects of niclosamide on glucose metabolism, the transcriptome and proteome, and quorum sensing to identify the mechanism by which niclosamide inhibits bacterial leaf blight in rice. further investigations of the effects of niclosamide on mitochondrial oxidative phosphorylation, changes in transcriptome and ros levels, and changes in hormone signals (including sa and ja) also are required to fully characterize its functions as a broad-spectrum signaling regulator in plants. the use of antibiotics and chemicals in plant agriculture is a subject of some concern. the use of antibiotics and chemicals in open fields over large expanses of land may increase the frequency of antibiotic resistance in gene pools, or possible chemical accumulation in plant tissues and the food chain. niclosamide is a teniacide, which is effective against cestodes such as tapeworms that infect humans and many other animals (although it is not effective against as pinworms and roundworms). niclosamide also is used as a piscicide. recent work reported that it appeared to be safe and well tolerated in humans, even when used in mass treatment campaigns in several countries, although it has known adverse effects including nausea, retching, abdominal pain, light headedness, pruritus, vomiting, and dizziness . there are no previous reports of niclosamide effects on plant growth, other organisms, or the food chain. we cannot state that niclosamide treatment of agricultural crops is safe for other organisms or for the food chain because research in this area is still lacking. therefore, further studies on the function and stability of niclosamide under natural environmental conditions and when applied to crop plants are required to determine whether niclosamide has adverse effects on other organisms and human health when applied to plants. in conclusion, the results presented herein indicate that niclosamide protects rice plants from bacterial leaf blight by inhibiting xoo growth, inducing sa accumulation, and/or by inducing the expression of defense-related gene pathways. further functional studies on niclosamide-mediated growth inhibition of bacterial and fungal pathogens, and elucidation of its role in plant signaling pathways, will provide information about the mechanism underlying niclosamide activity in plants. field tests evaluating the effects of niclosamide application on rice plants will provide information on whether it has adverse effects on other organisms or the food chain. examining the effect of niclosamide on xoo bacterial growth. the effect of niclosamide on the growth of xoo strains pxo , , and k a, and a xag strain, and the mic of niclosamide against the pxo and strains were investigated. xoo and xag strains were obtained from the rural development administration, korea. bacterial strains were cultivated in peptone-sucrose broth containing μ g/ml of the antibiotic cephalexin (sigma-aldrich), and grown to an optical density of . at nm. then, μ l of each culture was grown on agar medium containing different concentrations of niclosamide ( − μ g/ml) to examine the effects of niclosamide on bacterial growth. inoculated plates were incubated at °c for h and then photographed. the same method was used to determine whether parthenolide had bactericidal activity against the pxo and strains. the bactericidal activity of niclosamide on three different e. coli strains, top , rosseta , and dh b, also was examined. the e. coli strains were cultivated in lb medium to an optical density of . at nm. then, μ l of each culture was grown on agar medium containing different concentrations of niclosamide. inoculated plates were incubated at °c for h and photographed. to determine the mic, μ l of each culture were inoculated onto peptone-sucrose agar (psa) medium containing − . μ g/ml niclosamide. bacterial growth was not detected at a concentration of . μ g/ml niclosamide. thus, to determine a more specific mic value, the mic of niclosamide against both xoo strains was narrowed down from . to . μ g/ml. the lowest concentration of niclosamide ( . μ g/ml) with no visible growth was taken as the mic. to examine the effects of niclosamide on fungal growth, m. oryzae strains ( , , , , , , , , , , ki , were inoculated onto potato dextrose agar (difco) medium containing different concentrations of niclosamide ( − μ g/ml). the plates were then incubated at °c for h and photographed. to determine the minimum amount of niclosamide needed to inhibit the development of leaf blight in rice, the rice cultivar nipponbare and xoo strain pxo were used. pxo cells were prepared as follows. a single pxo colony was suspended in peptone-sucrose broth and plated onto fresh psa medium. after days of culture at °c, the cells were collected and centrifuged to remove exopolysaccharides. the cells were then resuspended in double-distilled water, producing more than cell forming units (cfu) per ml. the dosage effect of niclosamide on pxo lesion development was evaluated by measuring the lesion length (cm) and bacterial growth in the leaves of niclosamide-treated rice. to treat rice with pxo , plants were grown for days in a greenhouse at °c in the light and °c in the dark under a h photoperiod until the booting stage. the greenhouse humidity was maintained over %. the fully expanded uppermost leaves were inoculated with pxo by the leaf-clipping method, followed by treatment with various doses ( , , , , and μ g/ml) of niclosamide applied by foliar spraying every days. as a control, mock inoculation was conducted with distilled water. after days, the leaves were photographed and lesion length was measured using imagej software. all data are expressed as the mean values from leaves per treatment. the experiment was repeated five times under the same conditions. to estimate bacterial growth, leaves were cut off the plants and ground with a mortar and pestle. after grinding, each sample was suspended in sterile water and plated onto psa medium containing μ g/ ml cephalexin. the bacterial population was scored by counting the number of colonies every days after inoculation. the lesion lengths and bacterial populations were expressed as the mean value plus/minus the standard deviation. niclosamide effects on disease responses in rice. rice plants were inoculated with xoo and sprayed with niclosamide. pxo cells and plants were prepared as described above. to treat rice with pxo , the fully expanded uppermost leaves of -day-old rice plants were inoculated with pxo by the leaf-clipping method. the samples were incubated for days until leaf blight lesions with a length of cm developed, after which the leaves were treated with μ g/ml niclosamide by foliar spraying every days. as a control, mock inoculation was conducted with distilled water. leaves were photographed after days, and lesion length and bacterial growth estimated as described above. all data are expressed as the mean values of leaves per treatment. the experiment was repeated five times under the same conditions. the lesion length and bacterial population were expressed as the mean value plus/minus the standard deviation. systemic effect of niclosamide on rice disease responses. to examine the systemic effect of niclosamide on xoo-mediated leaf blight development, the fully expanded uppermost leaves of -day-old rice plants were inoculated with pxo by the leaf-clipping as described above. half of the leaves were completely covered with polythene bags and half were sprayed with μ g/ml niclosamide. the local leaves were then sprayed with μ g/ml niclosamide every days. both local and systemic leaves were photographed after days. lesion length and bacterial growth were estimated as described above. the experiment was repeated five times under the same conditions. the lesion length and bacterial population were expressed as the mean value plus/minus the standard deviation. quantification of systemically translocated niclosamide. half of the leaves of -day-old rice plants were covered by polythene bags and the remaining systemic leaves were sprayed with μ g/ml niclosamide. after treatment, the bag-covered leaves were harvested after incubation for the indicated time periods. niclosamide was extracted from . g of each sample using absolute meoh, and the niclosamide concentration was determined by hplc separation and fluorescence detection. the relative niclosamide concentration in each sample was compared with the ppb niclosamide standard. niclosamide levels were expressed as the mean value plus/ minus the standard deviation. determination of salicylic acid (sa) and its conjugate. sa and the sa conjugate glucosyl-sa were extracted from . g of rice leaves that were treated with μ g/ml niclosamide for h. samples were collected at five time points ( , . , , , and h). the concentrations of sa and glucosyl-sa were measured by hplc as described previously . estimation of pathogen-related gene transcript levels in niclosamide-treated rice. the rice cultivar nipponbare was sprayed with μ g/ml niclosamide as described above. total rna was extracted from niclosamide-treated and untreated leaves at , . , , and h, quantified, and diluted to equal concentrations. all niclosamide-treated or untreated plants were used for total rna extraction. first-strand cdna was synthesized using μ g of total rna and an iscript cdna synthesis kit (bio-rad). an equal volume of cdna was amplified by quantitative real-time rt-pcr (myiq, bio-rad) according to the manufacturer's protocol. then, nm of specific primers and template cdna were combined with μ l iq sybr green super mix (bio-rad) and the amplification reaction performed under the following thermal cycling conditions: °c for min; cycles of °c for s; °c for s; and °c for s. the ct values of target genes were normalized to the ct value of the actin gene and analyzed with icycler iq software (bio-rad). the experiments were repeated three times. pcr primers were designed using primer plus (http://www.bioinformatics.nl/ cgi-bin/primer plus/primer plus.cgi/). primer specificity was verified by cloning into the pgem t-easy vector (promega) and sequencing with an abi × l dna analyzer (applied biosystems). the primers used for quantitative pcr were as follows: ospr forward, ′ -ttatcctgctgcttgctggt- ′ ; ospr reverse, ′ -gatgttctcgccgtacttcc- ′ ; ospr forward, ′ -ggcaccatctacaccatgaa- ′ and ospr reverse, ′ -ttgtcggctgtgatgaatgt- ′ ; oswrky forward, ′ -ccggcatggagttcttcaag- ′ and oswrky reverse, ′ -tat t tctgtacacacgcgtggaa- ′ ; oswrky for ward, ′ -aggatgggtaccaatgga- ′ and oswrky reverse, ′ -acgagttgatggagatgga- ′ ; o s w r k y f o r w a r d , ′ -ag c c c a ag at c t c c a ag c t c - ′ a n d o s w r k y re v e r s e , ′ -acgaggatcgtgttgtcctc- ′ ; oshi-lox forward, ′ -gcatccccaacagcacatc- ′ and oshi-lox reverse, ′ -aataaagat t tgggagtgacatat tgg- ′ ; osacs for ward, ′ -ggaataaagctgctgccgat- ′ and osacs reverse, ′ -tgagcctgaagtcgttgaagc- ′ . niclosamide effect on rice growth and development. three-week-old nipponbare leaves were sprayed with μ g/ml niclosamide at -day intervals until seed maturation. the phenotypic characteristics of untreated and niclosamide-treated plants were measured at days after planting. grain phenotypes were examined after seed maturation. current status and future prospects of research on bacterial blight of rice anatomical studies of rice plant affected with bacterial leaf blight, xanthomonas oryzae (uyeda et ishiyama dowson) studies of bacterial leaf blight of rice mutants of xanthomonas oryzae pv. oryzae deficient in general secretory pathway are virulence deficient and unable to secrete xylanase genetics of xanthan production in xanthomonas campestris: the xana and xanb gene are involved in udp-glucose and gdp-mannose biosynthesis the xanthomonas oryzae pv. oryzae eglxob endoglucanase gene is required for virulence to rice role of an in planta expressed xylanase of xanthomonas oryzae pv. oryzae in promoting virulence on rice functional interplay between two xanthomonas oryzae pv. oryzae secretion systems in modulating virulence on rice plant growth-promoting rhizobacteria mediate induced systemic resistance in rice against bacterial leaf blight caused by xanthomonas oryzae pv biological control of rice diseases field efficacy of stable bleaching powder to control bacterial blight of rice in rice chemical control of bacterial leaf blight of rice caused by xanthomonas oryzae pv induction of systemic resistance in rice to bacterial blight by , , -benzothiadiazole -carbothioic acid-s-methyl ester (bth) treatments probenazole-induced accumulation of salicylic acid confers resistance to magnaporthe grisea in adult rice plants antibiotic use in plant agriculture new pcr-based sequence-tagged site marker for bacterial blight resistance gene xa of rice identification and fine-mapping of xa , a novel gene for resistance to xanthomonas oryzae pv evolution of the rice xa disease resistance gene family construction of a bac contig containing the xa locus in rice r gene expression induced by a type-iii effectors triggers disease resistance in rice xanthomonas oryzae pathovars: model pathogens of a model crop a novel bacterial blight resistance gene from oryza nivara mapped to kb region on chromosome l and transferred to oryza sativa l expression of xa , a bacterial blight resistance gene in rice, is induced by bacterial inoculation resistance genes complexes: evolution and utilization host and pathogen factors controlling the rice-xanthomonas oryzae interaction controlling rice bacterial blight in africa: needs and prospects pyramiding three bacterial blight resistance genes (xa , xa and xa ) using marker-assisted selection into indica rice cultivar pr expression profiling of rice genes in early defense responses to blast and bacterial blight pathogens using cdna microarray code-assisted discovery of tal effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene transgenic expression of cecropin b, an antibacterial peptide from bombyx mori, confers enhaced restance to bacterial leaf blight in rice evaluation of bacterial blight resistance in rice lines carrying multiple resistance genes and xa transgenic lines oswrky is a transcriptional activator that enhances rice resistance to the xanthomonas oryzae pathovar oryzae constitutive expression of rice wrky gene increases the endogenous jasmonic acid accumulation, pr gene expression and resistance to fungal pathogens in rice rice wrky plays a crucial role in benzothiadiazole-inducible blast resistance genome-wide identification of wrky -regulated genes that mediate benzothiadiazole-induced defense responses in rice development of disease-resistant rice by pathogen-responsive expression of wrky inhibition of severe acute respiratory syndrome corona virus replication by niclosamide quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death antihelminth compound niclosamide downregulates wnt signaling and elicits antitumor responses in tumors with activating apc mutations screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mtorc signaling the anti-helminthic niclosamide inhibits wnt/frizzled signaling the autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in k cells structure-activity analysis of niclosamide reveals potential role for cytoplasmic ph in control of mammalian target of rapamycin complex (mtorc ) signaling mechanism of action of reagents that uncouple oxidative phosphorylation studies on tolerance and rate reducing bacterial pustule of soybean cultivars/lines characteristic expression of twelve rice pr family genes in response to pathogen infection, wounding, and defense-related signal compounds ( / ) identification of an ospr a promoter region responsive to salicylic acid a comprehensive expression analysis of the wrky gene superfamily in rice plants during defense response oswrky , a rice transcription factor, is involved in rice defense response antineoplastic mechanisms of niclosamide in acute myelogenous leukemia stem cells: inactivation of the nf-kappa b pathway and generation of reactive oxygen species novel effect of antihelminthic niclosamide on s a -mediated metastatic progression in colon cancer making sense of hormone-mediated defense networking: from rice to a pair of allelic wrky genes play opposite roles in rice-bacteria interactions effect of probenazole on the activities related to the resistant reaction in rice plant a benzothiadiazole derivative induces systemic acquired resistance in tobacco benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat benzothiadiazole induces disease resistance in arabidopsis by activation of the systemic acquired resistance signal transduction pathway new life for an old drug: the anthelmintic drug niclosamide inhibits pseudomonas aeruginosa quorum sensing a review of the safety of niclosamide, pyrantel, triclabendazole and oxamniquine arabidopsis nitrate reductase activity is stimulated by the e sumo ligase atsiz we are grateful to dr. hong-gu kang in texas state university for critical reading of the manuscript. this work was supported by a grant from the next-generation biogreen program (plant molecular breeding center no. pj ), rural development administration, republic of korea. key: cord- -rc h drd authors: li, xuanyi; sigworth, elizabeth a.; wu, adrianne h.; behrens, jess; etemad, shervin a.; nagpal, seema; go, ronald s.; wuichet, kristin; chen, eddy j.; rubinstein, samuel m.; venepalli, neeta k.; tillman, benjamin f.; cowan, andrew j.; schoen, martin w.; malty, andrew; greer, john p.; fernandes, hermina d.; seifter, ari; chen, qingxia; chowdhery, rozina a.; mohan, sanjay r.; dewdney, summer b.; osterman, travis; ambinder, edward p.; buchbinder, elizabeth i.; schwartz, candice; abraham, ivy; rioth, matthew j.; singh, naina; sharma, sanjai; gibson, michael k.; yang, peter c.; warner, jeremy l. title: seven decades of chemotherapy clinical trials: a pan-cancer social network analysis date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: rc h drd clinical trials establish the standard of cancer care, yet the evolution and characteristics of the social dynamics between the people conducting this work remain understudied. we performed a social network analysis of authors publishing chemotherapy-based prospective trials from to to understand how social influences, including the role of gender, have influenced the growth and development of this network, which has expanded exponentially from fewer than authors in to , in . while . % of authors were directly or indirectly connected by , our results indicate a tendency to predominantly connect with others in the same or similar fields, as well as an increasing disparity in author impact and number of connections. scale-free effects were evident, with small numbers of individuals having disproportionate impact. women were under-represented and likelier to have lower impact, shorter productive periods (p < . for both comparisons), less centrality, and a greater proportion of co-authors in their same subspecialty. the past years were characterized by a trend towards increased authorship by women, with new author parity anticipated in . the network of cancer clinical trialists is best characterized as strategic or mixed-motive, with cooperative and competitive elements influencing its appearance. network effects such as low centrality, which may limit access to high-profile individuals, likely contribute to the observed disparities. the modern era of chemotherapy began in , with publications describing therapeutic uses of nitrogen mustard , . over the next years, the repertoire of available cancer treatments has expanded at an ever-increasing pace. chemotherapeutics have a notably low therapeutic index, i.e., the difference between a harmful and beneficial dose or combination is often quite small . consequently, a complex international clinical trial apparatus emerged in the s to study chemotherapeutics in controlled settings, and prospective clinical trials remain the gold standard by which standard of care treatments are established , . discoveries made by successive generations have led to overall improvement in the prognosis of most cancers . while social network analysis has been used to study patterns of co-authorship in scientific settings , , the social component of clinical trial research is not well characterized. little is known about how social factors have shaped the progress of the field, as cancer care has become increasingly subspecialized, and how social network baseline characteristics. n = of reviewed publications with an aggregate of n = , authors met the inclusion criteria (consort figure s ). cumulatively, most authors in the network (n = , , %) published at least one randomized trial, with n = , ( . %) participating in the publication of a "positive" trial (table s ). most of the included authors (n = , , . %) participated in the primary publication of a clinical trial, while a smaller subgroup (n = , , . %) participated in the publication of updates. the most common venues for publication were high-impact clinical journals: the journal of clinical oncology (n = , . %), the lancet family (n = , . %), the new england journal of medicine (n = , . %), and the blood family (n = , . %). co-authorship has changed in a non-linear fashion over time: the median number of authors per publication increased from n = in to n = (iqr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in ( figure s ). across subspecialties, the median number of co-authors per publication varied somewhat, from a low of n = (iqr - ) in gynecologic oncology to a high of n = (iqr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in dermatologic oncology. median longevity is < year at all times, although the number of authors with multiple years in the field grows substantially over time ( figure s ). a small number of individuals maintained the highest impact over time-nearly years each in the case of chemotherapy pioneers sidney farber and james f. holland ( figure s ). in any given year, most authors had a betweenness centrality of < % of the maximum; conversely, a very small number of authors had an exceptionally high score, with % of authors accounting for % of the total in recent years ( figure s ). accordingly, an increasingly smaller proportion of authors were both very highly connected and highly impactful; in , the % highest-impact authors (n = ) account for . % of links and . % of impact; in , the same proportion (n = ) account for . % of links and . % of impact. first/last authorship has also become concentrated; in publications, % of authors had at least one such role, whereas prior to it was on average > % ( figure s ). the structure of the network changes considerably over time, from relatively dense and connected to sparse and modular (fig. b) . the final network is very sparse ( . % of possible links are present); nevertheless, n = , ( . %) authors are in a single connected component; the next-largest component comprises authors. each of the cancer subspecialties developed at different rates, with clear influence of seminal events in several subspecialties, e.g., the introduction of adjuvant therapy and tamoxifen for breast cancer, completely new classes of drugs for plasma cell disorders, and immunotherapy for melanoma (fig. c) - . network visualization and cumulative metrics. the final cumulative network visualization is shown in figs. & s . the impact score of authors is unevenly distributed, median . (range - . ); however, the log-transformed impact scores approximate a normal distribution ( figure s ). authors with longevity ≥ year who changed primary subspecialty at least once (n = ) had nearly twice the median impact and longevity of those who remained in one subspecialty (n = , ), . (iqr . - . ) versus . (iqr . - . ) and years (iqr - ) versus years (iqr - ), respectively (p < . for both comparisons). cumulatively, subspecialized authors with calculable homophily (n = , ) have a median proportion of co-authors sharing the same subspecialty of % (iqr - %); , ( . %) of these authors' outlinks are within-subspecialty. this is reflected by a high assortativity by subspecialty since the mid- s (fig. b) modularity follows a sigmoid pattern with a period of linear increase between - followed by a plateau at high modularity; assortativity rapidly increases in early decades; median normalized pagerank decreases to a low plateau from the s onward; (c) subspecialties develop at different but broadly parallel rates, with seminal events apparently preceding accelerations of individual subspecialties, e.g.,: ( ) in the four years after , combination therapy (ac ), adjuvant therapy , and tamoxifen were introduced in breast cancer; ( ) thalidomide and bortezomib were reported to be efficacious for multiple myeloma; and ( ) immunotherapy (ipilimumab , ) was introduced in the treatment of melanoma. www.nature.com/scientificreports/ sensitivity analysis. normalized score distributions did not change significantly, although modulation of the trial design coefficient led to a bimodal peak ( figure s ). correlation of assortativity and modularity was high, ranging from . - . for the former and . - . for the latter (table s ; figure s ). the remarkable gains in the fields of hematology and oncology can be ascribed to the tireless work of numerous trialists and the generosity of countless patient participants. as a result, systemic antineoplastics now stand beside surgery and radiotherapy as a pillar of cancer care. our analysis of clinical trialists as a social network, particularly with respect to the density distribution of pagerank, reveals a mixed-motive network that differs only authors assigned to a subspecialty are visualized; these account for % of all authors in the database. this figure highlights various clustering trends by subspecialty, such as the apparent sub-clusters of sarcoma research (yellow) and the two dominant clusters of breast cancer research (pink). it is clear as well that certain subspecialties are more cohesive than others, such as the tightly clustered dermatology (black) compared to the spread-out head and neck cancer authors (red). www.nature.com/scientificreports/ substantially from "collegial" and "friend-based" online social networks. while clinical trials are conducted towards a collaborative goal-improved outcomes for all cancer patients-there are significant competitive pressures. examples of these pressures include resource limitations (e.g., funding and patients available for accrual), the tension between prioritization of cooperative group versus industry-funded trials, personal motivations such as academic promotion or leadership opportunities, and institutional reputation. the emergence of formal and informal leaders in scientific networks has been shown to facilitate research as well as create clusters . as fig. shows, there is a strong tendency for clustering based on subspecialty in the complete network, although some subspecialties (e.g., lymphoid and myeloid malignancies) have many more interconnections than others (e.g., sarcoma and neuro-oncology). many of these clusters appear to be organized around an individual or group of individuals who have high impact and centrality. as an organizational principle, these individuals appear to rarely be in direct competition, but their presence is a clear indicator of scale-free phenomena within the network. the facts that betweenness centrality follows a power law cumulative distribution bolsters this theory. scale-free phenomena, which are defined by a power law distribution of connectedness, are very common in strategic networks, especially when they become increasingly sparse, as this network does . the two related theories for this network behavior are preferential attachment and fitness. the former observes that those with impact tend to attract more impact; the latter postulates that such gains for the "fittest" come at the expense of the "less fit" . seminal events (fig. c) are likely a driver of preferential attachment , and may the network is overwhelmingly dominated by men until , when a trend towards increasing authorship by women begins to be seen; however, representation by women in first/last authorship remains low; gray shaded lines are % confidence intervals of the loess curves; (b) men tend on average to have a longer productive period and to achieve a higher author impact score than women (p < . for both comparisons); (c) men tend on average to be more central and have more collaborations outside of their subspecialty. note that the homophily calculation requires a subspecialty assignment, which explains the slightly lower numbers in (c) as compared to (b). www.nature.com/scientificreports/ partially explain why some authors change their primary subspecialty at least once over time (e.g., through a "bandwagon" effect driven by the diffusion of ideas ). given that these authors were observed to have nearly twice the impact and longevity of their single subspecialty peers, this dynamic will be a focus of future study, including calculation of the q factor, a metric developed to quantify the ability of a scientist to "take advantage of the available knowledge in a way that enhances (q > ) or diminishes (q < ) the potential impact p of a paper" . in the analysis of network dynamics (fig. b) , the field as a whole appears to emerge in the s, which is also when medical oncology and hematology were formally recognized through board certification. measurements of field maturity are by their nature subjective, but the pessimism of the late s was captured by sidney farber: "…the anticancer chemicals, hormones, and the antibiotics…marked the beginning of the era of chemotherapy of cancer, which may be described after years as disappointing because progress has not been more rapid…" . these concerns prompted the us national cancer act of , which was followed by the leveling of modularity at a very high level from onwards, suggesting that the subspecialties generated in the s have remained stable. the assortativity by subspecialty has increased as well, with recent levels approximately twice those seen in a co-authorship network of physicists . while median pagerank has decreased markedly, indicating decreasing influence for the average author, the distribution in is broadly right-skewed ( figure s ). these findings reveal a high level of increasing exclusivity, suggesting that it is becoming progressively more difficult to join the top echelon of the network. this has major implications for junior investigators' mobility, and potentially for the continued health of the network as a whole. while there is much to be applauded in the continued success of translating research findings into the clinic, we observed clear gender disparities within the cancer clinical trialist network: women have a statistically significantly lower final impact score, shorter productive period, less centrality, and less collaboration with those outside of their primary subspecialty. these findings are consistent with and build upon previous literature on www.nature.com/scientificreports/ the challenges facing women in pursuing and remaining in academic careers , , , . they are also consistent with more recent gender disparity findings, such as those observed in research published on covid- . other studies investigating the basis for such a gender gap have identified several layers of barriers to the advancement of women in academic medicine. these include sexism in the academic environment, lack of mentorship, and inequity with regards to resource allocation, salary, space, and positions of influence , . our study suggests that additional network factors such as relatively low centrality, which indicates a lack of access to other individuals of influence, and high homophily, which indicates a lack of access to new ideas and perspectives, also perpetuate the gender gap-corroborating recent findings from graduate school social networks . it is somewhat encouraging that there has been a steady increase in the proportion of authorship by women since (fig. a) . this increase is observed approximately a decade after the passage of title ix of the us civil rights act in . given that the majority of authors in this network are clinicians, a partial explanation could be that us-based women began to attend previously all-male medical schools in the early s, completed their training, and began to contribute to the network as authors approximately years later. if the nearly linear trend continues, we predict that gender parity for new authors entering the network will be reached by the year , years after us medical school enrollment approached parity . however, the proportion of first/last authors who are women is growing much more slowly, and parity may not be reached for + years, if at all. given that senior authorship is a traditional metric of scholarly productivity, it may be particularly difficult for clinical trialists who are women to obtain promotion under the current paradigm. one possible solution is to increase the role of joint senior authorship, which remains vanishingly rare in the clinical trials domain (furman et al. is one of very few examples that we are aware of)-although this is predicated on the acceptance of these roles by advancement and promotion committees. the field itself may also suffer from slow entry of new talent and a lack of broad perspectives. while the gender mapping algorithm and manual lookups are imperfect, our approach is consistent with prior work in this area , . unisex names posed a particular challenge . it should be noted that we could not account for all situations where an author changed their name (e.g., a person assumed their spouse's surname); this could have led to overestimation of representation by women and underestimation of impact, since this practice is more common with women. it is also possible that an individual's gender identity does not match the gender assignment of their given name. future work will include further analysis of gender disparities, factoring in institutional affiliation and highest degree(s) obtained, which are both likely to have significant influence on publication and senior authorship , . there are several additional limitations to this work, starting with the fact that co-authorship is but one way to measure social network interactions and this study reports results from published trials, which induces publication bias. although hemonc.org aims to be the most comprehensive resource of its kind, non-randomized trials and randomized phase ii trials are intentionally underrepresented, given that findings at this stage of investigation infrequently translate to practice-changing results (e.g., approximately % of oncology drugs fail during phase ii) [ ] [ ] [ ] . the effect of any biases introduced by this underrepresentation is unclear, given the confounding influence of publication bias, which may itself be subject to gender disparity . some older literature which no longer has practice-changing implications may have been overlooked. during name disambiguation, some names could not be resolved, primarily because neither medline nor the primary journal site contained full names. this effect is non-random, since certain journals do not publish full names. the choice of coefficients and their relative weights was based on clinical intuition and consensus; given that the "worth" of metrics such as first/last authorship is fundamentally qualitative, there must be some degree of subjectivity when formulating a quantitative algorithm. while the sensitivity analysis demonstrated that neither normalized author impact score distribution, assortativity, nor modularity are majorly changed by variation in the trial design and author role coefficients, it remains possible that other combinations of coefficients and relative weightings could lead to different results. furthermore, our impact algorithm weighs heavily on first and last authorship, but the definition of senior authorship has changed over time. for example, in the article by goodman et al. , the authors were listed in decreasing order of seniority (personal communication). in general, the impact score used in this paper, although similar to others proposed in the academic literature, is not validated and should be interpreted with caution. finally, the majority of authors in this database publish extensively, and their impact as measured here should not be misconstrued to reflect their contributions to the cancer field more broadly. in conclusion, we have described the first and most comprehensive social network analysis of the clinical trialists involved in chemotherapy trials. we found emergent properties of a strategic network and clear indications of gender disparities, albeit with improvement in representation in recent decades. the network has been highly modular and assortative for the past years, with little collaboration across most subspecialties. as the field pivots from an anatomy-based to a precision oncology paradigm, it remains to be seen how the network will re-organize so that the incredible progress seen to date can continue. - and referenced on hemonc.org were considered for inclusion. hemonc.org is the largest collaborative wiki of chemotherapy drugs and regimens and has a formal curation process . in order for a reference to be included on hemonc.org, it generally must include at least one regimen meeting the criteria outlined here: https ://hemon c.org/wiki/eligi bilit y_crite ria. as such, the majority of references on hemonc.org are randomized controlled trials (rcts) or non-randomized trials with at least participants and/or practice-changing implications. one of the main goals of hemonc.org is creating a database of all standard of care systemic antineoplastic therapy regimens. this is difficult as there is no universally accepted definition of standard of care except in a www.nature.com/scientificreports/ legal capacity. for example, the state of washington, in its legislation on medical negligence, inversely defines the standard of care as "exercis[ing] that degree of skill, care, and learning possessed at that time by other persons in the same profession". we currently employ four separate definitions that meet the threshold of standard of care: . the control arm of a phase iii randomized controlled trial (rct). by implication, this means that all phase iii rcts with a control arm must eventually be included on the website. . the experimental arm(s) of a phase iii rct that provide(s) reasonable evidence (p-value less than . ) of superior efficacy for an intermediate surrogate endpoint (e.g., pfs) or a strong endpoint (e.g., os). . a non-randomized study that is either: . any study (including case series and retrospective studies) that is specifically recommended by a member of the hemonc.org editorial board. all section editors of the editorial board with direct oversight of diseasespecific pages are board-eligible or board-certified physicians. in order to identify new regimens and study references for inclusion on hemonc.org, we undertake several parallel screening methods: as part of the process of building hemonc.org, we have also systematically reviewed all lancet, jama, and new england journal of medicine tables of contents from to december , . in addition, the citations of any included manuscript are hand-searched for additional citations. for any treatment regimen that has been subject to randomized comparison, we additionally seek to identify the first instance in which such a regimen was evaluated as an experimental arm; if no such determination can be made, we seek the earliest non-randomized description of the regimen for inclusion on the website. in order or prioritization, phase iii rcts are added first, then smaller rcts such as randomized phase ii, followed by non-randomized trials, followed by retrospective studies or case series identified by our editorial board as relevant to the practice of hematology/oncology. when a reference is added to hemonc.org, bibliographic information including authorship is recorded. the usually coincides with medline record details, although some older references in medline are capped at ten authors and are manually completed based upon the publication of record. for trials that do not list individual authors (e.g., the elderly lung cancer vinorelbine italian study group ), the original manuscript and appendices are examined for a writing committee. if a writing committee is identified, the members of this committee are listed as authors in the order that they appeared in the manuscript. if no writing committee is identified, the chairperson(s) of the study group are listed as the first & last authors. if no chairpersons are listed, the corresponding author is listed as the sole author. www.nature.com/scientificreports/ publications solely consisting of the evaluation of drugs not yet approved by the fda or other international approval bodies were not included. trials that appeared in abstract form only, reviews, retrospective studies, meta-analyses, and case reports were excluded, as were trials reporting only on local interventions such as surgery, radiation therapy, and intralesional therapy. non-antineoplastic trials (table s ) and trials of supportive interventions (e.g., antiemesis; growth factor support) were also excluded. disambiguation of author names. for each included publication, author names were extracted and disambiguated. author names on hemonc.org are stored in the medline lastname_firstinitial (middleinitial) format, which can lead to two forms of ambiguity: ( ) the short form, e.g., smith_j, can refer to two or more individuals, e.g., julian and jane smith; ( ) two short forms can refer to the same individual, e.g., kantarjian_h and kantarjian_hm. additionally, names can be misspelled and individuals can change their name over time (e.g., a person assumes their spouse's surname). we undertook several steps to disambiguate names: ( ) full first and middle names, when available, were programmatically accessed through the ncbi pubmed eutils application programming interface; ( ) when not available through medline, full first names were searched for on journal websites or through web search engines; ( ) automatic rules were developed to merge likely duplicates; and ( ) some names were manually merged (e.g., misspellings: benboubker_lofti and benboubker_lotfi; alternate forms: rigal-huguet_francoise and huguet-rigal_francoise; and subsumptions: baldotto_clarissa and serodio da rocha baldotto_clarissa). transformation algorithms are available upon request, and the full mapping table is provided in supplemental file . gender mapping. once the name disambiguation step was complete, we mapped authors with full name available to gender. we first mapped names to genders using us census data, which includes the relative frequencies of given names by gender in the population of us birth from to . we calculated the gender ratio for names that appeared as both genders. for names with gender ratio > . for one gender (e.g., john, rebecca), we assigned the name to that gender. to expand gender mapping to include names that are more frequently seen internationally (e.g., jean, andreas), we used a program that searches from a dictionary containing gender information about names from most european countries as well as some asian and middle eastern countries . for unmatched first names (e.g., dana, michele), we manually reviewed for potential gender assignment. for some names that are masculine in certain countries and feminine in others (e.g., andrea, daniele, and pascale are masculine in italy and feminine elsewhere), we mapped based on surnames. finally, we performed manual internet searches to look for photographs and pronouns used in online content such as faculty profiles, book biographies, and professional social media accounts for the remaining unmapped full names associated with a longevity of greater than one year. a total of , ( %) authors were assigned to the categories of woman (n = ; . %) or man (n = , ; . %). the gender of most of the people with unassigned names could not be determined because they only appeared with initials (n = ; . %) in the primary publication and medline. the remaining n = ( . %) were ambiguously gendered names that could not be resolved through manual searching, and were excluded in the gender-specific analyses. the full mapping table is provided in supplemental file . author impact score. we considered existing metrics for measuring author impact - , but ultimately proceeded with our own formulation given some of the unique considerations of prospective clinical trials and their impact. every author was assigned an impact score, using an algorithm calculated per manuscript using four coefficients: ( ) author role; ( ) trial type; ( ) citation score; ( ) primary versus updated analysis. the coefficients are multiplied to arrive at the score, and the total author impact score is summed across all of their published manuscripts. author role: first and last author roles are assigned a coefficient of three; middle authors are assigned a coefficient of one. when joint authorship is denoted in a medline record, there is an additional attribute "equalcontrib" that is set to "y" (yes). we look for this during the parsing process and treat these authors as first or last authors when the attribute is detected. trial type: any prospective trial with randomization is denoted as randomized and the authors of any manuscript reporting on such a trial are assigned a coefficient of two. non-randomized trials are assigned a coefficient of one. for manuscripts that reported on more than one trial with mixed designs (i.e., one or more randomized and one or more non-randomized trials), the randomized coefficient was used. citation score: we programmatically obtained a snapshot of citation counts from google scholar from september and used unadjusted total citations as the citation score coefficient for the years - . as more recent publications are still accruing citations, raw citation count is not an appropriate measure of their impact. therefore, we have calculated a blended citation score for articles published between - , adding the phased in median citation count for the journal tier in which the article was published for the years - (see tables s & s and figure s ). the citations scores are normalized to the manuscript with the maximum number of citations (stupp et al. , with , citations), such that the maximum citation score is one. primary publications vs. updates: the baseline coefficient is one. for updates, this score is multiplied by a half-life decay coefficient; i.e., scores for the first update are multiplied by %; scores for the second update by %; and so forth. this rule is applied equally to updates and subgroup analyses. for manuscripts that reported on pooled updates of more than one trial, the score was multiplied by the half-life coefficient corresponding to the update that resulted in the maximum score. see examples in supplemental methods. www.nature.com/scientificreports/ subspecialty designation of each publication. each publication was assigned to one of diseasespecific cancer subspecialties based on the cancer(s) studied (table s ). the majority of publications report on a clinical trial carried out in one disease or several diseases mapping to the same subspecialty. for publications studying diseases that map to more than one subspecialty, each author's impact score for that publication was divided evenly across the subspecialties. several clinical trials employ a site-agnostic approach, e.g., to a "cancer of unknown primary" or to biomarker-defined subsets of cancers (e.g., a basket trial ); for these, impact across subspecialties was split manually (table s ) . subspecialty designation based on authorship. authors were eligible for assignment to a primary subspecialty based on whether they were a first or last author at least once in the subspecialty, or whether they had a cumulative impact of at least one standard deviation below the mean of the author impact score of all authors in the subspecialty. authors who met either of these criteria were assigned to a primary subspecialty based on where the majority of their impact lay; if an author had equal impact in two or more subspecialties they were assigned equally to the subspecialties. this assignment was recalculated on an annual basis if the author had new publications, and primary subspecialty was re-assigned if a new subspecialty met either of the criteria and the impact in that subspecialty was higher than in the previous primary subspecialty. authors not meeting either of these criteria were assigned a primary subspecialty of "none" and were not included in the homophily analysis or the network visualization. social network construction and metrics. a dynamic social network was created with nodes representing authors and links representing co-authorship. the dynamic social network was discretized by year and the authors, scores, and links were cumulative (e.g., the th network was cumulative from - ). therefore, once an author is added to the network, they remain in the network, with their impact score cumulatively increasing as they publish and remaining constant if publication activity ceases. the following temporal metrics were calculated: ( ) network density (the number of actual connections/links present divided by the total number of potential connections); ( ) modularity by subspecialty (a measure of how strongly a network is divided into distinct communities, in this case subspecialties, defined as the number of edges that fall within a set of specified communities minus the number expected in a network with the same number of vertices and edges whose edges were placed randomly); ( ) assortativity by subspecialty (a measure of the preference of nodes in a network to attach to others that are similar in a defined way, in this case the same subspecialty; assortativity is positive if similar vertices tend to connect to each other, and negative if they tend to not connect to each other); ( ) betweenness centrality (a measure reflecting how important an author is in connecting other authors, calculated as the proportion of times that an author is a member of the bridge that forms the shortest path between any two other authors); ( ) pagerank (another measure of centrality, this time considering the connection patterns among each author's immediate neighbors; its value for each author is the probability that a person starting at any random author and randomly selecting links to other authors will arrive at the author); and ( ) proportion of co-authors sharing either the same primary subspecialty designation or the same gender (hereafter referred to as homophily). network density, modularity, and assortativity are calculated at the network level, while betweenness centrality, pagerank, and homophily are calculated at the author (node) level. further definitions of these metrics are provided in the supplemental glossary. all metrics incorporated the weighted co-authorship score, which takes into account each co-author's impact modified by the number of authors of an individual publication. for each pairwise collaboration, as defined by co-authorship on the same manuscript, a co-authorship score was calculated and used as the edge weight; duplicated edges were allowed to reflect the fact that weights could be distributed in a non-even fashion (e.g., two co-authors could be middle authors on a lower-impact publication as well as senior authors on a separate high-impact publication). this score was first calculated by multiplying the individual authors' manuscriptspecific impact scores together. in order to acknowledge the role of middle authors in large multi-institutional studies, this preliminary score was divided by the total number of authors on the manuscript. this has the effect of decreasing the weight of any individual co-authorship relationship in a paper with many authors, while allowing the overall weight of the neighborhood consisting of all co-authorship connections to increase linearly with the number of authors (see examples in supplemental methods). in order to visualize the final cumulative network, layout was determined using the distributed recursive graph algorithm . nodes were sized by author impact score rank and colored by primary subspecialty designation. edge width was determined by the weighted co-authorship score. statistical analysis. non-independent network metrics including growth, density, assortativity, modularity, and pagerank are reported descriptively with medians and interquartile ranges (iqr). gender proportion over time was fit with locally estimated scatterplot smoothing (loess) regression using default settings of degree = with smoothing parameter/span α = . . for the final cumulative network, the independent variables author impact score and longevity were compared ( ) between genders and ( ) by whether the author changed subspecialties over time; only those authors with longevity ≥ year were included in the second comparison. these comparisons were made with the two-sided wilcoxon rank sum test; p value < . was considered statistically significant. www.nature.com/scientificreports/ sensitivity analysis. to determine whether the scoring algorithm was robust to modifications, we conducted a sensitivity analysis where the author role and trial design coefficients were varied by ± % and ± %, respectively. normalized density distributions for the final cumulative network under each permutation were calculated, and temporal assortativity and modularity were compared to baseline with pearson's correlation coefficient. a version of this manuscript is posted on the medrxiv preprint server, accessible here: https ://www.medrx iv.org/conte nt/ . / v . a very early version of the work was presented in poster format at the visual analytics in healthcare workshop (november ). there are no other prior presentations. the datasets generated and analyzed in this study are available at harvard dataverse . received: january ; accepted: september scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ the biological actions and therapeutic applications of the b-chloroethyl amines and sulfides nitrogen mustard therapy; use of methyl-bis (beta-chloroethyl) amine hydrochloride and tris (beta-chloroethyl) amine hydrochloride for hodgkin's 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network analysis vanderbilt university) conducted and are responsible for the data analysis. we declare the following interests gibson are members of the editorial board of hemonc.org. rozina a. chowdhery, ronald s. go and eddy j. chen were members of the editorial board of hemonc.org. all positions at hemonc.org are voluntary and uncompensated, and the stock of hemonc.org llc has no monetary value none of the funders had any direct role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.l.w.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -acqiyqwz authors: jeong, hye jin; min, sein; chae, heelim; kim, sarah; lee, gunwoo; namgoong, sung keon; jeong, keunhong title: signal amplification by reversible exchange for covid- antiviral drug candidates date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: acqiyqwz several drug candidates have been proposed and tested as the latest clinical treatment for coronavirus pneumonia (covid- ). chloroquine, hydroxychloroquine, ritonavir/lopinavir, and favipiravir are under trials for the treatment of this disease. the hyperpolarization technique has the ability to further provide a better understanding of the roles of these drugs at the molecular scale and in different applications in the field of nuclear magnetic resonance/magnetic resonance imaging. this technique may provide new opportunities in diagnosis and research of covid- . signal amplification by reversible exchange-based hyperpolarization studies on large-sized drug candidates were carried out. we observed hyperpolarized proton signals from whole structures, due to the unprecedented long-distance polarization transfer by para-hydrogen. we also found that the optimal magnetic field for the maximum polarization transfer yield was dependent on the molecular structure. we can expect further research on the hyperpolarization of other important large molecules, isotope labeling, as well as polarization transfer on nuclei with a long spin relaxation time. a clinical perspective of these features on drug molecules can broaden the application of hyperpolarization techniques for therapeutic studies. ritonavir/lopinavir ( , -thiazol- -ylmethyl n-[( s, s, s) - -hydroxy- -[[( s)- -methyl- -[[methyl-[( - propan- -yl- , -thiazol- -yl)methyl]carbamoyl]amino]butanoyl]amino]- , -diphenylhexan- -yl]carbamate/ ( s)-n-[( s, s, s)- -[[ -( , -dimethylphenoxy)acetyl]amino] - -hydroxy- , -diphenylhexan- -yl]- -methyl- -( -oxo- , -diazinan- -yl)butanamide), the combination drug marketed as kaletra, is a relatively new medication for the treatment and prevention of hiv. this compound has also been suggested as a potential drug candidate against covid- due to its role as a proteinase inhibitor in association with the polyprotein processing of the coronavirus , . recent reports have provided evidence in favor of this drug for the treatment of covid- , however, its beneficial effects remain a topic of controversy . it is known to lessen viral loads and improve clinical symptoms , . however, compared to chloroquine and hydroxychloroquine, the interactions of this drug in the body at the molecular scale have not been elucidated so far. favipiravir ( -fluoro- -oxo- h-pyrazine- -carboxamide), which has been approved in japan for the treatment of influenza since , , has also shown effectiveness in accelerating viral clearance in chinese trials of hundreds of patients . to further understand the interactions of drugs with proteins, their metabolism, and other activities, nuclear magnetic resonance (nmr) spectroscopy has been widely used in pharmacokinetics. however, nmr is an inherently insensitive technique due to the small population differences in the spin states. hyperpolarization, which generates a non-boltzmann distribution of the spin state populations, may provide a breakthrough in addressing this challenge. moreover, magnetic resonance imaging (mri) is used as a solution to understand drug distribution throughout the body in vivo and study its activity. to visualize mri signals, the drug must be tagged with specific compounds, such as chelating agents (e.g., gd-chelate and mn-chelate) with t /t contrast. however, the fusion of additional compounds with the drug may have unpredictable effects due to their different molecular structures. this limitation may be overcome by using the hyperpolarization technique, which enhances the visualization of the hyperpolarized signal through mri. the use of this state-of-the-art technology can address several challenges and could be a key to visualizing and understanding the in vivo real-time distribution and activity of drugs. this may provide an opportunity to further elucidate the antiviral activities of drugs used in the covid- therapy. of the several methods to hyperpolarize drugs, the para-hydrogen-based signal amplification by reversible exchange (sabre) method is the most promising for the hyperpolarization of several key structures with nitrogen. although a remarkably high signal amplification is achieved using dynamic nuclear polarization, it requires extreme conditions (low temperature and high magnetic field) and a long hyperpolarization time (more than - h). para-hydrogen-induced polarization provides a much higher signal enhancement than sabre; however, molecules cannot be hyperpolarized continuously by parahydrogen. in this context, sabre does not require harsh conditions, and substrates can be constantly hyperpolarized without any structural changes during the polarization transfer. furthermore, in this method, the polarization can be real-time transferred from protons to other isotopes such as recently, several breakthrough studies that develop hyperpolarized drugs or metabolites [ ] [ ] [ ] [ ] [ ] , including those in pharmacokinetics, confirmed that sabre could be useful for a wide-scale application. this is particularly important at a time when scientific remedies are the only means to conquer this pandemic. however, hyperpolarized drugs or metabolites are limited to small-sized molecules, which constrain their practical applications in many studies. hyperpolarization studies using sabre may be useful and important in facilitating future applications of mri scanning using hyperpolarized covid- drug candidates, which mostly have high molecular weights. it is anticipated that sabre-based hyperpolarization studies on such large molecules may enable further research on a higher number of drugs and metabolites. to the best of our knowledge, this study is the first to evaluate the sabre-based hyperpolarization of specific covid- drug candidates in real-time. favipiravir sabre. the pyrazine moiety in favipiravir is recognized as the polarization source for sabre . however, its complex structure with several functional groups (fluorine, alcohol, and amide) has not been previously examined for sabre hyperpolarization (fig. ) . figure depicts an estimate of its complex with an ir catalyst, forming an exchangeable bond between ir(i) and the pyrazine moiety. this form is expected for favipiravir to transfer polarization from para-hydrogen. as predicted, we successfully obtained a hyperpolarized signal from the aromatic proton of favipiravir with a ~ -fold enhanced signal after sabre. an additional polarized signal was observed around - ppm and was attributed to the protons in the hydroxy and amide groups of the structure. the sabre-based polarization trend with the magnetic field was maximized at approximately g, which is consistent with previous reports (fig. ) - . chloroquine sabre. chloroquine (hydroxychloroquine) contains a quinoline structure, which may potentially be hyperpolarized using sabre. however, chloroquine has a long attachment (fig. ) , which could not be operable as sabre is dependent on the chemical exchange reaction. it is assumed that the transfer of its binding and kinetics to hyperpolarization with sufficient time from para-hydrogen may be challenging. (fig. ) . interestingly, the polarization transfer from para-hydrogen to chloroquine (hydroxychloroquine) was noteworthy as the polarization transfer occurred across a long distance of bonds (from nitrogen to hydrogen number ). if nitrogen in quinoline is the only group ligating with the ir catalyst, this is the first observation of such long-range hyperpolarization via sabre (fig. ). it will be worth conducting further studies on the polarization transfer mechanism in sabre with other molecules, as discussed later in this study. to understand the mechanisms at play, the enhancement of chloroquine (hydroxychloroquine) by hyperpolarization was measured by changing the magnetic field during the polarization transfer period (fig. ). www.nature.com/scientificreports/ although the degree of the polarization enhancement was similar, the protons in carbon (h- in hydroxychloroquine and h- in chloroquine) had the highest and second-highest enhancement, respectively, which differed greatly from the nitrogen in the quinoline moiety. this difference may stem from the direct polarization transfer from the hydride, which bonds to the ir catalyst through h- (hydroxychloroquine) and h- (chloroquine) via a dipolar coupling or through the polarization transfer from h- to h- /h- by a dipolar coupling or by a j-coupling network , . the clarification of this mechanism requires further detailed future research. similarly, the h- proton in quinoline on both structures showed high enhancement. these results indicate that the mechanism of polarization transfer is dependent on even a small change in structure and solubility. it is assumed that the relatively small polarization enhancement can be increased by using higher parahydrogen concentrations, higher partial pressures, and optimal sabre catalysts . these modifications might www.nature.com/scientificreports/ drastically improve the enhancement. as fig. indicates, the polarization was maximized around g, and exhibited a similar trend as that revealed by previous reports on polarization transfer mechanisms . ritonavir/lopinavir sabre. ritonavir/lopinavir does not contain any well-known functional groups in the structure, which can be harnessed to efficiently undergo polarization transfer from para-hydrogen. furthermore, lopinavir does not contain sp nitrogen in the structure, which has been widely used for polarization transfer. however, both structures contain a carbonyl group, which can transfer polarization from para-hydrogen to the carbonyl group with the ester group/amide group in the neighborhood. a recent study demonstrated that the carbonyl group can bind to the ir catalyst, and polarization can be transferred to pyruvate . moreover, we demonstrated that the ir catalyst was binding with carbonyl and phenyl ether by detecting the chemical shift of the lopinavir protons of (fig. ) after the formation of the complex. www.nature.com/scientificreports/ this is the first case in which a binding trend was identified among many functional groups in the structure and, interestingly, polarization was transferred to nearly all protons of lopinavir through this binding site. this behavior should be explored further to understand the polarization transfer mechanism because other functional groups could also participate in binding with the ir catalyst via a fast exchange, which would mitigate the chemical shift. however, it is noteworthy that the important functional group of lopinavir for polarization transfer could be identified from the chemical shift difference. the polarization transfer from para-hydrogen to ritonavir/lopinavir is noteworthy as the polarization transferred across a long distance of more than bonds in lopinavir. this is also the first case in which extremely long-range hyperpolarization via sabre was observed after binding with the carbonyl group (fig. ) . www.nature.com/scientificreports/ the enhancement of the proton nmr signal on lopinavir by hyperpolarization was measured by changing the magnetic field in the polarization transfer period to understand the sabre mechanism (fig. ) . this enhancement was also relatively small; however, it was expected to increase when higher para-hydrogen concentrations and partial pressures were used. as fig. shows, polarization was maximized around g, which exhibits a similar trend to previous reports on polarization transfer mechanisms, including chloroquine. interestingly, h- and h- exhibited the highest polarization enhancement during sabre. this may be attributed to the dipolar coupling, the polarization transfer through space, and the sabre-relay mechanism. understanding the exact mechanism will require further detailed studies in the future . in terms of the results associated with ritonavir sabre, - g was the optimum external magnetic field to match the polarization transfer (fig. ). this is a low magnetic field compared to the results of the previous antiviral drug sabre, which indicates that the optimum magnetic field for polarization transfer is dependent on the binding site with the ir catalyst and the structure, which can induce different scalar coupling constants. www.nature.com/scientificreports/ as such, we need to optimize the magnetic field to yield the maximum polarization enhancement in different materials, including the target materials, the solvent, and the catalysts. it was difficult to identify the binding site of ritonavir with the ir catalyst, as there were several possible binding sites to consider. we observed a small chemical shift in almost all protons of ritonavir after mixing with the ir catalyst. this implies that there may be more than one binding site for ritonavir with the ir catalyst. this finding is supported by that there are various possible binding sites, such as two thiazole moieties and three carbonyl sites. despite the presence of several binding sites in ritonavir, note that hyperpolarization occurred on all protons in the structure, and its enhancement of polarization was almost identical in the higher field. this offers a good explanation for the presence of several binding sites with the ir catalyst. ritonavir's polarization characteristics in the whole structure were only attributable to long-range polarization transfer, the main polarization transfer mechanism for sabre. these long-range polarization transfers imply ritonavir's potential use with hyperpolarized signals in nmr/ mri. this may provide a better understanding of the molecular dynamics of targeted proteins and pharmacokinetics. importantly, it is anticipated that it may be used for tracking the hyperpolarized signal through mri. examples of this include isotope labeling (such as carbon to c isotope and nitrogen into n), in the structures of the studied drugs, which may have a long t time. this is not only due to the lower gyromagnetic ratio but also because of its smaller relaxation effect from the ir catalyst owing to its relatively unstable complex structure. www.nature.com/scientificreports/ favipiravir, hydroxychloroquine sulfate, and ritonavir were purchased from shanghai alkynechem co., ltd. (shanghai, china) and were used without further purification. chloroquine diphosphate salt ( . %) and lopinavir ( %) were acquired from sigma-aldrich and were used as received. methanol-d (cd od, . atom % d, eurisotop) was also used in the form it was obtained. h nmr spectra used for the characterization of favipiravir, chloroquine, hydroxychloroquine, lopinavir, and ritonavir were acquired on a bruker avance Ш nmr spectrometer operating at a h resonance frequency of mhz, and were referenced to the residual ch peak of cd od (δ = . ). hyperpolarization studies were conducted in the same manner. a home-built instrument was designed as a para-hydrogen generator, in which hydrogen gas (hanmi gas, > . %, a mixture of the spin isomers ortho-hydrogen and para-hydrogen) was allowed to pass through a heat exchanger filled with a feo(oh) catalyst (sigma aldrich) . this structure was filled with liquid nitrogen in a dewar flask and produced ca. % para-hydrogen. in each experiment, para-hydrogen continuously flowed into the drug sample at a rate of www.nature.com/scientificreports/ ml/min at °c and atm. to obtain various magnetic field data, the following system was established and developed: the power supply was gps- d (bench power supply, linear dc). a shielded coil wound with copper-coated wire and a shielded coil outside the first consisted of a mm diameter and mm height. the magnetic field through the shielded coil was regulated by setting the current, which was in the range of - a. the magnetic field generated by the controlled current was measured using a lakeshore gaussmeter. favipiravir ( mg, . × − mmol) and the pre-catalyst ([ir(imes)(cod)cl], mg, . × − mmol) were dissolved in cd od ( μl) . chloroquine and hydroxychloroquine samples for hyperpolarization were prepared by mixing a solution of the substrate ( . × − mmol) and [ir(imes)(cod)cl] ( mg, . × − mmol) in cd od ( μl). lopinavir and ritonavir ( . × − mmol) were added to the cd od ( μl) solution of the pre-catalyst ([ir(imes)(cod)cl], mg, . × − mmol). the mixture of each drug and the pre-catalyst was bubbled by para-hydrogen for min in the nmr tube under the earth's magnetic field for activation. afterwards, in order to induce hyperpolarization on each sample in each magnetic field for sabre, the sample was www.nature.com/scientificreports/ swiftly (less than s) moved directly into a mhz nmr spectrometer and each hyperpolarized proton signal was obtained. all the nmr spectra were then acquired with scan (during s) in a varying magnetic field after polarization of the substrate for min by % para-hydrogen bubbled at ~ °c under atm. (earth's magnetic field, g, g, g, g, and g, respectively). the diverse experiments of the substrate were conducted in the same manner as mentioned above. subsequently, for calculation of the h signal enhancement factor (fold), the following equation was used , : signal(a) = signal of the amplified sample through hyperpolarization, signal(non-a) = signal of the nonamplified sample (normal h nmr signal, i.e., figs. , , ) . spectra were acquired on the same sample using duplicate conditions, such as acquisition parameters and receiver gain except para-hydrogen usage. the raw integrals of the hyperpolarized and non-hyperpolarized spectra were used to calculate the amplification. to determine the exact integral of the signal through the same chemical shift region, the solvent peak was compared. antiviral drugs, such as chloroquine, hydroxychloroquine, ritonavir, lopinavir, and favipiravir have been investigated as drug candidates in response to the covid- pandemic situation. spin hyperpolarization may open new opportunities in the diagnosis and biomedical research of covid- via mri and pharmacodynamics, metabolomics, and binding dynamics with proteins. this can be achieved by using enhanced signal intensity in nmr/mri. furthermore, even in case these drug candidates, which are under clinical investigation, may not be useful for the covid- , this sabre-based hyperpolarization study based on high molecular weight structures has not been previously conducted due to the special polarization transfer mechanism. in this study, high molecular weight chloroquine, hydroxychloroquine, ritonavir, lopinavir, and favipiravir were successfully tested for sabre-based hyperpolarization, and polarizations over long distances were detected, which may result in that the technique will be used for other materials. understanding the exact polarization transfer mechanism is important for future studies. hence, other polarization transfer mechanisms, such as those involving binding to other functional groups that are closer to the hyperpolarized spins, sabre-relay should also be investigated through the solvent or relaxation through dipolar effects. each polarization transfer-maximized external magnetic field was slightly different, which clearly implies that each structure has different optimal polarization transfer matching conditions. therefore, matching the magnetic field should be controlled to obtain efficient polarization enhancement in each structure. importantly, this method can be used to monitor the distribution and activity of a drug in vivo by mri. it may be harnessed to further investigate the molecular interaction of additional drug candidates with key proteins and unveil unknown activity on covid- , including pharmacokinetics. the polarization of the proton may be transferred to other isotope nuclei using pulse sequence, field cycling, and the matching of the spin energy. in the future, this will address the ultimate objective of obtaining hyperpolarized antiviral drugs to study their effect on covid- with a sufficiently long time of t . this study was unable to shed significant light on the real applications of the treatment of covid- . however, many related studies have reported applications in drug discovery, detecting tumors in vivo, as well as polarization transfer into many other isotopes along with pulse sequence development. future work on isotope labeling and further polarization transfer on long t time nuclei, including clinical perspectives, may open new opportunities to overcome this global pandemic. the datasets generated during and/or analyzed during the current study are available from the corresponding author (k. j.) on reasonable request. all methods were performed in accordance with the relevant guidelines and regulations. received: april ; accepted: august clinical features of patients infected with novel coronavirus in wuhan remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro response to 'is there a future for hydroxychloroquine/chloroquine in prevention of sars-cov- infection (covid- )? by moiseev et al could chloroquine /hydroxychloroquine be harmful in coronavirus disease (covid- ) treatment? anti-malaria drug chloroquine is highly effective in treating avian influenza a h n virus infection in an animal model covid- : hydroxychloroquine does not benefit hospitalised patients, uk trial finds chloroquine is a potent inhibitor of sars coronavirus infection and spread chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/ aids dose refinements in long-term therapy of rheumatoid arthritis with antimalarials cov- : an emerging coronavirus that causes a global threat a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease- treatment option clinical characteristics and therapeutic procedure for four cases with novel coronavirus pneumonia receiving combined chinese and western medicine treatment a trial of lopinavir-ritonavir in adults hospitalized with severe covid- case of the index patient who caused tertiary transmission of coronavirus disease in korea: the application of lopinavir/ritonavir for the treatment of covid- pneumonia monitored by quantitative rt-pcr the first case of novel coronavirus pneumonia imported into korea from wuhan, china: implication for infection prevention and control measures the mechanism of resistance to favipiravir in influenza influenza virus polymerase inhibitors in clinical development favipiravir versus arbidol for covid- : a randomized clinical trial selective hyperpolarization of heteronuclear singlet states via pulsed microtesla sabre over % n hyperpolarization in under one minute for metronidazole, an antibiotic and hypoxia probe iodonitrene in action: direct transformation of amino acids into terminal diazirines and n -diazirines and their application as hyperpolarized markers microtesla sabre enables % nitrogen- nuclear s n hyperpolarization by reversible exchange using sabre-sheath using parahydrogen to hyperpolarize amines, amides, carboxylic acids, alcohols, phosphates, and carbonates quasi-resonance fluorine- signal amplification by reversible exchange using signal amplification by reversible exchange (sabre) to hyperpolarise sn and si nmr nuclei the detection and reactivity of silanols and silanes using hyperpolarized si nuclear magnetic resonance hyperpolarising pyruvate through signal amplification by reversible exchange (sabre) using hyperpolarised nmr and dft to rationalise the unexpected hydrogenation of quinazoline to , -dihydroquinazoline nmr signal enhancement by effective sabre labeling of oligopeptides n mri of slic-sabre hyperpolarized n-labelled pyridine and nicotinamide hyperpolarization of pyridyl fentalogues by signal amplification by reversible exchange (sabre) pharmacokinetics of the sabre agent , -d -nicotinamide and also nicotinamide in rats following oral and intravenous administration determination of long-range scalar h- h coupling constants responsible for polarization transfer in sabre spontaneous transfer of parahydrogen derived spin order to pyridine at low magnetic field reversible interactions with para-hydrogen enhance nmr sensitivity by polarization transfer single-scan multidimensional nmr analysis of mixtures at sub-millimolar concentrations by using sabre hyperpolarization the feasibility of formation and kinetics of nmr signal amplification by reversible exchange (sabre) at high magnetic field ( . t) spin relays enable efficient long-range heteronuclear signal amplification by reversible exchange rational ligand choice extends the sabre substrate scope optimisation of pyruvate hyperpolarisation using sabre by tuning the active magnetisation transfer catalyst monitoring of hydrogenation by benchtop nmr with parahydrogen-induced polarization squid-based ultralow-field mri of a hyperpolarized material using signal amplification by reversible exchange analysis of -aminoisoquinoline using the signal amplification by reversible exchange hyperpolarization technique organic reaction monitoring of a glycine derivative using signal amplification by reversible exchange-hyperpolarized benchtop nuclear magnetic resonance spectroscopy the authors declare no competing interests. key: cord- -zpstb h authors: cui, tingting; theuns, sebastiaan; desmarets, lowiese m. b.; xie, jiexiong; de gryse, gaëtan m. a.; yang, bo; van den broeck, wim; nauwynck, hans j. title: establishment of porcine enterocyte/myofibroblast co-cultures for the growth of porcine rota- and coronaviruses date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: zpstb h a stable culture of primary porcine enterocytes is necessary to study porcine enteric virus replication characteristics. because the direct cultivation of primary porcine enterocytes is difficult, alternatives have to be considered. as subepithelial myofibroblasts secrete extracellular matrix and growth factors contributing to the attachment, proliferation and differentiation of epithelial cells, co-cultures of primary porcine enterocytes (ileocytes and colonocytes) with myofibroblasts were developed and evaluated for their susceptibility to enteric viruses. first, it was demonstrated that the co-cultured ileocytes and colonocytes were susceptible to an archival rotavirus strain rva/pig-tc/bel/rv / /g p[ ] and different other rotavirus genotypes (fecal samples containing g p[ ], g p[ ], g p[ ], g p[ ]). next, the tgev purdue strain infected both ileocytes and colonocytes whereas the miller strain only infected ileocytes. last, the pedv cv vero adapted and non-adapted (fecal suspension) strains could infect co-cultured ileocytes but not colonocytes. the infectivity of the cv vero adapted strain was higher when the cells were cultured without fetal bovine serum and the cv fecal suspension only infected the ileocytes cultured without fetal bovine serum. in conclusion, a novel co-culture of porcine enterocytes with myofibroblasts was established, which can be used for the investigation of the replication of enteric viruses. diarrhea and dehydration in piglets and destroy villous enterocytes in the small intestine. the severity and lethal outcome are strain dependent . different non-intestinal cell lines have been used in the past for virus cultivation in vitro , , but at low efficiency. tgev can be propagated in swine testicle (st) and porcine kidney (pk- ) cells. upon adaptation, pedv may infect african green monkey kidney (vero) cells. like tgev and pedv, rotavirus is mainly transmitted by fecal-oral route and virus infection causes the destruction of mature small intestinal enterocytes. similarly, most rotavirus research was conducted on non-polarized ma cells (african green monkey kidney epithelial cells) which are easy to culture and permissive for certain rotavirus strains of different genotypes. but there are still a lot of genotypes of rotavirus strains that do not grow in ma cells . data concerning the replication cycles of these enteric viruses in their target cell (mature intestinal enterocytes) are scarce. to this end, it is essential to obtain cultures of porcine intestinal enterocytes. the gastrointestinal tract is lined with a rapidly proliferating simple columnar epithelium. the epithelial cells migrate from the crypts where mitosis takes place towards the top of the villi (small intestine) or towards the top of the intercrypt (large intestine) as they mature. mature epithelial cells are replaced by a steady supply of crypt cells. in suckling piglets, intestinal epithelial cells renew every - days. because of the highly dynamic and rapid renewal properties of intestinal epithelial cells, it is difficult to successfully culture them in vitro. in , a porcine mid-jejunum epithelial cell line was established from neonatal piglets by immortalization upon transfection/ transduction with human telomerase reverse transcriptase (htert) gene . because the immortalization alters the biology of the original cells, field viruses replicate more efficiently in primary cells than in continuous cell lines. therefore, it is advisable to use primary cells for virus research. intestinal enteroids have been developed by culturing intestinal crypts onto matrigel which is enriched with laminin α and α . enteroids enhanced the viability of the cells and were already used for the study of rotaviruses , noroviruses and enteroviruses . the successful cultivation of enteroids is dependent on many growth factors, critically including wnt a, r-spondin, and noggin, which is an expensive method. intestinal myofibroblasts, one of the intestinal mesenchymal cells, are directly subjacent to the basement membrane of epithelial cells and they have been reported to support the proliferation and differentiation of epithelial cells . myofibroblasts are identified by the expression of intracellular cytoskeletal microfilament α-smooth muscle actin (α-sma). myofibroblasts contribute to the growth and differentiation of intestinal enterocytes by secreting several growth factors (hepatocyte growth factor, transforming growth factor beta (tgf-β ) , , insulin-like growth factors (icfs) ), extracellular matrix proteins (collagen type iv, laminin-β and γ , and fibronectin), cytokines and chemokines. to date, a mouse colonic myofibroblast cell line established by hirokawa has been reported to stimulate colonoid formation and human myofibroblasts isolated from small intestine were able to support human intestinal epithelial cell growth in vitro . all the information suggested the potential role of myofibroblasts in intestinal enterocytes cultivation in vitro. in this study, a porcine co-culture system of primary intestinal enterocytes with intestinal myofibroblasts was established which mimics the enterocytes growth in vivo. the morphological and functional features of co-cultured enterocytes were characterized. to determine the usability of this co-culture system, enteric rotaand coronaviruses were used to infect the co-cultured enterocytes. after euthanasia of a three-day old piglet, ileum and colon tissues were embedded immediately and cryosections were made to visualize the distribution of epithelial cells and myofibroblasts in vivo. immunofluorescence stainings were performed against the epithelial cell marker cytokeratin and myofibroblast marker α-smooth muscle actin. in the porcine ileum, a lot of myofibroblasts were located in the lamina propria directly underneath the epithelial cell layer of the villi, representing the largest cell population in the lamina propria. fewer myofibroblasts (< %) were observed in the lamina propria underneath the epithelial cell layer in colon. in contrast, the myofibroblasts formed an integral line underneath the epithelial cell layer in the colon crypts (fig. ) . these results show that porcine small and large intestinal epithelial cells grow in close contact to myofibroblasts in vivo. the contact communication between these two cell types is supposed to be an important element for the support of myofibroblasts in the attachment, proliferation and migration of epithelial cells. myofibroblasts were isolated from the subepithelial layer of porcine ileum. cell clusters were observed days post isolation and had a cobblestone-like morphology with stellate edges. the clusters continued to grow into larger structures which contain more than cells. meanwhile, the fibroblasts started to expand. the biggest cluster was marked by making a circle with a pen on the bottom of the plate and other cells were scraped away using a sterile tip. afterwards, this circled cluster was split using trypsin. cells maintained their morphology and could be continuously passaged ( fig. a) . the obtained cells were characterized by immunofluorescence staining. cells were stained positive for α-smooth muscle actin (α-sma), vimentin and fibronectin, which are all markers of myofibroblasts. the cells were negative for desmin, cytokeratin and sucrase-isomaltase (fig. b) . these results confirmed that the cells isolated from the ileum subepithelial layer were myofibroblasts. primary ileum epithelial cells were used as control, which were positive for cytokeratin and sucrase-isomaltase (fig. c ). myofibroblasts serve as supporting cells for porcine enterocytes. twenty-four hours post seeding, most of the ileum epithelial cells were attached and became confluent (> %) when seeded on porcine type i and iii collagen coated wells. however, days post seeding, the epithelial cells started to detach, and days post seeding, most of the epithelial cells were dead. only a few big epithelial cell clusters could be visualized. six days post seeding, fibroblasts took over the wells and a few tiny epithelial clusters were still present (fig. a) . the use of % conditioned medium did not give an improvement: most of the epithelial cells were dead at day (fig. b ). when monolayers of myofibroblasts were used as supporting layers for ileum and colon epithelial cell cultivation, epithelial clusters attached to the myofibroblasts and became visible at hours post seeding. the epithelial cells continued to expand and grew into monolayers days post isolation. they maintained their polygonal morphology and confluent layers for more than week (fig. c,d) . characterization of enterocytes co-cultured with myofibroblasts. cells isolated from ileum and colon co-cultured with myofibroblasts were characterized by immunofluorescence staining days post co-cultivation. antibodies against cytokeratin and vimentin were used to determine the epithelial nature. in the co-culture system, most of the cells were found to be cytokeratin positive ( ± . % of all cells in the ileum epithelial cell co-cultures and ± . % of all cells in the colon epithelial cell co-cultures), confirming the epithelial nature of the polygonal cells (fig. ) . interestingly, the vimentin positive cells (myofibroblasts) clustered into aggregates in both co-cultures, which suggests that the expansion of epithelial cell growth squeezed the myofibroblasts into aggregates. and after co-culture with myofibroblasts (in vitro). the differentiation status of porcine intestinal epithelial cells of days old piglets and the co-cultured enterocytes were analyzed by scanning electron microscopy. as shown in fig. , the epithelial cells of the ileum and colon had a different appearance in vivo. a few epithelial cells on the tip of a villus of the ileum were fully covered with microvilli, while most of the cells were immature without microvilli on the surface. in contrast, almost all epithelial cells of the colon had short microvilli. in the co-cultured ileum and colon epithelial cells, a lot of microvilli were present on the cell's surface at three days post co-cultivation and showed a different appearance (some microvilli were longer). these results demonstrate that myofibroblasts could support the differentiation of both ileum and colon epithelial cells in vitro. replication kinetics of rotavirus rva/pig-tc/bel/rv / /g p [ ] strain in co-cultured enterocytes. to determine the percentage of rotavirus infected cells in primary porcine enterocytes, cells were fixed at different time points ( , , , , , h) post inoculation with a low-passage archival rva strain. viral antigens were stained by immunofluorescence (fig. a ). the first antigen-positive ileum epithelial cells appeared at h p.i and increased over time. the percentage of rotavirus infection in ileum epithelial cells increased to . ± . % at h p.i. in colon epithelial cells, the first antigen-positive cells appeared at h p.i and the percentage of infection increased to . ± . % at h p.i (fig. b) . to determine the kinetics of virus production in ileum and colon epithelial cells, viral titers and rna copies of supernatant (extracellular virus) and cells (intracellular virus) were assessed (fig. c,d) . the intracellular and extracellular virus titers were determined from to h post inoculation. the intracellular virus titer of ileum epithelial cells increased from . ± . ccid /ml to . ± . ccid /ml. the extracellular virus titer of ileum epithelial cells increased from . ± . ccid /ml to . ± . ccid /ml. for colon epithelial cells, the intracellular and extracellular virus titers increased from . ± . ccid /ml to . ± . ccid /ml and from . ± . ccid /ml to . ± . ccid /ml, respectively. the rt-qpcr showed that viral rna started to be synthesized in ileum and colon epithelial cells from h p.i. viral rna increased up to . ± . log /ml in ileum epithelial cells and . ± . log /ml in colon epithelial cells. the viral rna started to be released into the supernatant between and h p.i; at h p.i, . ± . log /ml rna copies were detected in both ileum and colon epithelial cell cultures. susceptibility of primary porcine enterocytes to different rotavirus genotypes present in fecal suspensions of diarrheic pigs. a major restriction of rotavirus research is the lack of cell cultures supporting the growth of different genotypes of field strains. therefore, the susceptibility of primary porcine enterocytes co-cultured with myofibroblasts to four different genotypes of rotavirus a contained in fecal suspensions was tested by rt-qpcr (fig. ) . the results showed that these four genotypes of rotavirus could infect both ileum and colon epithelial cells. rotavirus r (g p [ ] ) strain demonstrated the highest infectivity to primary enterocytes, followed by the r (g p [ ] ) strain. for rotavirus r (g p [ ] ) strain, an increase of more than fold rna copies/ml was detected at h post inoculation of ileum and colon epithelial cells. ileum and figure . evaluation of different methods to support the growth of primary porcine ileum and colon epithelial cells. (a) porcine ileum epithelial cells were cultured without myofibroblasts on a porcine collagen type i/iii coated plate. they could live approximately days before detaching and dying, fibroblasts took over days after seeding. (b) porcine ileum epithelial cells were cultured without myofibroblasts on a porcine collagen type i/iii coated plate with medium supplemented with % conditioned medium collected from ileum myofibroblasts. epithelial cells could be maintained for less than days and a few tiny epithelial clusters were present at days post seeding. porcine ileum (c) and colon (d) epithelial cells were cultured in the presence of ileum myofibroblasts. epithelial cells grew into a monolayer days post co-cultivation and maintained their morphology for more than days. colon epithelial cells were less susceptible to rotavirus r (g p [ ] ) strain. these results demonstrated that the primary enterocytes could be infected by rotavirus field strains containing different genotypes but that the susceptibility of enterocytes to rotavirus differed among the different genotypes. susceptibility of co-cultured enterocytes to transmissible gastroenteritis virus. next, the susceptibility of primary enterocytes in co-cultures with myofibroblasts was tested for the tgev purdue and miller strains. tgev antigen expression kinetics were assessed in primary enterocytes (fig. ). tgev purdue infected both ileum and colon epithelial cells. at h p.i, the purdue strain had infected . ± . % and . ± . % of the ileum epithelial cells and colon epithelial cells, respectively, and the virus titer in both ileum and colon epithelial cells increased to . ± . ccid /ml. tgev miller only infected ileum epithelial cells. the highest infection ( . ± . %) appeared at h p.i. no tgev antigens of miller strain were found in colon epithelial cells on h post inoculation. pedv cv vero adapted strain and pedv cv positive fecal suspensions were used to infect co-cultured primary ileum epithelial cells to confirm the usability of primary enterocytes. twenty-four hours post inoculation, viral antigens of vero adapted cv were observed in a low number of ileum epithelial cells cultured with/without fbs. two times more infection was found in epithelial cells cultured without fbs. for cv fecal suspension, virus infection was only observed in ileum epithelial cells which were cultured without fbs (fig. ). in this study, a co-culture system of porcine ileum subepithelial myofibroblasts with porcine small (ileum) and large (colon) intestinal epithelial cells was established and the use of this co-culture system for enteric virus research was assessed. the cross talk between epithelium and subepithelial myofibroblasts has been reported to promote epithelial cell proliferation and differentiation in both mice and humans. in this study, a myofibroblast cell line was established from porcine ileum which was identified by the presence of α-smooth muscle actin, vimentin and fibronectin. the in vivo distribution of epithelial cells and myofibroblasts shows that a lot of myofibroblasts directly grow underneath the epithelium in porcine ileum and that myofibroblasts form an integral line along colon crypts. this initial contact may be an important factor for the support of myofibroblasts towards epithelial cells. at present, many mechanical and enzymatic seperation methods have been used for the isolation of intestinal epithelial cells from human, mice, rat, bovine, porcine and feline intestines. however, the successful cultivation of intestinal epithelial cells still poses a big challenge because of the rapid death/apoptosis of isolated epithelial cells which in vivo renew every - days. this apoptosis may be triggered by the disruption of the epithelial cell contact with extracellular matrix. a dispase and collagenase combination was used for epithelial cell isolation in the present study, which preserves more cell-to-cell interactions and reduce the damage of cell-matrix adhesions . the contamination with stromal cells is a huge problem for epithelial cell cultivation. in order to decrease the contamination with mesenchymal cells, we removed these cells by d-sorbitol density centrifugation and plastic adhesion for hours. according to the specific property that stromal cells attach to plates faster than epithelial cells, most stromal cells were separated from epithelial cells after hours incubation. in the presence of ileum myofibroblasts, both ileum and colon epithelial cells are growing longer than one week and maintain their polygonal, cobblestone-like morphology. in the absence of myofibroblasts, epithelial cells died after - days, even when supplemented with % conditioned medium collected from myofibroblast cultures. our data indicate that the supporting effect of myofibroblasts for epithelial cell growth is very dependent on the direct contact between these two cell types. we also demonstrated that myofibroblasts not only support the growth of intestinal epithelial cells from newborn piglets, but also the epithelial cells of weeks old pigs (data not shown), which confirms the important role of myofibroblasts on epithelial cell proliferation independently of the age of the donor. the epithelial cells in co-cultures were identified by the presence of cytokeratin which is regarded as an important marker of epithelial cells. most of the cells (> %) preserved their epithelial nature with a positive staining of cytokeratin after days of co-cultivation. remarkably, the myofibroblasts clustered into aggregates in this co-culture system. it seems that myofibroblasts retracted into aggregates during the expansion of epithelial cells growth. in earlier reports, it was shown that myofibroblasts can migrate to wound tissue and demonstrate high contractile activities to generate tissue contractures, which help wound healing and organ remodeling by secretion of extracellular matrix proteins and exerting strong contraction force [ ] [ ] [ ] . in addition, human and porcine myofibroblasts express s a proteins which have been demonstrated to be implicated in cancer cell migration , . taken together all this information, we hypothesize that myofibroblasts first secrete extracellular matrix proteins, such as collagen and laminin, coordinating the attachment and proliferation of epithelial cells and migration of myofibroblasts in clusters. epithelial cells co-cultured with myofibroblasts showed microvilli after days of co-cultivation which is in accordance with the reported data that myofibroblasts not only support the growth of epithelial cells, but also stimulate the differentiation of epithelial cells . rotavirus research is hampered by the lack of susceptible enterocyte cell lines. although some cell lines, including ma , marc, ipec-j and caco- cells [ ] [ ] [ ] are susceptible to some rotavirus strains, a lot of genotypes of rotavirus, such as p [ ] , p [ ] , p [ ] , p [ ] do not grow efficiently in these cell lines. therefore, enterocyte cultures are an essential tool to investigate rotavirus-cell interaction. in this study, the susceptibility of primary enterocytes to different rotavirus genotypes were explored. rotavirus rva/pig-tc/bel/rv / /g p [ ] strain, which was first isolated in belgium in from a pool of watery diarrhea of pigs , is a typical g porcine rotavirus. g is the most common vp genotype of human group a rotavirus, but is rarely found in porcine rotavirus strains . it is suggested that all human g vp genes originate from porcine rotavirus transmission to humans and that this interspecies transmission was followed by human-to-human transmissions . although rotavirus was reported to have an exclusive tropism for small intestinal enterocytes , rotavirus rva/pig-tc/bel/rv / /g p [ ] strain could infect both primary ileum and colon epithelial cells with a trypsin treatment. the antigen expression kinetics did not show significant differences in cell tropism of this rotavirus strain. studies showed that some animal group a rotaviruses, especially porcine rotaviruses recognize sialic acid as host receptor for virus attachment. on porcine small and large intestinal epithelium, sialic acid receptors were clearly detected and colon crypts showed even a greater abundance of sialic acid than small intestines . rotavirus genotype p [ ] has been classified as a sialic acid-dependent genotype , which may explain why rotavirus g p [ ] strain could also infect colon epithelial cells in vitro. both ileum and colon epithelial cells were also susceptible to four fecal suspensions containing different rotavirus genotypes (g p [ ] , g p [ ] , g p [ ] and g p [ ] ). g p [ ] and g p [ ] rotaviruses, which are the predominant g/p genotype combination of circulating rotaviruses in belgium, were more infectious than the other two strains, which was also observed on ma cells . the higher infectivity in enterocytes in vitro may explain the wide prevalence of these genotypes in the field. rotavirus p [ ] genotype is one of the major human rotavirus genotypes. it is associated with symptomatic infection in children and is frequently detected in africa. in pigs, p [ ] rotaviruses are also regularly detected and most of these strains display a high genetic similarity to human p [ ] rotavirus strains , . animal rotaviruses are regarded as a potential reservoir for the genetic and antigenic diversity of human rotaviruses and interspecies transmission from swine to humans has been reported increasingly for p [ ] genotype. eight hungarian human g p [ ] rotavirus strains were supposed to originate from pigs by independent events of zoonotic transmission . therefore, studying porcine rotavirus is a key step to deeply understand the evolution of human rotavirus. recent studies demonstrated that p [ ] rotavirus strains recognize human histo-blood group antigens (hbgas), which explains why most human rotavirus strains cannot be cultured in ma cells. in our study, rotavirus g p [ ] could infect both ileum and colon epithelial cells which demonstrates that the primary enterocytes contain the cellular receptor for rotavirus p [ ] , likely containing the right hbgas. this receptor is still not identified. rotavirus almost exclusively infects mature enterocytes of the small intestinal villi in vivo , while in vitro, the large intestinal epithelial cells could also be infected, which leads us to think that other factors in the large intestines block the rotavirus infection of colon epithelial cells in vivo. in the enterohepatic circulation, bile salts are synthesized in the liver, travel to the gall bladder and are secreted into the descending part of the duodenum helping in the digestion of fats and other substances. up to % of secreted bile salts are collected in the ileum and return back to the liver via the portal system. we hypothesize that the lack of bile salts in the lumen of the large intestines may be the reason why rotavirus cannot infect the epithelial cells of large intestines in vivo. in order to test this hypothesis, we collected bile from the porcine gall bladder and used it for the infection of rotavirus to primary ileum epithelial cells. the results show that the infectivity of rotavirus in ileum epithelial cells increased after treatment with bile ( supplementary fig. s ), which is in accordance with the results that bile/bile acids are essential for porcine enteric calicivirus, hepatitis c virus and human norovirus infection in cells , , . porcine epidemic diarrhea virus and transmissible gastroenteritis virus primarily replicate in the villous enterocytes of the small intestine. porcine aminopeptidase n (papn), one of the type ii cell surface metalloproteases, was reported to be a cellular receptor for both pedv and tgev [ ] [ ] [ ] . although these two viruses bear similarities in structure and disease, they are clearly distinct. in vitro, pedv does not grow in continuous cell cultures which are permissive to tgev. therefore, it is of interest to investigate the susceptibility of the target cell, primary porcine enterocytes, to both pedv and tgev. interestingly, we found that tgev purdue can infect both ileum and colon epithelial cells, whereas the virulent miller strain only infects ileum epithelial cells and that the infectivity of the purdue strain is much higher than the miller strain in ileum epithelial cells. the purdue and miller strains are two virulent american tgev strains, which originally caused % mortality of less than -weeks-old piglets due to the lytic infection of enterocytes. the purdue strain used in this study has been passaged times in primary porcine kidney cells and then adapted to grow in st cells by one more passage . a nucleotide mutation (t to g at nucleotide position ) of the purdue s protein, which causes a serine (s) to alanine (a) mutation at aa was reported . this mutation is present in the domain of the s protein encoded by nucleotide - which is used for the cell receptor papn recognition . this mutation may make the virus more suitable to grow in papn negative cultures/primary porcine kidney cells due to the recognition of other cell receptors. it may also be responsible for the growth of the purdue strain in primary porcine colon epithelial cells and porcine myofibroblasts which are both negative for papn ( supplementary fig. s ). due to the absence of papn, the miller strain cannot infect the primary colon epithelial cells. pedv cv strain has been adapted to grow in the apn negative vero cells by several blind passages and has been widely used for pedv research. we found that primary ileum epithelial cells were more susceptible to the cv vero adapted strain when the cells were grown in absence of fbs and the cv fecal suspension only infected the cells which were cultured without fbs. fbs is usually added in culture medium for cell growth because it contains various biological factors. but its compositional complexity also causes a lot of side-effects, such as the inhibition of cell differentiation and virus replication. fbs may inhibit viral replication by blocking cellular proteins, as shown for another nidovirus porcine reproductive and respiratory syndrome virus (prrsv) . cv strain shows a lower infectivity in ileum epithelial cells cultured with fbs suggesting that fbs contains factors that interfere with pedv infection or that without fbs the epithelial cells could differentiate better which makes them more susceptible to pedv infection. the susceptibility of primary ileum epithelial cells to a pedv fecal suspension represents a big step for further investigating pedv field isolates. in this study, the primary epithelial cells were susceptible to rota-and coronaviruses, however the infection efficiency was relatively low. this might be caused by the antiviral response of interferons, especially type iii ifns (ifn-λ). interferons belong to the class of cytokines, which are made and released by host cells to trigger protective defenses to several pathogens, such as viruses, bacteria and parasites. intestinal epithelial cells abundantly produce type iii ifns and elicit antiviral defenses in response to viral infections. zhang and colleagues demonstrated that ifn-λ possesses a strong antiviral effect on pedv replication in the porcine intestinal epithelial cell line ipec-dq, a subclone obtained from the nontransformed ipec-j cells by limited serial dilutions . a similar strong ifn-λ might have been rapidly induced in the currently described co-cultures. furthermore, rotavirus, pedv and tgev infect mature villous epithelial cells. in our co-culture system, not only the mature villous epithelial cells were cultured but likely also the less differentiated intestinal crypt epithelial cells. these undifferentiated crypt epithelial cells might cause the low infection efficiency. in future work, efforts will be made to stimulate the differentiation of primary epithelial cells and control the ifn antiviral response in order to increase the infection efficiency. in conclusion, porcine enterocyte/myofibroblast co-cultures were successfully established in the present study, demonstrating that myofibroblasts are necessary for small and large intestinal epithelial cell attachment, proliferation and differentiation in vitro. primary enterocytes were susceptible to both cell line adapted and non-adapted rota-and coronaviruses. this co-cultivation system will be a great assett in future research on rota-, coronavirus and other enteric viruses. ethical statement. euthanizing piglets was done in agreement with the european legislation on animal experiments. all experimental procedures were approved by the local ethical committee of the faculty of veterinary medicine, ghent university, and all methods were carried out in accordance with the approved guidelines. piglets and virus samples. healthy conventional -day-old suckling piglets were purchased from a conventional pig farm. using tissues of euthanized animals was in accordance of the requirements of the local ethical committee of the faculty of veterinary medicine. the piglets were euthanized by intravenous injection of % sodium pentobarbital ( ml/ . kg, kela laboratories, hoogstraten, belgium). rotavirus rva/pig-tc/bel/ rv / /g p [ ] strain grown on ma cells, tgev purdue and miller strain grown on st cells and pedv cv strain grown on vero cells were used in this study. in addition, four diarrheic fecal suspensions containing rotavirus of suckling pigs less than weeks old (table ) and pedv cv fecal suspension from an experimentally inoculated -day-old sucking piglet were also included. twenty percent fecal suspensions were prepared in phosphate buffered saline (pbs) containing u/ml penicillin (continental pharma, puurs, belgium), mg/ml streptomycin (certa, braine l' alleud, belgium), mg/ml gentamicin (gibco brl, merelbeke, belgium) and . % v/v fungizone (bristol-myers squibb, braine l' alleud, belgium) . establishment of porcine intestinal subepithelial myofibroblasts. the ileum was collected from a euthanized -day-old piglet and brought in ice-cold dulbecco's modified eagle medium (dmem; gibco brl, merelbeke, belgium), containing u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin (flushing medium) and % fetal bovine serum (fbs; gibco brl). subsequently, the intestine was cut into - cm long segments and turned inside-out, mucosal side facing outwards. intestinal contents were removed by one washing with ice-cold flushing medium containing % fbs and two washings with flushing medium without fbs. the intestinal segments were incubated in pbs containing mm edta and shaken at rpm/min for min at °c. the edta suspension was removed and this edta incubation step was repeated times. next, intestinal segments were incubated in dmem containing dispase ii ( . mg/ml, sigma, st. louis, mo, usa) and collagenase i ( . mg/ml, invitrogen, paisley, uk) for min at °c. subsequently, the digested mucosa was gently scraped with a sterile scalpel blade, scrapings were collected and incubated in dmem containing dispase ii ( . mg/ ml) for min whilst pipetting. after centrifugation for min at × g and °c, the pellet was resuspended in dmem containing % d-sorbitol (sigma) and . % fbs, and centrifuged at × g for min. the pellet was resuspended in dmem/f- (gibco brl) culture medium supplemented with u/ml penicillin, . mg/ml streptomycin, % fbs, and % non-essential amino acids (gibco brl) and incubated at °c and % co . culture medium was refreshed on day , afterwards medium was changed every days. morphology of the cells was evaluated daily by light microscopy (olympus). once myofibroblasts (cobblestone-like clusters) grew into big clusters (≈ cells), they were marked and other cells (e.g. epithelial cells, fibroblasts) were removed by scraping. then the cobblestone-like clusters were detached by trypsinization with μg/ml trypsin- . % edta, and sub-cultured in a -well plate (split ratio : ) with culture medium containing % fbs and evaluated daily for cobblestone-like features by light microscopy. subsequently, the cobblestone-like cells were digested by trypsinization and further expanded in flasks to generate a long-term semi-continuous culture. to characterize the obtained primary cells, immunofluorescence stainings were performed to visualize cytokeratin, vimentin, α-smooth muscle actin, fibronectin, desmin and sucrase-isomaltase. a third passage of cells was fixed with % paraformaldehyde for min at room temperature (rt) followed by permeabilization with . % triton x- for min at rt. the cells were respectively incubated with mouse monoclonal anti-human cytokeratin antibodies (dako, denmark a/s), mouse monoclonal anti-human vimentin antibodies (bio-rad), mouse monoclonal anti-human α-smooth muscle actin antibodies (dako), mouse monoclonal anti-human desmin antibodies (dako) sheep polyclonal anti-human fibronectin antibodies (bio-rad) or mouse monoclonal anti-human sucrase-isomaltase (santa cruz) for h at °c. afterwards, cells were washed and incubated with goat anti-mouse-igg fitc labeled antibodies (molecular probes) or rabbit anti-sheep-igg fitc labeled antibodies (molecular probes) for h at °c. nuclei were stained with hoechst (molecular probes) for min at rt. the slides were mounted using glycerin solution with . % , -diazabicyclo[ . . ]octane (janssen chimica, beerse, belgium) and analyzed using fluorescence microscopy (dm b fluorescence microscope, leica microsystems gmbh, heidelberg, germany). of supporting cells. two days before enterocyte isolation, myofibroblasts were seeded in -well plates at a density of cells/ml in dmem/f- culture medium containing u/ml penicillin, . mg/ml streptomycin, and % fbs, and % non-essential amino acids and cultured at °c and % co . monolayers of myofibroblasts were used as support layer for enterocytes growth and differentiation. preparation of conditioned medium collected from myofibroblasts. when myofibroblasts grew to % confluency, their medium was refreshed by dmem/f- culture medium. next, the refreshed medium was collected after h, centrifuged at × g for min and the supernatant was collected and used as conditioned medium. the conditioned medium was stored at − °c until later use. isolation of porcine enterocytes. after euthanasia, around cm long segments of ileum and colon were removed from a piglet. the intestinal segments were turned inside-out, mucosal side facing outwards. the intestinal contents were removed by one washing with ice-cold flushing medium with % fbs and two washings with ice-cold flushing medium without fbs. in order to fully expand the contact area of intestinal mucosa layer to digestion enzymes, one side of the intestinal piece was closed with a surgical clamp and the lumen was filled with warm ca + -and mg + -enriched pbs containing penicillin and streptomycin. then, the other side was also closed with a surgical clamp. the intestinal mucosa was digested in dmem containing dispase ii ( . mg/ml) and collagenase i ( . mg/ml) for min at °c. then, the pbs was released from the lumen by removing one clamp. the mucosa was gently scraped with a sterile scalpel blade. afterwards, the intestinal lumen was filled again with pbs and the mucosa was digested in dmem containing dispase ii ( . mg/ml) and collagenase i ( . mg/ml) again for another min (ileum) or min (colon) at °c. subsequently, the digested mucosa was deeply scraped with a sterile scalpel blade. the scrapings were incubated in dmem containing dispase ii ( . mg/ml) for min whilst pipetting. after centrifugation ( × g, min at °c) the pellet was resuspended in dmem containing % d-sorbitol and . % fbs and centrifuged at × g for min at °c to separate single stromal cells. this sorbitol process was repeated at least times. afterwards, the pellet was resuspended in dmem supplemented with antibiotics and filtered using a μm cell strainer (falcon). after centrifugation ( × g, min), the pellet was finally resuspended in dmem/f supplemented with u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin, % fbs, . % fungizone, ng/ml epidermal growth factor (sigma), % insulin-transferrin-selenium-ethanolamine (gibco brl) and % non-essential amino acids and seeded in -well plates in order to let the mesenchymal cells attach and separate enterocytes from mesenchymal cells. after h incubation at °c and % co , the non-adherent cell clusters in the -well plates were collected and reseeded on top of the monolayer of myofibroblasts. to confirm the support effect of myofibroblasts, cell clusters were also seeded on porcine collagen type i/iii (gentaur, kampenhout) coated plates and cultured with/without % conditioned medium. the cells were further incubated at °c and % co and the medium was refreshed every days. morphological features of primary enterocytes were evaluated by light microscopy (olympus). to characterize the origin of enterocytes which were co-cultured with myofibroblasts, immunofluorescence staining was performed against cytokeratin and vimentin. three days post isolation, co-cultured enterocytes grown on coverslips were fixed with % paraformaldehyde for min at rt followed by permeabilization with . % triton x- for min at rt. the cells were incubated with mouse monoclonal anti-human cytokeratin or mouse monoclonal anti-human vimentin antibodies containing % goat serum for h at °c, followed by goat anti-mouse-igg fitc labeled antibodies for h at °c. nuclei were stained with hoechst for min at rt. the percentage of cytokeratin positive cells were analyzed by fluorescence microscopy (leica microsystems gmbh). immediately after euthanasia of a -day-old piglet, mm square pieces of the ileum and colon were collected. tissues were embedded in methocel (fluka, sigma) and μm thick cryosections were made. immunofluorescence staining with markers for epithelial cells and myofibroblasts were performed. in brief, cryosections of ileum and colon tissues were fixed with methanol for min at − °c and then incubated with mouse monoclonal anti-human α-smooth muscle actin antibodies for h at °c, followed by goat anti-mouse-igg fitc labeled antibodies for h at °c. afterwards, the sections were incubated with rabbit polyclonal anti-bovine cytokeratin antibodies, followed by goat anti-rabbit-igg texas red labelled antibodies for h at °c. after washing, nuclei were stained with hoechst for min at rt. the slides were analyzed by fluorescence microscopy. scanning electron microscopy. in order to determine the differentiation status of intestinal epithelial cells, the presence of microvilli was assessed by scanning electron microscopy (sem). the protocol for sem was performed as described by glorieux and colleagues . tissue samples (ileum and colon from -day-old piglet) and cell samples (porcine enterocytes days post co-cultivation) were fixed in hepes-buffered glutaric aldehyde for hours. then, the samples were treated with % osmiumtetroxide for hours at rt, followed by ascending grades of alcohol dehydration. in order to avoid the water vaporization obstructing the electron beam and interfering with image clarity, the dehydrated samples were transferred to a critical point drier (cpd, bal-tec, balzers, liechtenstein) for complete drying. finally, the dried samples were mounted on a metal stub and sputter-coated with platinum. the microvilli of all the samples were acquired with a jeol jsm lv scanning electron microscope (jeol ltd., tokyo, japan). replication of ma grown rotavirus in co-cultured enterocytes. three days post seeding, primary enterocytes co-cultured with myofibroblasts were inoculated with μl rotavirus rva/pig-tc/bel/ rv / /g p [ ] at an m.o.i. of . after h of inoculation ( °c and % co ), cells were washed times with warm dmem and further incubated in serum-free dmem/f culture medium containing μg/ml trypsin. at different time points ( , , , , and h) post inoculation, cells were fixed with % paraformaldehyde for min and permeabilized with . % triton x- for min at rt. double-immunostainings were performed as described above and the percentage of infected enterocytes was counted by fluorescence microscopy. in addition, the supernatant and cells were collected at different time points ( , , , , and h) post inoculation for virus titration and qpcr quantification. cell culture supernatant was collected and centrifuged for min at × g. supernatant was collected (extracellular virus) and the pellet was collected together with cells that were scraped with serum free dmem/f culture medium (intracellular virus). the cells were lysed by freeze-thaw cycles to release virus particles. the virus titer was determined using virus titration and rt-qpcr. titration of intraand extracellular virus was performed. monolayers of ma cells growing in -well plates were inoculated with -fold dilution of supernatant and cells ( − - − ). five days after inoculation, the cytopathogenic effect (cpe) was visualized using a light microscope and the cell culture infective dose (ccid susceptibility of co-cultured enterocytes to different rotavirus genotypes present in fecal suspension of diarrheic pigs. four fecal suspensions containing different genotypes of rotavirus collected from less than -week-old diarrheic suckling pigs were used to determine the susceptibility of porcine primary enterocytes to rotavirus field strains. these samples were collected at a private diagnostic laboratory for etiology diagnosis as described before . three days after co-cultivation, enterocytes were inoculated with these four fecal suspensions at viral rna copies/ml using the method described above. the supernatant was harvested at h and h post inoculation. rt-qpcr was performed to determine the increased number of viral rna copies. susceptibility of co-cultured enterocytes to transmissible gastroenteritis virus. three days post seeding, monolayers of primary enterocytes co-cultured with myofibroblasts were inoculated with μl tgev strains (miller and purdue) at an m.o.i. of . after h inoculation at °c and % co , cells were washed times with warm dmem. serum free dmem/f supplemented with u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin, ng/ml epidermal growth factor, % insulin-transferrin-selenium-ethanolamine and % non-essential amino acids was added for further incubation. at different time points ( , , , , and h) post inoculation, cells were fixed with % paraformaldehyde for min and the supernatant was collected. double-immunostainings against both tgev antigens and cytokeratin were performed to specifically visualize the infected enterocytes. the cells were permeabilized with . % triton x- for min at rt and incubated with swine polyclonal anti tgev antibodies containing % negative goat serum for h at °c, followed by goat anti-swine-igg fitc labelled antibodies for h at °c. afterwards, cells were incubated h at °c with mouse monoclonal anti-human cytokeratin antibodies, followed by h at °c with goat anti-mouse-igg texas red labelled antibodies. nuclei were stained with hoechst for min at rt. the percentage of infected enterocytes was determined by fluorescence microscopy. virus titration of supernatant was performed using st cells. monolayers of st cells were inoculated with -fold dilutions ( - − ) of supernatant. five days after inoculation, cytopathogenic effect (cpe) was visualized and cell culture infective dose (ccid ) was calculated using the formula of reed and muench. hours post seeding, ileum epithelial cells were refreshed with culture medium with/without % fbs. three days post cultivation, monolayers of primary ileum epithelial cells cultured with/without fcs were inoculated with μl pedv cv vero adapted strain at . ccid /ml or viral rna copies/ml of fecal suspension with µg/ml trypsin. after h inoculation at °c and % co , cells were washed times with warm dmem and serum free dmem/f supplemented with u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin, ng/ml epidermal growth factor, % insulin-transferrin-selenium-ethanolamine and % non-essential amino acids was added for further incubation. twenty-four hours post inoculation, cells were fixed with % paraformaldehyde for min and permeabilized with . % triton x- for min at rt. the cells were incubated with mouse monoclonal anti-pig pedv antibodies containing % normal goat serum, followed by h at °c with goat anti-mouse-igg fitc labelled antibodies. nuclei were stained with hoechst for min at rt and the infection was analyzed by fluorescence microscopy. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) candidate new rotavirus species in sheltered dogs vp -sequence-based cutoff values as a criterion for rotavirus species demarcation species h rotavirus detected in piglets with diarrhea, brazil complete genome analyses of the first porcine rotavirus group h identified from a south african pig does not provide evidence for recent interspecies transmission 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interaction of porcine viruses with the respiratory tract serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization a simple method of estimating fifty per cent endpoints we are grateful to prof j. t. patton for supplying the antibody against rotavirus. special thanks go to ytse noppe and marthe pauwels for the excellent technical assistance. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -kj zi authors: coleman, kristen k.; nguyen, tham t.; yadana, su; hansen-estruch, christophe; lindsley, william g.; gray, gregory c. title: bioaerosol sampling for respiratory viruses in singapore’s mass rapid transit network date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: kj zi as a leading global city with a high population density, singapore is at risk for the introduction of novel biological threats. this risk has been recently reinforced by human epidemics in singapore of sars coronavirus, pandemic h n influenza a virus, and enterovirus . other major threats to singapore include mers-coronavirus and various avian and swine influenza viruses. the ability to quickly identify and robustly track such threats to initiate an early emergency response remains a significant challenge. in an effort to enhance respiratory virus surveillance in singapore, our team conducted a pilot study employing a noninvasive bioaerosol sampling method to detect respiratory viruses in singapore’s mass rapid transit (mrt) network. over a period of weeks, aerosol samples were collected during peak mrt ridership hours. nine ( %) tested positive for adenovirus, four ( . %) tested positive for respiratory syncytial virus type a, and one ( %) tested positive for influenza a virus using real-time rt-pcr/pcr. to our knowledge, this is the first time molecular evidence for any infectious respiratory agent has been collected from singapore’s mrt. our pilot study data support the possibility of employing bioaerosol samplers in crowded public spaces to noninvasively monitor for respiratory viruses circulating in communities. a high population density, international tourism and trade traffic puts singapore at a high risk of novel emerging respiratory epidemics. this risk has been recently reinforced by human epidemics in singapore of severe acute respiratory syndrome-associated coronavirus (sars-cov) , pandemic h n influenza a virus , and enterovirus , (which causes hand, foot and mouth disease). other major threats to singapore include middle east respiratory syndrome-related coronavirus (mers-cov) , and various avian (h n , h n , h n , h n , etc.) and swine (h n , h n , h n ) influenza a viruses, especially if they become highly transmissible between humans. for instance, the first human case of a(h n ) was confirmed in february , and a total of , human cases of a(h n ) have been reported since , with % ( ) in the th wave of the epidemic from october to september . compared to the estimated . % case fatality rate for the global pandemic h n virus , a(h n ) (including both high and low pathogenic strains) has an alarming % case fatality rate, which has resulted in known deaths since its emergence in , , . international travel can play a significant role in the spread of infectious diseases. for example, during the influenza h n pandemic, countries receiving the highest volume of passengers (> , in two months) from mexico (the country of origin of the h n virus), had a significantly higher risk of virus importation associated with travel to mexico . china (the country of origin of a(h n ) and a(h n ) viruses) is the third largest market of travelers for singapore changi airport, which receives ~ , passengers from china per week. with singapore's close connection to china, and recent evidence suggesting the potential for airborne transmission of highly pathogenic avian influenza a(h n ) , it is prudent for singapore to ramp up disease surveillance. the ability to quickly identify and robustly track respiratory threats is fundamentally important for an early emergency response. to better understand the dynamics of microbes in cities in relation to human health, and to create healthier environments in areas where people frequently visit, researchers have begun studying the microbiome in and around public transportation vehicles , . an international project named metasub was launched to map the microbiome of transit networks in the world's largest cities. over cities, including singapore, boston, shanghai, paris, stockholm, and são paolo are participating in the project to improve city planning and public health. although baseline metagenomic maps created from these studies are said to be useful for mitigating bioterrorism and infectious disease outbreaks, most of them focus largely on mapping surface-borne bacterial dna and neglect to address the threat of weaponized or global catastrophic biological risk-level (gcbr-level) agents, both of which would likely be aerosolized or respiratory-borne rna viruses . bioaerosol studies have demonstrated that concentrations of airborne bacteria in underground subway systems tend to fluctuate seasonally and are linked to human presence and activities , . such information demonstrates the possible role of public transportation in disease transmission, but again, only few studies have focused on the existence of airborne viruses in public transportation systems [ ] [ ] [ ] [ ] . mathematical models have been developed to assess the risk of airborne and droplet transmission of respiratory viruses in buses, airline cabins and cities [ ] [ ] [ ] , and although these simulation data are useful, we have sparse field data to support them. in this study, we sought to monitor for respiratory viruses in singapore's mass rapid transit (mrt) network, a likely area of virus incursion as well as a pathway for virus transmission. specifically, we asked, could noninvasive bioaerosol sampling detect respiratory viruses in singapore's mrt network? using real-time polymerase chain reaction (pcr) and real-time reverse transcription pcr (rt-pcr), fourteen ( %) of the aerosol samples collected from the singapore mrt tested positive for one or more respiratory viruses. nine ( %) of the virus-positive aerosol samples were collected from the north east mrt line (nel; purple) and five ( %) from the east west mrt line (ewl; green). nine of the virus-positive aerosol samples ( %) tested positive for adenovirus, four ( %) tested positive for rsv-a, and one ( %) tested positive for influenza a virus. one sample tested positive for both adenovirus and rsv-a. one additional sample with a ct value of was suspected to be positive for influenza a virus. aerosol samples did not test positive for any other target pathogens including influenza b viruses, enteroviruses, coronaviruses, and rsv-b. temperature, relative humidity (rh) and light intensity remained relatively consistent inside the purple and green mrt lines throughout the study, with averages of °c and °c, % rh, and . and . lum/m², respectively (table ). there were no significant differences in these environmental conditions on sampling days yielding virus-positive aerosol samples. adenovirus-positive droplets (> μm in aerodynamic diameter) and droplet nuclei (≤ μm in aerodynamic diameter) were retrieved from six ( %) and four ( %) of the adenovirus-positive samples, respectively. in one instance, adenovirus-positive particles of both size ranges were retrieved from the same sample. rsv-a droplets and droplet nuclei were both retrieved from four ( %) of the rsv-a-positive samples. only influenza a virus-positive droplets (not droplet nuclei) were retrieved from the influenza a virus-positive sample. adenovirus-positive samples were collected in january , and february, march, may, june, september and october . rsv-a-positive samples were collected in february , and the influenza a virus-positive sample was collected in september . the additional sample that was suspected to be positive for influenza a virus was collected in november . see table for a monthly summary of pcr-positive aerosol samples. attempts to culture and subtype adenovirus and influenza a virus from pcr-positive aerosol samples were unsuccessful. insufficient volumes of the original samples were leftover to attempt to culture rsv-a from pcr-positive aerosol samples. to our knowledge, this is the first time molecular evidence for any infectious respiratory agent has been collected from singapore's mrt. our results suggest that bioaerosol sampling might have a practical application for pathogen detection in public areas such as subway systems. bioaerosol sampling in the field provides a noninvasive way to monitor and characterize the community of aerosolized respiratory viruses that regularly infect the public, as well as potentially detect or discover novel pathogens with pandemic potential, such as the influenza a(h n ) virus. additionally, a bioaerosol sampling system is advantageous as it does not require collecting individual samples from human subjects, nor informed consent. although this pilot study provides important qualitative data (i.e., molecular presence of respiratory viruses in the mrt), it is important to note that solely detecting viral dna/rna in the air does not determine that a virus is successfully transmitted through the air. cultures in this study were negative and therefore the viruses detected may have been non-viable. however, studies have demonstrated that upon the first ~ minutes of aerosolization, viruses tend to decay the most rapidly , , and therefore it is possible that the viability of the viruses captured in our study rapidly decreased during sample collection and transport. moreover, temperature and rh have been widely understood to influence the viability of aerosolized viruses . however, novel research has recently demonstrated that the viability of aerosolized influenza virus is independent of rh when the aerosol composition includes human bronchial epithelial (hbe) extracellular material (ecm) , indicating that the influence of rh is more so on the rate of aerosol deposition after expulsion, which in turn, influences the overall concentration of viral aerosols in the environment. aerosols tend to deposit onto surfaces more quickly at higher rh levels (> %), suggesting that greater time spent in controlled environments with lower, more comfortable rh levels, could increase the risk of acquiring a respiratory virus via inhalation and respiration. therefore, the average rh level ( %) on the singapore mrt is not theoretically conducive to heavy airborne transmission of influenza viruses. a likely contributor to the lack of data regarding airborne transmission of viruses is the difficulty of detecting respiratory pathogens in the air. standard detection methods are designed to target viral nucleic acids which are subject to degradation from various environmental stressors (e.g., temperature, humidity, and uv light) as well as physical stress from aerosol sample collection methods, potentially deterring downstream detection. therefore, high rh levels, in addition to sample degradation, could explain the low recovery of virus-positive aerosol samples in our study. additionally, viral bioaerosols have notoriously low airborne concentrations, reducing the sensitivity of bioaerosol sampling. some bioaerosol samplers, such as the niosh sampler used in this study, are compact and require a low flow rate which also may reduce sensitivity. although the air pump flow rate and sample collection times used in our study have been demonstrated to efficiently capture aerosolized influenza virus and rsv rna [ ] [ ] [ ] , it is possible that these parameters are not optimal for capturing the other respiratory virus dna/rna targeted in our study. for example, it is possible that when compared to rna viruses (e.g., influenza virus, rsv), adenovirus (a more durable dna virus) can withstand a higher flow rate and/or longer sampling duration. furthermore, although each of the assays used to detect viral dna/rna in our aerosol samples are highly sensitive, validated, singleplex real-time rt-pcr/pcr assays, specific detection limits (viral copies per volume of air) were not tested in our laboratory. therefore, our dna/rna detections should not be mistaken as a true representation of the prevalence/densities of these viruses circulating in the air of the mrt, but rather a representation of the viruses our bioaerosol sampling technique is capable of detecting in this environment. to improve the sensitivity and specificity of bioaerosol sampling, more virus-specific controlled studies are needed to determine optimal sampling parameters for multiple respiratory viruses. airborne microbial concentrations have been demonstrated to fluctuate seasonally in underground subway systems . however, the singapore mrt is a controlled environment and therefore environmental conditions remained consistent throughout the study and no significant differences were recorded on days yielding virus-positive samples. furthermore, unlike temperate climates with distinct seasons, singapore has a tropical rainforest climate with high temperatures and humidity levels year-round, with little monthly variation. therefore, singapore experiences influenza outbreaks year-round, with two peak periods (april-june and september-december). although we were able to stratify our pilot study data by month, our results are too limited to properly assess a correlation between virus-positive aerosols and singapore's peak influenza periods. however, it is notable that the influenza a virus-positive and suspect-positive aerosol samples were both detected during singapore's second peak influenza period in . additionally, rsv-a-positive aerosols were collected during weeks of increased polyclinic attendances for acute respiratory infections in singapore . adenovirus-positive aerosols were collected during weeks of increased, average and decreased polyclinic attendances for acute respiratory infections . a larger study yielding more aerosol samples over a longer duration would be needed to further characterize the relationship between bioaerosols in the mrt and community health data in singapore. quickly identifying and robustly tracking respiratory threats are fundamental steps in an early emergency response to a pandemic. a bioaerosol sampling system is advantageous in this regard as it does not require the timely acquisition of ethical/irb approvals and informed consent needed to collect individual samples from human subjects. if an outbreak is suspected or underway, bioaerosol samplers can be immediately deployed in high-risk areas, yielding results within a minimum of ~ hours. moreover, if bioaerosol samplers are proactively used to monitor high-risk areas, initial results turnaround times are nearly cut in half, as sample collection time is ~ - hours. proactively monitoring for respiratory viruses also eliminates the risk of missing the time window of exposure to pandemic viruses in high-risk areas and would allow a more robust surveillance, which could be beneficial when the etiologic agent is known but the route of exposure is not fully understood. our bioaerosol sampling method also size-fractionates virus-laden particles, which can help measure the proportion of exposure to droplets versus droplet nuclei (i.e., inhalable versus respirable particles) in multiple environments and climates, which is important when assessing the type or severity of disease (e.g., an upper versus lower respiratory tract infection) that might follow. however, the full extent of exposure cannot be determined without measuring viral load. our study falls short in that we did not quantify the viral load in our aerosol samples, making it difficult to compare our results with quantitative aerosol studies. in the future, we will implement a dilution series of positive controls into our real-time rt-pcr/pcr assay protocols to create a standard curve that can be used to accurately quantify the viral load in each aerosol sample. when compared to a mobile/personal bioaerosol sampling method such as the one used in our study, a stationary sampling method is arguably a more practical approach to conducting disease surveillance. however, the use/installation of stationary samplers on the mrt would likely require permission from governmental authorities. strapping the bioaerosol samplers to our bodies was the least invasive way for our team to recover molecular evidence of aerosolized respiratory viruses on the singapore mrt. as this type of research is new to singapore, the wellbeing of singapore's civilians was carefully considered and therefore, researchers wore their duke-nus employee badges while sampling, and our laboratory's name was stitched onto the sampling backpacks (fig. ) for transparency. similar to a study measuring the risk of exposure to aerosolized influenza virus among healthcare workers in an emergency department during influenza season , strapping the samplers to personal backpacks allowed us to detect personal exposure to aerosolized viruses and provide insight into the efficacy of potential interventions (e.g., air/surface decontamination, and wearing face masks when infectious). moreover, with evidence validating the notion that mrt riders are at risk of exposure to respiratory viruses, we hope to motivate scientists to conduct similar field studies to unveil the true risk of exposure while using public transportation, as data on this topic are scarce. field studies may also inspire bioengineers and scientific instrument companies to design and test improved bioaerosol sampling such that it might be employed in a more widespread fashion to surveil for respiratory threats. in conclusion, our study suggests that when combined with molecular diagnostics, aerosol sampling has promising potential to work as a noninvasive tool to monitor for respiratory pathogens in public areas. additional studies are needed to assess a possible contribution of aerosol sampling to public health surveillance during periods of increased risk. bioaerosol sampling. from january through january , national institute for occupational safety and health (niosh) bc -stage aerosol samplers were used to collect aerosol samples from the following singapore mass rapid transit (mrt) heavy rail lines: east west line (ewl; green), and north east line (nel; purple). mrt lines were selected for their high capacity (~ , passengers per -car train), frequent use, and connection to high-traffic public areas. the ewl was specifically chosen for its connection to changi airport, and the nel for its connection to downtown singapore. aerosol samples were collected weekly, with occasional interruptions, during peak ridership (~ passengers/car or pax/m ) from : - : and : - : on wednesdays. sampling days varied occasionally due to public holidays and the researchers' availability. using a -foot ( cm) long piece of ¼" ( . cm) tygon tubing, niosh samplers were connected to airchek ® touch sample pumps (skc, eighty four, pennsylvania) carried inside personal backpacks worn by the researchers. both straps were tightened around the shoulders to secure the backpack against the researcher's back. niosh samplers were securely fastened to the front of the backpacks (on or above the researcher's heart) using velcro (fig. ) . for each separate mrt line (i.e., green and purple), the same assembled niosh sampler was used for both am and pm sampling sessions. to ensure all researchers followed the same sampling protocol, each researcher sampled one of the middle train-cars and remained standing with intermittent walking throughout each sampling session. between am and pm sampling sessions, sampler inlets were covered with electrical tape and the assembled samplers were refrigerated. all of the material collected by one niosh sampler during hours of sampling was counted as one aerosol sample. before aerosol sampling, air pumps were calibrated to sample at . l min − collecting a total of l of air per mrt line each week. calibration was performed by inserting the sampler into a calibration adaptor (designed for niosh samplers) attached to an skc check-mate calibrator. niosh samplers were engineered to separate collected particles into three size fractions: > μm, - μm, and < μm in diameter . prior to molecular analysis, the particles collected in the - μm and < μm size fractions were combined (see sample processing below). therefore, our study reports molecular results for two particle size fractions (droplets > μm and droplet nuclei ≤ μm). if processed directly, the minimum time duration between the start of sample collection and reporting of assay results (for one, hr aerosol sample) was hours. however, most of the dna/rna extracted from the aerosol samples was stored at − °c and analyzed in monthly batches. niosh samplers were rinsed with deionized water and soaked in % ethanol between each sampling day and a swab sample of the sterilized niosh sampler interior was used as a negative control sample for each sampling day. negative control swabs were transferred to tubes containing ml of . % bsa solution, vortexed for seconds, and discarded. negative control sample solutions were stored at − °c prior to further analyses. environmental data collection. ambient temperature, relative humidity (rh) and light intensity were logged during each sampling session using portable hobo data loggers (onset; bourne, ma, usa) tied to the sampling backpacks worn by the researchers. using bluetooth low energy technology, each hobo data logger wirelessly transmitted data to the researcher's mobile device using the hobomobile application. once sampling was complete, researchers used the application to stop the data logger and convert the data file to an excel file before sending the data to a designated research assistant's email address. data were then recorded onto a master excel file. sample processing. a uv-sterilized biological safety cabinet class ii and sterile consumables and equipment were used to process samples. using a filter-handling kit ( - ; skc), each polytetrafluoroethylene (ptfe) filter was removed from the niosh sampler cassettes and transferred to a ml falcon tube and vortexed while dry for seconds. to minimize cross-contamination, forceps were sterilized with % ethanol in between each filter transfer. one milliliter of . % bsa solution was then added to each ml falcon tube containing a filter and vortexed again for seconds. one milliliter of . % bovine serum albumin (bsa) solution was added to each . ml conical tube from the niosh samplers and vortexed for seconds. using cryotube vials, the bsa solutions from the ml falcon tubes containing filters were pooled together with their respective . ml conical tube sample. two milliliters of bsa solution were added to each ml falcon tube from the niosh samplers, vortexed for seconds, and transferred to cryotube vials. nucleic acid extraction and real-time rt-pcr/pcr. dna/rna was extracted from . ml of each aerosol sample and negative control sample solution using the qiaamp viral rna kit and qiaamp dna blood kit (qiagen) following the manufacturer's instructions. using previously validated probe-based molecular assays [ ] [ ] [ ] [ ] [ ] adapted to the duke-nus laboratory of one health research, extracted rna was tested for the presence/absence of influenza a and b viruses, enteroviruses, coronaviruses, and rsv subtypes a and b using superscript iii one-step real-time rt-pcr with platinum taq polymerase. rsv-a and rsv-b assays were not adapted to our laboratory during the time of sampling and therefore were performed months after the last aerosol sample was collected. extracted dna was tested for the presence/absence of adenoviruses by real-time pcr using a quantinova probe pcr kit (qiagen). three negative control reactions (no template) were included in each real-time rt-pcr/pcr assay. a positive aerosol sample was one in which virus was detected in one or more of the niosh sampler stages, with a ct value < . stage ( . ml tube) and stage (filter) were combined to represent respirable particles (i.e., droplet nuclei ≤ μm in diameter) compared with particles that are only inhalable (i.e., droplets > μm in diameter) collected in stage ( ml tube). cell culture and dna sequencing. virus-positive samples were tested for viability using cell culture. first, μl of each adenovirus-positive aerosol sample was inoculated into a cells (atcc) with dulbecco's modified eagle medium (dmem) % (v/v) fetal bovine serum (fbs) and incubated at °c. mdck cells were used to culture the influenza a virus-positive and suspect-positive aerosol samples. inoculated shell vials were then observed for cytopathic effect hours after inoculation, and daily for ten days afterwards. observation of cytopathic effect was used to score positive or negative cultures. rsv-positive samples were not tested for viability as rsv assays were not performed until after all of the original aerosol sample material had been used for dna/rna extraction, leaving us with no original sample material to perform cell culture on rsv-positive the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention. singapore ministry of health outbreak of pandemic influenza a (h n - communicable diseases surveillance in singapore epidemic hand, foot and mouth disease caused by human enterovirus middle east respiratory syndrome coronavirus (mers-cov). health advisory detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient a cross-sectional study of primary-care 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environment and urban systems high humidity leads to loss of infectious influenza virus from simulated coughs airborne micro-organisms: survival tests with four viruses the effect of environmental parameters on the survival of airborne infectious agents influenza virus infectivity is retained in aerosols and droplets independent of relative humidity. the journal of infectious diseases measurement of airborne influenza virus in a hospital emergency department development of an improved methodology to detect infectious airborne influenza virus using the niosh bioaerosol sampler distribution of airborne influenza virus and respiratory syncytial virus in an urgent care medical clinic singapore ministry of health healthcare personnel exposure in an emergency department during influenza season. plos one surveillance for respiratory and diarrheal pathogens at the human-pig interface in sarawak, malaysia increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms cdc protocol of realtime rt-pcr for influenza h n . world health organization evaluation of three influenza a and b real-time reverse transcription-pcr assays and a new h n assay for detection of influenza viruses performance of different mono-and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel improved real-time pcr assay for detection and quantification of all known types of human adenoviruses in clinical samples. medical science monitor: international medical journal of experimental and clinical research this study was supported by start-up funding for dr. gregory gray's laboratory of one health research at duke-nus medical school, singapore. the datasets generated and/or analyzed during this study are available from the corresponding author on reasonable request. k.c., t.n., c.h.e., and g.g. designed the study. k.c., t.n., s.y., and c.h.e. performed aerosol sample collection and t.n., s.y., and c.h.e. performed laboratory works. k.c., t.n., s.y., and c.h.e. analyzed and interpreted the data. w.l. provided the niosh aerosol samplers and k.c. and w.l. guided the researchers in their use. k.c. and g.g. supervised the study. k.c. drafted the manuscript and w.l. and g.g. contributed to writing and reviewing the manuscript. all authors read and approved the final manuscript. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -g vcchrh authors: agrawal, anurodh shankar; ying, tianlei; tao, xinrong; garron, tania; algaissi, abdullah; wang, yanping; wang, lili; peng, bi-hung; jiang, shibo; dimitrov, dimiter s.; tseng, chien-te k. title: passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: g vcchrh middle east respiratory syndrome coronavirus (mers-cov) has repeatedly caused outbreaks in the arabian peninsula. to date, no approved medical countermeasures (mcm) are available to combat mers-cov infections. several neutralizing human monoclonal antibodies (mabs), including m , a germline-like human mab, have been chosen as promising mcm for mers-cov. however, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. we assessed the prophylactic and therapeutic efficacy of m by using well-characterized transgenic mice shown to be highly sensitive to mers-cov infection and disease. we found that mice treated with m prior to or post lethal mers-cov challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. taken together, these results support further development of m and other human monoclonal antibodies as potential therapeutics for mers-cov infection. scientific reports | : | doi: . /srep and long-term threat to people, particularly those who interact closely with camels in the arabian peninsula. even though mers-cov presently has limited human-to-human transmission , , the high mortality rate of this virus and limited information on the mechanism able to confer increased human-to-human transmission have raised concerns of a potential mers pandemic. indeed, the recent outbreaks in korea and the appearance of super-spreading events indicate that mers-cov has the ability to cause large outbreaks outside of the arabian peninsula [ ] [ ] [ ] . currently, no approved vaccines or drugs are available to treat this viral infection. these facts highlight an urgent need to develop potent prophylactic and therapeutic agents to fight this lethal virus. similar to other coronaviruses, mers-cov uses the envelope spike (s) glycoprotein, a class i transmembrane protein, for interaction with its cellular receptor for binding, fusion and entry into the target cell . the receptor binding domain (rbd) located in the s domain of the mers-cov spike is responsible for binding to the well-characterized cellular receptor identified as dpp (cd ) and is, therefore, critical for binding and entry of the virus [ ] [ ] [ ] . therefore, neutralizing antibodies capable of blocking such interaction could be promising preventive and/or therapeutic candidates. recently, human monoclonal antibodies (mabs) capable of neutralizing mers-cov have been identified and characterized by several research groups [ ] [ ] [ ] [ ] [ ] [ ] . these antibodies have been isolated from naive human antibody libraries, from transgenic "humanized" mice, or from b cells of an infected individual, and they recognize different epitopes on mers-cov rbd. one of the most potent mabs, m , is a germline-like antibody identified from a very large (~ size) phage-displayed antibody library derived from b cells of healthy donors. this mab exhibits exceptionally potent neutralizing activity (ic = . μ g/ml) in vitro . moreover, because its epitope almost completely (~ %) overlaps with the receptor-binding site of dpp on mers-cov rbd, as is evident by its recently solved crystal structure , the probability of generation of resistant mutants may be absent or very low. notably, although the functions of these mabs have been extensively characterized in vitro, their further clinical development has been hindered by the lack of an effective animal model of mers-cov infection. mers-cov cannot infect small laboratory animals (e.g., mice, hamsters and ferrets) as a consequence of species-specific differences in dpp , while only causing mild-to-moderate symptoms in rhesus macaques. marmosets, which are more susceptible to mers-cov, developed a moderate-to-severe disease, but limited availability and high cost have hampered their use . rabbits can be infected, but the infectious virus is challenging to detect , . it was found that the expression of human dpp could overcome the lack of susceptibility in normal mice. with prior transduction of adenoviral human dpp -expressing vectors, mice became susceptible to mers-cov infection without revealing any measurable clinical manifestations . in contrast, transgenic (tg) mice with the human dpp gene integrated into the genome readily developed acute morbidity (weight loss), and uniform death occurred within a week , , making it an ideal preclinical model for the development of vaccines and treatments against mers. some of the aforementioned human neutralizing monoclonal antibodies have been shown to protect engineered human dpp -expressing mice and the naturally permissive rabbits, entirely based on their ability to inhibit mers-cov infection and/or alleviate histopathology of the lungs , , , . to further verify the protective efficacy of these human monoclonal antibodies, particularly m , against mers-cov infection, it is highly desirable to use the well-characterized human dpp tg mice known to result in acute disease (weight loss) and death , . by using this highly permissive tg mouse model, we evaluated the prophylactic and therapeutic efficacy of m mab in vivo. we report in this study for the first time that treatment of tg mice with a single-dose of m antibody prior to or after challenging with , ld of mers-cov protected mice from the lethality in a dose-dependent manner, thereby representing the first antibody tested for its protective efficacy against lethal mers-cov infection. prophylactic efficacy of mers-cov rbd-specific human monoclonal antibody, m . we established a tg mouse model which is profoundly sensitive and susceptible to mers-cov infection, as determined by high viral titers in the lungs, as well as a high rate of morbidity and mortality , . equipped with this small animal model of human mers-cov, we investigated the protective efficacy of mab m . to accomplish this, each group (n = ) of mice was treated via the intraperitoneal (i.p.) route with two different doses: . mg and mg per mouse diluted in μ l pbs, and challenged intranasally (i.n.) at h post treatment with tcid (i.e., , ld ) of mers-cov in a volume of μ l . challenged mice were monitored daily for clinical manifestations (weight loss) and mortality. as shown in fig. , the group treated with mg mab survived viral infection without showing any clinical symptoms. these mice initially showed either no weight loss or recovered from mild weight loss within three days (fig. a) . on the other hand, the group treated with . mg mab showed a gradual weight loss ( - %) until day just before starting to recover (fig. a ). all surviving mice (one died on day in mice treated with . mg of m ) continued to recover and appeared well up to dpi when the experiment was terminated (fig. b) . all mers-cov-challenged mice pretreated with a high dose ( mg) of irrelevant mab m . exhibited profound weight loss (> %) and succumbed to infection with % mortality by day p.i. (fig. a,b ). therapeutic efficacy of mers-cov rbd-specific human monoclonal antibody, m . to determine the therapeutic potential of this human monoclonal m antibody, groups of mice (n = per group) were challenged (i.n.) with tcid of mers-cov (i.e., , ld ) in a volume of μ l and then treated (i.p.) hours later with a single dose of either mg or . mg of m or mg of m . antibody (control) in μ l per mouse, followed by monitoring daily for wellbeing (weight loss and other clinical manifestations) and mortality of mice. we noted that whereas treatment with mg of m antibodies was effective in the protection against the lethality caused by mers-cov infection, it failed to protect mice fully from the onset of clinical illness (weight loss). specifically, all of the challenged mice treated with mg of m antibody suffered an attenuated (< %), and transient weight loss until day , and gradually recovered to day when the experiment was terminated (fig. ) . similarly, challenged mice treated with a low dose of . mg of m antibodies suffered from attenuated and transient weight loss until day p.i. and gradually recovered. however, we noted a single death at day in this low dose treatment group (fig. ) . as expected, all mice treated with a single dose of mg of control m . antibody exhibited profound weight loss (> %) and succumbed to mers-cov infection with % mortality by day p.i. (fig. ) . taken together, these results indicate that this mers-cov rbd-specific human m antibody can be highly effective as prophylactic or therapeutic modalities in protecting highly permissive transgenic mice against mers-cov infection and disease. we also investigated the protective mechanism of m against mers-cov by determining the lung virus titers in challenged mice at day after treatment. specifically, we sacrificed two mice (out of ) in each group, as described above for figs and and their lung specimens were harvested for determining viral titers by using via vero e cell-based infectivity assay and quantitative pcr (q-pcr)-based assay targeting the upstream e gene of mers-cov. as shown in fig. a we were unable to recover infectious virus from any mouse treated with mg of m antibody either before or after challenge with mers-cov. however, we were able to detect a barely detectable infectious virus, with the limit of detection (lod) of . log ticd /g, from a single mouse receiving . mg of m prior to viral challenge. these results indicated that mab m most likely confers protection from lethal challenge by restricting viral replication within the lungs, thereby preventing viral infection in the brains and other organs. titers of viral rna copy number, as shown by qrt-pcr assays, were also compared among groups having different doses of mabs. lungs of infected mice were harvested on day post-and pre-virus challenge group. all groups exhibited detectable viral rna. titers were significantly lower than those in the control group in all m -treated groups. in the pretreatment group, mice treated with mg of m showed a -log reduction in viral rna detection, while a ~ log reduction in viral numbers was seen in mice treated within . mg m when compared to mice receiving control mab m . . in the post-treatment group, a smaller (~ log) difference in viral rna copy number (compared to that in the pretreatment group) was observed between mice treated with mg antibody compared with those receiving control antibody, while a more than log reduction in viral rna number was seen in mice treated with . mg m when compared to mice receiving control mab (fig. b) . these data indicate that m confers significant protection to mice when administered pre-or post-viral challenge. taken together, these results suggest to us that the epitope targeted by this exceptionally potent rbd-specific m antibody has a great potential for further development as a potent preventive and therapeutic agent in the future. treatment with m attenuates lung pathology associated with mers-cov infection. the effect of m antibody treatment on the pulmonary pathology associated with mers-cov infection was evaluated by using formalin-fixed, paraffin embedded, and hematoxylin/eosin (h&e)-stained lung specimens harvested at day p.i. pulmonary pathology was noted in all mice that were treated with different doses of m or control m . antibodies either before or after viral infection. on a severity scale of to (none, mild, moderate, severe), h&e-stained samples from mice pretreated with mg and . mg of m antibody were graded and , respectively, for perivascular and intra-alveolar infiltration of mononuclear cells, including lymphocytes, macrophages/monocytes (fig. , middle panel) , whereas those obtained from mice that received post-infection lung specimens collected at day after viral challenge were processed for assessing the viral titers by using both vero e -based infectivity assay and qrt-pcr targeting upstream e gene of mers-cov, and expressed as log tcid /gram and log tcid equivalent (eq.)/gram, respectively. (a) prophylactic and therapeutic efficacy of human m antibody treatment in reducing the lung titers of infectious virus. (b) prophylactic and therapeutic efficacy of human m antibody in reducing the titers of viral rna. the data shown are representative of at least two independently conducted assays using the same samples. data is presented as mean ± standard error (se). * * * p < . as determined by using student's t test. scientific reports | : | doi: . /srep treatment with either dose of m were graded (fig. , right panel) , compared to the grade assigned to mice received control antibody treatment prior to infection (fig. , left panel) . mers-cov has attracted significant basic research and clinical studies since it was first discovered in early . even though the transmissibility of mers-cov among humans remains low at present, as a mutation-prone rna virus, it could eventually evolve into a highly communicable and more virulent human pathogens. this emphasizes the urgent need for the development of an effective antiviral therapy which could restrict the spread of this deadly disease. in other viral infections, neutralizing antibodies have been shown to protect the host from disease progression and/or reduce the severity of clinical symptoms. passive immunotherapy for prophylaxis and treatment of infectious viral diseases has been widely used for many decades [ ] [ ] [ ] [ ] [ ] . passive transfer of neutralizing antibodies is also a promising strategy for both prophylaxis and treatment against mers-cov infection. to this end, we and others have successfully demonstrated the protective efficacy of specific human neutralizing monoclonal antibodies in animal models of mers-cov infection , , , . among a panel of mers-cov-specific mabs generated by using a vast phage display library , we identified three mabs which specifically bind to the mers-cov rbd with very high affinity. among these three identified, we noted that mab m exhibited the highest potency in neutralizing live mers-cov. here, we further characterized this novel human mab in our tg mouse model of mers-cov infection and showed prophylactic and therapeutic protection of mice treated with m before and after a lethal challenge with the virus, respectively. thus, mab m is highly promising as a potent inhibitor for urgent prophylaxis in adjunctive treatment for patients infected with mers-cov. in our studies, we noted that passively transferred with mg and . mg of m monoclonal antibodies to individual mice h prior to challenge with , ld of mers-cov resulted in % and % protection against lethality, respectively (fig. ) , suggesting that using . mg m /mouse as a prophylaxis is suboptimal to completely neutralize viral infection, thereby allowing residual viruses to replicate within lungs during the course of infection. these data demonstrate that m confers a dose-dependent reduction of mers-cov infection, corroborating lower viral rna levels and live virus isolation determined for these mice when compared to control mice. our study also confirmed the therapeutic efficacy of m in a dose-dependent manner. similar to the prophylactic studies, administration of a single-dose of m antibody at a concentration of either or . mg per mouse at h after mers-cov challenge provided % and % protection, respectively, against infection-induced lethality, accompanied by reduced viral loads (both infectious virus and viral rna) within the lungs. however, we also noted the recovery of bodyweight loss and the reduction of viral loads in mice treated with mg of m at hrs after infection were slower than those treated with . mg of m , as shown in figs a and b, respectively. while there is no clear evidence showing an adverse impact on the overall wellbeing of mice imposed upon treatment with mg of m antibody before mers-cov challenge (fig. ) , it is difficult to completely rule out the existence of subtle "yet-to-be investigated" high-dose drug toxicity. we speculate that such a subtle high-dose drug toxicity in the phase of acute and dynamic mers-cov infection initiated at hrs before treatment with mg of m could exacerbate drug toxicity, resulting in reduction of appetite and antiviral capacity. however, such a negative impact imposed upon high-dose treatment of virally infected mice appeared to be transient and did not irreversibly alter the final outcome of infection, as judged by the mortality (fig. b) . additional studies, especially the pharmacokinetics and the dosing frequency of m are warranted in the future to optimize preventive and therapeutic strategies with this promising antibody. the transgenic mice that we used for evaluating the prophylactic and, especially, the therapeutic efficacy of this m antibody are extremely sensitive to mers-cov infection and disease, with ld and id of . and . tcid of mers-cov, respectively (data not shown), titers which are lower than our original estimations . such a striking ability of this m antibody, as a prophylactic or therapeutic agent, to significantly protect these transgenic mice against challenge with ld of mers-cov is highly impressive. the rbd of the mers-cov, targeted by this m antibody, is highly conserved among various clinical isolates and the mutation rate of this rbd appears to be extremely low, compared to that of other rna viruses , , thereby making the development of escape mutants to m unlikely. however, a combination treatment with multiple neutralizing mabs targeted at different epitopes or the mers-cov-specific hr p fusion inhibitor targeting the hr domain of the s subunit of the mers-cov s protein , could be desirable. by immunizing mice with rbd of mers-cov s protein, li, y. et al. recently developed a humanized mab, named c h, that exhibited strong neutralizing activity with nd of ~ . and ~ . μ g/ml against the pseudotyped and live mers-cov, respectively , which are about -fold less potent than m (nd = . and . μ g/ml against the pseudotyped and live mers-cov, respectively) . using ad -hcd -transduced mouse model , they demonstrated that intravenous administration of a single dose of c h one day before or after the mers-cov challenge resulted in reduction of viral titer by log at dpi. however, intraperitoneal administration of m to our hdpp tg mice lead to the reduction of viral titer as high as log at dpi. since mers-cov challenged ad -hcd -transduced mice showed no severe disease, the effect of c h on the weight loss and mortality in these mice is unavailable. additionally, unlike hdpp transgenic mice that we used in this study with well-defined hdpp expression as well as % lethal dose (ld ) and infectious dose (id ), the intensities of hcd expression among the ad -hcd -transduecd mice are variable, ranging from undetectable to a high level . although both c h and m bind to the rbd of mers-cov s protein, some of the critical amino acid residues recognized by these two mabs are different , . the epitope of m overlaps extensively with the dpp -binding site, which is composed of mers-cov rbd residues n -k , s , f , d , e , w -r , w , v , s and s . the epitope of mab c , the parental mouse mab of c h, only overlaps with partial of dpp -binding site, which is composed of five rbd residues, w -e and d -r . most of other rbd amino acids recognized by c , including y -n , k , l -k , p and v -s , are not located on the dpp -binding site, indicating that the neutralization efficacy of c h is largely attributed to the steric hindrance created by its binding with mers-cov rbd .these results suggest that combinational use of c h and m may exhibit synergistic antiviral effect against both wild-type strains and escape mutants (if any) of mers-cov. taken together, these results suggest to us that the mers-cov rbd protein-specific m mab is an excellent candidate for passive immunotherapy to provide immediate and effective protection to individuals who may be exposed to mers-cov and to treat patients who have been exposed. testing in humans is needed for its potential use as a therapeutic for the treatment of mers-cov-infected patients. monoclonal antibody production. for expression of m igg , the previously described m igg vector was used to infect cho-k cells (atcc, manassas, va) with polyfect transfection reagent (qiagen, valencia, ca). after screening of clones for antibody productivity by elisa and subsequent characterization, a stable cell line was generated and inoculated into a bioflo bioreactor (new brunswick scientific, nj) for large-scale production of m igg . purification was carried out by using a protein g column (ge healthcare), and endotoxin was removed by detoxi-gel endotoxin removing columns (thermo scientific) according to the manufacturer's instructions. transgenic mice expressing human dpp established by us were used throughout the study. animals were housed in on-site animal facilities at galveston national laboratory under a : light/dark cycle with room temperature and humidity kept between - °c and - %, respectively, and with ad libitum access to food and water. all experiments were performed in accordance with the guide of nih and aaalac and were approved by the institutional animal care and use committee at the university of texas medical branch, as described previously . briefly, groups of - -weeks tg mice were challenged intranasally (in) with tcid /ml (~ , ld ) of mers-cov-emc/ , originally provided by heinz feldmann (nih, niaid rocky mountain laboratories, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, netherlands). the titers of individual virus stocks, stored at − °c, were determined by using vero e -based infectivity assays and expressed as % tissue culture infectious doses (tcid )/ml. scientific reports | : | doi: . /srep viral infections and isolation. all of the animal studies involving infectious mers-cov were conducted within approved animal biosafety level (absl- ) at the galveston national laboratory. experimental designs and strategies in different tg mouse groups involving intranasal challenge with live mers-cov were described in individual experiments in the results section. for live virus isolation, lung tissues were collected at day post mers-cov challenge, weighed, and homogenized in phosphate-buffered saline (pbs) containing % fetal calf serum (fcs) by using tissuelyser (qiagen, retsch, haan, germany), as previously described . the resulting suspensions of infected tissues were tittered in the standard vero e cell-based infectivity assays to quantify yields of infectious virus expressed as log tcid per gram (g) of tissue. rna extraction and viral titers determination by real-time q-pcr. lung tissue samples from each group of mice were transferred to individual vials having rna later solution (qiagen) and subsequently homogenized and subjected to total rna isolation, by using trizol reagent (life technologies), to assess mers-cov-specific genome targeting of virus-specific upstream e gene (upe) and endogenous control gene (mouse β -actin) by using a one-step rt-pcr kit (invitrogen), as previously described . ct values for each sample were analyzed against ct values generated in our lab from the standard curve of mers-cov mrna copy number. relative mers-cov upe mrna expression value was calculated for each replicate and expressed as the equivalent of log tcid per gram (g) of the tissue by the standard threshold cycle (∆∆ct) method. ct value analysis was done by using bio-rad cfx manager . software. is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sarslike pandemic? hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku and pipistrellus bat coronavirus hku comparative analysis of twelve genomes of three novel group c and group d coronaviruses reveals unique group and subgroup features evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia family cluster of middle east respiratory syndrome coronavirus infections the first case of the korean middle east respiratory syndrome outbreak middle east respiratory syndrome coronavirus outbreak in the republic of korea environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc molecular basis of binding between novel human coronavirus mers-cov and its receptor cd structure of mers-cov spike receptor-binding domain complexed with human receptor dpp exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germlinelike antibody infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits prophylaxis with a mers-cov-specific human monoclonal antibody protects rabbits from mers-cov infection rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the growth and potential of human antiviral monoclonal antibody therapeutics the spike protein of sars-cov-a target for vaccine and therapeutic development passive immunity in prevention and treatment of infectious diseases prophylaxis and therapy for chikungunya virus infection post-exposure treatment of ebola virus using passive immunotherapy: proposal for a new strategy structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme virus receptor we thank dr. heinz feldmann, national institute of health at hamilton, montana, and dr. ron a. fouchier, erasmus medical center at rotterdam, the netherlands for the mers-cov. this research was supported in part by a national institutes of health grant, r ai - (to c-t.k.t), and pilot grants from the center for biodefense and emerging infectious diseases and from the galveston national laboratory (grant number: uc ai - . project title: national biocontainment laboratories (nbls) operations support), university of texas medical branch, galveston, tx (to c-t.k.t.), intramural funding of nci (to dsd), and the national natural science foundation of china (# to sj, # to ty). t.g was supported in part by a t biodefense training program ( t ai - ) awarded to utmb by nih. histopathology. mice were necropsied, lung tissues were inflated and fixed in % neutral buffered formalin for days before paraffin-embedded and processed for routine hematoxylin and eosin stain (h&e) for assessing the histopathology . key: cord- -rmnck f authors: theuns, sebastiaan; vanmechelen, bert; bernaert, quinten; deboutte, ward; vandenhole, marilou; beller, leen; matthijnssens, jelle; maes, piet; nauwynck, hans j. title: nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: rmnck f enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. the capacities of nanopore sequencing for viral diagnostics were investigated here. first, cell culture-grown porcine epidemic diarrhea virus and rotavirus a were pooled and sequenced on a minion. reads were already detected at seconds after start of sequencing, resulting in high sequencing depths ( . to . x) after h. next, diarrheic feces of a one-week-old piglet was analyzed. almost all reads ( %) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. contigs matched bacteroides, escherichia and enterococcus phages. moreover, porcine kobuvirus was discovered in the feces for the first time in belgium. suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding ( ( . )– ( . ) copies/swab) and diarrheic signs was not observed during a follow-up study. retrospective analysis showed the widespread (n = , . % positive) of genetically moderately related kobuviruses among belgian diarrheic piglets. minion enables rapid detection of enteric viruses. such new methodologies will change diagnostics, but more extensive validations should be conducted. the true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample . most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. third-generation sequencing using minion (oxford nanopore technologies, ont) might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. the flowcells used for sequencing consist of a membrane containing multiple csgg nanopore proteins from escherichia coli . an ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. this signal is converted into a nucleotide sequence by computational algorithms (basecalling). since the release of minion technology, major advances have been made in terms of the number and the quality of reads generated . in the field of virology, the technology has mainly been applied in human medicine. using nanopore sequencing, it was possible to distinguish three poxviruses with % nucleotide similarity at strain level . minion has also been used as a diagnostic tool during recent ebolavirus outbreaks in west africa, allowing fast on-site characterization of circulating strains , . coupled to a laptop-based bioinformatics workflow, minion was able to detect chikungunya virus, ebola virus and hepatitis c virus in less than hours using earlier versions of the technology . a multiplex pcr method for complete on-site zikavirus genome sequencing in samples with low viral loads has recently been developed by quick and coworkers . partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes . in veterinary virology, the use of nanopore sequencing is growing. a novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing . the entire genome of a parapoxvirus isolated from a seal was obtained by combining data from illumina next-generation sequencing with nanopore sequencing data . one study has reported the detection of venezuelan equine encephalitis virus from unamplified cdna created from poly-a tailed rna using cell culture grown viruses . to the author's knowledge, the present study is the first using minion as an aid in porcine health management. this study was aimed to explore the possibilities of minion as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. the ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. in a second case, the ability to detect (novel) viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. no gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the minion's sensitivity. a porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. moreover, archival ( ) fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in belgium. be performed for , reads with a mean length of nucleotides. reads with a q-score lower than were filtered out, resulting in , remaining sequences (mean length nt) for use in downstream analyses. results of the sequencing run, including taxonomical classification and mapping of reads against pedv and rotavirus a (rva) reference genomes are shown in fig. a . after hours of sequencing, a total of , reads were classified as viral by sensitive tblastx comparison against a complete viral database. of these, . % (n = , ) and . % (n = , ) were assigned to viral families comprising porcine epidemic diarrhea virus (family coronaviridae) and rotavirus (family reoviridae, subfamily sedoreovirinae), respectively. a fraction of the reads ( . %, n = , ) were assigned to order caudovirales. these reads originated from the lambda phage dna used in a previous control run on the same flowcell. at . and . seconds after the start of sequencing, respectively, the first reads matching pedv and rva were translocated through a nanopore. most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing (fig. b) . pedv and rva sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time (fig. c) . after one hour, sequencing depths were higher for pedv ( . x) than for rva ( . to . x). high sequencing depths were acquired after three hours of sequencing for pedv ( . x) and for most rva gene segments ( . to . x). de novo assembly was executed on the quality-filtered reads prior to identification (tblastx) to recover viral genomes. this resulted in the recovery of the almost complete pedv genome and rva gene segments with identities varying between and % compared to the reference genes (table ) . higher assembly accuracies ( to %) were obtained when only the reads matching against rotavirus and pedv were included for de novo assembly (table ) . however, execution of de novo assembly prior to taxonomical classification (tblastx) reduced the time to identify entire viral genomes in the dataset. virome composition of a young diarrheic piglet using nanopore sequencing. a total of , reads were generated by sequencing the diarrheic fecal sample for three hours. of these, , reads (q-score > , mean read length nt) were used for further analyses. different methods were used to compare the reads against a viral database using the hpc cluster of ghent university and results are shown in fig. . comparison against a complete viral database resulted in the detection of , to , potential viral reads, depending on the blast settings. blastn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tblastx. however, there was a very high difference between wall times on the hpc cluster, with only seconds of analysis time for blastn, versus almost hours for tblastx. the majority of sequences were assigned to bacteriophages within the order caudovirales and families siphoviridae (n = , to , reads), podoviridae (n = , to , reads) and myoviridae (n = to , reads). a de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for virsorter analysis. nineteen contigs were classified as sure (n = ; category ), somewhat sure (= ; category ) and not so sure (= ; category ) to be phage-like contigs (fig. b) . comparison of these contigs against the genbank database using blast allowed classification into four different groups. ten contigs showed moderate to high nucleotide similarities to the bacteroides phage b - , suggesting that they all belonged to one phage genome. this was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of bacteroides phage b - (data not shown). the longest contig with a size of , nucleotides, together with four other contigs showed similarities ( % nt identity) to different escherichia phages. as they also mapped nicely distributed across the reference genome of escherichia phage vb_ecop_phapec , it seems that they must also belong to one phage genome (data not shown). two contigs showed poor similarity to both the enterococcus phage vb_efas_ime_ , isolated from hospital sewage in china from an enterococcus faecalis strain, and the enterococcus hirae bacterial genome. the latter might be a prophage inserted in the bacterial genome. interestingly, three contigs were identified for which no similarities were found with existing viruses in genbank, but contig mapped to the reference genome of the enterococcus phage vb_efas_ime (data not shown). these might be novel phages or divergent variants from existing phages present in genbank. three eukaryotic porcine viruses, porcine kobuvirus (n = to reads), enterovirus g (n = to reads) and astrovirus (n = reads) were found at much lower abundancies. the genera kobuvirus and enterovirus belong to the family picornaviridae, whereas the genus mamastrovirus belongs to the family astroviridae. kobuvirus reads were mapped against a european reference strain s- /hun/ /hungary, as shown in fig. c . however, full-genome coverage at high sequencing depth was not obtained. shedding of porcine kobuvirus and rotaviruses in suckling piglets. the shedding of porcine kobuvirus, rva and rotavirus c (rvc) was quantitatively investigated in suckling pigs of the same farm from which the diarrheic feces originated. the fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in fig. a . all piglets started shedding porcine kobuvirus at the end of the first week after parturition. in two piglets (a and d) the shedding was sustained and lasted for at least weeks (above the limit of quantification). peak shedding titers of the porcine kobuvirus varied between . and . log copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and pedv. moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. diarrheic signs were only noticed in two piglets (a and b). in piglet b, an association between high rvc shedding and diarrheic episodes was observed. in contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. interestingly, a peak in kobuvirus shedding was observed in piglet c at day post-farrowing. this animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. acute rva shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. retrospective analysis of porcine kobuviruses shedding in belgian diarrheic suckling pigs and phylogenetic analysis. a total of diarrheic fecal samples collected in were screened for the presence of kobuvirus using the new rt-qpcr. of these, samples ( . %) tested positive and samples showed quantifiable viral loads ( . to . log copies/swab). seven samples were positive, but viral loads were too low to allow accurate quantification. the presence of rva and rvc had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in fig. b . kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. twelve samples contained a single rotavirus infection with a high rva load and in four of these, a high kobuvirus load ( . to . log copies/g) was observed. a single rvc infection was found in seven samples and in four of these tested positive for kobuvirus at high loads ( . to . log copies/g). a dual rva/rvc infection was seen in two samples, but neither contained quantifiable kobuvirus loads. many (n = ) of the rotavirus-negative samples contained high kobuvirus loads. strain v showed high similarity to other belgian porcine kobuvirus isolates from ( . to . % nucleotide sequence identity) and the hungarian reference strain s- /hun/ ( . %). furthermore, there was a high level of genetic variability between the belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between . and . %. a phylogenetic analysis, using the d gene of v and twelve belgian isolates from ( fig. c) , shows the belgian strains clustering between strains from different geographical locations. prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. only the most likely cause(s) of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. however, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses , , , , , . detection of nucleic acids from pathogens using ngs-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. however, most ngs platforms require large investments and processing of the reads can only start at the end of the sequencing run. viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present . furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before ngs analysis. amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. all these factors lead to a long turnover time between sample collection and diagnostic reporting. the third-generation sequencing device minion (ont), holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all dna/rna in a sample, theoretically without needing pre-amplification of viral nucleic acids. it was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. in a first experiment, cell culture-grown pedv and rva, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. sequencing of this pooled sample with the minion resulted in rapid identification of both viruses. real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by greninger and colleagues using the surpi analysis pipeline for rapid identification of human viruses from different clinical matrices . interestingly, the first reads matching pedv and rva were generated respectively after and seconds of sequencing. high sequencing depths ( . x) were acquired within one hour of sequencing for pedv and within three hours for most of the eleven rva gene segments ( . - . x). overall, higher sequencing depths were generated for pedv that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as pedv has a genome size of approximately kb, and rva gene segments are shorter ( . to . kb). this bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. it can be hypothesized that adapters are more easily attached to longer dna fragments, and bias should be avoided by standardization of viral nucleic acid input length. rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. while the technology can be useful for giving fast readouts of viruses (< hours) present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices (e.g. feces) and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between and %, hindering the precise analysis of subtle but important mutations in the viral genome. after the successful identification of the cell culture-grown viruses, the performance of the minion was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. real-time pcr analyses were conducted for rva, rvc, pedv and tgev. enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs . viral metagenomics was conducted on this sample using the minion and two different blast search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. overall, tblastx with an e-value of − was able to identify the most viral reads compared to other search options conducted. however, blastn search options also reached high sensitivity, but at much lower time cost: seconds instead of almost hours. for rapid read analysis and searching for closely related non-divergent viral sequences, blastn or another fast methodology should thus be preferentially used. however, tblastx might pick up more divergent or novel viruses, improving overall sensitivity. three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus g, were identified in sample v . astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of belgian pigs and in feces from pigs around the globe , , . in a recent study from thailand, the difference in prevalence of astrovirus in diarrheic ( . %) versus non-diarrheic ( . %) piglets less than -weeks-old was not statistically significant. also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear . in contrast, associations between diarrhea and human astrovirus infections have been made . a recent study in european countries (hungary, spain, germany, austria and sweden) have indicated the widespread of porcine astroviruses in the swine population. a one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from austria and spain. porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from hungary, and in -week-old pigs and sows in the united states , . the gut might be a hypothetical entry port for such neurological astrovirus infections. enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well , , [ ] [ ] [ ] . in a study from vietnam, no significant correlation was found between diarrhea status and presence of enterovirus g in feces . the involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. furthermore, while sensitive tblastx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. however, reporting of a porcine kobuvirus in belgian piglets with minion is unique. in belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in belgium . in the present study, a novel rt-qpcr assay, targeting the conserved d gene encoding the rna-dependent-rna-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. similar kinetics were also analyzed for porcine rotavirus a and c. while suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding ( . to . log copies/swab) and diarrheic signs was not observed. in one pig, an association was made between diarrheic episodes and the peak of rotavirus c shedding, a well-known enteric pathogen , . very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as pedv and rotavirus [ ] [ ] [ ] . similar viral loads for porcine kobuvirus were also found in case ( . ± . copies/qpcr reaction) and control pigs ( . ± . copies/qpcr reaction) during a recent danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. the study demonstrated that kobuvirus, astrovirus, rotavirus a, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome . the finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. the presence of kobuvirus rna in serum has also been demonstrated in hungarian pigs, but it was not known if the virus is also replicating in other organs . both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries [ ] [ ] [ ] . in our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. a brazilian study demonstrated the presence of kobuvirus rna in serum from -day-old piglets, which had disappeared by day , indicating viral clearance from the blood and excluding systemic persistence . a complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus . of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. in vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. attempts were made to isolate the virus in different cell lines (ma , st and sk), and peripheral blood mononuclear cells. there was no evidence of cytopathogenic effect after several days of incubation. antibodies to visualize antigen expression were not available and therefore the possibility of replication without scientific reports | ( ) : | doi: . /s - - - evident cytopathogenic effect cannot be ruled out. efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. to assess more broadly the prevalence of kobuvirus in the belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. a high proportion ( . %) of the samples (n = ) contained quantifiable viral loads ranging between . to . log copies/g feces. viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from denmark, demonstrating the endemic presence of the virus in the belgian swine population . in the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. however, the prevalence of kobuvirus has been widely described in pigs from several european countries (the netherlands ( . %), slovakia ( . %), hungary ( . %), czech republic ( . %), austria ( . %), italy ( . %), germany ( . %) and sweden ( . %)), american countries (the united states ( . %) and brazil ( . %)), african countries (kenya ( . %) and uganda ( . %)) and asian countries (thailand ( %), south korea ( . %) and vietnam ( . %)) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in hungary ( . % prevalence in healthy pigs vs . % prevalence in diarrheic pigs), spain ( . % healthy vs . % diarrheic), brazil ( % vs . %), thailand ( . % vs . %) and vietnam ( . % to . %) , , , . indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between . and . %. furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. because most ( %) of the reads generated during sequencing of the fecal sample v matched bacteriophages upon analysis with blast, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. de novo assembled contigs were analyzed using virsorter, a software package for mining viral signals from microbial genomic data. such tools allow maximizing the possibility of detecting dsdna phages . several contigs showed high similarities to the bacteroides phage b - , found in municipal wastewater and human fecal samples. it was shown to be absent in samples collected from different animal species, including pigs, and is therefore considered a human-specific phage , . the finding of several contigs, genetically similar to phage b and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. however, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. interestingly, several of the contigs found also showed similarities to escherichia phages. two of the contigs were similar to escherichia phages phapec and phapec , isolated from belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic escherichia coli. electron microscopic images of the phages phapec and phapec indicated that they belonged to the family podoviridae . two other contigs were similar to two closely related escherichia phages, st ph and g c, found in sewage and horse feces, respectively . finally, one contig showed limited similarity to an enterococcus phage, isolated from hospital sewage in china, while a last contig showed moderate similaraties to the bacterial enterococcus hirae genome. this region may be a prophage, inserted in the bacterial genome. the phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as enterococcus hirae to proliferate and to start secreting toxins. it is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. the new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case v and it may be that bacteriophages are involved in the pathogenesis of this syndrome. so far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. it is clear that new technologies will change the way diagnostics are be performed in the near future. pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. however, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. viruses. porcine rotavirus a (rva) strain rva/pig-tc/bel/ r / /g p [ ] was isolated from a diarrheic piglet and grown for three successive passages in ma cells to an infectious virus titer of . ccid / ml. the nucleotide sequences of the gene segments of this strain were resolved earlier using sanger sequencing (genbank accession numbers: km (vp ), km (vp ), km (vp ), km (vp ), km (vp ), km (vp ), km (nsp ), km (nsp ), km (nsp ), km (nsp ) and km (nsp )) . a porcine epidemic diarrhea virus strain (pedv, cv ) isolated in belgium in the s was adapted for growth in vero cells in the s . in our laboratory, the virus was grown to an infectious virus titer of . ccid /ml (genbank accession number: af ). origin of a fecal sample from diarrheic suckling piglets. a diarrheic fecal sample was collected from a belgian pig on a farm housing a total of sows and using a -week batch-production system, with a weaning age of days. topigs norsvin sows were crossed with piétrain boars, producing . toxins (suiseng, hipra). rotavirus a vaccination was done off-label with an inactivated bovine rotavirus a vaccine (lactovac, zoetis). until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages ( . - . %) were observed. since the spring of , enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in -days-old suckling piglets. a diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory (dialab, belsele, belgium) and labeled v . no virological cause was found to explain the diarrheic problems on the farm. the only isolated bacterium was enterococcus hirae. this bacterium was thereon added to the sow vaccination schedule (inactivated autovaccine). no other pathogens were found in this sample. as the clinical picture hinted at a viral cause for the disease, the sample was sent to the laboratory of virology at the faculty of veterinary medicine (ghent university) for further analysis. the sample tested negative for rva, rvc, pedv and tgev using in-house rt-qpcr assays , , . therefore, it was decided to perform a metagenomics analysis with minion described in this study. purification of viral nucleic acids. first, viral enrichment was done based on the netovir protocol to obtain pure viral nucleic acids for sequencing library preparation . minion analyses of cell culture grown viruses rva and pedv were conducted at the laboratory of clinical virology (rega institute, ku leuven), whereas the diarrheic fecal sample was analyzed at the laboratory of virology (faculty of veterinary medicine, ghent university). rva and pedv stocks were centrifuged at , × g for min. the supernatant of both suspensions was diluted to log ccid /ml and µl of each suspension was mixed to reach an equal concentration of both viruses. this mixture was filtered using a . µm polyethersulphone filter for min at , × g, followed by a nuclease treatment for hours at °c to digest free nucleic acids in the suspension: µl of the sample was added to µl of home-made buffer ( m tris, mm cacl and mm mgcl , ph ), µl of benzonase nuclease (millipore) and µl micrococcal nuclease (neb) as described earlier . fourteen microliters of edta were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the qiaamp viral rna mini kit (qiagen). the manufacturer's instructions were followed but no carrier rna was added and elution was done in µl of ave to concentrate the viral nucleic acid extract. the diarrheic fecal sample v was processed similarly as the cell culture grown viruses, with some minor modifications. a % w/v suspension of the diarrhea was made in minimum essential medium and centrifuged. the supernatant was filtered through a . µm syringe filter (sarstedt) and treated with benzonase nuclease for hour to speed up the diagnostic pipeline. viral nucleic acids were extracted using the qiaamp cador pathogen mini kit according to the manufacturer's instructions without addition of carrier rna. elution was done in a volume of µl. cdna and second strand synthesis for nanopore sequencing. nucleic acids were heated at °c for min and chilled on ice to resolve secondary rna structures and to denature double-stranded rna. superscript iv reverse transcriptase (thermoscientific) was used to generate cdna. ten microliters of template nucleic acids were mixed with . µl random hexamer primers (random primer , new england biolabs), µl dntp mix (neb) and . µl nuclease-free water. primer annealing was conducted at °c for min, after which µl superscript iv reaction buffer (thermoscientific), µl dithiothreitol (thermoscientific) and µl superscript iv reverse transcriptase (thermoscientific) were added in a total reaction volume of µl. the reaction conditions were as follows: °c for min, °c for min, °c for min and an infinite hold step at °c. a second strand of dna was generated from single stranded (c)dna molecules using the nebnext second strand synthesis kit (neb). twenty microliters cdna reaction mixture were added to µl nebnext second strand synthesis reaction buffer, µl nebnext second strand synthesis enzyme mix and µl nuclease-free water ( µl total reaction volume). isothermal amplification was done at °c for h and double-stranded nucleic acids were purified using µl of magnetic ampure xp beads (beckman coulter). two washing steps with freshly prepared % ethanol were conducted before eluting in µl nuclease-free water. nanopore sequencing library preparation. a deoxyadenosine was ligated to the ′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. fifty microliters of (un) amplified dna were mixed with µl ultra ii end-prep reaction buffer (new england biolabs) and µl ultra ii end-prep enzyme mix (new england biolabs), and incubated at °c for min and °c for min. next, nucleic acids were purified using µl ampure xp beads and eluted in µl nuclease-free water. sequencing adapters, provided with the ligation sequencing kit d (r . ) (sqk-lsk , ont), were ligated to the da-tailed nucleic acids. end-prepped dna ( µl) was mixed with µl adapter mix (amx, ont) and µl blunt/ta ligation master mix (new england biolabs) in a total reaction volume of µl and incubated at room temperature for min. the sequencing library, containing double-stranded dna with adapters ligated to the ′ ends, was then purified using µl ampure xp beads. two washing steps were conducted using µl adapter bead binding buffer (abb, ont) before eluting in µl of elution buffer (elb, ont). (exp-llb , ont), µl adapted and tethered library and . µl nuclease-free water. sequencing was done using the software programme minknow software (ont). bio-informatics analyses. raw reads were produced by minknow. live basecalling was enabled for the first experiment using minknow version . . . in the second experiment, basecalling was done after the sequencing run using albacore (version . . ., ont). quality scores and read lengths were visualized using nanoplot, followed by quality filtering with nanofilt . reads with a q-score lower than were omitted. sequences were then analyzed using different blast methods including blastn and tblastx (blast version . . ; e-value cut-off e − - e − ) to compare sensitivity and run-times to detect viral sequences among the reads. a complete viral database was composed of all virus sequences in genbank (taxonomy id , containing sequences up to th of september ). the best hit (lowest e-value) was visualized using kronatools . reads matching viruses were extracted using seqtk (https://github.com/lh /seqtk) and used in downstream analyses. graphmap (version . . ) and samtools (version . ) were used for mapping of reads against reference sequences, while canu . was used for de novo assembly of viral genomes [ ] [ ] [ ] [ ] . virsorter was run using the 'viromes' database to look for phages, with the virome decontamination mode on to identify phage contigs . bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of ghent university. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. sanger sequencing of porcine kobuvirus polymerase gene. a porcine kobuvirus was discovered in the sample v using the minion. the sequence of the d gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. the exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by sanger sequencing, as low coverage was obtained with minion. rt-pcr was executed using the onestep rt-pcr kit (qiagen) with the newly designed primers kobu_ fw and kobu_ rv (idt dna technologies) ( table ). the rt-pcr reaction contained µl × qiagen onestep rt-pcr buffer, µl dntps, µl of each primer ( µm), µl nuclease-free water, µl onestep rt-pcr enzyme mix and µl template rna or water (total reaction volume of µl). rt-pcr conditions were as follows: °c for min, °c for min, followed by cycles of amplification ( °c for s, °c for s and ° for s) and a final extension step at °c for min. reactions were held at °c prior to loading µl pcr product with µl of loading dye in a . % agarose gel. electrophoresis was conducted for min at v and pcr product was visualized by ethidium bromide staining and uv light. the amplicon was sent to gatc (constance, germany) for sanger sequencing using an abi xl dna analyzer system. quality control of the raw chromatograms was done using peaks (nucleobytes bv, the netherlands) and blastn (ncbi, united states). specific rt-qpcr primers (table ) for the porcine kobuvirus polymerase-encoding gene were designed using primerquest and oligoanalyzer (idt dna technologies) to allow exact quantification in feces of piglets. each rt-qpcr reaction consisted of µl precisionplus onestep qrt-pcr mastermix containing sybr green, rox and an inert blue pipetting dye (primerdesign, southampton, united kingdom), . µl of each primer ( nm) and . µl nuclease-free water. three microliters of template rna or water were added to each tube containing µl mastermix. a synthetic rna positive control ( nt) was generated by rt-pcr using the primers kobu d_qpcr +t _fw and kobu d_qpcr_rv, followed by in vitro transcription of this pcr product using a t rna polymerase. the positive control was measured using nanodrop and used to setup a standard curve over a linear dynamic range (ldr) from six to one log copies/reaction. reaction conditions were as follows: °c for min and °c for min, followed by cycles of denaturation ( °c for s) and annealing ( °c for s). detection of sybr green fluorescence was done at the end of each annealing phase. a melt curve analysis was executed to assess specificity of the amplicons generated. each dilution point in the standard curve and each sample was tested in duplicates. amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. assays were valid if the efficiency over the ldr was between and %, and r of the standard curve replicates was > . . quantification of the viral loads was possible if the cq-values of two qpcr replicates fell within the ldr of the assay. both replicates had to be positive for a sample to be considered as positive. if the cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. upon characterization of the virome with the minion, a longitudinal follow-up study was setup between august and september . to warrant the health status of the pig stock, entrance to the farm was strictly regulated. sampling was performed by the farmer. detailed instructions and sampling materials were provided to the farmer. sample collection in the longitudinal field study was done in agreement with the european legislation on animal experiments. sample collection was approved by and done in accordance to the requirements of the local ethical committee of the faculty of veterinary medicine and bioscience engineering of ghent university. one day after parturition of the sows, five litters were selected at random. within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. a dry cotton rectal swab (copan) was collected from each individual piglet at days , , , , , , and after birth. the swab was placed immediately in ml of viral transport medium (phosphate buffered saline containing u/ml penicillin (continental pharma, puurs, belgium), mg/ml streptomycin (certa, braine l′alleud, belgium), mg/ml gentamicin (life technologies) and . % v/v fungizone (bristol-myers squibb, braine l′alleud, belgium)) in a sterile ml falcon tube (sarstedt) and stored at − °c. every week, samples were collected from the farm and transported to the laboratory of virology. the farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. upon arrival in the laboratory of virology, the samples were thawed and placed on a shaker for min at °c to release viral particles in the transport medium. samples were extracted using the qiaamp cador pathogen mini kit according to the manufacturer's instructions and purified nucleic acids were eluted in µl of ave and stored at − °c until rt-qpcr analysis. rt-qpcr analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. furthermore, rva and rvc shedding was assessed using previously described in-house rt-qpcr assays , . belgian suckling pigs. fecal samples (n = ) of diarrheic suckling piglets less than weeks old were sent to a private laboratory by veterinarians (dialab, belsele, belgium) for etiological diagnosis, as described earlier. these samples were collected in and stored at − °c in the laboratory. they had previously been evaluated for the presence of rotaviruses using rt-qpcr . rna extraction was conducted using the qiaamp cador pathogen mini kit (qiagen) as described above and rt-qpcr was done to quantify the load of kobuvirus rna copies. samples with a quantifiable viral load were subjected to rt-pcr to amplify the d polymerase gene, after which sanger sequencing was performed. the sequences encoding the polymerase of 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funded by the belgian federal public service health, food chain safety and environment (rf / ) and an agricultural trajectory of the flemish agency innovation and entrepreneurship (vlaio, la ). bv ( s n) and lb ( s n) were supported by a phd scholarship of the research foundation flanders (fwo vlaanderen). pieter ganseman is acknowledged for his excellent technical assistance. philip vyt (dialab, belsele) is acknowledged for providing the diarrheic fecal sample of the suckling piglet. we appreciate the collaboration with the farmer from the case farm and are grateful for providing us fecal samples from his pigs. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -certy v authors: ruscitti, piero; di benedetto, paola; berardicurti, onorina; panzera, noemi; grazia, nicolò; lizzi, anna rita; cipriani, paola; shoenfeld, yehuda; giacomelli, roberto title: pro-inflammatory properties of h-ferritin on human macrophages, ex vivo and in vitro observations date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: certy v ferritin is an iron-binding molecule, which comprises subunits, heavy (feh) and light (fel) subunits, suggested to have a pathogenic role by the ‘hyperferritinemic syndrome’. in this work, we tested ( ) feh and fel in bone marrow (bm) and sera in patients with macrophage activation syndrome (mas); ( ) pro-inflammatory effects of ferritin, fel, and feh on macrophages; ( ) ability of feh-stimulated macrophages to stimulate the proliferation of peripheral blood mononuclear cells (pbmcs); ( ) production of mature il- β and il- p in extracellular compartments of feh-stimulated macrophages. immunofluorescence analysis and liquid chromatography mass spectrometry (lc–ms/ms) based proteomics were performed to identify fel and feh in bm and sera, respectively, in the same patients. macrophages were stimulated with ferritin, feh, and fel to assess pro-inflammatory effects by rt-pcr and western blot. the proliferation of co-cultured pbmcs with feh-stimulated macrophages was tested. immunofluorescence showed an increased feh expression in bms, whereas lc–ms/ms identified that fel was mainly represented in sera. feh induced a significant increase of gene expressions of il- β, il- , il- , and tnf-α, more marked with feh, which also stimulated nlrp . feh-stimulated macrophages enhanced the proliferation of pbmcs. the elisa assays showed that mature form of il- β and il- p were increased, in extracellular compartments of feh-stimulated macrophages. our results showed feh in bm biopsies of mas patients, whereas, lc–ms/ms identified fel in the sera. feh showed pro-inflammatory effects on macrophages, stimulated nlrp , and increased pbmcs proliferation. liquid chromatography mass spectrometry identification of fel in sera of patients with aosd and mas. in the same patients, who underwent bm biopsies, liquid chromatography mass spectrometry (lc-ms/ms) analysis has successfully identified both feh and fel along with many other proteins from the assessed bands. the samples were analysed using all taxonomy in uniprot and also an in-house curated database containing the feh protein sequence only. following analysis against all taxonomy at % ci probability, a mixed population of proteins was identified from each assessed band, a filtering of the data to highlight the macrophages genes expression after stimulation with ferritin, feh, fel. after stimulation of macrophages with ferritin, for and min, respectively, the mrna expression of feh, fel, il- , il- , tgf-β, vegf and nlrp remained unchanged, when compared with ut cells. the ferritin significantly increased the mrna levels of il- β, il- and tnf-α genes, when compared with ut cells [il- β mrna levels in ferritin treated cells for min . ( . - . ) vs il- β mrna levels in ut treated cells . ( . e − to . ), p = . ; il- mrna levels in ferritin treated cells for min . ( . - . ) vs il- mrna levels in ut treated cells . ( . - . ), p = . ; tnf-α mrna levels in ferritin treated cells for min . ( . e − to . ) vs tnf-α mrna levels in ut treated cells . ( . e − to . , p = . ] (fig. ) . furthermore, as reported in fig. , after stimulation with feh, for and min, respectively, the mrna expression of tgf-β and vegf remained unchanged, when compared to ut cells. concerning feh and fel genes, the levels of mrna expression significantly decreased after min of feh stimulation, when compared with ut cells, but after min the levels of mrna expressions were returned comparable to those of ut cells [feh mrna levels, in feh treated cells for min . ( . - . ) vs feh mrna levels, in ut treated cells . ( . - . ), p = . ; fel mrna levels, in feh treated cells for min . ( . - . ) vs fel mrna levels, in ut treated cells . ( . - . ), p = . ]. the feh stimulation, for both and min, induced a significant increase of mrna levels of expressions of il- β, il- , il- , il- and tnf-α, when compared with ut cells [il- β mrna levels in feh treated cells for min . ( . - . ); il- β mrna levels in after stimulation of macrophages with ferritin, for and min, the proteins expression of il- β, nlrp and il- remained unchanged when compared to ut cells (fig. ) . interestingly, when macrophages were stimulated with feh for min, the levels of proteins expression of il- β, nlrp and il- were significantly increased when compared with ut cells (p = . ) (fig. ). finally, we tested macrophages proteins expression of il- and tnf-α, after stimulation with ferritin and feh, for and min, but non-significant results were obtained (data not shown). showed that the levels of mature form of il- β, following stimulation with feh for both min and min, were significantly increased when compared with ut cells [mature il- β levels in feh treated cells for min . pg/ml ( . - . ) vs mature il- β levels in ut cells . pg/ml ( . - . ); p = . . mature il- β levels in feh treated cells for min . pg/ml ( . - . ) vs mature il- β levels in ut cells . pg/ml ( . - . ); p = . ] (fig. a) . similarly, il- p levels, following stimulation with feh for both min in this work, the results showed the presence of feh in bm biopsies of aosd patients complicated with mas patients, whereas fel was the predominant form in the sera of those. furthermore, pro-inflammatory effects of feh on human macrophages were observed in vitro, increasing pro-inflammatory cytokines and nlrp . finally, www.nature.com/scientificreports/ feh-treated macrophages enhanced the proliferation of co-cultured pbmcs. taking together all these results and considering that aosd and mas could be included in the so-called "hyperferritinaemic syndrome" , our data could reinforce the hypothesis that higher levels of ferritin may not only be considered a consequence or an epiphenomenon of the inflammation, but it may actively play a role in pathogenic mechanisms of those diseases, thus enhancing the inflammatory burden. our results showed that feh was more represented than fel in bm biopsies of patients with aosd and mas, as previously reported in affected tissues , , , . conversely, in sera of these patients, fel was the predominant form, as reported by lc-ms/ms. based on that discrepancy, we tested the inflammatory properties of ferritin, feh, and fel on human macrophages, and we observed that ferritin and, as particularly, feh induced the expression of pro-inflammatory cytokines. specifically, an increased gene expression of il- β, il- , il- , and tnf-α was observed. in this context, pro-inflammatory cytokines are largely overexpressed in patients with aosd complicated with mas [ ] [ ] [ ] and may induce preferentially the expression of feh, via fer . the latter, after activation, stimulates the synthesis of feh and the production of many pro-inflammatory cytokines, perpetuating a vicious pathogenic inflammatory circle [ ] [ ] [ ] [ ] [ ] . in addition, the assessment of protein expressions showed the stimulation of macrophages with feh induced a significant increase of il- β, and il- , when compared to ut cells, whereas ferritin and feh did not. these discrepancies among ferritin, feh, and fel could be related to a different effect of these molecules on macrophages, as observed for gene expression. furthermore, the production of mature form of il- β, in the extracellular compartment, was induced by feh stimulation on macrophages. this finding could suggest a specific pathogenic link between feh and il- β, which is a crucial mediator in aosd and mas, also because of clinical usefulness of il- inhibition in those patients , . in fact, multiple lines of evidence suggested the efficacy of il- inhibition in the context of the hyperferritinaemic syndrome [ ] [ ] [ ] . furthermore, the lack of confirmation of protein expression of both il- and tnf-α could reinforce this hypothesis. moreover, the efficacy of il- and tnf-α inhibition reported conflicting results , . additionally, paralleling with il- β, feh induced a significant expression of nlrp , a cytosolic innate immune signalling receptor, which is the main factor associated with the maturation and production of this cytokine . interestingly, our data could suggest a vicious cycle by feh, as a further stimulator of nlrp , since it could be an additional danger signal in triggering this factor. the activation of nlrp begins with the recognition of the danger or stressor, pathogen/damage-associated molecular patterns (pamps/damps), by the sensor pattern recognition receptors (prrs) . once activated, nlrp nucleates the assembly of an inflammasome, by interacting with an adaptor apoptosis speck-like protein (asc), recruits and activates procaspase- to generate active caspase- and then converts the cytokine precursors pro-il- β into mature and biologically active il- β , . after that, a series www.nature.com/scientificreports/ of inflammatory mechanisms and pyroptotic cell death are triggered . taking together all these observations and considering its involvement in aosd , , the direct stimulation of nlrp by feh could provide further insights to the pathogenesis of these diseases, linking the typical hyperferritinemia with the production of a crucial pathogenic mediator. additionally, feh induced a significant expression of intracellular il- as well as promoted its release in the extracellular compartment. it has been reported that il- is a pro-inflammatory cytokine produced by dendritic cells, macrophages and b cells in response to microbial pathogens . on this basis, we could speculate that il- , increased in our experimental conditions, could play a pro-inflammatory role. interestingly, it has been shown that after over-expression of il- , the phenotype of m macrophages could be re-directed to that of m -like macrophages . although it is presently known that functional polarization of macrophages is an over-simplified description of macrophage heterogeneity and plasticity, two classical different phenotypes of macrophages were described, considered the end-stage phenotypes of a continuum of functional states, classically activated (or inflammatory) macrophages (m ) and the other alternatively activated (or wound-healing) macrophages (m ) , . in this context, a differential cytokine production is a key feature of polarized macrophages . the m phenotype is typically il- high and il- low , whereas m macrophages are typically il- high and il- low . furthermore, the stimulation of macrophages with feh, enhanced the proliferation of co-cultured pbmcs. taking together these results and previous observations [ ] [ ] [ ] [ ] , it could be possible to hypothesize that the stimulation with feh could orientate the macrophages toward an m phenotype, suggesting the need of further studies to entirely clarify these issues. in addition, in inflammatory infiltrate of aosd and mas, a specific subset of macrophages was reported, displaying a specific cd /h-ferritin phenotype expressing il- , which cannot be observed in normal tissues , . finally, considering that the protein expressions did not confirmed the gene expressions observed following stimulation with ferritin on pro-inflammatory mediators, it could be possible to attribute the pro-inflammatory effects to feh subunits of the ferritin. in this study, the effects fel on pro-inflammatory cytokines were also tested, but non-significant results were obtained. in fact, it has been recently reported that a compensatory increase of fel, after deletion of feh, could reduce the cytokine levels, the multi-organ dysfunction and the mortality in a murine model of sepsis . in fact, an inhibitory action of fel was shown on nf-kb activation, a key signalling pathway which is implicated in the pathogenesis of sepsis but also of other inflammatory diseases . on the contrary, a stimulatory effect of feh on nf-kb was described acting as a pro-inflammatory cytokine on hepatic stellate cells . additionally, it has been reported that feh could modulate macrophage response to immune stimuli . taking together all these findings, it is possible to suggest that the pro-inflammatory effects of ferritin could be mainly attributed to feh than fel, suggesting also a possible therapeutic target to be investigated in future specific designed studies. taking together all these data, a contributory role of ferritin as a pathogenic mediator rather than being a product of inflammation could be suggested, but, additionally, ferritin is proposed to be a biomarker for the disease in early diagnosing and in monitoring the clinical response to therapies . in fact, hyperferritinemia, may identify a more aggressive subset of diseases, and, as observed in caps and mas, its reduction, after treatment, is associated with a lower mortality [ ] [ ] [ ] . in addition, recent evidence from coronavirus disease (covid- ), identified hyperferritinemia and il- as predictors of poor prognosis, suggesting that the mortality of these patients is related to a hyper-inflammatory process [ ] [ ] [ ] . this finding could thus hypothesize the inclusion of covid- , at least for a more severe subset of patients, in the "hyperferritinaemic syndrome", since sharing pathogenic mechanisms, clinical features, and possibly therapeutic targets. in spite of suggesting possible pro-inflammatory properties of ferritin and particularly of feh, our work is affected by some limitations, such as the relative low number of assessed patients, which could limit the external validity. however, it must be pointed out that aosd and mas are very rare diseases and it is very challenging to get matched bm biopsies and peripheral blood samples. an additional challenge is the severity of the diseases rapidly evolving into a life-threatening clinical picture, complicating even more the collection of biologic samples from affected patients. taking together these observations, further confirmatory and mechanicistic studies are needed to fully elucidate these issues. in conclusion, pro-inflammatory effects of feh on human macrophages could be suggested, since it increased the expression of the pro-inflammatory cytokines and nlrp , and enhanced the proliferation of co-cultured pbmcs. considering these results and previous observations [ ] [ ] [ ] , it could be possible to hypothesize that the stimulation with feh could orientate the macrophages toward an m phenotype, suggesting the need of further studies to entirely clarify this issue. taking together all these results and considering that aosd and mas could be included in the so-called "hyperferritinaemic syndrome", our data could reinforce the hypothesis that higher levels of ferritin may not only be considered a consequence of the inflammation, but it may actively play a role in the pathogenic mechanisms of those diseases enhancing the inflammatory burden. in addition, given that these pro-inflammatory effects could be mainly attributed to feh, it could be also possible to speculate a possible new therapeutic target to be tested to improve the management of these patients. patients. four patients with aosd complicated with mas were assessed at the time of diagnosis, collecting bm biopsies and sera, which were analysed at the same time. all these patients were admitted to the rheumatology clinic of l' aquila university, italy, and fulfilled the diagnostic criteria proposed by for aosd and mas in rheumatic diseases [ ] [ ] [ ] . in this study, we also evaluated bm biopsies derived from bm-donors, used as hcs. the local ethics committee approved the study (asl avezzano-sulmona-l'aquila, l' aquila, italy, protocol number / ) that was performed according to good clinical practice guidelines and declaration of helsinki. each patient provided informed consent for purposes of the study. scientific reports | ( ) : | https://doi.org/ . /s - - -w www.nature.com/scientificreports/ histological analysis of biopsies. four bm biopsies were evaluated, which derived from patients with aosd complicated by mas. the immunofluorescence analysis was performed on paraffin sections (thickness µm) and antigen retrieval was carried out using target retrieval solution (dako, usa). samples were stained with anti-feh, anti-fel antibodies (santa cruz biotechnology, usa), as reported previously , . the immunoreaction was revealed by using a secondary antibody (alexa fluor, sigma-aldrich, usa). cell nuclei were visualized using ′, -diamidino- -phenylindole. the fluorescence was assessed by using an olympus bx fluorescence microscope. enzymatic digestion. four sera of patients with aosd were mixed with mm sodium acetate (sigma aldrich, germany), ph . , heated at °c for min and centrifugated at g for min at °c. maintaining the solution at ph . ( °c), ammonium sulfate (sigma aldrich, germany) ( % of saturation) was added and the final solution was centrifuged at g for min at °c. the final pellet was re-suspended in pbs (ph . ). proteins contained in the final solution and recombinant feh (abcam, uk) were separated by % sds-page gel. the band with a molecular weight - kda, with the same weight of recombinant feh were collected. in-gel reduction, alkylation and digestion with trypsin were performed on the four gel bands before to a subsequent analysis by a mass spectrometry, as previously reported . lc-ms/ms. peptides were extracted from the gel pieces, the peptides were resolved by reversed phase chromatography and the evaluate was ionised by electrospray ionisation using an orbitrap velos pro (thermo fisher scientific, uk) operating under xcalibur v . , as previously reported . the report of lc-ms/ms analysis is reported in additional material . database searching. raw mass spectrometry data were processed into peak list files using proteome dis- monocytes isolation and differentiation. peripheral blood monocytes were obtained from healthy donors by direct isolation using whole blood collected in mm ethylenediaminetetracetic acid (edta) (sigma-aldrich, usa) and mixed with µl/ml rosettesep human monocytes enrichment cocktail (stemcell, usa), according to the manufacturer's protocol. the derived enriched human monocytes were plated × /cm in roswell park memorial institute (rpmi) , medium (euroclone, europe), supplemented with % foetal bovine serum (fbs; gibco, usa), mmol/l l-glutamine (euroclone, europe) and u penicillin, , u streptomycin (biochrom ag, germany). additionally, these cells were cultured for days with ng/ml macrophage colony-stimulating factor (m-csf) (promokine, germany), changing the medium every days. cells were incubated at °c in a humidified atmosphere consisting of % co . purity of cells was assessed by immunofluorescence staining. briefly, the cells were fixed with % paraformaldehyde (ems, pa), incubated min with protein block (dako, usa) and successively with anti-cd -antibody (invitrogen, usa) and anti-cd antibody (santa cruz biotechnology, usa). the visualization of the anti-cd -antibody was performed using an alexa fluor -conjugated (invitrogen, usa) and the visualization of the anti-cd -antibody was performed using an alexa fluor -conjugated (invitrogen, usa). after counterstained with ′, -diamidino- -phenylindole (dapi), images were obtained using an olympus bx fluorescence microscope. the number of cd + and cd + cells was counted to assess their purity. at day , before m-csf stimulation cd + cells were . % and cd + cells . %, out of the total percentage of cells, respectively. after days of stimulation with m-csf, cd + cells were . % and cd + cells . %, out of the total percentage of cells, respectively (additional material ). to establish the optimal concentration of ferritin, feh and fel, in our system, a dose/response curve was performed, evaluating the 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with mild systolic and diastolic contractile dysfunction, increased phospholamban thr phosphorylation, and exacerbated ischaemia-reperfusion injury blocking cd molecules in perivascular stromal cells of patients with systemic sclerosis strongly inhibits their differentiation toward myofibroblasts and proliferation: a new potential target for antifibrotic therapy mesenchymal stem cells (mscs) from scleroderma patients (ssc) preserve their immunomodulatory properties although senescent and normally induce t regulatory cells (tregs) with a functional phenotype: implications for cellular-based therapy piperine inhibits cytokine production by human peripheral blood mononuclear cells the authors thank mrs. federica sensini for her technical assistance. all authors made substantial contributions to the conception or design of the work, the acquisition and interpretation of data. all authors contributed to the critical review and revision of the manuscript and approved the final version. all the authors agreed to be accountable for all aspects of the work. p.r.: study conception and design, data interpretation, literature search, figures creation, writing, paper revision and acceptance; p.d.b.: study conception and design, data interpretation, literature search, figures creation, writing, paper revision and acceptance; o.b.: data collection, data interpretation, literature search, paper revision and acceptance; n.p.: performed experiments, data interpretation, paper revision and acceptance; n.g.: performed experiments, data interpretation, paper revision and acceptance; a.r.l.: performed experiments, data interpretation, paper revision and acceptance; p.c.: data collection, data interpretation, literature search, paper revision and acceptance; y.s.: data interpretation, data interpretation, literature search, writing, paper revision and acceptance; r.g.: data interpretation, data interpretation, literature search, writing, paper revision and acceptance. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - -w.correspondence and requests for materials should be addressed to p.r.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - jsnu authors: zhang, zhao; shen, libing; gu, xun title: evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: jsnu middle east respiratory syndrome coronavirus (mers-cov) belongs to beta group of coronavirus and was first discovered in . mers-cov can infect multiple host species and cause severe diseases in human. we conducted a series of phylogenetic and bioinformatic analyses to study the evolution dynamics of mers-cov among different host species with genomic data. our analyses show: ) potential recombinant sequences were detected and they can be classified into seven potential recombinant types; ) the spike (s) protein of mers-cov was under strong positive selection when mers-cov transmitted from their natural host to human; ) six out of nine positive selection sites detected in spike (s) protein are located in its receptor-binding domain which is in direct contact with host cells; ) mers-cov frequently transmitted back and forth between human and camel after it had acquired the human-camel infection capability. together, these results suggest that potential recombination events might have happened frequently during mers-cov’s evolutionary history and the positive selection sites in mers-cov’s s protein might enable it to infect human. acid substitutions in its s protein receptor binding domain along with its switching host from civet to human . moreover, two amino acid substitutions in the s protein's c-terminal of hku , a bat beta-coronavirus, enable its entry to human cells and the same amino acid substitutions are also found in mars-cov . furthermore, heptad repeat regions in c-terminal of mers-cov and related coronaviruses also play important roles in viral adaptive evolution . in summary, those studies introduced above suggested that s protein plays a vital role in mers-cov's cross-species transmissibility. however, the evolutionary mechanism of how mers-cov's s and other proteins facilitated the cross-species transmission of mers-cov remains to be investigated. here, we performed a series of phylogenetic and bioinformatic analyses for mers-covs. we systematically investigated the recombination events in mers-covs, the potential transmission route of mers-covs in five different host species and the evolutionary pressure of each mers-cov's protein during cross-species transmission. our study might offer some insight in explaining the possible mechanism in mers-cov's adaptive evolution. epidemic description and phylogenetic analysis of mers-cov. by far, the largest mers-cov outbreak is in saudi arabia and almost all human cases have a direct or indirect link to arabian peninsula. in this study, we collected human mers-cov whole genome sequences from countries (supplementary table ). the geographic distribution of these samples is shown in supplementary fig. a . the majority of them are from the countries in arabian peninsula ( . %, / ) and more than half of them are from saudi arabia ( . %, / ) (supplementary fig. a) . the peak season for mers is between april ( . %) and may ( . %) (supplementary fig. b) . based on the whole-genome alignment of our collected sequences (supplementary table ), we performed the phylogenetic analysis for these sequences with two sras-covs serving as the outgroup. our phylogenetic tree shows that all camel and human mers-covs are clustered together. the bat and hedgehog mers-covs formed a basal paraphyletic group to all camel and human mers-cov clade (fig. a) . a single camel mers-cov isolated in egypt (gi: ) forms a single basal clade to human and the other camel mers-covs (fig. b) . the human-camel mers-cov cluster can be further divided into two clades-clade a and clade b, as previously reported . clade a contains four human strains isolated in jordan and saudi arabia while human and camel mers-covs are mixed in clade b. there are five groups in clade b and we named them as group i to group v as the previous study . there are , , , and mers-cov sequences in group i to group v, respectively (fig. b) . we performed the recombination analysis on the collected full-length mers-cov sequences. we find that there are of them experienced potential recombination events ( . %, / ), including three camel mers-covs and human mers-covs (supplementary table ). we divided potential recombinant sequences into seven different types and named them as type to type ( fig. b- table ). type means the recombination happened between group ii and group v, which includes sequences and is about % of total recombinant sequences. type means the recombination happened between group iii and group v, which includes sequences ( %). interestingly, the mers-covs newly found in in south korea and china are type recombinants , . type means the recombination happened between group i and group iii, which includes sequences ( %). type , and are the recombination happened between different genomic regions of group iv and group v, which include , and sequences ( %, % and %), respectively. type is the recombination happened among three groups (group i, iv and v), which includes sequences ( %). our phylogenetic analysis showed type belongs to phylogenetic group ii while type and belong to phylogenetic group iii, and type to belong to phylogenetic group v. there is no recombination found in phylogenetic group i and group iv (fig. b) . we also reconstructed the phylogenetic tree using non-recombinant sequences only and found that its topology is consistent with the tree based on all sequences (supplementary fig. ) . we also performed the snp (single-nucleotide polymorphisms) analyses for each recombinant types and found the large recombination segments in type , , , are conspicuous but in type , , are obscure (supplementary fig. ). in order to explore the selection pressure on the mers-cov proteins when it transmitted from animal host to human, we performed the adaptive evolution analyses for all mers-cov protein in absence of recombinant strains. firstly, we set camel and human mers-covs as the foreground branch and bat and hedgehog mers-covs as the background branch to preform branch-site test in codeml of paml program (see fig. a ). the strong positive selection is detected in spike (s) glycoprotein between these two branches (p < . ), while there is no significant positive selection in the other mers-cov genes ( table ) . we find nine positive selection sites in mers-cov spike (s) glycoprotein and eight of them are statistically significant (table ) . six significant positive selection sites are located in the receptor binding domain of s protein (fig. a) . we utilized a published crystal structure (pdb id l in rcsb protein data bank), the receptor binding domain (rbd, aa - , fig. b ) of mers-cov spike glycoprotein complexed with the human receptor dipeptidyl peptidase (ddp ), to demonstrate their locations in a d environment (fig. b) . the receptor binding domain of mers-cov s protein can be further divided into a receptor-binding sub-domain and a core sub-domain. two significant positive selection sites, k r and g n, are in the receptor-binding sub-domain and k r is in direct contact with human receptor ddp . q s, g n, d s and r l are in the core sub-domain. moreover, we also detected a positive selection site in s protein's c-terminal, l s. secondly, we screened the positive selection sites among human-camel mers-covs (table ). five significant (table ). the genome-wide average nucleotide substitution rate of camel and human mers-covs was . × − substitutions per site per year. open reading frame (orf ) has the fastest substitution rate while orf has the slowest substitution rate. the orf b, nucleocapsid (n) glycoprotein, and spike (s) glycoprotein, have a similar substitution rate which is faster than the whole-genome substitution rate. in order to study the temporal and spatial pattern of mers-cov transmission, a maximum clade credibility (mcc) tree was constructed using mers-cov whole genome sequences without recombinant strains (fig. c) . the ancestral host state with time reference was estimated for each tree node and marked with different colors. we named six important nodes in mers-cov divergence on mcc tree for node a to f (fig. c ). the possible transmission time for each node and its % highest posterior density (hpd) are shown in fig. b . we found that the origin time of human-camel mers-cov is relatively late (node d). furthermore, the tmrca for clade b is in ~ (fig. b , node f) and clade a and clade b are divergent in ~ (fig. b , node e). interestingly, the mcc tree shows that there are six cross-species transmission events with high posterior probabilities in clade b. five of them are human-to-camel transmission events and one of them are camel-to-human transmission events (fig. c ). additionally, with the mers-cov of human/camel and bat/hedgehog mers-cov together, we inferred the ancient mers-cov exists for decades of years (fig. b,c) . the tmrca of the mers-covs for vespertilio superans, neoromicia capensis or erinaceus europaeus can be traced back to (node c), (node b) and (node a), respectively. before the emergence of human-camel mers-cov, the estimated tmrca for all mers-covs appeared in ~ (fig. c , node a). we also preformed root-to-tip analysis using the consistent dataset (fig. a) , and the result shows that the origin time of tmrca is in ~ with high statistical supports (r = . , p value < . ). together these results suggest that the ancient mers-cov should have existed for decades in animal host and got the ability to infect human or camel recently. mers-cov belongs to coronavirus, beta-coronavirus, lineage c. since it was discovered in , mers-cov has attracted extensive attention due to its human-to-human infection capability and high mortality rate. recombination events have been confirmed in human mers-cov . the fact that mers-cov can be found in multiple species proposes its cross-species transmissibility [ ] [ ] [ ] [ ] . by far, the evolutionary details of how mers-cov transmitted to human are still unknown. based on the most comprehensive collection of mers-cov genome sequences so far, we tried to elucidate the evolution and transmission of mers-cov among different species. mers-cov has been reported in five species including european hedgehog, two species of bats, dromedary camel, and human. we used the ml method to reconstruct the whole-genome phylogenetic tree of mers-covs isolated from these species. the ml tree shows that the hedgehog mers-covs are basal to all the other mers-covs and two bat mers-covs are basal to camel and human mers-covs. this result suggests that the ancestor of camel and human mers-covs may be from other animal host, such as the hedgehog or bat. we also reconstructed the phylogenetic tree of mers-cov using nj method or based on each mers-cov protein. these trees show a consistent topology, which proposes that the phylogenetic relationship estimated in our study is credible (supplementary fig. ) . we divided clade b into five groups as pervious study to detect the recombination of mers-cov . because the evolutionary distances among mers-covs are close (table ) , no large segment recombination could be detected among them. thus, according to discontinuous recombination segments, we defined potential recombination events in mers-covs. this method has been used in the previous study to label potential recombination . in our study, we found strains form seven recombinant types, which took more than % of all isolated mers-covs in human and camel. among them, we found strains in six recombinant types (type to type ) between two phylogenetic groups and two strains in one type (type ) among three phylogenetic groups. for now, the recombination of mers-cov was confirmed in previous study, but no report about the recombination among more than two groups of mers-cov. interestingly, most recombinant types (type , , , , and ) are related to group v and they make up . % of total recombinant strains ( / ). the result suggests that recombination events might happen frequently and the recombinant types involving group v might happen broadly. additionally, multiple recombination events indicate that double infection and super infection likely existed during the transmission history of mers-cov. we failed to detect possible large recombination segments in type table . evolutionary distance, nucleotide substitution rate and coefficient of variation of substitution rate for human and camel mers-cov proteins. , and . by comparing the snps (single-nucleotide polymorphisms) of reference sequences with recombinant sequences, we reckoned that specific nucleotide mutation might influence the results of recombination analysis. this problem can be solved by discovering more mers-cov sequences or developing more detailed genotype classification for mers-covs in the future. we also performed phylogenetic analyses for potential recombinant region and got similar results (supplementary fig. ) . interestingly, the east asian mers-cov strains (china and south korea) belong to type recombinant and the previous study show that their tmrca might be a result of potential recombination event , which indicates the recombinant strains have transmitted broadly. moreover, one recombinant mers-cov lineage has led to the large-scale outbreak in both camel and human . it proposes that recombinant mers-covs have experienced cross-species infection. additionally, our study reveals that the number of recombinant strains is large and the potential recombinant types are abundant. together these findings highlight that we should take more attention to recombinant mers-cov transmission. although how the mers-cov transmitted from its natural host to human is still unknown, it is confirmed the mers-cov have been found in many animal hosts, such as bats and hedgehog. to study the evolutionary pressure on each mers-cov's protein during its potential cross-species transmission, we conducted a comprehensive scan for positive selection sites in mers-cov's proteins. recombinant strains were excluded in this analysis. we set camel-human mers-covs as the foreground branch and hedgehog-bat mers-cov as the background branch and estimated the relative evolutionary pressure on the foreground branch compared to the background branch. we only found that mers-cov's s protein underwent strong positive selection and there are nine significant positive selection sites in s protein. it suggests that s protein was under strong evolutionary pressure during the transmission from its natural host to human. among significant positive selection sites, six of them are located in s protein's receptor binding domain (rbd). rbd is crucial for virus to enter host cells and it comprises of one binding region and one core region. based on the rbd's d model, we find that two sites are located in the binding region of rbd, which suggests that these amino acid substitutions might change mers-cov's binding capability to host cells and thus facilitate its cross-species transmission. the other four sites are located in the core region of rbd. these amino acid substitutions might change the structure of core region and indirectly influence mers-cov's cross-species capability. in order to eliminate the sample bias between human/camel group and bat/hedgehog group, we also did random sampling for the non-recombinant human and camel sequences. we tried , and random sampling, respectively. using random sampled sequences together with bat and hedgehog mers-cov sequences, we performed branch-site analyses and got the same results as our aforementioned (data not show). we also estimated the nucleotide substitution rates of camel-human mers-covs to investigate its evolutionary dynamics after it infected camel and human. our estimated nucleotide substitution rate for mers-cov's whole genome is . × − , which is slower than the previous estimation . one explanation for the phenomenon is that we used a larger dataset than the previous study, which includes all available mers-cov whole genome sequences. our estimated confidence interval of the substitution rate of mers-cov genome is largely overlapped with the result from another study . the estimated nucleotide substitution rates show that four proteins experienced the accelerated evolution. through evolutionary pressure analysis, we found camel-human mers-cov's four proteins underwent positive selection and detected five significant positive selection sites. one of them is located in m proteins. there is evidence that m proteins are powerful interferon antagonist , which proposes that the evolutionary pressure on m proteins are from host's immune system. two out of five significant sites are found in n protein which is fundamental for mers-cov self-assembly. coronavirus n protein is able to bind to different host cell proteins and demonstrated to have various functions, one of which is also counteracting host interferon as shown in sars-cov [ ] [ ] [ ] [ ] . it is reasonable to speculate that mers-cov n protein under intensive selection because its functions were similar to those of sars-cov n protein. the results above suggest that the arm race between mers-cov proteins and host's immune system might be the main evolutionary driving force behind mers-cov's adaptive evolution after it began to infect camel and human. mers-cov spike (s) glycoprotein evolves slightly faster than the genome-wide average rate, which indicates that the nucleotide substitution rate of mers-cov s protein still maintains a fast speed even after it crossed the species boundary. the positive selection site we found in mers-cov s protein with site-specific test is identical to the previous study's result . this site is located in heptad repeats which is a key component in membrane fusion architecture and required for mers-cov entering host cells . in absence of recombinant strains, we performed the mcc analysis using mers-cov whole-genome sequences in order to infer the time and source species when mers-cov crossed the species boundary. the topology of the mcc tree is highly congruent with that of the whole-genome phylogenetic tree. we defined six nodes (a-f) to explain transmission. the posterior probability for the ancestral sate of node a, b or c is not very high in our mcc analysis and the % highest posterior density (hpd) of the divergence time for these three nodes is quite long. so these results are weak for demonstrating the exact origin time or ancestor state of mers-cov. however, these estimations still provided the evidence that the ancestor mers-cov should have been infected a number of animal hosts, such as bat or hedgehog, for decades of years (supplementary fig. ) . the x-intercept (tmrca) in root-to-tip is ~ with high statistical supports, which is close to the estimated time of tmrca in mcc analysis. this hypothesis is in agreement with the result of serological studies , . the appearance of the common ancestor of human-camel mers-cov is in and the appearance of the tmrca of clade a and b is in , which are exactly the same as the previous report . in clade b, we detected five possible human-to-camel transmission events and one camel-to-human transmission event. it suggests that mers-cov frequently transmitted back and forth between human and camel after it acquired the capability of infecting both hosts. actually, there is at least one confirmed case of camel-to-human mers-cov transmission . scientific reports | : | doi: . /srep in conclusion, we found that potential recombination events are common in mers-cov's evolutionary history and potential recombinant mers-covs can be divided into seven types. the amino acid sites under positive selection in mers-cov s protein, especially those in its receptor binding domain, might have facilitated its cross-species transmission from animal host to human. we detected the strong positive selection in four proteins of camel-human mers-covs, which indicates that they probably experienced strong adaptive evolutionary pressure from host's immune system. additionally, we also found six possible cross-species transmission cases between human and camel. our study investigated the evolutionary dynamics of mers-cov, which shall provide a basis for mers-cov control and treatment. sequence data. the complete genomic nucleotide sequences of mers-covs and two sars-covs were downloaded from ncbi nucleotide database. among mers-cov genomic sequences, of them are from human, of them are from dromedary came, two of them from two bat species neoromicia capensis and vespertilio superans, and of them are from european hedgehog erinaceus europaeus. two sars-cov genomic sequences are from human and bat rhinolophus ferrumequinum, respectively. we used sequences as reference to extract open reading frames from each mers-cov genome in this study. genomic sequence alignment and phylogenetic analysis. total collected genomic sequences were aligned using the muscle software with default parameters . clustalw and mafft used to validate the muscle result , . alignments were refined manually in bioedit (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). only unambiguously aligned positions were used for subsequent phylogenetic analyses (supplementary files). we used the jmodeltest . to estimate the best nucleotide substitution model for our alignment , which is gtr+ i+ g. we used the phyml . to perform the phylogenetic analysis for collected genomic sequences based on their genomic sequence alignment . the branch support values were calculated with shimodaira-hasegawa test integrated in phyml. in clade b, we estimated the consensus sequences for every phylogenetic group using cons tool in emboss explorer (http://bioinfo.nhri.org.tw/cgi-bin/emboss/cons). five consensus sequences were set as reference and every sequence in clade b was used as the query to detect the possible recombination using simplot software . the window is set to bp and the step is set to bp. positive selection analysis. we extracted the coding region of each mers-cov protein using mers-cov strain as a reference template. the codeml program implemented in paml . package was used to detect the positive selection in the codon alignment of each mers-cov protein set . in the branch-site model, the group of human-camel mers-covs was set to be foreground, the group of bat-hedgehog mers-covs was set to be background, and model a with estimated ω value was compared with the null model (model a') with fixed ω value. to reduce the bias from sample size, we performed random sampling on human and camel mers-covs (clade a and b) which are all non-recombinant sequences. we used , and as the random sample size with and without replacement. the random sampled sequences together with bat and hedgehog mers-covs were used to generate the datasets for branch-site model analysis as aforementioned method (script see supplementary files). moreover, for each random sample size, we repeatedly drew five times in order to make results robust. we also used the site-specific model to detect positive selection in the human-camel clade. the site-specific model was performed by comparing the models m a (positive selection) and m (beta & ω ) vs. the null models the crystal structure of the receptor binding domain (rbd) of mers-cov spike (s) glycoprotein in complex with the human receptor dipeptidyl peptidase (ddp ) was displayed using jmol (jmol: an open-source java viewer for chemical structures in d we estimated the transmission of mers-cov among different hosts or geographic areas. the sampling time and host/geographic location of each sequence were also used in analysis. the nucleotide substitution rates and the origin time of most recent common ancestor (mrca) on various nodes of mcc tree were also estimated using the beast package. a relaxed molecular clock with an uncorrelated log-normal distribution, and a constant population size model were used in bayesian coalescence analysis. according to the outcome of jmodeltest . , the gtr+ gamma+ i model of nucleotide substitution was employed in mcc analysis. statistical uncertainty in parameter estimations was reflected by the % highest posterior density (hpd) values. mcmc analysis was run for / million generations for hosts/geographic transmission with sampling every , / , generations to achieve parameter convergence and adequate effective sample sizes (ess > ). we summarized the trees using treeannotator implemented in the beast v . . package. the initial % samples were discarded as burn-in, leaving % trees per run to produce the consensus tree middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group mers coronavirus neutralizing antibodies in camels antibodies against mers coronavirus in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat mers-related betacoronavirus in vespertilio superans bats characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs isolation of a novel coronavirus from a man with pneumonia in saudi arabia spread, circulation, and evolution of the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description evidence for camel-to-human transmission of mers coronavirus molecular epidemiology of human coronavirus oc reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku , from domestic rabbits origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptor-binding domain complexed with human receptor dpp recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus the heptad repeat region is a major selection target in mers-cov and related coronaviruses the recent ancestry of middle east respiratory syndrome coronavirus in korea has been shaped by recombination full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia the structural and accessory proteins m, orf a, orf b, and orf of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a sars-cov nucleocapsid protein binds to hubc , a ubiquitin conjugating enzyme of the sumoylation system sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus evidence for camel-to-human transmission of mers coronavirus muscle: multiple sequence alignment with high accuracy and high throughput clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform jmodeltest: phylogenetic model averaging new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . paml : phylogenetic analysis by maximum likelihood mega : molecular evolutionary genetics analysis version . bayesian phylogenetics with beauti and the beast . this work was supported by the grant from the national science foundation of china ( ). we thank the anonymous reviewers. z.z., l.s. and x.g. designed the experiments; z.z. and l.s. collected viral sequences, performed phylogenetic analyses, adaptive evolutionary analyses and transmission analyses. z.z., l.s. and x.g. wrote the manuscript. all authors reviewed the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep key: cord- - qin ak authors: song, cong-ying; xu, jia; he, jian-qin; lu, yuan-qiang title: immune dysfunction following covid- , especially in severe patients date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: qin ak the coronavirus disease (covid- ) has been spreading worldwide. severe cases quickly progressed with unfavorable outcomes. we aim to investigate the clinical features of covid- and identify the risk factors associated with its progression. data of confirmed sars-cov- -infected patients and healthy participants were collected. thirty-seven healthy people and confirmed patients, which include severe patients and mild patients, were recruited. covid- patients presented with dysregulated immune response (decreased t, b, and nk cells and increased inflammatory cytokines). also, they were found to have increased levels of white blood cell, neutrophil count, and d-dimer in severe cases. moreover, lymphocyte, cd (+) t cell, cd (+) t cell, nk cell, and b cell counts were lower in the severe group. multivariate logistic regression analysis showed that cd (+) cell count, neutrophil-to-lymphocyte ratio (nlr) and d-dimer were risk factors for severe cases. both ct score and clinical pulmonary infection score (cpis) were associated with disease severity. the receiver operating characteristic (roc) curve analysis has shown that all these parameters and scores had quite a high predictive value. immune dysfunction plays critical roles in disease progression. early and constant surveillance of complete blood cell count, t lymphocyte subsets, coagulation function, ct scan and cpis was recommended for early screening of severe cases. | ( ) sars-cov- -infected patients have problems with liver and kidney function, showing elevated levels of alanine aminotransferase (alt), aspartate aminotransferase (ast), and creatinine (cr). comparing data between mild and severe patients (table ) , more than half ( . %) of all confirmed patients were male, and the proportion of males in severe group was higher than that in the mild group; however, there was no statistically significant difference ( . % vs. . %, p = . ). patients in the severe group had a higher age distribution than in the non-severe group ( white blood cell count (× /l) . ( . - . immune status of patients with covid- . immune cell count and inflammatory factors were recorded to know the immune status of covid- patients. they had a decreased immune cell count, including total t cell, cd + t cell, cd + t cell, and nk cell and b cell count as in comparison with healthy people (fig. ) , and the levels of these immune cell count were also lower in severe than in mild cases (fig. ). there are higher levels of inflammatory factors, including il- , il- , il- , il- , tnf-α, and ifn-γ, found in covid- patients than in healthy people (fig. ) . however, these levels have no significant difference between severe and mild cases (fig. ). we also compared immune status between intubation group (n = ) and non-intubation group. intubated patients had lower cd + cell and cd + cell counts than patients without intubation. while, inflammatory factors had no significant differences between two groups. after initial analysis, variables with p < . were selected, and multivariate logistic regression analysis was performed with the use of the forward stepwise method. afterward, the cd + cell count (p = . ), nlr (p = . ) and d-dimer (p = . ) were considered the independent risk factors of the severe covid- cases (table ) www.nature.com/scientificreports/ to evaluate the predictive value of ct score, cpis, and three independent risk factors, the roc curve analysis was performed (fig. ) . to better distinguish severe and non-severe patients, we have defined the new threshold value of these parameters by calculating the cut-off value. table has shown that ct score had the greatest predictive value with an auc of . ( %ci, . - . ). cpis as well as the combination of cd + t cell count, nlr, and d-dimer had an auc of . ( % ci, . - . ) and . ( %ci, . - . ), respectively. the optimal cut-off values of the ct score and cpis were . and . , respectively. they all had a quite high sensitivity and specificity at the optimal cutoff value. these results have shown that the said parameters had quite a high predictive value. the worldwide outbreak of covid- has worsened . as the main battlefield during the first stage, early detection and effective quarantine of patients and close contacts have allowed the epidemic in china so far to be under effective control. however, the mortality rate of covid- patients in the severe group remains to be quite high because of the rapid progression of the disease and because there is no specific drug against the virus. www.nature.com/scientificreports/ in-depth research on the characteristics of severe cases was urgently needed to identify severe individuals earlier and quicker. in our study, we compared clinical characteristics between healthy people and covid- patients, and then compared these features between severe and mild cases. we found immune dysfunction in covid- patients, and immunosuppression was more obvious in severe cases than mild cases. parameters including cd + t cell count, nlr, and d-dimer, ct score, and cpis had quite great value for predicting disease severity, which could be considered in early warning of severe patients. severe cases usually have mild symptoms in the first week. the time point of aggravation was usually days to days after illness onset, after which the disease progressed quickly . with the characteristics of the disease figure . ct score and cpis in mild and severe covid- patients. ct score and cpis were calculated and compared between mild and severe covid- patients. ct score and cpis were higher in severe patients and these two clinical scores were positively related to disease severity. www.nature.com/scientificreports/ course, the first week was regarded as the early stage of the disease. so, we collected early clinical data days from illness onset. in this study, we have identified the independent risk factors for severe cases, such as decrease of cd + t cell count and increase of nlr and d-dimer. nlr was a great indicator of the overall immune status and was a widely used marker to assess the severity of bacterial infections and the prognosis of patients with pneumonia and tumors [ ] [ ] [ ] . several studies have shown that severe sars-cov- -infected patients have a higher nlr , , an independent risk factor for mortality in covid- patients . d-dimer was a molecular marker of hypercoagulable state and hyperfibrinolysis, and it could be used in the prognosis of patients with infection or sepsis . in patients with sepsis, inflammatory cells were activated, leading to the activation of the coagulation cascade and then causing the activation of the fibrinolytic system . increased coagulation could be found in covid- patients, and increased d-dimer was associated with poor prognosis in covid- patients , . in our study, we also found increased d-dimer was an independent risk factor for intubation. consistent with a previous study, we found lymphocyte, cd + t cell, cd + t cell, nk cell, and b cell counts to be negatively correlated to the severity of covid- , suggesting that immune suppression could be more likely found in severe patients and sars-cov- may directly or indirectly damage the lymphocytes or nk cells and thus further aggravate the disease progression , . even though inflammatory cytokine storms were thought to be a mechanism for covid- progression [ ] [ ] [ ] , there was only an increase in il- , il- , il- , il- , tnf-α, and ifn-γ when comparing pneumonia patients with healthy people, but no significant increase in severe patients compared to mild patients. thus, the role of inflammatory cytokine storms in the progression of the disease was still unclear and controversial. however, these findings might also be due to the limitation in size and the large heterogeneity at the time points of the first detection. il- β is a key proinflammatory cytokine in pyroptosis, which played an important role in various infectious diseases. it was reported that il- β increased in covid- patients and was associated with the disease severity , . however, this item was not detected in our hospital, thus, we could not explore the predictive value of il- β in covid- patients. and it could be considered to detect the level of il- β in further clinical work or research in order to explore its potential clinical value. there are similarities of other clinical features of patients in our medical center and those reported in previous studies [ ] [ ] [ ] [ ] [ ] . males were more susceptible to covid- but had no significance to predict disease severity in our study which may be because of the small sample size, although the male-to-female ratio was quite high in the severe group ( . however, they were all in the normal range, so we did not select this parameter as a predictive factor. it did not mean oxygen saturation had no clinical value. in our study, all data of basic vital signs we selected was the first records on admission, and the first records of oxygen saturation (spo ) was detected by noninvasive pulse oximeter, which was easy to get but could be affected by various interference factors. so it might bring a slight error in this data. continuous oxygen saturation monitoring or referring to arterial oxygen saturation (sao ) might better reflect the real hypoxemia status of the patient. as for the laboratory results, the higher levels of esr, crp, un, ldh, and hbdh were found in severe covid- patients, suggesting that there is an association between disease progression and the injury of cellular immunity, cardiomyocytes, the liver, and the kidney. even though these parameters were not independent risk factors based on our analysis, they could be used in severe case screening, and their predicted value should be assessed by using a larger amount of data in further studies. to investigate whether there was any clinical scoring tool used in early warning for severe cases, we have calculated the ct score and cpis. because of its good imaging data reflecting pulmonary inflammation, ct was often used to know whether the covid- case was severe or not. in order to quantify the image data, we have chosen a commonly used ct scoring method to calculate the specific value. cpis was initially developed as a diagnostic tool for ventilator-associated pneumonia, and it has been used as a predictor of prognosis recently as well . severe patients had higher ct score and cpis, and there was a good correlation between these two clinical scores and disease severity. the roc curve analysis has shown that ct score, cpis, and combination of clinical parameters had a good predictive value of distinguished severe cases in early stage. intubated patients also showed higher ct score and cpis, which suggested these two clinical scoring tools can also be used in pre-intubation evaluation. there are currently no specific antiviral therapies for sars-cov- infection. since immune status play important role in disease severity, immunotherapies are used in severely ill patients . recent immunotherapies included drugs target specific inflammatory molecules and pathways, intravenous immunoglobulin therapy, convalescent plasma infusion, and immune cell-targeted therapies (treg cell and nk cell) . immunotherapies could regulate abnormal inflammatory response and prevent lung damage. several researches have suggested immunotherapies can bring clinical benefits, including reduction of viral loads, and improved survival , . however, the evidence was limited, and the efficacy of these treatments was still not clear, and more researches are needed. there were several limitations in our study which should be considered. firstly, it was a retrospective study, which might contain selection bias, but we tried to avoid the bias by abiding strictly to the inclusion and exclusion criteria. besides that, additional multicenter, multi-ethnic, and prospective studies are expected to revise our diagnostic model, and we also plan to have a multicenter study with a larger sample size so as to further validate and optimize the model. moreover, it is our hope that better statistical algorithms will make the diagnostic model even more practical. now, we are trying to develop a covid- -related database, making data share and management more efficient. thus, data from the multicenter study could be used for further analysis. the combination of cd + t cell count, nlr, and d-dimer, ct score, and cpis could be used as covid- disease severity predictors. we have recommended that these parameters be surveyed earlier and constantly for early warning of severe covid- patients. immune dysfunction plays a critical role in disease progression. the | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ underlying mechanism of covid- development and progression might be complex, and so further research was urgently needed to help better understand and control this epidemic. laboratory confirmation. laboratory confirmation was achieved using the real-time reverse transcription-polymerase chain reaction (rt-pcr) assay for sars-cov- in accordance to the protocol established by the who . in our hospital, sputum samples were the sample of choice for rt-pcr assay within h. two target genes of sars-cov- were tested during the process: open reading frame ab (orf ab) and nucleocapsid protein (n). ct score and cpis. the ct scores were analyzed retrospectively by two radiologists without the knowledge of the patient's diagnosis and other clinical features. the first ct imaging on admission was selected and calculated using the method introduced by casarini et al. . in order to assess lung infection more rapidly, we have used the simplified version of cpis , and it was calculated by using the first clinical results in the hospital. statistical analysis. continuous variables were expressed as medians with interquartile ranges (iqr), while categorical variables were expressed as numbers and percentages in each category. the mann-whitney u-test evaluated continuous data, and the chi-square test was used for categorical variables. performed multivariate logistic regression analyses with forward stepwise method identified independent risk factors. spearman's rank-order correlation investigated whether the two clinical scores (ct score, cpis) and disease severity were associated. the predictive powers of these parameters and two clinical scores were known by calculating the area under the receiver operating characteristic curve (auc). all statistical analyses were done by the spss statistical software package (version . ). a p value < . means statistically significant. ethics approval and written informed consent. this study was approved by the ethical committee of the first affiliated hospital, school of medicine, zhejiang university (code number iit a). written informed consent was obtained from each patient or his/her authorized representatives following a full explanation of the study. all methods and procedures in this study were carried out in accordance with relevant guidelines and regulations. the datasets used and analyzed during the current study are available from the corresponding author on reasonable request. scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia importation and human-to-human transmission of a novel coronavirus in vietnam coronavirus -ncov: a brief perspective from the front line clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study coronavirus disease (covid- ) situation reports the neutrophil-to-lymphocyte ratio: a narrative review neutrophil-to-lymphocyte ratio in 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management of severely ill covid- patients deployment of convalescent plasma for the prevention and treatment of covid- treatment options for covid- : the reality and challenges national health commission of the people's republic of china. the notice of launching guideline on diagnosis and treatment of the novel coronavirus pneumonia (ncp) coronavirus disease (covid- ) technical guidance: laboratory testing for -ncov in humans cytokine levels correlate with a radiologic score in active pulmonary tuberculosis resolution of ventilator-associated pneumonia: prospective evaluation of the clinical pulmonary infection score as an early clinical predictor of outcome this research was supported by the foundation of key discipline construction of zhejiang province for traditional chinese medicine (no. xk-a ). the authors declare no competing interests. key: cord- -q b a authors: sato, shintaro; matsumoto, naomi; hisaie, kota; uematsu, satoshi title: alcohol abrogates human norovirus infectivity in a ph-dependent manner date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: q b a alcohol-based disinfectants are widely used for the sanitization of microorganisms, especially those that cause infectious diseases, including viruses. however, since the germicidal mechanism of alcohol is lipolysis, alcohol-based disinfectants appear to have a minimal effect on non-enveloped viruses, such as noroviruses. because there is no cultivation method for human norovirus (hunov) in vitro, murine norovirus and feline calicivirus have been used as surrogates for hunov to analyze the efficacy of disinfectant regents. therefore, whether these disinfectants and their conditions are effective against hunovs remain unknown. in this study, we report that ethanol or isopropanol alone can sufficiently suppress gii. genotype hunov replication in human ipsc-derived intestinal epithelial cells. additionally, ph adjustments and salting-out may contribute toward the virucidal effect of alcohol against other hunov genotypes and cancel the impediment of organic substance contamination, respectively. therefore, similar to sodium hypochlorite, alcohol-based disinfectants containing electrolytes can be used for hunov inactivation. virucidal activity of alcohols against hunovs. although the efficacy of alcohol-based disinfectants against non-enveloped viruses is controversial, whether these disinfectants show virucidal effects on hunovs remains to be elucidated. recently, costantini et al. demonstrated that % (v/v) ethanol or chlorine inactivated gii. hunov slightly or completely, respectively . therefore, we first investigated the virucidal effect of % ethanol and sodium hypochlorite (naclo) on several hunov genotypes, including gii. , which is a predominant genotype responsible for hunov epidemics. we used a threefold volume of disinfectant solution for the virus solution. consistent with the previous report, although all the studied hunov genotypes were well propagated in our ipsc-derived iecs without any disinfectant condition, a min pretreatment with . % ( ppm) naclo was sufficient to kill all of them (fig. a) . however, only % ethanol could effectively suppress gii. genotype hunov replication in our system, but it had no significant effect on the other genotypes tested in this mono-layered human ipsc-derived iecs were inoculated with × (gii. ; × , gi. ; . × ) genome equivalents of the pre-treated hunovs. inoculation and sampling were performed as described in the materials and methods. viral genome rna was extracted from both supernatants (i.e., those taken at and h post-infection [hpi]), and then genome equivalents were quantified with rt-qpcr. samples at hpi were used as references. each value is representative of at least three independent experiments and is shown as the mean ± sd from - wells of supernatants for each culture group. *, p ≤ . ; **, p ≤ . ; ***, p ≤ . ; ****, p ≤ . . dashed lines represent the limit of detection. a ). similar to % ethanol treatment, treatment with % isopropanol inhibited the propagative ability of gii. but not of gii. hunov (fig. b) . these data indicated that isopropanol could be used as an additive or substituted for ethanol as a gii. hunov disinfectant. virucidal activity of acid-alcohols against hunovs. it has been reported that low-ph ethanol enhances the virucidal effect on non-enveloped viruses . therefore, we next tested whether acid-ethanol has potential as a disinfectant for the inactivation of hunovs. similar to ethanol alone, % (w/v) citric acid alone (ph is about . ) did not influence the propagation of gii. hunov; however, the addition of at least . % citric acid to ethanol completely inactivated the virus ( fig. a) . furthermore, % ethanol containing % citric acid (ph ~ . ) was sufficient for the inactivation of other hunov genotypes, such as gii. , gii. , and gi. (fig. b ). in addition, isopropanol could be substituted for ethanol for the acid-alcohol sanitization of hunovs (fig. c) , indicating that acid-alcohols, similar to naclo, might be an effective disinfectant for hunovs. pure ( %) lemon juice from concentrate contains about % (w/v) citric acid. we examined the virucidal effect of % ethanol- % lemon juice, which contains % citric acid, against hunov. as shown in fig. d , % ethanol- % lemon juice inhibited gii. hunov propagation, indicating that concentrated lemon juice can be used as a source of citric acid to generate an acid-alcohol disinfectant. considering the use of acid-ethanol as a hand sanitizer for hunovs, a short incubation time is likely required for inactivation. therefore, we next examined how much time is needed for the inactivation of hunovs with acid-ethanol. in our in vitro hunov propagation system, both gii. and gii. hunovs were completely inactivated by % ethanol containing % citric acid within s of incubation (fig. ). virucidal activity of alkaline-alcohol against hunovs. next, we investigated whether alkaline-alcohol also shows a virucidal effect on hunovs. to adjust the ph and generate a weak alkaline (ph ~ ), we used sodium bicarbonate buffer because both sodium hydrogen carbonate and sodium carbonate are recognized as food additives, and thus its safety has been confirmed similar to citric acid. when hunovs were incubated in an alkaline solution (carbonate buffer), both gii. and gii. hunovs were not inactivated to the same extent as that observed in a low-ph solution (fig. a ). in addition, a high ph condition did not affect the virucidal effect of % ethanol on gii. genotype hunov (fig. a) . although alkaline-ethanol (carbonate buffer plus www.nature.com/scientificreports/ % ethanol) also appeared to have a virucidal effect on gii. hunov, which could not be inactivated by % ethanol alone, it was less effective than acid-ethanol ( figs. a and a ). however, when we used an increased volume of alkaline-ethanol in a ratio of : virus solution, gii. hunov was completely inactivated (fig. b) . these data indicate that ethanol, and probably isopropanol, have an actual virucidal effect on hunovs that is dependent on the ph. impact of organic substances on the virucidal activity of acid-alcohol against hunovs. as mentioned above, naclo has a powerful virucidal effect, even on non-enveloped viruses. however, naclo also strongly reacts with other organic substances contained in feces and vomit. therefore, we next investigated the virucidal effect of acid-ethanol on hunovs under conditions containing beef extract as an extra-organic substance . similar to the experiments shown in fig. , gii. or gii. hunov was mixed with acid-ethanol ( % etoh- % citric acid). interestingly, % beef extract was sufficient to abolish the virucidal effect of acidethanol on gii. , which could be inactivated by % ethanol only (fig. a ), indicating that soiled organic substances can inhibit the virucidal activity of acid-ethanol against hunovs. as electrolyte solutions, some mineral salts are used to precipitate proteins and low molecular organic compounds, which is a process termed coagulation or salting-out. it is speculated that if organic substances are removed from virus particles, acid-ethanol may fig. . each value is representative of at least three independent experiments and is shown as the mean ± sd from - wells of supernatants for each culture group. *, p ≤ . ; **, p ≤ . ; ***, p ≤ . ; ****, p ≤ . . dashed lines represent the limit of detection. | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports/ inactivate hunovs even in a solution containing extra organic substances. in a % beef extract solution, . % (w/v) magnesium sulfate (mgso )-containing acid-ethanol almost completely inactivated the propagation of the gii. genotype (fig. a ). although gii. hunov was also inactivated with mgso -containing acid-ethanol, it was at a low level, and virus propagation potency was retained. therefore, we used an increased volume of these disinfectants, similar to the experiments shown in fig. b . a nine times volume of mgso -containing acid-ethanol but not just acid-ethanol exhibited complete virucidal effects on both gii. and gii. in a % beef extract solution (fig. b ). this activity was also maintained in a % beef extract solution. these data suggest that mgso -containing acid-ethanol can be used as a disinfectant for hunovs with the same reliability as naclo, even as hand sanitizer because all of the components in mgso -containing acid-ethanol are safe for the human body. genomic rna reduction of acid-alcohol treated hunovs. many reports have investigated the effects of alcohol-based disinfectants against murine norovirus and feline calicivirus as surrogates for hunov , , . viral genome rna degradation by alcohol has also been reported in hunov , . when hunov was incubated with . % naclo at room temperature for min, the viral genome could not be detected by real-time pcr (fig. a) , indicating that naclo could disrupt the capsid and/or genome of hunovs. in contrast, the genomes of gii. and gii. hunovs could be detected after incubation with % ethanol-or citric acid-containing ethanol (fig. a) . although a small decrease in gii. genome copies was observed even in acid-ethanol (~ . log ), gii. hunov was more sensitive to % etoh (~ . log for - ; . log for - ) and acid-ethanol (~ . log for - ; . log for - ). we further examined the morphological change in virus particles after the incubation with disinfectants. to this end, we used virus-like particles (vlps), which comprise the vp capsid proteins of gii. . negative stain transmission electron microscopy analysis revealed that . % naclo could burst vlps (fig. b ). it appeared that acid-ethanol also destroyed vlps, but the vlps did not bust. in addition, % ethanol appeared to have no effect on the morphological change of vlps (fig. b) * * ** ** * **** **** * figure . salting-out efficacy for the contamination of extra organic substances. the solutions of the indicated hunov genotypes containing % or % beef extracts were suspended with threefold (a) or ninefold (b) volumes of the indicated disinfectants for min. inoculation and sampling were performed as in fig. . each value is representative of at least three independent experiments and is shown as the mean ± sd from - wells of supernatants for each culture group. *, p ≤ . ; **, p ≤ . ; ***, p ≤ . ; ****, p ≤ . . dashed lines represent the limit of detection. (fig. ) . interestingly, under the same conditions, products b and c failed to inactivate these hunovs, even gii. , which is inactivated by % ethanol alone (fig. a) . finally, we performed experiments to determine whether -fold dilution of all the disinfectants analyzed in this study would be sufficient for the attenuation of their cytotoxicity for iecs used for virus propagation (i.e., cytotoxicity control) and would also be able to neutralize their virucidal activity toward gii. hunov (i.e., neutralization control). as shown in supplementary fig. , -fold dilution of all disinfectants resulted in the appropriate neutralization of both cytotoxicity ( supplementary fig. a ,b) and virucidal activities (supplementary fig. c,d) . taken together, the findings presented in this study suggested that the virucidal effect of each disinfectant against hunovs should be validated using an in vitro propagation system. in , estes and her colleagues first reported that several hunov genotypes could propagate in iecs prepared from human small intestines . thereafter, our group also reported that human ipsc-derived iecs could be used for the cultivation of hunovs . in this study, we investigated whether alcohol-based disinfectants have actual hunov incubated with tenfold volumes of % ethanol for min were significantly but not completely lower compared with non-treated viruses, whereas incubation with % isopropanol had no effects . importantly, titers of input genomic rna from hunov treated with % ethanol were severely reduced compared with the level of replication , . however, our results in this study were partially inconsistent with their reports. although neither threefold volumes of % ethanol nor isopropanol could reduce the replication levels of gii. , gii. , gii. , and gi. hunovs, the replication of two batches of gii. was almost completely inhibited (fig. ) . one possible explanation for this discrepancy is that we used more diluted fecal filtrates for the experiments to modulate the genome copies of hunov to be × /well. therefore, it is possible that more organic substances, which can interfere with the virucidal ability of % alcohol, were contained in the virus solutions in the previous report . in fact, - % beef extract contamination prevented the virucidal effects of acid-ethanol on gii. hunov in our system (fig. ) . acidification is one of the inactivation methods for foodborne viruses. for example, rotaviruses lose their infectivity when incubated with a ph . solution . although feline calicivirus is also inactivated by incubation with a ph . solution for min at °c , , murine norovirus is stable under the same condition , . hunov (gii. genotype) appears to be resistant to low ph conditions since it can be propagated in iecs after min incubation with % citric acid (ph . ) ( fig. a) , suggesting that the capsid protein of hunov is more similar to that of murine norovirus than feline calicivirus. nevertheless, we revealed that the addition of % citric acid to % ethanol or isopropanol dramatically increased its virucidal effect against hunovs (fig. ) . the virucidal effect of acid-alcohols on other non-enveloped viruses , , including murine norovirus and feline calicivirus , has been reported. although murine norovirus and feline calicivirus are resistant to low-ph and alcohol, respectively, both are sensitive to acid-alcohol. from this point of view, hunovs exhibit both characteristics since they were stable in low ph alone and alcohol alone conditions. given that noroviruses are a group of non-enveloped and hydrophilic rna viruses, the mechanism underlying the virucidal effect of alcohol on hunovs likely involves protein denaturation rather than de-lipidation. this may also explain why either % ethanol or isopropanol alone was capable of inhibiting the replication of gii. hunov. the capsid protein of gii. genotype may be more sensitive to the protein denaturing effects of alcohol compared with other genotypes of hunov. the alkaline-alcohol but not alkaline solution with a ph of also showed a virucidal effect on hunovs, although it was milder than acid-alcohol (fig. ) . it was previously reported that feline calicivirus is inactivated by both a high ph and low-ph solution . given that murine norovirus is sensitive to % alcohol, whereas feline calicivirus is sensitive to the non-neutral ph range, it is possible that a change in ionic and/or hydrogen bonds between alcohol and water is needed for % alcohol to exert its virucidal effect on hunovs, except for the gii. genotype. soil containing stool and vomit would also affect the hydrate condition of alcohol, leading to a decrease in its denaturation effects on capsid proteins. in fact, a % beef extract solution almost completely inhibited the virucidal activity of acid-ethanol (fig. ) . these unexpected effects apply for inert ingredients such as moisturizers and swelling agents, present in commercially available hand-sanitizers. it might be explained by the fact that some commercially available acid-ethanol disinfectants did not show a virucidal effect on hunovs, including gii. (fig. ) . therefore, whether the final product of disinfectants, including acid-alcohol-based ones, has an actual virucidal effect on hunovs in soil load conditions should be considered. because the electrolyte solution (i.e., mgso ) used in this study, has been known to precipitate proteins via the effects of ions on biological and chemical processes in solutions, the addition of such salts (ions) could remove proteins adhering to virus surfaces. the order of the above effects is known as the hofmeister series , and both www.nature.com/scientificreports/ mg + and so are one of the strongest cations and anions, respectively. in addition, citrate ions are higher than phosphate ions and so in the hofmeister series . given that hunovs are usually excreted in feces and vomit containing various amounts of proteins, acid-alcohol disinfectants containing citric acid would be preferable. unlike murine norovirus and feline calicivirus, the genomic rna of hunovs, especially the gii. genotype, was stable when the virus solution was incubated with % ethanol or acid-ethanol (fig. a) . electron microscopic analysis revealed that acid-ethanol and naclo could change the morphology of gii. vlps similar to "apoptosis" and "necrosis" observed in animal cells, respectively. these findings also suggest that acid-ethanol could denature the capsid protein of hunovs, leading to a loss of their ability to infect iecs, whereas it had no impact on genomic rna. the significant advantage of acid-alcohol disinfectants is their good biological safety profiles as they can be generated using alcohol and some food additives. thus, mineral salts, including acid-alcohols, could be used as hunov disinfectants for tableware, cookware, hands, and even foods. however, since it is difficult to evaluate the cytopathic effects of hunovs on iecs in our system, it is also challenging to determine the median tissue culture infectious dose (tcid ) for hunovs to assess their virucidal activity. therefore, future studies are required to develop standard guidelines for the determination of the virucidal activity of hunovs. preparation of disinfectants. ultrapure dnase/rnase-free distilled water (thermo fisher scientific), ethanol, isopropanol, citric acid monohydrate, magnesium sulfate heptahydrate, sodium hydrogen carbonate, sodium carbonate (all purchased from nacalai tesque, japan), and concentrated lemon juice (pokka sapporo food and beverage ltd., japan) were used to prepare the indicated disinfectants. commercially available disinfectants and bleach ( % sodium hypochlorite) were purchased from a local retail store. after preparation, solutions were sterilized with . μm filters. cells. the differentiation of human ipscs into iecs and their culture were performed as described previously , . each conditioned medium (cm) was prepared as described previously ). after days of culture in a % co incubator at °c, the medium was changed to differentiation medium (advanced dulbecco's modified eagle medium/f supplemented with -mm hepes [ph . ]; mm glutamax; units/ml penicillin plus -μg/ml streptomycin; × b- ; . % human r-spondin and human noggin [rn] cm; ng/ml mouse egf; and nm a - ). after another days, the medium was changed to differentiation medium with . % porcine bile (sigma-aldrich). the cells were incubated for another days and then used for subsequent experiments. hunov preparation and infection. all virus samples used in this study were chosen from the hunovpositive stool specimens collected from osaka prefecture, japan during the - endemic season. preparation and infection of hunov were done as described previously , . briefly, hunov-positive stools were suspended in phosphate-buffered saline (pbs) at % (w/v) by vigorous vortexing. the suspensions were centrifuged at , g for min, and the supernatants were serially filtered with . μm and . μm filters. the filtered samples were aliquoted and stored at − °c as an undiluted virus solution (see table for strain details). just before use, each virus solution was diluted to × (gii. ), . × (gi. ), or × (other genotypes) genome equivalents/µl for a : suspension or × genome equivalents/µl for a : suspension with pbs. in the case of soil load experiments, a solution of % (w/v) beef extract (nacalai tesque) was used for virus dilutions, which contained a final % or % beef extract. the virucidal suspension assays were performed using the similar method as that of the astm e - . four microliter of diluted virus solution were suspended in µl (for : ) or µl (for : ) of each disinfectant solution for the indicated times at room temperature. then, each suspension was subjected to a -fold dilution with base medium (advanced dulbecco's modified eagle medium/f supplemented with mm hepes [ph . ], mm glutamax, and units/ml penicillin plus μg/ml streptomycin). the prepared iecs ( - wells per sample) were inoculated with μl ( × www.nature.com/scientificreports/ genome equivalents) of pre-treated virus solutions and then incubated for h in a % co incubator at °c. the inoculum was then removed, and the cells were washed twice with μl base medium. one hundred microliters of differentiation medium with . % bile were added to the cells, which were then pipetted lightly twice and collected. this step was performed again, and the samples were collected as h post-infection (hpi) reference samples (total μl). another μl of differentiation medium with . % bile were added into each well, and the mixtures were then cultured for h in a % co incubator at °c. the supernatants were collected with one wash in the same way as hpi reference samples (total μl). to assess "virus control" and "neutralization control, " we used distilled water and -fold dilutions of each disinfectant, respectively, to substitute for the full-strength disinfectant solution. for the "cytotoxicity control, " cells were pretreated with -fold dilutions of each disinfectant and incubated for h at % co and °c. after washing the cells twice with the base medium, the pretreated cells were inoculated with μl ( × genome equivalents) of gii. hunov followed by incubation for h. subsequent washing, culture, and sampling steps were performed as described above. a purelink viral rna/dna mini kit (invitrogen) was used to prepare rna from diluted virus solutions and samples collected at and hpi. rt-qpcr was performed using a qpcr norovirus (gi/gii) typing kit (takara) and lightcycler system (roche) following the manufacturers' protocols. statistical analysis. results were compared using either unpaired two-tailed student's t-test or one-way anova followed by dunnett's multiple comparisons test. statistical significance was established at p < . . all statistical analyses were conducted using graphpad prism . aqueous molecular clusters isolated as liquid fragments by adiabatic expansion of liquid jets updated classification of norovirus genogroups and genotypes replication of human noroviruses in stem cell-derived human enteroids human norovirus propagation in human induced pluripotent stem cell-derived intestinal epithelial cells tenacity of human norovirus and the surrogates feline calicivirus and murine norovirus during long-term storage on common nonporous food contact surfaces comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and gii. norovirus comparative virucidal efficacy of seven disinfectants against murine norovirus and feline calicivirus, surrogates of human norovirus comprehensive comparison of cultivable norovirus surrogates in response to different inactivation and disinfection treatments strain-specific virolysis patterns of human noroviruses in response to alcohols virucidal efficacy of olanexidine gluconate as a hand antiseptic against human norovirus human norovirus replication in human intestinal enteroids as model to evaluate virus inactivation japanese ministry of health, labour and welfare. the research report for the inactive condition of norovirus virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations rapid inactivation of rotaviruses by exposure to acid buffer or acidic gastric juice inactivation of caliciviruses surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus development and virucidal activity of a novel alcohol-based hand disinfectant supplemented with urea and citric acid lehre von der wirkung der salze what is the fundamental ion-specific series for anions and cations? ion specificity in standard partial molar volumes of electrolytes and electrostriction in water and non-aqueous solvents a refined culture system for human induced pluripotent stem cell-derived intestinal epithelial organoids a heterodimeric antibody fragment for passive immunotherapy against norovirus infection under the informed consent with providing a means to opt out, all stool samples were collected and provided by the osaka institute of public health. the study was performed in accordance with the relevant guidelines and was approved by the human ethical committee of research institute for microbial diseases, osaka university (approval # - ) and the osaka institute of public health (approval # - - ). s.s. designed and directed the overall research, performed the experiments, analyzed the data, and wrote the manuscript; n.m. and k.h. performed most of the experimental infections; and s.u. discussed the data. s.s. was employed by the research foundation for microbial diseases, osaka university. s.s has filed a patent application related to the content of this manuscript. the remaining authors disclose no conflicts. supplementary information is available for this paper at https ://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to s.s. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - toxlskr authors: lanave, gianvito; capozza, paolo; diakoudi, georgia; catella, cristiana; catucci, leonardo; ghergo, paola; stasi, fabio; barrs, vanessa; beatty, julia; decaro, nicola; buonavoglia, canio; martella, vito; camero, michele title: identification of hepadnavirus in the sera of cats date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: toxlskr hepadnaviruses infect several animal species. the prototype species, human hepatitis b virus (hbv), increases the risk of liver diseases and may cause cirrhosis and hepatocellular carcinoma. recently a novel hepadnavirus, similar to hbv, has been identified through transcriptomics studies in a domestic cat with large cell lymphoma in australia. herewith, a collection of feline serum samples was screened for hepadnavirus. overall, the virus was identified in . % of the sera with a significantly higher prevalence ( . %) in the sera of animals with a clinical suspect of infectious disease. upon genome sequencing, the virus was closely related ( . % nt identity) to the prototype australian feline virus sydney . the mean and median values of hepadnavirus in the feline sera were . × ( ) and . × ( ) genome copies per ml (range . × ( )– . × ( ) genome copies per ml). for a subset of hepadnavirus-positive samples, information on the hemato-chemical parameters was available and in / animals a profile suggestive of liver damage was present. also, in / animals with suspected hepatic disease, virus load was > ( ) genome copies per ml, i.e. above the threshold considered at risk of active hepatitis and liver damage for hbv. overall, we detected hepadnavirus dna in / ( . %) sera. dch dna was detected in ( . %) out of sera collected with a request for diagnosis of infectious diseases (collection a) and in ( . %) out of sera submitted to the laboratory for pre-surgical evaluation or for suspected metabolic or neoplastic disease (collection b) that were used to generate a baseline. the difference for dch prevalence was statistically extremely significant (p-value = . , or = . , ci % = [ . ; . ]) when compared animals from collection a and collection b (fig. a) . moreover, there was no significant difference in terms of prevalence (p = . ) between male ( . %, / ) and female ( . %, / ) individuals (fig. b) . we also analysed the distribution of hepadnavirus across various age groups (fig. c) . the prevalence of dch was higher in cats aged to months ( . %, / ) although without statistically significant difference (p = . ) compared to other age groups. we reconstructed the complete genome of the dch strain ita/ / - , of approximately . kb (fig. ) , which had . % nucleotide sequence identity to the australian reference strain aus/ /sydney across the entire genome. some differences were observed in the tree topologies between our analysis and previous studies , likely accounted for by the alignment complexity. in the consensus phylogenetic tree (fig. ) , a large group included bat and rodent viruses. the two feline hepadnaviruses segregated together in a defined branch within www.nature.com/scientificreports www.nature.com/scientificreports/ this large group. all the primate viruses were basal to this bat/rodent/feline group. also, there were two additional well-defined groups, encompassing avian and amphibian viruses, respectively. the findings of this study confirm that the novel hepadnavirus is a common component of the feline virome. the pathogenic role of this hepadnavirus, if any, is not yet known, but thus far, all hepadnaviruses have been reported to replicate preferentially in hepatocytes , . in this study we observed a marked and significantly higher prevalence ( . %, / ) in the cohort of cats with suspected infectious diseases (collection a) with respect to a group of animals (collection b) used as baseline. almost half of the sera positive for dch ( / , . %) of this cohort were collected from cats with retroviral infection (fiv and/or feline leukemia virus, felv). in turn, the overall prevalence of retroviral infection in collection a was . % ( / ), with . % ( / ) being co-infections with dch with a statistically significant (p = . ) correlation between dch and retrovirus positive cats. these findings echo what has been documented for hbv, which is more frequently observed in immunocompromised individuals . reactivation of hbv is common in patients with immunosuppression . even more interestingly, hepatopathy has been described in felv-infected cats, with icterus and various inflammatory and degenerative liver diseases . feline retroviruses have been demonstrated to impair severely the immune system in infected cats and diseases associated with immune-suppression account for a large portion of the morbidity and mortality observed in felv-infected cats [ ] [ ] [ ] . transmission of hbv in humans occurs through blood and other body fluids and contagion can also occur during sexual contact and by maternal/fetal route , . transmission of fiv/felv in cats occurs with similar modalities, as the virus is present in blood and body fluids . similar modalities of transmission might also be hypothesized for dch, since we found viremia in . % of the cats. importantly, the presence of dch in the sera may pose unexpected risks in transfusion medicine. dch, along with feline retroviruses, bartonella spp. and feline hemoplasma, should be considered in the screening of donor subjects . in cats there are not known viral agents strictly associated with hepatic disease, unlike what observed in human medicine where there are five major types of viral hepatitis (a to e) . for diagnosis of hbv infection and for predicting the stage of infection in human patients, antigens, antibodies and viral genome are profiled/ quantified and this information is coupled with haematological and blood chemistry tests. in the case of dch, we can only use the molecular diagnostics and hematological and blood chemistry data, since there are no immunological reagents available for the diagnostics. out of dch-infected cats, we could retrieve information on hematologic and serum biochemical parameters for animals (table ). in of these, increased levels of markers indicative of structural or functional liver damage (i.e. ast, alt, alp, ggt and total bilirubin) were present. hbv load is considered relevant in infected human patients and varies markedly across the phases of hbv infection . the lower threshold for risk of active hepatitis and liver damage is viral genome equivalents of hbv per ml, equivalent to about iu (international unit)/ml . usually, high hbv dna load in blood is found in acute infection, or in active chronic stages of disease, but the virus can reactivate after long periods of apparent remission, chiefly in immunosuppressed patients . the mean and median values of dch viremia in feline sera figure . results of the screening for dch in the feline sera. the prevalence of dch was evaluated in sera collected for the diagnosis of infectious diseases (collection a) and sera submitted to the laboratory for presurgical evaluation or for suspected metabolic or neoplastic disease (collection b) and used for comparison (panel a). the prevalence of dch was also evaluated in relation to sex (panel b) and age (panel c) of the animals. www.nature.com/scientificreports www.nature.com/scientificreports/ were . × and . × dna copies per ml (range . × - . × dna copies per ml). in out of animals with suspected hepatic disease, dch load was > genome copies per ml. although this parallelism between hbv and dch is intriguing, whether a correlation also exists between dch replication and liver damage should be assessed in structured, larger observational studies. for instance, we also found the virus in cats with unaltered hepatic markers. this condition occurs in human patients with chronic hbv infection during the inactive "immune-control" phase, defined as the presence of hbv antigens in serum without alt elevation and with low viremic hbv-dna levels . however, only in / cats with non-altered hepatic markers the virus titer was lower than . the actual patho-biology of dch in cats should be determined in order to understand better the patterns of dch infection. several novel viruses have been discovered in cats in recent years , . optimizing the diagnostic algorithms and gathering epidemiological data will help assessing the possible pathogenic role of these viruses in cats and eventually conceive strategies to protect their health. a total of sera were collected from two different veterinary clinic laboratories located in apulia region, southern italy and tested upon request of the veterinarian practitioners after anamnesis, medical history and clinical examination. one-hundred seventy-four sera (collection a) had been collected for diagnosis of infectious diseases (fiv, felv, feline coronavirus (fcov), toxoplasmosis, hemoplasmosis, bacterial and fungal infections). a total of / ( . %) sera were submitted with a suspect/request of diagnosis for fiv/felv, with sera being positive for retrovirus. fisher's exact test was performed to the collection a to evaluate the correlation between dch positive cats and retrovirus positive cats. the significance level of the test was set at . . of the other sera ( . %), were sent for a suspect/request of diagnosis for coronavirus (with / being positive), for toxoplasmosis (with / being positive), for giardia (with / being positive). five sera were collected from animals with suspected bacterial/fungal infections and serum (negative) was from a cat with suspected hemoplasmosis. a total of sera (collection b) submitted to the laboratory for pre-surgical evaluation (n = ) or for suspected metabolic (n = ) or neoplastic (n = ) disease was used for comparison to generate a baseline. information on the sera analyzed in the study is included in fig. . the study was approved by the ethics committee of the department of veterinary medicine, university of bari (authorization / ). all experiments were performed in accordance with relevant guidelines and regulations. www.nature.com/scientificreports www.nature.com/scientificreports/ total dna was extracted from collected sera by using qiaamp cador pathogen mini kit (qiagen, hilden, germany), according to the manufacturer's instructions. we performed sample screening using a pcr with consensus pan-hepadnavirus primers and a pcr with primers specific for dch . also, we screened sera using a quantitative pcr (qpcr) designed based on the sequence of the australian reference strain aus/ /sydney (genbank accession nr. mh ) ( table ) . for qpcr, we calculated dch dna copy numbers on the basis of standard curves generated by -fold dilutions of a plasmid standard topo xl pcr containing a . kb long fragment of the polymerase region of the australian reference strain aus/ /sydney (iq supermix; bio-rad laboratories srl, segrate, italy). we added μl of sample dna or plasmid standard to the -μl reaction www.nature.com/scientificreports www.nature.com/scientificreports/ master mix (iq supermix; bio-rad laboratories srl, segrate, italy) containing . μmol/l of each primer and . μmol/l of probe. thermal cycling consisted of activation of itaq dna polymerase at °c for min and cycles of denaturation at °c for s and annealing-extension at °c for s. we evaluated the specificity of the assay using a panel of feline dna viruses (parvovirus, herpesvirus and poxvirus). the qpcr assay was able to detect as few as dna copies per ml of standard dna and . × dna copies per ml of dna template extracted from clinical samples. dch quantification displayed acceptable levels of repeatability over a range of target dna concentrations, when calculating the intra-and inter-assay coefficients of variation within and between runs, respectively , . we carried out inferential statistical analyses using the chi-squared test with yates' correction, the evaluation of the odds ratio (or) and % confidence interval (ci %) with the online software medcalc easy-to-use statistical software (https://www.medcalc.org/calc/odds_ratio.php). the significance level of the test was set at . . full genome sequences of hepadnaviruses were retrieved from the genbank database and aligned using geneious version . . (biomatters ltd, auckland, new zealand) and the mafft algorithm . a set of genome sequences used in a previous study was integrated with additional genome sequences of hepadnaviruses of recent identification in mammalian, avian, amphibian species and in fish. the final dataset included hepadnavirus genomes. phylogenetic analysis was performed using jmodel test (http://evomics.org/resources/software/ molecular-evolution-software/modeltest/) to evaluate the correct best-fit model of evolution for the entire dataset. bayesian analysis , was therefore applied using four mcmc chains well-sampled and converging over www.nature.com/scientificreports www.nature.com/scientificreports/ one million generations (with the first trees discarded as "burn-in") and supplying statistical support with subsampling over replicates. the identified program settings for all partitions, under the akaike information criteria, included six-character states (general time-reversible model), a proportion of invariable sites and a gamma distribution of rate variation across sites (gtr + i + g). we also tried to perform phylogenetic analyses using other evolutionary models (maximum likelihood, neighbor joining) to compare the topology of phylogenetic trees. we could observe similar topologies with slight difference in bootstrap values at the nodes of the tree. accordingly, we did prefer to retain the bayesian tree. we deposited the nucleotide genome sequence of strain ita/ / - (mk ) in genbank. all data generated or analyzed in this study are included in this published article. fields virology detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in china a novel hepadnavirus identified in an immunocompromised domestic cat in australia hepatitis b virus: virology, molecular biology, life cycle and intrahepatic spread chronic hepatitis b: update diseases associated with spontaneous feline leukemia virus (felv) infection in cats isolation of a t-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome clinical and immunologic aspects of felv-induced immunosuppression clinical aspects of feline retroviruses: a review blood transfusion in cats: abcd guidelines for minimising risk of infectious iatrogenic complications viral hepatitis in principles and practice of infectious diseases update on prevention, diagnosis, and treatment of chronic hepatitis b: aasld hepatitis b guidance seroprevalence for -like vesiviruses in italian household dogs identification of a novel parvovirus in domestic cats novel parvovirus related to primate bufaviruses in dogs development and validation of a real-time pcr assay for specific and sensitive detection of canid herpesvirus mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform mrbayes : bayesian phylogenetic inference under mixed models mrbayes: bayesian inference of phylogenetic trees this study was supported by the grant ricerca corrente "nuovi flussi diagnostici in sanità animale: dalla ngs alla banca antigeni". reference grant number b e -izs am / rc (italian ministry of health). in addition, the authors would like to thank professor donatella ferraro, university of palermo for providing hbv positive controls. the study was conceived by v.m. experiments were performed by g.l., p.c., g.d. and c.c. data analysis was performed by g.l., m.c. and p.c. sample collection was accomplished by l.c., p.g., f.s. support for experiments and scientific feedback were provided by v.b., j.b., n.d., c.b., v.m. the manuscript was written by v.m., g.l., p.c. and m.c. and reviewed and approved by all authors. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - sk wc authors: wu, jianping; mok, chee-keng; chow, vincent tak kwong; yuan, y. adam; tan, yee-joo title: biochemical and structural characterization of the interface mediating interaction between the influenza a virus non-structural protein- and a monoclonal antibody date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: sk wc we have previously shown that a non-structural protein (ns )-binding monoclonal antibody, termed as h , can significantly reduce influenza a virus (iav) replication when expressed intracellularly. in this study, we further showed that h binds stronger to the ns of h n than a/puerto rico/ / (h n ) because of an amino acid difference at residue . a crystal structure of h fragment antigen-binding (fab) has also been solved and docked onto the ns structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. in one of the models, the predicted molecular contacts between residues in ns and h -fab correlate well with biochemical results. taken together, residues n and t in h n ns act cooperatively to maintain a strong interaction with mab h by forming hydrogen bonds with residues found in the heavy chain of the antibody. interestingly, the pandemic h n - and the majority of seasonal h n circulating in humans since has n in ns , suggesting that mab h could bind to most of the currently circulating seasonal influenza a virus strains. consistent with the involvement of residue t , which is well-conserved, in rna binding, mab h was also found to inhibit the interaction between ns and double-stranded rna. residues - in h n -ns are sufficient for its interaction with mab h . as described previously , the deletion of residues - of ns abolished its interaction with mab h . these residues lie in the helix α (residues - ) of h n -ns (rbd) and are well conserved between h n , h n and h n viruses (fig. a) . this is consistent with the ability of mab h to bind to both avian h n and seasonal iavs . in order to determine if the helix α of h n -ns (rbd) is sufficient for the interaction with mab h , enzyme-linked immunosorbent assay (elisa) was performed by using a synthetic peptide h n -ns - mer corresponding to helix α . as shown in fig. b , mab h bound to h n -ns - mer in a dose dependent manner, indicating that the helix α of ns (rbd) is sufficient for its interaction with mab h . in contrast, there was no binding between mab h and an irrelevant control peptide of similar molecular weight. within helix α , there is only one amino acid difference between h n and h n -pr , namely n in h n -ns and s in h n -pr -ns (fig. a) . to determine if residue in ns is involved in its interaction with mab h , recombinant h n -ns (rbd) and h n -pr -ns (rbd) proteins were bacterially expressed and purified for elisa. the elisa readings were similar for h n -ns (rbd) and h n -pr -ns (rbd) when high concentrations of proteins were coated on the plate (fig. c) . however, the readings were significantly higher for h n -ns (rbd) at lower protein concentrations, indicating that that the single amino acid difference between helix α of h n -ns (rbd) and h n -pr -ns (rbd) affects their interactions with mab h (fig. c ). residue in ns is critical for its interaction with mab h . to further define the contribution of residue in h n -ns to the interaction with mab h , two mutant proteins in which n was mutated to a and s respectively, were generated. comparative elisa showed that mab h bound to ns (rbd)-wild-type (wt) stronger than ns (rbd)-n s when different amounts of protein were coated on the plate and analyzed with μ g/ml of mab h ( fig. a) . similarly, when a fixed amount of protein ( μ g/ml) was coated on the plate and analyzed with different concentrations of mab h , the binding to ns (rbd)-n s was significantly lower than ns (rbd)-wt (fig. b ). furthermore, when n was substituted with a at residue , the interaction between mab h and ns (rbd) was totally abolished in all the conditions tested ( fig. a,b) . this result suggests that the difference at residue is the main reason for the stronger binding of mab h to h n -ns (rbd) when compared to h n -pr -ns (rbd) (fig. ). since both n and s are polar amino acids while a has no side chain, it is probable that the formation of hydrogen bonds is important for the interaction between mab h and ns . mab h binds differently to h n -pr virus when compared with mutant viruses carrying substitution at residue in ns . since mab h binds to bacterially-expressed ns protein of h n and h n -pr with different affinities, it is important to investigate whether this holds true for ns expressed in infected cells. thus, recombinant pr (rgpr ) viruses expressing the ns protein containing a single amino acid substitution at residue (rgpr -ns -s a and rgpr -ns -s n) were generated using a reverse genetics system. subsequently, a cells were infected with each virus at a low multiplicity of infection (moi) of . respectively and plaque assay was used to determine the amount of virus secreted at different time points post-infection. as shown in fig. a , although the viral titer was slightly lower in the case of rgpr -ns -s n infection, the overall growth rates of wt and mutant viruses were similar from to hours post-infection (h.p.i.). this is consistent with a previous report showing that substitution of residue in ns does not affect viral replication in vitro . next, t cells were infected with moi of viruses and cell lysates were collected at and h.p.i. to determine the rate of viral protein synthesis. consistent with the virus growth kinetics, the expressions of structural proteins np and m were similar for all viruses at both time-points (fig. b ). the level of ns , as determined by using a rabbit anti-ns polyclonal antibody, was also comparable for all viruses. however, mab h bound to rgpr -ns -s n virus significantly stronger than rgpr -ns -wt virus containing s residue in mismatches are shown in white letters. ns (rbd) is composed of α -helices as shown above the sequence. the region corresponding to helix α (residues - ) is boxed. (b) peptide elisa was performed to determine the region of ns sufficient for binding to mab h . wells were coated with serially diluted h n -ns - mer or a negative control peptide and probed with μ g/ml mab h . * indicates statistically significant difference of p < . when compared with control peptide. (c) comparative elisa was performed to determine the ability of ns (rbd) of h n -pr and h n to bind to mab h . wells were coated with serially diluted proteins and probed with μ g/ml mab h . data shown represents result from three independent experiments and error bars represent standard deviation (sd) of the experiment carried out in duplicates. *indicates statistically significant difference of p < . when compared with h n -pr -ns (rbd). scientific reports | : | doi: . /srep ns , supporting the above results that n in ns is preferred over s for the interaction with mab h . as expected, mab h did not bind to rgpr -ns -s a virus. to further define the interaction interface between mab h and ns , attempts were made to co-crystallize the complex but failed. however, the h -fab alone was found to crystallize in space group p ( ) and the structure was solved by molecular replacement using the structure of bl - (pdbid: q q) as the starting model and refined to the crystallographic r-factor of . % (deposited in the pdb under accession code b m). the refinement statistics are shown in table . previously, we demonstrated that the intracellular expression of h -scfv in mammalian cells reduced the replication of pr virus. since the three dimensional structure of h -fab has been solved, a commercially available lipodin-ab reagent (abbiotec), which is a protein transfection reagent dedicated to the transport of antibodies into living cells, was used to deliver h -fab into a cells. the cells were then subjected to infection so as to determine if h -fab has an impact on viral replication. when either h -fab or a -fab (which is derived from a negative-control antibody binding to the spike glycoprotein of sars coronavirus) was transfected into a cells using lipodin-ab reagent, intracellular accumulation of fab was observed even up to h post transfection (fig. a) . the intracellular accumulation of fab was also observed at as early as h post transfection (data not shown). the transfection efficiency was about % and most of the fab molecules were evenly distributed in the cytoplasm but there were some punctate staining which could be due to aggregation of fab inside a cells. in contrast, no intracellular accumulation of fab was observed when a -fab or h -fab was added to the cells without lipodin-ab reagent. as shown in fig. b , the delivery of fab did not have any significant effect on cell viability at either or h post transfection. upon successful delivery of fab, its biological function was assessed in influenza a virus infected cells. as mab h binds strongly to rgpr -ns -s n virus (fig. ) , a cells were infected with rgpr -ns -s n virus after transfection of h -fab. cell lysates were collected at , , h.p.i. to determine the rate of viral protein synthesis. as shown in fig. c ,d, the level of viral m protein at h.p.i. was significantly reduced in h -fab transfected cells when compared to a -fab transfected cells. at h.p.i., the average reduction in normalized m expression in h -fab transfected cells was % when compared to a -fab transfected cells. at h.p.i., a slight reduction in m expression (~ %) was also observed in h -fab transfected cells but it is not statistically significant. this result suggests that the successful delivery of h -fab into living cells could reduce viral replication by affecting certain function(s) of ns in the infected cells. next, computational modelling was used to study the complex between this high resolution h -fab structure and the published structure of h n -ns (rbd) of h n /a/crow/kyoto/t / strain (pdb id: z a). this was conducted by using haddock on water-refined models, including an analysis of energy contributions from van der waals interaction, electrostatic interaction, restraints violation and buried surface area . as comparative elisa in this and previous studies showed that residues n and t in ns (rbd) are important for the interaction with mab h , they were defined as active residues involved in the binding interaction to generate a series of models of the ns (rbd) and h -fab complex. all the models of ns (rbd) and h -fab complex were found to cluster into groups, in which there were at least two conformations of the ensemble showing backbone root-mean-square deviations at the interface of less than . Å. as additional elisa results showed that residues, namely s , r , and g , in ns are not involved in interaction with mab h ( figure s ), this information was used to distinguish between the models in these clusters. of all energetically best models generated, predicted models were grouped into cluster . the average buried surface area was . ± . Å , and rmsd from the overall lowest-energy structure was . ± . Å. among them, the best predicted model from this cluster showed good agreement with our comparative elisa data, since only residues n and t were predicted to be involved in the interaction, while the side chains of s , r and g were either distal from the interface (s and r ) or inaccessible (g ) to the binding partner of h -fab (fig. ) . by analyzing the polarity of the amino acids and distance between them in this model, it is predicted that residues n and n in the variable domain of heavy chain (vh)-complementarity determining region (cdr ) of h -fab could form hydrogen bonds with the side chain of n in ns (rbd). in addition, the side chain of t in ns (rbd) could form hydrogen bonds with residue r in vh-cdr . in contrast, vh-cdr , vh-cdr and all the cdrs in the variable domain of light chain (vl) are unlikely to be involved in the interaction as they are distal to helix α of ns (rbd). on the other hand, predicted models were grouped into cluster . for these refined structures analyzed, the average buried surface area was . ± . Å , and rmsd from the overall lowest-energy structure was . ± . Å. based on the best predicted model from this cluster ( figure s ), it was predicted that t of ns (rbd) could form the hydrogen bond with n of vh-cdr while n of ns (rbd) could form the hydrogen bond with n of vl-cdr . these predictions are in agreement with the results shown in fig. and in our previous publication . however, this model also predicted that r of ns (rbd) could be involved in the interaction with h -fab because it was in close proximity to two residues in vl-cdr . in this model, the distance between r of ns (rbd) and y of vl-cdr was . Å while the distance between r of ns (rbd) and s of vl-cdr was . Å. thus, this model does not agree with the results from comparative elisa which showed that substitution of r of ns (rbd) with k did not affect its interaction with mab h ( figure s ). lastly, another predicted models were grouped into cluster . the average buried surface area was . ± . Å , and rmsd from the overall lowest-energy structure was . ± . Å. based on the best predicted model from this cluster ( figure s ), the contacts between n and t with residues in h -fab were the same as described above for the model from cluster . however, this model also predicted that r of ns (rbd) could be involved in the interaction with h -fab because it was in close proximity to three residues in vl-cdr . in this model, y , s and y of vl-cdr were found to be at . Å, . Å and . Å from r of ns (rbd) respectively. thus, this model does not agree with the results from comparative elisa which showed that substitution of r of ns (rbd) with k did not affect its interaction with mab h ( figure s ). overall, the predicted model from cluster is consistent with our comparative elisa data and suggests that residues n and t are important for the binding between ns (rbd) and h -fab because their side-chains could make hydrogen bonds with residues in the vh-cdr of the fab. in addition, r of ns (rbd) was distal from the antibody-antigen interface, which is consistent with the results from comparative elisa ( figure s ) showing that substitution of r of ns (rbd) with k did not affect its interaction with mab h . in contrast, the predicted models from cluster and cluster do not agree with the results from comparative elisa. mab h disrupts ns and dsrna interaction. ns (rbd) forms a symmetric six-helical homodimer, which binds to dsrna. the key residues in ns involved in the interaction with dsrna are t , d , d , r , r , r , l , s and t , most of which are positively charged residues and mainly clustered in the middle of helices α /α ′ of the rbd , . to investigate whether mab h hampers dsrna-ns interaction in vitro, an alphascreen assay was carried out. in this experiment, glutathione s-transferease (gst)-tagged h n -ns (rbd) protein, which was produced in e. coli, was incubated with synthetic -nucleotide sirna ( nt-sirna) followed by addition of streptavidin coated donor beads and anti-gst-conjugated acceptor beads which recognize biotinylated rna and gst-tagged protein respectively. if the interaction between ns and nt-sirna brings both beads to close proximity, transfer of excitation energy from donor beads into acceptor beads will yield a luminescent signal (fig. a ) . when ns (rbd) was pre-incubated with different concentrations of mab h followed by addition of nt-sirna, acceptor and donor beads, the luminescent signal decreased at high concentration of mab h (fig. b ). the luminescent signal was reduced by ~ % and ~ % at mab h concentrations of and μ m respectively (fig. b) , suggesting that the binding of mab h to ns (rbd) can block the interaction of ns with dsrna. on the other hand, the negative control mab a did not reduce the luminescent signal at all the concentrations tested. furthermore, when mab h , ns and nt-sirna were mixed simultaneously, μ m of mab h could reduce the signal by about % (fig. c) , which suggest that mab h also directly competes with dsrna to bind to ns . the ns protein of influenza virus is a multi-functional protein that is involved in key aspects of the virus replication cycle . the ns (rbd) consists of the first amino acids and contains residues critical for dsrna binding . the dimerization of the rbd is a prerequisite for its rna binding activity . since the ns protein is an intracellularly expressed viral protein, which is subjected to less host selective immune response and thus has lower mutation rate, antibodies targeting this protein could be helpful for the development of therapeutic treatment of influenza a infection. indeed, our previous study showed that mab h binds to the highly conserved t residue in ns and reduces viral replication of h n -pr in mammalian cell lines . in this study, comparative elisa showed that helix α of ns (rbd) is sufficient for its interaction with mab h (fig. ) . while mab h binds to ns (rbd) of both h n and h n -pr , the binding affinity to the homologous h n viral protein is higher. interestingly, a single amino acid difference at residue in helix α of ns (rbd) of h n and h n -pr is found to be critical for the interaction with mab h (figs and ). by using either purified ns protein or ns expressed in infected cells, our results showed that the interaction of mab h with ns is stronger when residue is n than when it is s and is abolished when the residue is an a. to understand the pattern of polymorphism at residue in ns , sequences were retrieved from niaid influenza research database (http://www.fludb.org) and analyzed ( table ). sequence analysis of avian h n isolates revealed that % of them have n and % have s suggesting that mab h has the ability to bind to the majority of avian h n isolates. interestingly, a recent computational study compared the viral proteins of highly and lowly pathogenic h viruses and identified s to n substitution as a potential marker of pathogenicity of avian influenza virus subtype h . as for human isolates, the majority ( %) of seasonal h n isolated before have s in ns like h n -pr . however, almost all the pandemic h n (pdmh n ) isolates have n in ns . as for seasonal h n , % of viruses isolated before have n and this percentage increased to % for those isolated from to . thus, mab h is expected to bind to the majority of circulating seasonal influenza viruses. by solving a crystal structure of h -fab and docking it onto the ns (rbd) of h n with the haddock program, molecular contacts made by residues t and n at the interface of antibody-antigen complex were predicted (fig. ) . based on one of the energetically best models generated, the side chain of residue n in ns (rbd) could form hydrogen bonds with the side chains of residues n and n of vh-cdr of h -fab. meanwhile, residue t in ns (rbd) seems to interact with residue r in vh-cdr . these predictions are consistent with the results of binding assays performed using substitution mutants of ns as described above and in our previous study . while the predicted d model of the antibody-antigen complex gives us some clues on how residues and in ns (rbd) interact with h -fab, it may be able to accurately reveal the contributions of all molecular contacts between ns (rbd) and h -fab. hence, further studies could focus on the use of other epitope mapping methodologies , besides crystallography, to obtain a more precise map of the molecular contacts at the antigen and antibody interface. furthermore, an alphascreen assay showed that mab h could inhibit the interaction between ns and dsrna (fig. ) . while both residues and are located in helix α of the ns (rbd), it has been shown that t , but not s , is one of the key residues involved in dsrna binding . thus, the interaction between t and h -fab predicted in the docked model is consistent with the ability of mab h to inhibit the interaction between ns and dsrna. although a previous study has demonstrated that mutation at residue did not affect the affinity of ns (rbd) for dsrna , our study suggests that the side chain of n makes crucial contacts with mab h to stabilize the antibody-antigen complex. as such, when the side chain is not present in the case of gst-tagged ns protein was incubated with biotinylated dsrna and the interaction between ns and dsrna could bring streptavidin-coated donor bead and anti-gst-conjugated acceptor bead close to each other. when excited at nm, singlet oxygen molecules ( o ) are produced from donor beads, which react with acceptor beads to produce light emission measured at - nm. (b) inhibition activity of mab h was determined by pre-incubating nm gst-tagged ns protein with serially diluted mab h or a negative control mab a . following that, luminescent signal was measured after the addition of nm biotinylated dsrna, acceptor and donor beads. (c) inhibition activity of mab h was determined by incubating nm gst-tagged ns protein, nm biotinylated-dsrna and serially diluted mab h or a simultaneously. following that, luminescent signal was measured after the addition of acceptor and donor beads. all readings obtained were normalized against that of samples in the absence of antibody. error bars represent sd of the experiment carried out in triplicates. *indicates statistically significant difference of p < . when compared to mab a . the n a substitution mutant, interaction with mab h is completely abolished (figs and ). this indicates that the interaction between the side chains of residue t in ns and residue r in h -fab is not sufficient to maintain the antibody-antigen interface. on the other hand, the t v and t a substitution mutants still retained some binding to mab h ( figure s ), presumably because of the hydrogen bonds between residue n and the heavy chain of the antibody. based the alphascreen assay, a high concentration of mab h (~ μ m) was required to reduce the interaction between ns (rbd) and dsrna by > % (fig. ) . on the other hand, the concentration of mab h required for binding ns (rbd) in elisa was in the nanomolar range (fig. ) . this discrepancy could be due to the differences between the two assays but it also suggests that the binding of mab h to ns could have other effects on ns besides disrupting its interaction with dsrna. indeed, our previous gel filtration and dynamic light scattering results suggest that the complex between h -fab and ns (rbd) is multimeric in nature and each oligomer could consist of molecules of ns (rbd) and molecules of h -fab . in contrast, ns (rbd) eluted out the gel filtration column as a dimer, as would be expected, in the absence of h -fab . in addition, the delivery of h -fab into a cells also caused a reduction in the replication of rgpr -ns -s n recombinant virus (fig. ) . while it is difficult to precisely define the biologically relevant quaternary structures of ns , several studies have shown that the ns has conformational plasticity and dynamic changes in the quaternary structure of ns are likely to be important for the different functions of ns in infected cells . hence, it is possible that the binding of h -fab to the ns expressed in infected a cells could affect the conformational plasticity of ns and/or its ability to interact with certain cellular factor, thus resulting in a reduction in viral replication. in future studies, advanced fluorescence microscopic techniques could be used to determine the effect of h -fab on the dynamic of ns inside infected cells. in summary, we have used biochemical and structural methods to characterize the interaction between ns and mab h . our results showed that helix α in ns (rbd) is sufficient for interacting with mab h and residues n and t in this helix are likely to make hydrogen bonds with the cdr of the antibody heavy chain. helix α is highly conserved and this is consistent with the ability of mab h to bind to different subtypes of iav. after solving a high resolution crystal structure of h -fab, a haddock-derived model of the antibody-antigen complex has been obtained and the molecular contacts predicted from this model are in agreement with results obtained from comparative elisa performed using ns mutants. this model may be used in antibody engineering experiments to increase the affinity of interaction between ns and mab h so as to increase mab h 's potency in viral inhibition. in addition, it may be useful in structure-based rational drug design to identify small molecule inhibitors of ns . cells. a , t and mdck cells were purchased from american type culture collection (manassas, va, usa). a cells were cultured in minimum essential medium (mem) (gibco). t and mdck cells were cultured in dulbecco's modified eagle's medium (invitrogen). both media were supplemented with % fetal bovine serum (hyclone), penicillin ( , units/ml)-streptomycin ( mg/ml) solution (sigma aldrich). all cell lines were maintained at °c with % co . ascites and rabbit polyclonal antibodies production. ascites were produced by injecting hybridoma cells into peritoneal cavities of pristine-primed balb/c mice. the protocol was approved by institutional animal care and use committee (iacuc) of the biological resource center, a*star, singapore (protocol number: ). in order to generate rabbit anti-h n -ns polyclonal antibody, gst-fusion ns protein was purified using the method as previously described . new zealand white rabbits were then immunized with this protein and bled as previously described . the protocol was approved by iacuc of the biological resource center, a*star, singapore (protocol number: ). all the animal procedures were performed in strict compliance with the recommendations of the naclar guideline in singapore. all efforts were made to minimize the suffering and euthanasia was performed using carbon dioxide. protein expression and purification. ns (rbd) (residues - ) of a/chicken/hatay/ (h n ) was pcr amplified from a full-length ns gene (accession no.: caj . ) and the ns (rbd) (residues - ) of a/puerto rico/ / (h n ) was pcr amplified from a full-length ns gene (accession no.: abd . ). the expression constructs were generated by inserting pcr product into pet sumo expression vector (invitrogen). ns mutants expression construct was generated by overlap pcr as described previously . all proteins were expressed and purified as previously described . the purified proteins were dialyzed against dialysis buffer ( mm tris-hcl, ph . , mm nacl) and concentrated to mg/ml in a centrprep- (amicon) for subsequent assays. crystallization of h -fab. mab h was purified from the ascites by using affinity chromatography and h -fab was obtained by papain cleavage as described previously . the purified h -fab was dialyzed against dialysis buffer and concentrated to mg/ml for subsequent crystallization. crystallizations were performed with the hanging drop vapor diffusion method at °c by mixing μ l of h -fab with μ l of reservoir solution and the mixture was equilibrated against μ l of reservoir solution. crystals of h -fab were grown against crystallization buffer containing % peg , . m nacl and . m bis-tris, ph . . these crystals grew to a maximum size of . mm × . mm × . mm over the course of days. single crystals were obtained by dissecting from multiple crystals. crystals were flash frozen ( k) in the above reservoir solution supplemented with % glycerol. a total of frames of a native data set with oscillation at . Å wavelength were collected for h -fab. all data sets were processed by hkl . most of the crystals diffracted rather weak and the scaled data sets were anisotropic with strong ice rings and high mosaicities. nevertheless, one of the data sets displaying scientific reports | : | doi: . /srep weak ice ring was able to scale to . Å at the mosaicity of . °. this data was used for structure determination and refinement with the statistic table listed as table . of h -fab were predicted using the online igblast tool (http://www.ncbi.nlm.nih.gov/igblast/). as the three-dimensional structure of ns (rbd) of a/chicken/hatay/ (h n ) has not been solved, the structure of ns (rbd) of a/crow/kyoto/t / (h n ) (pdb id: z a) was used instead. as shown in fig. a , there are amino acid differences between the ns (rbd) of these two strains but the sequence of helix α in the ns (rbd) is % identical. mutagenesis data from comparative elisa was used to generate a series of models for the complex between h -fab and ns (rbd) by using version . of haddock webserver , . along with the available individual structure, haddock utilizes the experimentally derived data to predict the complex structure. to achieve this, ns (rbd) was docked to h -fab using the easy interface of haddock webserver, where both residues n and t of ns (rbd) and amino acids in the cdrs of h -fab were defined as active residues involved in the interaction. in this docking experiment, only the variable domains of h -fab were used. enzyme-linked immunosorbent assay (elisa). elisa was performed as described previously . briefly, a synthetic peptide corresponding to residues - in h n -ns (apfldrlrrdqkslrgrgntlgld, chemically synthesized by gl biochem) or purified wt and mutant ns (rbd) proteins were serially diluted into . m carbonate-bicarbonate buffer, ph . . a -mer peptide corresponding to a fragment of the hepatitis c virus core protein (rpswgpidprrrsknlgkvidtltcgfap, chemically synthesized by genway biotech) was used as a negative control. proteins or peptides ( μ l) were then coated onto -well elisa plates (nunc) overnight at °c. the wells were blocked in % milk in pbs with . % tween (pbst) for h at °c followed by addition of μ l of mab h as primary antibody to each well and incubated at °c for - h. the wells were then washed in pbst followed by the addition of goat anti-mouse horse-radish peroxidase (hrp)-conjugated antibody (pierce) as secondary antibody and incubated at °c for h. tetramethylbenzidine substrate (pierce) was added and reaction was stopped using . m sulfuric acid. absorbance at nm was recorded using an absorbance reader (tecan infinite m ). recombinant viruses were generated with phw reverse genetic system as described previously . residue in ns was changed from s to a or n by pcr mutagenesis and the resulting dna were inserted into phw vector. this plasmid was co-transfected with another seven phw plasmids containing the other pr genomic dnas into t cells. days post transfection, culture supernatant was collected and used to infect mdck cells. when cytopathic effect (cpe) was visibly detected, culture supernatant was collected and used to infect naïve mdck cells. individual plaque was then amplified and viral titer was determined by plaque assay. virus infection and western blot analysis. % confluent t cells were infected with moi of wt or mutant viruses at °c for h. the medium was discarded and cells were rinsed with pbs. cell lysates were harvested at and h.p.i. in ripa buffer ( mm tris-hcl ph . , mm nacl, . % np , . % sodium deoxycholate, . % sds). then, μ g of total lysate were resolved using electrophoresis on an sds-polyacrylamide gel and transferred to a nitrocellulose membrane (bio-rad). antibodies against gapdh (santa cruz), np (millipore), and m (as described previously) were used. mab h and rabbit anti-h n -ns polyclonal antibody (as described above) were also used. after washing, the membrane was incubated with a hrp-conjugated secondary antibody (pierce) at room temperature for h. the membranes were then washed and detected with enhanced chemiluminescence substrate (pierce) using chemidoc ™ mp imaging system (bio-rad). multiple-cycle growth kinetics of recombinant virus. plaque assay was applied to determine the growth kinetics of rgpr -ns -wt, rgpr -ns -s a and rgpr -ns -s n recombinant viruses. % confluent monolayers of a cells were rinsed with pbs and subsequently adsorbed with . moi of wt and mutant viruses respectively for h at °c. the medium was discarded and the cells were rinsed using pbs and cultured in mem without serum at °c. supernatant containing virus was collected at , , , and h.p.i. respectively and subjected to plaque assay to determine viral titer. plaque assay in mdck cells. % confluent mdck cells were adsorbed with serially diluted supernatants containing viruses for h at °c. the medium was discarded and the cells were rinsed using pbs. the cells were overlaid with ml of dmem supplemented by . % agar and μ g/ml tpck-trypsin (thermo scientific). after incubation at °c for days, the cells were fixed using % formalin for h and stained using . % crystal violet solution. delivery of a -fab and h -fab into a cells. a cells were cultured in -well plate. the a -fab and h -fab were then transfected into % confluent cells by using lipodin-ab reagent (abbiotec) according to manufacturer's protocol with slight modification. briefly, μ l of fab solution ( μ g) was mixed thoroughly with μ l lipodin-ab solution and incubated for min at room temperature. μ l of serum-free mem medium was added to fab/lipodin-ab solution and then immediately added to the cells. the cells were washed with pbs once before the fab/lipodin-ab solution was added. the cells were incubated at °c in % co for h, washed once with pbs and subjected to viral infection and western blot analysis as described above. cell viability assay. cell viability was determined using wst- reagent (roche) according to manufacturer's protocol. briefly, μ l of wst- reagent was diluted in the μ l of culture media and added to a cells cultured in a transparent -well microplate and incubated for h, followed by measuring absorbance at nm. immunofluorescence assay. a cells grown on coverslip were transfected with fab as described above. approximately h after transfection, the medium was aspirated, washed with pbs once and replaced with serum-free mem medium and incubated at °c in % co for another h. the cells were then washed with pbs once and fixed with % paraformaldehyde for min and permeabilized with . % tritonx- for min, followed by blocking with % bsa in pbs for min. the cells were incubated with alexa fluor -conjugated goat anti-mouse igg antibody (invitrogen) for h. after washing, cells were stained with dapi before mounting. images were captured using an olympus fluoview fv laser-scanning confocal microscope. alphascreen-based dsrna binding inhibition assay. a nt-sirna previously reported to form complex with ns (rbd) was purchased from thermo scientific dharmacon (dharmacon, lafayette, co). the sirna was biotinylated at the ′ end of the sense strand and the sirna sequences were as follows: ′ -biotin-agacaccauuaugcugucuuu- ′ (sense) and ′ -agacagcauaauggugucuuu- ′ (antisense). the lyophilized sirnas were reconstituted in rnase-free water to a final concentration of nm. to express gst-tagged ns (rbd) of a/chicken/hatay/ (h n ) was pcr amplified from a full-length ns gene (accession no.: caj . ) and cloned into pgex- p- vector (ge healthcare). the expression and purification were performed as described previously . the purified gst-fusion protein was dialysed against pbs and the final concentration was determined using the coomassie plus protein assay reagent (thermo scientific). in vitro rna binding inhibition assay was carried out in -well proxiplate by using the alphascreen anti-gst kit (perkinelmer). in the first experiment, μ l of nm gst-tagged proteins were mixed with same volume of serially diluted mab h and incubated at room temperature for h. then, μ l of nm biotinylated nt-sirna was added into the binding mixture and incubated at room temperature for h before the addition of μ l of detection mixture containing . μ l of anti-gst (glutathione s-transferase) acceptor beads and . μ l of streptavidin-coated donor beads (perkinelmer). after another incubation at room temperature for h, luminescent signal was measured using an enspire multimode plate reader (perkinelmer) . in the second experiment, μ l serially diluted mab h was mixed with μ l of nm gst-tagged proteins and μ l of nm biotinylated nt-sirna simultaneously. after incubation at room temperature for h, detection mixture was added and measurement was made similarly. statistical analysis. two-tailed student's t test was applied to evaluate the statistical significance of differences measured from the data sets obtained in independent experiments. p < . was considered statistically significant. influenza-who cares orthomyxoviridae: the viruses and their replication new world bats harbor diverse influenza a viruses mixed infections of pandemic h n and seasonal h n viruses in outbreak advances in the development of influenza virus vaccines toward a universal influenza virus vaccine: prospects and challenges evasion of influenza a viruses from innate and adaptive immune responses viral m ion channel protein: a promising target for anti-influenza drug discovery influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance avian flu: isolation of drug-resistant h n virus influenza a virus strains that circulate in humans differ in the ability of their ns proteins to block the activation of irf and interferon-beta transcription virulence of h n avian influenza virus enhanced by a -nucleotide deletion in the viral nonstructural gene rna binding by the novel helical domain of the influenza virus ns protein requires its dimer structure and a small number of specific basic amino acids binding of influenza a virus ns protein to dsrna in vitro the influenza virus ns protein is a poly(a)-binding protein that inhibits nuclear export of mrnas containing poly(a) the influenza virus ns protein binds to a specific region in human u snrna and inhibits u -u and u -u snrna interactions during splicing the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the ′ - ′ oligo (a) synthetase/rnase l pathway influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i structural basis for a novel interaction between the ns protein derived from the influenza virus and rig-i binding of the influenza a virus ns protein to pkr mediates the inhibition of its activation by either pact or doublestranded rna loss of function of the influenza a virus ns protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol -kinase (pi k) influenza a virus ns protein activates the pi k/akt pathway to mediate antiapoptotic signaling responses identification of influenza virus inhibitors targeting ns a utilizing fluorescence polarization-based high-throughput assay synthesis and evaluation of quinoxaline derivatives as potential influenza ns a protein inhibitors novel influenza virus ns antagonists block replication and restore innate immune function antiviral activity of baicalin against influenza virus h n -pdm is due to modulation of ns -mediated cellular innate immune responses small interfering rna targeting the nonstructural gene transcript inhibits influenza a virus replication in experimental mice a new panel of ns antibodies for easy detection and titration of influenza a virus a monoclonal antibody binds to threonine in the non-structural protein of influenza a virus and interferes with its ability to modulate viral replication roles of the phosphorylation of specific serines and threonines in the ns protein of human influenza a viruses monoclonal antibodies targeting the hr domain and the region immediately upstream of the hr of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus haddock: a protein-protein docking approach based on biochemical or biophysical information conserved surface features form the double-stranded rna binding site of non-structural protein (ns ) from influenza a and b viruses structural basis for dsrna recognition by ns protein of influenza a virus assay optimization and screening of rna-protein interactions by alphascreen the multifunctional ns protein of influenza a viruses a complete map of potential pathogenicity markers of avian influenza virus subtype h predicted from expressed proteins current approaches to fine mapping of antigen-antibody interactions conformational plasticity of the influenza a virus ns protein comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza a (h n ) virus expressed in insect cells and bacteria simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies haddock versus haddock: new features and performance of haddock . on the capri targets a dna transfection system for generation of influenza a virus from eight plasmids the pymol molecular graphics system, version we are grateful to r.g. webster for the pr based reverse genetics system. we thank members of the monoclonal antibody unit at institute of molecular and cell biology for technical assistance. this work was supported by the singapore ministry of health's national medical research council under its nmrc-cbrg scheme [grant no. nmrc/cbrg/ / ]. key: cord- -okmkgsbr authors: ohno, marumi; sekiya, toshiki; nomura, naoki; daito, taku ji; shingai, masashi; kida, hiroshi title: influenza virus infection affects insulin signaling, fatty acid-metabolizing enzyme expressions, and the tricarboxylic acid cycle in mice date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: okmkgsbr although the severity of influenza virus infections has been associated with host energy metabolism, the related mechanisms have not yet been clarified. here we examined the effects of influenza virus infection on host energy metabolism in mice. after infecting mice with intranasal applications of plaque-forming units of a/puerto rico/ / (h n ; pr ) virus, the serum levels of most intermediates in the tricarboxylic acid (tca) cycle and related metabolic pathways were significantly reduced. these data suggest that substrate supply to the tca cycle is reduced under these conditions, rather than specific metabolic reactions being inhibited. then, we focused on glucose and fatty acid metabolism that supply substrates to the tca cycle. akt phosphorylation following insulin injections was attenuated in the livers of pr virus-infected mice. furthermore, glucose tolerance tests revealed that the pr virus-infected mice showed higher blood glucose levels than the vehicle-inoculated control mice. these results suggest that influenza virus infection impairs insulin signaling, which regulates glucose uptake. however, increases in the hepatic expressions of fatty acid-metabolizing enzymes suggest that fatty acids accumulate in liver cells of infected mice. collectively, our data indicate that influenza virus infection dysregulates host energy metabolism. this line of investigation provides novel insights into the pathogenesis of influenza. www.nature.com/scientificreports/ investigated metabolic changes by determining the serum levels of metabolites, insulin sensitivity in the liver, glucose availability, and hepatic gene expressions in the early stages of symptom onset as well as the lethal phase of influenza in a mouse model. the results of this study indicate that influenza virus infection dysregulates glucose and fatty acid metabolism and decreases tricarboxylic acid (tca) cycle activity, leading to enhanced degradation of adenosine triphosphate (atp) and guanosine triphosphate (gtp). we believe that this research provides novel insights into the pathogenesis of influenza and contributes to the development of novel influenza drugs. metabolome analysis indicated reduced tca cycle activity and enhanced purine degradation in mice with severe influenza. upon infection of plaque-forming units of pr virus, the mice showed significant body weight loss starting at day post infection ( dpi, . % ± . % in control mice, . % ± . % in pr virus-infected mice; dpi, . % ± . % in control, . % ± . % in pr virus-infected mice; dpi, . % ± . % in control mice, . % ± . % in pr virus-infected mice). mice were sacrificed when weight loss reached a humane endpoint ( % at dpi). to investigate the systemic effect of influenza virus infection on the host metabolic system, metabolome analyses were performed with serum samples collected at , , and dpi for samples at very early-stage, onset of symptoms, and lethal phase during influenza, respectively. metabolome analyses identified metabolites in the serum samples of the control and pr virus-infected mice at all time points. peak areas and relative ratios of all metabolites are provided in supplemental tables s and s . samples collected at dpi showed the greatest effect of pr virus infection on serum metabolomes, as indicated by principle component analyses with high contribution rates of principle component (pc ; . %; supplemental fig. s ). moreover, pc scores for the control and pr virus-infected mice were negative and positive, respectively, suggesting that the pc score was positively related to the effects of pr virus infection. accordingly, metabolites with high positive and negative factor loading in pc tended to increase and decrease with pr virus infection, respectively (table ) . relative increases in the serum levels of hypoxanthine, xanthine, and inosine were . -, . -, and . -fold, respectively, in the pr virus-infected mice. these molecules are intermediates of purine degradation, and their increased serum levels are considered indicative of enhanced adenosine triphosphate (atp) and guanosine triphosphate (gtp) catabolism. in agreement, the relative serum levels of the upstream purine metabolites adenosine monophosphate (amp), adenosine, guanosine monophosphate (gmp), and guanosine, were decreased by . -, . -, . -, and . -fold, respectively, at dpi. among them, the serum levels of amp and adenosine did not differ significantly between the control and infected groups at any time point owing to large individual differences in the control groups (fig. ) . thus, the lethal phase of influenza is considered to be characterized by increased degradation of atp and gtp. metabolites that were present at reduced levels in the sera of pr virus-infected mice were mainly related to the tca cycle, urea cycle, and amino acid metabolism, as indicated by the serum levels of metabolite in these pathways at , , and dpi (fig. ) . the effects of -day pr virus infections on the serum levels of metabolite are summarized in fig. . the levels of tca cycle metabolites pyruvic acid, -ketoglutaric acid, fumaric acid, and malic acid were significantly reduced in pr virus-infected mice at dpi ( . -, . -, . -, and . -fold, respectively). succinic acid levels were significantly increased by . -fold at dpi but decreased by . -fold at dpi in pr virus-infected mice. in addition, -aminobutyric acid, which is synthesized from glutamic acid and converted to succinic acid, was present at reduced in pr virus-infected mice at and dpi ( . -and . -fold, respectively). the levels of urea cycle metabolites argininosuccinic acid, ornithine, and citrulline were decreased by . -, . -, and . -fold, respectively, in pr virus-infected mice at dpi, whereas the level of arginine was not altered significantly at any time point. given that the levels of most metabolites were significantly reduced in the tca cycle and related pathways, we suggest that pr virus infection reduces flux through these pathways, particularly through the tca cycle. as for amino acids, while the serum levels of ketogenic amino acids did not show any apparent decreases, those of glucogenic amino acids were significantly reduced in pr virus-infected mice at dpi. specifically, the serum levels of asparagine, glycine, proline, serine, and tyrosine were significantly decreased by . -, . -, . -, . -, and . -fold, respectively. furthermore, to estimate the effect of pr virus infection on glutaminolysis in which glutamine is converted to -ketoglutaric acid via glutamic acid and enters the tca cycle, we compared the ratio of the peak areas of glutamic acid to those of glutamine and the ratio of the peak areas of ketoglutaric acid to those of glutamic acid. while the ratio of glutamic acid to glutamine was not changed by the infection, the ratio of -ketoglutaric acid to glutamic acid was significantly decreased by . -and . -fold, respectively, at and dpi (supplemental table s ). therefore, it was indicated that glutaminolysis contribution to the tca cycle was attenuated by influenza virus infection. pathway analysis using metaboanalyst further confirmed that the tca cycle and related pathways were significantly affected by pr virus infection at dpi. the top pathways are listed in table . given that the tca cycle generates gtp and contributes electrons to mitochondrial oxidative phosphorylation to generate a substantial amount of atp, suppression of the tca cycle in pr virus-infected mice is considered to cause imbalanced synthesis and degradation of atp and gtp. moreover, these data suggest that suppression of the tca cycle was due to reduced substrate supply to the pathway due to pr virus infection, rather than inhibition of specific metabolic reactions. tca cycle flux is associated with glucose and fatty acid metabolism because both these energy sources are eventually converted to acetyl-coa, which enters the tca cycle. because the liver plays a predominant role in whole-body energy metabolism, we further investigated the effect of pr virus infection on liver function in terms of glucose and fatty acid metabolism. www.nature.com/scientificreports/ insulin regulates cellular glucose uptake from the blood and intracellular glycolysis , , which supplies substrates to the tca cycle. upon insulin binding to the insulin receptor on the cell surface, the signal is transduced to downstream molecules and phosphorylates akt. phosphorylated akt activates glucose transporters to increase glucose uptake. therefore, phosphorylation of akt is a good indicator of activation of insulin signaling. here we examined the effect of influenza virus infection on insulin sensitivity in the livers according to ratios of insulin-induced phosphoryla- table . increased or decreased serum levels of metabolites in pr virus-infected mice at dpi. metabolites with high factor loading (> . ) and significant differences (p < . , t-test) were selected as significant metabolites and are listed here. the relative serum levels of each metabolite from pr virus-infected mice are presented as fold changes relative to those from control mice. factor loading was defined for each metabolite as correlation coefficients between pc scores and metabolite levels that were normalized by autoscaling in each sample. www.nature.com/scientificreports/ tion of akt. western blotting analyses ( fig. a ) of phosphorylated and total akt in whole liver lysates of control and pr virus-infected mice at dpi revealed that the ratios of phosphorylated akt to total akt were increased by . -fold after insulin treatments in control mice, but this ratio was increased by only . -fold in pr virusinfected mice (fig. b) . similar experiments were performed with the livers collected at other time points. at dpi, akt phosphorylation was clearly inhibited by pr virus infection, but no clear differences were observed in samples collected at dpi (supplemental fig. s ). taken together, these results demonstrated that influenza virus infection impairs insulin actions in the liver. we also performed glucose tolerance tests (gtt) in control and pr virus-infected mice at dpi to investigate whether pr virus infection affected glucose uptake in response to high doses of glucose (fig. c) . we found no significant differences in fasting blood glucose levels between the groups. moreover, at min after glucose injections, the control and pr virus-infected mice showed similar blood glucose levels ( . ± . mg/dl vs. . ± . mg/dl). subsequently, blood glucose levels were rapidly decreased in control mice ( min, . ± . mg/dl; min, . ± . mg/dl; min, . ± . mg/dl). conversely, decreases in blood glucose levels were delayed and higher blood glucose levels were observed in pr virus-infected mice, compared with the control mice, at all time points ( min, . ± . mg/dl; min, . ± . mg/dl; min, . ± . mg/dl). significant differences in blood glucose levels were identified at and min between the groups (p < . , two-way anova). these results indicate that pr virus infection impairs insulin signaling in the liver and induces a tendency toward glucose intolerance, potentially reflecting reduced glucose uptake. fatty acid accumulation in the liver of infected mice was suggested by gene expression assays. we also investigated the effects of pr virus infection on the hepatic expressions of genes that are transcriptionally regulated by insulin signaling (fig. ) . pck , which encodes a gluconeogenic enzyme, was expressed dpi. mice were intranasally inoculated with pbs alone or pbs comprising pr virus, and serum samples were collected for metabolome analysis at , , and dpi. the serum levels of purine metabolites in pr virusinfected mice were expressed relative to those in control mice at each time point. bars represent mean ± sem of or animals. in each panel, white, gray, and black bars indicate data from pr virus-infecetd mice at , , and dpi, respectively; *p < . , unpaired t-test using the holm-sidak correction method with an alpha value of . , control vs. pr virus-infected mice at each time point. the right panel shows a schematic of the effects of pr virus infection on purine metabolism at dpi. upward and downward black arrows indicate significant increases and decreases, respectively. downward gray arrows indicate observed but not significant decreases. www.nature.com/scientificreports/ at slightly but significantly increased ( . -fold) levels in the liver of infected mice at and dpi (fig. a ). this gene is negatively regulated by insulin signaling through inactivation of a transcriptional factor forkhead boxcontaining protein o sub-family (foxo ) which reduces glycolysis and activates gluconeogenesis . therefore, the increase in pck expression in the present study also suggests reduced insulin activity or sensitivity in pr virus-infected mice. in addition to glucose, fatty acids are important sources of acetyl-coa for the tca cycle. fatty acid metabolism was previously reported to be inhibited by influenza virus infection , . therefore, we investigated whether pr virus infection altered the hepatic expressions of genes encoding fatty acid-metabolizing enzymes (fig. b) . cd and pnpla play important roles in fatty acid transport and lipolysis, respectively, and their gene expressions are downregulated by insulin signaling , . here, cd was expressed at significantly increased levels in the livers of pr virus-infected mice, with . -and . -fold increases at and dpi, respectively. in addition, pnpla was significantly induced by . -and . -fold at and dpi, respectively. significant increases in the expressions of figure . relative serum levels of intermediates of the tca cycle and related pathways in control vs. pr virusinfected mice at , , and dpi. mice were intranasally inoculated with pbs alone or pbs comprising pr virus, and serum samples were collected for metabolome analysis at , , and dpi. the serum levels of tca cycle metabolites in pr virus-infected mice are presented relative to those in control mice at each time point. each panels show metabolites of the tca cycle, urea cycle, and amino acid metabolism, respectively. bars represent mean ± sem of or animals. in each panel, white, gray, and black bars indicate data from pr virus-infected mice at , , and dpi, respectively; *p < . , unpaired t test using the holm-sidak correction method with an alpha value of . , control vs. pr virus-infected mice at each time point. www.nature.com/scientificreports/ cd and pnpla as well as of pck suggested the attenuation of insulin signaling in the liver of pr virus-infected mice. conversely, we observed no changes in the expressions of acox and cpt β, which encode key enzymes of fatty acid β-oxidation. in addition, the serum levels of carnitine were significantly decreased by . -fold at dpi (fig. ) . because carnitine is involved in fatty acid transport into mitochondria, the observed decrease would possibly reduce fatty acid β-oxidation in mitochondria. these changes suggest fatty acid accumulation in the liver cells of pr virus-infected mice, although further analyses on protein expressions and enzymatic activities are warranted in the future. in the present study, metabolome analyses demonstrated that pr virus infection decreases the serum levels of most tca cycle intermediates and metabolites in the related metabolic pathways, suggesting reduced flux through the tca cycle. because the tca cycle greatly contributes electrons for mitochondrial oxidative phosphorylation, suppression of the tca cycle leads to reduced atp synthesis. hence, the rates of atp and gtp degradation were possibly greater than the respective rates of their synthesis in pr virus-infected mice, leading to significant increases in the serum levels of metabolites, such as xanthine, hypoxanthine, and inosine. energy depletion indicated by the reduced levels of atp and gtp has been described in several studies on infectious diseases and endotoxemia [ ] [ ] [ ] . reduced atp levels in blood and various organs, including the liver, have been previously associated with influenza virus infection in a mouse model . decreased gtp levels have been shown www.nature.com/scientificreports/ in the lung tissues of rabbits treated with bacterial endotoxin lipopolysaccharide . increases in the serum levels of hypoxanthine and inosine have also been reported in patients with primary dengue virus infection at the febrile stage, suggesting an imbalance of atp and/or gtp synthesis and degradation during the acute stage of the dengue fever . these studies and ours indicate that regulatory energy depletion is a common symptom of infectious diseases. tca cycle flux is associated with glucose and fatty acid metabolism because these energy sources are eventually converted to acetyl-coa for entry into the tca cycle. if either pathway is inhibited, the other pathway becomes activated to compensate for the reduced substrate supply to the tca cycle. in the case of hfd-induced obese mice with insulin resistance, fatty acid oxidation is increased in a leptin-dependent manner, and tca cycle activity is enhanced , . conversely, db/db mice, a well-known mouse model of type diabetes, reportedly developed insulin resistance without activation of fatty acid oxidation due to lack of leptin receptor and showed reduced serum levels of the tca cycle intermediates citric acid, -ketoglutaric acid, malic acid, and fumaric acid with disease progression . in the present study, no changes in the expression of cpt or acox in the liver suggest that fatty acid oxidation was not enhanced in pr virus-infected mice despite insulin resistance. although the effect of pr virus infection on fatty acid oxidation was not evaluated in the present study, reduced fatty acid oxidation during influenza has been demonstrated previously , . if both insulin signaling and fatty acid oxidation are inhibited during influenza, it might result in suppression of the tca cycle due to reduced substrate supply. given significant body weight loss in pr virus-infected mice, however, decrease in food intake during influenza could affect the serum levels of the tca cycle and related pathways. further investigations, such as studies with pair-fed control and lower titer of virus, will provide helpful information to confirm the underlying association. here we demonstrated for the first time that influenza virus infection impairs insulin signaling in liver tissues, which critically regulates glucose metabolism , , and induces a tendency toward glucose intolerance. importantly, nagao et al., reported a high blood glucose level (over mg/dl) as one of the worst prognostic factors in pediatric patients with influenza-associated encephalopathy . in addition to the elevated glucocorticoid levels, as speculated by nagao et al., our study suggests the impairment of insulin signaling as a mechanism of elevated blood glucose levels in children with severe influenza. conversely, another study reported that glucose uptake was activated in the lungs of patients with respiratory viral infections, including influenza virus infection . this discrepancy may be associated with tissue-specific metabolic changes in response to influenza virus infection. however, given the direct activating effect of influenza virus proliferation on glucose metabolism in cultured mice were intranasally inoculated with pbs alone or pbs comprising pr virus. at dpi, the mice were intraperitoneally injected with pbs or insulin after overnight fasting, and liver samples were collected after min for whole lysate preparation. western blotting was performed to quantitate phosphorylated and total akt protein levels in lysates. (a) a representative western blotting analysis shows total akt and akt phosphorylated at ser on the same membrane that was sequentially immunoblotted with corresponding antibodies. (b) relative akt phosphorylation levels were calculated from band densities and expressed relative to data from pbs-treated control mice. bars represent mean ± sem of animals. white and black bars indicate data from pbs-and insulin-treated mice, respectively; p < . , two-way anova, pbs vs. insulin (*), control vs. pr virus-infected mice (#). (c) mice were intranasally inoculated with pbs alone or pbs comprising pr virus. at dpi, gtt was performed after overnight fasting. to this end, mice were intraperitoneally injected with glucose, and their blood glucose levels were sequentially measured at , , , and min post injection. dots represents mean ± sem of animals; *p < . , unpaired t test using the holm-sidak correction method with an alpha value of . , control vs. pr virus-infected mice at each time point. www.nature.com/scientificreports/ madin-darby canine kidney cells , the hepatic insulin resistance demonstrated here could have been induced by host factors and not the virus itself. upon intranasal infection of pr virus, proliferation of the virus occurs mainly in the lung and not in the liver of mice . hence, the hepatic insulin resistance observed herein is possibly induced by host factors, such as systemically secreted cytokines. the direct roles of cytokines on insulin signaling have been demonstrated in a study showing that the proinflammatory cytokine interleukin- (il- ) inhibited insulin-dependent phosphorylation of akt in hepg cells and human primary hepatocytes . furthermore, il- treatments reportedly impaired insulin signaling and inhibited insulin-induced decreases in blood glucose levels in mice ; in this previous experiment, the serum levels of il- reached approximately pg/ml, which was comparable to that observed herein in pr virus-infected mice at and dpi when reduced insulin-induced phosphorylation of akt was observed (supplemental fig. s , . and . pg/ml at and dpi, respectively). thus, we suggest that this cytokine is one of host factor candidates that affect insulin resistance, particularly in the liver. in addition to il- , interferon-gamma (ifn-γ) was reportedly induced by murine cytomegalovirus infections and was recently shown to induce insulin resistance in skeletal muscle . besides il- , the serum levels of ifn-γ were elevated in pr virus-infected mice at dpi but were very low at dpi in our study (supplemental fig. s ; . and . pg/ml at and dpi, respectively). therefore, we propose that influenza virus infection induces hepatic insulin resistance through systemic cytokine secretion. as stated above, in addition to glucose, fatty acids are important sources of the tca cycle substrate acetyl-coa. reduced fatty acid metabolism during influenza has been reported in previous reports showing mitochondrial abnormalities, decreases in the expressions of relevant enzymes, and hepatic steatosis in mice , . consistently, our gene expression data indicate altered fatty acid metabolism in the livers of pr virus-infected mice. in particular, the transcription levels of the fatty acid transporter cd and the lipolytic enzyme pnpla were upregulated at and dpi, possibly elevating extracellular fatty acid delivery and stored triglyceride lysis in liver cells. however, no changes were observed in the mrna expressions of acox and cpt b. these encoded enzymes are rate limiting factors for fatty acid β-oxidation in peroxisomes and mitochondria, respectively. taken together, our data suggest that fatty acids are accumulated in the cytoplasm of liver cells in influenza virus-infected mice. although anorexia due to influenza virus infection may induce similar alterations in gene expressions and subsequent hepatic steatosis, the findings of pair-fed studies on influenza eliminated the possibility that starvation www.nature.com/scientificreports/ is solely responsible for these changes , . moreover, fasting generally increases the mrna expressions of acox and cpt b, thereby discriminating the effects of influenza virus infection on fatty acid metabolism from those of starvation , . fatty acid accumulation can induce mitochondrial dysfunction, as reported previously in cultured hepatocytes . mitochondrial dysfunction increases oxidative stress via reactive oxygen species production. reactive oxygen species and diacylglycerol, converted from triacylglycerol by adipose triglyceride lipase (encoded by pnpla ), inhibit insulin signaling . it is suggested that accumulation of intracellular fatty acids triggers and complicates insulin resistance in the liver of infected mice. given the higher influenza-associated mortality and morbidity rates in patients and mice with energy metabolism disorders - , energy metabolism dysregulation induced by influenza virus infection is possibly associated with pathogenicity. increased mortality rates with influenza have been reported in diet-induced obese mice which probably had insulin resistance as well as in long-chain acyl-coa dehydrogenase-knockout mice, which had reduced mitochondrial fatty acid oxidation , . interestingly, both these mouse models presented with subdued immune responses to virus infection and similar or lower lung virus titers compared with the control mice. hence, increased influenza severity in animals and patients with energy metabolism disorders can be explained neither by the increased levels of inflammatory cytokines nor by the reduced clearance of pathogens , . as described above, dysregulation of energy metabolism appears to be a downstream response to cytokine and inflammatory signaling and thus could be more directly associated with the pathogenesis and severity of influenza. previous cohort studies have demonstrated that antiinflammatory corticosteroids have no beneficial effects and that these corticosteroids can exacerbate outcomes in patients with severe influenza . given that glucocorticoids inhibit insulin signaling as well as cytokine-dependent pathogen clearance , , corticosteroid treatments could confound pathogenicity. thus, improvements in host energy metabolism would be a novel therapeutic target for severe influenza rather than immune suppression. moreover, this concept could be expanded to other infectious diseases because energy metabolism dysregulation is possibly induced by host factors, such as proinflammatory cytokines, and not by the viruses themselves. given the recent emerging infectious diseases such as the pandemic influenza in and severe pneumonia by the novel corona virus in , the importance of developing novel therapeutic drugs that target host factors against common symptoms of various diseases is important. infants and children younger than years of age are generally at high risks of mortality and morbidity due to influenza compared with middle-aged adults . previously, a negative correlation of nasal lavage and plasma inflammatory cytokine levels with age has been reported and thought to be a reason for the high mortality and severe illness caused by influenza virus infection in young children , . in addition to cytokine responses, age-specific features of energy metabolism could provide insights into the pathogenesis of influenza at different ages. in pediatric patients with type i diabetes, the youngest population ( - years of age) reportedly showed the highest prevalence of diabetic ketoacidosis, which has an impact on morbidity and mortality , indicating that attenuation of insulin signaling by virus infection could result in more severe symptoms in such populations. the present study provides novel insights into host responses during influenza. based on the findings of the present and previous studies, we conclude that influenza virus infection induces dysregulation of both insulinregulating glucose metabolism and fatty acid oxidation, leading to decreased tca activity. in future studies, we aim to investigate the effect of influenza virus infection at lethal and sublethal doses of various virus strains for further understanding of influenza pathogenesis and examine the therapeutic effects of interventions that improve host energy metabolism. materials. phosphate-buffered saline (pbs) was purchased from gibco/life technologies (carlsbad, ca). tris-buffered saline (tbs) tablets were purchased from takara bio (otsu, japan). urea, tween , and glucose were purchased from sigma-aldrich (st louis, mo). sodium dodecyl sulfate (sds) was purchased from wako (tokyo, japan). mouse. male c bl/ mice were purchased from hokudo (sapporo, japan) and were kept at a bsl- laboratory at the research center for zoonosis control, hokkaido university, under standard laboratory conditions (room temperature °c ± °c, relative humidity % ± %) and a / -h light/dark cycle. the mice were administered a standard ce- chow diet purchased from clea japan (sapporo, japan) with water ad libtum. experiments were performed on - week-old mice. virus infection and sample collection. pr virus particles at plaque-forming units in µl of pbs or pbs only (control) were intranasally inoculated into the mice under inhalation anesthesia with isoflurane. at , , or dpi, the mice were euthanized, and their liver and blood samples were collected. blood samples were incubated at room temperature for h to clot and were then centrifuged at , g for min. supernatants were collected as serum and were stored at − °c until further analysis. tissue samples were stored at − °c until further analysis. to evaluate insulin sensitivity, the mice were administered insulin (humulin r, eli lilly, indianapolis, in) at u/kg body weight intraperitoneally at dpi after overnight fasting. after min, the mice were euthanized, and their liver samples were collected and frozen immediately in liquid nitrogen. liver samples were stored at − °c until further analysis. research (kyoto, japan) using liquid chromatography-tandem mass spectrometry (lc-ms/ms). briefly, after the addition of an internal standard ( -isopropylmalic acid) to the serum samples, sample preparation was conducted using a series of hydrochloride and acetonitrile extractions. after centrifugation at , rpm for min at room temperature, the supernatants were divided into two tubes: one was used for analyses of amp, gmp, aconitic acid, citric acid, and fumaric acid, whereas in the other one, the supernatant was mixed with hydrochloride and used for analysis of other compounds. chromatographic separations were performed on a discovery hs f - column ( . mm × mm, μm, sigma-aldrich). the oven temperature for the column was maintained at °c. mobile phases a and b were . % formic acid-water solution and . % formic acid-acetonitrile, respectively. separation was performed using gradient elution at a flow rate of . ml/min with a nexera uhplc system (shimadzu). compounds were eluted by changing proportions of mobile phase b as follows: % ( - min), %- % b ( - min), %- % b ( - min), %- % ( - min), % ( - min), %- % ( - . min), and % ( . - min). compounds were detected using an lcms- (shimadzu) instrument with electrospray ionization. the peak areas of each compound were measured using labsolutions (shimadzu) and were normalized to that of an internal standard. the metabolome analyses identified molecules in our sample sets. among them, cysteine, cytosine, and methionine sulfoxide were not detected in some samples from pr virus-infected mice at and dpi (supplemental table s ). to examine the effects of pr virus infection on the serum levels of various compounds, the relative peak areas for pr virus-infected mice were expressed as fold changes relative to the average peak areas for the control mice at corresponding time points. the peak areas and relative ratios are provided in supplemental table s and s . serum metabolome data analysis using metaboanalyst. utilizing peak areas of molecules, principal component analysis and pathway analysis were performed with metaboanalyst (https ://www.metab oanal yst.ca). the values were normalized by autoscaling function. missing values in cysteine, cytosine, and methionine sulfoxide were replaced by small values (a half of the minimum positive values in the original data). factor loading was defined for each metabolite as the correlation coefficient between the pc score and the level of the metabolite after normalizing by autoscaling for each sample. metabolites with high factor loading (> . ) and significant differences (p < . , t-test) were selected as significant metabolites and are listed in table . pathway analyses were performed based on the kyoto encyclopedia of genes and genomes (https ://www.genom e.jp/ kegg/), and the most significantly altered metabolic pathways in pr virus-infected mice were identified. the top pathways identified are listed in table . evaluation of phosphorylated akt by western blotting. liver samples were homogenized in tbs comprising m urea and % sds using an ultrasonic homogenizer (q sonicator, qsonica, newtown, ct) and centrifuged at , g for min at °c to obtain supernatants as whole liver lysates. protein levels were measured using pierce bca protein assay kits (thermo fisher scientific, waltham, ma). samples for western blotting were prepared by mixing whole liver lysates with the nupage lds sample buffer (thermo fisher scientific) and heating at °c for min. subsequently, μg aliquots of total protein were separated using % sds-polyacrylamide gel electrophoresis (sds-page) and were transferred to polyvinylidene difluoride membranes. after blocking with % nonfat dry milk in tbs buffer comprising . % tween (tbst), the membranes were probed with an anti-akt phosphorylated at ser antibody ( : , , # , cell signaling technology, beverly, ma) or an anti-akt antibody ( : , , cell signaling technology) in tbst buffer comprising % bovine serum albumin overnight at °c. the membranes were then treated with secondary horseradish peroxidase-conjugated antirabbit antibody ( : , , sc- , santa cruz) for h at room temperature. protein bands on membranes were detected using supersignal west femto maximum sensitivity substrate (thermo fisher scientific) and an imagequant las system (ge, buckinghamshire, uk). the bands were quantified using the imagequant tl analysis system (ge). full-length images are provided in supplemental fig. s . pbs at g/kg body weight after overnight fasting. blood was collected from tail veins at , , , and min after injections, and plasma glucose concentrations were measured using an accu-chek glucose meter (roche diagnostic, mannheim, germany). www.nature.com/scientificreports/ amount of assay buffer for serum samples or serum matrix for standards and controls. magnetic beads coated with antibodies against the target cytokines were added to each well, and the plates were incubated on a plate shaker overnight at °c. after washing with washing buffer in the kit, the samples were reacted with biotinylated detection antibodies for h and then with streptavidin-phycoerythrin for min. after washing and addition of loading buffer from the kit, the samples were analyzed by the magpix system (luminex, austin, tx, usa). the results are presented in supplemental fig. s . statistical analysis. statistical analyses were performed using prism (graphpad software, san diego, ca, usa). differences were identified using unpaired t-tests or two-way anova and were considered significant when p < . . data are presented as mean ± standard error of the mean (sem). all experiments were performed at least twice to confirm the reproducibility. ethical statement. all mouse experiments were performed with approval from the animal care and use committee of hokkaido university following the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology in japan. body weight losses were monitored daily after infection, and mice were humanely euthanized when weight loss reached %. all data generated or analyzed during this study are included in this published article and its supplementary information files. received: december ; accepted: june influenza-who cares diet-induced obese mice have increased mortality and altered immune responses when infected with influenza virus impact of diabetes mellitus on mortality 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metabolites in serum and urine reveals metabolic perturbation of tca cycle in db/db mice involved in diabetic nephropathy prognostic factors in influenza-associated encephalopathy targeting metabolic reprogramming by influenza infection for therapeutic intervention metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling multiorgan distribution of human influenza a virus strains observed in a mouse model interleukin- induces cellular insulin resistance in hepatocytes chronic exposure to interleukin- causes hepatic insulin resistance in mice virus-induced interferon-γ causes insulin resistance in skeletal muscle and derails glycemic control in obesity nutrient-sensing nuclear receptors coordinate autophagy extrahepatic pparα modulates fatty acid oxidation and attenuates fasting-induced hepatosteatosis in mice prevention of free fatty acid-induced hepatic lipotoxicity by carnitine via reversal of mitochondrial dysfunction inflammation and lipid signaling in the etiology of insulin resistance corticosteroids for severe influenza pneumonia: a critical appraisal glucocorticoid-induced insulin resistance: dexamethasone inhibits the activation of glucose transport in rat skeletal muscle by both insulin-and non-insulin-related stimuli interleukin- limits influenza-induced inflammation and protects against fatal lung pathology age-specific mortality risk from pandemic influenza mucosal immune responses predict clinical outcomes during influenza infection independently of age and viral load cytokine and chemokine response in children with the pandemic influenza a (h n ) virus infection ketoacidosis at diagnosis of type diabetes in french children and adolescents we thank the national institute of infectious disease in japan for kindly providing influenza virus a/puerto rico/ / (h n ). this work was supported by japan science and technology agency basic research programs, jsps kakenhi (grant number k ), and northern advancement center for science and technology, hokkaido japan (grant number k ). the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to h.k. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -ykog j authors: stewart, h.; bingham, r.j.; white, s. j.; dykeman, e. c.; zothner, c.; tuplin, a. k.; stockley, p. g.; twarock, r.; harris, m. title: identification of novel rna secondary structures within the hepatitis c virus genome reveals a cooperative involvement in genome packaging date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ykog j the specific packaging of the hepatitis c virus (hcv) genome is hypothesised to be driven by core-rna interactions. to identify the regions of the viral genome involved in this process, we used selex (systematic evolution of ligands by exponential enrichment) to identify rna aptamers which bind specifically to core in vitro. comparison of these aptamers to multiple hcv genomes revealed the presence of a conserved terminal loop motif within short rna stem-loop structures. we postulated that interactions of these motifs, as well as sub-motifs which were present in hcv genomes at statistically significant levels, with the core protein may drive virion assembly. we mutated of these predicted motifs within the hcv infectious molecular clone jfh- , thereby producing a range of mutant viruses predicted to possess altered rna secondary structures. rna replication and viral titre were unaltered in viruses possessing only one mutated structure. however, infectivity titres were decreased in viruses possessing a higher number of mutated regions. this work thus identified multiple novel rna motifs which appear to contribute to genome packaging. we suggest that these structures act as cooperative packaging signals to drive specific rna encapsidation during hcv assembly. hepatitis c virus (hcv) is the leading cause of chronic liver disease, hepatocellular carcinoma and liver transplants in the developed world. it is estimated that million people are chronically infected worldwide, approximately % of whom will eventually present with liver cirrhosis. hcv therefore represents a significant health and economic burden. direct-acting antivirals (daas) targeting viral non-structural proteins are now available for treatment of hcv infection. the rates of viral clearance, however, differ significantly between genotypes and a low genetic barrier to resistance for certain daas means that viral escape is highly likely. additionally, the pricing of the most recently available daa (sofosbuvir; a polymerase inhibitor) makes it unfeasible to treat the majority of patients in many countries. it is therefore apparent that further research into the basic virology of hcv is warranted. hcv is of the family flaviviridae, genus hepaciviridae and possesses a ~ . kb single-stranded positive-sense rna genome, encompassing highly structured untranslated flanking regions (utrs). the ′ utr includes an internal ribosome entry site (ires), which directs cap-independent translation of a single polyprotein that is subsequently cleaved into constituent proteins: core, e , e , p , ns , ns , ns a, ns b, ns a and ns b. although the non-structural (ns) proteins contribute to some degree to the assembly and release of the virion, core, e and e play mostly structural roles . the five c terminal ns proteins (ns , ns a, ns b, ns a and ns b) were shown in to represent the minimal essential requirement for rna replication of a subgenomic replicon . these proteins and the ensuing rna replication process have been available for targeted drug design much longer than the processes of virion assembly, rna packaging and viral egress. the availability of a fully infectious viral molecular clone (jfh- ) means that these later stages can also be investigated in detail . the role(s) of genomic rna structures during genome encapsidation has not, however, been the subject of extensive research. genomes are packaged during formation of the nucleocapsid by the core protein. core is encoded by the ′ region of the orf and is released from the initially-translated polyprotein by host signal peptidase cleavage at the c terminus. core is then further processed by signal peptide peptidase, resulting in a mature kda protein which forms homodimers and localises to cytosolic lipid droplets (for review see ) . the ns proteins forming the rna replication complex remain membrane-bound within an er-derived membranous web where rna replication occurs. there is accumulating evidence that the delivery of a nascent rna by its cognate replication complex to the site of virion assembly is required for assembly initiation (for review see , ) . however whether nucleocapsid formation and genome packaging occur at the lipid droplet, or whether this organelle merely acts as a storage site for core prior to its relocalisation back to the cytoplasmic face of the er (where assembly may occur), is a controversial topic . following assembly the nascent nucleocapsid buds into the er, acquiring the lipid envelope and the associated e -e viral glycoproteins prior to cellular egress (for review see ) . it is apparent that the rna replication and virion assembly processes are spatially distinct within the cellular microenvironment and interactions between the replication complex and the core protein are required for efficient packaging . consequently, the packaging of replication-defective genomes (which are not presented by a replication complex) is notoriously inefficient in hcv. it is this inability to study assembly without concurrent replication which has hindered the identification of rna structures contributing solely to assembly. the requirement for rna presentation by the replication complex may prevent encapsidation of defective and/or partial hcv genome fragments by core. the fact that cellular rnas are excluded from this process indicates additional selective factors must contribute. for many positive-sense rna viruses, the nucleocapsid assembly and genome packaging events occur simultaneously through recognition of a large stable rna structure (the packaging signal, ψ ) by their capsid protein. the essential role of these structural motifs within the viral rna in directing genome encapsidation has long been recognised . the fact that pre-formed "empty" hcv capsid-like particles have not been identified suggests that hcv utilises a similar mechanism of rna recognition to initiate assembly , although rna-free capsid-like structures have been isolated from in vitro translation systems . however, unlike prototypic packaging signals such as those found in the retroviruses, alphaviruses and coronaviruses, a single rna structure which is both essential and sufficient to target non-viral rnas to a nascent hcv nucleocapsid particle has not been identified. it is possible that hcv utilises a recently discovered novel mechanism of genome encapsidation during nucleocapsid assembly, utilising multiple, relatively low-affinity structures termed packaging signals (pss) , . we wished to investigate the possibility that similar specific secondary structures are present within hcv rnas destined for packaging, and whether their interactions with core cooperatively drive the rna encapsidation and nucleocapsid assembly processes. systematic evolution of ligands by exponential enrichment (selex) of rna aptamers against core. mature core consists of two distinct domains: the n terminal domain (d , residues), which is highly basic and binds rna, and the hydrophobic c terminal domain (d , residues) which possesses a membrane binding region. d stabilises core on the surface of lipid droplets and exposes the hydrophilic d to the viral rna, therefore it is essential for nucleocapsid formation in mammalian cells . however, in vitro d is sufficient to form stable proteinase-resistant nucleocapsids when stabilised by interactions with nucleic acids [ ] [ ] [ ] . we therefore prepared d as a selex target by recombinant protein expression. d hcv core (jfh- ) was expressed in e. coli and purified by nickel affinity chromatography against an n-terminal hexahistidine tag. the identity of the his-tagged protein was confirmed by lc-ms/ms of coomassie stained bands excised from an sds-page gel. the antigenicity of the final purified protein was also confirmed by immunoblotting for core d and the hexahistidine tag (fig. ) . this protein was used for selex with a ′ oh rna aptamer library encompassing a n random region. selex is an established process for the in vitro isolation of high-affinity aptamers: in vitro selected nucleic acid molecules which bind with high affinity and specificity to their target , . each aptamer within a randomised library possesses a unique tertiary structure, depending on the series of stems, pseudoknots, kinks and/or bulges which are present in its most stable conformation. each aptamer will bind with varying affinity to the ligand of interest. selex is therefore likely to enrich for aptamers with conserved conformation rather than a unique primary sequence. locations and consensus motif of putative packaging signals (ppss). the sequences present in the final round of selex were determined by next generation sequencing (ngs) of the pool and the individual aptamer sequences were aligned to the jfh- genome (genbank id ab . ). it should be noted that known pss in other ssrna viruses are sequence/structure degenerate and their capsid/coat protein recognition sequences are discontinuous and minimal , , . a consequence is that the pool of binders from selex should include a majority of oligonucleotides that, although matching essential features of hcv pss, are unlikely to match extended regions of the genome. despite this constraint, there are aptamers that match multiple sites within the genome and occur with statistical significance (a bernoulli score of or more; red peaks, fig. ) compared to the unselected naïve library (grey peaks, fig. ). this outcome suggests that the hcv genome does contain multiple ps sites. in order to test this hypothesis, the same procedure was applied to an additional genotype hcv genomes, i.e. they were compared to the aptamers selected against jfh- , and a similar picture emerged. this suggests that these ps sites are evolutionarily conserved. such peaks, within nucleotides of the scientific reports | : | doi: . /srep peaks in fig. , which were conserved for at least % of these additional strains, are highlighted by green arrows (fig. ) . for each of these conserved peaks the nucleotides ′ and ′ to the peak nucleotide in the jfh- genome were extracted, and a range of possible secondary structure folds were considered via mfold . a similarity analysis revealed the potential formation of stem-loops with similar loop motifs in each of these fragments. an alignment of the loop portions of these stem-loops is displayed in fig. a . this analysis reveals a bias towards a grrgr loop motif, r denoting a purine, suggesting that pss should exhibit this motif or close variations thereof. in order to establish the statistical significance of this motif, we compared its occurrence in the loop portion of stem-loops in the wild-type genome with that in randomised versions of the genome, both for jfh- and for the other strain variants considered here. this confirmed a significant statistical bias in the wild-type genomes for this motif. we then interrogated sub-motifs of grrgr in order to identify those with an even higher statistical significance, which revealed a ggrgg motif. the positions of stem-loops with this motif across the jfh- genome are displayed in fig. b . modeling indicates that an ensemble of packaging signals with different degrees of affinity for capsid protein is required to promote efficient assembly , . lower-affinity motifs may not necessarily be easily identifiable through a selex screen, yet their presence at statistically significant numbers within the wild-type genome statistically significant alignments of the aptamer pool with the jfh- genome correspond to peaks in the red curve that are above the corresponding alignment of the naïve library (grey curve). peaks occurring within nucleotides of statistically significant peaks in at least % of the representative set of hcv genomes analysed here are marked by a green arrow. would be indicative of a potential role in virus assembly. based on previously-published models, the statistical bias observed for the ggrgg motif would be predicted to encompass at most % of the putative packaging signals (ppss) . variations of the motif with statistical bias are the hallmark for pss with intermediate or weaker affinity for the capsid protein. the same statistical analysis was therefore used to interrogate other variations of the ggrgr motif, in an effort to identify sub-motifs that were more frequent in the wild-type genome than in the randomised version. the motif gxrxr fulfilled these criteria (see methods for details). mutagenesis to disrupt pps. since modeling suggests that ppss act collectively to promote virus assembly, an ensemble of ppss was selected for mutagenesis. the region encoding ns b was avoided as numerous studies have reported the existence of essential structures required for rna replication; mutations in this region would render the virus replication-defective , . modeling moreover implies that a combination of high and low affinity binders is important, i.e., a combination of ppss following the ggrgg motif and those that exhibit other variations of the grrgr motif: e.g. gxrxr . eight stem-loops were selected for mutagenesis, three of which contained the predicted highly conserved motif (ggrgg). the remaining five stem-loops contained the lower-affinity gxrxr motif identified above (fig. ) . the stem-loops are numbered according to the first paired nucleotide within the context of the jfh- genome, according to standard nomenclature. a range of silent mutations were introduced into the orf of hcv, using the infectious molecular clone pjfh- . these were designed to disrupt the base pairing within the stems of the ppss (fig. ) . it was not possible in the majority of cases to completely ablate favourable folding energies for the formation of stem-loops and even mutant sites with unfavourable folding free energy might still form in the presence of a protein ligand such as core. eight mutant pss were created, each possessing between and silent single nucleotide polymorphisms, collectively targeting an individual pps (fig. ) . these are referred to as the "single" pps mutants, Δsl , Δsl , Δsl , Δsl , Δsl , Δsl , Δsl and Δsl . all silent mutations were also combined into a single viral genome, termed Δ xsl, in order to examine co-operative effects between the altered ppss. replication and translation are unaffected in pps mutants. rnas encoding all single pps mutant viruses, the Δ xsl mutant, jfh- (wild-type) and jfh- -gnd (a polymerase-deficient control that is unable to undergo viral genome replication) were electroporated into huh- cells. the intracellular rna was quantified by qrt-pcr, at both and hours post electroporation (h.p.e) (fig. a) . impaired replication was not observed in mutant viruses containing impaired ppss, although Δsl possessed a slightly decreased replication rate which was not statistically significant. this was further confirmed by detection of the viral ns a protein compared to cellular gapdh expression via western blot (fig. b) . was titrated upon naïve huh- cells and the infectious titre calculated in infectious units per millilitre (iu/ml) as previously described . this reflects the number of infectious rna-containing virions which had formed and/ or been released from within the electroporated cells. non-rna containing particles, or aberrantly-assembled defective particles, therefore do not contribute to the viral titre as they are non-transmissible. at h.p.e., only the Δ xsl mutant virus displayed significantly decreased titres compared to the jfh- wt control, although Δsl and Δsl also appeared non-significantly decreased (fig. a) . the reduction in Δ xsl infectious titre was consistent across the entire hours, as evidenced by time course assays during which virus was harvested every hours post-electroporation to assess the rate of virion formation and release (fig. b) . this provides evidence that although mutating each pps independently has no apparent effect, the combined disruption of all ppss affects the late stages of the viral life cycle (assembly and/or egress) whilst not affecting rna replication. rna secondary structures are altered within the packaging signal mutants. to confirm that the introduced mutations caused structural alterations, in vitro transcripts from a section of the ns b-coding region from jfh- (wt) and Δ xsl mutant genomes were synthesised. rna was folded and selective ′ -hydroxyl acylation analysed by primer extension (shape) mapping was performed as previously described . briefly, folded rna was treated with a compound which reacts preferentially with single-stranded, flexible regions. this reaction forms an irreversible adduct which causes premature termination during the subsequent reverse transcription of the rna . primer extensions using radiolabelled primers were conducted and the resulting fragments were resolved on a % polyacrylamide denaturing gel. the terminations, indicative of a flexible nucleotide, were visualised and their reactivity normalised to that of a dmso-treated control rna. the specific location of such terminations is determined by comparison to ddntp sequencing ladders. a high shape reactivity is indicative of a flexible nucleotide, available for acylation by the compound; hence only the terminal loop of a prototype stem-loop structure would appear as "reactive" nucleotides. the hcv genome is dynamic and multiple rna conformations are potentially able to form, including kissing-loops, pseudoknots, g-quadruplexes and other long-range interactions . additionally the active folds of ppss may only form in the presence of an rna chaperone such as core or ns a, which is absent during shape mapping. the shape data therefore indicates whether the engineered mutations altered the rna flexibility, rather than confirming the mfold-predicted structure of a particular motif. the reactivity profile across the region encompassing sl provided evidence that the silent mutations had altered the flexibility of the mutant rna compared to wildtype (fig. ) . together our data indicates the putative packaging signals which we identified may form within the hcv genome, presumably during assembly. a mutant genome unable to form such structures displays impaired viral infectivity whilst rna replication is unaffected, indicating these motifs may interact specifically with core. the synergistic effect of these individual interactions may ensure encapsidation of a single viral genome occurs during virion assembly, thereby preventing non-specific packaging of cellular rnas. the mechanism by which the hcv core protein selectively binds and encapsidates viral genomic rna during virion assembly is currently unknown. in contrast to other positive-strand rna viruses , there is no evidence for a single cis-acting rna structure capable of directing packaging of both viral and non-viral rnas to a nascent capsid particle. therefore it is plausible that multiple rna structures contribute to rna-core binding and act cooperatively to drive encapsidation and assembly. the presence of multiple, weak-affinity packaging signals in other rna virus genomes has recently been reported , , which represents a novel mechanism for viral packaging in viruses which do not possess a readily identifiable prototypic packaging signal. here, we provide evidence that the abrogation of short motifs (ppss) located across the hcv genome has a significant effect on rna encapsidation, manifested by a decrease in infectious titre. this is only apparent in the multiple pps mutant (Δ xsl), consistent with rna packaging being a cooperative process in the hepaciviridae. the fact that depletion of all ppss was required before a significant packaging phenotype was observed may explain why previous mutagenic screens have been unsuccessful in identifying these novel motifs -if the ppss act synergistically, a threshold of ablation must be reached before significant phenotypic changes are observed. additional low-affinity ppss presumably also exist which were not mutated in this analysis. there are multiple occurrences of our described motifs within the jfh- genome which were not identified by our original aptamer screen; it may be that a large number of functionally redundant ppss exist and hence are able to easily compensate for many mutations which may occur during the error-prone rna replication process in vivo. in vitro transcripts of the ~ . kb region encompassing sl and sl were synthesised from both jfh- and mutant templates. rna was folded and subjected to structural analysis via shape mapping. following nmia (+ ) or dmso (− ) treatment and reverse transcription, a primer designed to bind approximately nucleotides downstream of sl was used for primer extension. products were resolved on a % denaturing polyacrylamide gel (upper panel) and the relative reactivity was quantified using safa . sequencing ladders were included to identify individual nucleotides, based upon the wildtype genome sequence. previous models of assembly which utilise multiple contributing packaging signals predict that highly efficient capsid assembly occurs in the presence of one higher-affinity rna structure; initial binding of this motif to the structural protein then instigates additional multimerisation and concurrent interactions with the lower-affinity packaging signals . the relative affinities of each of the ppss described in this study for the core protein would therefore be of significant interest. prior to the development of the infectious jfh- cell culture system, models of hcv virion assembly relied upon either self-assembled capsid-like particles generated from purified recombinant protein , , , or by expression of core in yeast , insect cells or bacterial systems followed by purification of the intracellular capsid-like particles. the formation of such regular capsid-like particles in these protocols requires the presence of structured rna, proving that protein-protein interactions are insufficient to drive core multimerisation. it is now recognised that these models do not reflect the assembly process in mammalian cells, therefore although investigating the binding kinetics of each pps and its respective mutant would be of significant interest, future work may have to utilise alternatives to such in vitro assembly models. additionally, the fact that intact hcv genomes cannot be packaged in in vitro assembly systems supports our model wherein the structures of interest may be formed temporarily and potentially only in certain scenarios, such as during presentation of nascent rna within a replication complex. the ns a protein (an essential component of the viral replication complex) possesses a broad rna binding spectrum and interacts specifically with core protein ; this combined with the fact that core exhibits extensive rna chaperone activity , suggests a highly plausible model of rna conformational alterations occurring during rna transfer and subsequent assembly. it is well established in the literature that core binds to the viral ′ utr , [ ] [ ] [ ] [ ] , although this interaction is not sufficient to drive genome packaging . we did not focus upon this region as its interactions with core are already well-explored; the preferred core-rna binding motif is the iiid loop and this interaction inhibits ires-mediated translation , . this motif is the only region of the ires which possesses similar features to the stem-loops selected for mutagenesis; specifically, the internal bulge and a g-rich stretch within the unpaired terminal loop. this suggests that multiple structures, each containing this motif in their terminal loop, may be required to interact with core during rna encapsidation. as this ires subdomain also forms long-range interactions with cres in the ns b coding region , , a model may be envisaged in which packaging, rna replication and translation are mutually exclusive events, partially dictated by the ligand bound to the ires iiid loop (core, cre rna or the ribosomal complex). the majority of our mutated ppss are within the non-structural protein coding regions, with the exception of sl . trans-packaging of viral subgenomic replicons has been extensively documented (for review see) and it has been noted by other research groups that the functionality of these systems reflects the lack of essential cis-acting rna elements within the core to ns genomic region . this is supported by our data wherein only of the mutated ppss are located in this area (sl and sl ); their removal may not reach a threshold level to affect packaging efficiency. trans-packaging studies utilising baculovirus-mediated structural protein expression also found that although the presence of the ns protein improves the production of replicon-containing particles, this was most apparent when ns is expressed in cis (within the replication-cassette), rather than in trans (within the structural protein construct) . although this may be due to the protein-protein interactions required for virion assembly , the fact that the pps sl is present upon this particular replicon may increase the efficiency of packaging, resulting in higher titres of virus-like particles in this system. in a parallel scenario, naturally occurring mutants with deletions spanning e -p have been found in multiple patient samples; as these subgenomic mutants may be packaged and released , an essential packaging signal is definitively not located in those coding regions. given the well-recognised roles of rna structure in the hcv life cycle, it is important to highlight the novelty of the mutated structures in this report as many previously-annotated structures were not altered by our silent mutations, including the ns b-located cres , , . in addition, the genome of hcv possesses genome-scale ordered rna structure (gors); a phenomenon amongst particular rna viruses which exhibit extensive rna structure across the entire genome . consequently the genome is constrained in its plasticity and evolutionary potential, in contrast to the predicted behaviour of a rna virus. it has been suggested that gors contributes to viral persistence or modulates host innate defence mechanisms. it therefore must be considered that these pps structures may additionally contribute to the overall architecture of the hcv genome and may not be solely involved in the core-rna interactions during packaging -they may have multiple functions, requiring distinct structural conformations to be adopted at precise points during the viral replication cycle. the Δ xsl mutant may represent a virus which has reached a threshold level of gors interruption. however this is unlikely; an equivalent effect upon replication and assembly would be predicted if this were the case. it is therefore apparent that our analysis does not merely reflect rna structures already annotated within the hcv genome, but rather the identification of novel structures utilised for core-rna binding. recombinant protein expression. core (d ) of jfh- was cloned into the pet b(+ ) construct (novagen), in-frame with a n-terminal hexahistidine tag. recombinant protein was expressed in bl [de ] cells (f-ompt hsdsb(rb-mb-gal dcm (de )). luria broth media ( % glucose) was inoculated with a : dilution of starter culture and maintained at °c until an od of . - . was reached. protein expression was then induced by the addition of mm isopropyl β -d- -thiogalactopyranoside and cultures were maintained at °c for hours whilst shaking, before being harvested by centrifugation. to an equilibrated ni + sepharose resin column (ge healthcare). resin was washed ( mm tris-hcl ph . , mm nacl, % glycerol, . % β -mercaptoethanol, mm imidazole) and the protein was eluted into wash-based buffer with mm imidazole. protein was dialysed overnight into pbs and quantified by spectrometry. sds-page was conducted upon both bacterial lysates and eluted protein fractions according to previously described methods using % gels. silver staining was conducted according to manufacturer's instructions (silverquest kit, invitrogen). immunoblots were conducted with an in-house rabbit anti-core polyclonal sera ( : ) , commercial anti-his monoclonal antibody ( : , invitrogen) and hrp-labelled secondary antibodies. for protein identification, coomassie stained bands were excised, reduced, alkylated and digested with trypsin. the recovered peptides were analysed by lc-ms/ms and subsequent searching of the tandem ms data against the uniprot sequence database. recombinant d ( mg) was immobilised via the his-tag to mg of dynabeads his-tag isolation and pulldown magnetic beads (thermofisher scientific) following the manufacturers protocol. excess protein was removed with washes of mm sodium phosphate ph , m nacl, . % (v/v) tween- . bead immobilised protein was then washed times with selection buffer (pbs, . % (v/v) tween- , × roche complete protease inhibitor per ml). a biomek laboratory automation work station (beckman coulter) was used to perform rounds of in vitro selex as described previously , using a n ′ oh rna library. this starting library was synthesised according to an experimentally and iteratively optimised protocol. it was purchased from aptait (munich, germany) who performed three rounds of synthesis, next generation sequencing (ngs) and statistical analysis of ngs data from the library using the compas software. in doing so the random region of the library has been optimised in respect of an equal distribution of nucleotide building blocks ( % of g, c, a & t, at each position), as well as in respect of a gaussian distribution of motifs of length - nucleotides. an equal distribution of nucleotides increases the sequence space on the level of shorter motifs, which finally mediate binding to the target of interest. consequently, the chance for a successful selex experiment increases with a homogeneous random region. here the library was initially transcribed with a hiscribe t high yield rna transcription kit (new england biolabs). negative selections were carried out at each round of selex using bare his-tag isolation and pulldown beads. the stringency of the aptamer-target interaction was raised by increasing the number of washes from to (slower off-rate) and by decreasing the amount of bead-immobilised protein by half (faster on-rate). selections were carried out at °c. following the final round of selex, aptamers were analysed by ngs using a miseq desktop sequencer (illumina). the selex library was prepared using the miseq reagent kit following the manufacturer's protocol. raw sequencing reads were processed using in-house scripts to identify sequences that contained correct ′ and ′ primers. processed sequences were used for packaging signal identification (see below). potential packaging signal identification. the following hcv genotype genome sequences were extracted from genbank: accession numbers ay ; ay ; ay ; d ; d ; dq ; ab . ; ab . ; hm ; ab ; af ; af ; af ; ay ; ab . . the final selected aptamer library contained , unique sequences, each nucleotides in length that were aligned against the jfh- genome as follows: each aptamer sequence was slid along the genome in increments of nucleotide. for each such position of the reference frame, the subset of the aptamer sequence with the best alignment to the genome was identified according to the bernoulli score b, which benchmarks the probability of a non-contiguous alignment to that of a contiguous alignment of b nucleotides. the bernoulli scores for all reference frames of a given aptamer sequence in the library were rank-ordered, starting from the largest score, and all matches with the genome up to a bernoulli score of counted. the procedure was then repeated for the other aptamer sequences, and corresponding matches added, resulting in the peaks in fig. . identification of a consensus motif. this analysis was repeated for all genomes listed above. peaks which occurred for at least % of the genomes within < nucleotides of each other were marked by a green arrow to indicate their conservation. these were used as a basis to identify the highly conserved pps recognition motif. for this, sequences of nucleotides, centred around the peak nucleotide, were extracted and all possible mfold predicted folds were considered. a similarity analysis of these stem-loops was performed, comparing both sequence and structure elements. the foldings of each peak area which had the highest degree of similarity with secondary structure elements in the other peaks was identified. this returned a stem-loop for each peak; an alignment of the corresponding terminal loop sequences is displayed in fig. . in order to identify which features of the consensus motif are statistically significant, the number of occurrence of stem-loops with different types of sub-motifs in the genome was calculated and benchmarked against their occurrence in randomised versions of the genome. the consensus motif was grrgr; this motif was . times more likely to occur in the wildtype than randomised genomes. in order to refine this motif, we performed similar statistical tests for submotifs of this consensus motif; we identified ggrgg and gxrxr motifs as being more likely to occur in the wildtype genome than in a randomised version, by a factor of . and . , respectively, suggesting that ppss with the former motif could correspond to high affinity pss, and those with the latter motif could correspond to lower affinity pss. mutagenesis of pjfh- . the dna construct containing the jfh- viral genome (pjfh- ) and a replication-defective control mutant of this plasmid (termed pjfh- -gnd, possessing a gdd > gnd mutation within the ns b rna-dependent-rna-polymerase active site) have been described previously . mutagenesis of pjfh- to introduce silent mutations was performed by either site-directed mutagenesis (quikchange, agilent) or through overlap pcr utilising mutagenic primers (primer sequences available upon request). the entire genome of all mutant viruses was confirmed through sanger sequencing to ensure additional mutations were not present. scientific reports | : | doi: . /srep in vitro transcription. dna constructs were linearised with xbai, briefly treated with mungbean nuclease to degrade ′ overhangs (new england biolabs) and purified by phenol-chloroform extraction. linearised dna was used as a template for in vitro transcription to produce full-length hcv genomic rna (ribomax express; promega, as per the manufacturer's instructions). following dnase digestion, rna transcripts were purified using silica-gel columns (rneasy, qiagen) and quantified by absorbance at nm prior to transfection into mammalian cells. mammalian cell culture. huh- cells were maintained in dulbecco's modified eagle's medium (dmem; sigma) supplemented with % foetal bovine serum (fbs), iu/ml penicillin, μg/ml streptomycin, mm hepes and % (v/v) non-essential amino acids in a humidified incubator at °c in % co . infectious hcv assays. . × huh- cells were electroporated with μg of viral rna at μf and v for ms. cells were resuspended in complete medium and seeded into multiple well plates ( . × cells per well) (corning). hours post electroporation (h.p.e.), cells were harvested with trizol (invitrogen life technologies) for rna quantification. h.p.e., all remaining wells were washed with pbs to remove excessive extracellular input rna and the media replaced. h.p.e., cells were harvested in trizol, protein lysis buffer or pbs, for rna quantification, protein detection and intracellular virus titration, respectively. virus supernatant was harvested at h.p.e. for rna quantification and released infectious virus titrations. intracellular virus was isolated by repeated freeze-thaw cycles of pbs cell lysates followed by clarification. virus titres were calculated according to previously reported methods . briefly, either viral supernatant or cellular lysates were serially diluted two-fold upon naïve huh- cells in well plate format, seeded hours prior to infection. at hours post-infection, cells were washed, fixed with % paraformaldehyde and the viral ns a protein was detected via indirect immunofluorescence. automated counting was performed using the incucyte zoom (essen bioscience) using previously-described parameters . quantitative reverse-transcriptase pcr. rna was purified by trizol extraction and diluted to ng/ul. ng was used in a taqman probe-based one-step qrt-pcr according to previously-reported methods . to quantify rna, cycle threshold (ct) values were converted to genome copies per ng of input rna by comparison to a standard curve of serially-diluted pjfh- ( to copies). virion assembly and release replication of subgenomic hepatitis c virus rnas in a hepatoma cell line production of infectious hepatitis c virus in tissue culture from a cloned viral genome dengue virus-and hepatitis c virus-induced replication and assembly compartments: the enemy inside-caught in the web hepatitis c virus rna replication and assembly: living on the fat of the land hepatitis c virus budding at lipid droplet-associated er membrane visualized by d electron microscopy hepatitis c virus: assembly and release of virus particles the lipid droplet is an important organelle for hepatitis c virus production cis-acting genomic elements and trans-acting 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trans-encapsidation of hepatitis c virus rnas into infectious virus-like particles expression of hepatitis c virus (hcv) structural proteins in trans facilitates encapsidation and transmission of hcv subgenomic rna hepatitis c virus p and ns proteins are essential for production of infectious virus genetic analysis of hepatitis c virus with defective genome and its infectivity in vitro naturally occurring hepatitis c virus subgenomic deletion mutants replicate efficiently in huh- cells and are trans-packaged in vitro to generate infectious defective particles detailed mapping of rna secondary structures in core and ns b-encoding region sequences of hepatitis c virus by rnase cleavage and novel bioinformatic prediction methods detection of genome-scale ordered rna structure (gors) in genomes of positive-stranded rna viruses: implications for virus evolution and host persistence serine phosphorylation of the hepatitis c virus ns a protein controls the establishment of replication complexes growth of human hepatoma cells lines with differentiated functions in chemically defined medium real-time detection system for quantification of hepatitis c virus genome semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments the authors would like to thank dr iain manfield (university of leeds), dr james ault (university of leeds) and dr sally harrison (leeds institute of molecular medicine next generation sequencing facility) for their help and advice regarding protein expression, mass spectrometry and sequencing, respectively. we also wish to thank dr michael blank of aptait gmbh, munich, for the provision of the n ssrna library used for selex and helpful discussions about the preparation of unbiased starting libraries for selex. this work was supported by a wellcome trust investigator award to mh (grant number ) and by financial assistance from the universities of leeds and york. rt acknowledges funding via royal society leverhulme trust senior research fellowship (lt ) and epsrc grant ep/k / for rjb. ecd has been funded via a leverhulme trust research fellowship (ecf- - ). key: cord- -gtg hiu authors: prather, randall s.; wells, kevin d.; whitworth, kristin m.; kerrigan, maureen a.; samuel, melissa s.; mileham, alan; popescu, luca n.; rowland, raymond r. r. title: knockout of maternal cd protects fetuses from infection with porcine reproductive and respiratory syndrome virus (prrsv) date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: gtg hiu after infection of the porcine dam at about days of gestation, porcine reproductive and respiratory syndrome virus (prrsv) crosses the placenta and begins to infect fetuses. outcomes of include abortion, fetal death and respiratory disease in newborn piglets. cd is the receptor for the virus. in this study, cd -positive fetuses, recovered between days of gestation or days after maternal infection, were completely protected from prrsv in dams possessing a complete knockout of the cd receptor. the results demonstrate a practical means to eliminate prrsv-associated reproductive disease, a major source of economic hardship to agriculture. porcine reproductive and respiratory syndrome (prrs) is the most economically important disease of swine in north america, europe and asia, costing north american producers approximately $ million annually . losses are the result of respiratory disease in young pigs, poor growth performance, reproductive failure, and in utero infection . the reproductive form of the disease accounts for an estimated % of losses, the result of abortions, dead fetuses, and respiratory disease in newborns. in its severest form, reproductive prrs can result in % mortality of fetuses/neonates, along with increased mortality for the dams. pigs that survive in utero infection become continuous sources of virus in downstream production phases, resulting in endemically infected herds . the severest form of reproductive disease is associated with a group of highly virulent isolates referred to as atypical prrsv , . interestingly, many of the atypical prrsv isolates emerged from prrs-vaccinated farms . in , an atypical virus, called high pathogenic prrsv (hp-prrsv), appeared in china and continues to decimate pig populations in that country . since the standard commercial breeding facility contains about , sows, an outbreak of high mortality reproductive prrs can have a devastating impact. to ensure sustainability of pork production and food security, solutions for the control of reproductive prrs remain a priority. vaccines have been unable to control the disease, largely because of genetic diversity within the structural proteins of the virus . in practice, intensive biosecurity measures provide the only means of protecting the reproductive herd. along with lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv), prrsv belongs to the family, arterviridae. structurally, the arteriviruses resemble togaviruses, but similar to coronaviruses, replicate via a nested ′-co-terminal set of subgenomic mrnas, which possess a common leader and a poly-a tail. arteriviruses exhibit a tropism for macrophages and possess the capacity to establish subclinical persistent infections, as well as cause severe and fatal disease . the reproductive form of prrs occurs following the infection of pregnant gilts or sows at about days of the day gestation period , . after an initial phase of replication in maternal macrophages, the virus crosses the placenta and begins to productively infect fetuses. the virus initially infects only a small number of fetuses, followed by horizontal transmission of virus from fetus to fetus . the exact mechanism of how the virus crosses the placenta remains unknown, but could be similar to the infected "trojan horse" macrophage, previously described for ldv . unlike the alveolar macrophages in adult animals, the primary site of prrsv replication in the fetus is the thymus . since the pig fetus becomes immunocompetent at about days of gestation, prrsv infection occurs in a fetal immune environment containing functional b and t cells , . cd is a kda type membrane protein composed of nine scavenger receptor cysteine-rich (srcr) domains and two spacer domains along with a transmembrane domain and a short cytoplasmic tail. in addition to functioning as a virus receptor, cd exhibits several important functions related to maintaining normal homeostasis. for instance, following infection or tissue damage, cd functions as a scavenger molecule, removing haptoglobin-hemoglobin complexes from the blood . the resulting heme degradation products regulate the associated inflammatory response . cd as a receptor for prrsv was first described by calvert et al. . transfection of non-permissive cell lines with cd cdna from a variety of species, including simian, human, canine, and mouse can make cells permissive for infection. we recently showed that pigs with a complete knockout (ko) of the cd gene lack cd expression on macrophages and fail to support prrsv infection , . since cd expression is a dominant trait and inherited in a classic mendelian fashion, offspring possessing normal cd expression and function can be derived by crossing a ko cd −/− female pig with a wildtype (wt) cd +/+ male. for this study, cd ko gilts were bred with wt boars producing heterozygous, cd +/− fetuses. the hypothesis to be tested was that the presence of the cd ko genotype of the dam would be sufficient to protect fetuses following maternal infection with prrsv. a detailed description of the knockout alleles used in this study is shown in table . each knockout allele possessed a mutation in exon that was predicted to result in a codon frameshift followed by a premature stop codon in the mrna. the matings between wt and cd ko parents are summarized in table . the first group of three dams, which served as positive infection controls, were cd +/+ dams carrying cd +/+ fetuses (++/++ group). a second group (−/+−) were cd −/− dams carrying cd +/− fetuses. in this group, the cd −/− dams are unable to support prrs replication, while the cd +/− fetuses retain susceptibility to prrs infection. and finally, a third group (−/−) consisted of cd −/− dams carrying cd −/− fetuses. for the last group, both dams and fetuses should be resistant to infection. clinical signs in the infected wt dams included lethargy and transient inappetance. the ko dams showed no clinical signs. during the study period, one wt dam, no. , aborted on day of gestation ( dpi). prrsv nucleic acid, measured at dpi, showed a viremia level for dam no. of . log templates per reaction, demonstrating the presence of a productive prrsv infection. between and dpi, all remaining dams were euthanized and uterine horns immediately removed. beginning at the tip of each horn, fetuses and placentas were removed, assessed for the presence of anatomic pathology. a blood sample was obtained from each fetus. if blood was not obtainable, a sample of fluid was collected from the abdominal cavity. the number of fetuses recovered from each dam is listed in table . for the cd wt group (++/++) (including the dam that aborted), the number of fetuses were , and (mean = . ). the cd ko dams carrying the cd +/− fetuses (−/+− group) yielded , and fetuses (mean = . ). for the cd ko dams carrying cd ko fetuses (−−/−−group), the numbers of fetuses were and . the results for fetal viremia and gross pathology are summarized in fig. and table . at the anatomic level, % and % of fetuses derived from the two cd wt (++/++) dams, no. and no. , showed some degree of pathology, including smaller than normal fetuses ( % of all fetuses), fetuses with detached or necrotic placentas ( %), meconium staining ( %), and fetuses table ). below each dam in parentheses is the result for prrs pcr in serum, measured as log templates per reaction. "n" is negative for prrsv nucleic acid (ct > ). fetuses are identified by number and relative position within each uterine horn. asterisks identify fetal pcr samples obtained from abdominal fluid. the number below each fetus is the result for prrs pcr in fetal serum (log templates per reaction). the number within each circle refers to the presence of anatomical pathology: ) normal fetus; ) small fetus; ) placental changes, such as detached placenta and/or necrosis; ) meconium stained fetus; ) fetus is dead and necrotic. lower case letters identify the genotype of the individual fetuses (see table table and fig. . * viremia shown as log virus nucleic templates per pcr. * fetuses showing pathology as described in the legend in fig. . * gilt aborted prior to recovery of fetuses. infection. first, there was a wide variation between fetuses in the concentration of virus detected in serum, the result of fetuses becoming infected at different times. secondly, the level of viremia was not always correlated with pathology. for example, fetus no. from dam no. possessed a high level of viremia ( . log templates per reaction) and yet the fetus appeared unaffected. the reason for the discrepancy between viremia and the pathology is unclear. one possibility is that fetal pathology is the result of tissue damage that occurs on the maternal side and not related to the level of fetal viremia. in the field, these normal, but infected newborn piglets can function as "supershedders", which facilitate the rapid dissemination of prrsv throughout a production system. for the −−/+− group (dams no. , and ), all fetuses appeared normal, with the minor exception of two fetuses that were smaller than the other littermates. the smaller than normal size is likely a consequence of crowding within the uterine horn that decreases the surface area of the placenta, thus restricting the growth of the developing fetus. all dams and fetuses in the −−/+− group were negative for the presence of prrsv nucleic acid. for the last group, −-/−−, there was no visible pathology, and all dams (no. and ) and fetuses were negative for prrsv nucleic acid. the results from this study clearly demonstrate that the absence of cd in the dam is sufficient to protect the prrsv-susceptible fetus. although cd -positive offspring derived from cd ko dams are susceptible to virus immediately after birth, the protection from prrsv in utero provides a means to eliminate a major source of economic loss and animal suffering. cd gene modification. the crispr/cas methods used to generate all of the ko alleles are described in detail in whitworth et al. . the specific edits for alleles a, b, d and e are described in whitworth et al. . the specific edit in allele c ( bp insertion) is described in whitworth et al. . the alleles, described in table were identified based on dna sequencing. the knockout genotype was confirmed by the absence of cd expression, which was measured by staining alveolar macrophages with anti-cd mab, a , as described in wells et al. prrsv infection. the prrsv strain used in this study, nvsl - (nvsl), is a laboratory strain isolated in from a herd in southeast iowa, usa that was experiencing a prrs abortion storm . the virus, maintained as a low passage isolate, was propagated and titered on marc- cells. at to days of gestation, gilts were inoculated with tcid of virus diluted in ml of culture medium. one half of the inoculum was administered by intramuscular injection and the remainder administered intranasally. all gilts were maintained in an environment that allowed for the continuous exposure to virus shed by infected pen mates. blood samples were taken from the gilts prior to infection, days post-inoculation (dpi), and at the time of euthanasia. prrsv nucleic acid was measured by isolation of total rna from serum followed by reverse transcriptase real-time prrsv pcr (tetracore, rockville, md). a standard curve was generated using the quantification standards supplied in the rt-pcr kit. results were reported as log templates per µl reaction, which approximates the number of viral rna templates per ml of blood. ethics statement. experiments involving animals and virus were performed in accordance with the federation of animal science societies guide for the care and use of agricultural animals in research and teaching, the usda animal welfare act and animal welfare regulations, or according to the national institutes of health's guide for the care and use of laboratory animals, and were approved by the kansas state university and university of missouri institutional animal care and use committees and institutional biosafety committees. animals were humanely euthanized by pentobarbital overdose following the american veterinary medical association (avma) guidelines for the euthanasia of animals, and all efforts were made to minimize suffering. assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers reproductive failure of unknown etiology lymphotropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero update on abortion storms and sow mortality clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical genetic variation and phylogenetic analyses of the orf gene of acute porcine reproductive and respiratory syndrome virus isolates emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses lactate dehydrogenase-elevating virus and related viruses experimental reproduction of swine infertility and respiratory syndrome in pregnant sows the interaction between prrsv and the late gestation pig fetus genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus identification of the haemoglobin scavenger receptor the macrophage scavenger receptor cd cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus substitution of porcine cd srcr domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome (prrs) viruses use of the crispr/cas system to produce genetically engineered pigs from in vitro-derived oocytes and embryos funding for this project was from genus, plc and food for the stcentury at the university of missouri. r.p., k.d.w., k.m.w., m.k., m.s., a.m., l.p., r.r. wrote, critiqued, and edited the manuscript text. r.r. prepared the figure, r.r. and k.m.w. prepared the tables. all authors reviewed the manuscript. competing interests: alan mileham is employed by genus plc, the company that provided funding for the project. randy prather is an inventor on a patent related to the cd knockout pig. the remaining authors, kevin wells, kristin m. whitworth, maureen kerrigan, melissa samuel, luca popescu, raymond rowland declare no potential conflict of interest.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -xty m w authors: marrugal-lorenzo, josé a.; serna-gallego, ana; berastegui-cabrera, judith; pachón, jerónimo; sánchez-céspedes, javier title: repositioning salicylanilide anthelmintic drugs to treat adenovirus infections date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xty m w the repositioning of drugs already approved by regulatory agencies for other indications is an emerging alternative for the development of new antimicrobial therapies. the repositioning process involves lower risks and costs than the de novo development of novel antimicrobial drugs. currently, infections by adenovirus show a steady increment with a high clinical impact in immunosuppressed and immunocompetent patients. the lack of a safe and efficacious drug to treat these infections supports the search for new antiviral drugs. here we evaluated the anti-adenovirus activity of niclosanide, oxyclozanide, and rafoxanide, three salicylanilide anthelmintic drugs. also, we carried out the cytotoxicity evaluation and partial characterization of the mechanism of action of these drugs. the salicylanilide anthelmintic drugs showed significant anti-adenovirus activity at low micromolar concentrations with little cytotoxicity. moreover, our mechanistic assays suggest differences in the way the drugs exert anti-adenovirus activity. niclosamide and rafoxanide target transport of the hadv particle from the endosome to the nuclear envelope, whilst oxyclozanide specifically targets adenovirus immediately early gene e a transcription. data suggests that the studied salicylanilide anthelmintic drugs could be suitable for further clinical evaluation for the development of new antiviral drugs to treat infections by adenovirus in immunosuppressed patients and in immunocompetent individuals with community-acquired pneumonia. anti-adenovirus activity of salicylanilide anthelmintic drugs. in initial studies, we determined the inhibitory concentrations of the three anthelmintic drugs against hadv in a plaque assay measured as the percentage of plaque formation inhibition compared to a control cells infected in the absence of drugs. the three salicylanilide anthelmintic drugs showed a dose-dependent anti-hadv activity against both hadv and hadv , with % inhibition of plaques formation at . , and . μm for nic, oxy and raf, respectively ( fig. a,b) . the ic values for the three anthelmintic drugs are summarized in table . we also evaluated whether these salicylanilide anthelmintic drugs were able to inhibit virus production using hadv , hadv and hadv yield reduction assays. treatment with nic, oxy and raf was associated with overall reductions in virus yield from to -fold (table ) . in an assay measuring antiviral activity of the three anthelmintic drugs as a function of moi, inhibition was inversely proportional to the number of input particles (fig. ). while they were also inhibitory at high moi, the relationship between the number of infecting hadv particles and the drugs concentration is less marked, as expected. the cellular cytotoxicity of the salicylanilide drugs was also analyzed. the cc values for these molecules were in all cases significantly higher than the ic concentrations required for inhibition in our antiviral activity and mechanistic assays for both β cells (table ) and a cells ( . ± . µm, . ± . µm and . ± . µm for nic, oxy and raf, respectively). step in hadv replicative cycle. as the first step toward identifying the specific step in the hadv replicative cycle that was inhibited by these drugs, we measured the time dependence of the three anthelmintic drugs addition on their ability to block hadv infection. a previous report showed biochemically that hadv viral particles were internalized within min of binding and reach the nuclear pore after min . our results demonstrated that nic, oxy and raf exhibited a time-dependent decrease in their inhibitory activity (fig. a) . all of them showed high inhibition of hadv infection when added at the beginning of the min incubation at °c (− min), and when added immediately prior to warming ( min) or after min. however, when drugs were added at or min, only oxy still showed a significant inhibition higher than % which was lost for nic and raf (fig. a) . to support these findings a western blotting for hadv e a protein was run at h post infection. results obtained showed a significantly lower expression of e a protein in samples treated with nic and raf compared with oxy (fig. b) . impact on hadv genome accessibility to the nucleus. this assay evaluated the impact of the salicylanilide anthelmintic drugs on hadv endosomal escape. after the binding and internalization of the hadv viral particles, the exposure of protein vi inside the endosome triggers endosomolysis and the partially decapsidated hadv escape to the cytosol from where it is transported to the nuclear pore complex at the nuclear membrane by the microtubule network. we used a functional assay comprising hadv-mediated co-delivery of α-sarcin in cells as a marker of the ability of these salicylanilide drugs to alter virus-mediated endosomolysis , . no significant differences in the id ( % inhibitory doses) for hadv-mediated endosome penetration were detected in the presence of any of the salicylanilide drugs ( fig. ) or the dmso negative control. in contrast, the control virus ad ts , an endosome penetration-defective mutant, exhibited a significant increase in the id compared to the dmso control (fig. ) . once viral particles leave the endosome they are transported to the nuclear pore complex where hadv genomes are imported into the nucleus . we hypothesized that if any of the drugs examined was blocking a step in the hadv entry, as expected for nic, the number of genomes that reach the nucleus would be lower. we next evaluated the ability of these salicylanilide drugs to block hadv genome accessibility to the nucleus by the quantification of hadv genomes isolated from the nucleus . as reflected in fig. a , only nic and raf showed a significant block in the accessibility of hadv genomes to the nucleus (fig. a) . the dna copy number of the cellular gene gapdh was also evaluated in both the nucleus and the cytoplasm as a control for the purity of nuclear isolation (data not shown). since our results indicated that nic and raf hindered the accessibility of hadv genome to the nucleus while oxy did not, we resolved to determine if this anthelmintic drug affected later steps in hadv life cycle. impact on hadv replication. the next step was to examine the effect that oxy had upon efficient hadv dna replication using quantitative real-time pcr (qpcr). a cells were infected with hadv and incubated for h at °c. then, the unbound virions were washed-out and cell cultures were incubated for h. hadv dna was extracted at that point to limit the interference caused by subsequent rounds of infection occurring - h post-infection . we used quantitative pcr to quantify in a single round of infection the synthesis of new hadv dna copies as a measure of dna replication efficiency. the presence of oxy ( μm) significantly inhibited hadv dna replication by more than % (p < . , dunnett's multiple comparison test), whilst the quantification of the dna copies of the gapdh gene did not showed a significant effect (fig. b ). this reduction in the hadv dna copy number at the nucleus in the presence of oxy implicated two alternatives for its mechanism of action: i) oxy could inhibit hadv dna replication by interfering with a protein required in this process like hadv dna polymerase, dna binding protein (dbp) or terminal protein (tp) or, alternatively, ii) oxy could impact transcription of the hadv immediate early gene e a, which is a key step before dna replication. to assay the inhibition of hadv mrna transcription, we infected a cells in the presence of oxy ( μm) for h. after the infection, we quantified the rna copy number of the e a gene using quantitative reverse transcription (rt-pcr). as shown in fig. c , oxy exerted a significant decrease in the e a mrnas copy number compared with the control treated with dmso. based on previous works where piperazine derivatives and nucleotide and nucleoside analogues showed antiviral activity against multiple dsdna virus including cytomegalovirus (hcmv) and hadv , , we evaluated the inhibitory activity of nic, oxy and raf on hcmv dna replication. the presence of these anthelmintic drugs generated significant reductions in the quantification of total hcmv dna h after infection of mrc- cells (fig. d ). oxy showed a % decrease whilst nic and raf reached a . % and a . % decrease, respectively of hcmv dna replication, showing no significant differences between samples for the quantification of the gapdh gene (data not shown). salicylanilide anthelmintic drugs combination improves their antiviral activity. since we found different mechanisms of action for these three drugs we hypothesize that their combination should significantly improve their antiviral activity. to evaluate our hypothesis we conducted a combination study based on the chou-talalay method for drug combination using the calcusyn software , . the constant ration for each combination was selected based on the ic values for each drug. the data for all the combinations showed good conformity to the mass action law (r = . - . ) ( table ). all the combinations were classified as synergistic (table ). for the nic:raf (ratio : ) combination the ic and ic levels of inhibition were classified as synergism and the ic level as strong synergism (table ). finally, the combination raf:oxy (ratio : ) was classified as synergism at all the levels of inhibition (table ). the aim of this study was to evaluate the anti-hadv activity of nic, a salicylanilide anthelmintic drug of human use to set the basis for its further experimental and clinical development as a potential new treatment for hadv infections. moreover, we included in our evaluation other two chlorinated salicylanilide anthelmintic derivatives approved for animal use, oxy and raf, because of their closely related structure and mechanism of action with nic. it is well-known that these drugs act as uncouplers of the oxidative phosphorylation, involving dissipation of the membrane potential . in case of nic, jurgeitr et al. previously demonstrated that its antiviral mechanism of action was specifically associated with the neutralization of the endosomal ph, thus showing a broad antiviral activity against ph-dependent viruses. although hadv endosomal escape is not exclusively dependent of ph, a low ph has proven to be associated with the endosomal escape of hadv , . with these premises, we initially assumed a probable common anti-hadv mechanism of action for these three drugs and decided to characterize their anti-hadv activity as a previous step before the evaluation of their safety and efficacy in the syrian hamster model of hadv infection. here, we show that nic, oxy and raf exert significant anti-hadv and anti-hcmv activity at low concentrations. to put this data into perspective, it is noteworthy that the reported ic values for cidofovir, the drug of choice for the treatment of hadv infections, are higher than those shown by these three salicylanilide drugs , . in addition, following our own methodologies, the ic value for cidofovir was . ± . µm, significantly higher than the values obtained for the three salicylanilide drugs. as for their mechanism of action, the data obtained from this work demonstrates that one of these three closely-related drugs did not share the expected mode of action. we have confirmed in our work that nic, and as a novelty raf, acts to decrease the number of hadv genomes associated with the host nucleus, however, as demonstrated by our physiologic assay using the ribotoxin α-sarcin nic and raf may act in a later step after hadv endosomal escape. unlike nic and raf, oxy did not inhibit the access of hadv genomes to the nucleus. this observation was confirmed in their time-course assay where we found that, for nic and raf inhibition occurred at earliest times of the hadv replicative cycle than for oxy as well as in the western-blot assay at hpi that showed higher inhibition of the e a gene expression for nic and raf. as shown by our results, the high inhibition in the hadv dna replication generated by oxy may be the consequence of the previous and significant inhibition of the e a gene transcription, which is supported by our results showing a clear inhibition of the e a transcription at hpi, both by quantitative real-time pcr and by western-blot. it is well known that prior to hadv dna replication, transcription of e a by cellular rna polymerase ii takes place from the e a promoter . then, e a protein promotes its own transcription and is needed for the subsequent expression of the early genes e b, e , e and e from different promoters as well as for dna replication and that is probably why we observed that significant inhibition on hadv dna replication. the existence of different mechanisms for the antiviral activity of these three drugs is supported by the significant combinatory index values obtained using the calcusyn software for all the drug combinations ( table ). the three anthelmintic drugs showed a moderate synergistic to a strong synergistic activity when they were combined in pairs, which was especially strong at all the levels of inhibition evaluated for the nic-oxy combination. likewise, using the combination of the three of them they showed a strong synergistic activity at all the levels of inhibition evaluated. repositioning of drugs for diseases different than those they were approved for is a valuable alternative for drug discovery and development since it reduces significantly the high cost and the time-consumption of developing a new drug [ ] [ ] [ ] [ ] . in case of nic and other anthelmintic drugs, numerous reports support their repurposing as anti-cancer drugs due to their cancer inhibitory properties [ ] [ ] [ ] [ ] . nic is an fda-approved drug that has been used for many years to treat helminthic infections in humans and has previously demonstrated its antiviral potential against different viruses such as japanese encephalitis flavivirus, zika virus, coronavirus, human rhinovirus or influenza virus [ ] [ ] [ ] [ ] . in rats, nic is known to exert low in vivo toxicity, with a median lethal dose % (ld ) of , mg/kg body weight and generate peak serum concentrations between . µm and µm, depending on the dose and the administration route , . in healthy humans orally treated with - , mg nic per person, no signs of intoxication were seen , and the drug remained detectable for - days. peak serum concentrations between . - µm were reported , . as shown in our results, these serum concentrations will be enough to reach the ic for hadv (table ) . oxy and raf are other fda-approved drugs for veterinary use to treat helminthic infections. for oxy, high serum concentrations ranging from . µm to . µm have been reported in sheep and goats after a single oral dose of mg/kg . in the case of raf, an intravenous single dose of mg/kg in goats generated serum concentrations ranging from . µm to . µm registered at . h and h, respectively . after a single oral dose of . mg/kg also in goats, the maximum serum concentrations obtained were . µm and . µm at h and h, respectively . in both cases, oxy and raf serum concentrations are significantly higher than their ic for hadv ( table ) . as for their toxicity, ld of raf has been reported to be higher than , mg/kg of body weight in rats while for oxy, according to the european medicines agency (emea), this ld would be higher than , mg/kg of body weight (also in rats) . our findings show a significant anti-hadv activity for the three salicylanilide drugs targeting different steps on the hadv life cycle. nic and raf would mainly block hadv infection at some point between endosomal escape and dna release into the nucleus, whilst the activity of oxy would be specifically targeting hadv immediately early gene e a transcription. our findings support the evaluation of these three drugs in the syrian hamster model of hadv infection to evaluate their efficacy and safety. the pharmacokinetic profile of these salicylanilide drugs showing low water solubility and oral bioavailability may hamper its further clinical development . in this situation, the generation of derivatives of these salicylanilide drugs based on rational chemical approaches may improve their poor pharmacokinetics, increasing their solubility and bioavailability as potential clinical candidates for antiviral therapy. wild-type hadv (species c), hadv (species b), hadv (species d), and human cytomegalovirus (hcmv) ad were obtained from the atcc. the hadv -gfp and hadv -gfp used in this study are replication-defective viruses with a cmv promoter-driven enhanced green fluorescent protein (egfp) reporter gene cassette in place of the e /e region . hadv were propagated in β cells and isolated from the cellular lysate by cesium chloride (cscl) density gradient combined with ultracentrifugation. virus concentration in mg/ ml was calculated using the bio-rad protein assay (bio-rad laboratories) and converted to virus particles/ml (vp/ml) using × vp/mg. cytotoxicity assay. nic, oxy and raf cytotoxicity was evaluated using the alamar blue cell viability assay (invitrogen) according to the manufacturer's instructions. actively dividing a or β cells were incubated with the salicylanilide anthelmintic drugs for h. after this incubation, the alamar blue reagent was added to the cells ( / th alamar blue reagent in culture medium) for an extra h. their % cytotoxic concentration (cc ) was calculated as previously reported by cheng et al. . the selectivity index (si) was calculated as the ratio of cc to ic , where the ic is defined as the concentration of the anthelmintic drug that inhibits hadv infection by %. hadv plaque assay. anthelmintic drugs were tested in a dose-response assay using an moi of . vp/cell and drug concentrations ranging from to . μm in a plaque assay. briefly, a density of × β cells per well were seeded in -well plates. at - % confluency they were infected with hadv -gfp or hadv -gfp ( . vp/cell) and rocked for h at °c. after this incubation wells were washed-out with pbs. then, cells were carefully overlaid with ml/well of equal parts of . % (water/vol) difco agar noble (becton, dickinson & co., sparks, md) and × emem (minimum essential medium eagle, biowhittaker) supplemented with × penicillin/streptomycin, × l-glutamine, and % fbs. the mixture also contained the drugs in concentrations ranging from to . μm. after incubation for days at °c, virus plaques were scanned with a typhoon imager (ge healthcare life sciences), and quantified with imagej . to determine the time course of the three anthelmintic drugs-mediated inhibition, parallel samples of wild-type hadv were incubated with or without μm of nic and μm of raf or oxy in complete dmem at °c for h. virus ( vp/cell) was then added to a cells ( , cells/well in a -well plate) and incubated at °c. anthelmintic drugs were added at the indicated time points (− , , , , and min) before or during this incubation. after incubation at °c and % co for h dna was purified from the cell lysate using the qiaamp dna mini kit (qiagen, valencia, ca) following the manufacturer's instructions, and the dna was quantified by quantitative pcr following the above described protocol. analysis of anthelmintic drug combinations. the software packet calcusyn (biosoft, ferguson, mo, usa), which compares the drug concentrations required in combination to generate a given effect to the drug concentration that would be needed individually to achieve that same effect was used. for this assay a plaque dose-response assay was carried out using all the possible combination of the three drugs starting from twice the ic obtained previously for each compound and the ratio of those concentrations. calcusyn software interpolates the drugs concentrations needed in combination at the selected ratio to generate effects of %, % and % inhibition and compares these combined drug concentrations with the concentrations from the three drugs' individual dose-response curves required to achieve the same inhibition. the combination effect of the three drugs was reported by the combination index (ci) value, a quantitative estimation of the pharmacological interaction which uses the potency (ic ) and the shape of the dose-response curve of each individual drug and their combinations. the ci value was interpreted in accordance with matthews et al. . nuclear-associated hadv genomes. the nuclear accessibility of hadv genomes was evaluated by real-time pcr following a previously described protocol with a few modifications . briefly, × a cells in -well plates were infected with wild-type hadv (moi , vp/cell) in the presence of μm nic or μm oxy and raf, or the same volume of dmso for negative control. forty-five minutes after infection, a cells were trypsinized and collected, and then washed twice with pbs. then, nuclear and cytoplasmic fractions were separated using a hypotonic buffer solution consisting of mm tris-hcl ph . , mm nacl, and mm mgcl . the cell pellet was resuspended in μl of × hypotonic buffer and incubated for min at °c. then, μl of np- was added and the samples were vortexed. the homogenates were then centrifuged for min at g at °c. hadv dna was isolated from the nuclear (pellet) and the cytoplasmic fractions (supernatant) using the qiaamp dna mini kit (qiagen, valencia, ca). to measure endosome disruption, hadv-mediated ribotoxin (α-sarcin) delivery assays were performed as previously described hadv dna and mrna quantification by real-time pcr. for dna quantification, , a cells/ well in a -well plate were infected with vp/cell (wild-type hadv ) and incubated for h at °c in complete dmem. then, the excess of virus was washed-out, and the medium was replaced with μl of complete dmem containing μm of either anthelmintic drugs or the same volume of dmso (negative control). all samples were done in triplicate. after h of incubation at °c and % co , dna was purified from the cell lysate using the qiaamp dna mini kit (qiagen, valencia, ca) following the manufacturer's instructions. taqman primers, probes and pcr conditions were like those previously reported . for the evaluation of rna expression, same conditions of infection applied for the dna quantification were used. six hours after infection, rna was purified with the mircury rna isolation kit (exiqon inc., ma) following the manufacturer's instructions. quantification of rna copy numbers was performed using primers in conditions previously reported for e a . human glyceraldehyde- -phosphate dehydrogenase (gapdh) gene was used as internal control. primers, probes and conditions applied for gapdh were those previously reported by rivera et al. . for quantification, gene fragments of hexon and gapdh were cloned into the pgem-t easy vector (promega), and known concentrations of those vectors were used to generate a standard curve for each experiment. all assays were performed in a c thermal cycler apparatus (biorad). a burst assay was used to assay the effect of the three anthelmintic drugs on virus production. a cells were infected with wild-type hadv , wild-type hadv or wild-type hadv in the presence or absence of μm nic or μm oxy and raf. after incubation for h, cells were harvested and subjected to three rounds of freeze/thaw. then, serial dilutions of clarified lysates were titrated on a cells, and tcid values were calculated using a previously reported end-point dilution method . to test the anti-hcmv activity of these anthelmintic drugs, mrc- cells were seeded in a -well plate ( . × cells/well), infected with hcmv (moi of . vp/ cell) and incubated in complete dmem in the presence of μm nic or μm in case of oxy and raf or the same volume of dmso in triplicate. then, cells were incubated for h at °c and % co and hcmv dna was purified from the cell lysate using the qiaamp dna mini kit (qiagen, valencia, ca) following the manufacturer's instructions. real-time pcr primers, mixtures and protocols were the same as previously reported . statistical analyses. statistical analyses were carried out using the graphpad prism suite. data are presented as the mean of triplicate samples ± standard deviation (sd), unless otherwise indicated. p < . was considered 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levamisole-oxyclozanide combination in sheep and goats following per os administration the pharmacokinetics of rafoxanide following single dose intravenous and oral administration in goats extrapolation to all ruminants): summary report ( ) -committee for veterinary medicinal products direct evidence from single-cell analysis that human {alpha}-defensins block adenovirus uncoating to neutralize infection enhancement of gene transfer to human myeloid cells by adenovirus-fiber complexes antiherpes simplex virus type activity of casuarinin from the bark of terminalia arjuna linn nih image to imagej: years of image analysis inhibition of adenovirus infection by mifepristone mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo a simple method of stimating fifty percent endpoints supported by plan nacional de i + d + i - and instituto de salud carlos iii, ministerio de economía, industria y competitividad, spanish network for research in infectious diseases (reipi rd / / )co-financed by "a way to achieve europe" erdf, the instituto de salud carlos iii, proyectos de investigación en salud (pi / ) and proyectos de desarrollo tecnológico en salud (dts / ), and the spanish adenovirus network (adenonet, bio / -redt). j.s.c. is supported by the "contract to access to the spanish system of research and innovation of the program of r + d + i of the university of seville" (use- -d) grant. j.a.m.l. and a.s.g. have made substantial contributions to acquisition and analysis of data, as well as in the preparation of the manuscript. j.b.c. helped with the acquisition of data. j.p. has made substantial contributions to conception, design and interpretation of data. j.s.c. designed and coordinated the work and the preparation of the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -tthttyl authors: poirier, canelle; luo, wei; majumder, maimuna s.; liu, dianbo; mandl, kenneth d.; mooring, todd a.; santillana, mauricio title: the role of environmental factors on transmission rates of the covid- outbreak: an initial assessment in two spatial scales date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: tthttyl first identified in wuhan, china, in december , a novel coronavirus (sars-cov- ) has affected over , , people worldwide as of july , and was declared a pandemic by the world health organization on march , . influenza studies have shown that influenza viruses survive longer on surfaces or in droplets in cold and dry air, thus increasing the likelihood of subsequent transmission. a similar hypothesis has been postulated for the transmission of covid- , the disease caused by sars-cov- . it is important to propose methodologies to understand the effects of environmental factors on this ongoing outbreak to support decision-making pertaining to disease control. here, we examine the spatial variability of the basic reproductive numbers of covid- across provinces and cities in china and show that environmental variables alone cannot explain this variability. our findings suggest that changes in weather (i.e., increase of temperature and humidity as spring and summer months arrive in the northern hemisphere) will not necessarily lead to declines in case counts without the implementation of drastic public health interventions. since december , an increasing number of pneumonia cases caused by a novel coronavirus (sars-cov- ) have been identified in wuhan, china , . this new pathogen has exhibited high human-to-human transmissibility with approximately , , confirmed cases of covid- and , deaths reported globally as of july , . on january , , wuhan-a city in china with million residents-was forced to shut down both outbound and inbound traffic in an effort to contain the covid- outbreak ahead of the lunar new year. however, it is estimated that more than five million people had already left the city before the lockdown , which has led to the rapid spread of covid- within and beyond wuhan. in addition to population mobility and human-to-human contact, environmental factors such as absolute humidity (defined as the water content in ambient air) and temperature, have been found to be strong environmental determinants of transmissions for some viral pathogens , . for example, influenza viruses survive longer on surfaces or in droplets in cold and dry air, thus increasing the likelihood of subsequent transmission. for covid- , a recent study found that higher temperatures may have led to higher transmission in cities in china, concluding that there was no evidence supporting the hypothesis that case counts of covid- would decline when temperatures increase . in contrast, another study showed that higher transmission was observed in colder places when analyzing data from cities across the world, suggesting that temperature could potentially impact covid- transmission . a third study found that warm and dry weather was favorable to the survival of the virus whereas a fourth determined that transmission would decrease with the arrival of spring and summer . as discussed in a recent paper , quantifying the relationship between covid- transmission and weather variables is a challenging task for multiple reasons. first, characterizing the time evolution of covid- www.nature.com/scientificreports/ transmission from the available datasets produced by multiple public health agencies can yield very different temporal outbreak trajectories. second, estimating the instantaneous transmission rate, rt, using the dates of report as opposed to using the dates of onset of symptoms will invariably lead to significantly different results. third, the choice of methods to calculate r t using for example cori's method or wallinga and teunis' method, will lead to temporal shifts that complicate establishing causal relationships between weather and transmission . fourth, non-pharmaceutical interventions to contain covid- in china since january , significantly reduced the country-wide disease duration and outdoor transmission ; the environmental impact on transmission may have been eclipsed as a consequence. finally, differences in reporting practices across regions may complicate any efforts to compare relationships between weather and transmission from one location to another. despite these challenges and inconsistent conclusions from research on this topic to date, it is important to propose alternative methodologies that provide a complementary understanding of the effects of environmental factors on the ongoing outbreak to support decision-making pertaining to disease control. this is especially true for locations where the risk of transmission may have been underestimated, such as humid and warm places. our contribution. here, we propose a methodology that can be implemented in real-time during the early phase of an outbreak to examine variability in environmental factors, mobility, and transmission of covid- across provinces and cities in china. we show that the observed spatial patterns of covid- transmission are not explained by ambient temperature, absolute humidity or human mobility alone. our findings do not support the hypothesis that high absolute humidity in warmer environments may limit the survival and transmission of this new virus. epidemiological data. to conduct our analysis, we collected epidemiological data from the johns hopkins center for systems science and engineering website . incidence data were collected from various sources, including the world health organization (who); u.s. centers for disease control and prevention (cdc); china cdc; european cdc; the chinese national health center (nhc); as well as dxy, a chinese website that aggregates nhc and local china cdc situation reports in near real-time. daily cumulative confirmed incidence data were collected for each province in china from january , to february , . we also obtained epidemiological data for other affected countries, including iran, italy, singapore, japan, and south korea and cities in china. estimation of a proxy for the reproductive number. based on the cumulative incidence data for each province, city or country, we estimated a proxy for the reproductive number r in a collection of -, -and -day intervals . r is a measure of potential disease transmissibility defined as the average number of people a case infects before it recovers or dies. our proxy for r, designated as r proxy , is a constant that maps cases occurring from time (t) to time (t + d) onto cases reported from time (t + d) to time (t + d); where d is an approximation of the serial interval (i.e., the number of days between successive cases in a chain of disease transmission). for multiple time points, t, we obtained values of r proxy (t,d), given by: where c is the cumulative case count up to time t, and the values of d range from [ to ] . our measure is considered only a proxy for r because it does not use details of the (currently imprecise definition of the) serial interval distribution, but instead, simply calculates the multiplicative increase in the number of incident cases over approximately one serial interval. such proxies are at least approximately monotonically related to the true reproductive number and cross when the true reproductive number crosses , i.e. increases in our proxy typically signal increases in r. after computing these proxy values over a variety of subsequent moving time windows, for each serial interval ( , and days), a mean value was obtained and used as our estimated reproductive number r for each province, city, and country. time windows. our study was conducted from january , to february , to make sure that there was covid- activity across all the locations. indeed, the main outbreaks in chinese provinces took place from the beginning of january to the end of february. in addition, to characterize the temporal evolution of the covid- outbreak (a large decrease in transmission after the closure of wuhan and a subsequent flattening of the epidemic curve), the reproductive number r proxy was calculated for two different time periods. the first one, τ , was from january , to february , and the second one, τ , was from february , to february , . in our study, the reproductive numbers computed on the first and second time periods are labeled r τ and r τ , respectively. weather data. all meteorological data for this study were taken from the era reanalysis, a state-of-theart data product produced at the european centre for medium-range weather forecasts , . era is generated by using a vast range of meteorological observations to constrain a physics-based numerical weather prediction model. this procedure, referred to by atmospheric scientists as data assimilation, yields a globally complete gridded data set including many different meteorological variables. time resolution of era is quite high ( h) and it is also frequently updated (preliminary era data are available days behind real time), making it useful for studies of rapidly evolving disease outbreaks . furthermore, a conceptually similar but much less sophisticated www.nature.com/scientificreports/ data product (the national centers for environmental prediction-national center for atmospheric research reanalysis ) has been found useful for studies of influenza epidemics . we obtained relevant era data at a spatial resolution of . ° (~ km at the equator). we represented weather conditions in each city of interest by those in the era grid box containing the city. because we assumed that the majority of disease incidence for each province occurs in or near the capital due to increased population density in these areas, we chose to represent each province's weather conditions by those in the era grid box containing the provincial capital. near-surface air temperature, used in this study, is one of the standard era variables. absolute humidity (more specifically, near-surface water vapor density) is not one of the standard era output variables. instead, it must be computed from variables that are available, namely near-surface air temperature (t ) and near-surface dew point temperature (t d ) (see supplementary material for more details). we produced hourly time series of temperature and humidity and then computed time mean absolute humidities and temperatures over january - , and february - , , for comparison to τ and τ r proxy data, respectively. human mobility data. we obtained mobility data made publicly available by the chinese internet search engine baidu . from the full origin-destination matrix for each day, we created a dataset to get the percentage of people traveling from wuhan and going to the different chinese provinces from january , to january , (i.e., before the mandated lockdown in wuhan.) data analysis. given the potential noise contained in the reported case counts, we tested the robustness of our findings by gradually removing provinces and cities for which their data was deemed too noisy or missing from our analysis. this was done in three subsequent filtering steps as follows. first, we included all provinces and cities where r proxy could be properly calculated (i.e. enough cases were reported). second, we removed provinces where mobility data was not available. finally, we removed provinces and cities where the values of r proxy were unrealistically high (due perhaps to reporting biases), specifically above . the latter filter was used to further remove potential noisy values that would affect our analysis and responding to the fact that the world health organization has estimated that r values range from to . . for country-level transmission, we did not conduct any statistical analysis due to the extremely noisy values of r proxy. human mobility as a predictor of the reproductive number. to disentangle if our reproductive number estimates could be explained by importation of cases from wuhan, hubei, alone; and if they could be interpreted as indicators of local transmission, we formulated a linear model with the local r proxy as the response variable, and human mobility as a predictor at the province level. specifically, we used mobility data before the closure of wuhan (i.e. from january , to january , ) to explain r τ . where r τ (j) is the proxy for the reproductive number for the province j during the immediate time-period of two weeks after wuhan's lockdown; and x mobility is the percentage of people traveling from wuhan and ǫ ∼ n ( , ) residuals of the regression. relationship between reproductive number and temperature. we used a loess regression to visually represent the relationship between the reproductive number for each province and temperature (fig. ) . to identify the statistical relevance of this relationship we implemented a linear model using the log of the local reproductive number r proxy as our response variable, and temperature as predictor and log transformation was employed to improve gaussianity (supplementary figure s ). the linear model was computed for both time periods described above: depending on the time period explained, r proxy corresponds to r τ or r τ for the province and the city-level; x temperature corresponds to the temperature for the first and second time periods. relationship between reproductive number and absolute humidity. as for temperature, we conducted the same analysis for absolute humidity. the linear model was: where x abshumidity corresponds to the absolute humidity for the first and second time periods. reproductive number proxy. in both time periods, τ and τ , our estimates of r proxy for each province within china, appeared to be consistent across the range of serial intervals we analyzed (fig. ) . in the first timeperiod, most regions have a r proxy estimate well above , signaling sustained disease transmission. r proxy estimates across provinces decreased dramatically on the second time-period, many below , likely as a response to the multiple (non-pharmaceutical) interventions implemented by chinese authorities. relationship with mobility. because wuhan (provincial capital of hubei) was the origin of the covid- outbreak, and exported cases could only be calculated in the rest of the provinces, we excluded hubei from our mobility analysis. as shown in tables and , identifying the influence of mobility on r proxy can only be done after the third step of filtering. human mobility (prior to wuhan's lockdown) did not appear associated with r proxy across chinese provinces during time-period τ (p value = . ). however, in the same time-period, figure . visualization of the relationship between covid- transmission as captured by r proxy and temperature and humidity. the data points on the scatter plot represent the value of rproxy (with its associated % confidence intervals displayed as vertical lines, obtained from the collection of r proxy calculated in subsequent time windows of length d for each location) as a function of temperature and humidity. the black line corresponds to a loess regression aimed at capturing the relationship between rproxyand temperature and humidity. in addition, the color intensity (orange) of each data point shows the size of the outbreak in each location, as captured by the log of cumulative case counts. www.nature.com/scientificreports/ once we excluded r proxy values above (third step of filtering), mobility was found to be associated with r proxy (p value = . ). relationship with temperature. figure is a visualization of the relationship between covid- transmission as captured by r proxy and temperature and humidity. the data points on the scatter plot represent the value of r proxy (with its associated confidence interval) as a function of temperature and humidity. the black line corresponds to a loess regression aimed at capturing the relationship between r proxy and temperature and humidity. specifically, for the first time period, we can see that higher temperatures lead to lower rates of transmission. in addition, the color intensity (orange) of each data point shows the size of the outbreak in each location, as captured by the log of cumulative case counts. regarding the results of the linear regression models, after the first step of filtering, for the time-period τ , temperature appeared to be associated with r proxy at the % confidence level (table ) . specifically, temperature showed a negative relationship, indicating that higher temperatures appeared to have lower transmission (fig. ) . after the two additional steps of filtering, the association between temperature and r proxy became weaker or non-significant (with p values equal to . and . respectively; tables and ). weak to non-significant associations were observed when we conducted our analysis for the second time-period τ , with p values ranging from . to . (tables , , ). at the city-level in china the temperature appeared to be associated to r proxy for the first time-period and after removing cities with low number of cases (p value = . ; supplementary table s ). after removing r proxy above , the temperature was no longer associated with r proxy , with a p value equal to . (supplementary table s ). no associations were observed for the city-level analysis for the second time-period, with p values equal to . and . after the two steps of filtering (supplementary tables s , s ). relationship with absolute humidity. in all steps of filtering at the province-level, and for both time periods, τ and τ , absolute humidity was not associated to r proxy , with p values ranging between . and . (tables , , , , , , ) . this can also be observed in fig. , where the black curve (corresponding to the loess regression) is relatively flat. meanwhile, fig. allows us to visualize the values of r proxy and humidity across regions. for cities, for time-period τ , and after the first step of filtering, absolute humidity appeared to be associate with r proxy with a p value equal to . (supplementary table s ). specifically, absolute humidity showed a table . relationship between reproductive number for the first time period r τ , and mobility with the second step of filtering. table . relationship between log(r τ ) and temperature with the first step of filtering. www.nature.com/scientificreports/ table . relationship between log(r τ ) and temperature with the third step of filtering. table . relationship between log(r τ ) and temperature with the first step of filtering. www.nature.com/scientificreports/ negative relationship, indicating that locations with higher absolute humidity experienced lower transmission. nevertheless, after the third step of filtering, absolute humidity was not found to be associated with r proxy , with a p value equal to . (table s ) . for the second time period τ , no associations were found either, with p values equal to . and . after the two steps of filtering, respectively (tables s , s ). table . relationship between log(r τ ) and temperature with the second step of filtering. table . relationship between log(r τ ) and temperature with the third step of filtering. table . relationship between log(r τ ) and absolute humidity with the first step of filtering. table . relationship between log(r τ ), and absolute humidity with the third step of filtering. ambient temperature appears to be associated to covid- transmission (as captured by our proxy of r) during the first time-period (january , -february , ) in both spatial resolutions and in the absence of any data filtering. specifically, temperature showed a negative relationship, indicating that higher temperatures appeared to have lower covid- transmission. these results were not robust to filtering techniques aimed at removing noisy values such as unrealistically high values of r proxy (more than ). in an effort to identify if transmission rates could be explained by the rate of case importations at the province-level, we analyzed if mobility table . relationship between log(r τ ) and absolute humidity with the first step of filtering. www.nature.com/scientificreports/ from wuhan to each province could explain the spatial variability of r proxy during the first time-period. our results showed no associations between mobility and r proxy in the absence of data filtering but showed that r proxy could be explained by mobility when removing values of r proxy larger than . finally, our analysis suggests that absolute humidity was not robustly associated with r proxy , but these results need to be interpreted carefully given the monotonic functional relationship between humidity and temperature (clausius-clapeyron relation). in other words, if temperature were associated to covid- transmission, very likely absolute humidity would play a role. limitations. our estimates of the observed r proxy across locations were calculated using available and likely incomplete reported case count data, with date of reporting, rather than date of onset, which adds noise to the estimation. in addition, the relatively short time length of the current outbreak, combined with imperfect daily reporting practices, make our results vulnerable to changes as more data becomes available. we have assumed that travel limitations and other containment interventions have been implemented consistently across provinces and have had similar impacts (thus population mixing and contact rates are assumed to be comparable), and have ignored the fact that different places may have different reporting practices. further improvements could incorporate data augmentation techniques that may be able to produce historical time series with likely estimates of case counts based on onset of disease rather than reporting dates. this, along with more detailed estimates of the serial interval distribution, could yield more realistic estimates of r. in addition, while the low r values from our models show that each individual variable is not enough to explain the variability of covid- transmission rate, we considered that finding statistically significant relationships could help us achieve our goal. in fact, if the goal were to design a model to explain the variance of rt one would likely require more input variables, for example the density of population in each area, people's behaviour (regarding mask-wearing adoption, for example) or socio economic factors, etc. future studies should incorporate all these variables to further characterize transmission. finally, further experimental work needs to be conducted to better understand the mechanisms of transmission for covid- . mechanistic understanding of transmission could lead to a coherent justification of our findings. despite the above limitations, our early and near-real-time analysis regarding the impact of environmental factors on covid- transmission in china could provide useful implications for policymakers and the public worldwide. sustained transmission and rapid growth of cases were observed over a range of temperatures and humidity conditions ranging from cold and dry provinces in china, such as jilin and heilongjiang, to tropical locations, such as guangxi and taiwan during the first time-period (τ , from january to february , ). our results show that weather alone cannot explain, in a robust way, the variability of the reproductive number in chinese provinces or cities. moreover, drastic reductions in transmission were observed during the second half of february, likely due to the strict non-pharmaceutical interventions imposed across china. in addition, we can see that all these findings have been confirmed in these past few months. further studies on www.nature.com/scientificreports/ the effects of environmental factors on covid- will be possible as more 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dynamics in wuhan, china, of novel coronavirus-infected pneumonia how generation intervals shape the relationship between growth rates and reproductive numbers era : fifth generation of ecmwf atmospheric reanalyses of the global climate global reanalysis: goodbye era-interim, hello era . ecmwf newsl evidence for ultraviolet radiation decreasing covid- growth rates: global estimates and seasonal implications ( ) the ncep/ncar -year reanalysis project baidu mobility data for atmospheric science: an introductory survey ecmwf. part iv: physical processes. ifs documentation ms and cp were partially supported by the national institute of general medical sciences of the national institutes of health under award number r gm . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to c.p. or m.s.reprints and permissions information is available at www.nature.com/reprints. www.nature.com/scientificreports/ publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - wxwktck authors: zhang, baoshan; chao, cara w.; tsybovsky, yaroslav; abiona, olubukola m.; hutchinson, geoffrey b.; moliva, juan i.; olia, adam s.; pegu, amarendra; phung, emily; stewart-jones, guillaume b. e.; verardi, raffaello; wang, lingshu; wang, shuishu; werner, anne; yang, eun sung; yap, christina; zhou, tongqing; mascola, john r.; sullivan, nancy j.; graham, barney s.; corbett, kizzmekia s.; kwong, peter d. title: a platform incorporating trimeric antigens into self-assembling nanoparticles reveals sars-cov- -spike nanoparticles to elicit substantially higher neutralizing responses than spike alone date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: wxwktck antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. however, the development of such immunogens is often complicated by inefficiencies in their production. to alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the spytag:spycatcher system to display trimeric antigens on self-assembling nanoparticles, including the -subunit aquifex aeolicus lumazine synthase (lus) and the -subunit helicobacter pylori ferritin. lus and ferritin coupled to spytag expressed well in a mammalian expression system when an n-linked glycan was added to the nanoparticle surface. the respiratory syncytial virus fusion (f) glycoprotein trimer—stabilized in the prefusion conformation and fused with spycatcher—could be efficiently conjugated to lus-spytag or ferritin-spytag, enabling multivalent display of f trimers with prefusion antigenicity. similarly, f-glycoprotein trimers from human parainfluenza virus-type and spike-glycoprotein trimers from sars-cov- could be displayed on lus nanoparticles with decent yield and antigenicity. notably, murine vaccination with . µg of sars-cov- spike-lus nanoparticle elicited similar neutralizing responses as . µg of spike, which was ~ -fold higher on a weight-per-weight basis. the versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with sars-cov- spike-lus nanoparticles inducing particularly potent neutralizing responses. to construct a reliable plug-and-play platform for nanoparticle presentation of antigens, we chose aquifex aeolicus lumazine synthase (lus) and helicobacter pylori ferritin as nanoparticle scaffolds with spytag:spycatcher conjugation system to display antigens on nanoparticle surface. the spytag:spycatcher system is highly specific and stable with an isopeptide bond and has been used for conjugation of antigens on nanoparticle surfaces , (fig. a) . lus and ferritin have served as scaffolds for nanoparticle immunogens in several clinical studies: for lus see https ://www.clini caltr ials.gov/ct /show/nct ; for ferritin, see https ://www.clini caltr ials.gov/ct /show/nct , , . the n termini of both ferritin and lus are exposed to the nanoparticle surface and are thus accessible for spytag or spycatcher attachment (fig. b) . the c terminus of lus is also accessible on the nanoparticle surface and can be used for attachment or to display purification tags. we designed mammalian expression constructs expressing fusion proteins of spytag or spycatcher with lus or ferritin. the constructs included both his-and strep-tags for purification purposes, along with a signal peptide for secretion of the expressed proteins into supernatant medium (fig. b) . initial constructs yielded low levels of soluble proteins for the nanoparticle-spytag or spycatcher fusion proteins. to improve protein solubility and expression, we added glycans to the surface of the nanoparticles, designing a panel of lus and ferritin constructs with spytag and spycatcher (table and supplementary table s ). for lus constructs, we added a glycosylation site at position (pdb hqk numbering). for ferritin constructs, two potential glycosylation sites ( and ) were tested. the addition of n-linked glycosylation sites facilitated expression of soluble nanoparticles in the cell culture supernatant. three of the constructs produced appreciable yields of well-assembled nanoparticles, lus with n and spytag at n-terminus (hereafter referred to as lus-n -spytag), ferritin with n and spytag, and ferritin s (glycan at n ) and spytag (table ) . of the two ferritin constructs, the ferritin with n and spytag had a higher yield and was chosen for further studies (hereafter referred to as ferritin-n -spytag). size exclusion chromatography (sec) and electron microscopy (em) analyses indicated that lus-n -spytag formed a homogeneous nanoparticle population in solution (fig. c,d) . the ferritin-n -spytag sample comprised mainly intact nanoparticles with some minor unassembled species (fig. c,d) . negative-stain electron microscopy (em) images indicated both nanoparticles to be well-assembled with expected sizes , (fig. d) . two-dimensional class average revealed more detailed structural features of the nanoparticles, which were consistent with previously published structures of the two nanoparticles. these data indicated the ferritin and lus nanoparticles were compatible with the spytag and glycosylation site addition. these alterations were well tolerated, allowing for robust nanoparticle assembly. to verify the glycosylation of lus-and ferritin-spytag nanoparticles, we performed pngase f digestion and checked for glycan cleavage through sds-page (fig. e) . both nanoparticles showed a band shift in the presence of pngase f, indicating the presence of n-liked glycan on the nanoparticles and its removal by the amidase digestion. while the glycan cleavage in lus-n -spytag is distinct, it is less apparent in ferritin-n -spytag, likely due to incomplete glycosylation of ferritin-n -spytag and multiple bands of ferritin on sds-page. ferritin has been observed to exhibit a single band on sds-page in some studies but multiple bands in others , , presumably due to protease cleavage at the c terminus or incomplete glycosylation. however, these different sized ferritin molecules assembled correctly as nanoparticles with expected dimensions as indicated by sec and em (fig. c, www.nature.com/scientificreports/ cine capable of eliciting protective antibodies have resulted in the identification of rsv trimers stabilized in its prefusion conformation, rsv f ds-cav (ds-cav ), and rsv f ds (ds ) , . ds was shown to elicit higher rsv neutralization responses than ds-cav . as a test case for our nanoparticle spytag:spycatcher system, we investigated the feasibility of displaying ds in the context of nanoparticle immunogens. we prepared ds coupled to spycatcher (hereafter referred to as rsv f-spycatcher) by genetic engineering to append spycatcher to the c-terminus of rsv f after a residue (gsg) linker (supplementary table s ). after expression and purification, we conjugated the purified rsv f-spycatcher to the purified -mer a spytag was placed at the n-terminus of the nanoparticle sequence after the cleavable signal peptide. his and strep tags were placed at the c-terminus of the lus nanoparticle. an n-linked glycosylation site was engineered in the nanoparticle sequence to facilitate protein expression (see table and supplementary table s for (fig. b ). sds-page showed the appearance of species of larger molecular weight of ~ kda in the conjugation mixture, followed by bands of residual lus-n -spytag monomer and rsv f-spycatcher components at kda and kda, respectively (fig. c) , confirming the success of the conjugation reaction. to estimate the conjugation efficiency, we measured the intensity of each band on the sds-page gel image of the conjugated nanoparticle product (fig. c) , as a surrogate of mass for each component. taking into consideration the molecular weight of each component, we calculated the molar ratio of each component to total protein in the sample. we estimated that % of all the lus nanoparticle subunit was conjugated to rsv f trimer. to verify particle integrity after conjugation, we performed negative stain em following sec purification. lus-n -spytag conjugated with rsv f-spycatcher efficiently produced uniform particles with a core diameter of . ± . nm decorated with trimer spike of . ± . nm in length (fig. d) . we then confirmed the prefusion state of the lus-n -spylinked-rsv f nanoparticle through surface plasmon resonance using rsv prefusion f specific antibodies d (site Ø) and mpe (site iii) (fig. e ) . notably, rsv f on nanoparticles showed an enhanced on-rate to the apex-targeting d antibody and reduced on-rate to the equatorial targeting mpe versus trimeric rsv f, a crucial antigenic characteristic signifying appropriate nanoparticle display . having produced successfully the lus-n -spylinked-rsv f nanoparticle, we next set out to conjugate the -mer ferritin-n -spytag with rsv f-spycatcher in the same manner (fig. a) . sec of ferritin-n -spytag nanoparticle showed a peak at around - ml, slightly slower than rsv f-spycatcher (fig. b ). negative stain em revealed that ferritin-n -spytag formed nanoparticle of the expected size (fig. c) . the conjugation mixture of ferritin-n -spytag with rsv f-spycatcher exhibited a peak at ~ ml (void volume of the sec column), suggesting successful formation of the conjugation product (referred to as ferritin-rsv f) (fig. b ). sds-page demonstrated the appearance of a new band at ~ kda, the expected size of ferritin-n -spylinked-rsv f nanoparticle, with residual ferritin-n -spytag at around kda (fig. c ). using the same method as for lus-n -spylinked-rsv f above, we estimated % of all the ferritin nanoparticle subunit was conjugated to rsv f trimer. to confirm the formation of ferritin-n -spylinked-rsv f nanoparticle, we performed negative stain em, which showed well-formed nanoparticles with the expected size and shape, displaying trimer spikes around the ferritin nanoparticle (fig. d) . to verify the conserved prefusion state of the conjugated rsv f trimer, we measured the binding of ferritin-n -spylinked-rsv f to d and mpe iggs through spr (fig. e) . importantly, we observed the on-rate to increase for d , which recognizes an epitope at the trimer apex, but the on-rate to decrease for mpe , which recognizes an equatorial epitope on the trimer, similar to the observation for lus-n -spylinked-rsv f. to demonstrate the plug-and-play versatility of the spytag:spycatcher nanoparticle system, we produced piv f glycoprotein trimer as a fusion protein with spycatcher at the c terminus and conjugated with the lus-n -spytag nanoparticle ( fig. a -c, supplementary table s ), similar to that described above for the conjugation of rsv f-spycatcher. piv is a prevalent human parainfluenza virus that causes respiratory illnesses, especially in infants and young children , . the conjugation mixture of piv f-spycatcher and lus-n -spytag was loaded onto the sec column to purify the conju- table . lus-and ferritin-nanoparticles with spytag require the addition of n-linked glycans for expression. a these bolded constructs showed suitable expression levels. lus-spytag no glycan x none < . www.nature.com/scientificreports/ gated nanoparticle product lus-n -spylinked-piv f from unconjugated nanoparticles and the piv f-spy-catcher trimer (fig. b) . sds-page analysis revealed that the conjugated product had the expected molecular weight, and no unconjugated piv f-spycatcher remained in the conjugation mixture (fig. c ). using the same method as for lus-n -spylinked-rsv f in the previous section, we estimated % of all the lus nanoparticle subunit was conjugated to piv f trimer. the piv f conjugated nanoparticle was further verified through negative stain em, which showed well defined trimer spikes decorating the lus nanoparticle with the expected size ( fig. d) . having produced nanoparticles of lus-n -spytag conjugated with piv f-spycatcher, we next evaluated binding of lus-n -spylinked-piv f with antibodies pia and pia , using spr (fig. e) . the headtargeting antibody pia showed an improved binding to lus-n -spylinked-piv f relative to its binding conjugation of sars-cov- spike trimer to lus nanoparticle via spytag:spycatcher displays the spike trimers homogeneously on the nanoparticle surface. severe acute respiratory syndrome coronavirus (sars-cov- ) caused the covid- pandemic that is ongoing worldwide . an effective vaccine against sars-cov- and related coronaviruses is urgently needed. the sars-cov- spike glycoprotein trimer mediates virus-cell membrane fusion and is thus a target for vaccine development , . to test the versatility www.nature.com/scientificreports/ of our plug-and-play spytag:spycatcher nanoparticle system, we expressed and purified sars-cov- spike fused with a c-terminal spycatcher and conjugated to the lus-n -spytag nanoparticle (fig. a -c, supplementary table s ). for this construct, we used the prefusion stabilized version of spike developed by mclellan and colleagues , which included gsas and pp mutations and the t phage fibritin trimerization domain along with a single-chain fc tag for purification as described by zhou and colleagues . the conjugation mixture was loaded onto an sec column to purify the conjugated nanoparticle product lus-n -spylinked-cov- spike from unconjugated lus-n -spytag and sars-cov- spike-spycatcher (fig. b) . sds-page analysis revealed the conjugated product to have the expected molecular weight, and unconjugated spike-spycatcher was not observed after conjugation (fig. c) . using the same method as for lus-n -spylinked-rsv f, we estimated % of all the lus nanoparticle subunit was conjugated to the spike www.nature.com/scientificreports/ trimer. negative stain em showed lus-n -spylinked-cov- spike nanoparticle to exhibit the expected size with spike trimers displaying on the lus nanoparticle surface (fig. d) . spr measurements showed lus-n -spylinked-sars-cov- spike to bind to cr , , an antibody targeting the receptor-binding domain (rbd), indicating successful nanoparticle presentation of the spike trimer using the lus-spytag:spycatcher system. to assess immunogenicity, we injected mice with the lus-n -spylinked-cov- spike nanoparticle or spike trimers (stabilized by p mutation) , , or mock (lus-n -spytag) nanoparticles at weeks and (fig. a) . serum samples were collected two weeks after each immunization. after the first immunization, at the lowest immunogen dose of . µg, spike nanoparticle-immune sera exhibited an anti-sars-cov- spike elisa geometric mean titer of , , whereas only out of trimeric spike-immunized sera exhibited a measurable titer (fig. b) ; after a second immunization, titers for the spike nanoparticle-immune sera increased substantially, by approximately -fold. immunizations with higher doses of spike nanoparticle ( . and . µg) increased titers more incrementally, both at week and at week . by contrast, increases in dose of the spike trimer raised elisa titers more substantially, with two of the mice in the . µg spike-trimer immune sera reaching the assay upper limit of detection with a titer of , , (fig. b) . importantly, pseudovirus neutralization assays revealed the lus-n -spylinked-cov- spike nanoparticle to elicit potent neutralization responses with geometric mean id titers of , , and for immunization doses of . , . , and µg, respectively (fig. c) . in comparison, two doses of trimeric spike elicited neutralization titers at the . and µg doses with a geometric mean id of and , respectively, with no measurable neutralization at the . µg dose. in essence, . µg of spike nanoparticle elicited a neutralization response that was higher, though statistically indistinguishable from µg of trimeric spike. this indicated ~ -fold higher immunogenicity on a weight-by-weight basis for the spike nanoparticle versus spike alone, suggesting a substantial "dose-sparing" effect. overall, presentation of the sars-cov- spike on the lus nanoparticle-based immunogens can induce potent neutralizing antibodies , , and thus may be promising vaccine candidates. to develop nanoparticle vaccine immunogens, rapid and efficient methods would help produce nanoparticle scaffolds that can be mixed and matched with different immunogens. previous efforts utilizing the spontaneous isopeptide bond formation with the spytag:spycatcher system for nanoparticle surface display of immunogens [ ] [ ] [ ] [ ] [ ] [ ] have proven the versatility of this system for antigen display. however, none of these previously published reports utilized mammalian expression allowing for post-translational modifications, such as n-linked glycosylation. here, we describe two nanoparticle platforms, lumazine synthase and ferritin, for the display of trimeric viral protein immunogens using the spytag:spycatcher system. by adding n-linked glycosylation sites to nanoparticle monomers, we were able to produce spytag-coupled nanoparticles using mammalian cell culture. lus and ferritin nanoparticle platforms vary in the number of molecules displayed on the surface. lus-n -spytag contains spytags whereas ferritin-n -spytag has displayed on surface, available for spy-catcher-carrying molecules to couple to. both platforms showed efficient conjugation of trimeric immunogens and formed nanoparticle rapidly under physiological conditions for rsv f, piv f and sars-cov- spike trimers. one advantage of the lus nanoparticle is the high efficiency of its particle assembly. the glycosylated lus-n -spytag assembled into a homogenous particle that exhibited a single peak in size exclusion chromatography. to demonstrate the versatility of our spytag-displaying nanoparticles in immunogen development, we conjugated them to three viral antigens of vaccine interest, the ds -pref stabilized rsv f , a ds -stabilized version of piv f , and the p-stabilized version of sars-cov- spike . in each of these, we appended the spycatcher after the 'foldon' heterologous trimeric stabilization motif . conjugation of spytag-nanoparticles with spycatcher-coupled rsv f, piv f and sars-cov- spike trimers resulted in proper particle assembly. in all three cases, we observed high conjugation efficiency. our antigenicity analyses indicate that presentation of trimeric antigens from viral pathogens on self-assembling nanoparticles needs to take into consideration the accessibility of the antigenic epitopes. when a trimer protein is conjugated to a nanoparticle, such as lus or ferritin in this study, the trimer molecules are densely displayed on the nanoparticle surface. as a result of the dense display, the epitopes near the nanoparticle surface, such as those at the stem region of the trimers in this study, are less accessible to antibodies than the epitopes on the apex of trimer molecules. consequently, we observed an increased level of antibody binding to epitopes on the apex and a decreased level of antibody binding to epitopes on the equatorial or stem region of rsv f and piv f trimer molecules (figs. e, e and e) . the increased antigenicity of the apical epitopes on the trimer conjugated to nanoparticles is expected to yield increased immunogenicity-especially at lower dose, and we provide proof-of-principle for this with murine immunization studies with lus-n -spylinked-cov- spike as compared to soluble spike. as observed in prior studies , , nanoparticle immunogens elicited stronger immune responses than the corresponding trimers at low immunogen doses: at the . µg dose after two immunizations, spike nanoparticle elicited neutralization response with id of , whereas trimeric spike elicited an equivalent neutralization titer only at the -fold higher dose of µg. at . µg, spike nanoparticle elicited ~ -fold higher id than trimeric spike. however, at a high dose of µg, spike nanoparticle-elicited neutralization response appeared to plateau-at a level ~ fivefold higher in neutralization titer than the trimeric immunogen. similar increases in immunogenicity and with dose-sparing have been recently reported for nanoparticles incorporating the receptor-binding domain (rbd) of the spike . overall, multivalent presentation of trimeric antigens on nanoparticle can significantly improve their immunogenicity, allowing for elicitation of potent immune responses at a relatively low immunogen dose. our spytag:spycatcher system provides a versatile platform for preparation of such nanoparticle immunogens from trimeric antigens. it will be interesting to see if the plug-and-display technology described here will allow for the incorporation of different molecules on multiple nanoparticles. such molecules could include not only trimeric viral immunogens, but also immunostimulatory components, or molecules targeting antigen-presenting cells. thus, the lus-and ferritin-spytag displaying nanoparticles described here may be amendable to mix-and-match display of immunogens and of immunostimulatory or targeting components. protein production and purification. the amino acid sequences of protein expression constructs are listed in supplementary table s . for protein expression, ml of turbo transfection reagent (speed biosystems) was mixed with ml opti-mem medium (life technology) and incubated at room temperature (rt) for min. mg plasmid dnas was mixed with ml of opti-mem medium in a separate tube, and the mixture added to the turbo opti-mem mixture. the transfection mixture was incubated for min at rt then added to ml of expi cells (life technology) at . million cells/ml. the transfected cells were incubated overnight in a shaker incubator at % co , °c, and rpm. on the second day, about ml of expi expression medium was added. on day post transfection, supernatants were harvested, filtered. proteins were purified from the supernatant using ni-nta and strep chromatography. sars-cov- spike-spycatcher was expressed as a fusion protein with a single-chain fc purification tag and purified using protein a chromatography. sars-cov- spike-spycatcher protein was cleaved off from protein a column by hrv c protease. all proteins were further purified by size exclusion chromatography on superdex increase / gl in pbs. www.nature.com/scientificreports/ residual components. the conjugated nanoparticle product was then run through sds-page to verify conjugation and analyzed by negative-stain em. samples were diluted to . - . mg/ml with a buffer containing mm hepes, ph , and mm nacl. a . -µl drop of the diluted sample was applied to a glowdischarged carbon-coated copper grid for approximately s. the drop was removed using blotting paper, and the grid was washed three times with . -µl drops of the same buffer. adsorbed proteins were negatively stained by applying consecutively three . -µl drops of . % uranyl formate and removing each drop with filer paper. micrographs were collected using serialem on an fei tecnai t electron microscope operated at kv and equipped with an eagle ccd camera or using epu on a thermofisher talos f c electron microscope operated at kv and equipped with a ceta ccd camera. the pixel size was . and . nm for tecnai t and talos f c, respectively. particles were picked automatically using in-house written software (y.t., unpublished). reference-free d classification was performed with relion . enzyme-linked immunosorbent assay (elisa). elisa experiments were carried out as previously described . briefly, nunc maxisorp elisa plates (thermofisher) were coated with ng/well of stabilized soluble sars-cov- spike protein (with his-tag cleaved to remove potential cross-reactivity) in x pbs at °c for h. to eliminate fold-on-specific binding, µg/ml of fold-on protein was added to serial dilutions of heatinactivated sera for h at room temperature (rt). after blocking in pbs-tween (pbst) supplemented with % nonfat milk, plates were incubated with sera for h at rt. after blocking in pbs-tween (pbst) supplemented with % nonfat milk, plates were incubated with serial dilutions of heat-inactivated sera for h at rt. secondary antibody, goat anti-mouse igg conjugated to horseradish peroxidase (thermofisher), was then added, followed by excitation with , , ′ ′-tetramethylbenzidine substrate (kpl). each step in this procedure was followed by standard washes in pbst. endpoint titers were calculated as the dilution factor that resulted in an optical density exceeding × background (secondary antibody alone). lentivirus-based pseudovirus neutralization assay. the pseudovirus neutralization assay was performed as described previously , . to produce sars-cov- pseudovirus, a codon-optimized cmv/r-sars-cov- spike (wuhan- , genbank #: mn . ) plasmid, was constructed and co-transfected with plasmids encoding luciferase reporter, human transmembrane protease serine (tmprss ) , and lentivirus backbone into hek t/ cells (atcc #crl- ), as previously described . heat-inactivated serum was mixed with the pseudovirus, incubated at °c, and then added to ace- -expressing t cells. cells were lysed after h, and luciferase activity was measured. percent neutralization was calculated with uninfected cells as % neutralization and cells infected with only pseudovirus as % neutralization. id titers were determined using a log (agonist) vs. normalized response (variable slope) nonlinear function in prism v (graphpad). all relevant data are within the paper and its supporting information files. received: june ; accepted: september self-assembling protein nanoparticles in the design of vaccines self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies induction of potent neutralizing antibody responses by a designed protein nanoparticle vaccine for respiratory syncytial virus a respiratory syncytial virus (rsv) f protein nanoparticle vaccine 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improvement of a fusion-glycoprotein vaccine against rsv structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types - community-acquired pneumonia requiring hospitalization among u.s. adults community-acquired pneumonia requiring hospitalization among u.s. children who declares covid- a pandemic structure-based design with tag-based purification and in-process biotinylation enable streamlined development of sars-cov- spike molecular probes human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants a highly conserved cryptic epitope in the receptor-binding domains of sars-cov- and sars-cov sars-cov- mrna vaccine design enabled by prototype pathogen preparedness efficacy and safety of rts, s/as malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase , individually randomised, controlled trial fibritin encoded by bacteriophage t gene wac has a parallel triple-stranded alpha-helical coiled-coil structure elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for sars-cov- automated electron microscope tomography using robust prediction of specimen movements implementation of a bayesian approach to cryo-em structure determination spider and web: processing and visualization of images in d electron microscopy and related fields eman : an extensible image processing suite for electron microscopy immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen evaluation of the mrna- vaccine against sars-cov- in nonhuman primates proteolytic activation of influenza viruses by serine proteases tmprss and hat from human airway epithelium evaluation of candidate vaccine approaches for mers-cov b.z. and c.w.c. designed research with b.z. heading protein design and production; b.z., c.w.c., a.s.o., and r.v. produced nanoparticle and trimer proteins; c.w.c. and b.z. prepared trimer-coupled nanoparticles and performed antigenic assessments; y.t. performed negative-stain em; s.w. assisted with manuscript assembly; t.z. provided design for sars-cov- spike protein; g.s-j. provided the design for piv f protein; a.p., l.w. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to p.d.k. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -z xbn authors: namvar, ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: in silico/in vivo analysis of high-risk papillomavirus l and l conserved sequences for development of cross-subtype prophylactic vaccine date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: z xbn human papillomavirus (hpv) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. nowadays, the virus-like particles (vlps) based on l proteins have been considered as the best candidate for vaccine development against hpv infections. two commercial hpv (gardasil and cervarix) are available. these hpv vlp vaccines induce genotype-limited protection. the major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk hpv. in this study, we developed dna constructs (based on l and l genes) that were potentially immunogenic and highly conserved among the high-risk hpv types. the framework of analysis include ( ) b-cell epitope mapping, ( ) t-cell epitope mapping (i.e., cd (+) and cd (+) t cells), ( ) allergenicity assessment, ( ) tap transport and proteasomal cleavage, ( ) population coverage, ( ) global and template-based docking, and ( ) data collection, analysis, and design of the l and l dna constructs. our data indicated the -epitope candidates for helper t-cell and ctl in l and l sequences. for the l and l constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of . % and . %, respectively. in vitro studies showed high expression rates of multiepitope l (~ . %) and l (~ . %) dna constructs in hek- t cells. moreover, in vivo studies indicated that the combination of l and l dna constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against c tumor cells (the percentage of tumor-free mice: ~ . %). thus, the designed l and l dna constructs would represent promising applications for hpv vaccine development. www.nature.com/scientificreports www.nature.com/scientificreports/ charge and secondary structure. at first step, the conserved region sequences were analyzed by bepipred- server to predict potential b-cell epitopes (table ). in l protein, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (eatvylppvpvskvv-type ), l - (pppggtledtyrfv-type ) and l - (nfgvppppttslvd-type ) epitopes had the best b cell epitope identification scores. for l protein, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (kqsgtcppdvvpkv-type ), l - (psdpsivslveets-type ), l - (epvgptdpsivtli-type ) and l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (glgigtgsgtggrt-type ) epitopes showed the highest epitope identification score between their own protein sequences. since a linear form of t-cell epitopes are bound to mhcs, the interface between t-cells and ligands can be accurately modeled. in this study, we used three different algorithms (published motifs, ann and quantitative matrix) for mhc-i and two algorithms for mhc-ii (ann and quantitative matrix). prediction of mhc-i. at first step, the l and l conserved regions were analyzed to find the most immunodominant peptides using netmhcpan . , syfpeithi and propred i. in each protein, peptides with the highest binding affinity scores were determined as high-potential ctl epitope candidates (tables and ). the analysis showed that l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (ylppvpvskv-type and ylpppsvarv-type ), l - (dqfplgrkfll-type ) , l - (dqyplgrkflv-type ), l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (krasatqlyk-type and krasvtdlyk-type ), l - (dpdfldivalhr-type ) and l - (dsdfmdiirlhr-type ) epitopes had the highest binding affinity among their own protein sequences. in general, the results of three different algorithms confirmed each other. conservancy and allergenicity analyses were done on the selected epitopes. the sequence of all the epitopes were well conserved among high-risk hpv types and none of them were allergens (tables and ). in addition, there was no cross-reactivity between peptide and human proteome. in this study, we used netmhciipan and propred servers for mhc-ii epitope identification analysis (table ). since a suitable t-cell epitope should be predicted to bind to different hla alleles, epitopes with the maximum number of binding hla-dr alleles were selected as high-potential helper t-cell epitope candidates. among predicted epitopes, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (eatvylppvpvskvv-type ) , l - (tqrlvwacvgvevgrgq-type and tqrlvwacagveigrgq-type ), l - (dtyrfvtsqaiacqk-type ) , l - (dtyrfvqsvaitcqk-type ), l - (dpsivtliedssvvtsgap-type ) , l - (pdfldivalhrpaltsr-type ) and l - (sdfmdiirlhrpaltsr-type ) had the highest scores of binding affinity. also, the sequence of all the epitopes were well conserved among high-risk hpv types and none of them were allergen (tables and ) . also, there was no cross-reactivity between peptide and human proteome. population coverage analysis. hla distribution varies among the diverse geographic regions around the world. thus, while designing an effective vaccine, population coverage must be taken into consideration to cover the maximum possible populations. in this study, population coverage was estimated separately for each putative epitope in specified geographic regions of the world (tables and ) . for ctl epitopes, the highest population coverage of world's population was calculated for l a clear band of ~ bp and ~ bp on agarose gel for l and l , respectively (data not shown). the recombinant endotoxin-free plasmids (i.e., pcdna-l and pcdna-l ) had a concentration range between . and . mg/ml. into the eukaryotic cell line (hek- t) was performed by turbofect as a transfection reagent. the levels of dna expression were evaluated using fluorescence microscopy and flow cytometry at h post-transfection. the data indicated that pegfp-l and pegfp-l can effectively penetrate into hek- t cells in vitro. the cellular uptake of the l and l genes into the hek- t cells was ~ . % and ~ . %, respectively. the delivery of pegfp-n as a positive control was detected in approximately ~ . % of hek- t cells (fig. ) . moreover, the spreading green regions were observed for l and l dna delivery using turbofect carrier by fluorescent microscopy in hek- t cells. on the other hand, western blot analysis indicated the successful expression of l and l proteins fused to gfp (i.e., l -gfp and l -gfp) using anti-gfp antibody. the data indicated the clear bands of ~ , ~ and ~ kda for l -gfp, l -gfp and gfp, respectively using dab substrate (fig. ). to evaluate the prophylactic effects of the designed l and l dna constructs, tumor growth and survival percentage were assessed in all groups for days after challenging with c tumor cells. as shown in fig. a , all test groups immunized with dna constructs (g , g & g ) demonstrated significantly lower tumor growth than that in control groups (pbs and empty vector, g & g , p < . ). our data showed progressive tumor growth in control groups on approximately - days (survival rate or tumor-free mice percentage: %). it was interesting that groups vaccinated with l dna, l dna and l + l dna constructs similarly reduced the tumor growth (p > . ). as shown in fig. b , group vaccinated with the mixture of l + l dna constructs showed a higher survival rate (g , ~ . %) than l and l dna constructs, alone (g & g , ~ . %). antibody assay. the levels of total immunoglobulin g (igg), igg a and igg b in mice immunized with the mixture of l + l dna constructs (g ) were significantly higher than other groups (p < . , fig. a ,c,d). moreover, our data showed that the levels of igg were similar in all groups vaccinated with dna constructs (g , g & g , p > . , fig. b ). there are no significant differences in the secretion of igg a and igg b isotypes between groups receiving the l and l dna constructs, alone (g & g , p > . , fig. c ,d). no significant anti-(l + l ) antibody responses could be detected in the sera of control groups, thus, the seroreactivities were completely l + l antigen-specific responses in mice. cytokine assay. the results of cytokine assay in each group showed that the levels of (l + l )-specific ifn-γ, il- and il- secretions in groups immunized with l (g ), l (g ) and l + l (g ) dna constructs were significantly higher than control groups (p < . , fig. www.nature.com/scientificreports www.nature.com/scientificreports/ fig. ) . furthermore, our data indicated that the ratios of ifn-γ/il- and ifn-γ/il- were higher in all test groups as compared to control groups; therefore, they could trigger th immune response. granzyme b secretion. the secretion of granzyme b in all test groups was significantly higher than the control groups (p < . , fig. ). the group immunized with the l + l dna construct (g ) produced significantly higher concentrations of granzyme b than other groups (g & g , p < . ). the level of granzyme b in group receiving l dna construct was similar to that in group receiving l dna construct (p > . ). in recent years, development of bioinformatics tools applied in vaccine researches could potentially save time and resources. indeed, the immunoinformatics tools help to identify antigenic domains for designing a multi-epitope vaccine. with sequence-based technology advancement, now we have enough information about the genomics and proteomics of different viruses . thus, using various bioinformatics tools, we can design peptide vaccines based on a neutralizing epitope. for example, in silico design of an epitope-based vaccine against human immunodeficiency virus , , coronavirus , dengue virus , and saint louis encephalitis virus has already been reported. while around high-risk hpvs were recognized, current vaccines just protect humans from few types. an important limitation of the current vaccines is their narrow coverage. the accessibility of fully sequenced proteome from high-risk hpv strains provides a prospect for in silico screening of reliable peptide-based therapeutic vaccine candidates among billions of possible immunogenic peptides. in silico approaches are intended to reflect the possibilities for overcoming the above-mentioned difficulties in hpv multi-type vaccine. gupta and coworkers designed prophylactic multiepitopic dna vaccine using all the consensus epitopic sequences of hpvs l capsid protein. they also evaluated how engineering cpg motifs by bioinformatics tools could increase immunogenicity of dna vaccines . hosseini et al. applied in silico analysis of l and l protein of hpv , , , and types to identify universal peptide vaccine in order to protect against mentioned types . in , singh et al. analyzed e , e , e and e proteins of high-risk hpv types to identify cd + t-cell epitopes. they suggested a pool of peptides ( to amino acids) to provide the protection against high-risk hpv types . www.nature.com/scientificreports www.nature.com/scientificreports/ panahi and colleagues used a two-step method (consist of molecular docking and sequence-based approach) to determine immunogenic epitopes for induction of immune system against the oncoproteins of hpv , , and types . in this research, we designed a framework for the comprehensive analysis of l and l conserved regions of high-risk hpv types containing both mhc-i and mhc-ii epitopes. the framework begins with conservancy analysis of all high-risk hpv strains following with ( ) b-cell epitope mapping, ( ) t-cell epitope mapping (cd + and cd + ), ( ) allergenicity assessment, ( ) tap transport and proteasomal cleavage, ( ) population coverage, ( ) global and template-based docking and ( ) data collection, analysis, and design of the l and l dna constructs. for experimental analysis, the final l or l dna constructs were cloned into mammalian expression vector with green fluorescent tag (pegfp vector) and their expression was evaluated in the eukaryotic cells using flow cytometry, fluorescent microscopy and western blotting. moreover, the l /l -specific antibody and t-cell immune responses induced by l and l dna constructs were assessed in mouse tumor model. at first, l and l sequences obtained from high-risk hpv types were aligned using muscle algorithms. conservancy analysis showed that five regions of hpv , l protein ( - , - , - , - and - ) and four regions of hpv , l protein ( - , - , - and - ) were more conserved among other subtypes and could be analyzed as an immunoinformatics input. in b-cell epitope prediction, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , l - , l - , l - , l - , l - and l - had the highest epitope prediction scores. unfortunately, a reliable method for prediction of b-cell epitope has not been revealed up to now and the sensitivity and specificity of existing methods were very low (the specificity and sensitivity of this method were . and . , respectively). in the case of t-cell epitope prediction, in silico analysis has been significantly improved, thus, the results are more reliable. in this study, for mhc-i epitopes, l - (ylppvpvskv-type and ylpppsvarv-type ), l - (dqfplgrkfll-type ), l - (dqyplgrkflv-type ), l - (krasatqlyk-type and krasvtdlyk-type ), l - (dpdfldivalhr-type ) and l - (dsdfmdiirlhr-type ) epitopes had the highest binding affinity scores. in addition, above-mentioned epitopes had the highest t-cell epitope prediction scores which were obtained from proteasomal cleavage and tap transport analysis. high degree of conservancy was observed between subtypes for these epitopes ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (ffgglgigtgsgtggr-type ) epitopes had the highest binding affinity scores. among them, l - had the greatest degree of conservancy (high similarity with all of the high-risk hpv types). one of the remarkable points is that l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and l - epitopes are the same (or overlapping with little difference (among b-cell and mhc-ii selected epitopes. due to a limitation of mhc-peptide binding prediction such as the gap between the peptides that are predicted to bind to mhc and those that experimentally bind www.nature.com/scientificreports www.nature.com/scientificreports/ been employed to address this problem and raise the accuracy of mhc-peptide prediction. in the current study, template-based docking and also global docking were performed on the selected peptides to determine which peptide would get into the groove of mhc with the highest modeling scores. for mhc-i epitope, l - , l - and l - sequences had the highest interaction similarity and cluster density scores. for mhc-ii epitopes, l - , l - , l - and l - sequences had the highest docking scores. in this study, mhc-i-peptide docking scores confirmed mhc-i-peptide binding affinity scores because the same epitopes had the highest scores in both methods but in mhc-ii molecular docking, the results were slightly different. one of the reasons is the significant conformational changes during the process due to the longer epitope length. as a general rule: the longer the length of the query peptide, the more torsions and conformational flexibilities . herein, due to longer peptide sequences, docking results in mhc-ii were less accurate than mhc-i. for example, average similarity score in mhc-i was variable ( . - . ), but in mhc-ii was . - . after the completion of the analysis and according to all of the above-mentioned parameters, two separate constructs were designed. in addition, accumulative population coverage of helper t-cell and ctl epitopes for the designed constructs www.nature.com/scientificreports www.nature.com/scientificreports/ were estimated. for the l and l constructs, the combination of epitope candidates for helper t-cell and ctl in a single universal vaccine could involve all world population by the rate of . % and . %, respectively (fig. ) . in previous studies, ylppvpvskv (hpv l ) and krasvtdlyk (hpv l ) have been reported as potentially immunogenic epitopes. the ability of in vitro expression of the designed l and l dna constructs was determined in hek- t cells using flow cytometry and western blot analysis. the transfection efficiency of the l and l dna constructs was ~ . % and ~ . %, respectively indicating their high potency for delivery into the eukaryotic cells. as known, the use of a polytope dna vaccine containing multiple t-cell and b-cell epitopes is an attractive strategy for developing a therapeutic and prophylactic vaccine against hpv infections. after in vitro assay, immunological experiments were performed in mice to determine the efficiency of the designed l and l dna constructs without the use of adjuvant or delivery system for vaccine development. similarly, some studies used the pcdna vector harboring the gene of interest for immunization without any adjuvant , . our data indicated that the groups immunized with l , l and l + l dna constructs increased antibody and t-cell responses as compared to control groups. furthermore, the (l + l )-specific immunity in mice receiving the mixture of l + l dna constructs (g ) resulted in higher secretion of total igg, igg a, igg b, ifn-γ, il- and il- cytokines as well as granzyme b than other groups. the higher levels of igg a and igg b as well as ifn-gamma (as a th cytokine) in this group drive t-cell responses toward th -type immunity. the studies showed that immunoglobulin g (igg ) is related to a th -type response, while a th response is associated with the induction of igg a and igg b in mice . regarding to our observations in protective studies, this regimen (l + l dna construct: g ) could confer further protection against c tumor-challenged mice (survival rate: ~ . %) depending on stimulation of cd + t cell-dominated th responses as well as granzyme b secretion (indicating ctl activity) as compared to the l or l dna constructs, alone (survival rate: ~ . %). these data showed high potency of the combined l + l dna constructs versus each dna construct alone as a prophylactic hpv vaccine. taken together, immunoinformatics approaches have been emerged as a critical field for accelerating immunological researches. yet, the immunoinformatics techniques applied to t-cells have more advancement than those dealing with b-cells . moreover, recently, due to the limited options for choosing an adjuvant in clinical trials, bioinformatics analyses have been developed to predict the best adjuvant. in this way, in silico studies help researchers saving time and resources, and also can guide the experimental work with higher probabilities of finding the desired solutions and with fewer trial and error repeats of assays. the accessibility of hpv genomic sequences and functional characterization of the genes involved in the virulence has significantly improved our understanding of the molecular foundation for the pathogenesis of hpv and offered a wealth of data that can be used to design new plans for vaccine design. nowadays, powerful immune system simulators have been developed using bioinformatics tools which predict artificial immunity provided by the vaccine. these approaches could predict the best adjuvant for using in human vaccine studies. there is a multi-scale computational infrastructure approach which can stimulate the dynamics of the immune response induced by several vaccination formulations and predict optimal combination in terms of adjuvant type, dosage and timing. netlogo is an agent-based modeling of the immune system running different simulations with different parameter settings. it also can interact with different modeling strategies including the investigation of pathogen growth, life cycle modeling environment for simulation complex phenomena [ ] [ ] [ ] . therefore, using these methods can increase efficiency and reduce costs in vaccine studies. in this study, for the first time, comprehensively integrated methods (using sequence-based tools in combination with flexible peptide-protein docking) were used to design highly immunogenic and protective vaccine candidates which were able to boost both humoral and cellular table . mhc-ii -peptide docking scores of selected helper t-cell epitopes. *higher rate shows better quality of peptide-mhc interactions. www.nature.com/scientificreports www.nature.com/scientificreports/ immune responses against all high-risk hpv types. in addition, in vivo analysis demonstrated high potency of the designed l and l constructs as combined in dna-based vaccines without the use of adjuvant or delivery system. however, we will improve the efficiency of these dna-based vaccines using a delivery system and also will compare their efficacy with the designed peptide-based vaccines along with adjuvants in near future. table . physicochemical properties of l and l dna vaccine constructs. *higher rate shows high degree of peptide antigenicity. **higher rate shows high degree of peptide solubility. protein alignments and conservancy analysis. to determine conserved epitopes between different subtypes, l and l sequence datasets were first aligned using snapgene software . . (from gsl biotech; available at snapgene.com). after protein alignments analysis using muscle algorithms, the conserved epitopes of each protein were selected for immune-bioinformatics analysis such as b-and t-cell epitope prediction. also, to calculate the degree of variability and conservancy of each epitope, iedb epitope conservancy tools (http://tools.immuneepitope.org/tools/conservancy/) were used. linear b-cell epitope prediction. a successful vaccine must elicit a strong t-cell and b-cell immune response, but above all, provide protection against the disease being targeted. therefore, it is essential to show that constructed immunogens are able to induce protective cellular and humoral immunity. since the antibodies are induced against linear b-cell epitopes, it would be very difficult to synthesize long peptides with the native protein conformation resembling for the induction of protective antibodies. however, optimal peptide-based vaccines should be presented in a desired secondary structure of peptides in order to induce a specific humoral response , . for the b-cell epitope prediction of conserved regions in l and l proteins, bepipred- . server (http://www.cbs. www.nature.com/scientificreports www.nature.com/scientificreports/ dtu.dk/services/bepipred- . /) was employed. in this study, epitope threshold value was set as . (the specificity and sensitivity of this method are . and . , respectively) . t-cell epitope prediction. mhc-i epitope prediction: the initial step on applying bioinformatics to vaccine researches is to assess potentially immunoprotective epitopes. t-cell epitopes presented by mhc molecules are typically in a linear form containing to amino acids. this fact facilitates accurate modeling for the interaction of ligands and t-cells . thus, the most selective step in the presentation of antigenic peptide to t-cell receptor (tcr) is the binding of the mhc molecule . in this study, we tried to use three different algorithms including artificial neural networks (netmhcpan . server (http://www.cbs.dtu.dk/services/netmhcpan/), quantitative matrix (propred i (http://crdd.osdd.net/raghava/propred /) and published motifs (syfpeithi server (http://www.syfpeithi.de) to predict high-potential t-cell epitopes. for netmhcpan, percentile rank was set at . % for strong binders and % for weak binders and for propred i threshold was set at %. mhc-ii epitope prediction: for mhc class ii, netmhciipan . server (http://www.cbs.dtu.dk/services/ netmhciipan/) and propred (http://crdd.osdd.net/raghava/propred/) were employed to predict potential interaction of helper t-cell epitope peptides and mhc-ii. in this case, the threshold for strong and weak binders was set at % and %, respectively. prediction of mhc-i peptide presentation pathway. investigating the tap transport and proteasomal cleavage as well as affinity prediction of binding is essential in mhc-i presentation pathway. in this study, we used netctl . server combined with tap transport/proteasomal cleavage tools (http://www.cbs.dtu.dk/services/netctl/) to access the prediction of antigen processing through the mhc class i antigen presentation pathway. in this method, parameters of weight on the c-terminal cleavage, tap transport efficiency, and epitope identification were set to default ( . , . and . , respectively) . population coverage. since the response to t-cell epitopes is restricted by mhcs, the selection of epitopes with multiple hla-binding increases population coverage in defined geographical regions where the peptide-based vaccine might be employed. the coverage rate of population for each epitope was computationally validated using the iedb population coverage tool (/population/iedb_input). in this study, individual epitope and its binding to hla alleles were analyzed, and different geographic areas were also selected. allergenicity and cross-reactivity assessment. since proteins are very important in inducing allergenic reactions, the prediction of potential allergenicity is an important item in the safety assessment especially in the field of genetically modified foods, therapeutics, bio-pharmaceuticals etc. . the food and agriculture organization (fao) and world health organization (who) protocol includes three terms to evaluate the allergenicity of proteins which are defined as following: the term sensitivity refers to correctly predicted allergens (%), whereas www.nature.com/scientificreports www.nature.com/scientificreports/ specificity refers to correctly predicted non-allergens (%), and also accuracy refers to the proportion of correctly predicted proteins . the allergenicity of the epitopes was analyzed by the pa p (http://lpa.saogabriel.unipampa. edu.br: /pa p/pa p/pa p.jsp) using allergen online ( aa and wordmatch) and afds-motif algorithms based on amino acid composition. the specificity of these methods is . % ( aa), . % ( aa) and . % (adfs) . to assess cross-reactivity between peptide and human proteome, top-ranked epitope were analyzed by peptide matching program (https://research.bioinformatics.udel.edu/peptidematch/index.jsp) . peptide-protein flexible docking. computational docking methods have been known as an important tool for drug design . with the rapid development of peptide therapeutics in rational drug design, the use of new techniques such as protein-peptide docking is inevitable. in this study, two different algorithms (template-based docking and global docking) were performed by galexypepdock server (http://galaxy.seoklab.org/cgi-bin/ submit.cgi?type=pepdock) and cabs dock server (http://biocomp.chem.uw.edu.pl/cabsdock). to estimate the formation of mhc-peptide complex, the galaxypepdock server effectively models the structural d peptide-protein complexes from input peptide and protein sequences using the structure database and energy-based optimization (template-based docking). cabs-dock server performs global docking procedure which at first explicit fully flexible docking simulation and then clustering-based scoring. receptor flexibility was limited by default to small backbone fluctuation but could be increased to include selected receptor fragments , . this study presented an example of mhc-peptide docking performed by each individual epitope and available pdb file (table ) of hla alleles, separately. physicochemical properties of the designed l and l constructs. based on l and l top-ranked epitopes, two different constructs were designed. the physicochemical properties of top-ranked epitopes such as solubility, molecular weight, estimated half-time, instability index and antigenicity were determined by protparam (https:// web.expasy.org/protparam/) tools , vaxijen (http://www.ddg-pharmfac.net/vaxijen/vaxijen/vaxijen.html) and protein-sol (https://protein-sol.manchester.ac.uk/) server . www.nature.com/scientificreports www.nature.com/scientificreports/ experimental studies construction of the recombinant plasmids. after bioinformatics analysis, the selected peptides were assembled in two separated constructs (fig. ) . the puc -l and puc -l constructs were synthesized by biomatik company. for in vitro experiments, the puc -l and puc -l vectors were digested by xhoi/hindiii, and the l and l genes were subcloned into xhoi/hindiii sites of pegfp-n vector, individually (i.e., pegfp-l and pegfp-l ). all the recombinant vectors were transformed into escherichia coli (e. www.nature.com/scientificreports www.nature.com/scientificreports/ coli) dh α strain. after extraction of plasmids from single colonies using mini-kit (qiagen), the presence of inserted l and l fragments was confirmed by digestion with restriction enzymes and sequencing. for in vivo immunological assessment, the puc -l and puc -l vectors were digested by bamhi/hindiii and the l and l genes were subcloned into bamhi/hindiii sites of pcdna . (-) vector containing cytomegalovirus early promoter and enhancer sequence, individually (i.e., pcdna-l and pcdna-l ). indeed, we used the pcdna vector harboring cpg motif for in vivo studies. as a final point, the recombinant dna vectors harboring l and l genes were purified by an endotoxin-free plasmid extra ef kit (macherey nagel, germany). the concentration and purity of the recombinant l and l dna constructs were determined by nanodrop spectrophotometry . in vitro expression of l and l dna constructs in hek- t cells. human embryonic kidney cells (hek- t) were cultured in rpmi supplemented with % fetal bovine serum (fbs) at °c and % co atmosphere. after some passages, the cells were seeded in a -well plate. the optimal cell confluency for effective transfection was considered - %. for the generation of turbofect-plasmid dna complex, μl of turbofect (thermo scientific) and μg of each plasmid (pegfp-l , pegfp-l and pegfp-n as a positive control) were mixed and incubated for min at room temperature. then, the complex was added to each well in serum-free media. in addition, the non-transfected hek- t cells were used as negative control. after six hours, the media was replaced with the completed rpmi medium. finally, the cells were harvested, washed and resuspended in pbs buffer, to analyze the expression of l and l dna constructs using flow cytometry, fluorescent microscopy and western blotting at hr after transfection . western blot analysis. hek- t cells were scraped from their plates and washed with pbs x. after washing steps, the cells were lysed in whole-cell lysis buffer ( % glycerol, nm dtt, mm natrium fluoride, . % triton x- , . edta in pbs ph = . ). the extracted protein samples (l -gfp, l -gfp and gfp) were separated by sds-page in . % (w/v) polyacrylamide gel and transferred to nitrocellulose membrane (millipore). the membrane was equilibrated with tbst (tris-buffered saline tween- ) solution containing . % bsa (bovine albumin serum) overnight. the anti-gfp polyclonal antibody ( : v/v; acris antibodies gmbh) was used to recognize the expressed proteins under standard procedures. the immunoreactive protein bands were visualized by detection of peroxidase activity using a substrate named as , ′-diaminobenzidine (dab, sigma) . peptide constructs synthesis. for immunological assay (i.e., secretion of antibody, cytokine and granzyme b), two peptide constructs (l and l peptides, fig. ) were synthesized by biomatik co. with more than % purity. mice immunization. five groups of six female c bl/ mice (obtained from the breeding stocks maintained at pasteur institute of iran; mhc haplotype b/h- kb/h- db) were immunized on days , , and (i.e., three times with a -week interval) with µg of each plasmid dna (pcdna-l or pcdna-l : g or g ) or their combination (pcdna-l + pcdna-l : g ) at the right footpad as shown in table . the control groups (g and g ) received pcdna . and pbs, respectively. all mice were maintained under specific pathogen-free conditions . moreover, all of the animal experimental procedures were approved by animal care and use committee of pasteur institute of iran and carried out according to the animal experimentation regulations of pasteur institute of iran (national guideline) for scientific purposes (code: ). for in vivo protection assay, vaccinated mice were subcutaneously challenged in the right flank with c tumor cells ( × cells), two weeks after the last injection. the c tumor cells contain whole hpv genome, and the presence of l and l genes was confirmed in the previous studies . tumor growth and the percentage of tumor-free mice were monitored twice a week by palpation for days post-challenge. at each time, tumor volume was calculated by this formula: v = (a b)/ (a = the smallest diameter and b = the biggest diameter) . antibody assay secreted from b-cells. two weeks after the last injection, serum samples were collected from each group. the levels of goat anti-mouse immunoglobulin g (igg ), igg a, igg b and total igg antibodies (diluted : , in % bsa/pbs-tween, sigma) secreted from b-cells were measured in the pooled sera of each group by indirect elisa. the coated antigens were the mixture of l and l synthetic peptides ( μg/ml). moreover, mice sera were diluted : in % bsa/pbs-tween . cytokine assay secreted from t-cells. three mice from each group were sacrificed and the spleens were removed. the red blood cell-depleted pooled splenocytes ( × cells/ml) were cultured in -well plates for h in the presence of μg/ml of l + l peptides, rpmi % as negative control and μg/ml of concanavalin a (cona) as positive control in complete rpmi culture medium. the supernatants were harvested to assess the secretion of ifn-γ, il- and il- from t-cells using the sandwich-based elisa method (r&d systems) according to the manufacturer's instructions. all data were represented as mean ± sd for each sample . granzyme b assay (in vitro ctl activity). to measure granzyme b (grb) by elisa, the p target cells (t) were seeded into -well plates ( × cells/well) incubated with the mixture of l and l peptides (~ μg/ml) for h. then, the prepared splenocytes (effector cells: e, before section) were counted and added to the target cells at e: t ratio of : in complete rpmi culture medium for h incubation. finally, the supernatants were harvested to measure the concentration of grb by elisa (ebioscience kit) according to the manufacturer's instruction . table . immunization program for in vivo analysis. worldwide burden of cancer attributable to hpv by site, country and hpv type estimate of the global burden of cervical adenocarcinoma and potential impact of prophylactic human papillomavirus vaccination hpv vaccination: the promise & problems pros, cons, and ethics of hpv vaccine in teens-why such controversy? virus-like particles for the prevention of human papillomavirus-associated malignancies therapeutic human papillomavirus vaccines: current clinical trials and future 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multi-epitope vaccine the proteomics protocols handbook vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines protein-sol: a web tool for predicting protein solubility from sequence hpv l improves hpv l gene delivery as an important approach for vaccine design against cervical cancer whole recombinant pichia pastoris expressing hpv l antigen is superior in inducing protection against tumor growth as compared to killed transgenic leishmania immunogenicity of an hpv- l dna vaccine small heat shock protein : an effective adjuvant for enhancement of hiv- nef antigen-specific immunity recombinant leishmania tarentolae encoding the hpv type e gene in tumor mice model a.n. and a.b. conceptualized the work. a.n. performed the experiments. a.n., a.b., g.j. and z.n. analyzed the data. a.n. wrote the manuscript. a.b. edited the manuscript. all authors approved the final version of the paper. the authors declare no competing interests. correspondence and 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from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - wcqgaq authors: shen, zu t.; sigalov, alexander b. title: sars coronavirus fusion peptide-derived sequence suppresses collagen-induced arthritis in dba/ j mice date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: wcqgaq during the co-evolution of viruses and their hosts, the viruses have evolved numerous strategies to counter and evade host antiviral immune responses in order to establish a successful infection, replicate and persist in the host. recently, based on our model of immune signaling, the signaling chain homooligomerization (school) model, we suggested specific molecular mechanisms used by different viruses such as severe acute respiratory syndrome coronavirus (sars-cov) to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (mirrs). this family includes t cell receptor (tcr) that is critically involved in immune diseases such as autoimmune arthritis. in the present study, we provide compelling experimental in vivo evidence in support of our hypothesis. using the school approach and the sars-cov fusion peptide sequence, we rationally designed a novel immunomodulatory peptide that targets tcr. we showed that this peptide ameliorates collagen-induced arthritis in dba/ j mice and protects against bone and cartilage damage. incorporation of the peptide into self-assembling lipopeptide nanoparticles that mimic native human high density lipoproteins significantly increases peptide dosage efficacy. together, our data further confirm that viral immune evasion strategies that target mirrs can be transferred to therapeutic strategies that require similar functionalities. the severe acute respiratory syndrome (sars) coronavirus (sars-cov) is the etiological agent of sars that represents the life-threatening disease associated with a mortality of about % . lymphopenia is observed in most sars patients with t-helper (cd + ) and t-cytotoxic/suppressor (cd + ) cell levels reduced in % and % of the patients, respectively . most of these patients have reduced cd + and cd + cell counts during the early phase of the disease with the lowest cell count values on day and from disease onset , . like other enveloped viruses encoding class i viral fusion proteins such as human immunodeficiency virus (hiv) and ebola and avian sarcoma viruses , sars-cov is presumed to use membrane fusion mechanisms for viral entry , . it has been shown that the sars-cov viral spike protein (s ) is a class i viral fusion protein that is responsible for driving viral and target t cell membrane fusion . the putative sars-cov fusion peptide (fp) has been identified at the n terminus of the sars-cov s subunit . the fusogenic activity of this peptide has been shown to depend on its amino acid sequence . multichain immune recognition receptors (mirrs) play an important role in the host immune response (reviewed in [ ] [ ] [ ] . in mirrs, the extracellular ligand recognition domains and intracellular signaling sequences containing immunoreceptor tyrosine-based activation motifs (itams) are located on separate protein chains (subunits) bound together by noncovalent transmembrane (tm) interactions , . structurally, t cell receptor (tcr) is a member of the mirr family and has the α and β antigen-binding subunits that are bound by electrostatic tm interactions with three signaling homo-and heterodimers: ζ ζ , cd ε δ , and cd ε γ (fig. a) . short synthetic tm peptides capable of inhibiting tcr-mediated cell activation are known since when tcr-targeted immunomodulatory activity was first reported for the tcr core peptide (cp), a synthetic peptide corresponding to the sequence of the tcrα tm domain (tmd) known to interact with the tmds of cd ε δ and ζ , . similar activity was later reported for hiv fp found in the n terminus of the hiv envelope glycoprotein (gp ) , . intriguingly, the patterns of tcr-targeted inhibitory activity of tcr cp and hiv gp fp were very similar: both peptides inhibit antigen-but not anti-cd -stimulated t cell activation , . both peptides were shown to reduce inflammation and ameliorate t cell-mediated autoimmune diseases such as arthritis in animal models , , . however, despite extensive studies , , , [ ] [ ] [ ] [ ] [ ] [ ] , the mode of action of these clinically relevant peptides was enigmatic until a novel model of immune signaling, the signaling chain homooligomerization (school) model, was first introduced and applied to this field , , . previously, using the school model and comparative primary sequence analysis of proven and predicted immunomodulatory sequences of viral fusion protein regions, we not only suggested the specific molecular mechanisms of t cell activation inhibition by tcr cp and hiv gp fp [ ] [ ] [ ] but also predicted similar immunomodulatory activity for other viral fps such as sars-cov fp (fig. b) . in this study, we demonstrate that a synthetic amino acid-long peptide (mg ) derived from sars cov fp reduces inflammation in dba/ j mice with collagen-induced arthritis (cia) and protects mice against bone and cartilage damage. the effect is specific as administration of the control peptide has no effect. incorporation of mg into synthetic nanoparticles that mimic human high density lipoproteins (hdl) substantially reduces the effective peptide dosage. in summary, our data demonstrate for the first time that sars-cov fp does not only have fusogenic, but also immunomodulatory activity. this study provides compelling experimental in vivo evidence in support of our hypothesis and further confirms that viral immune evasion strategies evolved during host-virus co-evolution can be transferred to therapeutic strategies that require similar functionalities (e.g., in the treatment of autoimmune diseases). to evaluate a putative anti-arthritic activity of sars-cov fp, we used the sars-cov fp-derived peptide sequence mwktptlkyfg (mg ). this peptide includes the charge distribution pattern with two essential positively charged amino acid residues (underlined) spaced apart by four amino acids that is similar to that of the tcrα chain tmd either of human (vigfrilllkvagfnllmtl) or mouse (svmglrilllkvagfnllmtl) origin. based on the school model, this sequence has been previously hypothesized to have a similar immunomodulatory activity as tcr cp (glrilllkv) or hiv gp fp . a sars cov fp peptide mutant with two functionally important lysines replaced by glycines (mg - g) was used as a negative control peptide. we used the cia mouse model, the most commonly studied autoimmune model of rheumatoid arthritis (ra) , since a vast majority of the studies of immunomodulatory activity of tcr cp and hiv gp fp has been done in animal models of autoimmune arthritis , , , , , . when intraperitoneally (i.p.) administered daily at a dose of mg/kg, mg significantly suppressed arthritis severity compared with administration of vehicle or control peptide mg - g ( mg/kg/day). as shown in fig. a , the difference between the mg and vehicle groups started on day and continued until day . on day , the mean ± sem clinical arthritis score in mg -treated mice with cia was much lower than that in mg - g-treated mice ( . ± . versus . ± . ; p < . ). the effect is dose-dependent: no anti-arthritic activity was observed for free mg i.p. administered daily at a dose of . mg/kg (data not shown). the tcr α and β recognition subunits are shown in red and blue, respectively. the cd ε , cd δ , cd γ and ζ signaling subunits are shown as purple, dark orange, light orange and green, respectively. immunoreceptor tyrosine-based activation motifs (itams) are shown as spheres and are colored accordingly by subunit. the recognition and signaling subunits are bound together by electrostatic transmembrane (tm) interactions. these tm interactions occur between basic and acidic amino acid residues. the tcrα transmembrane domain (tmd) contains two basic residues: a lysine, which interacts with two acidic residues of aspartic acid present in the tmds of the cd ε δ heterodimer, and an arginine, which interacts with two aspartic acid residues present in the tmds of the ζ ζ homodimer. the tcrβ tmd contains a lysine, which interacts with one aspartic acid residue and one acidic residue of glutamic acid present in the tmds of the cd ε γ heterodimer. (b) tmtargeted school peptides such as the sars-cov fp or the tcr cp disrupt tm electrostatic interactions between the tcrα subunit and both cd ε δ and ζ ζ by competing with tcrα for binding to cd ε δ and ζ ζ . scientific reports | : | doi: . /srep previously, we reported that incorporation of another immunomodulatory peptide, gf , that employs the school mechanisms of action and targets triggering receptor expressed on myeloid cells (trem- ), into synthetic hdl-like nanoparticles of spherical shape (shdl) significantly reduces the effective therapeutic dosage of gf in animal models of sepsis, lung cancer, and ra , . to evaluate whether incorporation of mg into shdl may have a similar effect, shdl-bound mg was i.p. administered daily at a dose of . mg/kg mg . despite a -fold decrease in administration dose of mg , the arthritis inhibitory effect observed for . mg/kg/day mg -hdl was comparable to that observed for mg/kg/day peptide in free form (fig. a) . although the underlying molecular mechanisms of this phenomenon are not completely understood and need to be further investigated, one can suggest that this results from the prolonged circulatory half-life of shdl-bound mg : while the in vivo peptide half-life is short, typically a few minutes , shdl are characterized by much longer half-lives up to - days . interestingly, in contrast to vehicle-or mg - g-treated mice, administration of mg at a daily dose of mg/kg and shdl-bound mg at a daily dose of . mg/kg resulted in an increase in body weight comparable to that observed for non-arthritic naïve mice (fig. b,c) . in summary, these data collectively indicate that the sars-cov fp-derived peptide mg generates a strong anti-arthritic effect in the cia mouse model of ra, thereby providing the first experimental in vivo evidence of previously predicted immunomodulatory activity of sars-cov fp . incorporation of mg into spherical hdl-like synthetic particles substantially reduces the effective dosage of peptide probably because of the prolonged circulatory half-life afforded by this strategy. to further evaluate the effect of mg in suppressing cia and determine whether mg inhibits chronic inflammation of synovial tissue, pannus formation, cartilage destruction, and bone erosion, we next examined the all results are expressed as the mean ± sem (n = mice per group). * * p < . ; * * * p < . ; and * * * * p < . versus vehicle. histopathology of the animal joints (fig. ) . overall, mice treated with mg/kg/day free mg or . mg/kg/day shdl-bound mg had significantly lower joint histopathological scores than the vehicle-or mg - g-treated groups (p < . ) (fig. ) . in the vehicle-treated arthritic mice, the fore and hind paw joints had moderate inflammation and cartilage damage with moderate pannus and bone resorption, as well as mild periosteal bone formation, in all joints (fig. ) . the knee joints had marked inflammation and moderate cartilage damage with pannus formation, bone resorption, and periosteal bone formation (not shown) (fig. ) . the ankle joints had moderate inflammation and cartilage damage with minimal pannus and bone resorption, as well as mild periosteal bone formation (fig. ) . markedly thickened synovial membrane and capsule were observed as a result of pannus formation and inflammatory cell infiltration. as shown in fig. , the chronic inflammation destroyed the joint lining, including the cartilage and other nearby supporting structures, such as bone. the formation of pannus is probably a result of overgrowth of the synoviocytes and the observed accumulation of inflammatory cells that led to deformed cartilage and bone. this agrees with the observed clinical scores. similar histopathology of the joints was observed in the animals treated with mg - g at mg/kg/day (not shown). for mice treated with mg- at mg/kg/day, the fore and hind paw joints had no or very minimal inflammation and minimal cartilage damage (fig. ) . the knee and ankle joints had no or very minimal inflammation and no or mild evidence of synovial membrane thickening with pannus formation, which falls within normal limits (fig. ) . similar histopathology was observed in mice treated with shdl-bound mg at . mg/kg/day (fig. ) . in summary, histopathology examination showed greatly reduced joint inflammation and damage in mg -treated mice compared with the vehicle-treated mice or mice treated with mg - g suggesting a specific protective effect of the mg peptide. no significant difference was observed in the histopathological analysis of the joint limbs in mice treated with free mg ( mg/kg/day) or shdl-bound mg ( . mg/kg/day). as mentioned above, the prolonged half-life of the peptide incorporated into the hdl particle is probably one of the reasons why shdl-bound mg at a dose of . mg/kg/day is similarly effective to free mg at a dose of mg/kg/day. the sars-cov fp sequence mg reduces cytokine serum levels in mice with cia. to investigate potential mechanisms underlying the effect of mg , we examined the serum levels of different cytokines on day using a quantitative multiplex elisa array. in mice treated with free mg at mg/kg/day or shdl-bound mg at . mg/kg/day, the cytokine levels were significantly lower than in the vehicle-treated mice or those treated with mg - g at mg/kg/day (fig. ) . interestingly, treatment with mg reduced the serum level of macrophage colony-stimulating factor (m-csf) that plays an important proinflammatory role in cia and is known to be produced by a variety of cells including activated t cells , . to further elucidate the molecular mechanisms underlying the observed immunomodulatory effect of mg in vivo, we used confocal fluorescence microscopy and demonstrated that mg inserts into the t cell membrane and colocalizes with tcr in t cells in vitro (supplemental fig. ) . in summary, our data suggest that the molecular mechanisms of cia suppression by mg can include inhibition of cytokine and growth factor production mediated by inflammatory t cells that are thought to be central to the pathology of autoimmune arthritis . to successfully infect, replicate and persist in the host, viruses have evolved numerous strategies to take control of multiple cellular processes including those that target transmembrane signal transduction mediated by immune receptors including mirrs (reviewed in [ ] [ ] [ ] ). for t lymphotropic viruses, this approach allows the virus to inhibit tcr signaling to disarm the receptor and successfully enter the cell while for other viruses it allows for evasion from t cell response towards the infected cells , , . recently reported tcr-targeted immunomodulatory activity mediated by hiv gp fp , suggests that fusion peptides function not only to fuse the virion with the host cell , but also to silence the tcr signaling pathway. interestingly, the characteristic pattern of tcr-targeted inhibitory activity of hiv gp fp is strikingly similar to that of tcr cp: both peptides colocalize with tcr in the cell membrane, inhibit antigen-but not anti-cd -stimulated t cell activation in vitro, and suppress autoimmune arthritis in vivo [ ] [ ] [ ] [ ] , . both peptides were suggested for the treatment of t cell-mediated pathologies including inflammatory skin diseases and ra , [ ] [ ] [ ] . in addition, tcr cp has been shown in human studies to be a proper treatment for human t cell-mediated dermatoses that can substitute for corticosteroids . the molecular mechanisms of action for these clinically relevant peptides were first explained by the school model , , , . later, based on the school model and primary sequence analysis of a variety of viral fps including sars-cov fp, we hypothesized that similar to hiv gp fp, these fps may not only have fusogenic but also tcr-targeted immunomodulatory activity and that the school model, together with the lessons learned from viral pathogenesis, can be used practically for rational drug design and the development of new therapies for immune disorders . at the end of treatment on day , different groups of mice with collagen-induced arthritis (cia) intraperitoneally (i.p.) administered daily with either vehicle, mg ( mg/kg) or shdl-bound mg ( . mg/kg) were euthanized and sections were prepared using fore paws, hind paws, knees and ankles. individual joint photomicrographs from representative mice are shown for each group. for paws and ankles, arrows identify affected joints. for knees, large arrow identifies cartilage damage, small arrow identifies pannus, and arrowhead identifies bone resorption. w, wrist; s, synovium. scientific reports | : | doi: . /srep as mentioned above, the amino acid-long hydrophobic stretch corresponding to residues to (myktptlkyfggfnfsqil) has been recently identified as the putative fusion peptide of the sars-cov s subunit . in the present study, in order to provide compelling experimental in vivo evidence to support our hypothesis , we used the school model to design a -mer synthetic peptide mg (myktptlkyfg) derived from the sars-cov fp sequence with the positioning of two essential positively charged lysine residues (underlined) spaced by four amino acids. the model suggests that this charge distribution pattern is functionally important to provide tcr-targeted inhibitory activity , , , . if our hypothesis is correct, the mg peptide should demonstrate the immunomodulatory activity in vivo similar to that demonstrated earlier for tcr cp and hiv gp fp in animal models of autoimmune arthritis , , , . the sars cov fp peptide mutant with lysines replaced by glycines (mg - g) was used as a negative control peptide. according to the school model , , this peptide cannot compete with the recognition tcrα subunit for binding to ζ ζ and cd ε δ signaling homoand heterodimers (fig. ) , and thus cannot inhibit tcr signaling. because of discrepancies found in prior studies of the immunomodulatory activity of tcr inhibitory tm peptides between in vitro (no activity observed) and in vivo (anti-arthritic activity observed in rats with adjuvant-induced arthritis, aia) data , in this study, we moved directly to in vivo studies and tested the mg and mg - g peptides in the cia model of autoimmune arthritis. the circulatory half-life of peptides in vivo is very short, typically only a few minutes . in order to prolong the half-life of mg , we tested in the present study whether this peptide can be incorporated into shdl nanoparticles that mimic human hdl, a group of native lipoproteins that transport cholesterol from the peripheral tissues to the liver and can be readily reconstituted in vitro from lipids and apolipoproteins (apos) . due to the half-life of native shdl in normal subjects being - days , these particles represent a promising and versatile delivery platform for peptide therapeutics. synthetic (reconstituted) hdl have several competitive advantages as compared with other delivery platforms: ) apo a-i, the major hdl protein, is an endogenous protein and does not trigger immunoreactions, ) the small size ( - nm) allows hdl to enter and accumulate in tissue and organ areas of interest, and ) a variety of drugs and imaging agents can be incorporated into this platform , , . with respect to therapeutics, human apo a-i is a large protein, which is purified from human plasma. thus, in addition to the immense monetary cost in purification, further development of apo a-i-containing therapeutic agents would require a number of safety precautions followed by a complicated transition into clinical practice. previously, we demonstrated that synthetic apo a-i peptides can functionally replace the native apo a-i protein in hdl. this encourages the further development of the hdl-based delivery platform. in the present study, synthetic shdl that contain apo a-i peptides were successfully loaded with mg and subsequently purified and characterized using a variety of biophysical procedures. this is the first study to test previously predicted immunomodulatory activity of sars-cov fp . as expected from the anti-arthritic activities demonstrated in animal models of autoimmune arthritis for tcr cp , , and hiv gp fp , the sars-cov fp-derived peptide sequence mg significantly suppresses cia in mice: the peptide at mg/kg/day inhibits inflammation in cia as assessed by clinical evaluation and scoring of the disease (fig. ) . histological analysis of the joints reveals that mg substantially reduces joint inflammation, protects against cartilage damage, abrogates bone erosion and reduces systemic bone loss (figs and ) the effect is specific as the control mg - g peptide administered daily at the same dose of mg/kg does not affect cia. incorporation of mg into shdl reduces the effective dosage of the peptide: mg in free form at mg/kg/day and shdl-bound mg at . mg/kg/day show similar anti-arthritic effects in cia both clinically and histologically. interestingly, mice treated with free mg at a daily dose of . mg/kg did not exhibit any significant disease improvement as compared to vehicle-treated mice (not shown). at the molecular level, activated t cells mediate production of multiple cytokines and growth factors that are known to be involved in the pathogenesis of ra . many of these molecules serve as targets of cytokine-blocking therapies that are currently in development (e.g., il- , il- , and il- ), at different phases of clinical trials (e.g., il- , il- , il- , and m-csf) or approved (e.g., tnfα , il- , and il- blockers) . in the present study, significantly reduced serum cytokine levels were observed in mice treated with mg as compared to vehicle-treated arthritic mice or mice treated with mg - g (fig. ) . colocalization of mg with tcr in the t cell membrane (supplemental fig. ) further supports the suggested molecular mechanisms of the observed immunomodulatory activity of the peptide. these findings are consistent with those previously reported for tcr cp and hiv gp fp , . in summary, the data presented in this study provide the first experimental evidence of the previously predicted immunomodulatory activity of sars cov fp and demonstrate a strong anti-arthritic effect of the sars cov fp-derived amino acid-long peptide sequence in a mouse model of ra. interestingly, immunosuppressive activity of the influenza fp has been recently demonstrated in vitro . further, we suggested before that: ) short synthetic peptides (school peptides) can be designed in line with the school platform-based strategy for therapeutic inhibition and modulation of a variety of functionally unrelated multichain receptors expressed on various cells, and ) the molecular mechanisms of action of the school peptides is similar to those that viruses use to evade the immune system , , . to date, the school peptides that target trem- , glycoprotein receptor vi (gpvi), and tcr were demonstrated both in vitro and in vivo to represent promising therapeutic approaches to the treatment of a variety of diseases with unmet clinical need including sepsis, lung cancer, rheumatoid arthritis, dermatoses, and others [ ] [ ] , , . taken together, these findings further support our unifying hypothesis , that the viral immune evasion strategies developed and optimized during millions of years of evolution of virus-host interactions can be practically used for the rational drug design of new mechanism-based therapies. incubated at °c for min. after cooling to °c, the solution containing a : mixture of apo a-i peptides h and h in pbs, ph . was added and the mixture was incubated at °c for h, followed by extensive dialysis against pbs to remove sodium cholate. the obtained mg -shdl particles were then purified on a calibrated superdex hr gel filtration column (ge healthcare biosciences, pittsburgh, pa) using the biocad e workstation (applied biosystems, carlsbad, ca) and characterized by analytical rp-hplc and nondenaturing gel electrophoresis as described previously . final peptide compositions were determined in the prepared particles by analytical rp-hplc as previously described . the mean size of the particles was determined using electron microscopy as described . animal studies. animal studies were performed by bolder biopath (boulder, co). all animal experiments were performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (nih) and in the united states department of agriculture (usda) animal welfare act ( cfr, parts , , and ). the protocol (bbp- .b) was approved by the institutional animal care and use committee (iacuc) of bolder biopath for compliance with regulations prior to study initiation (animal welfare assurance number a - ) and all methods were performed in accordance with the approved protocol. male - week old dba/ mice from harlan (indianapolis, in) were anaesthetized with isoflurane (vetone, boise, id) and injected intradermally with μ l of freund's complete adjuvant (sigma aldrich company, st. louis, mo) ( . mg/ml final concentration) containing bovine type ii collagen (bolder biopath, boulder, co) ( mg/ml final concentration) at the base of the tail on day and again on day . on day , mice were randomized by body weight into treatment groups. mice weighed approximately - grams (mean g) at enrollment on day when treatment was initiated. mice were i.p. injected with mg/kg/day mg or mg - g, or . mg/kg/day mg -shdl, or with pbs for days beginning at day . arthritis onset occurred on days - . mice were weighed on study days , , , , , , and (prior to necropsy) . daily clinical scores were given on a scale of - for each of the paws (right front, left front, right rear, left rear) on days - using the following criteria: = normal; = one hind or fore paw joint affected or minimal diffuse erythema and swelling; = two hind or fore paw joints affected or mild diffuse erythema and swelling; = three hind or fore paw joints affected or moderate diffuse erythema and swelling; = four hind or fore paw joints affected or marked diffuse erythema and swelling; = entire paw affected, severe diffuse erythema and severe swelling, unable to flex digits. on day , mice were humanely euthanized for necropsy. mice were anesthetized with isoflurane and bled by cardiac puncture. serum was prepared and stored frozen at − °c for cytokine analysis. for histology, fore paws, hind paws, and knees were harvested and placed in % neutral buffered formalin (nbf). after - days in fixative and - days in % formic acid for decalcification, tissues were trimmed, processed for paraffin embedding, sectioned at μ m, and stained with toluidine blue (t blue). hind paws, fore paws, and knees were embedded and sectioned in the frontal plane. six joints from each animal were processed for histopathologic evaluation. the joints were then assessed for inflammation ( - scale), pannus formation ( - scale), cartilage damage ( - scale), bone resorption ( - scale), and periosteal new bone formation ( - scale). a summed histopathology score was also determined (sum of five parameters). cytokine analysis. serum samples were collected on day and cytokines were analyzed using quantibody mouse cytokine array q kits from raybiotech (norcross, ga) following manufacturer's instructions. statistics. data analyses were performed using prism . 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transduction in the immune system viral pathogenesis, modulation of immune receptor signaling and treatment viral modulation of t-cell receptor signaling identification of the fusion peptide of primate immunodeficiency viruses detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus the potential of liposomal drug delivery for the treatment of inflammatory arthritis peptides in the treatment of inflammatory skin disease t-cell antigen receptor (tcr) transmembrane peptides: a new paradigm for the treatment of autoimmune diseases therapeutic application of t cell receptor mimic peptides or cdna in the treatment of t cell-mediated skin diseases signaling chain homooligomerization (school) model reconstitution of high-density lipoproteins diagnostic magnetic resonance imaging of atherosclerosis in apolipoprotein e knockout mouse model using macrophage-targeted gadolinium-containing synthetic lipopeptide nanoparticles nature-inspired nanoformulations for contrast-enhanced in vivo mr imaging of macrophages the pathogenesis of rheumatoid arthritis cyclization enhances function of linear anti-arthritic peptides immune suppressive activity of the influenza fusion peptide novel mechanistic concept of platelet inhibition we are grateful to bolder biopath for animal experiments. we also owe a debt of gratitude to phillip bendele, dr. alison bendele, john galvin and kyle rothermel, who did an excellent job conducting mouse studies, for their important expertise, experience, and skills and for numerous valuable discussions. this work was partly supported by a grant r ar (zts, abs; alexander b. sigalov, principal investigator) from national institute of arthritis and musculoskeletal and skin diseases of the national institutes of health. the additional funding (abs) has come from signablok, inc. the funding sources have no role in the design of the study, conduction of the experiments, interpretation of the data, and writing of the manuscript. accession codes: accession numbers (uniprotkb/swiss-prot knowledgebase, http://www.expasy.org/sprot/) for the protein sequences discussed in this research article is as the follows: t cell receptor alpha chain, p (human) and p (mouse); sars-cov, p ; hiv- , p . key: cord- -ujbxsus authors: jiang, xiandeng; chang, le; shi, yanlin title: a retrospective analysis of the dynamic transmission routes of the covid- in mainland china date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ujbxsus the fourth outbreak of the coronaviruses, known as the covid- , has occurred in wuhan city of hubei province in china in december . we propose a time-varying sparse vector autoregressive (var) model to retrospectively analyze and visualize the dynamic transmission routes of this outbreak in mainland china over january –february , . our results demonstrate that the influential inter-location routes from hubei have become unidentifiable since february , , whereas the self-transmission in each provincial-level administrative region (location, hereafter) was accelerating over february – , . from february , , all routes became less detectable, and no influential transmissions could be identified on february and , . such evidence supports the effectiveness of government interventions, including the travel restrictions in hubei. implications of our results suggest that in addition to the origin of the outbreak, virus preventions are of crucial importance in locations with the largest migrant workers percentages (e.g., jiangxi, henan and anhui) to controlling the spread of covid- . www.nature.com/scientificreports/ been analyzed and compared over a rage of affected countries (e.g., australia , , germany , italy , and south korea ). among those emerging large volume of studies, mathematical and statistical modeling plays a non-negligible role. also, the classical susceptible exposed infectious recovered (seir) model with its various extensions is the most popular method [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . seir family models are effective in exploring the epidemic characteristics of the outbreak, forecasting the inflection point and ending time, and deciding the measures to curb the spreading. despite this, they are less appropriate in identifying transmission routes of the covid- outbreak, which is also not thoroughly investigated in existing literature. in this paper, we fill in this gap and perform a retrospective analysis using the publicly available data . rather than employing the seir, we develop a time-varying coefficient sparse vector autoregressive (var) model. using the least absolute shrinkage and selection operator (lasso) , and the local constant kernel smoothing estimator , our model is capable of estimating the dynamic high-dimensional granger causality coefficient matrices. this enables the detection and visualization of time-varying inter-location and self-transmission routes of the covid- on the daily basis. the resulting "road-map" can help policy-markers and public-health officers retrospectively evaluate both the effectiveness and unexpected outcomes of their interventions. such an evaluation is critical to winning the current battle against covid- in china, providing useful experience for other countries facing the emerging threat of this new coronavirus, and saving lives when a new epidemic occurs in the future. model. throughout this study, we are interested in the growth rate y i,t such that: where x i,t is the accumulated confirmed cases in the provincial-level administrative region (location, hereafter) i on day t ( i = , . . . , n and t = , . . . , t ). t and n define the number of days and number of locations under consideration, respectively. we then define y t = (y ,t , . . . , y n,t ) ′ , an n × vector of the growth rate on day t. to investigate a dynamic direct transmission of the growth rate among locations, we propose a time-varying coefficient sparse var model, namely the tvsvar model, which assumes that granger causality coefficients are functions of time, such that: where α t is an n-dimensional intercept vector at time t. b t is an n × n granger causality matrix at time t with a dynamic sparse structure, for which entries can be exactly zero and the locations of zeros can vary with time. ǫ t is an n × vector of error terms. the sparsity of b t is assumed because n could be even larger than t in our case, which leads to very unstable estimations and problematic interpretations of b t . one important benefit of using the proposed tvsvar to model the transmissions is that the granger causality matrix, b t , can provide both the direction and strength of the route on day t. for example, the ijth entry in b t measures the strength of the transmission from location i to location j on day t. the ith diagonal of b t represents the self-transmission in location i that captures the relationship between the growth rate in the current and previous days. more critically, the sparse structure eases the interpretation of b t because many weak transmissions may be of a random nature. the corresponding coefficients, therefore, can be treated as noises and are shrunk to zeros exactly. moreover, a time-varying design of b t allows us to investigate changes in the identified transmissions over time. for instance, let , and indicate hubei, jiangxi and shanghai, respectively. on day t = , the estimated β , , and β , , are . and . , respectively. this suggests on that day, moderately strong transmission routes of confirmed covid- cases are detected from huber to jiangxi and from jiangxi to shanghai, respectively. further, estimated β ,i, for all i = , . . . , are zeros, suggesting that the confirmed cases in shanghai cannot spread to other locations on day . on day t = , we observe estimated β , , = . , β , , = . and all β ,i, = . thus, the two detected routes from huber to jiangxi and jiangxi to shanghai have become more influential, whereas the cases in shanghai are still yet to spread out on day . the above results cannot be derived using the classic epidemiological seir model. to capture both dynamic and sparse structure of the granger causality coefficients, we solve the following optimization problem: we use the epanechnikov kernel k(x) = . ( − x ) + and a unified bandwidth for each i ( b i ≡ b ) to avoid a large number of tuning parameters. the coefficients β i,j,t denotes the ijth entry of the granger causality matrix b t , and is the tuning parameter that aims to shrink insignificant β i,j,t to zero and thus controls the sparsity of b t . another essential feature of our proposed model is that the adaptive weights w i,j,t are employed to penalize β i,j,t differently in the lasso (l ) penalty , . the choice of weights w i,j,t takes account of the prior knowledge about the transmissions and can be specified by the users. in this study, we consider w i,j,t as the reciprocal of the accumulated confirmed in location i on day t − . that is, the growth rate of a location with a smaller accumulated confirmed cases is less likely to influence the growth rates of others, and thus, more likely to be shrunk to zero. the final sparsity structure of b t is still data-driven. ( ) www.nature.com/scientificreports/ the estimators as in ( ) can also be viewed as a penalized version of local constant kernel smoothing estimator . we utilize a modified version of the fast iterative soft thresholding algorithm (fista) to solve the optimization problem ( ) . given a bandwidth b and a penalty parameter , we can find the estimator ( α t , b t ) for each day t and observe the dynamic patterns of the transmission over time t for each pair of locations. the selection of b is critical to detecting the influential routes, which depends on the chosen criterion. among the existing literature, a popular approach is to adopt the cross-validation strategy, such that based on the estimated (α t , b t ) , the model will not 'overfit' y t . as for the time-series analysis, we use an expanding-window sample to implement the crossvalidation . this requires that the chosen b will minimize the cross-validated forecast error, which is measured by the one-step-ahead root mean squared forecast error (rmsfe), such that where [t , t ] is the evaluation period, which is given by the last third of the data in our study, y i,t+ denotes the one-step-ahead forecast for location i based on the data up to day t, and y i,t+ defines the observed growth rate at day t + for location i. note that rmsfe is analogous to the square root of the popular least squared errors. an interpretation is that the chosen b will lead to the minimized total out-of-sample forecast errors of the growth rates of confirmed cases over the last third of the sample period. data. the data studied in this paper include confirmed covid- cases which occurred in mainland china. the data are publicly available and sourced from the website of the national health commission of the people's republic of china . the data-coverage ranges from january , to february , , during which no missing data were recorded at location-level. the accumulated cases and the associated growth rates, grouped by the total national number, cases in hubei and cases in all other locations, are plotted in fig. a ,b, respectively. the total national (hubei) accumulated confirmed cases increased rapidly from , ( , ) on january , to , ( , ) on february , . note that on february , , confirmed cases in hubei included those confirmed by both laboratory and clinical diagnosis, leading to a one-time hump of the accumulated number. compared to those of hubei, confirmed cases of other locations took up a smaller proportion of the total national number, ranging from . % on january , to . % on february , . this suggests that the growth rate of other locations should be lower than that of hubei, which is consistent with fig. b . throughout our investigation period, except for the one-time hump on february , , growth rates of hubei and the rest steadily declined, from % and % to % and %, respectively. estimation results: transmission routes. by taking the difference of the logged accumulated cases and applying one lag, our estimated transmission routes are available from january to february , (two observations are lost). to avoid potential noises caused by small numbers, we only include data of locations, which had at least accumulated confirmed cases as of february , . altogether, our modeled sample contains location-level confirmed cases. we firstly test the stationarity of the growth rates separately. based on the augmented dickey-fuller test, only the rates of three locations (beijing, hainan and heilongjiang) are insignificant, which is in-line with the employed % significance level. the detailed results are available upon request. the model explained in "methods" is then fitted incorporating all the growth rates. a non-zero estimate of β i,j,t , the ijth entry of b t in ( ), indicates that on the tth day, the growth rate of location j is granger caused by that of location i. in other words, there is a transmission route from location i to location j. among the -day results, we noticed that the estimated transmission routes on days - changed considerably on daily basis. from the sixth day onwards, however, those estimated routes were more steady. hence, we plot the estimates on days - and those on the every fifth day thereafter, on fig. . be noted that estimates smaller than . (none-influential) are not presented a better visual illustration purpose. also, this research focuses on the analysis of mainland china only, which excludes taiwan, macau, hong kong and all important islands of china's territory, such as those located in the south china sea. the plots presented in fig. therefore do not present a complete map of the territory of china, nor should they be used for purposes other than displaying identified transmission routes of covid- in mainland china. the readers are directed to the national administration of surveying, mapping and geographic information of the people's republic of china, should they need to precisely explore the scope of maps, national boundaries and the drawing of important islands of the chinese territory. in fig. , we use color of light orange (small) to dark red (large) indicating the accumulated confirmed cases in each location, up to time t. estimated transmission routes are colored in blue. self-transmissions (indicated by β i,i,t ) are denoted by dots, and a larger size of dot suggests a larger estimated β i,i,t . inter-location transmission (indicated by β i,j,t , where i = j ) is represented by arrows, with the transparency indicating the magnitude of estimated β i,j,t . on the first day (january , ), there were influential inter-location transmissions from hubei to jiangxi, heilongjiang, zhejiang, henan, shandong, jiangsu and shaanxi, sorted by the magnitudes of strength (big to small). there were a few additional detected such transmissions on the second day, including those from hubei to guangxi, from jiangxi to fujian, and from guangdong to anhui, yunnan and hunan. the number of such identified inter-location routes, however, reduced rapidly over the next three days. on the fifth day (february , ), no influential transmission routes were found from hubei to directly affect other locations, and there were only three influential routes identified nationally, including zhejiang-shaanxi, www.nature.com/scientificreports/ zhejiang-jiangxi and jiangxi-shanghai. the number of those detected inter-location routes declined again in the next few days, and on day , only henan-heilongjiang was found influential. on days and (february and , ), there were no influential inter-location transmissions identified. the above findings suggest that the number of influential inter-location transmissions overall dropped quickly in the first five days and then reduced steadily for the rest days. this is consistent with the observations of fig. a, where the time-varying estimates of the granger causality of hubei on other locations are plotted. on each day, we report the mean, standard deviation (std. dev.), the % quantile ( q ) and % quantile ( q ) of those estimates in table , which also leads to consistent findings. www.nature.com/scientificreports/ as for the self-transmission, we firstly examine (fig. b) . it can bee seen that there were quite a few detected influential self-transmissions on the first two days. however, this number dropped quickly over days - , and only self-transmissions of heilongjiang, guangdong and zhejiang were found influential on day . since then, the number of influential self-transmissions increased quickly with growing magnitudes (influence). on the sixteenth day (february , ), out of the examined locations had an estimated β i,i, of at least . . those large self-transmissions, however, disappeared rapidly again in the next days. on february and , , there were no influential self-transmissions identified. this is consistent with our findings on fig. b , where time-varying estimated β i,i,t are plotted for each location. we report daily descriptive statistics of those estimates in table , which also results in consistent conclusions. since january, , many cities on mainland china started to introduce travel restrictions, including five cities (wuhan, huanggang, ezhou, chibi and zhijiang) of hubei . according to who's situation report , the average incubation period of covid- is up to days. thus, our estimated dynamic transmission routes supports the significant effectiveness of the interventions taken by the chinese authorities [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this is evidenced by fig. a-e, where the number of influential inter-location transmissions from hubei to other locations reduced very quickly. compared to multiple influential routes originating in hubei detected on the first two days (january and february , ), by february , (around days after the travel restrictions), there were already no such transmissions identified. on the other hand, from february to , , table suggests that the averaged magnitudes of self-transmission on each day were strengthening steadily. this may also be explained by the interventions, which have effectively blocked inter-location transmissions, such that the growth rate of each location could only be caused by its internal transmissions. we now focus on the inter-location transmission routes. since influential routes from hubei were no longer detected since day , we calculate the average β i,j,t of the locations affected by hubei over the first four days table . this is consistent with the fact that travel restrictions in hubei should not affect the connections among other locations. in all cases, jiangxi, henan, guangdong, zhejiang and anhui are the most influential origins other than hubei. it is worth noting that jiangxi, henan and anhui belong to both the top origins and destinations of the interlocation transmissions, excluding hubei. since the impact of hubei is not considered, this cannot be explained by the two influential transmission routes of hubei-jiangxi and hubei-henan listed in panel a of table . to see this, over days - , the transmissions out of hubei are no longer significant and thus should not affect routes from jiangxi and henan to another location. in contrast, one explanation is the large migrant workers from jiangxi, henan and anhui to other locations (excluding hubei). according to the report on china's migrant population development of , jiangxi ( . %), henan ( . %) and anhui ( . %) are among the top five locations in mainland china, ranked by the percentages of migrant workers in . www.nature.com/scientificreports/ conclusions coronaviruses have lead to three major outbreaks ever since the sars occurred in . although the exact origin is still debatable, the current shock, namely covid- , has taken place in wuhan, the capital city of hubei province in mainland china. as the fourth large-scale outbreak of coronaviruses, covid- is spreading quickly to all provincial-level administrative regions (locations, hereafter) in china and has recently become a world-wide epidemic. as a significant complement to existing research, this study employs a tvsvar model and retrospectively investigates and visualizes the transmission routes in mainland china. demonstrated in fig. , our baseline results review both the dynamic inter-location and self-transmission routes. since february , , the spread out of hubei was largely reduced, leading to no identifiable routes to other locations. simultaneously, the self-transmissions started to accelerate and peaked on around february , for most locations. given an average incubation period of days, those results support the argued effectiveness of the travel restrictions to control the spread of covid- , which took place in multiple cities of hubei on january , . on february - , , there existed no influential inter-location or self-transmission routes. thus, the growth rates of confirmed cases are of a more random nature in all locations thereafter, implying that the spread of covid- has been under control. for the detected inter-location transmissions, our findings demonstrate that jiangxi, heilongjiang, zhejiang, henan and shandong are the top locations affected mostly via routes directly from hubei. when the influence of hubei is excluded, jiangxi, henan and anhui are among both the top origins and destinations of transmission routes. our results have major practical implications for public health decision-and policy-makers. for one thing, the implemented timely ad-hoc public health interventions are proven effective, including contact tracing, quarantine and travel restrictions. for another, apart from the origin of the virus, as locations with largest migrant workers percentages, virus preventions are also of crucial importance in jiangxi, henan and anhui to controlling the epidemics like the outbreak of covid- in the future. with limited resources, taking ad-hoc interventions in such locations may most effectively help stop the spread of a new virus, from an economic perspective. the r code that supports the findings of this study is available from the author on request. www.nature.com/scientificreports/ publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. emerging coronaviruses: genome structure, replication, and pathogenesis history and recent advances in coronavirus 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coronavirus outbreak in china predictions of -ncov transmission ending via comprehensive methods preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak national health commission of the people's republic of china regression shrinkage and selection via the lasso the adaptive lasso and its oracle properties local polynomial regression: optimal kernels and asymptotic minimax effciency a fast iterative shrinkage-thresholding algorithm for linear inverse problems principles and practice national health and family planning commission of china. report on china's mi-grant population development the authors would like to thank southwestern university of finance and economics, australian national university and macquarie university for their support. xiandeng jiang acknowledges the research grants supported by the people republic of china ministry of education youth project for humanities and social science research (grant no. yjc ). we particularly thank the deputy editor (rafal marszalek) and two anonymous referees for providing valuable and insightful comments on earlier drafts. the usual disclaimer applies. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. x.j. collected data and designed the research. l.c. and y.s. performed the research and analyzed the data. x.j., l.c., and y.s. wrote the paper. the authors declare no competing interests. correspondence and requests for materials should be addressed to y.s.reprints and permissions information is available at www.nature.com/reprints. key: cord- -bqqyly authors: zhao, suhui; wan, chengsong; ke, changwen; seto, jason; dehghan, shoaleh; zou, lirong; zhou, jie; cheng, zetao; jing, shuping; zeng, zhiwei; zhang, jing; wan, xuan; wu, xianbo; zhao, wei; zhu, li; seto, donald; zhang, qiwei title: re-emergent human adenovirus genome type d caused an acute respiratory disease outbreak in southern china after a twenty-one year absence date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: bqqyly human adenoviruses (hadvs) are highly contagious pathogens causing acute respiratory disease (ard), among other illnesses. of the ard genotypes, hadv- presents with more severe morbidity and higher mortality than the others. we report the isolation and identification of a genome type hadv- d (dg _ ) from a recent outbreak in southern china. genome sequencing, phylogenetic analysis, and restriction endonuclease analysis (rea) comparisons with past pathogens indicate hadv- d has re-emerged in southern china after an absence of twenty-one years. recombination analysis reveals this genome differs from the s-era prototype and vaccine strains by a lateral gene transfer, substituting the coding region for the l / kda dna packaging protein from hadv- . dg _ descends from both a strain circulating in southwestern china ( ) and a strain from shaanxi causing a fatality and outbreak (northwestern china; ). due to the higher morbidity and mortality rates associated with hadv- , the surveillance, identification, and characterization of these strains in population-dense china by rea and/or whole genome sequencing are strongly indicated. with these accurate identifications of specific hadv types and an epidemiological database of regional hadv pathogens, along with the hadv genome stability noted across time and space, the development, availability, and deployment of appropriate vaccines are needed. human adenoviruses (hadvs) are highly contagious pathogens causing acute respiratory disease (ard), among other illnesses. of the ard genotypes, hadv- presents with more severe morbidity and higher mortality than the others. we report the isolation and identification of a genome type hadv- d (dg _ ) from a recent outbreak in southern china. genome sequencing, phylogenetic analysis, and restriction endonuclease analysis (rea) comparisons with past pathogens indicate hadv- d has re-emerged in southern china after an absence of twenty-one years. recombination analysis reveals this genome differs from the s-era prototype and vaccine strains by a lateral gene transfer, substituting the coding region for the l / kda dna packaging protein from hadv- . dg _ descends from both a strain circulating in southwestern china ( ) and a strain from shaanxi causing a fatality and outbreak (northwestern china; ). due to the higher morbidity and mortality rates associated with hadv- , the surveillance, identification, and characterization of these strains in population-dense china by rea and/or whole genome sequencing are strongly indicated. with these accurate identifications of specific hadv types and an epidemiological database of regional hadv pathogens, along with the hadv genome stability noted across time and space, the development, availability, and deployment of appropriate vaccines are needed. h uman adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory, ocular, gastrointestinal, and genitourinary systems , , and a metabolic disorder (obesity) . of the several respiratory disease-associated hadv pathogens, hadv- and hadv- are among the most commonly reported and associated with febrile respiratory disease, in particular, acute respiratory disease (ard) , . these two genotypes , particularly hadv- , circulate globally and frequently in civilian populations , [ ] [ ] [ ] . they are of such public health concerns that specific vaccines have been developed and deployed against them at two different time periods by the u.s. military , . these successful vaccine periods provided an unintentional experiment reinforcing the effectiveness of vaccines in public health . in between the two periods of highly effective vaccine deployments, the inexplicable suspension of the vaccination program resulted in a resurgence of ard cases to prevaccine-era levels . this demonstrates the effectiveness and supports the development, availability, and deployment of vaccines against the hadvs that affect certain populations routinely and predictably, particularly those under stressed and high-density conditions. two important considerations for this public health program are required, along with vaccines: ) the ability to monitor, and to identify circulating hadv types and ) the presence of molecular data quantifying and char-acterizing genome changes that may occur during viral evolution, particularly at the antigenic epitopes, e.g., the hexon hypervariable l /l regions [ ] [ ] [ ] [ ] . hadv- is of particular concern as it is associated often with illnesses presenting with more severe and higher levels of morbidity than other respiratory hadv pathogens, and also may result in higher levels of fatalities , [ ] [ ] [ ] [ ] [ ] . as an example, a higher mortality rate was reported for children infected by hadv- in china during and also in korea (seoul) during - , with mortality rates of %, caused by hadv- genome types d and l, as compared with . % noted for hadv- infections . hadv strains compete against each other in host populations, with the presumably more robust ones replacing the previously dominant circulating strains. in a survey of hadv strains from the u.s. and eastern ontario/canada , two genomic variants, that were previously absent in the population, were identified: hadv- d comprised % of the hadvs reported and hadv- h comprised % . hadv- d was first identified in the u.s. in and subsequently spread further in the u.s. and into eastern ontario . it was concluded that both genomic variants represented ''recent introduction[s]'' from ''previously geographically restricted areas… herald[ing] a shift in predominant [genome type] circulating in the [u.s.]'' . the origins of hadv- d, presumably the parent of hadv- d , was noted as china, where it circulated from to , having ''replaced hadv- b as the predominant circulating virus'' , over a period of years ( - ) . hadv- d, in particular hadv- d , from china subsequently spread ''beyond [its] formerly geographically restricted regions'', e.g., to south korea ( ) ( ) ( ) and also japan, israel and the u.s. and canada , , . as another example of the severity of type hadv pathogens, the genomic variant hadv- h also produced more severe symptoms, including higher fatality levels, when it emerged and circulated in south america in the s . novel and re-emergent strains of highly contagious hadv pathogens identified within mainland china are of public health concerns to the global community, and vice versa. therefore, we call attention to and report the apparent re-emergence of hadv- d, after an approximately nineteen years absence in mainland china ( china ( to , and twenty-one years in southern china ( ), with the isolation, identification, and analysis of the genome sequence of an adenovirus from a child afflicted with ard during an outbreak in a primary school located in dongguan of the guangdong province in southern china. this genome is nearly identical to two other recently characterized genomes, one isolated from shaanxi province in northwest china ( ) and the other from chongqing in southwestern china ( ) (unpublished; jx ). given the caveat that the viruses may have been circulating earlier but had not been identified properly nor reported, the re-emergence of this genomic variant of hadv- has potentially serious consequences in china, and globally, if it follows a similar trajectory as the earlier had- d and hadv- d genome types that emerged in other countries and caused higher morbidity and mortality rates , , . adenovirus identification and genome annotation. all specimens collected were amplified ''pcr-positive'' for adenovirus and identified as hadv- by type-specific pcr analysis. of these, two, isolated from hospitalized and presumably more severe cases, produced visible cpe upon culturing. they were archived as dg _ and dg _ . sequence analysis revealed identical hexons, which were identified as hadv type by blastn analysis. the genome from dg _ was then sequenced, assembled, annotated, and analyzed. figure presents the genomic organization and transcription map of dg _ . this genome contains , bp with a gc content of . %. a total of coding sequences were identified. these genome data, noted formally as ''human adenovirus strain chn/dg / / [p h f ]'' and in this report as ''dg _ '', were deposited in genbank (accession number kc ). genome type determination of hadv- strains dg _ , hz/shx/ , and cq _ . the genome type of dg _ was determined by comparing its in silico rea profiles with other hadv- genome types reported in the literature , , , , . although seemingly antiquated in comparison to genome sequencing, rea profiles are still useful for comparisons with unsequenced but previously reported genome types and strains, and also as rapid and less-expensive alternatives for large-scale characterizations of viruses given a correct reference strain. using the genome type denomination of li, et al. , dg _ is identified as hadv- d, evidenced by the rea patterns and identical with the first reported hadv- d , as shown in figure . the rea patterns generated from dg _ , hz/shx/ , which caused acute bronchitis and pneumonia in an ard outbreak comprising cases amongst young children in the shaanxi province in , including one fatality , and cq _ , which was associated with an epidemic in chongqing, southwestern china (ni, k., et al., unpublished; jx ), are identical to each other and also identical with those of hadv- d reported earlier in israel ( ) and japan ( ) , . the rea patterns of cq _ in this study provide evidence to amend the less-descriptive designation of ''mutant hadv- d '' noted by ni, k., et al. in the genbank entry for cq _ (jx ). for reference, the in silico rea profile for the prototype gomen hadv- is provided; the rea patterns for these recent isolates differ clearly from the prototype, as shown in figure . hadv- prototype is the correct reference genome as three rea profiles, bcli, sali, and xhoi, showed identical patterns and complement the rea patterns that differ, along with sequence similarities across the genome. phylogenetic analysis of hexon genes and whole genomes confirms the genome types. phylogenetic analysis of archived hadv- hexon genes showed that dg _ has an origin common to strains hz/shx/ , cq _ , hebei_sjz_ , and tw_ . these hexons form a subclade that is on the same branch with another subclade containing several non-china isolates, including hadv- d from the u.s., as shown in figure a . the bootstrap value of indicates the hexons from the china genomes are highly similar to each, but are separate from the u.s. hadv- d subclade (bootstrap value ). furthermore, the phylogenetic analysis of available hadv- whole genomes revealed dg _ , hz/shx/ , and cq _ forming a subclade comprising hadv- d, and confirming the close relationships with each other, reaffirming a common lineage ( figure b ) that is distinct from the hadv- d strains of the u.s.a. (bootstrap value ). all of the genome types form subclades that are separate from the clade containing the prototype (gomen; ), with hadv- h forming a separate subclade in the genome phylogenetic analysis in contrast to the hexon gene phylogenetic analysis. comparative genomic analysis and single nucleotide differences of hadv- strains causing ard outbreaks in china. comparative genomics analysis showed dg _ has near genome identity with an earlier hadv- isolate, hz/shx/ ( . %) and also with cq _ ( . %). comparative genomics analysis documented seven single nucleotide substitution and one single base insertion differences between the dg _ and hz/shx/ genomes. of these, two single nucleotide substitutions were localized in the itrs and one non-synonymous substitution each was located in the dna polymerase, penton base, and kda protein coding sequences (table ) . one synonymous nucleotide substitution each was present in the -kda hexon assemblyassociated protein and virus-associated (va) rna ii. the single nucleotide insertion was in a non-coding region of dg _ . there were three single nucleotide substitutions in coding sequences and seven base deletion differences in the itrs between cq _ and hz/shx/ genomes. one synonymous substitution (c to t) was located in hexon assembly-associated protein (a a) and the other two non-synonymous substitutions g to c and g to t were located in dna polymerase (s c) and kda protein (p q), respectively. the nucleotide deletions in itrs of cq _ may be sequencing errors given that the left itr was not identical with the right itr, or may represent recent mutations. if exclusive of itr differences, there were only three single nucleotide substitutions between the cq _ and hz/shx/ genomes ( . %). for strain dg _ , it had a higher genome identity with cq _ ( . %) than hz/shx/ ( . %) if exclusive of itr difference. there were only four single nucleotide substitutions and one single nucleotide insertion in non-coding region between both genomes, which led to three non-synonymous substitutions in dna polymerase (d e, s c) and penton base gene (v a), respectively. nucleotide substitution rates and selection pressures for hadv- d strain dg _ major capsid protein genes. the selective pressures at the protein level for the three hadv- capsid protein genes, hexon, penton base and fiber, were examined by comparing synonymous and non-synonymous mutations. all three genes have ka/ks ratios of less than ( table ). this is in accordance with the hypothesis that organismal evolution is dominated by negative selection, i.e., ones removing mutations harmful to fitness . specifically, both hexon and penton base genes have less non- synonymous substitutions per site, which leads to the low ratios of ka/ks. although the non-synonymous substitutions and ka/ks ratio of the fiber gene is also low, it is relatively higher than for the hexon and penton base genes. this may indicate that the fiber gene has less negative selection pressures, likely due to tissue tropism being determined and constrained by the fiber gene. overall, the majority of mutations are synonymous and do not affect the integrity of the hexon, penton base, and fiber proteins. genome recombination analysis of hadv- d. genome recombination analysis using simplot software reveals a lateral transfer of a small portion of the genome upstream of the penton base gene. this recombination contains the entire l / kda gene from hadv- into hadv- d, as shown in figure a . its importance remains to be revealed. the gene transfer is also found in the genomes from the earlier strains cq _ (southwestern china; ; unpublished) and hz/shx/ (northwestern china; ) , respectively, shown in figure b , but not found in the prototype gomen hadv- genome, as displayed in figure c . among the two hadv species b respiratory pathogens most frequently associated with ard outbreaks globally, hadv- is reported to cause a higher mortality rate than hadv- in one long-term survey , as well as in a recent shorter term survey of adenoviral pneumonia cases in beijing ( - ) . genome type hadv- d apparently originated and circulated in china from - , becoming the predominant strain during the period of - . it was also the prevalent genome type found in korea during two outbreaks in - and - , accounting for - % all of the type hadv strains assayed . interestingly, despite reports of global circulation, hadv- , and in particular hadv- d, epidemics had not been reported in mainland china from to . in , hadv- was identified as the respiratory pathogen in an outbreak that included a fatality in shaanxi and also in a outbreak in chongqing (unpublished), signaling a reemergence. thorough characterization of these pathogens is evidenced by the availability of two genome sequences (jf and jx ), both of which are further identified as the hadv- d genome type in this report, and shown to be nearly identical to this report of an isolate from a ard outbreak in guangdong province (strain dg _ ) by comparative genomics and, in particular, in silico rea pattern analysis, as presented in figure . although not ideal and largely replaced by whole genome sequencing, rea patterns can still provide rapid and relatively inexpensive characterizations of the genomes of large number of pathogens in an outbreak [ ] [ ] [ ] [ ] [ ] [ ] . for hadv comparisons, the caveat is to use the correct reference genome; for example, hadv- contains a partial hexon gene from hadv- , comprising approximately only . % of the length of the genome, in a chassis of hadv- , comprising approximately . % of the length of the genome . using the genome of hadv- as a reference yields meaningless patterns that are subject to researcher-biased interpretations and leads to erroneous conclusions that hadv- is a genome type of hadv- . using the hadv- genome as a reference provides a closer approximation of the genome identities , . however, the recombination event revealed by whole genome sequencing, with the conflicting ''trojan horse'' renal pathogen epitope observed with ard symptoms, indicates this was a novel and emergent pathogen , , [ ] [ ] [ ] [ ] . in contrast, for hadv- d, the prototype hadv- genome provides the correct reference: three rea patterns are identical (bcli, sali, and xhoi); four are obviously different (bamhi, bgli, hpai, and smai); and four are highly similar with a few differences in the band patterns (bglii, bsteii, hindiii, ecori, and xbai), shown in figure . the major advantages of rea comparisons are the value and abundance of earlier molecular epidemiology studies, prior to the genome sequencing era, presenting rea data, and, in many cases, relating particular genome types to clinical, epidemiological, and pathogenicity observations. all of these historical strains are physically lost and no longer available for further genomic or laboratory characterization. in essence, however, the value and knowledge of the outbreaks, pathogens, and researchers of the past are not entirely lost if genomes of current pathogenic strains of interest may be compared with published rea patterns of past pathogens, as demonstrated in the genome type identities presented in this report. whole genome characterization of hadv provides a higher-resolution perspective of understanding this pathogen, which may or may not lead to better public health strategies and measures to prevent outbreaks. as noted for two species b ard pathogens, hadv- and hadv- , ''restricted use'' but effective vaccines can be and are deployed currently in the u.s. military to prevent ard outbreaks [ ] [ ] [ ] . however, even if there were no viable strategy to manage hadv outbreaks, knowing the genome type, either by rea or by whole genome sequencing, allows an understanding of the epidemiology, including potential morbidity and mortality profiles, of the circulating pathogens. as discussed earlier, genome types may have different pathogenicity, infectivity, and virulence profiles; for example, a higher mortality rate was reported for children infected by genome types d and l in korea, with mortality rates of %, compared to . % for hadv- infections . another genome type, hadv- h, also resulted in more severe symptoms, including fatalities in south america . for their molecular epidemiological studies of hadv- , wadell and colleagues presented numerous rea patterns generated with restriction endonucleases (bamhi, bcli, bgll, bglii, bsteii, hindiii, hpai, smai, ecori, sali, xbai, and xhoi), parsing hadv- isolates from various regions and across many years to divide them into more than genome types , , [ ] [ ] [ ] . adenoviruses contain relatively stable double-stranded dna genomes , , . there are seven single base substitutions and a one-base insertion between strains dg _ and hz/shx/ , which led to three non-synonymous substitutions in the dna polymerase, penton base, and kda protein coding sequences. interesting, there are only three single base substitutions between strains cq _ and hz/shx/ , exclusive of the nucleotide deletions in itrs of cq _ which may be due to possible sequencing errors. the high genome percent identity between strains cq _ and hz/shx/ and the adjacent locations of chongqing and shaanxi ( kilometers apart) where strains cq _ and hz/shx/ were isolated indicate strain hz/shx/ may be the origin of strain cq _ . strain dg _ has a higher genome identity with cq _ than hz/shx/ , which also supports the hypothesis that strain cq _ is be the ancestor of strain dg _ . although hadv genomes appear stable in terms of single base changes, as expected for double stranded dna viruses and as observed in pairs of hadv genomes examined to date, e.g., the prototype versus circulating strains of hadv- and - , separated by approximately fifty years , , less common but biologically and clinically significant larger genome changes are observed either as a single, small recombination event, such as the lateral transfer of the renal pathogen epsilon epitope (hadv- ) providing a ''trojan horse'' effect to the recombinant hadv- , an emergent acute respiratory disease (ard) pathogen in a putatively immune naive host population , or as multiple and larger recombination events, such as the lateral transfer of the non-pathogen epsilon epitope (hadv-d ) along with multiple other sequences to an emergent recombinant resulting in the highly contagious ocular pathogen causing epidemic keratoconjunctivitis (ekc), hadv-d . additionally, the presence of the epsilon epitope of a nonpathogenic type, hadv- , found in several recently reported emergent recombinant ekc pathogens, hadv- , support the hypothesis that recombina-tion amongst hadvs is an important mechanism driving the molecular evolution and genesis of hadv pathogens . in both of these latter examples, newly emergent hadv pathogens have the ''serotype'' of nonpathogens but are potent, significant, and highly contagious human pathogens. recombination appears to play another novel and major role in the molecular evolution of hadvs and genesis of human pathogens. recent reports of hadv genomes containing genome segments, including near-entire genomes, derived from simian adenoviruses (sadvs) indicate zoonosis is an avenue of lateral gene transfer. thus, nonhuman primates may be a wellspring of emergent human pathogens , , and vice versa . a novel third type of lateral gene transfer is revealed in this newly reported genome of hadv- d strain dg _ , that of a ''moderate-sized'' single whole gene recombination. this serendipitous insight into the molecular evolution of these respiratory pathogens from hadv species b demonstrates the genomes of individual hadv types, such as type , contain changes revealed only by high-resolution genome sequences and may be important in the context of hadv molecular evolution, viral fitness, origins and bases of clinical and pathogenicity differences, and account for emergent and re-emergent pathogens. blast analysis reveals the recombinant region to encode the entire l / kda gene of hadv-b with flanking non-coding sequences. the blast scores indicate the first highly similar sequence, aside from several type sequences, is that from the hadv-b prototype (max. score , total score , query cover %, e-value . , ident. %) and a hadv-b recombinant ( , , %, . . %), with additional homologous and highly similar sequences found in hadv-b ( , , %, . , %) and hadv-b strains ( , , %, . , %). this encodes a dna-binding protein that is expressed in both the early and late stages of infection, suggesting it could play multiple roles in the adenoviral life cycle. the l / kda protein interacts with the iva protein and is an essential protein that is absolutely required for dna packaging as well , . effects of this particular moderatesized recombination from hadv-b into the hadv-b genome chassis and the resultant emergent pathogen are unknown pending wet-bench investigations and additional clinical reports. the hadv- prototype strain (ay ) analyzed is also known as the gomen strain, which was isolated as a clinical specimen from a throat washing of a u.s. military recruit with pharyngitis . this strain is nearly contemporaneous with the greider strain (ay ) , aka hadv- a, which was used to develop the vaccine strain , . although there are minor genome differences, e.g., point mutations, between the prototype and the vaccine strains, pairwise genome dot blot analysis (pipmaker) indicated no recombination events . these observations strongly support and validate the recent paradigm change of using the genome data along with biological and clinical profile changes to recognize, characterize, type, and name novel hadvs rather than relying solely on the epsilon and/or gamma epitopes , , determined either by serology or imputed by limited dna sequencing, in the past. with the exception of sporadic hadv- infections reported in children in guangzhou ( ) and the three recent outbreaks, the apparent absence of type ard pathogen circulating in the population of southern china before leads to a concern that the dense city populations in china are now immunogenically naïve with respect to hadv- . in northern china, recently, isolates were typed as hadv- by pcr and sequencing of hexon genes from hadv-positive specimens during - ; hav- was associated with most of the severe lower respiratory hadv infections . coupled with increased opportunities for travel, a ''perfect storm'' for present and near-future outbreaks of the apparently more severe disease-causing hadv- d strains is foreboding. . in the former outbreak, a total of patients were sampled, with all of the patients being males, with ages between - years . in the latter, patients aged between - years were reported as afflicted with ard . in taiwan, there was a large community outbreak of hadv- in . in this instance, an abrupt increase in percentage of hadv- infections occurred, from . % in - to % in . the hexon nucleotide sequences of five hadv- isolates collected in taiwan were identical to the sequence of hadv- strain hz/shx/ , which was also identical to dg _ . in the context of the data in this report, these ''taiwan'' hexon genes formed the same subclade with strains hz/shx/ , cq _ and dg _ (fig. a) . given that only the hexon genes were sequenced, the exact genome types of these strains in the two outbreaks remain unknown. however, the possibility of a hadv- d genome type circulating is foreboding. further data, including complete genome sequencing and in silico rea, are important to confirm this possibility. in the interest of global public health, with these recent outbreaks and the identification of nearly identical contemporary hadv- d genome types, we strongly urge molecular surveillance and genotyping of newly isolated hadv strains in china by whole genome sequencing and/or in silico rea. additionally, the newlyredeveloped vaccines, which are now only accessible to the u.s. military , should be made available to the civilian ''at-risk'' public to prevent ''preventable'' highly contagious outbreaks involving hadvs associated with high morbidity rates and fatalities , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , , , , . in particular, the vaccine against hadv- is urgently needed in china, due to the apparent decades-absence of circulating hadv- , which presumably resulted in a corresponding lower level of herd immunity in today's population. given the higher severity of diseases and fatality rates caused by hadv- , especially hadv- d, extensive surveillance and corresponding molecular investigation, including genotyping, genome typing, and genome sequencing, should be carried out when confronting outbreaks of hadv pathogens in the high-density populations of china to protect the public and the global community. specimen collection and handling. during february to march of , twentythree primary school children under the age of (dongguan; guangdong province) presented with flu-like symptoms, including fever, pharyngalgia, and coughing as well as other indications of ard. two were hospitalized with severe symptoms. eleven throat swab specimens were collected into -ml viral transport media; transported at uc- uc; and preserved at uc for virus isolation and nucleic acids extraction. this study protocol was approved by the institutional ethics committee of the center for disease control and prevention of guangdong province (guangdong cdc) and was carried out in accordance with the approved guidelines. the guardians of all under-aged participants gave signed informed consent for participation in the study. data records of the samples and sample collection are de-identified and completely anonymous. detection of respiratory pathogens. total nucleic acids were extracted from the specimens using the qiaamp minelute virus spin kit (qiagen; hombrechtikon, germany). human adenovirus, respiratory syncytial virus, influenza virus a and b, parainfluenza virus types - , human rhinovirus, human metapneumovirus, and human coronavirus oc and e were detected by real-time pcr as described earlier . for hadv identification, type-specific primers were used to characterize the type by pcr, as described in an earlier report . adenovirus isolation and genomic dna extraction. adenovirus-positive throat swab specimens, identified by pcr analysis, were inoculated into a cell cultures, and grown in dulbecco's minimum essential medium supplemented with iu penicillin ml , mg streptomycin ml , and % (v/v) fetal calf serum, at an atmosphere of % (v/v) carbon dioxide. cytopathic effect (cpe) was monitored for at least ten days. viral genomic dna was extracted from infected cells for genomic analysis, as described by le, et al. . genome sequencing and annotation. the genome of hadv strain dg _ was sequenced using a sanger chemistry-based, primer-walking method by pcramplification, with overlapping regions sequenced , . both -and -ends (including both inverted terminal repeats) were sequenced directly by primers ad -ltrs a ( -gcctcttgacggaactcg- ) and ad -ltrs ( -ggtccctctaaatacacataca- ), respectively, using genomic dna as template; this ensured the accurate determination of the end sequences , . the sequence data, collected with an abi genetic analyzer, provided an average genome coverage of -to -fold, with both strands represented. gaps and ambiguous sequences were pcr-amplified using different primers and resequenced. these sequencing ladders were assembled with the seqman pro software . . (dnastar, inc.; madison, wi. usa). nucleotide and amino acid sequences were aligned with clustal and blast software. the genome sequence was annotated based on the previous annotation of hadv- prototype strain (gomen) and deposited into genbank with the accession number kc . in silico restriction endonuclease analysis (rea). the specific adenovirus genome type was determined using in silico rea analysis of the whole-genome sequences in accordance with the in vitro protocol described by li, et al . this was performed using the software vector nti advance . (invitrogen corp.; san diego, ca. usa). twelve restriction enzymes were used for this analysis, as performed by li, et al. : bamhi, bcli, bgll, bglii, bsteii, hindiii, hpai, smai, ecori, sali, xbai, and xhoi. phylogenetic analyses of hadv- hexon genes and the whole genome sequences. the molecular evolutionary genetics analysis (mega) version . . software was used for phylogenetic analyses of the hadv- hexon genes and the whole genomes, with additional sequences retrieved from genbank database, as described previously , . neighbor-joining phylogenetic trees with , boot-strap replicates were constructed using a maximum-composite-likelihood method with default parameters. bootstrap numbers shown at the nodes indicate the percentages of , replications producing the clade, with a value of noted as robust and significant. archived hadv- genome sequences from genbank were used for phylogenetic analysis. these are as follows (for reference, the names include the corresponding genbank accession number, country of isolation, strain name, year of isolation (if available), and genome type (if available)): ay _gomen_ _ p, jx _chn_cq _ _ d, jf _chn_ hz/shx_ _ d, jx _usa_ak _ _ b, jx _usa_arg/ak _ _ h, jx _usa_ak _ _ d , jx _usa_ak _ _ d , jn _usa_fs _ _ d , jn _jpn_takeuchi_ _ , jn _ar_ - _ _ h, gq _chn_gz _ , hq _chn_gz _ , ay _usa_vaccine_ , ay _chn_vaccine, ay _usa_nhrc_ _ , and kc _chn_dg _ _ d. the hadv- hexon complete sequences used for these analyses are as follows: ab _gomen_ _ p, jn _jpn_takeuchi_ _ , af _usa_ _vaccine_ _ a, ay _usa_vaccine_ , af _chn_beijing, ay _chn_vaccine, jn _ar_ - _ _ h, af _jpn_ _ _ d, af _jpn_bal_ _ d , ay _kr_ - _ _ d, jx _usa_ak _ _ d , jx _usa_ak _ _ b, ay _usa_nhrc_ _ , af _jpn_s- _ _ a, ay _kr_ - _ _ l, ab _jpn_ _ dx, ab _jpn_osaka_ _ dx, jx _usa_arg/ak _ _ h, jx _usa_ak _ _ d , hq _chn_gz _ , gq _chn_gz _ , gu _chn_ hz/shx_ _ d, jn _usa_fs _ _ d , jx _chn_cq _ _ d, jq _chn_hebei_ /sjz_ , jq _chn_hebei_ /sjz_ , jq _chn_hebei_ /sjz_ , jx _tw_tw _ , jx _tw_tw _ , jx _tw_tw _ , jx _tw_tw _ , jx _tw_tw _ , and kc _chn_dg _ _ d. , along with the prototype gomen genome were analyzed for sequence recombination events using the software tool simplot (http://sray.med.som.jhmi.edu/scroftware/simplot/) . for the recombination analysis, mafft software was used first to align the hadv-b species sequences using default parameters (http://mafft.cbrc.jp/alignment/server/). default parameter settings for the simplot software were used for analyzing the whole genomes, along with the following input: window size ( nucleotides [nt]), step size ( nt), replicates used (n ), gap stripping (on), distance model (kimura), and tree model (neighbor-joining). the following genomic sequences of hadv-b members were used: hadv-b p (ay ), hadv-b (ay ), hadv-b (ay ), hadv-b (ay ), hadv-b (ay ), hadv-b (ay ), hadv-b (ay ), hadv-b (ay ), hadv-b (ay ), and hadv-b (fj ). substitution rate analysis of the hexon, penton base and fiber genes in hadv- . the numbers of non-synonymous (ka) and synonymous (ks) substitutions per site from between sequences were noted and the ka/ks ratios were calculated. this www.nature.com/scientificreports scientific reports | : | doi: . /srep hadv- analysis was conducted using the nei-gojobori model , and included nucleotide sequences from hexon genes, fiber genes, and penton base genes available from genbank. all positions containing gaps and missing data were eliminated automatically. evolutionary analyses were performed with mega . . . the hadv- complete hexon, penton base and fiber gene sequences available in genbank were achieved for analysis. the hadv- complete hexon gene sequences used for this analysis are same with previous those in phylogenetic analysis. the following hadv- complete fiber gene sequences were used: ay , ay , the hadv- complete penton base gene sequences diagnostic procedures for viral, rickettsial, and chlamydial infections adenovirus infections in immunocompetent and 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replication-competent infectious molecular clone of hiv- crf _bc simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions q.z. and d.s. conceived and designed experiments. s.z., c.w., c.k., j.s., s.d., l.zou, j.z., z.c., s.j., z.z., j.z., x.wan, x.wu, w.z., l.zhu, d.s. and q.z. performed the experiments and analyzed the data. s.z., c.w., d.s. and q.z. wrote the manuscript. all authors reviewed the manuscript. competing financial interests: the authors declare no competing financial interests. key: cord- -ckdx j authors: zheng, shou-yan; xiao, qiu-yan; xie, xiao-hong; deng, yu; ren, luo; tian, dai-yin; luo, zheng-xiu; luo, jian; fu, zhou; huang, ai-long; liu, en-mei title: association between secondary thrombocytosis and viral respiratory tract infections in children date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ckdx j secondary thrombocytosis (st) is frequently observed in children with a variety of clinical conditions. the leading cause of st is respiratory tract infection (rti) in children. nasopharyngeal aspirate samples were collected and assessed for common respiratory viruses. the relationships between virus infections and secondary thrombocytosis were analyzed retrospectively. the blood platelet count and the presence of respiratory viruses were determined for rti patients, and ( . %) cases with platelet ≥ × ( )/l were considered as the thrombocytosis group. compared with the normal group, the detection rates of respiratory syncytial virus (rsv) and human rhinovirus (hrv) were significantly higher in the thrombocytosis group (p = . and . , respectively). hrv single infection was a risk factor associated with thrombocytosis [odds ratio (or) = . , % confidence interval (ci) = . – . ]. furthermore, st was more likely to occur in younger patients who had clinical manifestations of wheezing and dyspnea and who had been diagnosed with bronchiolitis. furthermore, the course of disease lasted longer in these patients. st is associated with viral respiratory tract infections, especially rsv and hrv infections. hrv single infection is a risk factor associated with thrombocytosis. scientific reports | : | doi: . /srep ethics statement. all experiments were approved by the ethics committee of the children's hospital of chongqing medical university. the guardians of the patients signed informed consent for participation in this study and for the publication of the individual clinical details. the methods were carried out in accordance with the approved guidelines. the study was conducted in compliance with the principles of the declaration of helsinki. study subjects and samples collection. children with respiratory tract infections (rtis) that were treated at the department of respiratory medicine at the children's hospital of chongqing medical university in china between june and may were enrolled in the study. in all patients, the diagnosis of rti was based on clinical, laboratorial and radiological evidence. nasopharyngeal aspirate (npa) samples were collected when the patients were admitted to our department. the specimens were kept at °c for a maximum of h and stored at − °c until further processing. the viral dna and rna were extracted from -μl aliquots of the npa samples using a qiaampminelute virus spin kit (qiagen, hilden, germany). the rna was applied as the template for cdna synthesis using the superscript iii first-strand synthesis system (invitrogen, california, usa). the dna and rna extractions and cdna products were used for subsequent testing for respiratory viruses. all of the samples were analyzed using a commercial detection kit (takara biotechnology, dalian, china and applied biosystems, california, usa), according to the manufacturer's instructions. multiplex nested polymerase chain reaction (pcr) was used to detect the following common respiratory viruses, as described previously [ ] [ ] [ ] [ ] [ ] : rsv subtypes a and b (rsva, rsvb); influenza virus (ifv) subtypes a, b and c (ifva, ifvb, ifvc); human coronaviruses (hcov); metapneumovirus (mpv); parainfluenza virus type to (piv - ); adenovirus (adv); human bocavirus type (hbov ) and human rhinovirus (hrv) subtypes a and c (hrva, hrvc). real-time pcr (rt-pcr) was also used to detect hrv and hbov . hrv-positive samples were further amplified and sequenced to identify the hrv subtype. platelet counting and thrombocytosis definition. complete blood counts were performed in the clinical laboratory using an xe- blood autoanalyzer (sysmex, japan). we used clinical features and published reference standards to define normal platelet counts as being between × /l and × /l for children. platelet counts of ≥ × /l were defined as thrombocytosis. moreover, to better consider the clinical characteristics and implications of thrombocytosis, patients with platelet counts between - × /l were diagnosed with mild thrombocytosis and those with counts of ≥ × /l were considered as moderate to severe thrombocytosis; platelet counts above × /l were considered indicative of extreme thrombocytosis . a retrospective review of the medical records was performed on all patients, and those who had at least one platelet count ≥ × /l during hospitalization were recruited into the thrombocytosis group. statistical analyses. statistical analyses were performed using the statistical package for the social sciences version . (spss . ). the categorical variables were compared using the chi-square test or fisher exact test, and the continuous variables were compared using student's t-test or the nonparametric mann-whitney u-test. correlation was analyzed using logistic regression analysis. p-values < . were considered to be significant. demographic data and platelet count of total patients. during the five-year study period, the blood platelet count and presence of respiratory viruses were randomly assessed in children ranging in age from month to years (median = months). approximately . % ( ) of these children were male. based on the clinical data, ( . %) cases with platelet counts ≥ × /l (median × /l) were considered as the thrombocytosis group, and ( . %) cases with platelet counts ranging between and × /l (median × /l) were considered as the normal group. among thrombocytotic patients, ( . %) with counts between and × /l were considered as having mild thrombocytosis, and ( . %) patients with counts of ≥ × /l were classified as having moderate to severe thrombocytosis; this last group included patients with platelet counts > × /l. thrombocytosis was presented in . % ( / ) of patients and most frequently ranged from to × /l. we observed that the platelet count began to elevate on approximately the th day of illness. the age distribution of patients with st was month to years (median months); most of these patients were younger than two years of age, ( . %), and were markedly younger than patients without st (median months) (p < . ). moreover, ( . %) of the thrombocytotic patients were male, but the gender distributions were not significantly different from the normal platelet patients. virus detection of the normal and thrombocytosis groups. the virus detection rates of the normal and thrombocytosis groups were . % ( / ) and . % ( / ), respectively. the single viral infection rates were . % ( / ) and . % ( / ), respectively, and the co-infection rates were . % ( / ) and . % ( / ), respectively. there was no significant difference in the total virus detection rate between the two groups. among the thrombocytosis group, rsv ( . %) was the most common virus detected, followed by hrv ( . %) and piv ( . %). the detection results for each virus and their different subtypes for both groups are summarized in table . compared with the normal group, the total detection rates of rsv and hrv were both significantly higher (p = . and . , respectively) in the thrombocytosis group, and adv and ifv were lower (p = . and . , respectively). the single viral infection rates of rsv and hrv in the thrombocytosis group were both significantly higher (p = . and . , respectively), and the single viral infection rates of adv and ifv in thrombocytosis group were significantly lower compared to the normal platelet group (p < . and p = . , respectively). however, the co-infection rate of viruses between two groups was not remarkably different except for rsv. the specific virus detection results are summarized in table . we further divided the thrombocytosis group into mild and moderate to severe groups and analyzed the viral infections of these subgroups. there was no marked difference of the virus detection rate between the two groups, with the exception that hbov co-infections were more common in the mild thrombocytosis group (tables and ). we also divided the normal platelet counts group into two subgroups: ( - ) × /l and ( - ) × /l, then analyzed the viral infections of the subgroups. the total detection rates of rsv and piv were both significantly higher (p = . and . , respectively) in the latter group, and adv and ifv were lower (p < . and . , respectively) ( table ) . clinical data in the normal and thrombocytosis groups. we compared the clinical conditions of the two groups, and the clinical manifestations of wheezing and dyspnea were significantly overrepresented in the thrombocytotic group patients compared to the normal group patients (p < . and p = . , respectively). however, the occurrence of fever in the normal group was remarkable higher than the thrombocytosis group. the most common clinical diagnoses of the two groups were lower respiratory tract infections, such as pneumonia, bronchiolitis, severe pneumonia, severe bronchiolitis and bronchitis. other conditions included upper respiratory tract infection, laryngitis, and amygdalitis. the incidence of bronchiolitis in the thrombocytosis group was significantly higher than in the normal group (p < . ). the days of illness before admission, length of hospitalization and course of disease in the thrombocytosis group were all longer compared with the normal group (p < . ). the c-reactive protein (crp) levels of ( . %) thrombocytotic patients were above mg/l. the percentage of patients with elevated crp levels was not significantly different between two groups, but the rate of anemia in the thrombocytosis group was higher (p = . ). in the thrombocytosis group, sputum culture-positive results were obtained in ( . %) cases, and virus detection and sputum cultures were both positive in ( . %) cases. the typical clinical findings are summarized in table . table . comparison of specific virus detection rate between the mild and moderate to severe thrombocytosis groups. moreover, we also investigated patients with very high platelet counts. there were patients with extreme thrombocytosis, defined as platelet counts of > × /l; the maximum count was × /l. of these patients, were less than one year old, were diagnosed with bronchiolitis, and were diagnosed with pneumonia. in of these cases, at least one viral infection was detected, and the primary viruses were rsva, piv , and hrva. the age distribution, occurrence of wheezing, prevalence of bronchiolitis, course of disease and viral infection were further analyzed using logistic regression models. in general, hrv single infection was a risk factor associated with thrombocytosis (or = . , % ci = . - . ) after considering the effects of age, course of the disease, occurrence of wheezing and diagnosis with bronchiolitis. hrva was especially strongly associated with thrombocytosis (or = . , % ci = . - . ) ( table ) . in the thrombocytosis group, no one developed thromboembolic or hemorrhagic complications. in total, ( . %) patients reached clinical recovery or improvement, ( . %) patients did not improve and ( . %) patients died of respiratory failure and septicemia. table . comparison of clinical data between the normal and thrombocytosis groups. *crp: c-reactive protein; crp level above mg/l were defined as increased. st is primarily found in pediatric patients with respiratory tract infections. in our study, st was identified in approximately . % ( ) of the inpatients with rti. st was especially common in patients with lower respiratory tract infections and occurred most frequently in the mild thrombocytosis group ( - × /l). previous studies showed that st occurs in - % of patients with respiratory tract infections [ ] [ ] [ ] . the different incidence rates may be explained by the cutoff platelet count, study population (inpatients, outpatients, or both), and median age of the enrolled subjects. additionally, the platelet count peaked on the th day after the onset of illness. this also supports the findings by other investigators who reported that platelet count in st peaked during the second and third week of the illness , . in our cases, the highest incidence of st occurred in patients who were under two years of age ( . %), which is consistent with the mastubara et al. study of japanese children with thrombocytosis, more than % of whom were aged less than two years . because bone marrow precursor cells of younger infants are more sensitive and rapid to external stimuli such as infections. our findings showed that childhood st is related to respiratory viral infections, but the degree of platelet elevation is unrelated to the species of virus. the total virus detection rate was . % ( / ) in patients with st. rsv ( . %) was the most commonly detected virus, followed by hrv ( . %) and piv ( . %). from the present data, we found that rsv single infection (especially subtype b) and hrv infection (especially subtype a) were prominent causative agents of thrombocytosis. to our knowledge, our study is the first to find that st occurrence is associated with hrv infection. in addition, hbov co-infection with other viruses in the mild thrombocytosis group is more common than in the moderate to severe thrombocytosis groups. this may be because this group of patients is frequently co-infected with rsv and hrv. according to the above results, we speculate that increased platelet counts may be indicative of a respiratory tract inflammatory reaction after rsv or hrv infection. the mechanism by which infection can promote thrombocytosis has not yet been fully elucidated. reports in the literature show that st is related to increased endogenous levels of several cytokines, such as thrombopoietin (tpo), interleukin- (il- ), interleukin- (il- ), interleukin- alpha (il- a) and tumor necrosis factor alpha (tnf-a) [ ] [ ] [ ] . it has been found that during rsv infection, many infants demonstrated rsv-specific cytokine responses, such as increased levels of il- and il- . however, st caused by hrv infection has not been described. bronchial epithelial cells secrete a wide variety of inflammatory cytokines, including il- , il- , il- , and gm-csf, and these cytokines are responsible for the development of thrombocytosis following rsv and hrv infections , . furthermore, tpo concentrations usually correlate with crp levels , . among our thrombocytotic patients, . % ( ) had elevated crp levels; this could indicate that crp participates in thrombocytosis. some authors have suggested that anemia may also play a role, and we observed a higher rate of anemia in the thrombocytosis group , . another theory confirmed that the number of megakaryocytes increased and that the pulmonary capillary bed released more platelets due to stimulation by tpo after infection. we believe the specific viral agents that cause rti induce inflammatory response and circulating cytokines that can then lead to st and perhaps other hematological disorders. patients that develop st have been more likely to also experience wheezing, dyspnea and a longer disease course. therefore, increased platelet counts may be a clinical marker associated with the severity of rti. our logistic regression analysis showed that patients who had clinical manifestations of wheezing, bronchiolitis and a longer course of disease were more inclined to develop thrombocytosis. additionally, hrv single infection was a risk factor associated with thrombocytosis. therefore, physicians should carefully monitor these patients. however, the mechanism through which this group of patients developed thrombocytosis has not been fully determined. the percentage of patients with fever in the normal group was higher than in the thrombocytosis group; this could possibly due to the presence of bacterial infections in patients with normal platelet counts. the great majority of our patients underwent clinical recovery or improvement. in childhood, st rarely results in thromboembolic or hemorrhagic complications, so treatment with platelet aggregation inhibitors is not required even when the platelet count is more than × /l. treatment should be targeted at the primary disease rather than the platelet count . we observed that the detection rate of adv and ifv was lower in children with st. generally, cytomegalovirus (cmv), epstein-barr virus (ebv) and parvoviridae often lead to thrombocytopenia , . one explanation for this observation could be that adv infection is more common in older children that do not frequently develop st. further work is needed to determine why adv and ifv seldom cause thrombocytosis. the limitations of our study include that the fact that this is a retrospective study rather than a cohort study, our study was performed in a single medical center, the subjects were limited to a young age group, and we had no follow-up study of platelet change. a large-scale cohort study is needed to investigate the causal relationship between viral infections and st in children. more specific studies are needed to further elucidate the inflammatory process involved in viral infection and the specific pathophysiology of thrombocytosis. in summary, research regarding viral respiratory tract infections and thrombocytosis is lacking, and we performed a large sample analysis that linked a specific viral agent to thrombocytosis in this clinical retrospective study. childhood st is related to respiratory tract infections, the occurrence rate is approximately . %, and the most common viruses are rsv and hrv, but infections with adv or ifv seldom cause thrombocytosis. hrv single infection is a risk factor associated with thrombocytosis. younger infants that are diagnosed with bronchiolitis and have a clinical manifestation of wheezing are more likely to develop st and undergo a longer course of disease. these observations suggest that increased platelet counts may be a retrospective marker of respiratory tract inflammatory reactions after viral infections and can indicate the severity of lower respiratory tract infections. aetiology and clinical significance of thrombocytosis: analysis of patients with an elevated platelet count guideline for investigation and management of adults and children presenting with a thrombocytosis reactive thrombocytosis in children with upper urinary tract infections primary and secondary thrombocytosis in childhood clinicohematological study of thrombocytosis in children thrombocytosis at an early stage of respiratory tract viral infection respiratory syncytial virus-positive bronchiolitis in hospitalized infants is associated with thrombocytosis thrombocytosis in pediatric patients is associated with severe lower respiratory tract inflammation incidence and clinical significance of reactive thrombocytosis in children aged to months, hospitalized for community-acquired infections simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay human bocavirus and acute wheezing in children use of an improved quantitative polymerase chain reaction assay to determine differences in human rhinovirus viral loads in different populations proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types thrombocytosis and thrombocythemia thrombocytosis after pneumonia and empyema and other bacterial infections in children age-dependent changes in the incidence and etiology of childhood thrombocytosis circulating levels of thrombopoietic and inflammatory cytokines in patients with clonal and reactive thrombocytosis stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand interleukin- stimulates thrombopoiesis through thrombopoietin: role in inflammatory thrombocytosis human infant respiratory syncytial virus (rsv)-specific type and cytokine responses ex vivo during primary rsv infection host immune responses to rhinovirus: mechanisms in asthma rhinovirus induces airway epithelial gene expression through double-stranded rna and ifn-dependent pathways elevation of serum thrombopoietin precedes thrombocytosis in acute infections clinicohematological study of thrombocytosis screening children with thrombosis for thrombophilic proteins. cui bono? virus-associated immune thrombocytopenic purpura in childhood virus-associated idiopathic thrombocytopenic purpura we acknowledge the assistance of the patients and their caregivers involved in the study, the staff of the e.m.l. and a.l.h. conceived and designed this study and revised the manuscripts. s.y.z., q.y.x., x.h.x., y.d., l.r., d.y.t., z.x.l., j.l. and z.f. collected the samples and performed the experiments. s.y.z. and q.y.x. analyzed the data and wrote the paper. all authors reviewed the manuscript. all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. key: cord- -x tt kx authors: kiyota, yasuhiro; muramatsu, hiroyasu; sato, yuiko; kobayashi, tami; miyamoto, kana; iwamoto, takuji; matsumoto, morio; nakamura, masaya; tateno, hiroki; sato, kazuki; miyamoto, takeshi title: smoking cessation increases levels of osteocalcin and uncarboxylated osteocalcin in human sera date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: x tt kx smoking is thought to be a risk factor for osteoporosis development; however, the consequences of stopping smoking for bone homeostasis remain unknown. here we conducted two separate human studies and show that bone mineral density was significantly lower in smokers than in non-smokers. the first was an observational study of pre- and post-menopausal healthy female smokers and non-smokers; the second included current smokers determined to stop smoking. in the second study, levels of bone formation markers such as osteocalcin and uncarboxylated osteocalcin significantly increased after successful smoking cessation, as verified by significantly reduced levels of serum cotinine, a nicotine metabolite. moreover, nicotine administration to mice reduced bone mineral density and significantly increased the number of osteoclasts in bone. reduced bone mass phenotypes seen in nicotine-treated mice were significantly increased following nicotine withdrawal, an outcome accompanied by significantly reduced serum levels of tartrate-resistant acid phosphatase, a bone resorption marker. taken together, our findings suggest that bone homeostasis is perturbed but can be rescued by smoking cessation. www.nature.com/scientificreports/ smoking is also a risk factor for fractures and osteoporosis development [ ] [ ] [ ] . smoking is reportedly associated with altered fracture healing resulting in delayed union seen in smokers compared to non-smokers . nicotine, which is taken in by smoking, disrupts bone homeostasis, and levels of the nicotine metabolite cotinine, are inversely correlated with bone mineral density (bmd) . nicotine acts via the alpha nicotinic acetylcholine receptor (α nachr), a ligand-gated ion channel, which is permeable to ca + and na + following acetylcholine or nicotine binding , . α nachr was demonstrated required for regulating levels of serum receptor activator of nuclear factor kappa b ligand (rankl) and osteoprotegerin (opg), which stimulate and inhibit, respectively, osteoclast differentiation . we showed that α nachr-deficient mice exhibit significantly increased bone mass due to inhibition of osteoclastogenesis rather than to stimulation of osteoblastogenesis compared to wild-type mice . however, it is still unclear whether smoking cessation has a positive effects on bones, and whether altered bone homeostasis can be reversed when individuals stop smoking. in the current study, we demonstrate that: ( ) in post-menopausal women, bmd is significantly lower in smokers than non-smokers; ( ) serum levels of osteocalcin and uncarboxylated osteocalcin, both markers of bone formation markers, significantly increase after smoking cessation; ( ) nicotine administration to wild-type mice reduces bone mass, a phenotype reversible by nicotine withdrawal; and ( ) in mice treated as in ( ) serum levels of tartrate resistant acid phosphatase b (trap b), a marker of osteoclastic bone resorption, significantly decrease following nicotine withdrawal. taken together, our results indicate that disturbance of bone homeostasis by smoking can be recovered by smoking cessation. bone mineral density is significantly lower in smokers than in non-smokers among post-menopausal females. for this analysis, we invited women over years of age ( fig. ) and obtained informed consent from all. of the subjects, and were current smokers and non-smokers, respectively ( fig. ) . we excluded subjects due to use of anti-osteoporosis or hormonal drugs, or due to pregnancy or lack of a complete data set ( fig. ). of the remaining subjects, and were smokers and non-smokers, respectively (fig. ). subjects were then subdivided into pre-( ) and post-menopausal ( ) women (fig. ) . among pre-menopausal women, and were smokers and non-smokers, respectively, and and postmenopausal subjects were smokers and non-smokers, respectively (fig. ) . the average age of smokers and nonsmokers was comparable in both pre-and post-menopausal groups (table ) . basic characteristics of subjects, including body weight, height and bmi, were also similar between smokers and non-smokers in both pre-and post-menopausal women, although non-smokers were significantly taller than smokers in the pre-menopausal group (table ) . interestingly, bone mineral density (bmd) was significantly lower in smokers than in nonsmokers among post-menopausal women (fig. a) . in pre-menopausal women serum levels of tracp b, a marker of osteoclastic bone-resorption, were significantly higher in smokers than in non-smokers (fig. b) . however, levels of serum n-terminal telopeptide of type collagen (ntx), a different marker of bone resorption, were significantly lower in smokers than in non-smokers (fig. b) . moreover, serum levels of osteocalcin and uncarboxylated osteocalcin (ucoc), both markers of bone formation, were comparable between smokers and non-smokers (fig. b ). smoking cessation elevates bone formation parameters in human subjects. for the second study, we invited current smokers who had visited smoking cessation clinics as outpatients (fig. ) . among them, and were male and female, respectively (fig. a) . among that , males and females reported success in stopping smoking (fig. a) . among that , we performed blood tests in subjects immediately before and then approximately days after smoking cessation to evaluate cotinine levels (fig. a) . blood cotinine levels significantly decreased after smoking cessation in of subjects (fig. a,b) . based on this analysis, we concluded that these subjects successfully quit smoking. www.nature.com/scientificreports/ www.nature.com/scientificreports/ we then monitored changes in various biochemical parameters in individuals who had successfully stopped smoking (fig. , fig. s and table ). first, we focused on bone markers and found that parameters associated with bone resorption, namely, urinary ntx and deoxypyridinoline and serum tracp b levels, were unchanged after smoking cessation (fig. a) . however, serum levels of some parameters associated with bone formation, namely osteocalcin and ucoc, significantly increased after subjects quit smoking, although levels of other bone formation factors such as total type procollagen-n-propeptide (p np) and bone specific alkaline phosphatase . changes in bone parameters after smoking cessation. (a) analysis of bone-resorption parameters such as urinary ntx and deoxypyridinoline, and serum tracp b before (before) and after (after) smoking cessation. data are presented as mean percent changes from baseline values ± s.e after relative to before smoking cessation of ntx, deoxypyridinoline, and tracp b. (b) analysis of bone formation markers evaluated in sera before (before) and after (after) smoking cessation. data represents mean percent changes from baseline values ± s.e after relative to before smoking cessation of total p np, bap, osteocalcin and ucoc (*p < . ; **p < . ; ns, not significant). dotted lines indicate baselines. www.nature.com/scientificreports/ (bap) did not (fig. b) . to identify potential regulators of osteocalcin and ucoc levels, we assessed other factors associated with bone metabolism (table ). however, serum levels of pentosidine, homocysteine, parathyroid hormone (pth), (oh)d, and estradiol (e ), all implicated in regulating bone homeostasis, were comparable in these subjects before and after they stopped smoking ( table ). bone mass increases in mice after cessation of nicotine administration. finally, we asked whether bone homeostasis would improve in nicotine-treated mice once drug was withdrawn (fig. ) . to do so, first we conducted bone mineral density analysis, and trap and toluidine blue staining of femoral bone and tibial bone sections, respectively, in mice that had been treated with and without nicotine cross sectionally (fig. ). these data indicated that daily subcutaneous administration of nicotine to wild-type mice over a -week period starting at weeks of age reduced bone mass relative to untreated controls, but those changes were not significant ( fig. a,b) . however, when we examined bone sections from these mice, nicotine-administered mice showed a significant increase in the number of osteoclasts relative to controls (fig. c,d) , while osteoblast number was comparable in nicotine-and pbs-treated mice (fig. e ,f). we then examined bone mass longitudinally in nicotine-administered mice weeks after nicotine withdrawal, when mice were weeks of age (fig. ) . these mice showed significantly elevated bone mineral density compared to mice evaluated immediately after weeks of nicotine treatment (fig. a,b) . twelve-week-old mice that had undergone week of nicotine withdrawal also showed decreased levels of serum tracp b than did control mice treated with nicotine for weeks (fig. c ). osteoporosis is a multifactorial disease brought on by various risk factors or their combination - . among them, aging, menopause and genetic factors cannot be controlled as a means to antagonize osteoporosis development; however, lifestyle-related factors such as excessive alcohol consumption and smoking are manageable. in this study, we addressed effects of smoking and smoking cessation in two human studies. in the first, we assessed bmd differences in female pre-and post-menopausal smokers versus comparable non-smokers. that analysis revealed bmd to be significantly lower in post-menopausal smokers compared to non-smokers. in a second study, we evaluated male and female smokers before and after they quit smoking and found that levels of bone formation markers significantly increased within days of smoking cessation. this analysis indicates that the negative effects of smoking on bone homeostasis can be reversed within a short period following smoking cessation, conferring a significant benefit for bone health. also, mouse studies reported here show that bmd increases within a short period after nicotine discontinuation accompanied by significantly reduced serum levels of trap b, a marker of bone resorption. smoking is a well-known risk for several diseases, including osteoporosis, lung cancer, chronic obstructive pulmonary disease (copd), and covid- ; our analysis adds to the body of evidence demonstrating that smoking cessation has positive effects on individuals' health. nicotine promotes various effects in smokers. among them, euphoria and decreased heart rate are due to vagal nerve stimulation. nicotine intake reportedly promotes peripheral vessel vasoconstriction, which is associated with osteoporosis development or delayed fracture healing . in mice, nicotine administration was demonstrated to elevate rankl and to decrease opg levels in sera to stimulate osteoclastogenesis . mice deficient in the nicotinic acetylcholine receptor α nachr exhibit a significantly decreased rankl/opg ratio and osteoclast number relative to controls and a significant increase in bone mass . in this study, we demonstrated that interrupting administration of nicotine to mice increased bone mass and decreased levels of the bone resorption marker tracp b. we also observed increased levels of some markers of bone formation in humans who had stopped smoking. thus, although we currently do not understand the reasons for differences in effects of smoking cessation on bone metabolism between humans and mice, it is clear that stopping smoking is a way to decrease the risk of osteoporosis development. at present, we do not know precisely why only osteocalcin and ucoc levels changed after smoking cessation in human subjects. nonetheless, these findings suggest that osteocalcin and ucoc respond more rapidly to smoking cessation than tracp b, making them potentially more appropriate markers of bone effects of smoking cessation. the osteocalcin-ucoc axis reportedly plays a role in regulating glucose metabolism ; however, since hemoglobin a c (hgba c) levels were unchanged after smoking cessation (table ) , we do not attribute changes in ucoc to improved glycemic control. relevant to study limitations, we did not collect bmd data from subjects who had stopped smoking, as most stopped smoking within days, a period too short to observe significant changes in bmd. we evaluated smoking habits as either "yes" or "no", and did not inquire about amounts of smoking. also, samples were collected www.nature.com/scientificreports/ at fasting but at various times of day. we note, however, that tracp b levels are reportedly unaffected by circadian rhythm and feeding . also, we did not follow up with subjects after successful smoking cessation, and samples were not collected before or after days. finally, the number of non-smokers evaluated exceeded the number of smokers: the proportion of smokers in our subjects was . %, which is lower than global averages . nonetheless, we feel that conclusions relevant to reported changes in bone density are valid and that our study makes a significant contribution to the field. in summary, we conclude that based on our findings, deleterious changes in bone homeostasis associated with smoking can be rescued by smoking cessation. such changes in smoking behaviors should be encouraged as a potential means to improve bone metabolism and reduce risk of osteoporosis development. www.nature.com/scientificreports/ human subjects. this study protocol was approved by an ethics committee at keio university school of medicine and carried out in accordance with guidelines of that committee. informed consent was taken from all subjects prior to the study. we conducted two separate human studies. in the first, we invited female medical workers in our university hospital, aged to years of age, and obtained informed consent from . all completed a self-reported questionnaire regarding current and/or previous menstruation status, smoking habits, past history of disease or drug usage. based on that questionnaire, we excluded due to medical complications and/or use of anti-osteoporosis or hormonal drugs, which are known to alter bone metabolism, or due to pregnancy or lack of a complete data set. the remaining were divided into pre-menopausal (n = ) and postmenopausal (n = ) women, and in each group, smokers and non-smokers were assessed for bone mineral density (bmd) and serum bone markers. the second study included current smokers who intended to stop smoking. of them, reported they had stopped smoking, and of those were blood cotinine-negative. in that group of subjects, we evaluated levels of bone markers and other parameters in sera before and after smoking cessation. in the first study, body height, weight, and serum levels of tartrate resistant acid phosphatase b (tracp b), n-terminal telopeptide of type collagen (ntx), osteocalcin and undercarboxylated osteocalcin (ucoc) were assessed in all subjects, and body mass index (bmi) was calculated based on body height and weight data. tracp b (nittobo, fukushima, japan) and ntx (abbott diagnostics medical co., ltd, tokyo, japan) were analyzed by enzyme immunoassay (eia) and enzyme-linked immunosorbent assay (elisa), respectively. osteocalcin (roche, basel, switzerland) and ucoc (sekisui medical, tokyo, japan) were analyzed by an electrochemiluminescent immunoassay (eclia). bmd was analyzed using an aos- system (aloka, tokyo, japan), as described . in the second study, parameters associated with bone resorption, namely, urinary ntx and deoxypyridinoline and serum tracp b were assessed in all subjects as well as bone formation parameters including serum osteocalcin, uncarboxylated osteocalcin (ucoc), total type procollagen-n-propeptide (p np), and bone specific eight-week-old wild-type female mice were administered nicotine for weeks. then, nicotine administration was stopped and mice were maintained for two more weeks without nicotine administration. bone mineral density of femurs divided equally longitudinally was evaluated serially from distal to proximal points in mice representing two time points: ( ) after nicotine had been administered for weeks (nicotine), and ( ) weeks after nicotine withdrawal (nicotine cessation). mice in group were weeks old, and those in group were weeks old. data represent mean bmd ± s.d. (*p < . ; nicotine, n = ; nicotine cessation, n = ). (c) eightweek-old wild-type female mice were administered nicotine for weeks. then, nicotine administration was stopped and mice were maintained for seven more days without nicotine administration. serum trap b levels were evaluated by elisa after nicotine had been administered for weeks (day ), and days after nicotine withdrawal (day ). data represent mean serum tracp b (mu/dl) ± s.d. (*p < . ; n = ). www.nature.com/scientificreports/ alkaline phosphatase (bap), as were pentosidine, homocystein, intact parathyroid hormone (pth), (oh)vitd, estradiol (e ), and cotinine levels. tracp b (nittobo, fukushima, japan), pentosidine (fsk, kagawa, japan) and deoxypyridinoline (sb bioscience co., ltd, osaka, japan) were analyzed by eia. pth (roche diagnostics, tokyo, japan), osteocalcin (roche), total p np (roche), e (roche), and ucoc (sekisui medical, tokyo, japan) were analyzed by eclia. bap (beckman coulter, pasadena, ca) and untx (alere medical co., ltd, tokyo, japan) were analyzed by radioimmunoassay. homocysteine (ymc co., ltd, kyoto, japan) was analyzed by high performance liquid chromatography (hplc). mice. c bl/ j wild-type mice were purchased from sankyo labo service (tokyo, japan). eight-week-old wild-type female mice were randomly assigned to each treatment group. nicotine (at a dosage of μg/day) or pbs were injected subcutaneously every day. in the first examination, mice were administered nicotine or pbs vehicle (control) for weeks. we then evaluated bone mineral density of femurs divided equally longitudinally. tibial bone sections were stained with trap or toluidine blue, and the number of osteoclasts per bone perimeter (n.oc/b.pm) and the number of osteoblasts per bone perimeter (n.ob/b.pm) were then evaluated (control, n = ; nicotine, n = ). in the second examination, mice were administered nicotine for weeks. then, nicotine administration was stopped, and mice were maintained for two more weeks without nicotine administration. we then evaluated bone mineral density of femurs divided equally longitudinally serially from distal to proximal points in mice representing two time points: ( ) after nicotine had been administered for weeks (nicotine), and ( ) weeks after nicotine withdrawal (nicotine cessation) (nicotine, n = ; nicotine cessation, n = ). in the third examination, mice were administered nicotine for weeks. nicotine administration was then stopped, and mice were maintained seven more days without drug. serum trap b levels were evaluated by elisa after nicotine had been administered for weeks (day ), and days after nicotine withdrawal (day ) (n = ). for bmd analysis, femurs were removed, fixed in % ethanol, and subjected to dual energy x-ray absorptiometric (dexa) scanning to measure bmd (mg/cm ) at proximal to distal points using a dcs- r system (aloka, co. ltd, tokyo, japan). bone morphometric analysis and tartrate-resistant acid phosphatase (trap) staining were performed in tibiae, as described . all animals were maintained under specific pathogen-free conditions in animal facilities certified by the keio university animal care committee. animal protocols were approved by that committee and carried out in accordance with the committee's guidelines. animals were housed up to mice per cage and kept on a h light/ dark cycle. water and food were available ad libitum. all animal studies were performed in accordance with the guidelines of the keio university animal care committee, as described . serum trap b assay in mice. eight-week-old wild-type female mice were subcutaneously injected nicotine (at a dosage of μg/day) every day for weeks, and then nicotine administration was stopped. sera were collected from nicotine-administered mice before and days after nicotine cessation. trap b levels in sera were assessed by elisa, according to the manufacturer's protocol (immunodiagnostic systems limited, boldon, tyne & wear, uk). biochemical markers of bone metabolism the pathophysiology and treatment of osteoporosis lifestyle and osteoporosis the role of cellular senescence in diabetes mellitus and osteoporosis: molecular pathways and potential interventions hif alpha is required for osteoclast activation by estrogen deficiency in postmenopausal osteoporosis human aldehyde dehydrogenase gene family a missense single nucleotide polymorphism in the aldh gene, rs , is associated with hip fracture aldehyde dehydrogenase (aldh) associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and aldh gene targeting mouse pharmacokinetic and pharmacodynamic basis for partial protection against alcoholism in asians, heterozygous for the variant aldh * gene allele aldehyde-stress resulting from aldh mutation promotes osteoporosis due to impaired osteoblastogenesis fracture risk associated with smoking: a meta-analysis cigarette smoking and risk of hip fracture in women: a meta-analysis of prospective cohort studies inclusion of tobacco exposure as a predictive factor for decreased bone mineral content smoking cessation and bone healing: optimal cessation timing dose-related effect of urinary cotinine levels on bone mineral density among korean females pharmacology of neuronal nicotinic acetylcholine receptor subtypes daidzein sulfonate sodium provides neuroprotection by promoting the expression of the alpha nicotinic acetylcholine receptor and suppressing inflammatory responses in a rat model of focal cerebral ischemia the nicotinic acetylcholine receptor alpha subunit is an essential negative regulator of bone mass smoking is associated with covid- progression: a meta-analysis cigarette smoking increases complications following fracture: a systematic review undercarboxylated osteocalcin: experimental and human evidence for a role in glucose homeostasis and muscle regulation of insulin sensitivity clinical performance of immunoreactive tartrate-resistant acid phosphatase isoform b as a marker of bone resorption health at a glance a serum metabolomics-based profile in low bone mineral density postmenopausal women tumor necrosis factor receptor-associated factor is required to inhibit foreign body giant cell formation and activate osteoclasts under inflammatory and infectious conditions elevation of pro-inflammatory cytokine levels following anti-resorptive drug treatment is required for osteonecrosis development in infectious osteomyelitis statistical analysis. statistical analysis was performed using a non-parametric test (*p < . ; **p < . ; ***p < . ; ns, not significant, throughout the paper). the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to k.s. or t.m.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -wmfwl bh authors: jung, eunhye; nam, sangwoo; oh, hyeryeon; jun, sangmi; ro, hyun-joo; kim, baek; kim, meehyein; go, yun young title: neutralization of acidic intracellular vesicles by niclosamide inhibits multiple steps of the dengue virus life cycle in vitro date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: wmfwl bh dengue fever is one of the most important mosquito-borne viral infections in large parts of tropical and subtropical countries and is a significant public health concern and socioeconomic burden. there is an urgent need to develop antivirals that can effectively reduce dengue virus (denv) replication and decrease viral load. niclosamide, an antiparasitic drug approved for human use, has been recently identified as an effective antiviral agent against a number of ph-dependent viruses, including flaviviruses. here, we reveal that neutralization of low-ph intracellular compartments by niclosamide affects multiple steps of the denv infectious cycle. specifically, niclosamide-induced endosomal neutralization not only prevents viral rna replication but also affects the maturation of denv particles, rendering them non-infectious. we found that niclosamide-induced endosomal neutralization prevented e glycoprotein conformational changes on the virion surface of flaviviruses, resulting in the release of non-infectious immature virus particles with uncleaved pr peptide from host cells. collectively, our findings support the potential application of niclosamide as an antiviral agent against flavivirus infection and highlight a previously uncharacterized mechanism of action of the drug. in this study, the anti-dengue activity of niclosamide was evaluated using four denv serotypes. after h post-infection (p.i), viral titres from the supernatants and the number of infected cells were measured by fluorescence-activated cell sorter (facs) analysis and focus-forming assay, respectively. the proportion of cells positive for denv antigen decreased in a dose-dependent manner in cells treated with niclosamide compared to that in dmso-treated controls as determined by facs analysis (fig. a) . specifically, the percentage of denv-positive cells was significantly reduced when infected cells were treated with niclosamide at a concentration of . μm or higher for all four serotypes of denv (fig. a) . the ec values of niclosamide against denv- , denv- , denv- , and denv- were approximately . μm, . μm, . μm and . μm, respectively. to rule out the possibility that virus infected cells were more sensitive to niclosamide treatment resulting in synergistic cytotoxicity compared to mock-infected cells, the percentages of live and dead cells from mock-and denv- infected cultures with increasing concentrations of niclosamide were determined by facs analysis. the data showed no difference in cell viability between mock-and denv-infected cells treated with increasing concentrations of niclosamide ( supplementary fig. s ). next, the % cytotoxicity concentration (cc ) value of niclosamide was determined by measuring cell viability using the mtt assay. the estimated cc value of niclosamide was > μm; however, minor cytotoxic effects at all sub-lethal doses were observed in huh- cells (supplementary fig. s ). similarly, niclosamide inhibited the production of infectious denv particles of all four serotypes in a dose-dependent manner as quantified by the focus-forming assay. significantly, no infectious denv particles were detected when infected huh- cells were treated with niclosamide at a concentration of μm or higher (fig. b) . these results together confirm that niclosamide effectively inhibits denv infection independent of the virus serotype within a non-cytotoxic range in huh- cells. demonstrating that niclosamide has an antiviral effect against denv independent of its serotype, the denv- ngc strain was employed in all subsequent experiments. we performed a time-of-addition experiment to investigate the stage of the denv life cycle during which niclosamide exerts its antiviral activity. huh- cells were infected with denv- and subsequently treated with μm niclosamide starting at , , and h p.i. until the medium and cell lysates were harvested at h p.i. (fig. a) . analysis of the infectious progeny released in the supernatant revealed that niclosamide not only affected an early stage of the viral life cycle but also reduced progeny titres when added at later stages. specifically, there was a complete inhibition of infectious virus production when niclosamide was added at h p.i., while a -log reduction was observed when niclosamide treatment was initiated at , or as late as h p.i. (fig. b) . similarly, niclosamide treatment was maximally effective (~ %) in reducing intracellular viral rna when added at h p.i., while the inhibitory effect markedly decreased when the drug was added at later time points ( , and h p.i.), suggesting that niclosamide interferes with viral rna replication (fig. c) . likewise, there was a marked reduction of viral genome copies released in the supernatant from samples that received niclosamide treatment at h p.i. (~ -log reduction), while the effect diminished www.nature.com/scientificreports www.nature.com/scientificreports/ when the drug was added at later time points such as , or as late as h p.i. (~ -log reduction, fig. d ). thus, the data together indicated that niclosamide possibly inhibits viral rna replication when it is added at early step of viral life cycle but it also affects a late stage of virion biogenesis, such as maturation, since a strong antiviral effect was consistently observed in infected cells with niclosamide treatment starting as late as h p.i. (fig. bd) . in addition, viral ns and e protein levels were significantly reduced upon niclosamide treatment at early post-infection time points, whereas the antiviral effect diminished when the drug was added after h p.i. (fig. e) . the data represent the means (±sd) of at least two independent experiments performed in duplicate. n.d., not detected. *p < . , ***p < . and ****p < . compared to dmso control. www.nature.com/scientificreports www.nature.com/scientificreports/ taken together, these data suggest that niclosamide potently inhibits the early stages of the denv life cycle, which impacts viral rna accumulation and protein expression, as well as the production of infectious viruses. in addition, the data revealed that niclosamide affects not only an early stage but also a late stage of the denv life cycle independent of the effects on intracellular viral rna accumulation and protein synthesis. neutralization of endosomal ph by niclosamide inhibits denv rna genome replication and viral polyprotein processing. niclosamide is a proton carrier and blocks endosomal acidification . we tested whether niclosamide neutralizes the low-ph compartments in huh- cells by using acridine orange (ao), which is a fluorescent ph-sensitive dye. as shown in fig. a , the low ph of endosomes (red) present in mock-treated huh- cells was completely absent in cells treated with niclosamide (green), suggesting that it effectively blocked endosomal acidification in huh- cells. bafilomycin a (bafa ) and ammonium chloride (nh cl) were used as controls. to further confirm the correlation of the antiviral effect with the neutralization of endosomal ph, lysotracker (a marker for acidic compartments) analysis was performed together with immunofluorescence staining for the detection of double-stranded rna (dsrna) in denv-infected cells treated with niclosamide at early (pre, − h to h p.i.) or late stages (post, h p.i. to h p.i.) (fig. b) . the results showed that viral dsrna expression, an intermediate product in replication, was significantly inhibited in infected cells treated with niclosamide at early and late stages compared to that in dmso-treated controls, indicating that viral rna replication is severely affected (fig. c) . notably, there was no difference in the levels of endosomal acidification in cells treated with niclosamide during the early stage and subsequently removed from the culture, whereas the endosomal ph was effectively neutralized in cells that received the treatment at the late stage and the treatment remained until lysotracker staining, indicating the reversible effect of the drug on the inhibition of endosomal acidification (fig. c) . to further corroborate that suppression of viral genome replication is specifically due to niclosamide-induced ph neutralization, denv replicon reporter bhk cells encoding the renilla luciferase gene were treated with niclosamide at the time points indicated in fig. b . ribavirin, a broad-spectrum antiviral compound known to impede rna virus replication, was used as a positive control. cell viability of replicon cells in the presence of niclosamide and ribavirin treatment was determined by the mtt assay ( supplementary fig. s ). as shown in fig. d , treatment of denv replicon reporter cells with μm niclosamide at early time points (pre, − h to h) resulted in a significant reduction in luciferase activity, comparable to that of ribavirin, whereas the inhibitory effect was almost restored to the level of dmso-treated cells when the cells were treated with niclosamide after h of culture (post, h p.i. to h p.i). in contrast, the reduction in luciferase activity reached a maximum when ribavirin was added at h p.i. and remained in the culture until harvest. thus, the replicon-based assay suggested that niclosamide inhibits the steps involved in viral rna replication independent of its effect on entry, membrane fusion and genome release. next, we evaluated the impact of niclosamide on the proteolytic activity of denv ns b-ns , which plays an essential role in viral polyprotein processing required for the initiation of viral replication. the proteolytic activity of denv ns b-ns protease, using the fluorogenic substrate, was evaluated in the presence of various concentrations of niclosamide. as a result, the half-maximal inhibitory concentration (ic ) for niclosamide was determined to be . μm, showing only a modest inhibition of ns protease activity (fig. e ). lastly, the expression level of denv non-structural (ns ) protein after niclosamide treatment at early (pre, − h to h p.i.) or late stages (post, h p.i. to h p.i.) was examined by western blot analysis (fig. f) . consistent with the immunofluorescence assay results, the expression of ns protein was significantly reduced in cells treated at early and late stages, although the inhibition was more pronounced in cells treated at the early stage of virus replication than in those treated after h p.i. (fig. f) . overall, the data indicate that the antiviral activity of niclosamide during the early stage of the denv life cycle correlates with the neutralization profile of the low-ph compartments, suggesting that blocking endosomal acidification results in the inhibition of viral genome replication and polyprotein processing, which further impedes viral protein expression and virus production. neutralization of low-ph compartments by niclosamide also impairs the denv maturation process. to further corroborate the effect of niclosamide-induced neutralization of low-ph compartments on virion biogenesis, particularly maturation, which is a ph-dependent process, we analysed the composition of prm and e proteins in the virus particles produced by infected cells treated with niclosamide. briefly, denv- -infected huh- cells were treated with μm niclosamide at h p.i. and supernatants were collected at h p.i. subsequently, tissue culture medium obtained from niclosamide-treated and mock-treated cells was pelleted by ultracentrifugation. samples were adjusted to equal viral genomic copy numbers, separated by sds-page, and viral proteins were detected by western blot using monoclonal antibodies against e and prm proteins. the data showed that virus particles obtained from niclosamide-treated and untreated (dmso) samples had comparable levels of denv e protein (fig. a) . in contrast, a clear prm protein band (approximately kda) was detected in denv released from niclosamide-treated cells, in which the pr peptide was not cleaved during maturation, resulting in the release of immature and non-infectious virus particles in the medium. in contrast, the prm protein band was barely detected from virions released from dmso-treated cells, indicating that they were mature and fully infectious (fig. a) . next, the morphology of denv particles released from dmso-and niclosamide-treated cells was examined by transmission electron microscopy (tem). as shown in fig. , denv particles derived from dmso-treated cells had relatively smooth and spike-less outer surfaces, which are characteristics of mature particles (fig. b,c [upper panels]) compared to those obtained from niclosamide-treated cells, which had the expected spiky appearance of immature particles (fig. b,c [lower panels]). similar results were observed in human monocytic u -dendritic cell-specific icam-grabbing non-integrin (u -dc-sign) cells suggesting that the antiviral effect of niclosamide is not limited to huh- cells (supplementary fig. s ). taken together, these results indicate that inhibition of endosomal acidification by niclosamide interferes with the ph-dependent denv maturation process by preventing cleavage of the pr peptide from the m protein on surface of the virus. www.nature.com/scientificreports www.nature.com/scientificreports/ the zikv particle maturation process is affected. we hypothesized that niclosamide-induced neutralization of low-ph compartments also impacts zikv, a member of the flavivirus genus, virion biogenesis. we first corroborated that niclosamide reduced the number of zikv-positive cells and infectious viral production in a dose-dependent manner in huh- cells with an ec of . μm as evaluated by facs analysis and focus-forming www.nature.com/scientificreports www.nature.com/scientificreports/ assay, respectively (fig. a,b) . next, the composition of prm and e proteins in zikv virions released in the medium of niclosamide-treated cells was examined to confirm whether niclosamide impacts the maturation process. as shown in fig. c , zikv particles released in the medium of dmso-treated cells were completely processed with no detectable prm protein band, while a prominent m protein band at approximately kda was observed, indicating that the virions are mature and fully infectious. in contrast, zikv particles released in the medium of niclosamide-treated cells had a prominent prm protein band, and almost no m protein was detected, indicating that cleavage of the pr peptide during the maturation process had been hampered (fig. c) . consistent with these results, the electron microscopy images also demonstrated that virions from dmso-treated cells had a smoother surface (fig. d , upper panels), while those obtained from niclosamide-treated cells had a spiky outer surface (fig. d , lower panels), indicating that conformational surface protein changes and pr peptide cleavage were blocked. taken together, our data suggest that niclosamide-induced neutralization of low-ph compartments interferes with the denv and zikv maturation process, resulting in the release of prm-containing immature and non-infectious viral particles into the extracellular environment. niclosamide is a well-established drug that has been safely used for antihelmintic therapy against tapeworm infection for approximately years . its antiparasitic activity is attributed to its ability to inhibit mitochondrial oxidative phosphorylation and anaerobic atp production, which affect the ph homeostasis of parasites , . recently, niclosamide has also been identified as an effective antiviral agent against a number of ph-dependent viruses. its inhibitory effect has been attributed to the neutralization of endo-lysosomal ph, which interferes with ph-dependent membrane fusion that is critical for virus entry . in this study, we found that neutralization of low-ph intracellular compartments by niclosamide not only inhibited the early stage of the denv viral life cycle, such as viral rna replication, independent of the entry step but also the late stage, specifically, the maturation of virus particles into infectious virions. similar to previous reports, our data showed that niclosamide neutralized the low-ph intracellular compartments in a reversible manner and suggested that its antiviral effect correlates with neutralization of these acidic environments, which are critical for denv replication , . here, we confirmed that niclosamide effectively inhibited intracellular viral rna synthesis, protein expression and production of infectious denv particles when the drug was added at an early time point during infection. the immunofluorescence assay coupled with staining of intracellular acidic compartments in live denv-infected cells showed that niclosamide-induced ph neutralization during the first h of infection was sufficient to completely block viral dsrna replication and its subsequent steps, such as viral protein expression and production of infectious viral particles. indeed, niclosamide treatment www.nature.com/scientificreports www.nature.com/scientificreports/ during the first h of infection reduced denv replication in bhk- cells harbouring dengue replicons to a level comparable to that of ribavirin, suggesting that the drug affects viral rna replication and/or translation independent of its effect on entry, membrane fusion and genome release. furthermore, we provide evidence that niclosamide impairs the proteolytic activity of denv ns b-ns , which is essential for the initiation of www.nature.com/scientificreports www.nature.com/scientificreports/ viral replication and may explain its effects on denv genome replication and/or translation, strengthening the findings of li et al. . however, these findings are somewhat contradictory to the results reported by kao et al. , which, using a replicon-based assay, indicated that niclosamide had no effect on denv genome replication. we speculate that the variance in the efficacy of niclosamide against viral genome replication using replicon-based cells could be due to a difference in the times of drug-addition used in each study. in this study, we identified a new potentially interesting mode of action of niclosamide, which impacts a late stage of the virus life cycle in huh- cells. our time-of-addition data demonstrated that the quantity of infectious virus particles released into the media was significantly reduced even when niclosamide was added to the cells several hours after infection, whereas intracellular and extracellular viral rna levels were not affected to the same extent as the production of infectious virus particles. these data led us to the hypothesis that niclosamide-induced intracellular ph neutralization affects the maturation of denv particles. it has been extensively described that the low-ph-induced conformational rearrangement of the prm and e proteins is critical for denv maturation and infectivity , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed, our data showed that denv, as well as zikv particles obtained from niclosamide-treated cells, contained high levels of uncleaved prm proteins compared to the untreated virions, suggesting that most of the virus particles were immature and non-infectious. similarly, transmission electron microscopy images showed that the denv and zikv particles secreted from niclosamide-treated cells were substantially larger than untreated control virions and had irregularities on the surface, indicating that the viral particles were immature and contained uncleaved prm on the surface. taken together, we believe that we have provided the first evidence that neutralization of the low-ph intracellular compartments by niclosamide prevents conformational changes to e glycoproteins on the virion surface during the flavivirus maturation process, which results in the release of immature non-infectious virus particles from host cells. in summary, we confirmed that the antiviral effect of niclosamide against flaviviruses is mostly associated with neutralization of the low-ph intracellular organelles. as a consequence, multiple ph-dependent steps of the flavivirus life cycle, including viral and host membrane fusion and uncoating, viral rna replication (by inhibition of viral polyprotein processing), and the maturation process of the progeny virions, are impaired, highlighting the complexity of the antiviral efficacy of niclosamide (fig. ) . collectively, the data presented in this study provide further evidence to support the repurposing of niclosamide as a potential therapeutic option against ph-dependent rna viruses, particularly flaviviruses. . all denvs were propagated in c / cells, and tissue culture fluid (tcf) supernatants were harvested at - days post-infection. tcf was clarified by centrifugation, and -ml aliquots were stored at − °c until further use. viral titres were quantified by focus-forming assay on vero cells as described previously . zika virus, an mr strain (atcc ® vr- tm ) purchased from atcc, was amplified, and viral titres were measured by plaque assay on vero cells. to determine the mechanism of action of niclosamide, huh- cells were inoculated with the denv- ngc strain at an moi of . at °c for h. unbound virus was removed by washing with ice-cold phosphate buffered saline (pbs), fresh medium was then added, and plates were shifted to °c to allow synchronous entry and infection. soon after the temperature shift, µm niclosamide was added at , , and h and maintained throughout the infection. at h p.i., cell culture supernatants were collected for virus titration and extracellular viral rna quantification by focus-forming assay and quantitative reverse-transcription polymerase chain reaction (rt-qpcr), whereas cell lysates were harvested and subjected to intracellular viral rna and viral protein analyses by rt-qpcr and western blot assays, respectively. rna quantification, total cellular rna was purified from cell lysates using an rneasy mini kit (qiagen, valencia, ca, usa) according to the manufacturer's instructions. viral rna in the tcf samples was extracted using a qiaamp ® viral rna mini kit (qiagen) according to the manufacturer's instructions. rt-qpcr was performed using a superscript iii one-step rt-pcr system with platinum taq polymerase (invitrogen), primers/probe sets targeting ns or e genes and a quantstudio real-time pcr system (applied biosystems, foster city, ca, usa) as described previously . the relative viral rna expression levels were calculated by the ΔΔc t method, and β-actin was used as an endogenous control. absolute viral rna genome copy number was calculated based on the in vitro-transcribed denv rna standard curve and reported as the absolute number of viral rna genome copies per ml of tcf . two biological replicates, each with technical duplicates, were used for quantification. western blot analysis. at h p.i., virus-infected cells were washed with pbs and lysed using m-per buffer (thermo fisher scientific, waltham, ma, usa) containing . % protease inhibitor cocktail (pierce, rockford, il, usa). the cell lysates were clarified by centrifugation, and the total protein content was determined by the bradford assay (bio-rad, hercules, ca, usa). equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and electro-transferred to a pvdf membrane. viral proteins were detected using primary antibodies specific for denv ns , denv e, denv prm, zikv e, and zikv prm followed by a horseradish peroxidase (hrp)-conjugated goat anti-mouse or anti-rabbit secondary antibody. as a loading control, cellular β-actin was detected with an anti-β-actin-specific primary antibody and hrp-conjugated goat anti-mouse secondary antibody. after the addition of a chemiluminescent hrp substrate (supersignal west pico chemiluminescent substrate; pierce), images were obtained using a las- luminescent image analyzer (fujifilm, tokyo, japan). denv replicon assay. bhk- cells encoding a luciferase-expressing denv- replicon (bhk-d -rluc) were used to determine denv rna replication efficiency. briefly, bhk- replicon cells were seeded in -well plates and incubated with different concentrations of niclosamide at the indicated time points at °c for h. after incubation, antiviral activity was measured using the renilla luciferase assay (promega, madison, wi, usa) in a microplate luminometer (tecan, männedorf, switzerland). in vitro protease activity assay. the denv- ns b-ns protease expression plasmid used in this study was kindly provided by dr. rolf hilgenfeld, university of lübeck, lübeck, germany. the in vitro denv- ns b-ns protease activity assay was performed as described elsewhere . the fluorogenic peptide substrate (boc-gly-arg-arg-amc) was purchased from bachem ag (bubendorf, switzerland). to measure ph changes in cytoplasmic membrane-enclosed vesicles, cells were stained with acridine orange (ao, thermo fisher scientific), as described previously . briefly, huh- cells ( × cells per well) were cultured at °c in mm glass-bottom dishes (greiner bio-one, frickenhausen, germany). on the following day, cells were treated for h with μm niclosamide, nm bafilomycin a (a v-atpase inhibitor), or mm ammonium chloride (an intralysosomal ph-neutralizing agent). acridine orange was added to the culture medium at a final concentration of μg/ml, and the cells were imaged with a confocal microscope zeiss lsm meta (carl zeiss, oberkochen, germany). the excitation wavelength was nm, and images were collected in two emission windows: - nm and - nm. for labelling and tracking of acidic organelles in niclosamide-treated denv-infected cells, a deep red-fluorescent dye, lysotracker dnd- (ltr, thermo fisher scientific), was used. briefly, huh- cells were grown on -well chamber slide and incubated overnight. the huh- cells were infected with the denv- ngc ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ strain at an moi of and treated with μm niclosamide at h prior to infection and left in culture until h p.i. (pre − to h) or treated at h p.i. and the treatment maintained throughout the course of the infection. after h of incubation, cells were washed and loaded with . μm lysotracker dnd- (invitrogen) for min at °c. subsequently, the cells were washed with pbs and fixed with % paraformaldehyde in pbs for min at room temperature. the fixed cells were incubated with antibodies against denv e protein or dsrna antibodies for h at room temperature. the cells were then incubated with af -conjugated goat anti-mouse igg antibodies for h at room temperature. nuclei were counterstained by incubation for min with μg/ml dapi, and the coverslips were mounted in prolong tm gold antifade reagent (invitrogen). image analysis was performed using a confocal microscope (zeiss lsm meta). electron microscopy. huh- cells were infected with denv- (ngc strain) or zikv (mr strain) at an moi of . for h at °c. at h p.i., μm niclosamide or dmso was added and infected cultures were further incubated for h. after a total of h of infection, virus particles were pelleted by ultracentrifugation at °c in a beckman type sw rotor at , × g for h. virion pellets were resuspended in nte buffer. five microliters of resuspended denv- and zikv were mounted on plasma-cleaned mesh, carbon-coated, copper grids (electron microscopy sciences, hatfield, pa). grids were washed once with distilled water and then negatively stained with % aqueous uranyl acetate (electron microscopy sciences, hatfield, pa) for s. the solution was blotted with filter paper, and the sample grids were rinsed briefly with distilled water three times. after drying in air, the grids were examined under a zeiss leo ab transmission electron microscope (carl zeiss) at an accelerating voltage of kv and an fei tecnai g t- s transmission electron microscope (fei company, hillsboro, or) at an accelerating voltage of kv. statistical analysis. all statistical analyses were performed using graphpad prism version . (graphpad software, la jolla, ca, usa). the % effective concentration (ec ) was calculated by non-linear regression analysis. data sets were analyzed using one-way anova by dunnett's test for multiple comparison with a significance of p < . . all data generated or analyzed during this study are included in this article (and its supplementary information file) . dengue/dengue haemorrhagic fever: history and current status the global distribution and burden of dengue dengue: a continuing global threat fields virology zika virus associated with microcephaly zika virus and birth defects-reviewing the evidence for causality rapid spread of zika virus in the americas-implications for public health preparedness for mass gatherings at the brazil olympic games guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a casecontrol study guillain-barre syndrome associated with the zika virus outbreak in brazil magnetic resonance imaging findings in guillain-barre 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for point-of-need diagnosis of dengue virus infection by use of the pockit nucleic acid analyzer protegrin- inhibits dengue ns b-ns serine protease and viral replication in mk cells intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin the authors are grateful to dr. rolf hilgenfeld (university of lübeck, lübeck, germany) for kindly providing denv- ns b-ns protease expression plasmid used in this study. we would also like to acknowledge dr. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -jc xklzk authors: chen, jun; wu, lianlian; zhang, jun; zhang, liang; gong, dexin; zhao, yilin; chen, qiuxiang; huang, shulan; yang, ming; yang, xiao; hu, shan; wang, yonggui; hu, xiao; zheng, biqing; zhang, kuo; wu, huiling; dong, zehua; xu, youming; zhu, yijie; chen, xi; zhang, mengjiao; yu, lilei; cheng, fan; yu, honggang title: deep learning-based model for detecting novel coronavirus pneumonia on high-resolution computed tomography date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: jc xklzk computed tomography (ct) is the preferred imaging method for diagnosing novel coronavirus (covid ) pneumonia. we aimed to construct a system based on deep learning for detecting covid- pneumonia on high resolution ct. for model development and validation, , anonymous images from admitted patients, including patients of laboratory confirmed covid- pneumonia and control patients of other diseases in renmin hospital of wuhan university were retrospectively collected. twenty-seven prospective consecutive patients in renmin hospital of wuhan university were collected to evaluate the efficiency of radiologists against -cov pneumonia with that of the model. an external test was conducted in qianjiang central hospital to estimate the system’s robustness. the model achieved a per-patient accuracy of . % and a per-image accuracy of . % in internal retrospective dataset. for internal prospective patients, the system achieved a comparable performance to that of expert radiologist. in external dataset, it achieved an accuracy of %. with the assistance of the model, the reading time of radiologists was greatly decreased by %. the deep learning model showed a comparable performance with expert radiologist, and greatly improved the efficiency of radiologists in clinical practice. in december , a new coronavirus infection disease (hereinafter referred to as covid- ) was first reported in wuhan. subsequently, the outbreak began to spread widely in china and even abroad [ ] [ ] [ ] . the clinical manifestations of the covid- pneumonia is complicated and could be characterized as fever, cough, myalgia, headache, and gastrointestinal symptoms onset . although the nucleic acid detection was considered determinant for identifying the covid- infection and more rapid detection kit for the novel coronavirus has come into mass production, computed tomography (ct) scan is still the most efficient modality for detecting and evaluating the severity of pneumonia . an update series demonstrate that ct findings were positive in all laboratory-confirmed covid- patients, even in the early stage , . in the fifth version of diagnostic manual of covid- launched by the national health and health commission of china, the radiographic characteristics of pneumonia was included the clinical diagnostic standard in hubei province www.nature.com/scientificreports/ of covid- were reported within day on feb , in wuhan, including , cases of clinical diagnoses . this highlighted the importance of ct in the diagnosis of covid- pneumonia. due to the outbreak of the covid- , thousands of patients waited in line for ct examination in the designated fever outpatient hospital at wuhan and other cities. as of feb , there are , suspected cases, , confirmed patients receiving treatment in hospital, and , cases under medical observation in hubei province . most of them need to undergo ct examination, however, there are less than , radiologists in cities of hubei according to the china health statistical yearbook ( ) . meanwhile, because the lung infection foci are small in the early stage of the covid- infection, thinner layer ( . mm, . mm or even . mm) scanning were usually needed instead of conventional ct scan ( mm) for diagnosis, which would be more timeconsuming. all these made radiologists overloaded, delay the diagnosis and isolation of patients, affect patient's treatment and prognosis, and ultimately, affect the control of covid- epidemic. deep learning, an important breakthrough in the domain of ai in the past decade, has huge potential at extracting tiny features by the basic unit of dcnn's sampling kernel in image analysis . our group also succeeded in recruiting this technique in minor lesion detection and real-time assistance to doctors in gastrointestinal endoscopy [ ] [ ] [ ] [ ] [ ] . in the present research, we construct and validate a system based on deep learning for identification of viral pneumonia on ct. our model has comparable performance with expert radiologist, but take much less time. the module and source code developed in this work were shared for global researches in https ://githu b.com/ endo-angel /ct-angel , and an open-access website has been made available to provide free provide to the present system (https :// . . . /znyx-ncov/index ). . for patients whose ct scans were stored in the retrospective databases, informed consent was waived by the ethics committee. a statement to confirm that all methods were carried out in accordance with relevant guidelines and regulations. diagnostic testing for covid- . patient's respiratory secretions were collected and transferred to a sterile test tube with a virus transport medium. fluorescent rt-pcr analysis of samples was performed using the covid- nucleic acid detection kit developed by shanghai geneodx biotechnology co., ltd. this detection kit was approved by the us national drug administration (nmpa) on january , and recommended by the centers for disease control and prevention (cdc) . the rapid, high-precision covid- detection kit greatly accelerated the confirmation of human covid- infection. fig. , a total of , ct scan images from covid- pneumonia patients and control patients of other disease from renmin hospital of wuhan university were collected for developing the model to detect covid- pneumonia. after filtering those images without good lung fields, , images were selected and split into training and retrospectively testing datasets. enrolled images in training dataset covered almost all common ct features of covid pneumonia, as presented in fig. . three radiologists with more than years of clinical experience labelled infection lesions of covid- pneumonia patients in training dataset, and selected images containing covid pneumonia lesions in testing set, and their labels were combined by consensus. for prospectively testing the model, , images of consecutive patients undergoing ct scans in feb , in renmin hospital of wuhan university were further collected. all ct scans were obtained in renmin hospital of wuhan university. to estimate the robustness of the system, an external dataset containing patients ( , images from covid- and , images from normal control patients) were retrospectively collected from qianjiang central hospital, china. the instruments used in this study included optima ct , revolution ct and bright speed ct scanner (all ge healthcare). training algorithm. this work is built on the top of unet++, a novel and powerful architecture for medical image segmentation , for the identification. resnet- was used as backbone of unet++ as previously described . resnet- was pretrained using imagenet dataset , and all the pre-training parameters of resnet- are loaded to unet++. the network architecture of unet++ was shown in fig. . briefly, unet++ consists of encoder and decoder connecting through a series of nested dense convolutional blocks. the semantic gap between the feature maps of the encoder and decoder is bridged prior to fusion. the encoder extract features by down-sampling; the decoder map features to the original image by up-sampling, make classification by pixels, and thus achieve the purpose of segmentation. we first trained unet++ to extract valid areas in ct images using randomly selected ct images and tested it in other randomly selected ct images. the prediction schematic of the model was shown in fig. . raw images were firstly input into the model, and after processing of the model, prediction boxes framing suspicious lesions were output. valid areas were further extracted and unnecessary fields were filter out to avoid possible false positives. to predict by case, a logic linking the prediction results of consecutive images was added. ct images with the above prediction results were divided into four quadrants, and results would be output only when three consecutive images were predicted to have lesions in the same quadrant. to evaluate the performance of the model on ct scan images, five metrics including the accuracy, sensitivity, specificity, positive prediction value (ppv) and negative prediction value (npv) were calculated as follows: accuracy = true predictions/total number of cases, sensitivity = true positive/positive, specificity = true negative/negative, ppv = true positive/(true positive + false positive), npv = true negative/(true negative + false negative). the "true positive" is the number of correctly predicted covid- pneumonia cases/images, "false positive" is the number of mistakenly predicted covid- pneumonia cases/images, "positive" is the number of cases/images of covid- pneumonia patients, "true negative" is the number of correctly predicted non-covid- pneumonia cases/images, "false negative" is the number of mistakenly predicted non-covid- pneumonia cases/images and 'negative' is the number of non-covid- pneumonia cases/images enrolled. representative images of covid pneumonia. more than six common computed tomography (ct) features of covid pneumonia were covered in selected images. (a-d), the lesions were mainly ground-glass-like, with thickened blood vessels walking and including gas-bronchial signs in (c); (a-d), the lesions were mainly ground glass changes, and paving stone-like changes were observed on (d); (a-c), the lesions become solid with a large range, and air-bronchial signs are seen inside; , the lesion is located in the lower lobe of both lungs, and is mainly grid-like change with ground glass lesion; (a,b), the lesions are mainly consolidation; (a,b), the lesions are mainly large ground glass shadows, showing white lung-like changes, with air-bronchial signs. the performance of the model in consecutive prospective patients. twenty-seven patients were enrolled in the prospective dataset in renmin hospital of wuhan university. sixteen ( . %) patients were diagnosed as viral pneumonia by the expert radiologist, and the other eleven patients were not. two other radiologists reviewed the ct imaging, approved the expert's results, and summarized that the ct characteristics of table . comparison between the efficiency of radiologist with or without the assistance of ai. in the first time the expert radiologist read ct scan images of the prospective patients, the average reading time for him to determine whether each patient has viral pneumonia was . s per case (iqr . - . ). after days of wash out period, the same expert radiologist re-read the ct images of the prospective patients with the assistance of the ai model. the results for determining whether each patient has viral pneumonia were not changed, while the average reading time of the expert was greatly decreased by %. this indicates that the efficiency of radiologist could be greatly improved with the assistance of ai. a website has been made available to provide free access to the present model (https :// . . . /znyxncov/index ) (fig. ) . ct scan images could be uploaded by both clinicians and researches as a second opinion consulting service, especially in other provinces or countries unfamiliar with the radiologic characteristics of covid- . cases of covid- pneumonia were also been made available on the open-access website, which might be a useful resource for radiologists and researchers for fighting covid- pneumonia. furthermore, the module and source code developed in this work were shared for global researches in https ://githu b.com/ endo-angel /ct-angel . table . the performance of the deep learning model on both retrospective and prospective dataset. ppv positive prediction value; npv negative prediction value. www.nature.com/scientificreports/ as of feb , , the national health commission had reported , confirmed cases, , deaths and , suspected cases . in the face of such large number of patients and high contagiosity of the novel coronavirus (with an estimated reproduction number r of . ~ . ), timely diagnosis and isolation are the keys to prevent further spread of the virus [ ] [ ] [ ] [ ] [ ] . ct scan is the most efficient modality for screening and clinically diagnosing covid- pneumonia , . however, compared to the needs of the patients, the number of radiologists is quite small, especially in hubei province, china, which could greatly delay the diagnosis and isolation of patients, affect patient's treatment and prognosis, and ultimately, affect the overall control of covid- epidemic. deep learning, a technology has shown great performance on extracting tiny features in radiology data, may hold the promise to alleviate this problem . recently, ardila d, et al. achieved end-to-end lung cancer screening on low-dose chest ct with an auc of . % . chae kj, et al. successfully used the convolutional neural network to classify small (≤ cm) pulmonary nodules on ct scan images . however, there was rare research being conducted to detect viral pneumonia , , . most previous studies detected pneumonia on x-ray using deep learning while not focused on viral pneumonia. furthermore, ct is more sensitive and commonly used than x-ray for identifying covid- . in our previous work, we succeeded in recruiting deep learning in minor lesion detection and real-time assistance to doctors in gastrointestinal endoscopy [ ] [ ] [ ] [ ] [ ] . here, we enrolled this technique in identification of covid- pneumonia in ct images. results from both retrospective and prospective patients showed that the model was comparable to the level of expert radiologist, and hold great potential to reduce diagnosing time. (fig. ) . early diagnosis and early isolation of suspected patients are the most important ways to prevent the spread of epidemic . due to the sudden outbreak of covid , the radiology department is overloaded and patients have to wait for long times for chest ct scan, which largely increase the risk of cross-infection. in recent days, radiologists' daily workload is huge in hubei province, and a ct scan report has to be awaited several hours to achieve. based on the number of suspected patients and close contacts in being, radiologists in the hardest hit, hubei province, china, may not be enough to resist the rapid spread of the virus, which holds high estimated r of . ~ . [ ] [ ] [ ] [ ] . it could be inferred that before radiologists fulfilling the demands of existing patients, newly infected cases would appear, and the overall burden of radiologists is more overwhelming like a growing snowball. relieving the pressure of radiologists is essential for the control of virus spreading. in the present study, our model achieved a comparable performance but with much shorter time compared with expert radiologists. it holds great potential to relieve the pressure of radiologists in clinical practice, and contribute to the control of the epidemic. timely diagnosis and early treatment of infected patients is important for patients' prognosis . the fatality rate of covid patients in hubei province is significantly higher than that of other regions, which probably figure . abstract diagram. computed tomography (ct) is the most efficient modality for screening and clinically diagnosing covid- pneumonia. however, compared to the needs of the patients, the number of radiologists is quite small. after enrolling artificial intelligence in identifying covid- pneumonia in ct images, the efficiency of diagnosis is greatly improved. the artificial intelligence holds great potential to relieve the pressure of frontline radiologists, accelerates the diagnosis, isolation and treatment of covid patients, and therefore contribute to the control of the epidemic. www.nature.com/scientificreports/ due to delayed treatment and shortage of medical resources , . accelerating diagnosis efficiency is significant for improving patient outcomes. in the present study, our model helped expert radiologists achieve the same work with much shorter time, which greatly accelerats the efficiency of diagnosis in clinical practice, and may contribute to the improvement of patient outcome. in addition to relieving radiologists' pressure and accelerating diagnosis efficiency, artificial intelligence also holds the potential to reduce miss diagnosis of covid- patients. the lung infection foci are sometimes mild in the early stage of the covid- infection , and requires careful observation under . mm layer scanning. radiologists vary in skills, and could be affected by subjective status and outside pressure. one miss diagnosis could lead to multiple spread. the model is highly sensitive and stable, and would never be affected by work burden and work time. as a preliminary screening tool, it might help radiologists improve the sensitivity and reduce miss diagnosis. notably, the sensitivity per patient is better while the other performance per patient is worse than the performance per image. each patient has a large number of ct images (about ), most of which were negative images without lesions. the specificity is equal to the true negative divided by all the negatives. the denominator increases hundreds of times when calculating specificity by image, while the numerator (false positive) does not increase so much, therefore, the specificity per image is higher than that of per patient. the same principles could be applied to accuracy and ppv. for sensitivity, a few images having suspicious lesions may be missed in covid patients (sensitivity per image), while the probability that all images having suspicious lesions in a patient would be much lower (sensitivity per patient). on the basis of the accuracy and efficiency of the model in detecting covid- pneumonia, a cloud-based open-access artificial intelligence platform was constructed to provide assistance for detecting covid- pneumonia worldwide. ct scan images could be uploaded freely by both clinicians and researches as an assistant tool, especially in other provinces or countries unfamiliar with the radiologic characteristics of covid- . this free open-access website can read images in batches, provide high-level auxiliary diagnostic services for different hospitals in free, and expand the boundaries of regions and manpower. cases of covid- pneumonia were also been made available on the open-access website, which might be a useful resource for radiologists and researchers for fighting covid- pneumonia. in summary, the deep learning-based model achieved a comparable performance with expert radiologist using much shorter time. it holds great potential to improve the efficiency of diagnosis, relieve the pressure of frontline radiologists, accelerates the diagnosis, isolation and treatment of covid patients, and therefore contribute to the control of the epidemic. the continuing covid- epidemic threat of novel coronaviruses to global health-the latest novel coronavirus outbreak in wuhan, china first case of novel coronavirus in the united states transmission of covid- infection from an asymptomatic contact in germany clinical features of patients infected with novel coronavirus in wuhan, china clinical and thin-section ct features of patients with the covid- in wuhan (in chinese) epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in 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three-dimensional deep learning on low-dose chest computed tomography deep learning for the classification of small (≤ cm) pulmonary nodules on ct imaging: a preliminary study diagnosis and treatment recommendations for pediatric respiratory infection caused by the novel coronavirus covid- outbreak in hubei province on yh, yl and cf conceived and supervised the overall study. wl and gd contributed to writing of the manuscript. yh, yl, hs and wy contributed to critical revision of the report. cj, zl, zj, gd, wh, dz, xy, zy, cx, zm, cq, hs, ym and yx contributed to collecting and analyzing the data of patients. cj, zl and zy contributed to label ct images. hs, hx, zb, zk and wy developed the system. all authors reviewed and approved the final version of the manuscript. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to l.y., f.c. or h.y. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /.