key: cord- -kr o hoc authors: tan, chee wah; wittwer, kevin; lim, xiao fang; uehara, anna; mani, shailendra; wang, lin-fa; anderson, danielle e. title: serological evidence and experimental infection of cynomolgus macaques with pteropine orthoreovirus reveal monkeys as potential hosts for transmission to humans date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: kr o hoc pteropine orthoreoviruses (prv) are emerging bat-borne viruses with proven zoonotic transmission. we recently demonstrated human exposure to prv in singapore, which together with previous reports from malaysia and vietnam suggest that human infection of prv may occur periodically in the region. this raises the question whether bats are the only sources of human infection. in this study, we screened cynomolgus macaques caught in singapore for evidence of exposure to prv m (also known as melaka virus), which was first isolated from human patients in melaka, malaysia. we found that serum samples were prv m positive by elisa and were also positive by virus neutralization assay. to investigate whether monkeys could act as hosts for prv transmission, we experimentally infected cynomolgus macaques with prv m and housed these animals with uninfected monkeys. although no clinical signs of infection were observed in infected animals, viral rna was detected in nasal and rectal swabs and all infected macaques seroconverted. additionally, one of the uninfected animals seroconverted, implying active shedding and transmission of prv m. we provide evidence that prv exposure in the macaque population in singapore occurs at a relatively high prevalence and this study suggests that cynomolgus macaques may be an intermediate or reservoir host for prvs. pteropine orthoreoviruses (prvs) are a group of emerging bat-borne viruses, belonging to the genus orthoreovirus within the family reoviridae. prv virions are non-enveloped, fusogenic, and contain doublestranded rna genomes with ten segments (s , s , s , s , m , m , m , l , l and l ). prv n (alternatively known as nelson bay virus) was first isolated in from a grey-headed flying fox (pteropus policephalus) in nelson bay, australia [ ] . the second prv isolated was prv p (alternatively known as pulau virus) in from pooled urine samples from a fruit bat (pteropus hypomelanus) in tioman island, malaysia [ ] . in , prv m (alternatively known as melaka virus) was isolated during the investigation of an outbreak of a severe respiratory and enteric disease among different members of a family in melaka, malaysia [ ] . prv infections in humans vary from asymptomatic or mild flu-like to severe respiratory diseases. conversely, in an experimental infection, nine-week old female balb/c mice intranasally infected with of × of prv-mb (prv m, alternatively known as miyazaki-bali / [ ] ) developed clinical signs of disease (piloerection, slowness in movement, anorexia and weight loss) from days post-infection and all mice died by days post-infection [ ] . appreciation of both the diversity and the zoonotic potential of prvs increased following the isolation of prv k (alternatively known as kampar virus), yet another prv isolated from humans with acute respiratory illness in kampar, malaysia [ ] . evidence of prv infection in humans has been reported in various locations in southeast asia (figure ). in a molecular surveillance study in rembau, malaysia, prv was detected in oropharyngeal swabs from % of outpatients suffering from acute respiratory tract infections [ ] and in serological studies on select populations, prv seroprevalence rates range from . - % [ ] [ ] [ ] . although direct bat-to-human transmission was likely the route of transmission in the prv m outbreak [ ] , it is less clear whether bats played a direct role in transmitting the viruses to humans in the other reported cases of human infection. the high prevalence in selected populations from the recent surveillance studies supports the possibility of transmission by reservoir or intermediate hosts, which may have closer contact with humans than bats. to date, there have been at least different accounts identifying prvs from either human outbreak investigations or bat virome studies [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is well known that interspecies transmission of bat-borne viruses is a source of disease outbreaks in the human population and indeed evidence of bat-to- human transmission of prv has been demonstrated [ , ] . in contrast to all previous discoveries, nonhuman primates were the source of the most recent prv detection. given the cross-species transmission of prvs and frequent human-monkey interaction in singapore and surrounding regions, we initiated this study to determine the seroprevalence of prv m in wild cynomolgus macaques and to examine the susceptibility of cynomolgus macaques to experimental infection with prv m and their potential role in acting as hosts facilitating cross-species transmission. non-human primates may serve as natural reservoirs and maintain prv, or act as intermediate hosts to provide a link for virus transmission between bats and humans. during the preparation of this manuscript, the isolation and characterization of an orthoreovirus from monkey faeces collected in lopburi province, thailand was reported [ ] . this supports our original hypothesis that animals other than bats may be susceptible to prv infection and in turn play a role for crossspecies transmission. vero cells (african green monkey, atcc#ccl- ) cells were maintained in dulbecco's modified eagle medium (dmem, invitrogen) supplemented with % foetal bovine serum (fbs, invitrogen) at °c and % co . prv m (gift from kaw bing chua from temasek lifescience laboratory, singapore) was amplified in vero cells. upon - % virus-induced cytopathic effect (cpe), viruses were harvested, clarified by centrifugation at , x g for min and the virus-containing supernatant was stored in − °c . titres were determined by limiting dilution. in brief, -fold serial diluted virus was added into a well plate containing × vero cells per well. cells were observed for cpe for days post-infection and the titres were expressed as tcid /ml. clarified prv m virus-containing supernatant was overlaid on ml of % sucrose in an ultra-clear centrifuge tube (beckman coulter) and was centrifuged at , × g for min (sw rotor in opitma xpn- ultracentrifuge). after centrifugation, the supernatant was gently removed and the pellet was resuspended with dpbs (invitrogen) and stored in − °c. to establish a prv elisa based on purified wholevirus antigen, μl of purified prv m was coated on maxisorp nunc immuno plates (nunc, usa) in carbonate coating buffer ( . m nahco , . m na hco , ph . ) overnight at °c. following coating, plates were blocked with μl of opteia (bd bioscience, usa) for h at room temperature. each monkey serum sample was diluted to : in opteia and μl was added to the well and incubated for h. wells were washed times with μl of pbst, then μl of hrp-conjugated anti-monkey secondary antibody (santa-cruz, usa) was added at a : , dilution for h. wells were washed times and μl tmb substrate (life technologies) was added and incubated at room temperature for min. following incubation, μl of stop solution (kpl) was added and absorbance readings were measured at nm using a cytation microplate reader (biotek, usa). serum neutralization tests were performed by incubating tcid of prv m with -fold serial diluted macaque serum for min at °c. the virus-antibody mixture was then added to a monolayer of vero cells and incubated for h at °c. after incubation, the inoculum was removed and replaced with dmem supplemented with % fbs. virus-induced cpe was observed at days post-infection and the titre was recorded at the highest dilution where cpe was absent. prv m western blot × tcid of purified prv m was resolved by sds-page on a % gel. the proteins were transferred onto a pvdf membrane, and blocked for h in % bsa in tris-buffered saline containing . % tween- (tbs-t). membranes were incubated with monkey sera diluted to : in % bsa in tbs-t for h at room temperature. membranes were washed times in tbs-t, then hrp-conjugated anti-monkey antibody (santa-cruz, usa) at a : , dilution was added for h. the immuno-signal was developed using amersham ecl western blotting detection reagent (ge healthcare, uk) and images were captured using che-midoc mp imaging system (bio-rad, usa). cynomolgus macaques (macaca fascicularis) were used for all studies. the experiments were approved by the singhealth institutional animal care and use committee of the singhealth experimental medicine centre (semc iacuc reference: /shs/ ). cynomolgus macaques were purchased from the sin-ghealth colony and free of antibodies against prv m as determined by vnt and elisa. in a pilot study, three animals were infected via the intranasal route with × tcid of prv m and three animals were uninfected. all animals were housed in the same room in individual cages. the cages were placed next to each other, so the animals could have physical contact through the cages. due to the nature of the open cages system, virus transmission could either be through contact or aerosol. animals were observed daily for activity and clinical signs. the animals were anaesthetized with - mg/kg ketamine and blood samples were collected from the femoral vein on days - , , , , , , and . each time the animals were anaesthetized, body temperature was measured rectally, weight recorded and oral and rectal swabs collected. chest x-rays were taken on - , and days post-infection using a fuji-film digital x-ray. powered air purifying respirators were worn by all personnel while handling monkeys. rna was isolated from monkey serum samples using zr viral rna (zymo research) according to the manufacturer's instructions. rna was isolated from oral and rectal swabs using e.z.n.a. total rna kit i (omega biotek) according to the manufacturer's instructions. templates for in vitro transcription were generated by digesting a plasmid containing a t promotor and the prv m s gene with bamhi. the digested plasmid was reverse transcribed using hiscribe t high yield rna synthesis kit (neb). dna was removed by dnase treatment with dnase i (neb) for min at °c and the rna purified using rneasy mini kit (qiagen). the total amount of rna was quantified using a nanodrop (thermoscientific). the number of rna molecules produced by in vitro transcription was calculated using the following formula: rna molecules = (amount of rna in ng × . × molecules/mole)/ (length × g/ mole × ng/g). quantitative pcr (qpcr) was performed using quan-titect probe rt-pcr kit (qiagen) reagents and with the cfx real-time system (bio-rad). each µl pcr reaction contained . µl x quantitect pcr master mix, µl of each mm primer, . μl . mm probe, . μl reverse transcriptase, µl rna template and . µl h o. every pcr was performed as follows: reverse transcription at °c for min, initial pcr activation at °c for min and amplification cycles consisting of a °c denaturation for sec and a °c annealing/extension for s. sequences of primers and probes are as follows; prv m-s -f: ′ -cat tgt cac tcc gat tat gg - ′ , prv m-s -r; ′ -tgg gag ggt gca gag cat ag - ′ , prv m-s -probe ′ -/ -fam/ gta ggc atg ccg ctc gtg gaa tcc a / bhq_ / - ′ . each pcr was performed in duplicate to obtain an average ct for each sample. amplicons were quantified by plotting the ct values against standard curves made using -fold dilutions of cdna a total of serum samples from wild-caught cynomolgus macaques were collected from to in singapore. an initial screening was performed using a prv m whole virus-based elisa. by using three standard deviations of the geometric mean as a cut-off, serum samples were identified as reactive to prv m (figure (a) , table ). to further confirm the positivity of the samples and increase specificity, a serum neutralization test against prv m was performed on all elisa positive serum samples. a total of serum samples were able to neutralize prv m at a dilution of at least : (table ) . to further confirm the specificity, western blot analysis was conducted using purified virions. shown in figure (b) is a representative western blot conducted with sample # , a known positive sample with a high neutralizing antibody titre, and a negative control sample, both used at dilution of : . the western blot analysis not only confirmed findings from elisa and vnt, but also revealed that the major outer capsid protein (mu ) was the most reactive viral protein (figure (b) ). to assess the clinical disease spectrum of prv m, we infected three adult cynomolgus macaques with × tcid prv m via the intranasal route. only animals seronegative to prv were used for this study. blood and swab samples were collected daily for the first days, and then on days , , , , and from anaesthetized animals. body temperature and weight were recorded every time animals were anaesthetized. infected animals did not experience a significant temperature rise, although the body temperature of the infected group of animals was slightly higher overall than the contact group for the duration of the study (figure (a) ). no significant weight loss occurred in either group, but there was a fluctuation in the infected group between days - post-infection ( figure (b) ). chest x-rays were normal in all animals when visualized on days - , and post-infection ( figure (c) ). no additional clinical signs such as rash, loss of appetite, diarrhoea, dehydration, depression or inactivity were observed throughout the experimental period. we investigated the viral kinetics in serum, and oral and rectal swabs. viremia was not evident in any of the time points measured. viral rna was detected in throat swabs of infected animals from day post-infection (table ) and was sporadically present until day post-infection. viral rna was detected in of the infected monkeys in rectal swabs. to evaluate the hypothesis that macaques can serve as an intermediate host for spillover of prv, we monitored the occurrence of transmission of prv m from experimentally infected to naïve monkeys. similar to the infected animals, none of the naïve animals displayed any clinical signs of disease and viremia was not detected (data not shown). in contrast to the infected animals, viral rna was not detected on rectal and oral swabs, with the exception of a trace reading on day post-infection in a rectal swab of one animal (monkey # ) and in the oral swab of another animal at day post-infection (monkey # ). virus isolation was not attempted in this pilot study. to confirm infection in the absence of clinical signs in both infected and contact animals, serum neutralization tests were performed from all monkeys on days , , , and post-infection (table ) . all infected bat-borne zoonotic viruses have been responsible for several important emerging zoonotic infectious disease outbreaks in the last two decades including hendra virus, nipah virus, ebola virus, sars and mers coronaviruses [ ] [ ] [ ] [ ] [ ] [ ] . it is important to note that, almost in all of the cases, an intermediate host has been identified to play a crucial role in amplifying and transmitting the virus from bats to humans such as horses for hendra virus, pigs for nipah virus and palm civets for sars-cov [ ] [ ] [ ] [ ] [ ] . with multiple reports of prv presence in bats in many parts of asia [ , , [ ] [ ] [ ] [ ] and a higher than expected prevalence in certain human populations [ ] [ ] [ ] , we raised the hypothesis that it is possible that a yet-to-be-identified intermediate host(s) may also play a role in the spillover of prvs from bats to humans. alternatively, an additional reservoir may play a role in zoonotic transmission of prv, as is the case with camels and mers-cov [ ] [ ] [ ] . in this study, we first performed a prv seroprevalence study on cynomolgus macaques residing in singapore. we identified out of ( . %) macaques that were seropositive for prv, and of these monkeys out of ( . %) had neutralizing antibodies specifically against prv m. this is a significant finding in the context of our recently published study indicating human exposure to prvs in singapore [ ] . for the first time, we have demonstrated evidence of exposure to prvs in both human and monkey populations co-residing in the same city/region in close proximity. we then extended the study to conduct experimental infection of monkeys by prv to firstly confirm the susceptibility of cynomolgus macaques to prv infection and secondly to test whether prv can be transmitted from infected to naïve animals under experimental conditions. we experimentally infected three seronegative, wild-caught monkeys with prv m. we demonstrated that monkeys were susceptible to prv m infection, but the infection was subclinical. this confirms the serological surveillance data in relation to susceptibility. the infection displayed no signs and this observation was also not unexpected as it has been reported before that human infection can occur without clinical manifestations [ ] . this was in contrast to experimental infection studies in mice, where % of infected animals exhibit disease signs and eventually succumb to the infection [ , ] . it is possible that certain risk factors absent in healthy individuals pre-dispose the development of clinical signs in monkeys and humans upon prv exposure, and further epidemiological studies in patient cohorts would help clarify this. furthermore, the demonstration of transmission between animals under experimental conditions supports our hypothesis that mammal(s) other than bats may also play a role in the spillover of prvs into human population. wild monkeys are often sighted near human populations in southeast asia [ ] [ ] [ ] , and our data raised the possibility that monkeys in the region may act as an intermediate host for transmission of prvs to humans and other animals. while our preliminary data is highly encouraging and significant, further research is needed in better understanding of the bat-monkey interaction and assessing whether other mammals, such as rodents, in the region are also susceptible to prvs and play a role in the overall transmission landscape. the infection of monkeys with prv without observable signs of disease is also important in the context of acting as a potential reservoir host as healthy animals are usually more active with greater area of movement, hence increasing the chance of transmission to other species via contaminated faeces or other routes. furthermore, as the immunobiology of humans is more closely related to monkeys than bats, host-adaptation within monkeys could serve to enhance the virulence or transmission efficiency of prv in humans. given the overlapping habitats of bats and monkeys, interspecies transmission to monkeys and subsequent pathogen adaptation could be a relevant theme in the broader context of bat-borne virus spillover to humans, and warrants further investigation. finally, the recent report by a group in thailand on the isolation and characterization of a prv from monkey faecal samples ruled out contamination of these samples with bat faeces. during analysis of the mitochondrial s rrna gene, bat dna was not detected in the faecal samples, emphasizing monkeys, especially a long-tailed macaque, as potential reservoirs of zoonotic viruses, such as prv [ ] . we believe that the data obtained from our current study supports the notion that the thai study represents a first isolation of a prv replicating in monkeys in this region. while the current data presented in this study and from previous publications are not sufficient to determine whether non-human primates may act as a reservoir or intermediate host for prvs, it is important to bear in mind that prvs have been detected in a wide geographic area and the first prv was isolated from bats in australia where non-human primates do not exist. in summary, our data provides strong support for the hypothesis that non-bat mammals can play a role in the transmission and spillover of prvs in regions where monkeys and humans co-exist in close proximity. due to the wide species tropism of prvs at the cell culture level, we would not be surprised to find other terrestrial mammals as potential hosts of prvs in the future. structure and cytopathic effects of nelson bay virus pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus virulence, pathology, and pathogenesis of pteropine orthoreovirus (prv) in balb/c mice: development of an animal infection model for prv identification and characterization of a new 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mass spectrometry-based identification and wholegenome characterisation of the first pteropine orthoreovirus isolated from monkey faeces in thailand isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of nipah virus from malaysian island flying-foxes fruit bats as reservoirs of ebola virus isolation and characterization of a bat sars-like coronavirus that uses the ace receptor discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus close relative of human middle east respiratory syndrome coronavirus in bat nipah virus infection in bats (order chiroptera) in peninsular malaysia isolation and characterization of viruses related to the sars coronavirus from animals in southern china fatal encephalitis due to nipah virus among pig-farmers in malaysia nipah virus: a recently emergent deadly paramyxovirus viruses in bats and potential spillover to animals and humans mers-cov spillover at the camel-human interface. elife mers coronaviruses from camels in africa exhibit regiondependent genetic diversity co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia lethal murine infection model for human respiratory diseaseassociated pteropine orthoreovirus the infectious diseases consequences of monkey business human infections and detection of plasmodium knowlesi the common monkey of southeast asia: long-tailed macaque populations, ethnophoresy, and their occurrence in human environments we thank all laboratory members, past and present, for continuing support and lively discussions. we wish to thank the environmental health institute, singapore, for the provision of resources and materials. we express our gratitude to dr no potential conflict of interest was reported by the authors. key: cord- -ejpblc y authors: wen, yu-mei; klenk, hans-dieter title: h n avian influenza virus - search and re-search date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: ejpblc y nan in february , two patients with severe pneumonia and pulmonary dysfunction were admitted to shanghai fifth hospital affiliated with fudan university. the first patient, an -year-old man became ill on february th and died on february th. the second patient, also male and years old, became ill on february th and died on march th. in both cases there were fever, cough, and respiratory tract infection during the early stages of the illness. five to ten days later, the patients developed severe pneumonia and progressive respiratory distress with lethal outcome. following the guidelines of the nation-wide alert network for diagnosis of respiratory infectious diseases established in , etiological studies were immediately carried out at the laboratory of pathogen diagnosis and biosafety of shanghai public health clinical center, which is one of the sites selected for survey in the alert network. blood, throat swabs and sputum samples were collected and examined by polymerase chain reaction using primers for amplification of severe acute respiratory syndrome coronavirus, new corona virus, and h n influenza virus, which all yielded negative results. the only positive results were observed by using universal primers for amplification of influenza a viruses. to identify the viral subtype, the gene segments were subjected to further amplification, sequencing and blast analysis. the results showed that the hemagglutinin fragment showed high homology to influenza a virus h ( . %) while the neuraminidase fragment was high homologue to influenza a virus n ( . %). mp, np, pa, pb , pb showed %- % homology with h n virus. specimens were sent to the chinese center for disease control and prevention for confirmation and h n avian influenza viruses were isolated. china national health and family planning commission organized experts to do a comprehensive analysis based on the laboratory results, clinical manifestations of patients, epidemiological data, and concluded that the patients were infected with strains of h n avian influenza virus. china national health and family planning commission notified the world health organization that a new h n avian flu virus had caused lethal human infections in china (gao rb et al., unpublished). epidemiological survey revealed no h n infection occurred among close contacts of the lethal cases. both lethal cases had a history of contacts with pigs, but not with chickens. in contrast, some of the h n cases observed subsequently in jiangsu province had a history of slaughtering chickens. the municipal government of shanghai has implemented stage iii regulations for survey and control for fever and acute respiratory infections in all hospitals, and up to april th, fourteen cases with six deaths have been reported. emerging viral diseases are a continuous threat to humans worldwide, and the majority of the emerging viruses originated from animal hosts. of particular concern is the transmission of avian influenza viruses to humans with or without pigs as intermediate hosts. though such zoonotic transmissions are usually self-limited, they always pose a pandemic threat, since the viruses may adapt to humans. the avian influenza viruses that have occasionally infected and caused disease in people without showing human adaptation comprise h n , h n , and a variety of h viruses. , the h viruses isolated from humans include both highly pathogenic and low pathogenic avian viruses. whereas highly pathogenic h n and h viruses may cause fatal disease in people, all human infections caused by low pathogenic h viruses so far had been mild. it is therefore concerning to see that the new h n virus which, according to the available evidence, is derived from a low pathogenic avian virus can be lethal for man. however, it has to be emphasized that it is not clear yet whether the h n virus is transmitted directly from birds or pigs to people. so far, this virus as an agent not causing disease in animals may escape detection in an animal reservoir more easily than the highly pathogenic h n viruses. it is also not clear whether the current h n human infection is a rare event, or the severe cases observed are just the tip of an iceberg of many asymptomatic human infections as has been suggested for h n and h n viruses. the identification of h n virus as a cause of human disease is only the beginning of a long journey aimed at the elucidation of the epidemiology, the host range, and the pathogenicity of this virus, and at the development of efficient vaccines and antiviral therapeutics. furthermore, adequate surveillance is the most important. the nation wide alert laboratory network established at selected sites in china significantly contributed to the immediate identification of the emerging h n virus, demonstrating that generous financial support from the government is necessary and fruitful. however, integrated surveillance of animal and human infections still needs to be improved. the barriers between physicians and veterinarians should be removed, and gaps should be filled between government agencies responsible for animal health (ministry of agriculture) and public health (ministry of health). while a close watch on h n is going on in china, we expect that at the same time, basic research and international collaboration will be supported and encouraged. while writing this news, up to april th, the number of patients infected with h n in mainland china has increased to , with six deaths, distributed in jiangsu and zhejiang provinces, and all are in the eastern part of china. only few patients had a history of contact with chickens. among them, one is a four-year-old child with very mild respiratory infection symptoms but showed h n positive by polymerase chain reaction in his throat swab. currently in shanghai and several other provinces, strict survey for h n cases has been implemented in outpatients with fever and respiratory infections in all hospitals. at the same time survey for h n among wild birds and poultries has been carried out by the animal centers for disease control and prevention, and confirmed by the national key laboratory of avian influenza institute in harbin. h n isolates with high homology have been detected in pigeons from songjiang district of shanghai, and sanitary measures including slaughtering pigeons in that region and shutting down of markets trading live poultries in shanghai have been implemented. since this new virus is susceptible to antiviral drug oseltamivir, patients were treated with this drug as early as possible after the diagnosis. avian influenza a h n virus: a continuous threat to humans avian influenza viruses in humans infection of immunocompromised patients by avian h n influenza a virus human illness from avian influenza h n , british columbia key: cord- -ekxq l a authors: yao, yanfeng; wang, huadong; chen, jianjun; shao, zhiyong; he, bin; chen, jie; lan, jiaming; chen, quanjiao; chen, ze title: protection against homo and hetero-subtypic influenza a virus by optimized m e dna vaccine date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: ekxq l a current influenza vaccines provide hemagglutinin strain-specific protection, but rarely provide cross-protection against divergent strains. it is, therefore, particularly important to develop a universal vaccine against conserved proteins or conserved regions of the virus. in this study, we used n-terminal extracellular region of the influenza virus m protein (m e) as the target antigen and constructed two optimized m e dna vaccines (p-tpa-p m e and p-p m e) with increased antigenic epitope density and enhanced antigen secretion. both vaccines induced high m e-specific humoral and cellular immune responses in the vaccinated mice. these two vaccines also conferred protection against a lethal infection of homo-subtypic h n virus, with p-tpa-p m e being the most effective. in addition, p-tpa-p m e also showed cross-protection against different subtypes of the influenza virus (h n , h n , and h n ) at varying rates ( %, %, and %, respectively). after passive immunization, m e dna vaccine-induced antibodies in the sera provided complete protection against homologous virus challenge. an analysis of the mechanism underlying this immunization-mediated protection indicates that m e-specific igg and t-cell immune responses may play critical roles in the prevention of infection and viral clearance. taken together, our results indicate that this optimized m e dna vaccine is a promising candidate for the development of a universal, broad-spectrum influenza virus vaccine. influenza is caused by the influenza virus, an important human respiratory infectious disease, which causes , - , deaths worldwide every year [ ] . in recent years, drug-resistant strains and new types of influenza are continuously emerging, causing the influenza epidemic to be a constant and serious issue [ , ] . vaccinations are the most effective way to control influenza. however, most of the influenza vaccines currently available are subtype-specific and fail to protect patients against other types or antigenic variants of the virus. furthermore, the influenza virus undergoes frequent and unpredictable mutations that necessitate annual vaccine updates [ , ] . this not only limits the applicability of the vaccine but also wastes manpower and material resources. thus, the development of universal vaccines that provide broad protection to control seasonal influenza epidemics and the occasional outbreak of pandemics is essential. influenza virus m protein is an integral membrane protein expressed on the viral surface in low quantities, while being abundantly present on the surface of infected cells [ , ] . this expression pattern plays an important role in viral replication. the m protein consists of an n-terminal extracellular region (m e), transmembrane region, and c-terminal cytoplasmic tail region. the m e is composed of amino acids and is highly conserved among different influenza a subtypes [ ] . thus, this region may be a good candidate epitope for the preparation of a universal vaccine. however, in its natural state, viral m e has low immunogenicity and abundance. to improve immunogenicity, previous studies have used multimeric forms of m e created by fusing m e to highly immunogenic carriers or by applying them in conjunction with adjuvants. the main types of vaccines include peptide vaccines, recombinant protein vaccines, and virus-like particles [ ] [ ] [ ] [ ] [ ] . currently, several m e universal vaccines have entered clinical trials. however, some problems surfaced during clinical trials, which prevented the commercialization of these vaccines [ , ] . some of the more prominent problems include unsatisfactory immune efficacy and certain side effects. these problems may be due to the vaccine containing multiple ingredients [ ] . moreover, this type of vaccine has a relatively high cost of production and a complex manufacturing technique, which further limits its development. therefore, further research on m e vaccine is necessary. among the various types of vaccines, dna vaccines are particularly attractive because of their simplicity, ease of production, and lack of anti-vector immune responses. though the development of novel cross-protective influenza vaccines based on m e dna is a promising strategy, few of the enhanced immunogenic vaccine strategies have been successful. thus, further research is necessary. in this study, we used a combination strategy to improve the immunogenicity of the m e dna vaccine. first, we increased m e epitope density using a combined gene construct containing three m e genes in tandem ( m e). next, m e gene codon optimization (p m e) was performed to increase expression. finally, we coupled p m e with the secretory signal sequence of tissue plasminogen activator (tpa) to form tpa-p m e to improve protein secretion. these enhanced m e dna vaccines were then tested in mice to determine the protective effects against the influenza virus. to our knowledge, this is the first time an optimized m e dna vaccine with these particular modifications has been tested in vivo against both homologous and heterologous viruses. three sequential repeats of the m e gene, conjugated with linkers, were joined to the c-terminus of tpa (p-tpa-p m e) or without tpa (p-p m e) (figure (a) ). to detect expression in vitro, p-p m e and p-tpa-p m e were transfected into t cells and western blotting was performed. as shown in figure (b) and (c), the p-tpa-p m e was more highly expressed than p-p m e in the cell lysates and supernatants. in fact, m e protein expression was undetectable in the supernatants of the cells transfected with p-p m e. these data indicate that the addition of the tpa signalling sequence greatly enhanced the expression and secretion of m e protein in t cells. enhanced humoral and cellular immune effects are observed following optimized m e dna construct immunization in mice balb/c mice were vaccinated with μg of the different dna constructs. figure shows the scheme followed for these experiments. the levels of m e-specific antibodies were determined with enzyme-linked immunosorbent assay (elisa) using synthesized m e peptide as the antigen. our results show that m e-specific antibodies are present in both the p-tpa-p m e and p-p m e immunized groups weeks after the initial immunization ( figure (a) ). furthermore, while antibody titer increased following each immunization for both groups, the levels were higher in the group immunized with p-tpa-p m e than those observed for the p-p m e group ( figure (a), (d), (g)). notably, no m e-specific antibodies were detected in the p-tpa-pm e, p-pm e, or pvax immunized groups. to further evaluate the t-cell-related immune effects of p-tpa-p m e and p-p m e, specifically the th /th immune response, we tested the igg and igg a titers in the serum samples. high levels of igg and igg a antibodies were induced in both the p-tpa-p m e and p-p m e immunized groups, suggesting that they both induce a th /th related immune response ( figure (b), (e), (h)). moreover, the m e-specific igg and m e-specific igg a titers schematic of the study. balb/c mice were immunized once, twice or three times with dna vaccine, however, the control groups were immunized three times with control plasmids. sera were collected at the indicated times to analyse the humoral immune response and spleen cells were isolated for elispot assay. in parallel experiments, the remaining mice were challenged with a lethal dose of mouse-adapted viruses to detect the protective effect. were similar, indicating that p-tpa-p m e and p-p m e induced a balanced th /th immune response. to determine whether the antibodies produced following m e dna vaccination recognize native m protein, we performed a whole-cell elisa assay. our results show that the serum of mice immunized with p-tpa-p m e and p-p m e specifically binds to h n -infected mdck cells, indicating that the m e-specific antibodies induced by p-tpa-p m e and p-p m e immunization recognize the m protein on the surface of cells infected with influenza virus ( figure s ). secretion of ifn-γ by spleen cells is known to reflect the cellular immune response after immunization. as shown in figure (c), the number of spots produced after two immunizations with p-tpa-p m e and p-p m e was significantly higher than that of the control group (p < . ). the number of spots also gradually increased with each immunization for both groups ( figure (f), (i)). these results further confirm the induction of an m e-specific th /th cell response following immunization with p-tpa-p m e and p-p m e. the control group also had several nonspecific spots (spot number ≤ / cells). to determine the protective effects of p-tpa-p m e and p-p m e vaccination, mice were infected with lethal mouse-adapted h n virus. as shown in figure (a), (d), (g), p-p m e and p-tpa-p m e dna was immunized once, twice, or three times. in the p-p m e, the survival rates of mice after viral challenge were %, %, and %, respectively, while those for the p-tpa-p m e group were %, %, and %, respectively. thus, the survival rate of the p-tpa-p m e group was better than that of the p-p m e group with the same number of immunizations and was particularly significant for single immunization (p < . ). in the control groups, all mice died within days (figure (a), (d), (g)). the above results indicated that the optimized m e dna vaccine significantly increased the survival rate of mice infected with homologous influenza virus. in the lungs, mice in the control groups all had a high titer of virus, while the lung virus titer of p-tpa-p m e and p-p m e immunized mice was significantly lower than that of the control groups (p < . ). in addition, as the number of immunizations increased, lung viral titer in both dna vaccine groups gradually decreased. moreover, for each number of immunizations, the lung viral titer was lower in the p-tpa-p m e group than it was in the p-p m e group ( figure we also evaluated weight loss after viral challenge. notably, the weight loss rates of the p-tpa-p m e and p-p m e immunized groups were lower than that of the control group, but the difference was not significant (p > . ). the surviving mice in both dna vaccination groups recovered after a slight loss of body weight, whereas mice in the control groups died within days post-infection and had body weight losses of over % (figure (c), (f), (i)). to further determine the potential universal effects of p-tpa-p m e dna vaccination against heterologous viruses, mice were infected with avian origin, mouseadapted h n , h n , and h n viruses. as shown in figure (a), (d), (g), we found that the protection rates of p-tpa-p m e immunization against heterologous viral infection with h n , h n , and h n were %, %, and %, respectively. however, this immunization-mediated protection was only significant for the h n infected group compared to the viral challenged control group that was immunized with empty pvax (p < . ). the differences between the immunized h n and h n groups and their respective control groups were not significant. similarly, the lung viral titer in h n challenged mice immunized with p-tpa-p m e was significantly lower than that in the control immunized group (p < . ), but the differences in the h n and h n challenged groups immunized with p-tpa-p m e compared to their respective control groups were not significant ( figure (b) , (e), (h)). the weight loss rates of the p-tpa-p m e immunization groups were also lower than those in the respective control groups. indeed, the surviving mice in the immunized groups began to recover by day post-viral challenge ( figure (c), (f), (i)), while mice in the control immunized groups died within days of the challenge and had body weight losses of over %. the above results demonstrate that vaccination with p-tpa-p m e protects mice from lethal infection with heterologous avian influenza strains, especially the h n virus. passive immunization with anti-serum from optimized m e vaccinated mice conferred protection against lethal viral infection passive immunization was also performed to examine the protective efficacy of the immune serum of p-tpa-p m e or naked pvax (controls) vaccinated mice. all mice injected with serum containing m e-specific antibodies presented some clinical symptoms (eg ruffled hair and flocking together) after the challenge, but none of them died ( figure (a)). in contrast, mice injected with the control serum all died within days of the challenge. in addition, mice injected with m especific antibodies also showed less weight loss than the control groups ( figure (b)). the above results suggest that vaccination-mediated protection is likely provided by circulating m e-specific antibodies. to better understand the relationship between protective efficiency and the humoral and cellular immune responses, correlation coefficients were calculated. with respect to the humoral immune response, the survival percentage induced by m e immunization was highly related to the total m e-specific igg antibody levels ( figure s a ). moreover, the survival percentage was also markedly related to the m e-specific cellular immune response ( figure s b) . thus, the protective efficacy of m e immunization appears to be related to both the humoral and cellular immune responses. at present, conventional influenza vaccines must be evaluated almost every year to follow the antigenic drift and shift of the target virus [ ] . mismatch between the circulating strains of influenza virus and vaccine strain may result in excessive influenza-related morbidity and mortality. this makes it necessary to develop a universal vaccine based on conserved epitopes, such as m e. in this study, we created a novel m e dna vaccine which induced significant humoral and cellular immune responses and reduced lung virus titer and weight loss rate. this vaccine not only provided better protection against homologous viruses but also had good cross-protection effect with other heterologous viruses. to our knowledge, this is the first time an optimized m e dna vaccine with these particular modifications has been tested in vivo against both homologous and heterologous viruses. m e is known to have low immunogenicity. previous studies have indicated that multiple tandem copies of m e in a vaccine construct elicit higher m e igg titers than constructs containing a single copy [ ] [ ] [ ] . in the present study, p m e was created and appears to induce higher production of m especific antibodies than single-copy vaccines, which is consistent with previous research result. it has also been reported that secretory antigens can induce stronger immune responses than their cell-associated counterparts [ ] [ ] [ ] [ ] . tpa is a signalling peptide located in the endoplasmic reticulum that is commonly used to promote the secretion of foreign proteins. therefore, we coupled p m e with tpa to increase protein expression and secretion. indeed, our results indicate that p-tpa-p m e vaccination induced higher expression and secretion of m e protein and also induced greater humoral and cellular immune responses than p-p m e ( figure ) . moreover, when challenged with homologous virus (h n ), p-tpa-p m e achieved complete protection after only two immunizations, while p-p m e failed to achieve complete protection even after three immunizations. taken together, these results not only demonstrate that increasing the expression of secreted m e protein enhances its immune effects but also highlight the effectiveness of the particular modifications used in this study. the mechanism underlying the observed m e vaccination-mediated increase in immune protection is not completely clear at present. however, other reports have also shown that m e-specific antibodies play an important role in protective immunity [ ] [ ] [ ] . indeed, anti-m e antibody titer has been closely correlated to dose-dependent disruption of the viral life cycle and death of the infected cells via antibody dependent cell-mediated cytotoxicity (adcc) [ , ] . in the present study, high antibody titers were induced in the p-tpa-p m e and p-p m e immunization groups, both of which demonstrated increased protection against challenge with homologous virus (h n ). in addition, mice with m e antibody-mediated passive immunity were also shown to be completely protected against h n challenge, further indicating that m e-specific antibodies play an important role in protective immunity. we also observed m e-specific ifn-γ-secreting lymphocytes in the spleens of vaccinated animals, and these levels were found to be significantly correlated with protection. notably, the p-tpa-p m e vaccine induced a higher t-cell response than p-p m e, which is likely related to the ability of the secreted protein to affect other cell types. in cross-protection experiments on p-tpa-p m e dna immunized mice, vaccination also appears to be effective against the heterologous viruses h n , h n , and h n . the difference in cross-protection rates ( %, %, and %, respectively) is likely due to the differences in the m e sequence between the heterologous viruses and the h n virus ( table ). the m e sequences of h n , h n , and h n differ from that of h n by four, six, and seven residues, respectively. these differences, which affect antibody binding to the heterologous m e protein, result in failure of the vaccine to effectively clear the virus. indeed, serum igg antibodies from mice vaccinated with p-tpa-p m e appear to recognize m e of these three viruses expressed on infected mdck cells, but bind to them more weakly compared to h n -infected mdck cells (data not shown). in conclusion, we have described a potential universal influenza vaccine that provides protection against homo-and hetero-subtypic influenza in mice. immunization with p-tpa-p m e, without any adjuvant, induced high level, m e-specific antibody production, humoral/cellular immune responses, and protected balb/c mice from lethal infections of homo-and hetero-subtypic viruses. the combined vaccine strategy offers promising prospects for further vaccine development. all of our animal studies were carried out in strict compliance with the guidelines for the care and use of laboratory animals of the people's republic of china. the protocols used in this study were approved by the committee on the ethics of animal experiments of the wuhan institute of virology, chinese academy of sciences. all procedures were performed under pentobarbital sodium anesthesia, and all efforts were made to minimize animal suffering. the influenza viruses used were a mouse-adapted a/ pr/ / (h n ), influenza virus a/chicken/jiangsu/ / (h n ), influenza virus a/environment/ dongting lake/hunan/ - / (h n ), and influenza virus a/duck/hb/ / (h n ). the h n , h n , and h n influenza viruses were passaged and adapted for mouse studies as described in our previous studies [ ] [ ] [ ] . the prepared viruses were then frozen at − °c until use. notably, the h n , h n , and h n viruses were all used in a biosafety level containment facility at the wuhan institute of virology, chinese academy of sciences. specific-pathogen-free female balb/c mice, - weeks old, were obtained from the center for disease control and prevention in hubei province, china. the animals were bred in the animal resource center at the wuhan institute of virology, chinese academy of sciences. all mice were maintained in specificpathogen-free conditions prior to infection. p m e was synthesized by genscript co., ltd. each m e sequence was linked with a gsg linker (gly-ser-gly-gly-gly-gly). the eukaryotic expression vector pvax (invitrogen, carlsbad, ca, usa) was used to construct the dna vaccine. for p-p m e, p m e was digested with ecori and xhoi and then cloned into pvax to create p-p m e. to construct p-tpa-p m e, the tpa signal sequence was amplified by pcr using the following primers: ′ -cccaagcttatg-gatgcaatgaagag agggctctgctgtgtgct gctgctg- ′ and ′ -ccggaattcgctgggcgaa acgaagactgctccacacagcagcagcacaca gcagag- ′ . the pcr product was then digested with hindiii and ecori, followed by ligation into p-p m e to create p-tpa-p m e. the single-copy m e plasmids (p-tpa-pm e and p-pm e) were constructed using similar methods. the plasmids were propagated in escherichia coli dh α bacteria and purified using nucleobond® xtra (macherey-nagel gmbh and co. kg). the m e peptide slltevetpirnewgcrcngssd was synthesized by shanghai sangon biological engineering technology and services co., ltd. (> % purity). t cells were plated onto six-well plates. approximately h after plating, the cells were transfected with the vector plasmids using lipofectamine (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the cells and supernatants were collected separately h after transfection. the proteins in the cell lysates and supernatants were separated using non-reducing sds-page ( % gel) and blotted onto pvdf membranes. the membranes were then immunoblotted with influenza a m monoclonal antibody c (abcam, uk). in vivo electroporation was performed according to the method described by aihara and miyazaki [ ] . to evaluate the capability of dna vaccination to protect against homologous influenza infection, a total of nine groups of -week-old balb/c mice (n = mice per group) were respectively immunized with μg of p-p m e (three groups immunized once, twice, and three times, respectively), p-tpa-p m e (three groups immunized once, twice, and three times, respectively), p-tpa-pm e ( group immunized three times), p-pm e ( group immunized three times), and empty pvax ( group immunized three times). this also allowed us to examine the effects of immunization time on the protective effects of p-p m e and p-tpa-p m e. the p-tpa-pm e, p-pm e, and pvax immunizations were used as a reference. notably, the different plasmids (in phosphate buffered saline (pbs)) were injected into the right quadricep muscle of each mouse. after injection, a pair of electrode needles were inserted into the muscle mm apart to cover the dna injection sites, and electric pulses were delivered using an electric pulse generator (ecm ; btx, san diego, ca). two weeks after the last immunization, the mice were anesthetized with pentobarbital sodium (c h n nao ) at a dose of mg kg ml − for each animal and challenged with μl of the viral suspension containing times the ld of h n , the homologous virus, by intranasal drip. after viral challenge ( days), three mice were taken from each group for blood collection, and their lungs were removed to prepare lung homogenates for measuring the virus titration. the remaining mice were observed for days to record the survival rates and weight loss. as p-tpa-p m e immunization induced high titer and homogenous m e-specific antibody production, we focused on this immunization group for the heterologous virus challenge experiment. a total of mice were divided into six groups (n = mice per group). three groups were immunized three times with μg of p-tpa-p m e at -week intervals. the other three groups were immunized with empty pvax as a control. two weeks after the third immunization, the p-tpa-p m e and pvax immunization groups were challenged with nasal drips of times the ld of h n , h n , and h n influenza viruses, respectively. the mice were observed for days after viral challenge, during which weight loss and survival rate were recorded. virus titers in the lungs collected at day post-challenge were tested via tcid assay. blood was collected days after each immunization, and serum was isolated for antibody detection. serum samples were stored at − °c until use. mice from each group (n = ) were sacrificed days after viral challenge with h n , h n , h n , or h n and their lungs were isolated. lung homogenates were centrifuged at rpm for min. after centrifugation, the supernatants were collected for viral titration via tcid assay. m e-specific antibodies were tested using plates coated with μg/ml of m e synthetic peptides in well plates by elisa as described previously [ ] . to identify the m e-specific antibody isotypes, the serum samples were diluted in % bovine serum albumin-pbs and incubated for h at room temperature (rt). after washing, μl of biotinylated anti-mouse igg, igg , and igg a (southern biotechnology associates, inc., usa) conjugated to streptavidin-horseradish peroxidase (hrp) were added and incubated for h at rt. the samples were then incubated with , ′ , , ′ -tetramethylbenzidine (tmb) substrate for min, and then the reaction was stopped with m phosphoric acid. the end-point elisa titers are expressed as the highest dilution that yielded an optical density greater than the mean plus two times the standard deviation of a similarly diluted negative control sample. whole-cell elisa for m e-specific antibodies was performed in well plates using a previously published procedure with some modifications [ ] . in brief, madin-darby canine kidney (mdck) cells were grown in well culture plates in minimum essential medium (mem) complete medium containing % fetal bovine serum (fbs) at °c until the cells were almost confluent. the cells were then incubated with eid of h n viruses ( μl) in pbs or with medium alone (for uninfected controls). after incubating for h at °c, μl of complete medium was added to each well and plates were incubated at °c for h. the plates were then washed with pbs and fixed with % formalin at rt for min. then, the cells were washed three times with pbs and blocked with μl/well pbs/ % bsa for h at °c. serial dilutions of tested sera were added to the wells and incubated for - h at °c. after incubation, the cells were washed and incubated with hrp-conjugated goat anti-mouse igg (boster, wuhan, china) for h at °c, followed by tmb horseradish peroxidase color development solution for elisa (beyotine biotechnology, shanghai, china) for min at °c. the reaction was stopped with the addition of μl of mol/l h so and the od was measured on a microplate spectrophotometer. data reflect the mean change in od (infected-uninfected) of triplicate wells per sample. mdck cells were seeded (n = ) at × cells per well in a -well plate. after being cultured for h, the cells were infected with μl of a / -dilution series of lung homogenate supernatant and incubated at °c for h. the supernatants were then replaced with μl of serum-free dulbecco's modified eagle medium (dmem) containing a cocktail of antibiotics. the cytopathic effects in the infected mdck cells were observed daily. after incubating for days, virus titer was assayed in the supernatant by measuring hemagglutinating activity and processing these data using the calculations of reed and muench. splenocytes were isolated from mice for elispot assays weeks after the final immunization. according to manufacturer's instructions (u-cytech, netherlands), the immunospot plates (millipore, bedford, ma) were coated with rat anti-mouse interferon (ifn)-γ monoclonal antibody and incubated at °c overnight. the plates were washed three times with sterile pbs and then blocked with µl of blocking solution r. next, × splenocytes were added to the wells in triplicate. after stimulating the splenocytes with µg/ml of m e peptide at °c for h, biotinylated anti-mouse ifn-γ antibody was added to each well and incubated at °c for h. subsequently, diluted streptavidin-hrp conjugate solution was added and incubated at rt for h. finally, the plates were treated with µl of aec substrate solution and incubated at rt for min in the dark. the reaction was stopped by washing with demineralized water. spots were quantified with an elispot reader (bioreader ; bio-sys, germany). to prepare a high titer of m e-immune serum, balb/c mice (n = ) were immunized with μg p-tpa-p m e vaccine construct as described above. a second group of mice (n = ) was immunized with empty pvax vector. mice were immunized three times at -week intervals. two weeks after the final immunization, mice were killed and the other mice from each group were used as controls. blood samples were collected, and serum was prepared by clotting the blood at °c for min followed by centrifugation. a total of naive mice were immunized by tail vein injection with μl of pooled serum from the mice immunized with p-tpa-p m e. another group of naive mice received the same amount of pooled serum from the mice immunized with only pvax . after h, the mice were anesthetized and challenged with h n influenza virus as described above. mortality and morbidity (weight loss) were monitored for days after challenge [ ] . statistical analyses were conducted by one-way analysis of variance with bonferroni post-test using spss for windows software (ver. , spss inc., chicago, il, usa). an unpaired two-tailed student's t-test was used to compare the means between different groups. a p-value less than . was considered statistically significant. the differences in survival rate between the groups were analysed using the kaplan-meier method. results are expressed as the means ± standard deviations (sd). recombinant m e outer membrane vesicle vaccines protect against lethal influenza a challenge in balb/ c mice influenza a (h n )pdm virus exhibiting enhanced cross-resistance to oseltamivir and peramivir due to a dual h y/g r substitution a highly pathogenic avian h n influenza virus isolated from a human is lethal in some ferrets infected via respiratory droplets an update on swine-origin influenza virus a/h n : a review world health organization. influenza vaccine viruses and reagents identification of a second protein (m ) encoded by rna segment of influenza virus influenza virus m protein has ion channel activity a distinct lineage of influenza a virus from bats a universal influenza a vaccine based on the extracellular domain of the m protein virus-like particles containing multiple m extracellular domains confer improved cross-protection against various subtypes of influenza virus an influenza a vaccine based on tetrameric ectodomain of matrix protein cta -m e-dd: a novel mucosal adjuvant targeted influenza vaccine safety and immunogenicity of a recombinant m e-flagellin influenza vaccine (stf . xm e) in healthy adults synthetic multiepitope peptides identified in silico induce protective immunity against multiple influenza serotypes m -based influenza vaccines: recent advances and clinical potential tissue plasminogen activator (tpa) signal sequence enhances immunogenicity of mva-based vaccine against tuberculosis chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice immunization with plasmid dna encoding a truncated, secreted form of the bovine viral diarrhea virus e protein elicits strong humoral and cellular immune responses dna vaccines against dengue virus based on the ns gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice influenza a virus m protein: monoclonal antibody restriction of virus growth and detection of m in virions universal vaccine based on ectodomain of matrix protein of influenza a: fc receptors and alveolar macrophages mediate protection therapeutic potential of a fully human monoclonal antibody against influenza a virus m protein human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza a viruses protection against avian influenza h n virus challenge by immunization with hemagglutinin-or neuraminidase-expressing dna in balb/c mice characterization of an h n influenza virus isolated from dongting lake wetland characterization of lowpathogenic h n avian influenza viruses in central china gene transfer into muscle by electroporation in vivo cross-protection against influenza virus infection by intranasal administration of m -based vaccine with chitosan as an adjuvant potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza m e to the tlr ligand flagellin cross-protection against influenza virus infection by intranasal administration of m -based vaccine with chitosan as an adjuvant we thank xuefang an, hao tang, yuzhou xiao, youling zhu and fan zhang (core facility and technical support at wuhan institute of virology, chinese academy of sciences, wuhan , china) for assistance with the mice experiments. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. no potential conflict of interest was reported by the authors. key: cord- -toompiz authors: gonzález-sanz, rubén; taravillo, irene; reina, jordi; navascués, ana; moreno-docón, antonio; aranzamendi, maitane; romero, maría pilar; del cuerpo, margarita; pérez-gonzález, carmen; pérez-castro, sonia; otero, almudena; cabrerizo, maría title: enterovirus d -associated respiratory and neurological illness in spain, – date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: toompiz during , enterovirus d (ev-d ) outbreaks were described globally, causing severe respiratory diseases in children and, in some cases, subsequent paralysis. in this study, the type characterization of enterovirus (ev) detected in respiratory illnesses and the epidemiology and clinical association of ev-d infections in spain over a five-year period were described. a total of ev-positive samples from hospitalized patients with respiratory infections were included. ev-d was the most frequently detected type ( . %, / typed ev). other ev from species a ( . %), b ( . %) and c ( . %) were also identified. ev-d infections were more associated with bronchitis while ev-a/b types were more frequent in upper respiratory illness (p < . ). ev-d was also detected in patients with neurological symptoms (nine meningitis/meningoencephalitis and eight acute flaccid paralysis cases). phylogenetic analysis of ′-vp region showed most spanish ev-d sequences from to belonged to subclades b /b , as other american and european strains circulating during the same period. however, those detected in and clustered to the emerged subclade d . in summary, different ev can cause respiratory infections but ev-d was the most prevalent, with several strains circulating in spain at least since . association between ev-d infection and neurological disease was also described. enteroviruses (ev) are responsible for a wide range of diseases in humans including febrile illness, myocarditis and neurologic illness, such as aseptic meningitis, encephalitis or paralysis [ ] . ev can be also found in the respiratory tract and some types, especially from species c and d cause upper and lower respiratory symptoms [ ] . since the first isolation in [ ] , enterovirus d (ev-d ) has been associated almost exclusively with respiratory diseases. it shares similar biological characteristics with rhinoviruses, which are currently classified together with ev in the genus enterovirus within the picornaviridae family [ ] . ev-d has acid sensitivity and grows at lower optimal temperatures in some cell lines than other ev [ ] . before , only a few outbreaks or sporadic cases of ev-d infections had been reported in different parts of the world [ ] [ ] [ ] . however, in the late summer of that year, a large-scale outbreak of severe respiratory illness associated with ev-d was noted in the united states (us) and canada [ , ] . moreover, there was a significant increase of cases with neurological complications after the respiratory episode, mainly acute flaccid myelitis [ ] . since , surveillance of ev-d infections is recommended and several studies have been published in europe and other parts of the world regarding ev-d circulation, some reflecting an increase in detection in recent years [ ] [ ] [ ] [ ] [ ] . additionally, cases of paralysis or severe myelitis in children, in which ev-d was the only etiologic agent detected, were reported in different countries, including spain [ ] [ ] [ ] [ ] [ ] . regarding respiratory diseases, in our country most of detected ev were not usually characterized, and the first reports of ev-d detection were published in [ ] [ ] [ ] . therefore, the objectives of the present study were first, to identify the genotype of the ev detected in clinical samples collected from hospitalized patients with different respiratory diseases over a -year period, and second, to describe the molecular epidemiology and clinical characteristics of ev-d infections in spain. enterovirus genotyping. rna was extracted from clinical samples using the qiaamp viral rna mini kit (qiagen, germany). genotyping was performed using a previously described rt-nested pcrs in '-vp region for ev species a, b and c [ ] and subsequent sequencing. in addition, a specific rt-nested pcr for ev-d detection in the same region `-vp was designed and evaluated. primers used in this case were: outer sense, '-cataccttagrttt-gatgctg and outer antisense, '-ccattgaat ycctggrccttc; inner sense, '-gtaccmac tggtgctcttac and inner antisense, '-ctg attgccartccacatag. pcr reaction mix, cycling programme, and sequencing conditions were the same as previously described for species a, b and c rt-pcr [ ] . an ev-d isolate cultured in rd cells was used to validate the designed rt-pcr. the virus titre was determined by the median cell culture infective dose (ccid /ml) and calculated by the kärber method [ ] . to determine the lower limit of detection (lod) of the assay, serial -fold dilutions of the virus, containing ccid /ml, were analysed. a set of ev strains from species a to c and four ev-d isolates (obtained in - by inoculating rd cells with respiratory samples from hospitalized patients with respiratory illnesses) was analysed for testing the rt-pcr efficiency (table ) . phylogenetic analysis. a total of ev-d sequences obtained in this study and detected between and were included in a phylogenetic analysis with different ev-d sequences available in genbank in the same '-vp region from american, asian and european countries (n = ). the prototype strain fer-monus/ was included as the outgroup. the phylogenetic tree was constructed with mega . software using a neighbour-joining distance method under the maximum composite likelihood substitution model with , bootstrap resamplings. the sequences obtained in this study have been deposited in genbank under accession numbers mn -mn . statistical analysis. clinical characteristics and laboratory variables were compared using the student's t test and fisher exact test. a -sided value of p < . was considered statistically significant. lod and validation of the designed ev-d rt-nested pcr. using serial dilutions of the assay ev-d -positive control, the minimal amount of virus detected by the specific pcr assay was ccid /ml. a set of ev strains from species a to c and four ev-d isolates was analysed for testing the rt-pcr efficiency. only ev-d -positive samples were amplified ( table ) . the cross-reactivity with other viruses was also studied. rhinoviruses (rv) or human parechoviruses (hpev) were not detected. ev genotype identification. from the total of ev-positive respiratory samples included in the study, ( . %) were successfully amplified with ev-a, b, c or d rt-nested pcrs. rv was identified in samples and in the remaining untyped ev, the presence of ev-d was excluded by using a rtnested pcr in ´-non-coding region of the ev genome [ ] and subsequent sequencing. ev-d was the most frequent serotype detected (n = , . %), followed by ev-a (n = , . %), coxackievirus (cv)-a (n = , . %), echovirus (e)- (n = , . %) and e- (n = , . %). other ev types were also identified in minor proportion ( figure ). overall, the distribution of ev species was . % for ev-d, . % for ev-b, . % for ev-a and . % for ev-c ( figure ). epidemiological and clinical characteristics of ev-d infections: comparison with ev-a or b infections. overall, ev-a and b types were detected throughout the year whereas ev-d was identified during the cold months (from october to march) ranged from to % of the total evs in those periods. in and , in addition, a high incidence of ev-d infections was detected between april and september (warm months) with and cases which represented . and . % respectively of the total ev typed in those months ( figure ). in / ( . %) ev-d infections, the virus was the only known respiratory infectious agent detected in the samples. rv, human adenovirus (hadv) and bocavirus (hbov) were co-detected in nine, three and two of the ev-d patients, respectively. in addition, one patient was co-infected together with hadv and hbov, and another with rv, hadv and human parainfluenza virus (hpiv). however, coinfection with one (n = ), two (n = ), three (n = ) or even four (n = ) respiratory viruses was identified in out of ev-a or b-positive patients ( . % vs. . % in ev-d infections, p = . ), rv being the most frequent (n = ). also identified were respiratory syncytial virus, human metapneumovirus, hadv, hbov, hpiv, human coronavirus oc and influenza a virus. clinical data were available in characterized ev infections: were ev-d -positive patients, children (mean age, . ± sd . years, interquartile range . ) and adults (mean age . ± sd . years, interquartile range . ); were patients with ev-a/b infections, children (mean age . ± sd . years, interquartile range . ) and adults (mean age . ± sd . years, interquartile range . ). bronchitis were more frequently diagnosed in ev-d -infected children compared to ev-a/ev-bpositive patients (p < . ), whereas ev-a or ev-b genotypes were mainly detected in upper respiratory tract infections (p < . ) ( table ). there were no significant differences in the proportion of patients who were admitted to the icu when comparing ev-d with ev-a or b-positive patients ( / , . % vs / . %, p = . ). all patients but two admitted to icu were children. regarding previous clinical history, out of patients had an underlying disease (leukemia, multiple myeloma, lymphoma, cystic fibrosis, liver transplantation, congenital heart disease, and asthma or recurrent wheezing). of them, / patients were infected by ev-d ( . %) and / by ev-a or b ( . %) (p = . ). only of these patients required admission to the icu. during the routine ev genotyping in non-respiratory diseases (neurological, mucocutaneous and systemic diseases), ev-d was identified in patients with febrile syndrome and with specific neurological symptoms. all patients except two were children between one month and years, and ev-d was the only pathogen detected. regarding patients with neurological symptoms, eight children and one adult were diagnosed with meningitis or meningoencephalitis and the remaining eight children were finally described as acute flaccid paralysis (afp) cases. in all but two patients, ev-d was detected in respiratory or/and stool samples. however, ev-d could be detected in csf samples from those two patients (one with aseptic meningitis and one with meningoencephalitis). afp cases were notified through the national afp surveillance system, all required admission to the icu and all but two presented respiratory symptoms previously. one child with severe meningoencephalitis was also in icu. ev-d afp cases were identified throughout the study period (one case in , four cases in , one case in and two cases in ). ev-d phylogenetic analysis. ev-d phylogenetic tree in vp region ( figure ) showed that almost all spanish sequences from to belonged to the clade b, according to the classification previously described [ ] , and were genetically related to the majority of the strains detected in the us and canada outbreaks during as well as sequences isolated in other european, american and asian countries during - . spanish sequences from and clustered in subclade b whereas those detected in belonged to the subclade b . however, most of the ev-d sequences from and ( / , . %) clustered in the recently described subclade d [ ] [ ] [ ] . finally, strains collected in our laboratory before ( - ) fell into clade a (figure ). regarding to clinical manifestations, most neurological cases were b strains although b or d were also detected in two patients with these symptoms. since , an increasing number of ev-d outbreaks associated with severe respiratory diseases, mainly in children, have been occurring in different countries, first in the us and canada, followed by europe and asia. concurrently, clusters of cases with neurological complications such as myelitis or acute flaccid paralysis were reported in the same geographical areas [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in spain, ev surveillance in respiratory infections was not used to be routinely carried out. during , however, the enterovirus laboratory of cnm started receiving ev-positive respiratory samples for genotyping. our laboratory had already developed tools for genotyping ev species a, b and c but not for species d, in which serotype d belongs. therefore, a specific rt-pcr for ev-d was designed. analysis of ev-positive specimens collected from april to december from spanish hospitalized patients with respiratory illnesses confirmed the presence of ev-d in almost half of the total characterized ev. other ev types from species a, b and c were also detected at different rates. ev-a and b are occasionally detected in respiratory samples and their involvement in the respiratory pathology is not as clear as in the case of ev-d and ev-c , ev-c or ev-c [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] . this is in agreement with the fact that in our study, in most cases, ev-d was the single infectious agent. in ev-a and b infections, however, the number of co-infections with other known respiratory virus was significantly higher which could mean that in those cases, ev could be an incidental finding. furthermore, most of the identified ev-a and b types were those predominantly circulating in spain during the same period of time (data not published). however, as there are few reports about which ev-a or b types are presented in respiratory symptoms, further studies are necessary to confirm or not their involvement in those pathologies. although hospitals did not refer any bacterial infection in these patients, a possible limitation of our study could be the lack of common criteria from all of them testing the samples searching for different viruses or bacteria. therefore, some coinfection positive cases could be missing. regarding seasonal distribution, ev-d cases were identified sporadically in cold months during the four years of study. in and , however, it circulated almost throughout the year, with a higher incidence in spring and summer. this seasonal distribution, in which ev-d has periods of low circulation followed by others with high incidence, has been reported in other countries [ , , [ ] [ ] [ ] , and suggest epidemics of ev-d in and , which was confirmed by phylogenetic analyses of partial vp sequences showing increased genetic diversity. the phylogenetic tree showed that most of the spanish ev-d sequences detected during clustered to the recently described lineage b [ ] , while strains from and belonged to subclade b . similar results have been obtained with other studies [ , ] , suggesting that all the outbreaks would be [ ] [ ] [ ] . these findings indicate that although ev-d epidemics can be due to the emergence of new lineages, multiple clades have been circulating simultaneously worldwide from to the present day. as previously reported [ ] , most of the ev-d infections were detected in young children. however, there were also adult patients infected, half of them older than . interestingly, % of sequences from - which belonged to subclade d were identified in adult patients with respiratory symptoms, suggesting a possible different epidemiology of this new strain. these findings had already been observed in previous studies [ ] . clinical data were available in most of the cases included in this study. in other studies [ ] ev-d was detected in patients diagnosed with different respiratory diseases, ranging from a common cold with fever to basal pneumonia, but in our series, ev-d infection seemed to be more associated with bronchitis than with other respiratory presentations. by contrast, ev from species a or b were more frequently detected in upper respiratory tract infections (p < . ). also, there was no significant difference in the proportion of patients from each group admitted to the icu in contrast to those reported by other authors who observed a higher incidence of severe disease among patients infected by ev-d [ , ] . in addition to respiratory infections, ev-d has been associated with neurological complications in recent years [ , [ ] [ ] [ ] [ ] [ ] . during our study-period, this virus was identified in children and one adult with neurological symptoms. of them, eight were diagnosed as afp cases. the association between ev-d and afp or afm is now accepted, although causality has not yet been proven. regardless, the recent publication of a mouse model, in which mice inoculated with ev-d developed symptoms of myelitis, as well as several reports evaluating the evidence for a causal relationship supported this association [ ] [ ] [ ] [ ] . indeed, the world health organization and the pan american health organization have recently recommended testing ev-d on respiratory samples in cases of afp/afm, both for case management and for surveillance purposes [ ] . furthermore, in the present study, ev-d was detected in csf specimens from two patients with signs of meningoencephalitis, reinforcing the neurotropism of this ev. finally, all neurological ev-d strains belonged to subclades b /b or d . these emerging genotypes are considered to have more neurotropic potential than a or c strains [ , , ] . in conclusion, ev-d infections have been confirmed as a cause of mild lower respiratory illnesses in spain, mainly in children, but also in adults. multiple clades of ev-d have circulated since in our country, although one (subclade b ) becomes prevalent in and other (d ) emerges in . in addition, ev-d has also been associated with severe neurological cases, indicating the need for better surveillance of this ev type in respiratory specimens at national levels. further studies are required to understand the mechanisms of this recent emergence and the severe disease presentations involved. rhinoviruses and respiratory enteroviruses: not as simple as abc a probable new human picornavirus associated with respiratory diseases ictv virus taxonomy profile: picornaviridae enterovirus is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses clusters of acute respiratory illness associated with human enterovirus -asia, europe, and united states worldwide emergence of multiple clades of enterovirus emergence and epidemic occurrence of enterovirus respiratory infections in the netherlands in outbreak of enterovirus d in north america detection of enterovirus d in canadian laboratories a novel outbreak enterovirus d strain associated with acute flaccid myelitis cases in the usa ( - ): a retrospective cohort study european surveillance for enterovirus d during the emerging north-american outbreak in emergence of enterovirus d in denmark the emergence of enterovirus d in a dutch university medical center and the necessity for routinely screening for respiratory viruses molecular epidemiology of enterovirus d from to in philippines global emergence of enterovirus d : a systematic review acute flaccid paralysis following enterovirus d associated pneumonia two cases of acute severe flaccid myelitis associated with enterovirus d infection in children cluster of atypical adult guillain-barre syndrome temporally associated with neurological illness due to ev-d in children enterovirus d infection in a cluster of children with acute flaccid myelitis first cases of severe flaccid paralysis associated with enterovirus d infection in spain molecular epidemiology of severe respiratory disease by human rhinoviruses and enteroviruses at a tertiary paediatric hospital in first enterovirus d (ev-d ) cases detected in hospitalised patients in a tertiary care university hospital in spain respiratory infections by enterovirus d in outpatients and inpatients spanish children molecular epidemiological study of hev-b enteroviruses involved in the increase in meningitis cases occurred in spain during world health organization. polio laboratory manual viral diagnosis of neurological infection by rt multiplex pcr: a search for entero-and herpesviruses in a prospective study molecular evolution and the global reemergence of enterovirus d by genome-wide analysis emergence of divergent enterovirus (ev) d sub-clade d strains, northern italy emergence of enterovirus d clade d , france enterovirus genotype ev- in humans newly identified enterovirus c genotypes, identified in the netherlands through routine sequencing of all enteroviruses detected in clinical materials from to upsurge of enterovirus d , the netherlands enterovirus-d (ev-d ) in pediatric patients with respiratory infection: the circulation of a new b clade in italy outbreak of enterovirus d of the new b lineage in a mouse model of paralytic myelitis caused by enterovirus d contemporary circulating enterovirus d strains have acquired the capacity for viral entry and replication in human neuronal cells enterovirus d and acute flaccid myelitis-evaluating the evidence for causality the association between acute flaccid myelitis (afm) and enterovirus d (ev-d ) -what is the evidence for causation? pan american health organization / world health organization. epidemiological alert: acute flaccid myelitis associated with enterovirus d thanks to the enterovirus laboratory staff and all clinicians and laboratories participating in the spanish enterovirus surveillance for providing ev positive samples and clinical data. no potential conflict of interest was reported by the authors. dr. r. gonzález-sanz is supported by a grant from the instituto de salud carlos iii [grant number pi ciii- ]. this study was partially supported by the same grant.orcid maría cabrerizo http://orcid.org/ - - - key: cord- -ikt r o authors: loeffelholz, michael j.; tang, yi-wei title: laboratory diagnosis of emerging human coronavirus infections – the state of the art date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: ikt r o the three unprecedented outbreaks of emerging human coronavirus (hcov) infections at the beginning of the twenty-first century have highlighted the necessity for readily available, accurate and fast diagnostic testing methods. the laboratory diagnostic methods for human coronavirus infections have evolved substantially, with the development of novel assays as well as the availability of updated tests for emerging ones. newer laboratory methods are fast, highly sensitive and specific, and are gradually replacing the conventional gold standards. this presentation reviews the current laboratory methods available for testing coronaviruses by focusing on the coronavirus disease (covid- ) outbreak going on in wuhan. viral pneumonias typically do not result in the production of purulent sputum. thus, a nasopharyngeal swab is usually the collection method used to obtain a specimen for testing. nasopharyngeal specimens may miss some infections; a deeper specimen may need to be obtained by bronchoscopy. alternatively, repeated testing can be used because over time, the likelihood of the sars-cov- being present in the nasopharynx increases. several integrated, random-access, point-of-care molecular devices are currently under development for fast and accurate diagnosis of sars-cov- infections. these assays are simple, fast and safe and can be used in the local hospitals and clinics bearing the burden of identifying and treating patients. cov, mers-cov and sars-cov- ). in temperate regions endemic hcovs usually display a winter seasonality, although hcov- e has been detected sporadically throughout the year [ ] . endemic hcovs are globally distributed and are maintained in the human population. the sars-cov pandemic came to an end in (https://www.who.int/csr/resources/ publications/cds_csr_aro_ _ .pdf?ua= . accessed february ), less than a year after the first reported case. in contrast, human cases caused by mers-cov continue to be reported at the time of writing, more than seven years after the first reported case. most laboratory-confirmed mers cases have occurred in the eastern mediterranean region, and the majority of those in saudi arabia. unlike the endemic hcovs, sars-cov and mers-cov are maintained in zoonotic reservoirs. the sars and mers outbreaks were driven in part by super-spreading events in which individuals directly infected a disproportionally large number of contacts [ ] . the sars-cov- -caused coronavirus disease (covid- ) epidemic originated in a wuhan, china market that sold exotic animals for consumption. based on genetic relatedness to other betacoronaviruses, sars-cov- likely has a zoonotic reservoir. however, the precise source of sars-cov- that initially infected humans remains to be confirmed. the sars-cov- appears to be substantially more contagious than sars-cov ( table ). the distribution of sars-cov- in different mammalian species is unknown. an interesting question is the susceptibility of farm animals and pets, and their role in the epidemiologic cycle as their angiotensin-converting enzyme (ace ) receptor shares similarity with human ace [ ] . infections caused by endemic hcovs have an incubation period of - days and are associated with mild upper respiratory symptoms (the "common cold"). endemic hcovs are among the most frequent cause of upper respiratory tract infections. lower respiratory tract infections (bronchiolitis, pneumonia) are rare. following an incubation period of usually - days, patients infected with sars-cov often present with symptoms of fever, headache, and myalgias. respiratory symptoms including cough and dyspnoea usually develop from several days to a week after illness onset. atypical pneumonia and respiratory deterioration occur in - % of cases. the incubation period and clinical course of mers are similar to that of sars, the exception being a higher proportion of cases progressing to respiratory deterioration and distress. the incubation period and clinical course of sars-cov- infection are probably similar to that of sars. li et al. first reported a mean incubation period of . days [ ] . fever and cough are frequently reported early in the course of illness [ , ] . infections are also characterized by dyspnoea, respiratory distress and positive chest x-ray [ ] . lower respiratory symptoms often develop about week from the onset of initial symptoms [ ] . globally over cases and over deaths due to sars-cov were reported, with a case-fatality ratio of approximately % (https://www.who.int/csr/sars/en/ whoconsensus.pdf. accessed february ). between september and november , there were laboratory-confirmed cases of mers, with deaths (https://www.who.int/emergencies/merscov/en/. accessed february ). the mers casefatality rate of . % is about triple that of sars, and persons in the - year age group are at highest risk for primary cases. in the short time from its emergence in december to march , the sars-cov- has been reported in countries. at the time of writing, the situation was evolving rapidly, with over , confirmed cases reported globally (over , in china) and deaths in china ( . % case-fatality rate) and over deaths outside of china. of countries and continents outside of china, south korea, iran, and europe (particularly italy) have experienced a high number of covid- cases (https://www.who.int/emergencies/diseases/novel-coro navirus- /situation-reports/. accessed march ). mortality rates vary widely, and depend on the age of patients, underlying risk factors, and the denominator definitionhospitalized cases, all symptomatic cases, only moderate to severe cases, etc. in a study of adult patients (mean age . y; % with chronic illnesses) with sars-cov- pneumonia admitted to the intensive care unit (icu), . % died within days [ ] . in contrast, a study of hospitalized patients (median age . years) across beijing showed [ , , , ] % of cases to be severe and % mild, with a fatality rate of . % [ ] . mortality is highest in older persons, with a median age of - years [ , ] . treatment for all severe hcov infections is supportive although a randomized, double-blinded, control clinical trial has been conducted on a gilead drug remdesivir [ ] based on one study focused on children, a total of children aged from month to years have been reported in china. all paediatric cases with laboratory-confirmed sars-cov- infection were mild cases with no deaths reported [ ] . during the first months of the current outbreak, covid- spread rapidly throughout china and caused varying degrees of illness with a death rate of . %. patients often presented without fever, and many did not have abnormal radiologic findings [ ] . the chinese centers for disease control and prevention team analysed more than , patient records, of which , were laboratory-confirmed cases, , suspected cases, , clinically diagnosed cases, and asymptomatic cases. of the confirmed cases, about % of the illnesses were severe, which included pneumonia and shortness of breath, and about % have the critical disease, marked by respiratory failure, septic shock, and multi-organ failure. the overall case-fatality rate was . %, and of deaths included in the study, the majority were in people age and older or those with underlying medical conditions http://www.cidrap.umn.edu/news-perspective/ / /more-outbreak-details-emerge-covid- -casestop- (accessed february ). it must be appreciated that no matter how accurate and fast laboratory testing methods are, the diagnosis of viral pneumonias such as caused by sars-cov- involves collecting the correct specimen from the patient at the right time. the endemic hcovs have been detected from a variety of upper and lower respiratory sources including throat, nasal nasopharyngeal (np), sputum, and bronchial fluid [ , , ] . wang et al have just reported that oropharyngeal (op) swabs (n = ) were used much more frequently than np swabs (n = ) in china during the covid- outbreak; however, the sars-cov- rna was detected only in % of op swabs, which was significantly lower than that in np swabs ( %) [ ] . the us centers for disease control and prevention (cdc) recommends collecting the upper respiratory np swab. collection of an op specimen is a lower priority, and, if collected, should be combined in the same tube as the np swab (https://www.cdc.gov/coronavirus/ -ncov/lab/guidelines-clinical-specimens.html. accessed march ). swab specimens should be placed in a universal or viral transport medium. nasopharyngeal aspirates are also suitable specimens for the detection of hcovs. for the most sensitive detection of sars-cov, mers-cov, and sars-cov- , the collection and testing of both upper and lower respiratory samples [sputum, bronchoalveolar lavage fluid (bal)] is recommended [ ] . however, the collection of sputum and particularly bal via bronchoscopy increases biosafety risk to healthcare workers through the creation of aerosol droplets. proper use of personal protective equipment (ppe) by healthcare workers is important. bronchoscopy is a highly technical procedure requiring well-trained staff and may not be available in many parts of the world. upper respiratory specimens are easy to collect, thereby increasing access to testing for patients with mild symptoms, and in the resource limited settings. sars-cov and mers-cov rna are also detected from stool, urine and blood specimens, although generally less reliably than from respiratory specimens [ ] [ ] [ ] ]. an exception is sars-cov rna which is consistently detected in feces at about two weeks after symptom onset [ , ] . for the most sensitive detection of endemic hcovs, upper respiratory specimens should be collected within the first few days of symptom onset. the dynamics of rna shedding in mers and sars patients may reflect the specimen source, severity of illness, as well as underlying risk factors. among hospitalized patients who did not require ventilator support, mers-cov rna levels in the upper respiratory tract usually peaked in the first week after symptom onset. among eventual fatal cases requiring ventilation, rna levels in lower respiratory tract specimens peaked between weeks and [ ] . similar shedding patterns were seen for sars-cov: rna positive rates peaked in upper respiratory tract specimens at - days after symptom onset and then steadily declined after that, while rna positive rates in lower respiratory tract specimens remained higher throughout weeks after onset of illness [ ] . in one study, diabetes was associated with prolonged mers-cov rna shedding in the respiratory tract [ ] . viral pneumonias typically do not result in the production of purulent sputum. thus, a nasopharyngeal swab/wash is usually the collection method used to obtain a specimen for testing. nasopharyngeal specimens may miss early infection; a deeper specimen may need to be obtained by bronchoscopy. alternatively, repeated testing can be used because over time, the likelihood of the sars-cov- being present in the nasopharynx increases. self-collected saliva specimens were tested positive in of covid- patients, suggesting it is a promising non-invasive specimen for diagnosis, monitoring, and infection control in sars-cov- infections [ ] . at the time of writing there was little data on the performance of upper vs. lower respiratory tract specimens for the detection of sars-cov- [ ] . serum is another source for the detection of sars-cov- . however, only % of patients hospitalized with pneumonia had detectable rna in serum [ ] . specimens collected for laboratory testing of hcovs should be maintained at refrigerated temperature for up to h, or frozen at − °c or below (https://www.cdc.gov/coronavirus/ -ncov/lab/guidelines-clinical-specimens.html. accessed march ). rectal specimens have been reported positive in patients infected with sars-cov- [ ] . if the patient's travel or exposure history or symptoms suggest possible infection with a high-risk, novel agent, sars-cov, or mers-cov, then the initial handling of the specimen should be performed under biosafety level (bsl- ) conditions until the specimen or an aliquot is rendered noninfectious by lysis or another method. virus isolation should not be routinely performed in this situation (https://www.asm.org/ articles/policy/laboratory-response-network-lrn-sentinel-level-c. accessed february ). the u.s. cdc biosafety guidelines state that routine diagnostic testing of specimens from suspected or confirmed sars-cov- patients, can be handled in a bsl- laboratory using standard precautions (https://www.cdc. gov/coronavirus/ -ncov/lab/lab-biosafetyguidelines.html. accessed march ). isolation of hcovs in cell culture is not routinely performed for diagnostic purposes due to the lack of permissive cell lines, time to results, labour and expertise requirements, and the lack of commercial antisera for culture confirmation (table ) . sars-cov and mers-cov and sars-cov- will grow in primary monkey cells and cell lines such as vero and llc-mk , but cell culture should not be performed for suspect cases in routine diagnostic laboratories for biosafety reasons [ , , , ] . however, virus isolation in cell cultures is critical to obtain isolates for characterization and to support the development of vaccines and therapeutic agents. rapid antigen tests would theoretically provide the advantage of fast time to results and low-cost detection of hcovs but are likely to suffer from poor sensitivity based on the experience with this method for influenza (flu) viruses [ ] [ ] [ ] [ ] [ ] (table ). in a pre-peer reviewed article, diao et al. reported that a fluorescence immunochromatographic assay is an accurate, rapid, early and simple method for detecting nucleocapsid protein of sars-cov- in np swab for diagnosis of covid- (https://www.medrxiv.org/content/ . / . . . v . accessed march ). the incorporation of colloidal gold-labeled immunoglobulin g (igg) as the detection reagent is an approach that may increase the sensitivity of rapid antigen tests for respiratory viruses [ ] . monoclonal antibodies specifically against sars-cov- have been under preparation. novel approaches to concentrate antigen, or to amplify the detection phase are needed if these methods are to have clinical utility. sona nanotech (halifax, canada) is developing a quick-response lateral-flow test to screen covid- patients targeting to produce results in - min (https://sonanano.com/sona-develops-rapidscreening-test-for-coronavirus/. accessed february ). timing of specimen collection, when viral titres are highest, may improve the diagnostic sensitivity of rapid antigen tests for hcovs [ ] . serological assays are not routinely used for diagnosis of hcov infections due to the lack of commercial reagents, let alone commercial reagents that have been vetted by clinical trials and the regulatory review process [ , ] (table ). serological assays, on the other hand, are important for understanding the epidemiology of emerging hcovs, including the burden and role of asymptomatic infections. it has been particularly important for antibody detection in the diagnosis of cases of novel and emerging hcovs, such as sars-cov and mers-cov [ , ] . in these situations, affected patients may not test positive for viral rna, particularly in the early phase of the disease, but retrospectively can be shown to have developed an immune response. when sars-cov- was identified, especially when rapid antigen testing and/or molecular assays are neither available nor stable, serology can be used as a supplementary diagnostic tool. a recent study demonstrated that both igm and igg antibodies were detected days after onset in all patients infected with sars-cov- infection. the authors recommended to use serology to facilitate the diagnosis of sars-cov- infections when an np swab specimen was collected inappropriately and the molecular assays were performed unsatisfactorily [ ] . in china, six serology devices have just received urgent approval from the national medical products administration (nmpa) by march (table ) . proper specimen handling and storage are important to maintain the integrity of specimens and the performance of serologic tests. random-amplification deep-sequencing approaches played a critical role in identifying mers-cov and sars-cov- [ , , [ ] [ ] [ ] [ ] [ ] . for the clinical diagnostic application, the genetic heterogeneity of hcovs precludes a single "pan-hcov" molecular assay [ ] [ ] [ ] [ ] ( table ). some pan-cov assays use degenerate primers [ ] , some utilize multiple primer sets [ ] , and others employ a single set of nondegenerate primers [ ] . current molecular respiratory panels that detect the endemic hcovs (hcov-nl , hcov-hku , hcov-oc , and hcov- e) require multiple sets of pcr oligonucleotides [ , [ ] [ ] [ ] . sars-cov- cases tested negative for endemic hcovs included in molecular respiratory panels [ ] . in china, at the time of revising, eleven molecular devices from shanghai zj bio-tech, shanghai geneodx biotech, bgi biotech (wuhan), mgi tech, da an gene, sansure biotech, shanghai biogerm medical biotech capitalbio (chengdu), beijing applied biological technologies, maccura biotechnology, and wuhan easydiagnosis biomedicine have received urgent approval from nmpa and their characteristics are contrasted in table . variable performance has been reported on these devices [ , ] . in their registration certificates, it was clearly indicated that the certificate was for urgent and supplemental diagnosis of pneumonia caused by sars-cov- . additional multi-centre clinical trial data are needed for extension after one year. among them, one (mgi tech) uses its ngs technique to detect all pathogens in a given specimen including sars-cov- and the other one (innovita) uses its isothermal amplification followed by chip detection. the other nine devices incorporated real-time pcr technique with hydrolysis probes. after nucleic acids get extracted (separated reagents and systems), the extracts are transferred to a real-time pcr thermocycler (e.g. abi fast dx real-time pcr instrument) for nucleic acid amplification and detection. several rt-pcr protocols for detection of sars-cov- rna have been posted by the world health organization at https://www.who.int/emergencies/ diseases/novel-coronavirus- /technical-guidance/ laboratory-guidance. (accessed march ). three of these protocols are listed below. the us cdc developed developed a rt-pcr diagnostic panel for universal detection of sars-like betacoronaviruses and specific detection of sars-cov- [ ] . three separate rt-pcr reactions target the n gene. one primer/probe set detects all betacoronaviruses, while two sets are specific for sars-cov- . all assays must be positive to report presumptive positive for sars-cov- (https://www.fda.gov/ media/ /download. accessed march ). specimen types included upper and lower respiratory specimens (such as np or op swabs, sputum, lower respiratory tract aspirates, bal, and nasopharyngeal wash/aspirate or nasal aspirate). it the charité algorithm (berlin, germany) begins with two rt-pcr assays that detect e and rdrp genes of subgenus sarbecovirus (sars-cov, sars-cov- , and bat-associated betacoronaviruses). both assays must be positive to advance to the next step in the testing algorithm. the second step consists of a [ ] [ ] [ ] naat, monoplex, specific-hcov high sensitivity and specificity for special species, potential quantification - h diagnosis (detection, differentiation, and limited typing) and research [ , ] naat, multiplex high sensitivity and specificity, covering other pathogens, filmarray rp ez is clia-waived - h diagnosis (detection, differentiation, and limited typing) and research [ , [ ] [ ] [ ] naat, poct rapid and safe, good sensitivity and specificity, some are clia-waived diagnosis (detection and limited differentiation) and research [ , ] note: eia, enzyme immunoassay; ifa, immunofluorescent assay; naat, nucleic acid amplification test; clia, clinical laboratory improvement act. sars-cov- specific rt-pcr that targets rdrp [ , ] . exclusivity testing showed that alphacoronaviruses (cov-nl and − e) and betacoronaviruses hcov-oc , hcov-hku and mers-cov were not detected (https://www.who.int/docs/default-source/ coronaviruse/protocol-v - .pdf?sfvrsn=a ef c_ . accessed february ). the university of hong kong li ka shing faculty of medicine protocol uses two assays (n gene screening assay followed by orf b assay for confirmation) to detect subgenus sarbecovirus [ , ] . since sars-cov is not circulating in humans currently, cases that are positive should be considered as sars-cov- infected cases. exclusivity testing showed that e, oc and mers, e, hku , nl , oc yielded negative results (https://www.who.int/docs/defaultsource/coronaviruse/peiris-protocol- - - .pdf? sfvrsn=af aac _ . accessed february ). all three novel coronaviruses are highly contagious. fast, safe, simple to use diagnostic devices performed at or near the point of care (poc) (figure ) which have been shown to impact patient management and control of infectious disease epidemics [ ], are extremely desirable in poc when biosafety facility is limited (table ) . several manufactures have been spending efforts to generate devices for poc testing (poct) [ ] . the id now™ (previously alere i) influenza a & b assay (abbott, san diego, ca) was cleared by the us fda for direct use on np swabs as the first-ever clinical laboratory improvement amendments (clia)-waived nucleic acid-based test in january [ , ] . similarly, the xpert® xpress flu/rsv (cepheid, sunnyvale, ca) and cobas® liat® flu a/b & rsv (roche molecular systems, pleasanton, ca) assays are integrated nucleic acid extraction-independent devices that have recently received fda clearance and clia-waiver for simultaneous detection and identification of flua, flub, and rsv in nasopharyngeal swabs [ ] . the filmarray® respiratory ez panel (biofire, salt lake city, ut) so far so far is the only clia-waived syndromic panel that covers a set of respiratory viral and bacterial pathogens including classical coronavirus species [ ] . considering the increased levels of mortality and infectivity associated with three novel-coronavirus outbreaks, these random-access, safe and simple tests, which offer fast and accurate detection and identification, are likely to have an immediate impact on prompt clinical and epidemiological decisions [ , ] . lysis buffer can be used to inactivate the infectivity of specimens so the testing can be run at poc when a biosafety cabinet is not available. fast near-patient and poct could help more efficiently triage of suspected cases of novel coronavirus, helping to focus limited resources on enabling appropriate use of quarantine. a handful of diagnostics developers are now striving to bring fast sars-cov- tests to market as soon as possible, with hopes of ultimately assisting with the ongoing outbreak in china. molecular diagnostic tests for use at the are in development from cepheid and hibergene (dublin, ireland). cepheid has some advantages in the molecular poct space because it already has instruments placed in china. mobidiag, meanwhile, may offer additional benefits with a multiplex test for coronavirus and flu viruses (https://www. genomeweb.com/pcr/diagnostics-firms-rush-developrapid-point-care-tests-novel-coronavirus#.xkea sgzy x. accessed february ). mjl and y-wt are employees of cepheid, the commercial manufacturer of the xpert xpress sars-cov- test. figure . evolutions in molecular testing procedures. the point-of-care test (poct) devices incorporate nucleic acid extraction, amplification and detection together into an integrated and sealed cartridge making it simple, rapid and safe. during end-point pcr, dna is detected or measured at the completion of pcr amplification, requiring post-pcr processing. real-time pcr is a closed-tube system in which dna is detected or measured during the exponential phase of amplification. epidemiology, genetic recombination, and pathogenesis of coronaviruses a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of cases of middle 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acute respiratory syndrome (sars)-associated coronavirus in cells derived from throat wash samples of patients with sars comparison of immunofluorescence with monoclonal antibodies and rt-pcr for the detection of human coronaviruses e and oc in cell culture evaluation of a commercial colloidal gold assay for detection of influenza a and b virus in children's respiratory specimens rapid detection and monitoring of human coronavirus infections examination of seroprevalence of coronavirus hku infection with s protein-based elisa and neutralization assay against viral spike pseudotyped virus seroepidemiology of group i human coronaviruses in children molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia rna based mngs approach identifies a 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evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b profile of the alere i influenza a & b assay: a pioneering molecular pointof-care test parallel validation of three molecular devices for simultaneous detection and identification of influenza a and b and respiratory syncytial viruses performance and impact of a clia-waived, point-of-care respiratory pcr panel in a pediatric clinic identification of a new human coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays key: cord- - ji g authors: chen, weilie; lan, yun; yuan, xiaozhen; deng, xilong; li, yueping; cai, xiaoli; li, liya; he, ruiying; tan, yizhou; deng, xizi; gao, ming; tang, guofang; zhao, lingzhai; wang, jinlin; fan, qinghong; wen, chunyan; tong, yuwei; tang, yangbo; hu, fengyu; li, feng; tang, xiaoping title: detectable -ncov viral rna in blood is a strong indicator for the further clinical severity date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: ji g the novel coronavirus ( -ncov) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time pcr in the clinical lab. unexpectedly, the -ncov rna was readily detected in the blood ( of patients) and the anal swabs ( of patients). importantly, all of the patients with detectable viral rna in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral rna with the disease severity (p-value = . ). meanwhile, of the patients with annal swab virus-positive was in severe clinical stage. however, the concentration of viral rna in the anal swab (ct value = + ) was higher than in the blood (ct value = + ) from patient , suggesting that the virus might replicate in the digestive tract. altogether, our results confirmed the presence of virus rna in extra-pulmonary sites. the novel coronavirus ( -ncov), originally outbreaking from wuhan china, has transmitted in an extremely short period to countries and infected over individuals as of feb , , causing an international alarm. basic scientific research has achieved significantly in the investigation of viral origination [ , ] , transmission and evolution [ ] , and unprecedented public health control actions in china have been activated and effectively prevented the otherwise dramatic spread. the -ncov virus seems more infectious in its public transmission capacity compared to the well-known sars virus in spite of the unavailability of convincingly scientific evidence. the mechanism of viral transmission is still worthy of further exploration. currently, one urgent and critical challenge is to treat infected patients and save their lives. several studies have roughly described the overall clinical features of -ncov patients [ , ] . however, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately - percent of totally confirmed cases based on the local data in our hospital. clinically, for those severe patients, the main symptoms of -ncov pneumonia are fever, decreased white blood cell and lymphocyte count, increased c reaction protein and abnormally expressed cytokines [ ] . one remaining question to be resolved is whether the -ncov virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. in this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. patients, who were confirmed to be infected by the -ncov virus, were firstly enrolled in or transferred to guangzhou eighth people's hospital for treatment purposes. this study followed the guideline of the ethics committee of guangzhou eighth people's hospital. all blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. viral rna was extracted with nucleic acid isolation kit (da'an gene corporation, cat: da ) on an automatic workstation smart (da'an gene corporation) following the guidelines. real-time reverse transcriptional polymerase chain reaction (rt-pcr) reagent (da'an gene cooperation, cat da ) was employed for viral detection per the protocol. in brief, two pcr primer and probe sets, which target orf ab (fam reporter) and n (vic reporter) genes separately, were added in the same reaction tube. positive and negative controls were included for each batch of detection. samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). all patients had pneumonia-based diseases but with diversified clinical manifestation. to simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by chinese government. patients who were with at least one of the following symptom should be diagnosed to be severe case, ) distress of respiratory with respiratory rate > = /min; ) oxygen saturation < = % in the rest state, and ) arterial oxygen tension (pao₂) over inspiratory oxygen fraction (fio₂) of less than mm hg. in the blood detection cohort (figure (a)), patients who had at less one serum sample measurement with the pcr method were included. in the , cases were detected to be blood positive, all of them ( %) were severe in symptom requiring special care attention, and the blood of the rest cases was without detectable virus in the blood, only of them ( . %) were severe cases. the ratio of severe symptoms between these two groups was significantly different (p value = . ). in the anal swab cohort (figure (b)), of cases were detected to be anal swab positive, of them ( . %) were with severe symptoms, which was significantly higher than that ( . %) of the rest cases without detectable virus in anal were severe cases. fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. patient (figure (a)) was admitted to icu after enrollment evaluation and was highly suspected infection with -ncov because of his recent travelling from wuhan and of confirmed pneumonia by radiographic diagnosis with -day fever and -day continuous dry coughing. he was then confirmed to be infected by the -ncov virus on illness day by cdc. high concentrations of the viral rna were detected in the pharyngeal swabs on illness days (ct = + ), , (ct = + ), and (ct = + ). in the blood, no viral rna was detected on day but the sample on day gave a weak positive signal (ct = neg+ ), and then the signal was gone again on day . on day , a low level of viral rna (ct = + ) was detected again in the blood. on day , the blood lost signal again. a high concentration of virus rna (ct = + ) was detected in the anal sample on day , on the day the -ncov virus was not detected in the pharyngeal swab. unfortunately, he was transferred out to another hospital after an emergency expert consultation. patient (figure (b)), who had a clear infection history and started fever -day ago and dry coughing -day ago, was admitted with clinically highly suspect of -ncov infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. the virus was detected in his blood on illness day (ct = + ) and (ct = + ). his infection was also informed by the cdc on day . because his disease advanced very fast, he was transferred to the icu ward for special medical care requirements on day , on which day high titers of virus (ct = + ) were detected in the pharyngeal sample. importantly, virus rna was detected in all pharyngeal (ct = + ), blood (ct = + ) and anal (ct = + ) samples on day . he was transferred out to another hospital after an emergency expert consultation. finally, we described here the four patients with detectable serum viral rna. patient (figure (a) ) was transferred to the icu directly on illness day because of his severe condition, the -ncov virus was laboratory detected both in pharyngeal (ct = + ) and blood samples (ct = + ) on day , and his infection was confirmed by cdc on day . pharyngeal samples were pcr positive on days and and became negative on day . patient (figure (b) ) was transferred to the icu ward on the illness day with a cdc confirmation. his disease advanced pretty fast and became severe on day and he was transferred to icu after his blood sample was detected to be virus-positive (ct = + ). on day , he was transferred out. patient (figure (c) ) was admitted on illness day and his blood sample was virus-positive (ct = + neg) on day . her disease progressed rapidly to a severe stage within the next days. patient ( figure (d)) with a clear history of virus infection was confirmed to be infected on infection day . viral rna was detected in his blood sample on day , one day ahead of his transfer into icu. as his condition worsens, he was transferred out on day . in this retrospective study, we analyzed the pcr data of virus detection in different tissues in our laboratory. firstly, our observation indicated that the presence of viral rna outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. in the blood test cohort, all the infected patients were in (or later progressed to) severe disease stage when serum viral rna became detectable, which showed a significant difference compared to the blood negative group (p = . ). patient (figure (b) ), (figure (c)) and ( figure (d) ) all had detectable viral rna in the serum before they progressed to the clinical severe symptom stage. unfortunately, we missed the earlier time points of patient (figure (a) ) and (figure (a) ) who were directly admitted to icu on transfer to our hospital because of severe condition, of patient (figure (b) ) who had serum sample collected one day post the diagnosis of severe illness. we, fortunately, observed high serum viral load in serum within their severe illness stage. in the anal swab cohort, we found that the presence of virus rna in the anal digestive tract was also positively correlated with disease severity (p = . ). the patients detected with anal virus rna but in mild stage should be monitored whether they will progress to the severe stage. we have summarized the information of approximately percent of the patients in guangzhou city, and the study represented nearly the whole picture of this region. however, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. secondly, a high concentration of viral rna in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. for patient , a high concentration of viral rna (ct = + , on day ) was detected in anal swab but not in pharyngeal (the same day) and blood ( d ahead). for patient , higher concentrations of viral rnas were detected in anal swab (ct = + ) and pharyngeal swab (ct = + ) than in the blood (ct = + ) on the same day. angiotensin-converting enzyme (ace ) still is one of the receptors for -ncov attachment and entry [ ] . intensive structural analysis of the s protein of -ncov with the sars-coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ace [ ] . of note, ace is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [ ] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. we propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ace receptor, which exacerbated the disease vice versa. bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [ ] . nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral rna in the stool. but we believe the existence of virus rna in the stool samples from these patients because that a large amount of viral rna was detected in anal swabs and that viral rna had also been detected in a case reported from the united states [ ] . also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with -ncov prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. in summary, we find that the presence of viral rna in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus rna in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction. a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin rna based mngs approach identifies a novel human coronavirus from two individual pneumonia cases in wuhan outbreak epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with novel coronavirus in wuhan analysis of clinical features of patients with novel coronavirus pneumonia receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin first case of novel coronavirus in the united states we declare no competing interest. we thank all the physicians and nurses who cared these patients. this work was supported by national natural science foundation of china grant (no. and ) and by the national grand program on key infectious disease control ( zx - - ). no potential conflict of interest was reported by the author(s). we declare no competing interest. we thank all the physicians and nurses who cared these patients. this work was supported by national natural science foundation of china grant (no. and ) and by the national science and technology major project ( zx - - ). key: cord- - ghtmhu authors: chua, kaw bing; gubler, duane j title: perspectives of public health laboratories in emerging infectious diseases date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: ghtmhu the world has experienced an increased incidence and transboundary spread of emerging infectious diseases over the last four decades. we divided emerging infectious diseases into four categories, with subcategories in categories and . the categorization was based on the nature and characteristics of pathogens or infectious agents causing the emerging infections, which are directly related to the mechanisms and patterns of infectious disease emergence. the factors or combinations of factors contributing to the emergence of these pathogens vary within each category. we also classified public health laboratories into three types based on function, namely, research, reference and analytical diagnostic laboratories, with the last category being subclassified into primary (community-based) public health and clinical (medical) analytical diagnostic laboratories. the frontline/leading and/or supportive roles to be adopted by each type of public health laboratory for optimal performance to establish the correct etiological agents causing the diseases or outbreaks vary with respect to each category of emerging infectious diseases. we emphasize the need, especially for an outbreak investigation, to establish a harmonized and coordinated national public health laboratory system that integrates different categories of public health laboratories within a country and that is closely linked to the national public health delivery system and regional and international high-end laboratories. infectious diseases have affected humans since the first recorded history of man. infectious diseases remain the second leading cause of death worldwide despite the recent rapid developments and advancements in modern medicine, science and biotechnology. greater than million (. %) of an estimated million deaths that occur throughout the world annually are directly caused by infectious diseases. millions more deaths are due to the secondary effects of infections. moreover, infectious diseases cause increased morbidity and a loss of work productivity as a result of compromised health and disability, accounting for approximately % of all disability-adjusted life years globally. , compounding the existing infectious disease burden, the world has experienced an increased incidence and transboundary spread of emerging infectious diseases due to population growth, urbanization and globalization over the past four decades. [ ] [ ] [ ] [ ] [ ] [ ] most of these newly emerging and re-emerging pathogens are viruses, although fewer than of the approximately pathogen species recognized to infect humans are viruses. on average, however, more than two new species of viruses infecting humans are reported worldwide every year, most of which are likely to be rna viruses. emerging novel viruses are a major public health concern with the potential of causing high health and socioeconomic impacts, as has occurred with progressive pandemic infectious diseases such as human immunodeficiency viruses (hiv), the recent pandemic caused by the novel quadruple re-assortment strain of influenza a virus (h n ), and more transient events such as the outbreaks of nipah virus in / and severe acute respiratory syndrome (sars) coronavirus in . [ ] [ ] [ ] [ ] [ ] in addition, other emerging infections of regional or global interest include highly pathogenic avian influenza h n , henipavirus, ebola virus, expanded multidrug-resistant mycobacterium tuberculosis and antimicrobial resistant microorganisms, as well as acute hemorrhagic diseases caused by hantaviruses, arenaviruses and dengue viruses. to minimize the health and socioeconomic impacts of emerging epidemic infectious diseases, major challenges must be overcome in the national and international capacity for early detection, rapid and accurate etiological identification (especially those caused by novel pathogens), rapid response and effective control (figure ). the diagnostic laboratory plays a central role in identifying the etiological agent causing an outbreak and provides timely, accurate information required to guide control measures. this is exemplified by the epidemic of nipah virus in malaysia in / , which took more than six months to effectively control as a consequence of the misdiagnosis of the etiologic agent and the resulting implementation of incorrect control measures. , however, there are occasions when control measures must be based on the epidemiological features of the outbreak and pattern of disease transmission, as not all pathogens are easily identifiable in the early stage of the outbreak (figure ). establishing laboratory and epidemiological capacity at the country and regional levels, therefore, is critical to minimize the impact of future emerging infectious disease epidemics. developing such public health capacity requires commitment on the part of all countries in the region. however, to develop and establish such an effective national public health capacity, especially the laboratory component to support infectious disease surveillance, outbreak investigation and early response, a good understanding of the concepts of emerging infectious diseases and an integrated country and regional public health laboratory system in accordance with the nature and type of emerging pathogens, especially novel ones, are highly recommended. traditionally, emerging infectious diseases are broadly defined as infections that: (i) have newly appeared in a population; (ii) are increasing in incidence or geographic range; or (iii) whose incidence threatens to increase in the near future. , six major factors, and combinations of these factors, have been reported to contribute to disease emergence and re-emergence: (i) changes in human demographics and behavior; (ii) advances in technology and changes in industry practices; (iii) economic development and changes in land use patterns; (iv) dramatic increases in volume and speed of international travel and commerce; (v) microbial mutation and adaptation; and (vi) inadequate public health capacity. , from the perspective of public health planning and preparedness for effective emerging infectious disease surveillance, outbreak investigation and early response, the above working definition of emerging infectious disease and its associated factors that contribute to infectious disease emergence are too broad and generic for more specific application and for the development of a national public health system, especially in the context of a public health laboratory system in a country. thus, in this article, emerging infectious diseases are divided into four categories based on the nature and characteristics of pathogens or infectious agents causing the emerging infections; these categories are summarized in table . the categorization is based on the patterns of infectious disease emergence and modes leading to the discovery of the causative novel pathogens. the factors or combinations of factors contributing to the emergence of these pathogens also vary within each category. likewise, the strategic approaches and types of public health preparedness that need to be adopted, in particular with respect to the types of public health laboratories that need to be developed for optimal system performance, will also vary greatly with respect to each category of emerging infectious diseases. these four categories of emerging infectious diseases and the factors that contribute to the emergence of infectious diseases in each category are briefly described below. [ ] [ ] [ ] [ ] [ ] [ ] factors that contributed to the occurrence of emerging infectious diseases in this subcategory include population growth; urbanization; environmental and anthropogenic driven ecological changes; increased volume and speed of international travel and commerce with rapid, massive movement of people, animals and commodities; and deterioration of public health infrastructure. subcategory b includes known and unknown infectious agents that occur in new host 'niches'. infectious microbes/ agents placed under this subcategory are better known as 'opportunistic' pathogens that normally do not cause disease in immunocompetent human hosts but that can lead to serious diseases in immunocompromised individuals. the increased susceptibility of human hosts to infectious agents is largely due to the hiv/acquired immune deficiency syndrome pandemic, and to a lesser extent, due to immunosuppression resulting from cancer chemotherapy, antirejection treatments in transplant recipients, and drugs and monoclonal antibodies that are used to treat autoimmune and immune-mediated disorders. a notable example is the increased incidence of progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system that is caused by the polyomavirus 'jc' following the public health laboratories in emerging infectious diseases kb chua and dj gubler increased use of immunomodulatory therapies for anti-rejection regimens and for the treatment of autoimmune diseases. examples of past emerging infectious diseases under this category are antimicrobial resistant microorganisms (e.g., mycobacterium tuberculosis, plasmodium falciparum, staphylococcus aureus) and pandemic influenza due to a new subtype or strain of influenza a virus (e.g., influenza virus a/california/ / (h n )). , - factors that contribute to the emergence of these novel phenotype pathogens are the abuse of antimicrobial drugs, ecological and host-driven microbial mixing, microbial mutations, genetic drift or re-assortment and environmental selection. accidental or potentially intentional release of laboratory manipulated strains resulting in epidemics is included in this category. factors that lead to the spillovers and emergence of these novel pathogens are human population expansion, economic development, changes in land use patterns, modifications to natural habitats, and changes in agricultural practices and animal husbandry. human behavior, such as wildlife trade and translocations, live animal and bush meat markets, consumption of exotic foods, development of ecotourism, access to petting zoos and ownership of exotic pets, also plays a significant role in the transfer of pathogens between species. [ ] [ ] [ ] [ ] [ ] [ ] examples of infectious diseases under category b are gastritis and peptic ulcers due to helicobacter pylori, kaposi sarcoma due to human herpesvirus and chronic hepatitis due to hepatitis virus c and g. [ ] [ ] [ ] [ ] advances in scientific knowledge and technology have contributed substantially to the discovery of these infectious etiological agents. regardless of the category, with some exception for category b, effective early detection, identification, characterization, containment, control and ultimately prevention of the emerging infectious diseases will require a good, functional national public health surveillance system. the system needs to be well supported by a network of primary public health and clinical/medical diagnostic laboratories that are coordinated by a national public health reference laboratory with real-time and harmonious communication between the laboratories and epidemiological surveillance units. confronted with the great diversity of these emerging pathogens and the equally diverse mechanisms and factors that are responsible for their emergence, there is an urgent need to develop a network of diagnostic laboratories, especially in countries where epidemic infectious diseases are likely to emerge. this network should include local laboratories with basic clinical laboratory capabilities, provincial and national public health diagnostic laboratories with greater capability to diagnose known pathogens and support effective laboratory-based surveillance, and a centralized national reference laboratory that can provide laboratory training and quality control for diagnostic assays for the network of diagnostic laboratories in the country. ideally, the national reference laboratory should have state-of-the-art laboratory technology and be able to identify and characterize novel pathogens with specialized university laboratories and foreign institutes that can provide backup capability, but more importantly, the national reference laboratory should be able to conduct research for the development of new diagnostic technologies to detect and identify novel pathogens, especially those classified as category . the us system, which includes local and state public health laboratories that conduct diagnoses of known pathogens, the centers for disease control and prevention and university laboratories that provide research and reference activities, is a good model. disease or pathogen-specific public health diagnostic laboratories established to support world health organization (who)-specific disease surveillance programs and vaccine-preventable diseases, e.g., national influenza, poliovirus and measles laboratories, led to the 'compartmentalization' of laboratory diagnostic services, segregation of functions, and duplication of facilities and equipment. the situation is further complicated by the siting of various pathogen-specific diagnostic laboratories in different buildings or institutes or different locations within a country, thus preventing more cost-effective measures of sharing common equipment and reagents, clinical samples, information and human resources. finally, the problem is compounded by policy makers and laboratory managers lacking flexibility and not allowing these disease or pathogen-specific laboratories to adopt a more generic approach in the investigation of infectious disease outbreaks. past incidents have shown that misdiagnosis or delay in the diagnosis of epidemics can cause substantial economic losses and social disruption and prevent containment or control as a result of the implementation of inappropriate control measures or a delay in implementing the appropriate control measures. the proposed integrated system of public health laboratories is not entirely new; public health laboratories are already in existence in most countries, but most are poorly equipped and are not adequately funded or staffed with trained professional staff. moreover, a lack of knowledge and coordination has led to ineffective operation in the support of infectious disease surveillance. the basic concept of realigning and harmonizing public health laboratories to optimize their roles and functions can be drawn from the system of medical practices. due to rapid and vast expansion of medical knowledge, technology and demand of specialized skills and therapy, medical practices have evolved into a number of specialties and subspecialties, such as infectious disease, cardiology, gastroenterology, neurology, radiology, anesthesiology and oncology. an excellent aspect of the medical system is the continual retention of the general physician (family physician) or general pediatrician as the initial or first entry point for patients seeking consultation for any medical problem before being subsequently referred to the appropriate specialist, if deemed necessary. it is not uncommon for patients, especially older individuals, to have more than one disease or pathology at the time of presentation to the doctor. in a similar manner, in outbreaks of infectious diseases, 'background' endemic pathogens are often present that are capable of similar disease manifestations. thus, public health analytical diagnostic laboratories (both primary and clinical) should adopt a generic approach and serve as the initial or first entry point for the investigation of the causative pathogens in the event of an infectious disease outbreak or the occurrence of any fatal illness with clinical suspicion of infectious etiology. in addition, public health laboratories must have the capability to support the expanded scope and sophistication of public health activities brought about by a rapid increase in population and social, demographic and ecological changes, in addition to the factors mentioned above. despite the presence of several types of health laboratories, they can be classified into three main categories: (i) public health research laboratories; (ii) public health reference laboratories; and (iii) public health analytical diagnostic laboratories. public health analytical diagnostic laboratories can be further subcategorized into primary public health (community-based) and clinical/medical (hospital and clinic-based) analytical diagnostic laboratories. a proposed organizational model to establish an integrated system of public health laboratories within a country to coordinate and link health laboratories under different ministries and in both public and private institutions based on their functional roles is shown in figure . the broken lines indicate the diagnostic laboratories that are not directly regulated by the ministry of health. a schematic flow chart illustrating the functional relationships and linkages between various types of public health laboratories in a country was described previously. a defined and harmonious linkage and collaboration will not only avoid duplication and redundancy, but also enhance and complement the function and output quality of each laboratory. bearing in mind that not all countries in the world have similar resources (financial, man-power and expertise), demography, geopolitical structure, needs and commitment, the proposed model can be appropriately modified to tailor each country's immediate needs with a provision for future upgrading and expansion. ultimately, it is recommended that all countries establish an integrated system covering all three categories of public health laboratories, with a cohesive centralized national public health reference laboratory. in countries with limited resources, an interim centralized national public health diagnostic laboratory can take on some of the roles and functions of a national reference laboratory, especially in supporting laboratory training and quality assurance. for countries without such an idealistic centralized public health reference laboratory, an in-place system of networking should be developed to link to regional and international high-end laboratories or who collaborative centers to rapidly identify and characterize novel pathogens and provide other specialized laboratory diagnostic reagents, assays or validation. in addition, each region should have a regional center for reference public health laboratories in emerging infectious diseases kb chua and dj gubler and research to help the national reference and/or diagnostic laboratories train and maintain laboratory quality control. the us centers for disease control and prevention is a major who collaborative partner and provides laboratory service not only for the american region, but also for many other countries in the world. investigations into the diagnosis of nipah virus, sars coronavirus, pandemic influenza a virus and hemorrhagic fever viruses are just a few examples illustrating its worldwide function. the placement of the centralized reference laboratory under the national center for disease control strengthens close communication and coordination among public health specialists, epidemiologist and laboratory personnel and serves as an important coordinating center to support the functions and activities pertaining to biorisk issues, centralized pathogen characterization and storage, laboratory-based surveillance and laboratory quality assurance, as shown in figure . a national reference laboratory will also be able to play an important role as part of a regional laboratory network to strengthen regional public health laboratory capacity in providing specific referral functions for public health diagnostic laboratories in other countries that do not have a reference laboratory. the public health research laboratories within the research institutes of ministries of health and universities or even private research institutions are best suited and can play a crucial role in collaborating with the national public health reference and diagnostic laboratories to discover novel pathogens of many human diseases under category , especially in subcategory b. the proposed network scheme will provide more cost-efficient laboratory services and ensure a regular flow of laboratory work to maintain the competency of technical staff to produce quality output. because of the increased likelihood of epidemic diseases caused by novel pathogens, diagnostic laboratories serving as the primary entry point of investigation should be able to take a more generic approach in pathogen detection, isolation and identification. the traditional existing system of 'compartmentalization' of national disease/pathogenspecific diagnostic laboratories should thus be reviewed and integrated into the national public health infectious disease diagnostic laboratory system. this proposed model would improve cost-efficiency and allow a more appropriate approach to infectious disease outbreak investigation and control. we thank tikki pang, lee kuan yew school of public policy, national university of singapore, for useful comments and suggestions in the preparation of this manuscript. emerging infections: getting ahead of the curve emerging infectious diseases: a -year perspective from the national institute of allergy and infectious diseases emerging viruses: 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viruses for old disease? a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections saffold virus, a human theiler's-like cardiovirus, is ubiquitous and causes infection early in life unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome molecular cloning and disease association of hepatitis g virus: a transfusion-transmissible agent this work is licensed under a creative commons attribution-noncommercial-noderivative works to view a copy of this license key: cord- -rhhmvnlp authors: joseph, sunitha; wernery, ulrich; teng, jade ll; wernery, renate; huang, yi; patteril, nissy ag; chan, kwok-hung; elizabeth, shyna k; fan, rachel yy; lau, susanna kp; kinne, jörg; woo, patrick cy title: first isolation of west nile virus from a dromedary camel date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: rhhmvnlp although antibodies against west nile virus (wnv) have been detected in the sera of dromedaries in the middle east, north africa and spain, no wnv has been isolated or amplified from dromedary or bactrian camels. in this study, wnv was isolated from vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in dubai. complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage a. there was no clustering of the present wnv with other wnvs isolated in other parts of the middle east. within lineage a, the dromedary wnv occupied a unique position, although it was most closely related to other wnvs of cluster . comparative analysis revealed that the putative e protein encoded by the genome possessed the original wnv e protein glycosylation motif nys at e – , which contained the n-linked glycosylation site at n- associated with increased wnv pathogenicity and neuroinvasiveness. in the putative ns protein, the a s substitution observed in other cluster wnvs and p , which has been implicated in neuroinvasiveness, were present. in addition, the foo motif in the putative ns a protein, which has been implicated in neuroinvasiveness, was detected. notably, the amino-acid residues at positions in the present dromedary wnv genome differed from those in most of the closely related wnv strains in cluster of lineage a, with the majority of these differences observed in the putative e and ns proteins. the present study is the first to demonstrate the isolation of wnv from dromedaries. this finding expands the possible reservoirs of wnv and sources of wnv infection. west nile virus (wnv) is a positive-sense single-stranded ribonucleic acid (rna) virus in the flaviviridae family. wnv is the leading cause of mosquito-borne encephalitis in humans in many parts of the world. in addition to humans, many animals are also susceptible to wnv infections. large outbreaks have occurred globally in both humans and other animals, such as horses and pigs. birds are the natural reservoirs of wnv. bird-to-bird, bird-to-mammal and bird-to-human transmissions are achieved by mosquito bites, with humans and other mammals serving as dead-end hosts because of low viral loads. camels are one of the most unique mammals on earth and have shown perfect adaptation to desert life. there are two surviving old-world camel species: camelus dromedarius (dromedary or onehumped camel), which inhabits the middle east and north and northeast africa, and camelus bactrianus (bactrian or two-humped camel), which inhabits central asia. among the million camels on earth, % are dromedaries. although wnv is known to infect some of the new world camels, such as llamas and alpacas, only antibodies against wnv have been detected in the sera of old-world camels, such as the dromedaries in the middle east, north africa and spain, with a seroprevalence of %- %. [ ] [ ] [ ] [ ] to date, no wnv has been isolated or amplified from either dromedary or bactrian camels. recently, the emergence of middle east respiratory syndrome (mers) and the isolation of the mers coronavirus (mers-cov) from dromedaries boosted interest in the search for novel viruses in dromedaries. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this article, we report the first isolation of wnv from a dromedary calf in the united arab emirates during the process of mers-cov screening and the results of the comparative genome and phylogenetic analysis. clinical samples were obtained during a necropsy of a dromedary calf at the central veterinary research laboratory in dubai, the united arab emirates, using standard procedures. the central veterinary research laboratory in dubai is the center for performing necropsies of dromedaries from sheikhs in the uae with the aim of finding the cause of death and preventing the spread of infectious diseases to other camels or herds. nasal swabs and pooled ground trachea/lung samples were inoculated onto vero cells for mers-cov screening. the pooled clinical samples were diluted -fold with viral transport medium and filtered. two hundred microliters of the filtrate was inoculated into μl of minimum essential medium (gibco, grand island, ny, usa). four hundred microliters of the mixture was added to -well tissue culture plates with vero cells by adsorption inoculation. after h of adsorption, the excess inoculum was discarded, the wells were washed twice with phosphate-buffered saline, and the medium was replaced with ml of minimum essential medium (gibco). cultures were incubated at °c with % co and inspected for cytopathic effects daily using inverted microscopy. negative-contrast electron microscopy was performed as previously described. tissue culture cell extracts infected with dromedary wnv were centrifuged at g at °c. then, the pellet was resuspended in phosphate-buffered saline and stained with % phosphotungstic acid. samples were examined with a philips em s electron microscope (philips scientifics, eindhoven, the netherlands). sample preparation for illumina sequencing rna was extracted from the isolated virus using the qiaamp viral rna mini kit (qiagen, hilden, germany). reverse transcription and polymerase chain reaction (pcr) were performed using the super-script iii reverse transcriptase (invitrogen, carlsbad, ca, usa) and a random primer containing a -base arbitrary sequence at the ′ end followed by a randomized octamer ( n) at the ′ end. a single round of priming and extension was performed using the klenow fragment polymerase (new england biolabs, ipswich, uk). pcr amplification with a primer consisting of only the -base arbitrary sequence of the random primer was performed with cycles of °c for s, °c for s and °c for min and a final extension at °c for min in an automated thermal cycler (applied biosystems, carlsbad, ca, usa). standard precautions were taken to avoid pcr contamination, and no amplified pcr product was observed in the negative control. the pcr product was purified using the minelute pcr purification kit (qiagen) following the manufacturer's protocol. the purified dna was eluted in μl of eb buffer and used as the template for library construction. library construction for illumina sequencing the metagenomics library was generated using a dna library prepared with the nextera xt dna sample preparation kit (illumina, san diego, ca, usa) according to the manufacturer's protocol. briefly, ng of input dna was tagmented by the nextera xt transposome at °c for min. this transposome simultaneously fragmented the input dna and added an adapter sequence to the ends, thereby allowing amplification by pcr in subsequent steps. the sequencing library with the tagmented dna was amplified in cycles of °c for s, °c for s and °c for s and a final extension at °c for min in an automated thermal cycler (applied biosystems). the amplified dna library was purified using . × ampure xp beads (beckman coulter, indianapolis, in, usa) to remove very short library fragments from the population. the amplified dna library was analyzed using the bioanalyzer instrument (agilent technologies, santa clara, ca, usa). the purified sequencing library was quantified using the kapa library quantification kit (kapa biosystems, wilmington, de, usa) and subsequently sequenced on the illumina hiseq with bp paired-end reads (rapid run mode). image analysis and base calling were performed with scs . /rta . (illumina). fastq file generation and the removal of failed reads were performed using casava ver. . . (illumina). genome assembly and complete-genome sequencing illumina sequence reads were quality trimmed by prinseq-lite, and de novo genome assembly was performed with mira . . the ′ and ends of the viral genome were confirmed by rapid amplification of cdna ends (race) using the ′/ ′ race kit (roche, penzberg, germany). the nucleotide sequence of the genome and the deduced amino-acid sequences of the open reading frames were compared with those of other wnv strains with complete genomes in the vipr sequence database (http://www.viprbrc.org/). n-linked glycosylation sites were predicted using the netnglyc . software (http://www.cbs.dtu.dk/ services/netnglyc/). complete polyprotein nucleotide sequences were aligned with muscle. a maximum-likelihood phylogenetic tree with bootstraps was constructed using the gtr substitution model; gamma distribution among sites was conducted in mega . the nucleotide sequence of the dromedary wnv genome isolated in this study has been lodged in the genbank sequence database under accession no ku . necropsy of the female -month-old kg fresh dromedary calf revealed pale muscles and myocard, massive lung congestion and colitis. the causes of death were white muscle disease, colisepticemia and candida enteritis. at the first passage, the vero cells inoculated with both the nasal swab and pooled trachea/lung samples showed identical cytopathic effects on day , with cell rounding, progressive degeneration and detachment ( figure a) . electron microscopy showed enveloped icosahedral virions - nm in diameter with a -to -nm electron dense core ( figure b ). both the cytopathic effects and viral morphology were inconsistent with those of mers-cov. the complete genome of the isolated virus was sequenced and assembled. the size, g+c content and genome structure were similar to other wnvs. the single polyprotein is putatively cleaved by proteases into three structural proteins (capsid (c), premembrane/ membrane (prm/m) and envelope (e)) and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ). phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage a (figure a ). the present dromedary wnv occupied a unique position within lineage a, although it was most closely related to other wnvs of cluster ( figure b ). comparative analysis based on the complete polyprotein sequence showed the highest ( . %- . %) overall amino-acid identities to wnv strains isolated from a mosquito in kenya (genbank accession number ay ), horses in italy and morocco (genbank accession numbers af , ay and ay ) and a human in italy (genbank accession number jq ). detailed annotation revealed that the putative e protein encoded by the genome possessed the original wnv e protein glycosylation motif nys at e - , which contained the n-linked glycosylation site at n- associated with increased wnv pathogenicity and neuroinvasiveness. in the putative ns protein, the a s substitution observed in other cluster wnvs and p , which was implicated in neuroinvasiveness, were also present. in addition, the foo (flavivirus overlapping orf) motif in the putative ns a protein that was implicated in neuroinvasiveness was detected. notably, amino-acid residues in the present dromedary wnv genome differed from those in most of the closely related wnv strains in cluster of lineage a (figure ) , with the majority of these differences observed in the putative e and ns proteins. we report the first isolation of wnv in a dromedary. the first evidence for the presence of hemagglutination inhibiting and complement-fixing antibodies in dromedaries from nigeria against wnv was published in . , recently, a study has shown the presence of neutralizing antibodies against wnv from dromedaries in north africa. in the united arab emirates, antibodies against wnv have been reported in % of dromedary sera and . % of tested equine sera, supporting the conclusion that wnv is present in the country. in the present study, we isolated a wnv from two independent respiratory samples from a dromedary calf using vero cells, which is one of the most widely used cell lines for the recovery of wnv. this finding was consistent with previous studies that showed that wnv could also be detected in respiratory specimens from birds, humans and other mammals. , notably, the glycosylation motif nys in the e protein, the a s substitution and p in the ns protein and the foo motif in the ns a protein, which are important for pathogenicity and neuroinvasiveness, were also detected in the present wnv isolate. [ ] [ ] [ ] however, because there were no clinical signs in the dromedary calf, the pathogenic role of wnv in dromedaries remains to be determined. viral cultures using other samples from the dromedary were not performed because the samples were initially intended for mers-cov isolation. the present dromedary wnv belongs to wnv lineage a. the whole-genome phylogenetic trees showed that the present dromedary wnv was most closely related to other wnvs in cluster of lineage a with high bootstrap support ( figure ). however, there was no clustering of the present wnv with wnvs isolated in other parts of the middle east, such as israel, egypt and cyprus (data not shown). indeed, two wnvs from israel (genbank accession number ay ) and cyprus (genbank accession number gq ) belong to lineage , another isolate from israel (genbank accession number hm ) belongs to cluster of lineage a, and two other isolates from israel (genbank accession number hm ) and egypt (genbank accession number eu ) belong to cluster of lineage a. moreover, amino-acid positions located in various regions of the genome encoding five different proteins showed marked differences with the corresponding amino acids in the genomes of the isolate's close relatives in cluster . interestingly, most of the amino-acid differences between the present dromedary wnv and the closely related wnvs from africa and europe are located in the e and ns genes. the e protein is generally the least conserved protein because it is exposed to external selection pressure, whereas the ns protein is highly conserved because it contains the viral methyltransferase and rna-dependent rna polymerase. complete-genome sequencing of more wnv strains and comparative genomic and phylogenetic studies are needed to ascertain whether dromedary wnvs form a unique cluster in lineage a. the mers epidemic and the discovery of dromedaries as the reservoir of mers-cov have boosted interest in the search for novel viruses in dromedaries. prior to , viruses from at least eight families were found to infect camels. in the last two years, we have reported the discovery of a novel dromedary camel cov (uae-hku ), a novel genotype of hepatitis e virus, a novel genus of enterovirus, a novel astrovirus, and novel picobirnaviruses and circoviruses in dromedaries. , [ ] [ ] [ ] [ ] these discoveries have remarkably widened the spectrum of viruses known to infect dromedaries. notably, after the description of the novel genotype of hepatitis e virus in dromedaries, another group found this dromedary camel hepatitis e virus as a cause of chronic hepatitis e infection in a liver transplant recipient with a habit of camel milk and meat consumption, highlighting the importance of camels as a source of infectious diseases. the present study is the first demonstration of the isolation of wnv in dromedaries and the first wnv complete-genome sequenced from a dromedary among the relatively few full-length wnv genome sequences from the middle east. further studies will help elucidate whether dromedaries can serve as another possible source of wnv infection and determine the tissue tropism of wnv in s joseph et al figure summary of amino-acid changes in the various putative proteins encoded by the dromedary wnv genome relative to those of its most closely related wnv strains in cluster of lineage a. only changes that occurred in ⩾ strains are shown. dots indicate no difference from the dromedary wnv. west nile virus in a dromedary s joseph et al viral diseases of new world camelids a transversal study on antibodies against selected pathogens in dromedary camels in the canary islands rift valley and west nile virus antibodies in camels emerging viral diseases in dromedary camels in the southern morocco seroepidemiological studies for the detection of antibodies against nine infectious diseases in dairy dromedaries (part- ) middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of a novel coronavirus from a man with pneumonia in saudi arabia a phylogenetically distinct middle east respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in dubai with rising seroprevalence with age a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease acute middle east respiratory syndrome coronavirus infection in livestock dromedaries isolation and characterization of a novel betacoronavirus subgroup. a coronavirus, rabbit coronavirus hku , from domestic rabbits metagenomic analysis of viromes of dromedary camel fecal samples reveals large number and high diversity of circoviruses and picobirnaviruses the challenge of west nile virus in europe: knowledge gaps and research priorities viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the new york strain of west nile virus loss of dimerisation of the nonstructural protein ns of kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of ns ns ' of flaviviruses in the japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness a survey for haemagglutination-inhibiting antibody to west nile virus in human and animal sera in nigeria west nile complement fixing antibodies in nigerian domestic animals and humans west nile fever in the united arab emirates west nile virus detection in kidney, cloacal, and nasopharyngeal specimens fatal hemorrhagic fever caused by west nile virus in the united states novel betacoronavirus in dromedaries of the middle east new hepatitis e virus genotype in camels, the middle east a novel astrovirus from dromedaries in the middle east a novel dromedary camel enterovirus in the family picornaviridae from dromedaries in the middle east chronic infection with camelid hepatitis e virus in a livertransplant recipient who regularly consumes camel meat and milk we are grateful to dr ismail hassab elrasoul mohammed khair (central veterinary research laboratory, dubai, uae) for sending the carcass. we also thank the members of the centre for genomic sciences, the university of hong kong, for their technical support. this work is partially supported by the theme-based research scheme (project no t / / ), the university grant committee, and the strategic research theme fund, the university development fund, the university of hong kong. dromedaries. other studies on the epidemiology, disease spectrum and ecology of wnv in this group of unique animals are also warranted. key: cord- -weat qs authors: kleine-weber, hannah; schroeder, simon; krüger, nadine; prokscha, alexander; naim, hassan y.; müller, marcel a.; drosten, christian; pöhlmann, stefan; hoffmann, markus title: polymorphisms in dipeptidyl peptidase reduce host cell entry of middle east respiratory syndrome coronavirus date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: weat qs middle east respiratory syndrome (mers) coronavirus (mers-cov) causes a severe respiratory disease in humans. the mers-cov spike (s) glycoprotein mediates viral entry into target cells. for this, mers-cov s engages the host cell protein dipeptidyl peptidase (dpp , cd ) and the interface between mers-cov s and dpp has been resolved on the atomic level. here, we asked whether naturally-occurring polymorphisms in dpp , that alter amino acid residues required for mers-cov s binding, influence cellular entry of mers-cov. by screening of public databases, we identified fourteen such polymorphisms. introduction of the respective mutations into dpp revealed that all except one (Δ - ) were compatible with robust dpp expression. four polymorphisms (k e, k n, a p and Δ - ) strongly reduced binding of mers-cov s to dpp and s protein-driven host cell entry, as determined using soluble s protein and s protein bearing rhabdoviral vectors, respectively. two polymorphisms (k e and a p) were analyzed in the context of authentic mers-cov and were found to attenuate viral replication. collectively, we identified naturally-occurring polymorphisms in dpp that negatively impact cellular entry of mers-cov and might thus modulate mers development in infected patients. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus with a single-stranded rna genome of positive polarity. it belongs to the coronaviridae family (genus betacoronavirus), which is part of the order nidovirales. mers-cov was isolated in from the sputum of a year old man suffering from acute pneumonia and renal failure in saudi arabia [ ] . since its discovery, mers-cov has caused , human infections of which ( . %) had a fatal outcome (as of may, ) [ ] . dromedary camels are reservoir hosts of mers-cov and display only common cold-like symptoms upon infection but constitute the main source of human infections. transmission to humans occurs via close contact to animals or contaminated animal products [ ] [ ] [ ] [ ] . human-to-human transmissions seem limited and were mainly observed in health care settings, leading to mers outbreaks in hospitals [ ] [ ] [ ] [ ] [ ] [ ] . finally, differences in the tissue specific expression of the cellular receptor for mers-cov, dpp , were recently suggested to account for the differences in mers-cov transmission and disease induction in camels and humans, respectively [ , ] . in order to infect a host (cell) and replicate, mers-cov has to deliver its genome into the cellular cytoplasm for gene translation and genome replication. this process is facilitated by the viral spike (s) glycoprotein, a type-i transmembrane protein embedded in the viral envelope. for host cell entry, the surface unit, s , of mers-cov s binds to the cellular type-ii transmembrane protein dipeptidyl peptidase (dpp , cd ) [ ] . the structure of the interface between dpp and mers-cov-s was resolved on the atomic level and fifteen residues in dpp were found to make direct contact with residues in the viral s protein [ ] . upon dpp engagement, mers-cov s undergoes proteolytic activation through the cellular serine protease tmprss or the endosomal cysteine protease cathepsin l [ ] [ ] [ ] , which allows the transmembrane unit, s , of mers-cov s to fuse the viral membrane with cellular membranes. dpp is a prolyl oligopeptidase that is expressed in various tissues [ ] and involved in multiple biological processes including t-cell activation [ ] , control of the activity of growth factors, chemokines and bioactive peptides [ ] [ ] [ ] , and regulation of the glucose metabolism [ ] . mature dpp is embedded in the plasma membrane as a homodimer and each monomer consists of an n-terminal cytoplasmic domain, followed by a transmembrane domain and a large ectodomain, which can be further subdivided into a short stalk domain, a glycosylation-rich and a cysteine-rich region as well as the c-terminal catalytic domain (α/ β-hydrolase domain) [ ] . polymorphisms in the dpp gene were implicated in several diseases and conditions, including diabetes [ , ] and myocardial infarction [ ] but their potential impact on mers-cov infection has not been analyzed. we asked whether naturally-occurring amino acid polymorphisms in dpp residues making contact with mers-cov s have an impact on mers-cov entry. we identified fourteen polymorphisms by screening public databases and introduced the respective mutations into a dpp expression plasmid. we identified four mutations that reduced mers-cov s binding to dpp and mers-cov s-driven host cell entry without affecting dpp expression at the cell surface. t cells were transfected with expression vectors for wt or mutant dpp , or empty expression vector (negative control). at h post transfection, the culture medium was replaced and the cells were further incubated for additional h. then, the cells were washed with pbs and mixed with x sds-sample buffer ( . m tris-hcl, % glycerol, % sds, . % bromophenol blue, mm edta). cell lysis was achieved by incubating the samples for min at room temperature followed by incubation at °c for an additional min. the samples were further loaded on polyacrylamide gels and sds-page (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed. next, the proteins were transferred onto nitrocellulose membranes (hartenstein gmbh) by immunoblotting. the membranes were further blocked by incubation in pbs-t (pbs containing . % tween and % skim milk powder) for min at room temperature. afterwards, the membranes were incubated overnight at °c with undiluted supernatant of a hybridoma cell line secreting anti-cmyc antibody e (for dpp detection) or pbs-t containing anti-ß-actin (actb) antibody (mouse, : , , sigma aldrich). following three washing intervals with pbs-t, the membranes were further incubated with pbs-t containing horseradish peroxidaseconjugated anti-mouse antibody (goat, : , , dianova) for h at room temperature before an in house-prepared enhanced chemiluminescent solution ( . m tris-hcl [ph . ], µg/ml luminol, mg/ ml para-hydroxycoumaric acid, . % h o ) was added and signals were recorded using the chemocam imaging system and the chemostar professional software (intas science imaging instruments gmbh). in order to quantify the signal intensity of the protein bands, the program imagej (fiji distribution) [ ] was used. to account for differences in the total protein content of the samples and variations, we normalized the dpp signals against the respective signals of the loading control (actb). t (human kidney cells, dsmz no. acc ), bhk- (hamster kidney cells, dsmz no. acc ) and vero (african green monkey kidney cells, kindly provided by andrea maisner, philipps-university marburg) were cultivated in dulbecco's modified eagle medium (pan-biotech) while caco- cells (human colorectal adenocarcinoma cells) were cultivated in minimum essential medium (thermofisher scientific). the media were supplemented with % fetal bovine serum (biochrom), u/ml of penicillin and . mg/ml of streptomycin (pan-biotech). all cell lines were incubated at °c and % co in a humidified atmosphere. for subcultivation and seeding, cells were washed with phosphate-buffered saline (pbs) and detached by incubation with trypsin/ edta solution (pan-biotech) (bhk- , vero and caco- ) or by resuspending the cells in culture medium ( t). transfection of t and bhk- cells was carried out by calcium-phosphate precipitation or with the help of icafectin- (in-cell-art) or fugene hd (promega). all dpp mutants were generated based on a pcdna . /zeo(+)-based expression vector in which the coding sequence for human dpp (genbank: xm_ . ) containing an c-terminal cmyc epitope was inserted into via bamhi/ecori restriction sites. the following aa (amino acid) substitutions were introduced via overlap-extension pcr: k e, k n, q k, t i, t s, a v, a p, a v, r k, y h, i t, i v and k n . in addition, a deletion mutant was generated that lacks aa residues - (Δ - ). information on dpp polymorphisms was retrieved from the ensembl database (https://www.ensembl.org/index.html) [ ] and the single nucleotide polymorphism database (dbsnp) of the national center for biotechnology information (ncbi) (https://www.ncbi.nlm.nih.gov/snp) [ ] , and is based on data provided by the gnomad database (genome aggregation database, https://gnomad. broadinstitute.org/), topmed program (trans-omics for precision medicine, https://www.nhlbiwgs.org/), exac consortium (exome aggregation consortium, http://exac.broadinstitute.org/) [ ] and the g project ( , genomes project, http://www. internationalgenome.org/) [ ] (for detailed information see supplementary table ) . we further utilized pcaggs-based expression vectors for vesicular stomatitis virus (vsv) glycoprotein (g), mers-cov s wildtype (wt) and mers-cov s (d g) (the latter two either untagged or equipped with a c-terminal v epitope) that have been described elsewhere [ ] [ ] [ ] . in addition, a previously described expression vector for angiotensin converting enzyme was employed [ ] . similar to the strategy used for the generation of dpp mutants, we employed the overlap-extension pcr technique to introduce a single mutation into the mers-cov s open reading frame, thus generating untagged and v -tagged mers-cov s (d n). soluble s comprising the s subdomain of mers-cov s (aa residues: - ) fused to a human igg fc tag was generated by inserting the pcr-amplified s sequences into the pcg fc vector [ ] (kindly provided by georg herrler, university of veterinary medicine hannover) making use of the bamhi/sali restriction sites. in addition, we generated an expression vector for the enhanced green fluorescent protein (egfp) by inserting the egfp coding sequence, which was pcr-amplified from the pegfp-c vector (clontech), into the pcaggs plasmid using the ecori/xhoi restriction sites. all pcr-amplified sequences were subjected to automated sequence analysis (microsynth seqlab) to verify their integrity. sequences of primers used for cloning of the different constructs are available upon request. analysis of dpp surface expression by immunofluorescence analysis bhk- cells were grown on coverslips and transfected with the different dpp constructs or empty expression vector using icafectin- (in-cell-art) at h post seeding according to the manufacturer's instructions. after changing the culture medium at h post transfection, the cells were incubated for additional h. then, the culture medium was aspirated and the cells were washed with pbs, before they were fixed by incubated with pbs containing % paraformaldehyde (pbs/pfa) for min at room temperature. subsequently, the cells were washed with . m glycine/ pbs solution followed by a washing step with pbs. next, the coverslips were incubated with anti-dpp antibody (mouse, diluted : in pbs containing % bovine serum albumin [pbs/bsa], abcam) for h at °c. for this, the coverslip was put on a drop ( µl) of antibody solution that was added on a sheet of parafilm inside a humidity chamber (a glass dish in which the parafilm was placed on wet paper tissue). thereafter, the cells were washed x with pbs before incubation with alexafluor -conjugated antimouse antibody (goat, : , diluted in pbs/bsa, thermofisher scientific) for min at °c was performed. subsequently, the cells were washed x with pbs. finally, the cells were incubated with dapi ( ', -diamidino- -phenylindole, carl roth) and mounted in prolong gold antifade mountant (ther-mofisher scientific) before they were analyzed using a zeiss lsm (zeiss) confocal laser scanning microscope and the zen imaging software (zeiss). analysis of dpp surface expression by flow cytometry bhk- cells were transfected with expression vectors for wt or mutant dpp , or empty expression vector (negative control). at h post transfection, the culture medium was replaced and the cells were further incubated for additional h. then, the cells were washed with pbs, resuspended in pbs/ bsa and pelleted by centrifugation ( x g, min, °c). after aspiration of the supernatant, the cells were resuspended in pbs/bsa containing anti-dpp antibody (mouse, diluted : , abcam) and incubated for h at °c. next, the cells were pelleted, washed with pbs/bsa, pelleted again, resuspended in pbs/bsa containing alexafluor -conjugated anti-mouse antibody (donkey, diluted : , thermo-fisher scientific) and incubated for h at °c. subsequently, the cells were washed (as described above) and resuspended in pbs/pfa for h at °c for fixation. finally, the cells were washed (as described above) and resuspended in pbs/bsa for flow cytometric analysis using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express flow research software (de novo software). for quantification of dpp surface expression, the mean fluorescence intensity (mfi) value of the negative control was subtracted from all samples. for normalization of dpp surface expression, values obtained for cells expressing dpp wt were set as % and the relative surface expression of the respective dpp mutants was calculated accordingly. in order to generate soluble mers-cov s for binding studies, t cells were transfected with an expression vector for the s subunit of mers-cov s fused to the fc fragment of human immunoglobulin g (solmers-s -fc). at h post transfection, the culture medium was exchanged and the cells were further incubated for h before culture supernatants were harvested and freed from cellular debris by centrifugation ( , x g, min, °c). the clarified supernatants were loaded on vivaspin protein concentrator columns with a molecular weight cut-off of kda (sartorius) and centrifuged at , x g at °c until the sample was -fold concentrated. for the binding studies with solmers-s -fc, a similar protocol was followed as described for the analysis of dpp surface expression with the exceptions that sol-mers-s -fc was used instead of the primary antibody ( : dilution in pbs/bsa) and that an alexafluor conjugated anti-human antibody (goat, : dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. bhk- cells transfected with expression vectors for wt or mutant dpp , ace or empty expression vector (both negative controls) were analyzed by flow cytometry for solmers-s -fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express flow research software (de novo software). for quantification of solmers-s -fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of solmers-s -fc to cells expressing dpp wt was set as % and the relative binding efficiencies to cells expressing the respective dpp mutants or ace were calculated accordingly. t cells (grown in -well plates) were cotransfected with expression plasmids coding for solmers-s -fc and wt or mutant dpp . cells transfected with empty expression vector instead of dpp or sol-mers-s -fc (or both) served as controls. at , x g at °c, before µl of the supernatant were mixed with µl of protein a-sepharose ( g protein a-sepharose [sigma-aldrich] in ml pbs) while the residual µl of the cell lysate were mixed with µl x sds-sample buffer and incubated for min at °c (these samples were later analyzed to confirm comparable total protein levels [via detection of actb] as well as comparable dpp and solmers-s -fc levels before the co-immunoprecipitation [co-ip] step.). following incubation of the lysate/protein a-sepharose mixtures for h at °c in an overhead shaker, the samples were centrifuged for min at , x g at °c to pellet the protein a-sepharose/solmers-s -fc/dpp -complexes. after aspiration of the supernatant, µl of np lysis buffer (without protease inhibitors) were added and the cells were mixed by vortexing, before being centrifuged again. this washing routine was repeated three times, before finally µl of x sdssample buffer were added to the pelleted complexes and the samples were further incubated for min at °c. thereafter, the samples were subjected to sds-page and western blot analysis (see above). detection of dpp (lysate and co-ip samples) and actb (lysate samples) was carried out as described above. solmers-s -fc was detected (lysate and co-ip samples) by incubation with a peroxidase-conjugated anti-human antibody (goat, : , , dianova). signal intensities of the protein bands were quantified as described above. further, signals obtained for dpp were normalized against the respective signals for solmers-s -fc in order to account for variations in transfection efficiency and sample processing. for the binding studies with soluble dpp , a similar protocol was followed as described for the analysis of binding of solmers-s -fc with the exceptions that a soluble dpp fused to the fc region of human igg (soldpp -fc, acro biosystems) was used instead of solmers-s -fc ( : dilution in pbs/bsa) and that an alexafluor -conjugated anti-human antibody (goat, : dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. t cells transfected with expression vectors for wt or mutant (d g and d n) mers-cov s, or empty expression vector (negative control) were analyzed by flow cytometry for soldpp -fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express flow research software (de novo software). for quantification of soldpp -fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of soldpp -fc to cells expressing mers-cov s wt was set as % and the relative binding efficiencies to cells expressing the respective mers-cov s mutants were calculated accordingly. we employed a previously described protocol for the generation of vsv pseudotype particles (vsvpp) that is based on a replication-deficient vsv vector that lacks the genetic information for vsv-g but instead contains the genetic information for egfp and firefly luciferase (fluc) as reporters of transduction efficiency (vsv*Δg-fluc, kindly provided by gert zimmer, institute of virology and immunology, mittelhäusern/switzerland) [ , ] . in brief, t cells transfected with expression vectors for mers-cov s, vsv-g (positive control) or empty expression vector (negative control) were inoculated with vsv*Δg-fluc for h before being washed with pbs and further incubated for h with culture medium that was supplemented with anti-vsv-g antibody (i , mouse hybridoma supernatant from crl- ; atcc) (except for cells expressing vsv-g). the produced vsvpp were inoculated onto bhk- cells expressing wt or mutant dpp , or no dpp (empty expression vector, negative control) and incubated for - h before fluc activity in cell lysates was quantified as an indicator for transduction efficiency using the beetle-juice kit (pjk) and a plate luminometer (hidex) [ ] . bhk- cells were transfected with expression vectors for wildtype or mutant dpp (k e or a p), or empty expression vector (negative control) using fugene hd (promega) according to the manufacturer's instructions. at h posttransfection, the cells were infected with mers-cov (human betacoronavirus c emc/ , mers-cov emc- , genbank accession number: jx ) at a multiplicity of infection of . for h. thereafter, the inoculum was removed and the cells were washed x with pbs before fresh medium was added and the first sample (time point h postinfection) was taken. the cells were further incubated and additional samples were taken at and h postinfection. viral titers in the culture supernatant were analyzed by quantitative reversetranscriptase pcr, using the upe assay according to a published protocol [ ] . in brief, viral rna was isolated from cell culture supernatant using the nucleospin rna virus kit (macherey-nagel), reversetranscribed into cdna using the superscript iii one step rt-pcr system (thermofisher scientific) and analyzed on a lightcycler qpcr cycler platform (roche) with primers and conditions as specified for the upe assay [ ] . in vitro-transcribed standard samples containing defined amounts of mers-cov fragments ( , , , and , copies) were included for absolute quantification as genome equivalents (ge). the dpp protein structure ( pv ) [ ] and the structure of the complex formed by the mers-cov s receptor binding domain bound to dpp ( l ) [ ] were retrieved from the research collaboratory for structural bioinformatics protein database (rscb pdb, https://www.rcsb.org/). structure visualization and colorization was performed using the yasara software (http://www.yasara.org/index.html) [ ] and ucsf chimera version . (developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco) [ ] . one-way or two-way analysis of variance (anova) with dunnett's posttest was used to test for statistical significance. only p values of . or lower were con- identification of polymorphisms in dpp that alter amino acid residues which make contact with mers-cov s the binding interface between mers-cov s and the cellular receptor dpp was resolved by wang and colleagues using crystallography, revealing the interacting amino acid residues for each binding partner [ ] : fourteen residues of mers-cov s (y , n , k , l , d , r , e , d g , d , y , r , w and v ) interact with a total of fifteen residues in dpp (k , f , q , t , a , a , l , i , h , r , y , r , q , i and k ) [ ] , which are distributed over the glycosylation-rich domain and the cysteinerich domain ( figure a-c) . in order to identify polymorphic residues in dpp that contact mers-cov s, we screened public databases that provide information on polymorphic amino acid residues based on data derived from different bio projects (i.e. gnomad, topmed, exac, g; more information is given in the materials and methods section). by this method we found that nine out of the fifteen dpp residues interacting with mers-cov s are polymorphic (k , q , t , a , a , r , y , i and k ) ( figure c ). while five of these residues can be replaced by only a single different amino acid residue (q [k], a . circles with sticks represent glycosylation sites, while small numbers indicate the amino acid residues. triangles below the domains highlight the positions of amino acid residues that directly interact with mers-cov s (grey triangles mark residues for which no polymorphism has been reported, while red triangles indicate polymorphic residues). (b) side (left) and top (right) view of homodimeric dpp (the dotted line indicates the border between the two monomers and the cellular plasma membrane is schematically depicted below the side view model of dpp ). the protein model was constructed on the published crystal structure ( pv ) deposited in rscb pdb and the binding interface with mers-cov s has been highlighted (green). (c) close-up on the dpp residues that directly interact with mers-cov s and for which no polymorphic (yellow) or polymorphic (red) residues have been reported. in addition, the specific residues in dpp (regular letters and numbers), including the respective polymorphic residues (letters in brackets), and the corresponding interacting residues in mers-cov s (italicized letters and numbers) are indicated. (d) frequency of polymorphic dpp residues in the human population. public databases (see supplementary table and the materials and methods section for detailed information) were screened for the frequency of the polymorphic residues under study (y-axis). error bars indicate standard error of the mean (sem) and refer to polymorphic residues found in more than one database. table ). finally, the frequency of these polymorphisms in the human population is low, ranging roughly from : , (a v) to : , (t i) ( figure d and supplementary table ). we next introduced the polymorphisms in a dpp expression plasmid. western blot analysis and signal quantification revealed that all resulting dpp variants were robustly expressed and total expression levels were comparable (figure a-b) . as dpp needs to be transported to the plasma membrane to be engaged by mers-cov s for host cell entry, we next investigated whether the presence of the polymorphic dpp residues has an impact on dpp cell surface localization. for this, we performed flow cytometry and confocal laser scanning microscopy, using transfected bhk- cells and an antibody targeting the dpp ectodomain. we found that all dpp variants but one, a deletion variant lacking amino acid residues - (Δ - ), displayed comparable cell surface expression levels ( figure a-b) . we next investigated whether polymorphic dpp residues impact mers-cov host cell entry. for this, we made use of vesicular stomatitis virus (vsv) pseudotypes (vsvpp) bearing mers-cov s or vsv g, which does not bind to dpp and served as negative control [ ] . as expected, vsvpp harboring vsv g were able to efficiently transduce bhk- target cells irrespective of dpp expression. in contrast, transduction of bhk- cells mediated by mers-cov s critically depended on ectopic expression of human dpp , in accordance with published findings [ ] ( figure ) . notably, four dpp polymorphisms -k e, k n, a p and Δ - -severely reduced mers-cov s-driven transduction compared to dpp wt (figure ). in order to analyze whether the reduction in mers-cov s-driven host cell entry would translate into attenuated mers-cov replication, we next investigated two dpp polymorphisms (k e and a p) in the context of infection with authentic mers-cov. when followed over a period of two days post infection it was observed that mers-cov replication in bhk- cells expressing human dpp was significantly reduced when dpp contained either k e or a p ( figure ). after the identification of dpp polymorphisms that reduce s-driven cellular entry of rhabdoviral vectors as well as mers-cov replication, we next sought to investigate whether the attenuating phenotype was due to reduced binding of mers-cov s to dpp . for this, we used soluble mers-cov s, produced by fusing the s subunit, which contains the dpp binding domain, to the fc portion of human immunoglobulin g (solmers-s -fc). co-immunoprecipitation analysis demonstrated that dpp variants harboring polymorphisms k e, k n or a p, which were not compatible with efficient mers-cov s-driven host cell entry, displayed significantly reduced ability to interact with mers-cov s as indicated by weaker dpp signals upon protein a-sepharosemediated pull-down of dpp /solmers-s -fc (as compared to dpp wt, figure a-b) . notably, dpp variant Δ - could be as efficiently coimmunoprecipitated as dpp wt, indicating that its inefficient receptor function was solely due to its defect in proper surface transport. the findings obtained by co-ip analysis were confirmed by flow cytometry. it was revealed that polymorphisms that reduced mers-cov s-driven host cell entry (k e, k n, a p and Δ - ) and spread of authentic mers-cov (k e and a p) also reduced mers-cov s binding to cells expressing dpp on the cell surface ( figure c ). in addition, polymorphism a v, which decreased mers-cov s-driven transduction to a lesser extent than the aforementioned polymorphisms (figure ) , also reduced mers-cov s binding to dpp . thus, dpp polymorphisms k e, k n and a p reduce mers-cov s-driven host cell entry and mers-cov infection by diminishing mers-cov s binding to dpp . host cell entry of mers-cov critically depends on the interaction between the viral s protein and the cellular receptor dpp . a link between obesity or underlying diseases like diabetes mellitus, which both can affect dpp expression levels [ ] , and the risk of fatal outcome of mers-cov infection has been made [ ] . moreover, alanine scanning mutagenesis identified dpp residues critical for mers-cov entry, including k , l , i , r and r [ , ] . however, the impact of natural-occurring variations on host cell entry of mers-cov has not been addressed so far. we identified dpp polymorphisms that reduce s protein-driven host cell entry and replication of authentic mers-cov by lowering the binding efficiency of mers-cov s to dpp , suggesting that the dpp phenotype may impact the course of mers-cov infection. western blot analysis, flow cytometry and confocal laser scanning microscopy revealed that none of the polymorphisms studied, except deletion of amino acids - , had a significant impact on total or cell surface expression of dpp , at least in the context of dpp transfected cells. four polymorphisms located at three different sites in dpp (k e, k n, a p and Δ - ) severely reduced s protein-driven host cell entry. as dpp Δ - was shown to be incompatible with robust cell surface transport but able to interact with mers-cov s in co-ip analysis, we conclude that the reduction in entry efficiency is solely due to insufficient dpp surface levels. in contrast, reduction of host cell entry by k e, k n and a p could not be explained by reduced dpp expression and these polymorphisms were thus further investigated. mers-cov infection of bhk- cells transfected to express dpp wt and variants k e or a p revealed that k e or a p were not compatible with robust mers-cov replication. finally, co-ip analyses and binding studies with soluble mers-cov s showed that these dpp polymorphisms reduced s protein binding to dpp . when looking at the crystal structure of the complex consisting of the mers-cov s receptor binding domain bound to dpp , these observations do not come as a surprise. dpp residue k has been reported to contact mers-cov s residues g and d , including a salt bridge interaction with d [ ] . the exchange of k to either glutamate (e) . whole cell lysates (wcl) were prepared and analyzed for dpp expression by sds-page under non-reducing conditions and wb using a primary antibody targeting the c-terminal cmyc epitope and a peroxidase-conjugated secondary antibody. further, expression of beta-actin (actb) was analyzed as a loading control. shown are the expression data from a representative experiment. numbers at the left indicate the molecular weight in kilodalton (kda). (b) quantification of total dpp expression in wcl. after normalization of dpp band intensities with that of the corresponding actb bands. dpp wt expression was set as % and the relative expression of mutant dpp was calculated accordingly. presented are the combined data of three independent experiments with error bars indicating the sem. no statistical significance for differences in total expression between wt and mutant dpp was observed by one-way analysis of variance with dunnett's posttest (p > . , not significant [ns]). . surface expressed dpp was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the dpp ectodomain and an alexafluor -conjugated secondary antibody. fluorescent signals representing surface-expressed dpp were analyzed by flow cytometry and the mean fluorescence intensity (mfi) values for each sample were calculated. for normalization, the mfi value of the negative control was subtracted from all samples. further, surface expression of dpp wt was set as % and the relative surface expression of the dpp mutants was calculated accordingly. shown are the combined data of three experiments with error bars indicating the sem. statistical significance for differences in surface expression between wt and mutant dpp was tested by one-way analysis of variance with dunnett's posttest (p > . , not significant; p ≤ . , *). (b) dpp surface expression was further analyzed by immunofluorescence analysis. for this, dpp wt or dpp mutants were expressed in bhk- cells grown on coverslips (cells transfected with empty expression vector served as negative control). after fixation of the cells, surface expressed dpp was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the dpp ectodomain and an alexafluor -conjugated secondary antibody. in addition, cellular nuclei were stained with dapi. finally, images were taken using a confocal laser scanning microscope at a magnification of x. or asparagine (n) likely abolishes/decreases the interaction with mers-s due to the different biochemical properties of k (positively charged, basic) versus e (negatively charged, acidic) and n (not charged, acidic) (supplementary figure ) . for dpp residue a , which has been reported to contact the mers-cov s residue e , no information on the type of interaction is available [ ] . here, we speculate that the bulky and distorted side chain of proline (in comparison to the small side chain of alanine) abolishes/decreases interaction with mers-cov s residue e (supplementary figure ) . in contrast to that, valine contains a small side chain and also has identical biochemical properties as alanine and thus might be efficiently contacted by e of mers-cov s, which is why we did not observe any impact of polymorphisms a v on mers-cov s-driven entry and mers-cov s mers-cov s binding/interaction (supplementary figure ). the observation that certain polymorphisms in dpp reduced mers-cov s binding and viral entry triggered the question whether residues in mers-cov s that are in direct contact with the respective dpp residues are also polymorphic. indeed, we obtained initial evidence to support such a concept. thus, we found that residue in mers-cov s which contacts dpp residue is polymorphic, with certain mers-cov variants harboring an asparagine instead of an . reduced mers-cov s-driven host cell entry is caused by inefficient s protein binding to dpp harboring polymorphic amino acid residues. in order to investigate whether reduced mers-cov s-driven host cell entry and mers-cov replication is due to inefficient mers-cov s binding to dpp harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-ip) as well as binding experiments with a soluble s protein comprising the s subunit of mers-cov s fused to the fc region of human igg. (a) t cells were cotransfected with expression plasmids coding for soluble, fc-tagged mers-cov s (solmers-s -fc) and the indicated dpp variant containing a c-terminal cmyc-tag. cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solmers-s -fc or dpp served as controls. at h posttransduction, cells were lysed and incubated with protein a sepharose. next, samples were subjected to sds-page and western blot analysis. dpp levels were detected via antibodies specific for the cmyc-tag, whereas solmers-s -fc was detected using a peroxidase-coupled anti-human antibody. similar results were obtained in three individual experiments. analysis of whole cell lysates (wcl) for expression of solmers-s -fc, dpp and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solmers-s -fc and dpp . (b) for quantification of mers-cov s/dpp interaction we first normalized the dpp signals against the respective solmers-s -fc signals. then, mers-cov s/dpp interaction was set as % for wildtype (wt) dpp and the relative interaction efficiency for each dpp mutant was calculated accordingly. presented are the mean data from three independent experiments. error bars indicate the sem. statistical significance of differences in mers-cov s/dpp interaction between wt and mutant dpp was analyzed by one-way analysis of variance with dunnett's posttest (p > . , ns; p ≤ . , *; p ≤ . , ***). (c) soluble mers-cov s -fc was incubated with bhk- cells expressing wildtype (wt) or mutant dpp , or cells transfected with empty expression vector or an ace -expression plasmid (controls). to detect bound s protein, the cells were subsequently incubated with an alexafluor -conjugated anti-human antibody directed against the fc-tag. fluorescent signals representing bound solmers-s -fc were analyzed by flow cytometry and mfi values for each sample were calculated. for normalization, the mfi value of the negative control (empty expression vector) was subtracted from all samples. further, binding of sol-mers-s -fc to cells expressing dpp wt was set as % and the relative binding to cells expressing the dpp mutants was calculated accordingly. shown are the combined data of five independent experiments with error bars indicating the sem. statistical significance of differences in solmers-s -fc binding to cells expressing wt or mutant dpp was analyzed by one-way analysis of variance with dunnett's posttest (p > . , ns; p ≤ . , *; p ≤ . , **; p ≤ . , ***). aspartate residue at this position. d n reduced entry into cells expressing relatively low amounts of dpp but had no effect on entry into cells expressing high amounts of dpp (supplementary figure ) . moreover, and more interestingly, d n slightly rescued mers-cov s-driven entry from the negative effect exerted by dpp polymorphism k n (supplementary figure ) . similarly, residue in mers-cov s, which is known to interact with dpp residues and , was found to be polymorphic, and previous studies demonstrated that polymorphism d g reduced dpp binding but also increased resistance to neutralizing antibodies [ ] . notably, d g slightly increased entry via dpp harboring polymorphism y h and allowed mers-cov s to use dpp with polymorphism r k with the same efficiency as wt dpp . it should be stated that none of these effects was statistically significant and that dpp and mers-cov s polymorphisms occur with low frequency. although it is unlikely that the dpp polymorphisms have emerged as a result of evolutionary pressure from mers-cov infections, our results suggest that certain existing dpp polymorphism(s) might foster the emergence of mers-cov variants with altered biological properties. the polymorphisms studied here occur with relatively low frequencies of one per ∼ , (a v) to ∼ , (t i) individuals. however, detailed information on the geographic distribution or incidence in certain ethnical groups is largely missing. thus, dpp polymorphisms could contribute to the perplexing absence of mers cases in africa, where the virus circulates in camels [ ] [ ] [ ] [ ] [ ] [ ] . however, recent evidence suggests that sequence variations between african and arabian mers-cov might be a factor [ , ] . more importantly, it remains to be analyzed how frequent dpp polymorphisms that affect s protein binding occur in the middle east and whether they are associated with the clinical course of mers. isolation of a 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coronavirus receptor in the upper respiratory tracts of humans and dromedary camels dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptor-binding domain complexed with human receptor dpp the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss , and is targeted by neutralizing antibodies role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss physiology and pharmacology of dpp- in glucose homeostasis and the treatment of type diabetes revisiting an old acquaintance: cd and its molecular mechanisms in t cell function dipeptidyl-peptidase iv (cd )-role in the inactivation of regulatory peptides cd / dipeptidylpeptidase iv-chemokine interactions: double-edged regulation of inflammation and tumor biology inhibitors of dipeptidyl peptidase iv (dp iv, cd ) induces secretion of transforming growth factor-beta (tgf-beta ) in stimulated mouse splenocytes and thymocytes dpp- inhibitors and their potential role in the management of type diabetes cut to the chase: a review of cd /dipeptidyl peptidase- 's (dpp ) entanglement in the immune system association of dpp gene polymorphisms with type diabetes mellitus in malaysian subjects association between dpp gene polymorphism and serum lipid levels in chinese type diabetes individuals polymorphisms in dipeptidyl peptidase iv gene are associated with the risk of myocardial infarction in patients with atherosclerosis fiji: an open-source platform for biological-image analysis ensembl variation resources. database (oxford) dbsnp: the ncbi database of genetic variation analysis of protein-coding genetic variation in , humans a global reference for human genetic variation the glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses sialic acid binding properties of soluble coronavirus spike (s ) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon the hemagglutinin of bat-associated influenza viruses is activated by tmprss for ph-dependent entry into bat but not human cells detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction discovery of dipeptidyl peptidase iv (dpp ) inhibitors based on a novel indole scaffold yasara: a tool to obtain structural guidance in biocatalytic investigations ucsf chimera-a visualization system for exploratory research and analysis ldl receptor and its family members serve as the cellular receptors for vesicular stomatitis virus host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase high circulating plasma dipeptidyl peptidase- levels in non-obese asian indians with type diabetes correlate with fasting insulin and ldl-c levels, triceps skinfolds, total intra-abdominal adipose tissue volume and presence of diabetes: a case-control study epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study identification of residues on human receptor dpp critical for mers-cov binding and entry dipeptidyl peptidase- levels are increased and partially related to body fat distribution in patients with familial partial lipodystrophy type middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses from camels in africa exhibit region-dependent genetic diversity antibodies against mers coronavirus in dromedary camels mers coronavirus neutralizing antibodies in camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate emc we are grateful to thank g. herrler, a. maisner and g. zimmer for providing plasmids and reagents. we further thank a.-s. moldenhauer for excellent technical support. no potential conflict of interest was reported by the authors. this work was supported, including the efforts of stefan pöhlmann and christian drosten, by the bundesministerium für bildung und forschung [grant numbers ki d and ki a], network project rapid (risikobewertung bei präpandemischen respiratorischen infektionserkrankungen). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord- - p qxb authors: jiang, shibo; du, lanying; shi, zhengli title: an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: p qxb nan . both bat-sl-cov zc and zxc were found in chinese horseshoe bats (rhinolopus sinicus) in zhoushan city of zhejiang province, china between and [ ] , which can infect suckling rats and cause disease. given that there were some bats and live animals in the seafood market, -ncov may be originated from bats or live animals exposure to the materials contaminated with bat droppings in the seafood market or surrounding area. the rapid identification of this novel coronavirus is attributed to recent advances in the detection of respiratory virus infection, including reverse transcription pcr (rt-pcr), real-time reverse transcription pcr (rrt-pcr), reverse transcription loop-mediated isothermal amplification (rt-lamp), and real-time rt-lamp as well as multiplex nucleic acid amplification and microarray-based assays [ ] . these methods are useful for detecting novel coronaviruses not only in humans, but also in animals for identification of animal reservoir or intermediate host of -ncov. who recommended that if there is no clue about the putative pathogen from the pneumonia outbreak, a pan-coronavirus assay should be used for amplification followed by sequencing of the amplicon for characterization and confirmation (https://apps.who.int/iris/bitstream/ handle/ / /who- -ncov-laboratory- . -eng.pdf). by aligning -ncov s protein sequence with those of sars-cov and several bat-sl-covs, we predicted that the cleavage site for generating s and s subunits is located at r /s ( figure ). s subunit contains two functional domains, the n-terminal domain (ntd) and a receptor-binding domain (rbd), both of which are responsible for the binding of the virion to the receptor on the host cell. they also contain several conformational neutralizing epitopes, serving as a target for developing neutralizing antibodies and vaccines [ ] . s subunit contains three functional domains, fusion peptide (fp), and heptad repeat (hr) and . after binding of rbd in s to the receptor, the s subunit changes conformation by inserting the fp into the host cell membrane and association between hr and hr to form six-helical bundle ( -hb), resulting in the fusion between viral and cellular membranes. the viral genetic materials enter into the host cell through the fusion pore for replication in the cell [ ] . a peptide derived from the hr domain of sars-cov s protein (sc- ) can interact with hr region in viral s protein to form heterologous -hb, resulting in the inhibition of homologous -hb formation between hr and hr domains in viral s protein and thus blocking the viral fusion with the host cell [ ] . since -ncov s-hr sequence is % identical to that of sars-cov, while there are only a few mutations of non-critical amino acids in s-hr region, sc- peptide is expected to be also effective against -ncov infection. we have recently designed and engineered a pan-cov fusion inhibitor, ek peptide, which could inhibit infection of five human coronaviruses, including sars-cov and mers-cov, and three bat-sl-covs [ ] . intranasal application of ek peptide before or after viral challenge, ek peptide can protect human dpp -transgenic mice from mers-cov infection, suggesting its potential prophylactic and therapeutic effect against -ncov infection. once confirmed, we will develop ek peptide as a t prophylactic or therapeutic for intranasal application to prevent or treat infection by -ncov and other emerging coronaviruses in the future. the rbds of sars-cov and mers-cov contain multiple conformation-dependent neutralizing epitopes that induce more potent neutralizing antibodies and protective efficacy against sars-cov and mers-cov infections, respectively, than other regions in s protein [ , , ] . modification of mers-cov s-rbd amino acid residues based on the structure design could improve its protection against mers-cov infection [ ] , suggesting that -ncov s-rbd or modified s-rbd of other coronavirus may be applied for developing -ncov vaccines. of course, the rbd-containing s and s of a coronavirus, e.g. -ncov, can also be used for vaccine development [ ] . the recently developed sars-cov and mers-cov neutralizing monoclonal antibodies (mabs) and nanobodies with protective efficacy are specific to the s subunit of s protein, particularly the rbd [ , [ ] [ ] [ ] . therefore, the -ncov s-rbd is anticipated to be a key target for developing -ncov neutralizing mabs. the neutralizing mabs targeting non-rbd regions, including ntd and s of sars-cov and/or mers-cov s could also be identified [ , , , ] , although their neutralizing potency is generally lower than that of rbd-specific mabs. it may take several months or even years for researching and developing neutralizing antibodies against -ncov infection. one of the rapid approaches is to evaluate the currently available sars-cov neutralizing antibodies with cross-neutralizing and protection activity against -ncov infection. we have shown that sars-cov s-rbd-specific neutralizing mabs and sera could cross-neutralize bat-sl-covs, such as bat-sl-cov-w v and bat-sl-cov-shc [ ] , suggesting that they may also cross-neutralize -ncov. once identified, these cross-neutralizing antibodies can be promptly developed for urgent prevention and treatment of -ncov infection. outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle genomic characterization of the novel human-pathogenic coronavirus isolated from patients with acute respiratory disease in wuhan genomic characterization and infectivity of a novel sars-like coronavirus in chinese bats recent advances in the detection of respiratory virus infection in humans the spike protein of sars-cov-a target for vaccine and therapeutic development interaction between heptad repeat and regions in spike protein of sarsassociated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors a pan-coronavirus fusion inhibitor targeting the hr domain of human coronavirus spike mers-cov spike protein: a key target for antivirals advances in mers-cov vaccines and therapeutics based on the receptor-binding domain introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines evaluation of candidate vaccine approaches for mers-cov a novel neutralizing monoclonal antibody targeting the n-terminal domain of the mers-cov spike protein cross-neutralization of sars coronavirus-specific antibodies against bat sars-like coronaviruses we thank dr ben hu at wuhan institute of virology, chinese academy of sciences, wuhan, china for phylogenetic analysis of -ncov s gene. no potential conflict of interest was reported by the author(s). this work was partially supported by the nih r ai to l.d. shibo jiang http://orcid.org/ - - - lanying du http://orcid.org/ - - - zhengli shi http://orcid.org/ - - - x key: cord- - pgy authors: yang, yue-lin; meng, fandan; qin, pan; herrler, georg; huang, yao-wei; tang, yan-dong title: trypsin promotes porcine deltacoronavirus mediating cell-to-cell fusion in a cell type-dependent manner date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: pgy porcine deltacoronavirus (pdcov) is a newly emerging threat to the global porcine industry. pdcov has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. here, we systematically investigated the role of trypsin in pdcov replication including cell entry, cell-to-cell membrane fusion and virus release. using pseudovirus entry assays, we demonstrated that pdcov entry is not trypsin dependent. furthermore, unlike porcine epidemic diarrhea virus (pedv), in which trypsin is important for the release of virus from infected cells, pdcov release was not affected by trypsin. we also demonstrated that trypsin promotes pdcov replication by enhancing cell-to-cell membrane fusion. most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during pdcov transmission, and the role of trypsin in pdcov replication in cells with different virus spreading types. overall, these results clarify that trypsin promotes pdcov replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. this knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments. porcine deltacoronavirus (pdcov) (genus deltacoronavirus; family coronaviridae) is a newly emerging swine pathogen [ ] [ ] [ ] [ ] . acute cases of pdcov infection exhibit watery diarrhea in sows and nursing piglets, resulting in severe gastrointestinal disease which may have a lethal outcome [ ] [ ] [ ] . pdcov poses a major threat to the swine industry, and is currently epidemic in several countries; first reported in hong kong in [ ] , it has since been found in the united states [ ] [ ] [ ] , canada [ ] , south korea [ ] [ ] [ ] [ ] , mainland china [ ] [ ] [ ] [ ] , thailand [ ] [ ] [ ] and vietnam [ ] . importantly, porcine aminopeptidase n (papn) has been reported to serve as a functional receptor for pdcov in two recent studies [ , ] , and the virus may engage apn from diverse species to facilitate its interspecies transmission [ ] . recently, pdcov has been reported to successfully infect chickens and calves [ , ] . thus, pdcov must be studied more extensively to better understand its emergence, lifecycle, evolution and pathogenesis in order to facilitate future control of the virus. despite the many reports of pdcov outbreaks, very few viruses have been successfully recovered, showing the difficulty of virus isolation [ , ] . pdcov was first isolated in swine testicular (st) and llc porcine kidney (llc-pk) cells by adding trypsin or pancreatin [ ] . although trypsin was used for pdcov isolation and propagation, its role in the virus lifecycle remains unclear. to address this aspect, we evaluated the importance of trypsin for the pdcov infection in two different cell lines (llc-pk and st). we developed a pdcov pseudotype virus system to investigate the impact of trypsin on viral entry. our findings indicate that pdcov entry was not promoted by trypsin. we further illustrate that virus release was also not influenced by this protease. our findings provide evidence that trypsin plays an important role in pdcov-mediated cell-to-cell membrane fusion, which facilitates virus spread. the st cell line (swine testicle; atcc crl ), llc-pk cell line (porcine kidney; atcc cl- ), hek t and hek (human embryonic kidney) cells were maintained in dmem (gibco, usa) with % foetal bovine serum (hyclone, usa). the trypsin used in this study was purchased from gibco (lot: ). the hek -apn cell line (stably expressing papn) was generated by the piggybac (pb) transposon system [ ] . papn was amplified by pcr including a flag tag in the forward primer (f: catagaagattctagacaccatggattaca-aggacgacgatgacaaggccaagggattctacatttc, r: atttaaattcgaattcttagctgtgctctatgaacca) and then cloned into the pb b vector to generate pb b-apn (system biosciences, mountain view, usa) [ ] . then, hek cells were co-transfected with μg pb b-apn and μg helper vector expressing pb transposase (system biosciences, mountain view, usa). forty eight hours later, cell media were replaced with growth media containing µg/ml puromycin, (gibico, usa) and replaced every days. the pdcov s gene was cloned into pcaggs-ha by the following primers with ecor i and xho i (f: ctgaattcctcgagatgcagagagctc, r: aactcgagctaccattccttaaacttaaagg). pdcov chinese "hunan" strain was used as in our previous described study [ ] . pdcov was isolated and prepared in llc-pk cells (less than passages) in the presence of μg/ml trypsin and without foetal bovine serum. pdcov in the current study was passaged fewer than times, and titred by plaque assay in st cells. briefly, when st cells reached up to % confluence, they were washed with pbs three times and subsequently infected with pdcov in the presence of μg/ml trypsin. two hours later, the cells were overlaid with % low-melting agarose and maintained with μg/ml trypsin in dmem at °c with % co for - days. the cells were then stained with . % crystal violet, and the plaques were counted. the pdcov pseudovirus was produced in hek t cells as previously described [ ] . briefly, hek t cells were seeded in -well plates and when cell confluency reached - %, hiv- based luciferase reporter plasmids were co-transfected (by calcium phosphate) with the helper plasmids pspax (addgene, usa) and pdcov-s to generate pseudotyped viruses. after h, cells were washed with pbs and then serum-free medium was added. the pseudovirus in the supernatant was collected at h posttransfection, and μl was used to infect llc-pk and st cells. these were washed and subjected to luciferase analysis at h post-infection (hpi). llc-pk and st cells were seeded in -well plates, and when they reached % confluence, cells were infected at moi = . of pdcov in the presence of indicated trypsin concentrations ( , , and μg/ml) at °c with % co . two hours later, the cells were washed three times with pbs, and rna was extracted and quantified by qpcr as described previously [ ] . releasing assay assay . llc-pk and st cells were infected with pdcov at a multiplicity of infection (moi) of in the presence or not of trypsin, and the virus released to the supernatant was collected at and hpi. samples were centrifuged at , × g for min at °c to remove cell debris, and centrifuged again at , × g for h at °c to pellet the virions. meanwhile, the virus-infected cells were washed once with pbs and then lysed in radio immunoprecipitation assay (ripa) lysis buffer containing a protease inhibitor cocktail (roche, usa). floating and necrotic cells were centrifuged at × g for min at °c, and pelleted cells were included in the experiment. n proteinspecific antibody was prepared and stored in our lab. the virions in both the supernatant and cell lysate were analyzed by western blot. assay . llc-pk cells were infected with pdcov (moi = . and ) in μg/ml trypsin for h, and the cells were further cultivated without trypsin for h, then infected cells were treated with indicated concentration ( and μg/ml) of trypsin at °c for min. floating and necrotic cells were centrifuged at × g for min at °c, and pelleted cells were included in the experiment. virus titre was quantified by plaque assay as described above. immunofluorescence assay llc-pk and hek -apn cells were plated in -well plates, and when confluency reached %, cells were washed three times with pbs and infected with pdcov at different moi in the presence or not of trypsin. after h, cells were fixed in % paraformaldehyde for h, washed three times with pbs and then permeabilized with . % triton x- for h. after washing with pbs three times, cells were blocked with % bsa for h, then incubated for h at room temperature with a monoclonal antibody specific for the pdcov n protein. alexa fluor -conjugated goat anti-mouse igg (sigma, usa) was used as the secondary antibody; for nuclear visualization, cells were stained with dapi (sigma, usa). cell-to-cell membrane fusion assay hek -apn cells were first plated in -well plates, and when confluency reached %, cells were transfected with the indicated plasmids: hek -apn effector cells were co-transfected with μg pgl -luc (promega, usa) and μg pdcov-s; target cells were transfected with μg pbind-id (promega, usa) and μg pact-myod (promega, usa). pbind-id and pact-myod generate fusion proteins containing the dna-binding domain of gal and the activation domain of vp , respectively. the pgl -luc vector contains five gal binding sites upstream of a minimal tata box, which in turn, is upstream of the firefly luciferase gene. pbind-id and pact-myod collaborate to initiate firefly luciferase expression of the pgl -luc vector only if cell fusion occurs. after h, both effector and target cells were detached with trypsin and washed with pbs for three times then the pellet was resuspended with culture medium and mixed at a : ratio, and seeded into fresh -well plates. after attachment, medium was replaced with or without trypsin, and luciferase activities were measured after two days of co-cultivation. after seeding in -well plates and the confluency of each cells reached around %, pdcov was used to infect llc-pk (moi = . , and ) and st cells (moi = , and ), washed twice with pbs at hpi, and then medium supplemented or not with μg/ml trypsin was added. infected cells were lysed and subjected to western blot at , and hpi. cleavage assay of s protein in virions: pdcov virions were purified by centrifugation at , × g for h at °c, and virions were incubated with the indicated concentrations ( , , , μg/ml) of trypsin at °c for h. n protein was used as a virus loading control. cleavage assay of s protein in virus infected cells: llc-pk and st cells were infected with pdcov (moi = . and , respectively) in μg/ml trypsin, and incubated for h in order to increase virus replication and bring s protein to a detectable level. then, the cells were further cultivated without trypsin for h, and both cell types were treated with the indicated concentrations ( , , , μg/ml) of trypsin at °c for h. floating and necrotic cells were centrifuged at × g for min at °c, and pelleted cells were included in the experiment. n protein was used as a virus loading control. llc-pk cells of . × were seeded in a -mm petri dish, and when the cells reached confluence, they were inoculated with pdcov at moi = in μg/ml of trypsin and incubated at °c in % co . these virus-infected cells were defined as effector cells. other llc-pk cells were seeded in -well plates at a density of . × cells/well for h, and then labelled with cell tracker dye deep red (invitrogen), which can label the cytoplasm of living cells. these naïve, pre-labelled cells were defined as target cells. at hpi, the effector cells were detached and washed with fresh culture medium twice to remove residual trypsin. afterwards, the collected effector cells were added directly to the target cells already growing in -well plates (contact cell model). simultaneously, the same number of effector cells as mentioned above were seeded on trans-well filters (corning, . mm, . μm pore size) at a density of . × cells. the filters were suspended in wells in a -well plate already containing target cells (uncontact cell model). in both infection models, medium supplemented or not with μg/ml trypsin was added. after h of interaction between effector and target cells, infection in target cells was detected as the presence of viral n protein by immunofluorescence assay, and both target and effector cells were collected for viral titration. origin graphpad prism . software was used for all graphical representations. statistical significance was analyzed by one-way-anova and tukey's multiple comparison test or the independent student's t-test. all p values < . were considered statistically significant. in previous studies, pdcov was successfully isolated in st or llc-pk cells by adding trypsin to the medium [ , , ] . however, the mechanism for how trypsin promotes pdcov replication is unknown. to explore whether it is essential for pdcov replication, we first infected llc-pk cells at a low virus/cell ratio (moi = . ) and determined the virus yield in the presence or absence of trypsin by western blot at different times post-infection. only a faint band of viral n protein was detectable at hpi. pdcov n protein production was significantly enhanced at or hpi in trypsin-treated samples as compared to the untreated control (figure (a) ). in the absence of the exogenous protease, only a weak band of n protein was detected at hpi, consistent with previous reports [ , ] . we further quantified viral titre by qpcr as described previously [ ] , revealing that trypsin significantly promoted pdcov replication in llc-pk cells at hpi (figure (b) ). next, we evaluated whether trypsin was essential for pdcov replication in st cells. it was difficult to detect n protein at a low moi (data not shown), and increasing the infectious dose up to moi = resulted in detection of only a weak band (figure (c) ). however, to our surprise, pdcov replication was no different in st cells regardless of the presence or absence of trypsin ( figure (c, d) ). taken together, these results indicate that trypsin significantly promotes pdcov replication in llc-pk cells but not in st cells. to elucidate which step of the viral replication cycle is being affected by trypsin in llc-pk or st cells, we first considered the initial stage of infection. to assay viral entry, we used a pseudovirus approach in llc-pk. briefly, μl of lentivirus-based pseudovirus containing the s protein of pdcov was incubated with each of the two cell types for h in the presence or absence of trypsin, washed three times with pbs and subjected to luciferase analysis. vsv-g pseudovirus was used as a positive control, and non-enveloped packaging group as a negative control. there was no significant difference in luciferase activity in the presence or absence of trypsin treatment in llc-pk cells ( figure (a) ). we got a similar result when st cells were infected with pseudovirus ( figure (b) ). this result indicates that trypsin treatment does not promote pdcov-s protein-mediated entry of pseudoviruses into llc-pk and st cells. next, we wanted to know whether trypsin treatment influences entry of real virus, and whether pdcov entry was influence by various concentrations of trypsin. llc-pk cells and st cells first were infected at moi = . with pdcov in the presence of the indicated concentration of trypsin, and at hpi, entry of pdcov was quantified by qpcr. the results indicated that entry of real pdcov into both cell lines was not influenced by trypsin, despite increasing the concentration of trypsin up to μg/ml in both cells (figure (c, d) ). cleavage of the coronavirus s protein by trypsin always plays a determinant role in virus entry. to test whether s protein was cleaved by trypsin in the current study, we first purified pdcov virions and then treated them with different concentrations ( , , , μg/ml) of trypsin at °c for h. we did not obviously detect s protein cleavage (figure (e)); thus, we think trypsin is not involved in the pdcov entry process in llc-pk or st cells. next, we analyzed whether trypsin supports the egress of pdcov from infected cells, as has been shown previously for pedv infection [ ] . to this end, we infected llc-pk and st cells at a high multiplicity (moi = ) to limit cell-to-cell spread of infection. we also demonstrated that when llc-pk and st cells were infected at a low multiplicity, the pdcov virus was more prone to cell-to-cell transmission rather than releasing the viruses ( figure s ). in order to differentiate between intracellular virus and virus released from infected cells, the cell lysates and supernatants were collected separately. the amount of virus in cell lysates or in the supernatant fraction at and hpi was not significantly altered by the presence of trypsin ( μg/ml) in either cell type (figure (a, b) ). to further confirm this, we performed a release assay in llc-pk cells as described previously [ ] . llc-pk cells were infected with pdcov (moi = . and ) with trypsin for h (to increase virus replication), the cells were further cultivated without trypsin for h, then both cells were treated with the indicated concentrations ( and μg/ml) of trypsin at °c for min. there was no significant difference in intracellular virus titre (figure (c, e) ) or titre in the supernatant (figure (d, f) ), regardless of trypsin treatment. these results indicate that unlike pedv, the release of pdcov is not substantially enhanced by the addition of trypsin [ ] . trypsin enhances pdcov cell-to-cell spread in llc-pk cells by promoting membrane fusion next, we investigated whether trypsin promotes pdcov replication by inducing cell-to-cell membrane fusion. we infected llc-pk cells at an moi of , and stained infected cells with antibodies directed against the n protein at hpi. pdcov induced cell fusion was detected in llc-pk cells treated with trypsin ( figure (a) ), indicating that the exogenous protease significantly promoted cellto-cell membrane fusion of llc-pk cells. to confirm this result, we used a luciferase reporter system to analyze cell-to-cell fusion with hek -apn cells [ ] [ ] [ ] . in previous studies, apn has been shown to serve as a pdcov receptor [ , ] ; therefore, we stably expressed papn in hek cells by applying the piggybac (pb) transposon system. after having confirmed that papn was well expressed in hek cells (data not shown), we analyzed whether pdcov could induce cell-to-cell fusion in hek -apn cells at moi = . . in the presence of trypsin, several virus-infected cells were located next to each other (figure (b) ), their contacting cell membranes having disappeared and their nuclei gathered in large conglomerates similar to what was observed in llc-pk cells. we next performed a cell-to-cell membrane fusion assay in hek -apn cells. hek -apn effector cells were transfected with pdcov s plasmid and pgl -luc, and co-cultivated with hek -apn target cells transfected with pbind-id and pact-myod plasmids. after mixing the effector and target cells, fresh medium with or without trypsin was added, and luciferase activity was measured after two days of co-cultivation ( figure (c)), revealing a dose-dependent effect. compared to the untreated control, fusion activity was increased at ng/ml, but it was most pronounced at ng/ml trypsin. these results indicate that trypsin significantly increases cell-to-cell fusion activity during pdcov infection of llc-pk cells. in a previous study, hu et al. successfully isolated pdcov in both llc-pk cells and st cells [ ] ; st cells in general are less susceptible to pdcov infection than llc-pk cells. in the current study, we analyzed the susceptibility of both cell lines to pdcov in the presence or absence of trypsin. we first infected llc-pk cells at different mois from . - , and evaluated pdcov replication by analyzing the cells at , and hpi for the presence of the viral n protein. at an moi of . and , trypsin did not have an effect at or hpi; however, it did significantly increase virus replication at hpi ( figure (a, b) ), which indicates that trypsin promotes pdcov replication at a late stage of the viral infection rather than at an early stage ( - h) . at high multiplicity (moi = ), the enhancing effect of trypsin was less pronounced (figure (c) ). this demonstrated that trypsin-mediated augmentation of pdcov infection in llc-pk cells is strongly moi dependent. next, we performed the experiment in st cells, using mois ranging from . to ; when an moi of . was applied to st cells, no bands were detectable (data not shown). when the moi was increased to and , only faint viral n protein bands were observed ( figure (d, e) ), and at an moi = , we detected more robust pdcov replication ( figure (f) ). however, trypsin treatment did not have a noticeable effect on viral replication at the times analyzed ( figure (f) ). furthermore, the amount of viral n protein in st cells at hpi (moi = ) was much lower than that in llc-pk cells (moi = . ). the same result was obtained at moi = in st cells (figure ). these results confirm that llc-pk cells are more susceptible to pdcov infection than st cells, and that trypsin promotes pdcov replication at a late stage in llc-pk cells but not in st cells. it seems that the different effects of trypsin on pdcov replication in llc-pk and st cells are responsible for the different spreading patterns observed in the two cell lines. to test this hypothesis, we infected both cell types with pdcov and performed ifa at hpi to visualize cell spread. pdcov infection in llc-pk cells exhibited a spreading pattern consistent with cell-to-cell fusion (syncytium formation as indicated by arrows) (figure (a)). however, in st cells, pdcov transmission was completely different, showing mainly single virus- infected cells and no obvious syncytium formation ( figure (b) ). taken together, the above results show that trypsin promotes pdcov infection in llc-pk cells by enhancing cell-to-cell fusion, whereas by contrast, trypsin does not facilitate transmission of pdcov infection in st cells. coronavirus s protein cleavage by trypsin always plays a critical role for cell-to-cell fusion. to test whether cleavage of s protein by trypsin was different in llc-pk and st cells, we infected both cell types with pdcov for h with trypsin (to increase virus replication and bring s protein to a detectable level). the cells were further cultivated without trypsin for h, then treated with indicated concentrations ( , , , μg/ml) of trypsin at °c for h. the results showed a clear cleavage of s protein in llc-pk cells ( figure (c) ), but was less efficient in st cells ( figure (c, d) ). the results indicated that differential cleavage of the s protein may be involved in the variable effects of trypsin on pdcov replication in llc-pk and st cells. next, we wanted to get information about the efficiency of the pdcov spread by cell-to-cell fusion. we designed an experiment to evaluate virus replication efficacy according to two spreading models, using two distinct culture models (figure (a) ). the first allowed pdcov-infected cells (effector cells) to directly interact with non-infected llc-pk cells (target cells), referred to as the contact-cell model. the second kept the effector cells and target cells separated across the membrane of a trans-well filter, termed the non-cellto-cell model, which allowed only free virus particles to cross the membrane and infect target cells. the results indicated that in the cell-to-cell model, with trypsin supplement, viruses transmission from effector cells to target cells was very efficient (figure (b) ). however, in the target cells not supplemented with trypsin, there were only a few single infected cells, and cell-to-cell fusion was rarely detected (figure (b)). in the non-cell-to-cell model treated with trypsin, the cell-to-cell spread between target cells was observable, though the number and size of fusion cells was less than in the contact-cell model (figure (b) ). to further confirm this, pdcov in both models with or without trypsin was quantified by qpcr, also demonstrating that pdcov spread by cell-to-cell fusion was significantly more efficient than the non-cell-to-cell model. furthermore, cell-to-cell spread of deltacoronavirus was slowed down if no trypsin was added (figure (c)). these results indicate that pdcov figure . llc-pk cells were more susceptible to pdcov infection than st cells. llc-pk cells were infected with pdcov at an moi of (a) . , (b) , or (c) , and st cells were similarly infected at an moi of (d) , (e) , or (f) . both infected cell types were cultured in the presence or absence of μg/ml trypsin and then cells were washed and lysed for western blot at , and hpi. pdcov n proteins were analyzed with a specific antibody against n protein, and actin was used as a loading control. experiments were repeated at least three times. transmission via cell-to-cell spread in llc-pk cells is very efficient. isolation and propagation of pedv and pdcov requires addition of exogenous trypsin to cell cultures, and thus it is commonly accepted that trypsin is essential for entry of these viruses into the cell [ ] [ ] [ ] [ ] . however, trypsin is not indispensable for all strains of pedv in vero cells, as cell entry and release of the vero cell culture-adapted dr (vaccine strain) was independent of trypsin [ ] . for pdcov, all previous studies have used live virus, which makes it difficult to differentiate between virus entry and the later steps of the virus lifecycle. to analyze pdcov entry independently from other replication steps, we applied a pdcov pseudovirus entry assay, demonstrating that trypsin failed to promote pdcov entry in the same way as pedv [ ] . a recent study reported that pdcov enters cells via two pathways: trypsinmediated entry at the cell surface or cathepsinmediated entry in the endosome [ ] . our results show that pdcov entry does not depend on trypsin; this is consistent with the fact that pdcov and pedv entry is greatly activated by lysosomal proteases [ ] [ ] [ ] . in the pedv lifecycle, trypsin plays a crucial role in viral release [ ] . however, in our study, we demonstrated that the amount of pdcov released into the supernatant was not influenced by trypsin ( figure ). this suggested that mechanisms of viral egress of pdcov is different from that reported for pedv ( ) . we demonstrated that trypsin contributes to cell-tocell membrane fusion in pdcov infection in vitro, and this step needs the interaction of s glycoprotein of pdcov and its receptor. papn has been reported to serve as a functional receptor for pdcov [ , ] . however, in another study, zhu et al. provided some evidence that papn may contribute to virus entry but does not serve as the primary receptor for pdcov [ ] . in this study, we found that hek cells which stably express papn are susceptible to pdcov infection, whereas normal hek cells are resistant, supporting an important role for papn regardless of whether it is the primary receptor or not. therefore, hek -apn cells were used in assays that could differentiate cell-to-cell fusion from other steps of the viral lifecycle. we found that trypsin mediated syncytium formation with cellular material exchange between effector and target cells. the mechanisms contributing to the difference in cell-to-cell fusion ability of pdcov in the different cell lines is unclear. one may speculate that variable expression of papn or other critical cellular factors may be responsible. firstly, llc-pk cells are more susceptible to pdcov infection than st cells under the similar condition where trypsin is supplemented in the cell culture medium, which may be one of the possible explanations for the different effects of trypsin on pdcov replication in llc-pk and st cells. however, in a recent study, zhang et al. demonstrated that the s glycoprotein could successfully induce cell-to-cell fusion in the presence of trypsin in st cells, facilitating virus replication [ ] , which is contrary to our results and another previous study [ ] . hu et al. demonstrated that pancreatin rather than trypsin can promote pdcov replication in st cells [ ] , whereas our results indicated that s protein cleavage in llc-pk cells was more pronounced than in st cells (figure (c, d) ). we speculate that the st cell line used by zhang et al. [ ] may have been a different lineage from that used in this study, possibly one with a greater receptor abundance than ours; receptor abundance is a critical switch for virus efficient replication [ ] . what contributes to this difference is unclear and needs to be further studied. trypsin promotes pdcov replication at a late stage of the infection in llc-pk cells, and the effect was more pronounced at low moi ( figure (a, b) ). this result supports our conclusion that trypsin promotes pdcov replication at the cell-to-cell fusion stage because syncytium formation occurs at a late stage of the virus lifecycle. as determined by western blot, there was no obvious increase in viral replication in cell lysates from trypsin-treated llc-pk cells at and hpi (figure (a)). we think this was because a high moi = was chosen for this experiment, and the expression of viral n protein may have become saturated, making it difficult to see significant differences by western blot. in fact, when we used a lower moi of . to inoculate llc-pk cells, we noticed a significant increase in virus replication ( figure s ). this notion is also consistent with the finding that the promoting effect of trypsin is less pronounced at high moi. if most cells are infected during the first round of infection, cellto-cell spread is not required for further spread of the virus. in summary, we identified that extracellular trypsin is required for pdcov cell-to-cell fusion in llc-pk cells. based on the efficiency of infection, we also recommend isolation and propagation of pdcov to be performed in llc-pk cells rather than in st cells. furthermore, infection of llc-pk cells should be more efficient at high confluency because it more easily allows pdcov spread by cell-to-cell fusion. these data may provide a basis for improving virus culture methods, leading to efficient isolation and propagation of pdcov for future development of vaccines and other therapeutic products. identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in china isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku , from domestic rabbits porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis origin, evolution, and virulence of porcine deltacoronaviruses in the united states emerging and re-emerging coronaviruses in pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus detection and genetic characterization of deltacoronavirus in pigs complete genome sequence of strain sdcv/usa/illinois / , a porcine deltacoronavirus from the united states full-length genome sequence of porcine deltacoronavirus strain usa/ia/ / swine enteric coronavirus disease: a review of years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the united states and canada prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in south korea complete genome sequences of porcine deltacoronavirus strains dh / and dh detection and phylogenetic analysis of porcine deltacoronavirus in korean swine farms complete genome characterization of korean porcine deltacoronavirus strain kor/ knu - 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wdwrttn authors: li, taisheng title: diagnosis and clinical management of severe acute respiratory syndrome coronavirus (sars-cov- ) infection: an operational recommendation of peking union medical college hospital (v . ): working group of novel coronavirus, peking union medical college hospital date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: wdwrttn since december , china has been experiencing an outbreak of a new infectious disease caused by severe acute respiratory syndrome coronavirus (sars-cov- ). the clinical features include fever, coughing, shortness of breath, and inflammatory lung infiltration. china rapidly listed sars-cov- -related pneumonia as a statutory infectious disease. to standardize the diagnosis and treatment of this new infectious disease, an operational recommendation for the diagnosis and management of sars-cov- infection is developed by peking union medical college hospital. severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel type of coronavirus of β genus that leads to an emerging infectious disease with remarkable pulmonary involvement in china since december . the clinical features include fever, dry cough, shortness of breath, normal or low levels of peripheral white blood cells, and inflammatory changes on chest x-ray. china has designated sars-cov- infected pneumonia as a statutory infectious disease. to standardize the clinical diagnosis and treatment, peking union medical college hospital (pumch) has established a working group and formulated the following operational recommendation regarding "diagnosis and clinical management of severe acute respiratory syndrome coronavirus infection" (v . ). selection of front-line personnel front-line medical personnel are qualified only after passing the physical examinations and professional training of sars-cov- . staff with the following conditions are exempt from sars-cov- related clinical/ laboratory work, including pregnancy, age over years old, a past history of chronic diseases such as chronic hepatitis, renal diseases, diabetes mellitus, autoimmune diseases and tumours. individuals affected with acute fever should also be excluded from sars-cov- related work. baseline tests should be arranged including the complete blood count, urine analysis, biochemical tests, creatine kinase, and chest x-ray. see "the guidelines for the diagnosis and treatment of severe acute respiratory syndrome coronavirus (sars-cov- )infection (pilot rd version)" published by the national health commission of the people's republic of china [ ] . isolation and observation of medical personnel after close contact with sars-cov- ( ) medical personnel in close contact with sars-cov- infected pneumonia patients should be relatively isolated, avoiding walking around and extensive contact with others. ( ) medical personnel should be isolated immediately and receive relevant examinations upon onset of fever, cough, shortness of breath and other symptoms. ( ) when work in the sars-cov- infection ward is finished, nasopharyngeal or oropharyngeal swabs for sars-cov- and a complete blood count should be carried out. those who have abnormal test results should undergo strict isolation and observation; while others will be generally isolated for observation and resume work after one week. screening criteria [ ] [ ] ( ) epidemiological history: history of travel or residence in hubei province within weeks prior to the onset of illness, or contact with patients from hubei province with fever and respiratory symptoms within days prior to onset, or presented with clustering onset. ( ) acute onset of fever within h without influenzalike symptoms, which could not attribute to other confirmed etiology. ( ) supportive epidemiological history ( ) clinical manifestation: fever; normal or low levels of white blood cells or decreased lymphocyte counts at onset. chest radiology at early stage is characteristic of multiple small patchy shadows and interstitial changes, more prominent in the extrapulmonary bands. multiple ground-glass opacities and infiltrations may develop bilaterally with disease progression, with possible consolidation in severe cases. hours) and normal chest imaging, if the absolute count of peripheral lymphocytes is less than . × /l, or the count of cd + and cd + t cells decreases significantly, isolation and close observation should be conducted at home even if the first sars-cov- nucleic acid test is negative. repeat of rrt-pcr should be considered after h, and a chest ct scan should be performed when necessary. screening cases on the day of visit nucleic acid examination of sputum or naso-/oropharyngeal swabs, complete blood count, urine analysis, arterial blood gas analysis, liver and kidney function, c-reactive protein (crp), procalcitonin, creatine kinase plus myoglobin, coagulation, and chest ct should be performed. inflammatory cytokines [such as interleukin (il)- , il- , and tumour necrosis factor (tnf)-α], tb lymphocyte subsets, and complement can be tested as appropriate [ ] [ ] [ ] . ( ) complete blood count, liver and kidney function, creatine kinase and myoglobin, coagulation function and crp can be checked on the rd, th and th days after admission and on discharge according to the disease status. pct and tb lymphocyte subsets can be repeated on days - if feasible [ ] [ ] [ ] . ( ) the chest x-ray or ct scan should be re-examined - days after the admission, and the time for subsequent re-examination depends on the disease status, no longer than days. ( ) complete blood count, chest x-ray, liver and kidney function, and all abnormal examinations on admission should be re-examined before discharge except for referrals. place of treatment according to the severity of the disease all cases with screening indications are subject to onsite medical isolation (single-room isolation), and once diagnosed, should be transferred to a designated hospital. according to the definition of the national health commission [ ] , patients in accordance with one of the following standards should be hospitalized and transferred to beijing designated medical institution as soon as possible; ( ) respiratory rate increased (≥ per min) or dyspnoea; ( ) oxygen saturation ≤ % when breathing ambient air, or arterial oxygen tension (pao₂) over inspiratory oxygen fraction (fio₂) of less than mm hg ( mm hg equals to . kpa); ( ) lung imaging indicating multilobular lesions or progression of lesions over % within h; ( ) quick sequential organ failure assessment (qsofa) score ≥ ; ( ) community-acquired pneumonia- (curb- ) score ≥ ; ( ) combined pneumothorax; ( ) other clinical conditions that require hospitalization. according to the definition of the national health commission [ ] , patients in accordance with respiratory failure, septic shock or other organ failure should be transferred to intensive care unit immediately and to designated medical institution as soon as possible when feasible. patients should be kept in bed and closely monitored for vital signs and levels of oxygen saturation. supportive treatment should be ensured, including enough supply of energy and fluid, maintenance of electrolyte and acid-base homeostasis. oxygen therapy patients with hypoxemia should be given oxygen therapy immediately and maintain a blood oxygen saturation level to no less than % in man and non-pregnant women, and between % and % in pregnant women. choice of oxygen therapy. patients with mild hypoxemia should be put on nasal cannula, l/min. if the patient is getting worse, high flow nasal cannula should be considered, starting with l/min and increasing to - l/min gradually. fraction of oxygen should be adjusted according to oxygen saturation. the way of respiratory support. non-invasive ventilation is only considered for patients who can tolerate. for the patients requiring invasive ventilation, endotracheal intubation should be performed by experienced physician with personal protective equipment. we recommend protective ventilation strategy for acute respiratory distress syndrome (ards). for those patients with most severe ards, extracorporeal membrane oxygenation or prone position is recommended. interventions should be implemented to prevent complications associated with critical illness. standard precautions should always be routinely applied in all areas of healthcare facilities. currently, there is no evidence to support the effectiveness of existing antiviral drugs against sars-cov- . lopinavir/ritonavir can be used when appropriate, tablets, twice daily for days. severe patients could receive glucocorticoid at early stage, e.g. intravenous methylprednisolone - mg, once daily for days, and the course of treatment can be prolonged according to the clinical condition and radiological manifestations. early intravenous infusion of human immunoglobulin is recommended for critically ill patients, based on their clinical condition, at . - . g/(kg·d), for - days [ ] [ ] . if bacterial infection is suspected according to the patient's clinical and imaging findings, patients of mild type can take oral antibiotics for communityacquired pneumonia, such as second-generation cephalosporins or fluoroquinolones. as regards patients of severe type, all possible pathogens should be covered when necessary. ( ) once the critically ill patients are diagnosed and endotracheal intubation is anticipated, they should be immediately transferred to the icu ward with negative pressure. all the procedures should be carried out in accordance with the requirements of comprehensive protection. ( ) oxygen storage mask is used to supply oxygen above l/min during transport, during which complete filling of the oxygen storage airbag should be ensured. ( ) endotracheal intubation should be induced by standard rapid procedure, and muscle relaxants should be used as much as possible to avoid droplet transmission caused by choking. ( ) reusable items such as goggles should be disinfected after intubation before taken out of the negative pressure ward. ( ) patients with intubation should use closed endotracheal suction to avoid airborne transmission caused by ventilator airflow. ( ) under certain circumstances when the ventilator must be disconnected for operation, the standby function of the ventilator should be set to avoid airborne transmission caused by ventilator airflow. once the standby function is not available, the y-tube port of the ventilator should be blocked to avoid air spread. criteria of isolation release and discharge [ ] patients could be discharged or transferred to the other departments for comorbidities, when the body temperature returned to normal for more than days, the respiratory symptoms significantly improved, the pulmonary lesions markedly absorbed, and respiratory nucleic acid is tested negative for sars-cov- for two consecutive times at least day apart. wrote by department of infectious diseases, peking union medical college hospital) department of infectious diseases, peking union medical college hospital) department of internal medicine icu, peking union medical college hospital) hongwei fan (department of infectious diseases, peking union medical college hospital) experts participating in the discussion (sorted by family name of chinese phonetic alphabet) wenzhao chai national health commission. the guidelines for the diagnosis and treatment of severe acute respiratory syndrome coronavirus (sars-cov- )infection (pilot rd version clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance rapid loss of both cd + and cd + t lymphocyte subsets during the acute phase of severe acute respiratory syndrome the alterations of t cell subsets of severe acute respiratory syndrome during acute phase characteristics and prognostic value of peripheral blood t lymphocyte subsets in patients with severe influenza prominent plasmacytosis following intravenous immunoglobulin correlates with clinical improvement in guillain-barré syndrome use of intravenous immunoglobulins for prophylaxis or treatment of infectious diseases no potential conflict of interest was reported by the author(s). key: cord- - j pb pu authors: yongchen, zhang; shen, han; wang, xinning; shi, xudong; li, yang; yan, jiawei; chen, yuxin; gu, bing title: different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid- patients date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: j pb pu effective strategy to mitigate the ongoing pandemic of novel coronavirus (covid- ) require a comprehensive understanding of humoral responses against severe acute respiratory syndrome coronavirus (sars-cov- ), the emerging virus causing covid- . the dynamic profile of viral replication and shedding along with viral antigen specific antibody responses among covid- patients started to be reported but there is no consensus on their patterns. here, we conducted a serial investigation on individuals infected with sars-cov- in two medical centres from jiangsu province, including non-severe covid- patients, and severe covid- patients and asymptomatic carriers based on nucleic acid test and clinical symptoms. the longitudinal swab samples and sera were collected from these people for viral rna testing and antibody responses, respectively. our data revealed different pattern of seroconversion among these groups. all non-severe covid- patients and severe covid- patients were seroconverted during hospitalization or follow-up period, suggesting that serological testing is a complementary assay to nucleic acid test for those symptomatic covid- patients. of note, immediate antibody responses were identified among severe cases, compared to non-severe cases. on the other hand, only one were seroconverted for asymptomatic carriers. the sars-cov- specific antibody responses were well-maintained during the observation period. such information is of immediate relevance and would assist covid- clinical diagnosis, prognosis and vaccine design. the ongoing outbreak of novel coronavirus (covid- ), known as severe acute respiratory syndrome coronavirus (sars-cov- ), was first reported in wuhan, china in dec [ ] . as the outbreak of coronavirus disease (covid- ) surges worldwide, this emerging pandemic has affected more than , , patients globally. the dynamic profile of viral replication and shedding along with viral antigen specific antibody responses among covid- patients started to be reported [ ] but there is no consensus on their patterns. the longitudinal profiles of viral rna and antibody response are urgently needed to guide clinical diagnosis, treatment, infection control and vaccine design [ ] . in this respective study, we serially analysed the virus rna test results in swab samples, along with anti-sars-cov- igm and igg responses among covid- patients at the second hospital of nanjing and the affiliated hospital of xuzhou medical university in jiangsu province, china. patients with suspected sars-cov- were confirmed after two sequential positive respiratory tract sample results. throat swab samples were collected every - days. anal swab samples were also obtained for rna testing since february , as anal swab samples with prolonged viral shedding were observed during clinical practice [ ] . viral rna was tested using real-time reverse transcriptional polymerase chain reaction (rt-pcr) kit (bgi genomics, beijing, china) as recommended by chinese center for disease control and prevention (cdc) following who guidelines [ ] . the serum samples retrieved from routine biochemical or immunological testing were inactivated at °c for min. these samples were later stored at − °c for later serological detection. the igg and igm antibody responses against sars-cov- spike protein and nucleocapsid protein were tested by gold immunochromatography assay supplied by innovita co., ltd, china (cfda approved). the demographic information and disease severity of covid- patients were obtained from their electronic medical records. patients who had any of the following features during covid- disease progression were classified as severe cases: (a) respiratory distress; (b) hypoxia (spo ≤ %); (c) abnormal blood gas analysis (pao /fio ≤ mm hg); or (d) severe disease complications including respiratory failure which requires mechanical ventilation, septic shock, or nonrespiratory organ failure. the illness severity was defined according to the chinese management guideline for covid- (version . ) [ ] . asymptomatic carriers were defined as individuals who were positive for covid- nucleic acid but without any symptoms during screening of close contacts. this study was approved by ethics committee of each medical centre, and information consent was waived as part of a public health outbreak investigation. between jan and march , , patients were enrolled including ( . %) non-severe covid- patients, ( . %) severe patients, and ( . %) asymptomatic cases with sars-cov- infection. as of march , all patients have been clinical recovered and discharged. the characteristics of each group were summarized in table . the dynamic viral shedding from throat swab and anal swabs were analysed ( figure ). for non-severe patients, the respiratory swab remained positive for a median of (range - ) days since symptom onset, whereas a median of (range - ) days for severe patients. for asymptomatic cases, the period of positive respiratory swab lasted for a median of (range - ) days. despite of no statistical difference between groups, non-severe covid- group was prone to become respiratory swab rna negative in shorter period, compared to the group of severe patients and asymptomatic cases. among patients who tested for anal swab samples, ( . %) anal swabs remained positive for sars-cov- since their respiratory swab samples turned negative for sars-cov- . our serial sars-cov- rna testing identified a prolonged viral shedding for asymptomatic cases compared to covid- patients, suggesting the importance of early identification and timely quarantine for these asymptomatic carriers. consistent with previous studies [ ] , we also found that the anal swab was able to maintain positive for weeks even after respiratory samples turned negative, validating the efficient replication in gastrointestinal tract during later stage of covid- and a possible faecal-oral transmission route. the longitudinal antibody responses were also determined in our cohort ( figure ). all of symptomatic patients ( %) were seropositive at the time point of discharge or during follow-up period. specifically, among non-severe patients, ( . %) patients seroconverted within week , ( . %) patients were anti-sars-cov- positive during week , while ( . %) patients showed positive antibody responses within week , and ( %) patients were seropositive within week . for ( . %) of patients, the first detection of antibody responses occurred during the period when their swab samples converted to rna negative, suggesting that antibody responses might facilitate the viral clearance especially for non-severe covid- patients. furthermore, for all severe covid- patients, antibody responses were detected within weeks. of note, out of severe patients generated viral specific igg responses prior to viral clearance. it is possible that significantly high level of sars-cov- viral load observed in severe cases [ , ] drives an early antibody response produced by immediate activation of extrafollicular b cells during acute infection [ , ] . moreover, we also observed well-maintained antibody responses for all seroconverted individuals during our observation period, at least lasting for weeks. only ( %) out of asymptomatic cases generated sars-cov- specific antibody responses (table and figure ), and this patient (patient ) was not seroconverted until week since her diagnosis. consistent with her delayed antibody responses, the throat swab sample converted to rna negative as late as week . for the remaining asymptomatic patients, patients were not seroconverted within week and week , respectively; while patients remained anti-sars-cov- negative during week . it is not known whether they become seropositive later. it is possible that such seronegative asymptomatic carriers were resulted from low level of data are presented as n (%) or median (range). viral load, but the false positive nucleic acid test results cannot be ruled out. our current study has revealed important implications to understand the dynamic interplay between sars-cov- and humoral responses. first, seroconversion was observed in % ( / ) of symptomatic patients during the observation period, suggesting that the serological test could serve as a complementary testing assay to nucleic acid test for those symptomatic covid- patients, especially given the potential false negative viral rna results using throat swab samples. nevertheless, we also noted only one person with humoral responses among asymptomatic carriers. additional viral and immunological analyses are warranted to understand this observation. better understanding on the biological significance of asymptomatic cases without seroconversion is urgently needed. although it is generally considered a beneficial role of specific antibody response during viral infection, we did not identify a strong association of seroconversion and disease severity in our cohort. for both non-severe and severe covid- cases, the viral specific antibody responses were detected. meanwhile, our study revealed an early induction of antibody responses in severe cases. consistently, a recent study also revealed that high level of antibody titer might be independently associated with a worse disease severity for covid- patients [ ] . a similar clinical observation was also observed from a previous study of severe acute respiratory syndrome coronavirus (sars-cov), in which deceased infected patients reached the peak of antispike neutralizing antibody much earlier than that of the clinical recovered patients [ ] . however, only measurement of peripheral antibody responses may not be sufficient. we can also speculate that high level of initial viral load early in infection may lead to severe covid- cases. such high viral load can drive strong extrafollicular b cell responses leading to rapid antibody responses which do not follow the sequence of igm to igg development stages [ , ] . such high quantity of antibodies produced from extrafollicular b cells can contribute greatly to the inflammatory responses by promoting monocyte and macrophage accumulation and the massive cytokine storm including il- and mcp- , which might be responsible for fatal acute lung injury, as indicated during sars-cov infection [ ] . also, antibody might facilitate the infection of target cells by promoting the uptake of virion-antibody complex via fc receptors (fcr) [ ] . whether the acute anti-sars-cov- antibody responses attributed to disease progression of covid- disease deserves further investigation. on the other hand, a graduate development of viral antigen specific b cells undergoes the somatic hypermutation and affinity maturation at the traditional germinal centre, which ultimately lead to high affinity protective antibody responses as observed in non-severe covid- patients. our study also has several limitations. first, our study is limited by small sample size, thereby it is not known whether our finding could be generalized. nevertheless, our cohort is representative of different covid- disease spectrum, including non-severe cases, severe cases, and asymptomatic carriers. furthermore, the quantitative viral load and titers of antibody response were not available, so the kinetics of viral shedding and the magnitude of antibody response during covid- disease progression remained unknown in this study. collectively, our study of serial nucleic acid and serological testing among various covid- patients indicated distinct dynamic patterns among three groups of sars-cov- infected individuals. our findings contribute to the evolving understanding of the sophisticated interaction between this emerging sars-cov- virus and host immune system. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study timely development of vaccines against sars-cov- molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance national health commission of the people's republic of china prolonged presence of sars-cov- viral rna in faecal samples viral dynamics in mild and severe cases of covid- detectable -ncov viral rna in blood is a strong indicator for the further clinical severity the multifaceted b cell response to influenza virus how specific is too specific? b-cell responses to viral infections reveal the importance of breadth over depth antibody responses to sars-cov- in patients of novel coronavirus disease antibody responses against sars coronavirus are correlated with disease outcome of infected individuals antibody-enhanced dengue virus infection in primate leukocytes anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection no potential conflict of interest was reported by the author (s). key: cord- -y cp wza authors: negrey, jacob d.; reddy, rachna b.; scully, erik j.; phillips-garcia, sarah; owens, leah a.; langergraber, kevin e.; mitani, john c.; emery thompson, melissa; wrangham, richard w.; muller, martin n.; otali, emily; machanda, zarin; hyeroba, david; grindle, kristine a.; pappas, tressa e.; palmenberg, ann c.; gern, james e.; goldberg, tony l. title: simultaneous outbreaks of respiratory disease in wild chimpanzees caused by distinct viruses of human origin date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: y cp wza respiratory viruses of human origin infect wild apes across africa, sometimes lethally. here we report simultaneous outbreaks of two distinct human respiratory viruses, human metapneumovirus (mpv; pneumoviridae: metapneumovirus) and human respirovirus (hrv ; paramyxoviridae; respirovirus, formerly known as parainfluenza virus ), in two chimpanzee (pan troglodytes schweinfurthii) communities in the same forest in uganda in december and january . the viruses were absent before the outbreaks, but each was present in ill chimpanzees from one community during the outbreak period. clinical signs and gross pathologic changes in affected chimpanzees closely mirrored symptoms and pathology commonly observed in humans for each virus. epidemiologic modelling showed that mpv and hrv were similarly transmissible (r( ) of . and . , respectively), but mpv caused . % mortality mainly in infants and older chimpanzees, whereas hrv caused no direct mortality. these results are consistent with the higher virulence of mpv than hrv in humans, although both mpv and hrv cause a significant global disease burden. both viruses clustered phylogenetically within groups of known human variants, with mpv closely related to a lethal variant from mountain gorillas (gorilla beringei beringei), suggesting two independent and simultaneous reverse zoonotic origins, either directly from humans or via intermediary hosts. these findings expand our knowledge of human origin viruses threatening wild chimpanzees and suggest that such viruses might be differentiated by their comparative epidemiological dynamics and pathogenicity in wild apes. our results also caution against assuming common causation in coincident outbreaks. respiratory viruses of human origin have caused disease in wild apes across sub-saharan africa and pose a significant and growing threat to wild ape health and conservation [ , ] . for example, respiratory disease is the leading cause of morbidity and mortality among chimpanzees (pan troglodytes) in gombe stream national park, tanzania [ , ] and in kibale national park, uganda [ ] , two populations that have been studied continuously for decades. mortality from anthroponotic respiratory pneumoviruses (family pneumoviridae) and paramyxoviruses (family paramyxoviridae) has been documented in western chimpanzees (p. t. verus) in cote d'ivoire [ , ] , eastern chimpanzees (p. t. schweinfurthii) in tanzania [ ] , mountain gorillas (gorilla beringei beringei) in rwanda, lowland gorillas (g. g. gorilla) in central african republic [ ] and bonobos (p. paniscus) in the democratic republic of the congo [ ] . rhinovirus c [ ] and coronavirus oc [ ] of human origin have also caused chimpanzee mortality in uganda and mild respiratory disease in cote d'ivoire, respectively. biological similarities between humans and apes predispose them to cross-species pathogen transmission [ ] , and habitat alterations may exacerbate inter-species contact and anthroponotic transmission risk [ ] . although simultaneous infections of apes and people with the same respiratory virus have rarely been confirmed directly [ , ] , viruses that are relatively benign in humans can cause lethal outbreaks in ape populations, indicating lack of resistance in apes. suitable prevention strategies have included improved hygiene and sanitation [ ] , reduced human visitation [ ] and large-scale vaccination of apes (if effective vaccines someday become available) [ ] . such policies could also benefit human public health by reducing zoonotic transmission risk [ , ] . here, we report simultaneous outbreaks of respiratory disease in two nearby chimpanzee communities in uganda, caused by two distinct negative-sense rna viruses of human origin. the outbreaks occurred from december to february in the ngogo and kanyawara chimpanzee communities in kibale national park [ ] (figure s ). only km apart, the communities are interconnected by contiguous moist evergreen forest, separated by only one intervening chimpanzee community that is not currently studied, and both communities generally experience low mortality rates [ , ] . although outbreaks of respiratory disease in wild chimpanzees are common, their causes often remain undiagnosed [ ] . fresh carcasses are often not recovered, and the remoteness of field sites complicates sample storage and analysis. non-invasive diagnostics (usually from feces) have proven highly informative [ ] , but negative results in such cases are inconclusive [ ] . in the present case, rapid recovery of carcasses, the presence of trained veterinarians and the availability of basic field laboratory capabilities on site provided an opportunity for etiologic diagnoses. furthermore, coordinated, prospective collection of observational data between the two sites offered an unusual chance to compare the clinical and epidemiologic features of the outbreaks directly. from december to february , the ngogo chimpanzee community of kibale national park, uganda ( figure s ), experienced an outbreak of severe respiratory disease. during the same period, the nearby kanyawara community ( figure s ) also experienced a respiratory disease outbreak. at the onset of the epidemics, ngogo community consisted of chimpanzees from < year to years old, and kanyawara community consisted of chimpanzees from < year to years old. epidemic curves ( figure ) show that the ngogo and kanyawara outbreaks each occurred in a single phase, with most cases occurring in january . at ngogo, . % of chimpanzees observed between december and february exhibited respiratory signs. at kanyawara, . % of chimpanzees observed during the same period exhibited respiratory signs. at ngogo, chimpanzees ( . %) died during the outbreak period (figure ). in contrast, no chimpanzees at kanyawara died during the outbreak period, with the exception of a female recovering from disease who died following conspecific aggression (see below). respiratory signs consisted of coughing, sneezing, dyspnea, and nasal exudate; other signs included lethargy, immobility, and dramatic loss of body condition ( figure s ). epidemiologic modelling of the ngogo and kanyawara outbreaks (table and figure s ) yielded daily transmission rate estimates of . and . , and durations of infectivity of . and . days, respectively. these parameters yielded basic reproductive numbers (r ) of . and . for ngogo and kanyawara, respectively. these are similar to values estimated from an outbreak of rhinovirus c in kanyawara in (table ) during which . % of chimpanzees died, and to published values for the human "common cold" [ ] . however, % confidence limits around these estimates were non-overlapping for daily transmission rates of all three outbreaks and duration of infectivity and r in ngogo (both lower than in kanyawara in or / ). risk factor analysis (table s ) showed that age significantly predicted morbidity at both ngogo (χ = . , df = , p = . ) and kanyawara (χ = . , df = , p = . ). in both communities, respiratory signs were least frequently observed among infants and increased through successive age categories. sex did not affect morbidity or mortality at ngogo, but females were significantly more likely to exhibit respiratory signs at kanyawara than were males (χ = . , df = , p = . ). at ngogo, age predicted mortality (χ = . , df = , p < . ), with mortality highest among infants (or . , % ci: . - . ) and individuals ≥ years old (or . , % ci: . - . ), compared to intermediate ages. at ngogo, the carcass of a -year-old female chimpanzee was recovered immediately after the onset of respiratory signs and subsequent death. post-mortem analysis of this individual revealed consolidation of the dependent lobes of both lungs and a serosanguinous pericardial effusion but no other gross pathologic abnormalities ( figure s ). at kanyawara, the carcass of a -year-old female chimpanzee was recovered approximately days after having recovered from respiratory signs (but still remaining weak), immediately after having been attacked by conspecifics (for unclear reasons). post-mortem analysis of this individual showed severe, diffuse pleuropneumonia with fibrinous adhesions to the thoracic wall and severe consolidation of all lobes of both lungs, as well as a serosanguinous pericardial effusion ( figure s ). analysis of paired fecal samples (prior to and during the outbreak period) using a luminex assay that tests for a suite of human respiratory agents revealed different viral etiologies for each community ( table ) . metapneumovirus (mpv, pneumoviridae: metapneumovirus) was detected in of individuals ( . %) from ngogo chimpanzees exhibiting clinical signs during, but not before, the outbreak period (fisher's exact p = . ). human respirovirus (hrv ; paramyxoviridae; respirovirus, formerly known as parainfluenza virus ) was detected in of individuals ( . %) from kanyawara chimpanzees exhibiting clinical signs during, but not before, the outbreak period (fisher's exact p = . ). adenoviruses (adenoviridae) were present in samples from both ngogo ( . %) and kanyawara ( . %) but showed no association with the outbreak period (fisher's exact p = . and . , respectively) and have been previously characterized in this population at comparable frequencies [ ] . enteroviruses (picornaviridae) were also present at low frequency in samples from both communities, similarly showed no association with the outbreak period (fisher's exact p = . in both cases), and have also been previously characterized in this population at comparable frequencies [ ] . metagenomic analysis of respiratory tract swab samples from the chimpanzee examined postmortem at ngogo yielded , , reads after trimming, of which , assembled to yield a coding-complete mpv genome of , bases with average coverage of , consistent with the luminex results described above. this genome (genbank accession number mh ) was most similar ( . %) to a human-derived variant from brazil (genbank accession number mg ). intriguingly, the virus was nearly as similar ( . %) to a variant from a mountain gorilla from rwanda in (genbank accession number hm ) detected during a lethal outbreak [ ] . mpv rna was present in all sections of the respiratory tract, including the lung parenchyma, with the proportion of viral sequence reads declining monotonically from the upper to the lower respiratory tract ( figure s ). sequencing of a nucleotide portion of the viral f gene from fecal samples was successful for three other luminex-positive chimpanzees at ngogo (ab, mi and wi; table ), yielding identical sequences within this variable genomic region (gen-bank accession numbers mh -mh ). by contrast, neither hrv nor any other virus was detected in the respiratory tract of the chimpanzee that died at kanyawara, likely reflecting prior infection and viral clearance. a coding-complete hrv genome ( , bases) was therefore reconstructed from a fecal sample from this same individual collected when she was coughing approximately weeks earlier, using pcr with virus-specific primers and sanger sequencing (table s ). this genome (genbank accession number mh ) was most similar ( . %) to a human-derived variant from the usa (gen-bank accession number ky ). sequencing of a variable and epidemiologically informative nucleotide portion of the viral f gene from fecal samples was successful for three other luminex-positive chimpanzees at kanyawara (al, an and az; table ), yielding identical sequences within this genomic region (genbank accession numbers mh -mh ). re-analysis of respiratory tract metagenomic data from this individual and the individual from ngogo revealed that a small proportion of reads in the upper respiratory tracts of both animals ( . % and . %, respectively) mapped to the reference genome of staphylococcus pneumoniae (genbank accession number nc_ ), which can infect chimpanzees secondarily during viral respiratory disease outbreaks [ , ] , indicating the presence of this or a related bacterium; however no reads mapped to this organism in the lungs of either animal. phylogenetic analysis revealed mpv from the ngogo outbreak to sort within a sub-clade of subtype b [ ] viruses from brazil, peru, rwanda and the usa ( figure ). this sub-clade contains recently collected viruses ( ) ( ) ( ) ( ) ( ) ( ) ( ) , including the mountain gorilla variant from rwanda. the mpv variant from ngogo belongs to a different subtype than a previously reported b virus from a chimpanzee outbreak in tanzania [ ] , and it is less closely related to a previously reported b subtype from a chimpanzee outbreak in cote d'ivoire [ ] than to other variants within its subclade, including the gorilla-derived variant, based on the available nucleotide region of the viral p gene (genbank accession numbers eu -eu ; not shown). phylogenetic analysis revealed hrv from the kanyawara outbreak to sort within a sub-clade of highly similar human-derived viruses from peru and the usa collected between and (no more recently than other sub-clades; figure ). notably, hrv from kanyawara was divergent from viruses from other non-human primates, including viruses from wild zambian baboons (papio cynocephalus) [ ] and simian agent , originally isolated from a blue monkey (cercopithecus mitis) in south africa [ ] . outbreaks of severe and sometimes lethal respiratory disease are common in chimpanzees across sub-saharan africa, but their causes often remain undetermined [ , ] . consequently, and because of differences in field protocols among research sites, it has proven difficult to compare these outbreaks directly. the simultaneity of the two / outbreaks at ngogo and kanyawara, coupled with coordinated data and sample collection efforts between the two sites, allowed a rare direct comparison to be made. analyses demonstrate that the two viruses displayed different pathogenicities and different epidemiological dynamics in the two chimpanzee communities. mpv at ngogo caused substantially higher mortality than hrv at kanyawara, despite causing apparently lower morbidity. however, the estimated daily transmission rate and duration of infectivity of mpv at ngogo differed from corresponding values for hrv at kanyawara (table ), suggesting that, despite comparable r values, the viruses displayed different epidemiologic dynamics. higher mortality due to mpv in infants and older adults compared to intermediate age categories is congruent with data from humans, where infants, young children and older adults are most susceptible to severe clinical manifestations of mpv infection [ , ] , as well as with demographic data from prior mpv outbreaks in chimpanzees [ , ] . neither virus was detected in chimpanzees immediately prior to the outbreaks ( table ) . mpv was detected in . % of affected ngogo chimpanzees sampled during the outbreak, and hrv was detected in . % of affected kanyawara chimpanzees sampled during the outbreak. viral sequences from affected individuals were identical within the variable region sequenced. mpv and hrv were therefore likely single-origin causes of their respective epidemics. adenoviruses and enteroviruses were present both before and during the outbreaks at statistically indistinguishable (adv) and low (ev) frequencies. adenoviruses occur in both healthy and ill wild apes [ ] [ ] [ ] [ ] and in chimpanzees during respiratory outbreaks [ ] , including at kanyawara [ ] , leading to debate about their etiologic role; however, our results support previous conclusions that these viruses are most likely benign. our results support a similar conclusion for enteroviruses, with the notable exceptions of polio virus [ , ] and rhinoviruses [ ] , which can cause deadly disease in chimpanzees. we note that, at the time of the outbreaks, the ngogo and kanyawara epidemics were assumed to have the same cause, with concomitant fears that an infectious agent was spreading rapidly across kibale national park. moreover, avian influenza had recently been reported in waterfowl in lake victoria, on the other side of uganda [ ] . our results highlight how heightened vigilance for emerging infectious diseases can sometimes lead to erroneous inferences. common environmental drivers (e.g. weather, human visitation patterns) may precipitate simultaneous outbreaks of distinct anthroponotic viruses in wild apes, creating the appearance of a single disease with a seasonal pattern. our results also suggest that the transmission of respiratory viruses between chimpanzee communities is probably uncommon, despite high rates of transmission within chimpanzee communities. chimpanzees from neighbouring communities rarely interact [ ] , which may limit opportunities for intercommunity transmission even of highly contagious microbes. mpv and hrv both represent significant burdens to global public health [ ] . mpv infection in humans table s . causes coughing, nasal discharge and weight loss [ ] ; in non-human models signs are often more severe than for other related viruses [ ] . mpv also causes clinical disease in experimentally infected chimpanzees [ ] and severe and sometimes lethal outbreaks of respiratory disease in wild chimpanzees and gorillas, often with similar demographic patterns of infection as we describe herein [ , , , ] , as does a related virus, respiratory syncytial virus (pneumoviridae, orthopneumovirus) [ ] . our results show that this comparatively high virulence of mpv is recapitulated in naturally infected chimpanzees. hrv causes respiratory disease and asthma exacerbation in people worldwide, especially in children and the elderly, but it is less virulent than mpv [ ] . hrv has a wide host range, including primates, ruminants and rodents [ ] . simian agent , first isolated in from an apparently healthy blue monkey [ ] , was later identified as hrv and attributed to anthroponotic transmission [ ] . hrv has been detected in the respiratory tracts of apparently healthy wild yellow baboons in zambia [ ] . experimental infection of marmosets (saguinus mystax) with hrv causes acute and transmissible disease [ ] , but experimental infection of other primates, including rhesus macaques (macaca mulatta) and chimpanzees [ ] , is milder. our results mirror these findings and, more generally, show that some negative-sense rna respiratory viruses of human origin cause only mild and self-limiting infections in wild chimpanzees. we cannot exclude the role of other co-infecting pathogens in modulating the pathogenicity of mpv and hrv in our study populations. for example, hrv can cause upper respiratory disease and predispose chimpanzees to invasive pneumococcal infection [ ] , and the bacterium streptococcus pneumoniae co-occurs with human metapneumoviruses and respiratory syncytial viruses in both wild and in captive apes [ , ] . our finding of a low percentage of metagenomic sequence reads matching s. pneumoniae (or a related bacterium) in the upper respiratory tracts of both necropsied individuals, but not in their lungs, supports the notion that wild chimpanzees host microbes that could easily invade the lower respiratory tract following primary viral infection. population surveys of endemic microbes and serologic assessments of prior exposure to agents that might predispose chimpanzees to infection or enhance pathogenesis could prove informative. clinical signs in ngogo chimpanzees were congruent with those in severely infected humans [ ] . one adult female at ngogo ("kidman") has exhibited persistent wheezing since the outbreak; in human children suffering severe mpv infections, subsequent wheezing episodes are not uncommon [ ] . post-mortem analysis of one individual at ngogo revealed surprisingly little gross pathology of the respiratory tract, consistent with acute infection and rapid death, and mpv was recovered from all sections of that individual's respiratory tract, including the lung parenchyma ( figures s and s ). in contrast, post-mortem analysis of an individual at kanyawara revealed advanced respiratory tract pathology, consistent with longer term infection ( figure s ), but we did not recover hrv from this individual's respiratory tract despite recovering a complete hrv genome from this same individual's feces collected approximately weeks earlier. this individual had likely recovered from the initial viral challenge, cleared the virus, and perhaps acquired a secondary bacterial infection [ , ] . notably, both individuals had pericardial effusions, which in humans are rare but serious sequellae of certain viral infections [ ] . phylogenetic analyses revealed that mpv from ngogo and hrv from kanyawara are each closely related to globally circulating human viruses, indicating independent anthroponotic sources for both viruses. apparent geographic associations in these phylogenies likely represent bias in available sequence data (e.g. high representation of viruses from peru and the usa; table s ). the ngogo mpv variant clusters within a sub-clade of viruses that is more recent ( - ) than other sub-clades, perhaps reflecting antigenic replacement of mpv strains over time. notably, the ngogo variant is very similar to a virus from a fatal infection of a mountain gorilla in rwanda in [ ] . this particular viral lineage may thus have a propensity for infecting apes, or it may simply circulate widely in east africa. the virus is divergent, however, from previously described, lethal viruses from wild chimpanzees in tanzania [ ] and cote d'ivoire [ ] , indicating that multiple mpv variants can infect and kill wild chimpanzees. the pathways by which these viruses entered chimpanzees from humans remain vexingly obscure. people frequent the habitats of both chimpanzee communities for research and associated activities (not solely focused on chimpanzees) and to travel or collect forest products [ ] . tourism is sometimes cited as a risk for anthroponotic transmission of viruses to great apes and helps inform current international union for the conservation of nature (iucn) recommendations for preventing such transmission, such as visitor vaccination requirements, minimum observation distances, alcohol-based sanitizing gel and facemasks [ ] . however, neither ngogo nor kanyawara is a tourism site. ngogo is bordered by a tourism site (kanyanchu) receiving thousands of visitors each year, but there were no reports of a concurrent respiratory disease outbreak in the kanyanchu chimpanzees, although similar events have occurred there at other times. at gombe national park in tanzania, provisioning chimpanzees with bananas (a discontinued practice) was, along with season, the strongest predictor of chimpanzee respiratory signs [ ] , which affected between % and % of chimpanzees per month between and [ ] . however, neither the ngogo nor the kanyawara chimpanzees are provisioned, and biosecurity measures based on iucn recommendations [ ] for visitors to both sites are in place. at kanyawara, data over years show a seasonal pattern of respiratory illness in chimpanzees, typically peaking in march, but no correlation between clinical signs in chimpanzees and seasonal patterns of respiratory illness in humans living nearby [ ] . the role of other species in transmitting these viruses from humans to chimpanzees is also unclear. kibale national park contains a diversity of primate species and social groups, none of which were observed with respiratory signs at the time of the outbreaks, but complex patterns of cross-species transmission from humans to chimpanzees cannot be discounted. such possibilities could be investigated through broad sampling of people (especially research staff) and wildlife during future outbreaks, including serologic assessments of prior exposure. overall, these findings broaden our understanding of human viruses that can cause disease in wild chimpanzees and show that their clinical manifestations can vary markedly. our study also shows that epidemiological analyses, enabled by prospective, coordinated observational data collection, may be able to distinguish among viruses based on early-stage infection patterns. if so, "real-time" epidemiologic analyses would be a valuable complement to molecular diagnostic testing, in that they could help guide response strategies as epidemics progress. epidemiologic analyses could also inform future monitoring efforts to detect and minimize the impact of anthroponotic transmission events on wild ape populations. the study was observational and non-invasive. protocols were approved by institutional animal care and use committees (iacuc) of harvard university (protocol - ) and university of new mexico (protocol - -mcc) and were exempt by boston university's and the university of michigan's iacuc. protocols followed the weatherall report, nih guide for the care and use of laboratory animals, usda animal welfare act, institute for laboratory animal research guide for the care and use of laboratory animals, us public health service and national academies of sciences national research council, and us centers for disease control and prevention. during the outbreaks, trained field assistants collected individual-level observational data on the chimpanzees daily. chimpanzees in both the ngogo and kanyawara communities are individually identifiable, making such data possible to collect. we compiled these data into measures of clinical signs (coughing or sneezing, further classified as mild or severe), and observation time per chimpanzee. we estimated dates of mortality events as the midpoint of the last date a chimpanzee was seen alive and the first date it was absent from a group containing frequent associates, including dependent offspring. to infer epidemiological transmission parameters, we constructed two sir (susceptible-infectious-removed) mathematical modelsone for each communityfollowing althaus [ ] , fitting curves to cumulative incidence data using the nelder and mead optimization algorithm in the optim package in r, version . . [ ] . for both communities, we assumed free admixture and did not incorporate heterogeneity of social interaction to minimize model complexity, as we did previously for a rhinovirus c outbreak in kanyawara [ ] . we analysed risk factors for morbidity and mortality using a generalized linear model (glm) with binomial distribution and log-link function g in r [ ] . age, sex and their interaction were included as predictor variables, and observation hours were included as a control variable. age was modelled as a categorical variable with four levels: infants (< years), juveniles ( - . years), adults ( - . years) and older (≥ years). at ngogo, where mortality occurred, individuals not observed with clinical signs during the outbreak period that disappeared were coded as having died and as positive for respiratory signs. because none of these individuals were adolescent females (the only age-sex category of chimpanzee that can migrate between communities), their sudden disappearance (and the fact that they have never been seen since) confirms that they had, in fact, died. we collected fecal samples from individual chimpanzees in both communities during the outbreak period. two millilitres of feces were placed immediately into rnalater buffer (thermo fisher scientific, waltham, ma, usa) at a : ratio, homogenized and stored at − °c until exported to the united states. paired fecal samples were available from clinically ill chimpanzees ( from ngogo and from kanyawara) during and before (fourth quarter of ) the outbreak period, and we tested these for a suite of respiratory pathogens using the nxtag respiratory pathogen panel (luminex corporation, austin, tx, usa) as previously described [ ] , which tests for influenza virus a (multiple subtypes), human respiratory syncytial viruses a and b, coronaviruses (multiple subtypes), human metapneumovirus, rhinovirus/enterovirus, adenovirus, parainfluenza viruses - , bocavirus, and the bacterial pathogens chlamydophila pneumoniae and mycoplasma pneumoniae. necropsies were performed on two chimpanzees recovered immediately after death (the only two carcasses recovered, despite extensive effort) by trained veterinarians wearing appropriate personal protective equipment. one chimpanzee from ngogo died on january ("stella," a -year-old female) and the other chimpanzee from kanyawara died on january , ("rwanda," a -year-old female). in the latter case, the individual had been coughing days earlier and was attacked repeatedly by conspecifics while still weak; the resulting trauma appeared to be the immediate cause of death. samples (sterile swabs with plastic shafts and dacron tips) were collected from the nose, larynx, trachea, bronchi and lung parenchyma of both individuals and stored in . ml rnalater buffer at − °c. swabs were homogenized and total rna was extracted and converted to cdna libraries in the field, then prepared for sequencing on an illumina miseq instrument (illumina, san diego, ca, usa) using bp paired-end read chemistry as previously described [ ] . polymerase chain reaction (pcr) and sanger sequencing were used to complete viral genomes and to characterize viruses detected by luminex assay in fecal samples (table s ). to infer phylogenetic relationships among the two chimpanzee viruses and published viral sequences, we analysed full genome alignments of all available viruses in genbank (alignment lengths , and , positions, respectively) using phyml . 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phyml . metagenomic assessment of adventitious viruses in commercial bovine sera we thank the uganda wildlife authority, and especially c. tumwesigye, for ongoing help and support. we are also grateful to the uganda national council for science and technology and makerere university biological field station for permission to conduct this research. we thank the field assistants of the ngogo and kibale chimpanzee projects for help in data and sample collection, and k. sabbi, r. donovan and y. bochkov for invaluable assistance, input and discussion. no potential conflict of interest was reported by the authors. key: cord- - hqqheho authors: ng, kim tien; oong, xiang yong; pang, yong kek; hanafi, nik sherina; kamarulzaman, adeeba; tee, kok keng title: outbreaks of enterovirus d in malaysia: genetic relatedness to the recent us outbreak strains date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: hqqheho nan temperature ( c), relative humidity (%), number of rain days, and the amount of rainfall (mm) collected from the nearest meteorological station (latitude: n, longitude: e) was analyzed using pearson's correlation. specimens positive for enteroviruses were further confirmed using standard molecular approaches that involved amplification and sequencing of the human enterovirus vp /vp gene using primers described previously. pcr amplification of the capsid (p ) region ( bp) of ev-d was performed when vp /vp gene was identified as ev-d . a combination of previously published primers, namely -forward, -reverse, fwd , rev , fwd , rev , and newly designed primers were used to amplify the p region by forming several overlapping fragments. these newly designed primers are ev p . f ( -tca aaa tty act gaa cca gt- ), ev p . r ( -gtt gcc atg aag ctv cca ca- ), ev p . r ( -gat atg ttt cct act ara gt- ), ev p . f ( -cca ggg car gtc cgy aac atg- ), ev p . r ( -cca ytt gwa aaa gtt ytt gtc- ), ev p . f ( -gtg gar tca atg gag at- ), and ev p . r ( -gct gat tta tca cyg tgc gag- ). a total of vp /vp , vp , and p of ev-d retrieved from genbank (accessed on february ) were analyzed using neighbor-joining method implemented in mega version to deduce the viral phylogenies. the statistical robustness of the branching orders was evaluated by bootstrap analysis of replicates. in this molecular epidemiological surveillance, approximately % ( / ) of patients had at least one viral pathogen detected by the multiplex assay, of whom . % ( / ) were infected with ev-d . these ev-d cases were detected in the second half of (june to december) and between december and january , of which september and january were the peak months of infection. no ev-d cases were detected in other months, suggesting transient outbreaks of ev-d . the patients (five males and seven females) from whom ev-d was detected ranged in age from to years old. during recruitment, most of the patients experienced mild sneezing and moderate-to-severe cough. correlation of ev-d infections with meteorological factors was not observed (correlation coefficient , . ). based on previously described ev-d classification, the newly sequenced strains from malaysia were found within clade a (my-cluster- ) and clade b (my-cluster- ). phylogenetic analysis of the p region indicated that . % ( / ) of the malaysian ev-d formed clusters, suggesting the transient ev-d outbreaks were most likely caused by at least two viral lineages ( figure ) . a sporadic ev-d strain ( mykl ) that did not cluster with the major lineages was also observed. of note, my-cluster- contains ev-d mostly sampled in , while my-cluster- contains ev-d sampled in / . such observation suggests an ongoing ''clade shift'' or lineage replacement of circulating ev-d in causing new outbreak, as observed in other enterovirus-associated outbreaks. sequence homology comparison based on the p region indicated that the malaysian ev-d strains shared . %- . % (nucleotide) and . %- . % (amino acid) sequence identities with the fermon strain (ay ), a prototype strain isolated in the usa in . phylogenetic tree also showed that the representative ev-d sequences sampled from recent outbreaks in the usa were grouped into three distinct lineages. these lineages are designated as outbreak lineage (consists of sequences isolated from missouri, mo), lineage (illinois, il and kentucky, ky), and lineage (ky) (figure ) . interestingly, malaysian ev-d sequences (my-cluster- ) was related to outbreak lineage while outbreak lineage (us/ky/ - ) was clustered with the my-cluster- , supported by strong statistical evidence (bootstrap values of % and %, respectively) at the internal tree nodes and genetic distance of f . . likewise, outbreak lineage was closely related to the ev-d sequences (cu and cu ) reported recently in thailand. all members of my-cluster- and outbreak lineage exhibited unique amino acid substitution at two residue positions (s a and t a), while members of my-cluster- and outbreak lineage sequences displayed unique amino acid substitutions at four residue positions (n e, g e, f y, and i v) relative to the fermon strain (figure ). these ''clade-defining'' substitutions differentiate outbreak lineage from other outbreak and non-outbreak lineages. a unique substitution t a discriminated outbreak lineage from outbreak lineage , further suggesting that the former is indeed closely related to the thai strains. contribution of these amino acid substitutions on virus pathogenesis however remains unclear. although the amino acid substitutions are located in the putative immunogenic bc and de loops, experiments in susceptible cell systems are needed to prove the importance of such substitutions. the divergence dates of the malaysian outbreak and other major lineages were estimated by the bayesian coalescent-based relaxed molecular clock model with constant population size, implemented in the beast software. . our data suggest that the recent ev-d strains associated with unprecedented severe respiratory outbreaks in the usa in were probably descended from the recent ev-d lineages circulating in thailand and malaysia. however such observation remains presumptuous largely because limited ev-d sequence data originating from the usa (due to inadequate surveillance) are available for genealogical analysis. given the close relationship of the southeast asian isolates with the us strains from recent outbreak, a more active enterovirus surveillance should be in place to monitor risks of impending outbreak. centers for disease control and prevention. enterovirus surveillance -united states clusters of acute respiratory illness associated with human enterovirus -asia severe respiratory illness associated with enterovirus d -missouri and illinois genome sequence of enterovirus d from genome sequence of enterovirus d and clinical disease seven strains of enterovirus d detected in the united states during the severe respiratory disease outbreak transmission of the common cold to volunteers under controlled conditions. i. the common cold as a clinical entity evaluation of nuclisens easymag for automated nucleic acid extraction from various clinical specimens comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus worldwide emergence of multiple clades of enterovirus mega : molecular evolutionary genetics analysis version . evolutionary genetics of human enterovirus : origin, population dynamics, natural selection, and seasonal periodicity of the vp gene can sequence data predict enterovirus d infection outcome? beast: bayesian evolutionary analysis by sampling trees key: cord- -r bbomvk authors: woo, patrick cy; lau, susanna kp; tsang, chi-ching; lau, candy cy; wong, po-chun; chow, franklin wn; fong, jordan yh; yuen, kwok-yung title: coronavirus hku in respiratory tract of pigs and first discovery of coronavirus quasispecies in ′-untranslated region date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: r bbomvk coronavirus hku is a deltacoronavirus that was discovered in fecal samples of pigs in hong kong in . over the past three years, coronavirus hku has been widely detected in pigs in east/southeast asia and north america and has been associated with fatal outbreaks. in all such epidemiological studies, the virus was generally only detected in fecal/intestinal samples. in this molecular epidemiology study, we detected coronavirus hku in . % of the nasopharyngeal samples obtained from pigs in hong kong. samples that tested positive were mostly collected during winter. complete genome sequencing of the coronavirus hku in two nasopharyngeal samples revealed quasispecies in one of the samples. two of the polymorphic sites involved indels, but the other two involved transition substitutions. phylogenetic analysis showed that the two nasopharyngeal strains in the present study were most closely related to the strains pdcov/chjxni / from jiangxi, china, and ch/sichuan/s / from sichuan, china. the outbreak strains in the united states possessed highly similar genome sequences and were clustered monophyletically, whereas the asian strains were more diverse and paraphyletic. the detection of coronavirus hku in respiratory tracts of pigs implies that in addition to enteric infections, coronavirus hku may be able to cause respiratory infections in pigs and that in addition to fecal-oral transmission, the virus could possibly spread through the respiratory route. the presence of the virus in respiratory samples provides an alternative clinical sample to confirm the diagnosis of coronavirus hku infection. quasispecies were unprecedentedly observed in the ′-untranslated region of coronavirus genomes. coronaviruses (covs) are found in a wide variety of animals, in which they can lead to enteric, hepatic, neurological and respiratory illnesses of differing severity. on the basis of genotypic and serological characterization, covs were traditionally divided into three distinct groups. in , the coronavirus study group of the international committee for taxonomy of viruses replaced the traditional cov groups , and with three genera, alphacoronavirus, betacoronavirus and gammacoronavirus, respectively. in the same year, we discovered three novel covs in avian cloacal swabs. these covs formed a distinct novel cov genus, named deltacoronavirus. subsequently, in a large epidemiological study, we discovered seven additional deltacoronaviruses. interestingly, one of these deltacoronaviruses, which was originally named porcine cov hku , was found in fecal samples of pigs in hong kong, and it is the only mammalian deltacoronavirus. in , the coronavirus study group of the international committee for taxonomy of viruses rectified the species name for this virus to coronavirus hku . over the past three years, coronavirus hku has been widely detected in pigs in east/southeast asia and north america and was found to be associated with fatal outbreaks. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in these epidemiological studies, coronavirus hku was generally only detected in fecal/intestinal samples. however, some covs, such as transmissible gastroenteritis cov (tgev)/porcine respiratory cov (prcv) of alphacoronavirus and bovine cov of betacoronavirus , could be detected consistently in both fecal and respiratory samples. , therefore, we hypothesized that coronavirus hku may also be present in respiratory samples of pigs, which has implications for its transmission and potential role in respiratory diseases and for the use of nasopharyngeal sampling as an alternative method of identifying infected pigs. to test this hypothesis, we performed a molecular epidemiology study on nasopharyngeal samples collected from pigs in hong kong. two complete genomes of the 'respiratory' coronavirus hku were sequenced, and comparative genomic and phylogenetic studies were performed. the implications of the presence of coronavirus hku in respiratory samples are also discussed. nasopharyngeal samples from pigs were collected in hong kong over a -month period (january -february ). these samples were obtained from slaughterhouses and pig farms in hong kong with the assistance of the veterinary public health section, food and environmental hygiene department; and the agriculture, fisheries and conservation department of the government of hong kong. immediately after sample collection, each nasopharyngeal swab was submerged in viral transport medium for viral transport and maintenance. rna extraction and reverse transcription viral rnas were extracted from the nasopharyngeal samples of pigs by using μl of inoculated viral transport medium for each sample and by utilizing the ez advanced xl system (qiagen, hilden, germany) and ez virus mini kit v . (qiagen) according to the manufacturer's protocol with rnase-free water as the eluent. reverse transcription (rt) was performed using the superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa) according to the manufacturer's protocol by random priming. coronavirus hku screening detection of coronavirus hku was performed by polymerase chain reaction (pcr) amplifying a -bp fragment of the rnadependent rna polymerase (rdrp) gene, using the specific primer pair lpw ( ′-aca cac ttg ctg taa cca aa- ′) and lpw ( ′-atc att aga gtc acc acg at- ′). pcr and dna sequencing were carried out following our previous publications with slight modification. , briefly, each pcr mixture contained pcr buffer ( mm of kcl, mm of tris-hcl at ph . and mm of mgcl ; applied biosystems, foster city, ca, usa), μm of each deoxynucleoside triphosphate (roche diagnostics, basel, switzerland), μm of each primer (invitrogen), . u of amplitag gold dna polymerase (applied biosystems) and cdna. the mixtures were subjected to thermocycles of °c for min, °c for min and °c for min, with an initial denaturation at °c for min and a final extension at °c for min for dna amplification using the geneamp pcr system automated thermal cycler (applied biosystems). standard precautions were taken to avoid contamination, and no falsepositive result was observed for the negative controls. pcr products were agarose gel-purified using the qiaquick gel extraction kit (qiagen). both strands of the pcr products were sequenced twice by the abi prism xl genetic analyzer (applied biosystems) using the two pcr primers. the genomes of two coronavirus hku strains detected in the nasopharyngeal samples of two different pigs were sequenced following our previous publications , with modifications. briefly, viral rnas were converted to cdnas using superscript iii reverse transcriptase with a combined random priming and oligo(dt) priming strategy. the cdnas were pcr-amplified by primers (supplementary tables s and s ) that were designed by multiple alignment of the genome sequences of other coronavirus hku strains with complete genomes available or from the results of the first and subsequent rounds of pcr-sequencing. pcrs were performed using the iproof high-fidelity pcr kit (bio-rad laboratories, hercules, ca, usa) according to the manufacturer's protocol, and dna sequencing was performed as mentioned above. when ambiguous peaks were observed consistently in the electropherograms after several attempts of pcr-sequencing for certain genomic regions, cloning followed by plasmid sequencing was performed according to our previous publication, except the zero blunt topo pcr cloning kit (invitrogen) was used to resolve the sequence ambiguities. pcrs using recombinant plasmids as templates were also performed to confirm that indels at mononucleotide polymeric regions were not the result of polymerase slippage. the ′ ends of the viral genomes were amplified and sequenced by the rapid amplification of cdna ends (race) using the smarter race ′/ ′ kit (clontech laboratories, mountain view, ca, usa) according to the manufacturer's protocol with the reverse primers lpw ( ′-tgg gta atg tgt ccg ctg acg ggc ggt g- ′), lpw ( ′-aga agt ggt gga tgg tca gag gaa cgg t- ′) and lpw ( ′-gtg gct ggt ttc cag gta atc ta- ′). the sequences of the pcr products were then assembled manually to obtain the genomes of the two nasopharyngeal strains. pairwise alignment was performed using bioedit . . (optimal global alignment) or emboss stretcher (nucleotide alignment); whereas multiple sequence alignment was performed using muscle . , where the aligned sequences were further manually inspected and edited. tests for substitution models and phylogenetic analysis by the maximum likelihood method were performed using mega . . . divergence times for the coronavirus hku strains were calculated based on the complete genome sequence data, utilizing the bayesian markov chain monte carlo method using beast . . with the substitution model gtr (general time-reversible model)+g (gammadistributed rate variation)+i (estimated proportion of invariable sites), a strict molecular clock, and a constant coalescent. fifty million generations were run with trees sampled every th generation to yield trees. convergence was assessed based on the effective sampling size after a % burn-in using tracer . . . the mean time to the most recent common ancestor (tmrca) and the highest posterior density (hpd) regions at % were calculated. the trees, after a % burn-in, were summarized as a single tree using treeannotator . . by choosing the tree with the maximum sum of posterior probabilities (maximum clade credibility) and viewed using figtree . . . the complete genome sequences of the two nasopharyngeal coronavirus hku strains were deposited into the international nucleotide sequence databases with accession numbers lc and lc . a total of nasopharyngeal samples from pigs were tested. rt-pcr for a -bp fragment of the rdrp gene of coronavirus hku was positive in ( . %) of the nasopharyngeal samples. the samples that tested positive were mostly collected during winter (december-march) ( figure ). dna sequencing showed that seven sequence variants were detected among the positive samples, and pairwise alignment showed that these seven sequence variants possessed . %- % sequence identity to the corresponding region in the rdrp gene of coronavirus hku strain hku - that we previously found in fecal samples of pigs in hong kong (supplementary figure s ). complete genome sequencing and genome analysis complete genome sequencing was performed for the coronavirus hku found in two of the positive nasopharyngeal samples (s n and s n) . excluding the ′ poly(a) tail, the genomes of s n and s n were - and nucleotides long, respectively. the genome organization of the two strains was the same as that of other coronavirus hku strains. the lengths of the seven open reading frames (orfs) of the two strains s n/ s n were / , , , , , and bp, respectively. the genomes of the two strains possessed . %- . % sequence identity to that of the representative isolate hku - . quasispecies were detected in one of the samples (s n) at the ′ genomic region via two independent nested pcrs targeting the nd- th bases of the genome using two different primer pairs for the first round and the same primer pair for the second round of reaction. for s n, direct sequencing of the pcr products yielded ambiguous peaks in the sequencing electropherograms, which could only be resolved after cloning (figure ). post-cloning dna sequencing revealed that there were six sequence variants, with four polymorphic sites, for this genomic region ( figure ) . two of the polymorphic sites, located at the th and th nucleotide positions, involved indels (Δt and Δa/c, respectively), whereas the other two polymorphic sites, located at the nd and th nucleotide positions, involved transition substitutions (t → c and g → a, respectively). additionally, pcr-dna sequencing using the recombinant plasmids as amplification templates did not generate the same sequence ambiguities observed in the pre-cloning experiment. phylogenetic analysis of the complete genomes of the two nasopharyngeal strains and other coronavirus hku strains showed that the outbreak strains in the united states possessed highly similar genome sequences and that they were all clustered together monophyletically, whereas the asian strains were more diverse and paraphyletic, with the lao and thai strains occupying the basal lineage; however, the south korean strain knu - was more similar to the us strains than to the other asian strains (figure and supplementary figure s the estimated mean evolutionary rate of the complete genome sequence data set was . × − ( % hpd: . - . × − ) substitutions per site per year, which is approximately . -fold higher than that estimated in a previous study. the root of the tree was september ( % hpd: june -march ). the tmrca of the diversity of coronavirus hku was dated to june ( % hpd: november -june ); and the tmrca of the thai/laos strains was traced back to september ( % hpd: may -january ). the tmrcas for the clade containing us/korean strains was estimated to be october ( % hpd: june -january ), which is slightly delayed compared with that estimated in a previous study. for the two nasopharyngeal strains characterized in this study (s n and s n) , they were estimated to have diverged from their respective mrcas in december ( % hpd: . six intra-strain quasispecies were found. post-cloning plasmid-dependent pcr-sequencing confirmed that the presence of indels at positions and was not due to polymerase slippage. quasispecies and were detected in both nested pcr using first round primers lpw /lpw and second round primers lpw /lpw as well as nested pcr using first round primers lpw /lpw and second round primers lpw /lpw . however, quasispecies and were only detected in nested pcr using first round primers lpw / lpw and second round primers lpw /lpw , whereas quasispecies and were only detected in nested pcr using first round primers lpw /lpw and second round primers lpw /lpw . coronavirus hku was detected in nasopharyngeal samples of pigs. although coronavirus hku has been widely detected in various locations around the pacific ocean, including canada, china, , , , hong kong, laos, , mexico, south korea, , thailand, , vietnam and the united states, [ ] [ ] [ ] [ ] [ ] [ ] , , , the virus has principally been found in fecal or intestinal specimens. there have been a few exceptional circumstances; in one study, the presence of coronavirus hku was reported in the blood, liver, lung and kidney of one pig, and in a few other studies, coronavirus hku was found to exist in the blood (n = ), mesenteric lymph node (n = ) and saliva (n = )/oral fluid (n = ) of pigs, , , implying that coronavirus hku can cause systemic infections in occasional cases. in this study, coronavirus hku was found in . % of the nasopharyngeal samples of pigs, which is similar to the . % positive rate of coronavirus hku in fecal samples of pigs that we reported previously. seasonal variation in the detection rate of coronavirus hku from pigs was noted, where most of the positive samples were collected in winter. this is similar to the pattern of seasonal variation in a surveillance study carried out in the united states, where the detection rate for coronavirus hku was much lower during summer. it has recently been confirmed that coronavirus hku is able to cause swine enteric infections by infecting gnotobiotic and conventional pigs with coronavirus hku . the detection of coronavirus hku in respiratory tracts of pigs has the following implications. first, in addition to enteric infections, coronavirus hku may be able to cause respiratory infections in pigs. second, in addition to fecal-oral transmission, the virus may be able to spread through the respiratory route. third, the presence of the virus in respiratory samples provides an alternative clinical sample to confirm the diagnosis of coronavirus hku infection. further studies will determine the full spectrum of clinical diseases and pathologies associated with coronavirus hku . from the data of the present study, both the 'enteric' and 'respiratory' coronavirus hku may possess similar properties. a number of animal covs possess dual or multiple tissue tropisms. for example, tgev, which is another enteropathogenic cov that infects pigs, could also be found in the nasopharynx of pigs as prcv, which is a deletion mutant of tgev. moreover, bovine cov is both an enteric and a respiratory pathogen in cattle. similar to tgev/ prcv, coronavirus hku is recovered from both respiratory and gastrointestinal samples. however, unlike tgev/prcv, in which there is a - nucleotide deletion at the ′ end of the spike (s) gene leading to a loss of - antigenic sites in prcv, comparative genome analysis of coronavirus hku from respiratory and fecal samples did not show any obvious difference in their s proteins or other parts of their genomes. phylogenetic analysis also did not reveal a separate clustering of fecal/intestinal and nasopharyngeal isolates ( figure ). further cell culture experiments are required to confirm whether all strains of this species possess intrinsic tropism to both enteric and respiratory tissues. this is also the first report of cov quasispecies in the ′untranslated region (utr). in one (s n) of the two coronavirus hku genomes that we sequenced in this study, variant sites were observed at four positions; two of them were due to nucleotide substitutions, and the other two were results of indels at mononucleotide polymeric regions ( th and th bases). these two indels were genuine variant sites instead of being due to polymerase slippage during the amplification process because recombinant plasmiddependent pcr-sequencing no longer resulted in sequence ambiguities in the electropherograms. although the existence of quasispecies has been reported in covs, the variant sites were found in coding regions or ′-utr. [ ] [ ] [ ] [ ] in the case of severe acute respiratory syndromerelated coronavirus, all of the variant sites observed in the quasispecies were located at the s gene. for bovine cov, one of the two strains with naturally occurring intra-isolate quasispecies had all seven variant sites located at orf a, whereas for the other strain with naturally occurring intra-isolate quasispecies, there were polymorphic sites scattered across orf a (n = ), orf b (n = ), kda-non-structural protein (nsp) gene (n = ), hemagglutinin esterase (he) gene (n = ), s gene (n = ), . kda-nsp gene (n = ), . kda-nsp gene (n = ), membrane (m) gene (n = ), nucleocapsid (n) gene (n = ) and ′-utr (n = ). similar to bovine cov, middle east respiratory syndrome-related coronavirus also possessed all the intra-host single nucleotide variations throughout its genome except the ′-utr. , in this study, all four variant sites ( Δt, t → c, g → a and Δa/c) were present in the ′-utr and were not located in the leader sequence or the transcription regulatory sequence. we speculate that the existence of quasispecies in covs may play a role in cov evolution, in addition to the more well-known high-recombination and mutation rates in cov genomes. virus taxonomy: ninth report of the international committee on taxonomy of viruses, international union of microbiological societies, virology division comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group c coronavirus discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus create new species in the family coronaviridae complete genome characterization of korean porcine deltacoronavirus strain kor/knu - 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- journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: lgkqwm u zika virus (zikv) is an emerging mosquito-borne flavivirus that can potentially threaten south china. a chinese family of four returning from venezuela to china was found to be positive for zikv when the youngest son's fever was first detected at an airport immigration inspection. they were isolated temporarily in a local hospital in enping city, guangdong province, where their clinical data were recorded and urine and saliva were collected to isolate zikv and to obtain viral sequences. all of them except the mother presented mild symptoms of rash and fever. envelope gene sequences from the father, daughter and son were completely identical. phylogenetic analysis demonstrated that this strain is similar to several imported strains reported in recent months, which are all clustered into a group isolated from zika outbreaks in brazil. together with the climatic features in venezuela, new york and guangdong in february, it can be concluded that our subjects are imported cases from venezuela. with the same viral sequence being shared between family members, neither direct human-to-human nor vector transmission can be ruled out in this study, but the former seems more likely. although our subjects had mild illness, epidemiologists and public health officials should be aware of the risk of further expansion of zikv transmission by local competent vectors. although zika virus (zikv) was first identified early in in uganda, africa, outbreaks in french polynesia in significantly accelerated the spread of this virus to other parts of the world. zikv is a reemerging mosquito-borne flavivirus circulating in a wide range of regions including africa, south america, and asia. zikv infection can cause serious damage to the central nervous system, such as infant microcephaly and guillain-barré syndrome. [ ] [ ] [ ] the virus has proven to be neurotropic in animals, and a recent experiment in vitro also showed that it can infect human neural progenitor cells derived from induced pluripotent stem cells. another study showed that human dermal fibroblasts, epidermal keratinocytes and immature dendritic cells are also permissive to the most recent zikv isolates. an animal model of zikv infection has been established in ag mice by foot pad injection. by far, aedes aegypti is considered the principal transmission vector of zikv, although aedes albopictus, which caused several outbreaks of dengue fever in guangdong province of south china in the last two decades, may play a role in the spread of this virus because a. albopictus may be a competent vector. there are over chinese in venezuela, which is one of the regions most heavily affected by zikv infection in south america. with frequent people shuttling between south america and guangdong, there is a potential risk of spreading zikv to south china, where a. albopictus are active in densely populated communities. in this study, a family of four flying from venezuela to guangzhou of guangdong province was found to be zikv positive in their peripheral blood. to gain a better understanding of transmission among communities, the phylogenetic relationship between the isolates from this family and others from diverse regions of the world was analyzed. because this virus may be transmitted directly by body fluids, [ ] [ ] [ ] it was also necessary to explore this possibility in this family. four hospitalized individuals from a family (father, mother, daughter and son) were diagnosed with zikv infection at enping people's hospital. this family had lived in venezuela for more than two months before february ; then they flew to new york on that day and stayed there for~ days. finally, they flew from new york to guangzhou, china on february ( figure ). these four infected individuals were first confirmed by real-time reversetranscription polymerase chain reaction (rt-pcr) in baiyun international airport of guangzhou, where the youngest one (the son) had developed fever, and the family was then isolated by the local department of public health. the infected individuals then lived in a shared ward (without other patients) in the infectious disease department of enping people's hospital. the room had been screened against mosquitoes, and each bed was also covered with a bed net to prevent spreading by local competent vectors. at the time of hospitalization, the subjects' clinical history and results of a general physical examination, blood tests and routine urine tests were documented. the youngest one (a -year-old boy) was the first case whose manifestation was fever and maculopapules. saliva, urine and peripheral blood were collected from the patients during the onset and recovery period and were stored at − °c. all samples were tested for zikv rna by real-time pcr, and some urine was utilized to isolate virus. informed consent was obtained from all patients before sample collection. the study protocols were reviewed and approved by the scientific and ethical committee of guangzhou women and children's medical center. before rna extraction, urine samples were concentrated with amicon ultra- . centrifugal filter units with ultracel- membrane (millipore, shanghai, china). the qiaamp viral rna mini kit (qiagen, hilden, germany) was used to extract viral rna from urine and saliva according to the manufacturer's instructions. rna was eluted in μl of ave buffer and stored at − °c until use. samples positive for zikv were selected to amplify genes encoding envelope protein (e) and nonstructural protein (ns ). complementary dna (cdna) was synthesized from viral rna by using the revertaid first strand cdna synthesis kit (thermo fisher, salt lake city, usa) according to the manufacturer's instructions. four pairs of primers were designed to generate overlapping dna fragments covering the e and ns gene regions by using the software oligo . (http://www.oligo.net/. supplementary table s ). polymerase chain reaction (pcr) was performed with ex taq hs dna polymerase (takara, dalian, china) under conditions of initial heating of °c for min, followed by cycles of °c for s, °c for s and °c for . min. the sequences of the pcr products were identified by using the sanger sequencing method. before phylogenetic analysis, the e and ns coding sequences of four individuals in our study were aligned with other reference sequences using clustal . software (http://www.clustal.org/clustal /). the reference sequences selected were those with highly similar blast (megablast, http://blast.ncbi.nlm.nih.gov/blast.cgi) scores. phylogenetic trees were drawn using the maximum likelihood method in the tamura-nei model with gamma-distributed evolutionary rates in mega . (www.megasoftware.net). an initial tree was made automatically with the nearest-neighbor-interchange method. the gaps/missing data treatment was set as complete deletions. bootstrap analyses with replications were utilized to determine confidence values for groupings within the phylogenetic trees. other parameters were set to default style. the clinical characteristics of four individuals with zikv infection are summarized and compared in table . in this family, whose members were living together for a long period before and after the first onset, the -year-old son was the first patient, and he had both fever and rash . before returning to china, they had no symptoms of any infectious disease, but the son and the daughter both had a history of mosquito bites in venezuela according to the father's memory, although the specific date of biting was not clear. the -year-old daughter was the second one who had symptoms, which included fever and rash, with the rash occurring on the second day after her brother had fever and rash. the -year-old father was the latest patient; the only symptom he had was a rash, and the rash was distributed uniformly on his neck and back ( figure ). his neck rash first occurred on the third day after his son showed fever and rash. the -year-old mother did not have any symptoms during the whole period of observation. a time axis based on rash indicates the sequence, the time interval and the date of positive detection for zikv ( figure ). the four family members had been isolated since february , and they were released on march . the clinical laboratory showed results that the overall symptoms of the four subjects were mild. the daughter and the son were subjected to laboratory blood tests twice and thrice, respectively (table ) . unlike most other viral infectious diseases, low white blood cell counts were not a common feature of our subjects. detection of zikv in saliva and urine from four hospitalized patients. real-time pcr based on taqman probes was used to detect zikv rna. among the four tested urine samples collected on february , patient (father), patient (daughter) and patient (son) were found to be positive for zikv. only patient was positive for zikv in a saliva sample (detected on the night of february , when the samples were collected). the urine and saliva samples from patient (mother) were all negative for zikv detection. nucleic acid sequencing, alignment and phylogenetic analysis pcr products were successfully obtained only from patient , patient and patient . after sequencing and fragment assembly, the e gene sequences of the three patients were found to be exactly the same. the e gene sequence of patient is identical to z (genbank: ku . , submitted by wu et al, guangdong provincial center for disease control and prevention), the viral sequence from the same patient. we found that all our sequences were similar to those of strains circulating in south america, especially brazil ( figure ). to further investigate the origin of the zikv isolated from our subjects, phylogenetic trees were constructed using the maxim likelihood method. zikv infection is becoming a global public health concern since microcephaly cases were linked to zikv infection during pregnancy. so far, the data suggesting that microcephaly cases in brazil might be linked with zikv infection are only epidemiological. , growing evidence has indicated that zikv can damage the fetus, causing intrauterine growth restriction. , a recent report that zikv can be detected in amniotic fluid of fetuses with microcephaly substantiated this point. with the establishment of a prevention and control system for emerging infectious diseases in china after the outbreak of severe acute respiratory syndrome in , imported cases from affected areas with fever will be strictly examined for specific pathogen infections. travelers with fever would usually be intercepted for further quarantine in an international airport when they have a history of being bitten by a mosquito in an area affected by zikv. in our study, when the youngest son had a fever, the whole family needed to be examined. because the four subjects in this family showed zikv positivity in their peripheral blood, isolation of the whole family was obligatory. south american countries closest to the equator have been deeply plagued by zikv infection in recent years. for example, zikv infection has been frequently reported in counties such as brazil, suriname, colombia and venezuela. neighboring caribbean countries including guatemala, haiti, puerto rico and dominica have also been affected. throughout the whole year, these areas have a warm and humid climate suitable for the survival of mosquitoes. the temperature in february in north america, especially northern regions such as new york, is below °c; thus, the family members in our study were unlikely to be bitten by mosquitoes during their -day stay in new york. thus, the zikv isolates carried by this family could only have come from venezuela (figure ). rash and low-grade fever seem to be the main symptoms for most zikv-infected individuals. rash was reported to be the most frequent symptom (presented in . % cases), followed by fever and arthralgia. in general, symptoms of zikv infection (mild flu-like symptoms) are milder than those of dengue virus (denv) infection; the latter often include high-grade fever with myalgia, headache, arthralgia and nausea. leukopenia (o /mm ) has been detected iñ % of dengue fever patients. in our study, one adult and two children had the symptoms of fever, rash and conjunctival congestion, and no one complained of any pain. unexpectedly, the three patients with fever all had normal leukocyte counts ( /mm ). whether the milder symptoms of zikv infection (milder than denv infection) mean a higher leukocyte count is unclear because of the lack of data obtained in past reports on zikv outbreaks. hence, if the mother without any symptoms in our study is considered only a carrier of zikv (not a patient), this proportion of patients in our study is almost consistent with previous reports, in which rashes were presented by almost all patients. information about pruritus, the second most common clinical symptom in the confirmed cases in previous study, was not obtained from the family while inquiring them regarding their case history. perhaps this symptom is transient and unnoticeable at the time of disease onset. rt-pcr based on taqman probes may be the most convenient tool to detect zikv infection in suspected patients , because enzyme-linked immunosorbent assays (elisas) for igm antibody against zikv would cross-react with other flaviviruses, , and the results must be validated by a plaque reduction neutralization test, a labor-intensive and costly method. moreover, compared with blood drawing, urine and saliva collection are more acceptable options for suspected individuals, and the latter have an advantage for viral detection and isolation because virus can be detected at higher titers and for a longer period in urine than in serum. a previous study figure phylogenetic tree based on e gene sequences of zika virus isolates. e gene sequences from the father, daughter and son (genbank: ku ) in our study were used for alignment with other reference sequences. all isolates are indicated with related information (genbank number, country, year and host, some with sample type). phylogenetic trees were drawn using the maximum likelihood method by the tamura-nei model with gamma-distributed evolutionary rates in mega . . an initial tree was made automatically with the nearest-neighbor-interchange (nni) method. gaps/missing data treatment was set as complete deletion. bootstrap analyses with replications were utilized to determine confidence values for groupings within the phylogenetic trees. other parameters were set to default style. first imported familial zikv cases in china y yin et al showed that a patient had prolonged shedding of viral rna in saliva and urine for up to days after symptom onset. nevertheless, positive taqman rt-pcr detection is not usually a guarantee of successful sequencing or isolation of target viruses. urine and saliva usually need to be concentrated before rna extraction or viral inoculation, which was performed in our study. like other flaviviruses, the structure of the single polyprotein encoded by zikv genomic rna is ′-c-prm-e-ns -ns a-ns bns -ns a-ns b-ns - ′, in which the e (envelope) protein is the main antigen recognized by the host immune system, and ns is the principal component of the replication complex of the virus. in previous research, e gene sequences of zikv isolates were usually utilized to construct phylogenetic trees based on experience from molecular study of dengue virus. our phylogenetic tree constructed based on the e protein coding gene is topologically similar to that based on the complete zikv genomic sequence (figure ) , meaning that the phylogenetic tree based on the e gene is strong enough to distinguish all clusters of zikv isolates from all over the world. because complete genome sequencing of an rna virus is a time-consuming procedure, constructing a phylogenetic tree using e protein gene sequences is a simpler method for molecular epidemiologic study, especially in an outbreak. in our phylogenetic tree constructed from e protein gene sequences, zikv can be divided into two lineages, african and asian. , although there is a very short evolutionary distance between our strains and the venezuela imported strain ku . -ve ganxian, the diversity of the strains is apparent, and the latter is nearer to kj . -h/pf/ french polynesian strains. , currently, all cases reported in china seem to be imported from south america and pacific islands, which also indicates there was a great diversity of strains circulating during early in venezuela. our strains are more similar to the strains circulating in south america in late , where there were more strains isolated from patients. a phylogenetic tree based on ns gene sequences cannot efficiently distinguish different subgroups of zikv from various sources of isolates (supplementary figure s ) because the ns gene sequence is more conserved than that of e or even ns , which is a gene sequence used more frequently to build phylogenetic trees. currently, the e gene sequence is the most commonly used sequence in molecular epidemiologic studies of flaviviruses because it has the greatest genetic diversity (supplementary figure s ) . it is worth noting that strains of the african lineage were almost solely isolated from aedes mosquitoes circulating in the african continent according to the phylogenetic tree, probably because zikv did not draw much attention before massive microcephaly cases were reported in brazil. zikv is spread primarily by a. aegypti, a dominant vector for several mosquito-borne viruses in the western hemisphere. zikv was detected in a. albopictus from gabon, a country in central africa. no direct evidence has demonstrated that this virus could be spread by a. albopictus, a local vector for dengue virus in south china, yet the threat is quite real. the fact that zikv can propagate in the c / cell line derived from a. albopictus suggests that this mosquito may be a competent vector and have the potential for spreading the virus, although there have not been any reported isolates from mosquitoes in south china. a previous experiment conducted in laboratory indicated that zikv could be detected in the midgut and salivary glands of a. albopictus ten days after oral infection. outbreaks of denv have proved that south china may be a permissible environment for transmission of emerging viral diseases such as zikv, and imported cases may be sources of subsequent autochthonous cases. , a key difficulty in preventing the spread of infection is that a high proportion of infected individuals have no symptoms. similar to the mother in our study, asymptomatic individuals with viremia may be a source of infection when they are exposed to competent vectors. if the son's fever had not been found at the airport entrance, our subjects would not have been suspected of being infected with zikv, and thus their viremia would not have been confirmed. it is difficult to convince zikv carriers to be examined even if they are returning from an affected region. furthermore, in addition to the mosquito-borne route, other complex modes of transmission such as body fluid transmission will make controlling zikv infection even more difficult than controlling dengue fever. the priorities for control include strengthening the education of travelers from infected areas to non-epidemic regions, identifying virus carriers, preventing local transmission caused by imported cases, and an effective vector management program. in our study, four subjects in one family had lived and traveled together for the past few months, so they had similar opportunities for exposure to mosquito bites. however, because they are a family, the probability of human-to-human transmission by close contact between family members should not be ignored. if their zikv infections were all caused by mosquito-biting, from a time axis showing when symptoms occurred, the incubation periods of three of the patients were all more than five days (figure ), which is consistent with previous reports ( - -day incubation period according to the world health organization website). denv transmission by non-blood routes is almost impossible, so vector transmission is nearly the only way. thus, as for clustered cases in one family, if the vector transmission is considered, zikv can be comparable to denv because they have the same transmission dynamics via vectors. until now, there have been no reports of denv case numbers in one family being more than two, especially when they share the same virus sequence. therefore, because vector transmission of denv between family members is less likely, so is that of zikv. because the zikv isolated from three members is identical and there was a to day interval between initial symptom occurrence, human-to-human transmission may also be a plausible explanation. moreover, viral isolation from urine and saliva samples (collected on february ) of three patients was performed successfully using inoculation of suckling mice (data not shown), indicating that during onset, there was viral shedding in these three patients' body fluids, which was infectious to their family members. regarding direct transmission, sexual contact may be a route, for a relative case had been reported previously but with no direct evidence. because an in vitro experiment demonstrated that human skin cells are permissive to zikv, it might be possible that zikv can be transmitted by other bodily fluids such as saliva and urine. rt-pcr positivity for zikv rna together with successful viral isolation from the three patients' urine samples proved that there were live viruses present in these samples. rt-pcr also indicated the existence of viral rna in the patients' saliva. however, currently there is no reliable evidence demonstrating zikv transmission via these types of bodily fluids. a bibliometric analysis of global zika research zika virus and guillain-barre syndrome: another viral cause to add to the list guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study neurological expertise is essential for zika virus infection zika virus infects human cortical neural progenitors and attenuates their growth biology of zika virus infection in human skin cells characterization of lethal zika virus infection in ag mice zika virus: the latest newcomer zika virus in gabon (central africa)- : a new threat from aedes albopictus the zika outbreak of the st century probable non-vector-borne transmission of zika virus potential sexual transmission of zika virus zika: 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the americas first report of autochthonous transmission of zika virus in brazil we thank dr de wu (guangdong provincial center for disease control and prevention, china) for helpful discussions on zika virus isolation. this work was supported by a grant ( y - ) from the science and technology program of guangzhou, china. key: cord- -csao flr authors: chen, cong; liu, zuliang; liu, liguo; xiao, yan; wang, jianmin; jin, qi title: broad neutralizing activity of a human monoclonal antibody against h n strains from to date: - - journal: emerg microbes infect doi: . /s - - - sha: doc_id: cord_uid: csao flr h n influenza virus has been circulating among humans for five epidemic waves since it was first isolated in in china. the recent increase in h n infections during the fifth outbreak in china has caused concerns of a possible pandemic. in this study, we describe a previously characterized human monoclonal antibody, hnigga , obtained by isolating rearranged heavy-chain and light-chain genes from patients who had recovered from h n infections. hnigga recognized multiple has and neutralized the infectivity of out of the h n strains tested, as well as three emerging hpai h n isolates. the only resistant strain was a/shanghai/ / (h n -sh ), which carries the avian receptor alleles v and q in the sialic acid-binding pocket. the mab broadly neutralized divergent h n strains from to and represents a potential alternative treatment for h n interventions. the h n influenza virus was first isolated in southeastern china in early . since then, five epidemic waves have been reported that have caused human infections, with~ % resulting in death . h n causes severe respiratory distress syndrome in patients and is frequently associated with secondary bacterial pneumonia caused by multidrug-resistant acinetobacter baumannii and klebsiella pneumonia . noticeable dysbiosis of the oropharyngeal microbiome in h n patients has also been observed . during the fifth epidemic alone, human infections were confirmed, making it the largest h n epidemic to date. although no sustained humanto-human transmission of the h n virus was confirmed, limited human-to-human transmission has been observed . because h n viruses are able to be transmitted by the airborne route between ferrets and can infect and replicate in the human lower airways , they have the potential for efficient human-to-human transmission and are an increasing pandemic threat. furthermore, unlike h n , a high pathogenicity avian influenza (hpai) virus that causes severe disease in birds and poultry, most of the currently isolated h n viruses are low pathogenicity avian influenza (lpai) viruses that typically elicit no observable signs of disease in birds after viral infection. early warning and disease control for an h n pandemic may be extremely difficult, although hpai h n viruses that are more pathogenic in birds and mammals have recently emerged [ ] [ ] [ ] . h n cases have spread to provinces and municipalities in mainland china . due to its ability to more readily be transmitted from birds to humans, avian-origin h n has raised concerns regarding its potential for increasing the possibility of a pandemic. thus, it is prudent to conduct clinical trials to identify an effective treatment against h n influenza. as a typical member of influenza a viruses, h n is classified into subtypes based on the two major surface glycoproteins, haemagglutinin (ha), and neuraminidase (na), which are responsible for viral recognition, attachment to the cellular receptor and viral release. ha and na are ideal targets for antiviral drug design. na inhibitors, including oseltamivir and zanamivir, are currently the primary therapeutic treatment against h n infection in clinical settings , . however, because of the emergence of escape mutants that are resistant to oseltamivir or zanamivir or even both , , alternative treatment options for human h n infection are urgently needed. vaccination is the most effective intervention against seasonal influenza. it has been demonstrated that the h n vaccine is able to induce the production of both neutralizing and nonneutralizing antibodies in humans , and an inactivated h n vaccine has entered clinical trials . it was very interesting and encouraging to discover that some nonneutralizing antibodies could also protect mice from h n infection through fc-fcgr interactions . vaccination with seasonal h n strains was also shown to elicit h cross-reactive antibodies, although the level of serum protection in the general population remains to be determined . however, in the event of a pandemic outbreak, massive vaccinations against an emerging virus cannot promptly achieve herd immunity. limited by antiviral drug resistance, neutralizing therapeutic antibodies are considered to be a potentially effective treatment for influenza infections. passive immunotherapy using convalescent plasma from patients to treat h n and h n infections has achieved encouraging results and has reduced mortality [ ] [ ] [ ] . however, the large-scale production of antiserum is not possible in response to an emergency epidemic. the production of neutralizing monoclonal antibodies (mabs) would provide a feasible solution to this problem. recent studies have characterized several neutralizing antibodies from human donors that target different epitopes on viral ha proteins, such as ct , h . , m , hniggd , and hnigga , all of which represent potential interventions in the event of an h n pandemic. hnigga was isolated by our lab by isolating rearranged heavy-chain and light-chain genes from human survivors who had recovered from a/anhui/ / (h n -ah) infections. the antibody exhibited potent neutralizing activity against h n influenza in vitro and in vivo. in this study, we determined the breadth of the effectiveness of hnigga against divergent h n strains isolated from march to january , as well as against three hpai h n variants. a series of representative viral isolates were tested in pseudovirus-based neutralization assays. we report that hnigga can neutralize the most prevalent h n strains. other than an early a/shanghai/ / (h n -sh ) isolate, all prevalent h n strains from to could be neutralized by this antibody. to trace the evolution of the h has in the five epidemic waves, we randomly selected ha genes of human h n viruses from april to january and conducted polygenetic analyses. as shown in fig. a , the results confirmed that all the human h s descended from the same ancestor. most of the has from to were very similar and formed a cluster. however, after the initial outbreak, a broad dissemination of the virus was observed that indicated the occurrence of more frequent amino acid changes in ha. previously, we reported that an h n -neutralizing antibody, hnigga , recognized the viral receptor binding site (rbs) on ha . to evaluate potential antigenic drift, we performed an alignment of randomly selected ha sequences and assessed the amino acid substitutions in the viral rbs. as shown in fig. b , many amino acid changes occurred, most of which were in the hypervariable region around the receptor-binding pocket. to determine the breadth of activity for hnigga , its binding affinity was tested against a panel of ha s. of the strains tested (fig. ), hnigga only failed to bind to the ha of h n -sh , whereas it bound the other strains with a high affinity (k d values from . e− to . e− m). thus, the mab hnigga presents strikingly strong, broad binding activity, suggesting it has a broad neutralizing activity against divergent h n strains. comparing the ha of h n -ah with that of h n -sh , five amino acid substitutions occurred, including a s, s n, v g, p t, and l q. to assess their roles in the antigenic drift of the virus, we constructed five mutants of h n -ah ha such that each carried one of the five corresponding amino acids present in h n -sh . all the mutants were confirmed, and as shown in fig. a , all five mutants retained the ability to bind hnigga . nevertheless, the v g or l q mutations led to a noticeably reduced affinity. dual mutations at v g and l q resulted in complete loss of binding to the mab (fig. b ). in contrast, when g and q in h n -sh were reversely mutated to v and l , the mutants were restored in their ability to bind hnigga (fig. c) . these findings were further confirmed with full-length viral ha by the mab was immobilized on a cm chip, and a series of concentrations of ha were flowed over the immobilized mab. binding affinity (k d ) values were calculated using a steady-state affinity model produced with the biacore analysis software immunofluorescence assay (ifa) detection. as expected, the ha of h n -ah but not h n -sh was recognized by hnigga (fig. d) . the v g and l q mutations disrupted binding between the h n -ah ha and the mab, whereas binding was observed when both g v and q l were introduced into the ha of h n -sh . hnigga was previously shown to neutralize h n -ah and -sh influenza viruses in cells and in balb/c mice . in this study, to avoid the lethal pathogenicity of the live h n virus, we took advantage of a pseudovirusbased neutralization assay to evaluate the neutralizing activity of hnigga . pseudoviruses expressing the na of h n -ah and divergent has were produced in t cells, and the neutralizing activity of hnigga was tested on susceptible mdck cells. as in the neutralization assay with live h n -ah viruses, hnigga neutralized the h n -ah pseudovirus in a dose-dependent manner with an ic of . ng/ml (fig. a) . the neutralizing activity of hnigga against divergent h n strains was also tested. as expected, although the h n -sh strain was resistant to hnigga , the antibody could neutralize the other strains with estimated ic values from . to . ng/ml (fig. b) . the v g and l q mutations led to antigenic drift for viral ha and the fulllength ha protein, and the ability of the mutations to confer resistance against the mab was also determined c the mutations g v and q l restored the ability of the mab to bind the ha of h n -sh . d viral ha and the mutated ha proteins were expressed in hela cells and detected via ifa using pseudovirus entry assay. as shown in fig. c , when v g and l q were present in h n -ah, the pseudovirus successfully escaped from hnigga pressure. in contrast, when g and q were replaced by v and l , the mutated h n -sh was neutralized the mab. taken together, these results confirmed that v g and l q were responsible for antigenic drift in h n -sh . hnigga neutralizes the emerging hpai h n strains h n viruses have remained lpai viruses in poultry since their first appearance in . however, human infections with hpai h n viruses were reported in february , . a multibasic cleavage site motif in viral ha was identified that facilitates hpai h n virus replication in avian species. to assess the neutralizing activity of hnigga against hpai h n , pseudoviruses expressing the ha of three hpai h n isolates (a/ guangdong/ sf / , a/guangdong/th / , and a/taiwan/ / ) and the na of h n -ah were generated. as shown in fig. , hnigga was capable of neutralizing all three variants with ic values of~ ng/ ml. the avian influenza a h n virus continues to be a serious threat to public health and has raised concerns of a potential pandemic. thus, it is necessary to develop vaccines and antiviral drugs as well as neutralizing antibodies for the prevention and control of fatal h n infections in humans. in the present study, we characterized a previously isolated human h n -neutralizing antibody (hnigga ) and determined the breadth of its neutralizing activity. the significant role of neutralizing antibodies in protecting humans against viral infections has been well , , . because it is present on the surface of the influenza virion, the ha glycoprotein is the primary target for developing neutralizing antibodies. however, the high variability of viral ha makes it difficult to develop a highly cross-reactive neutralizing antibody. this is especially true since viral ha is able to continually evolve while maintaining surface sialic acid receptor binding capability, which leads to escape from the antibody response and is known as antigenic drift , . although different has display unique structural features, the sialic acid-binding pocket on the ha rbs is highly conserved for the recognition of the common receptor. a few rbs-directed antibodies have successfully achieved modest cross-reactivity , , by interposing a smaller footprint into the receptor binding groove. however, most rbs-directed antibodies have exhibited a limited breadth in their neutralization activity, including d and j , due to extra contacts with variable residues in the periphery of the conserved pocket. in this study, we tested the binding affinity of hnigga for different has of prevalent h n strains isolated from april to january . h n ha has been rapidly evolving (fig. a) , which is consistent with the previous result that h ha is continually mutating at a high rate . our results showed that hnigga utilizes avidity to extend its breadth of recognition and to increase its affinity against a large portion of divergent ha strains ( out of ), including the a v/r k variant, which has become the dominant circulating strain in the fifth wave . hnigga exhibited a loss of binding to the ha variant on the h n -sh strain, an earlier isolate from the shanghai epidemic carrying an avian h signature . the preference for avian α− , -linked sialic acid cellular receptors rather than human α− , linked sialic acids receptors limited its transmission to humans, and the sh strain was rarely observed in latter outbreaks. two amino acids substitutions (v g and l q) were responsible for the preference of different cellular receptors, as well as the antigenic drift of h n -sh (fig. ) . although hnigga recognized other has, the observed binding affinities were not completely consistent. an approximately tenfold decrease in binding affinity was observed for a/hebei/ / and a/guangdong/ sf / carrying the l q and v a mutations, respectively, indicating important roles of v and l in binding hnigga . the reason for the attenuated binding affinity to a/jiangsu/ / , a/fujian/ / , a/anhui/ / , and a/zhejiang/ / remains to be determined. however, the a v substitution in the rbs that the four strains share in common is surely worth studying further. all h n pseudoviruses, including three emerging hpai h n isolates, were neutralized by hnigga with the exception of h n -sh (figs. b and ) . however, because the pseudotyped virus particle entry assays may be overly sensitive, an accurate assessment of the in vitro and in vivo neutralizing activity of hnigga using live h n viruses is still needed. this is especially necessary since mutations on other genes besides ha are also associated with viral replication capacity, pathogenicity, and transmissibility. the e k mutation in the polymerase basic (pb ) gene enhances h n replication in mammals . the k r mutation in pb , which was identified in a hpai h n (a/taiwan/ / ) strain, also contributes to improve replication efficiency together with k . the na r k mutation confers resistance to both oseltamivir and peramivir . nevertheless, based on genetic and phylogenetic analyses, the who has recommended that h n -ah may serve as a viable vaccine candidate for broad protection against h , . a comprehensive survey of common resistance mutations among the many strains tested here suggests that mab hnigga may be a useful template for a therapeutic antibody. genetically and antigenically novel h n influenza viral strains will continue to emerge due to the genetic nature and broad host range of this virus. in the absence of an effective vaccine, direct administration of h n neutralizing antibodies could be used as an intervention to prevent h n infections in humans. this approach could be especially helpful for those at high risk for contracting h n . when administered prophylactically, hnigga conferred % protection in virus-infected mice , supporting this rationale. alternatively, the rbs-directed neutralizing antibody hnigga can be used together with other antiviral drugs (e.g., oseltamivir and zanamivir) to control the production of the escape mutants that limit the effectiveness of these na inhibitors , . a total of influenza a h n strains isolated during the - seasons throughout china were selected and genetically characterized. the retrieved full-length ha amino acid sequences of influenza a h n viruses were aligned with the reference sequences available in the gisaid database (http://platform.gisaid.org/) by "clus-talw" using molecular evolutionary genetic analysis (mega) version . multiple alignment sites with gaps in any of the sequences were excluded. phylogenetic trees were generated by the maximum-likelihood method in mega with bootstrap replicates. the constructed phylogenetic trees were analyzed for possible geographical linkages. the globular head ha protein (residues to , based on h numbering) was expressed using the bac-to-bac baculovirus expression system (invitrogen). briefly, the ha coding sequence was amplified by pcr and inserted into the pfastbaci vector (invitrogen). to facilitate protein secretion and purification, an n-terminal gp signal peptide and a c-terminal × his-tag were fused to the ha gene. the recombinant bacmid was transfected into sf insect cells, and the protein was purified by nickel-nitrilotriacetic acid (ni-nta) affinity chromatography followed by size exclusion chromatography on a superdex column (ge health care). the ha and na genes of h n -ah are stocked by our lab. the remaining viral ha genes were obtained by site-directed mutagenesis in the rbs with the h n -ah strain according to the gene alignment results in fig. b . human igg protein for the mab hnigga was purified as described previously. the heavy-chain and lightchain genes were cloned into the antibody expression vector and expressed by transient transfection in hek f cells using lipofectamine (invitrogen). the cell culture was collected h later and was centrifuged to remove cell debris. human igg protein was then purified by affinity chromatography using protein a agarose (transgen biotech) and was further purified by size exclusion chromatography. the protein concentration was measured spectrophotometrically (ge healthcare). the binding of hnigga to viral ha proteins was measured by enzyme linked immunosorbent assay (elisa). the -well plates were coated overnight at °c with viral ha protein ( ng per well). serial four-fold dilutions of antibodies (beginning with ng) in pbs were made and assayed for binding to recombinant ha proteins. after washing, bound antibodies were detected by horseradish peroxidase (hrp)-conjugated goat antihuman igg ab (sigma-aldrich) at a nm absorbance using an elisa plate reader (tecan). the immunofluorescence assay (ifa) was performed as described previously . first, hela cells were grown on glass slides as monolayers. next, the pcdna . vector expressing ha from h n -ah/h n -sh was transfected into cells using lipofectamine (invitrogen). at h post transfection, the slides were fixed with % paraformaldehyde and washed with pbs. the cells were then incubated with hnigga at °c for h. bound antibodies were detected with a rhodamine-conjugated goat anti-human igg antibody (invitrogen) and were observed using a fluorescence microscope. surface plasmon resonance measurements were performed using a biacore instrument with cm chips (ge healthcare) at room temperature ( °c). all proteins were exchanged into pbst buffer (phosphate-buffered saline with . % (v/v) tween- , ph . ) by gel filtration. the purified mab was first immobilized on a cm chip. the ha proteins were serially diluted to between . and . nm ( . , . , . , . , . , and . nm) and were then flowed over the mab. the binding kinetics were analyzed with biacore analysis software (biaevaluation version . ) using a : langmuir binding model. to generate h n pseudoviruses, viral ha-and naencoding sequences were cloned into the pcdna . expression vector and were cotransfected with a pnl - -luc-r-e-viral backbone plasmid into t cells using lipofectamine (invitrogen). supernatants were collected h after transfection. the h n pseudovirus was purified by ultracentrifugation and dissolved in phosphatebuffered saline (pbs) before being stored at − °c in aliquots until their use in the neutralization assay. the has were obtained by site-directed mutagenesis of the h n -ah ha-encoding gene according to the gene alignment results in fig. b . the ha genes of three hpai h n variants, including a/guangdong/ sf / , a/guangdong/th / , and a/taiwan/ / , were custom synthesized and cloned into the pcdna . vector. all the clones used were confirmed by sequencing. the viral titers were determined in mdck cells by luciferase activity as the median tissue culture infective dose (tcid ). neutralization assays were performed by incubating tcid of pseudovirus with serially diluted antibodies at °c for h. the mixture was added to the cultured mdck cells. viral infection was quantified by the luciferase activity h after infection. an irrelevant human igg protein (anti-hiv, immune technology) was used as a negative control. human infection with a novel avian-origin influenza a (h n ) virus. new engl human infection with avian influenza a (h n ) virus-china an analysis of microbiota-targeted therapies in patients with avian influenza virus subtype h n infection disordered oropharyngeal microbial communities in h n patients with or without secondary bacterial lung infection infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs tropism and innate host responses of a novel avian influenza a h n virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract human infection with 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a/h n infection: other treatment options and issues meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h n treatment? a potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza a virus h n influenza virus neutralizing antibodies that possess few somatic mutations a potent germline-like human monoclonal antibody targets a ph-sensitive epitope on h n influenza hemagglutinin characterization of two human monoclonal antibodies neutralizing influenza a h n viruses human monoclonal antibodies targeting the haemagglutinin glycoprotein can neutralize h n influenza virus structural insight into a human neutralizing antibody against influenza virus h n broad and potent neutralization of hiv- by a gp -specific human antibody broad neutralization coverage of hiv by multiple highly potent antibodies specificity, cross-reactivity, and function of antibodies elicited by zika virus infection molecular 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influenza a viruses by insertion of a single antibody loop into the receptor binding site the global circulation of seasonal influenza a (h n ) viruses the evolution of human influenza viruses cross-protective potential of a novel monoclonal antibody directed against antigenic site b of the hemagglutinin of influenza a viruses naturally occurring antibodies in humans can neutralize a variety of influenza virus strains, including h , h , h , and h structural basis of preexisting immunity to the h n pandemic influenza virus antibody recognition of the pandemic h n influenza virus hemagglutinin receptor binding site antigenic drift of h n viral hemagglutinin molecular basis for high virulence of hong kong h n influenza a viruses the k r substitution in viral protein pb enhances the effects of e k on influenza virus replication resistance to neuraminidase inhibitors conferred by an r k mutation in a human influenza virus h n isolate can be masked by a mixed r/k viral population evaluation of the immune responses to and cross-protective efficacy of eurasian h avian influenza viruses development of a high-yield reassortant influenza vaccine virus derived from the a/anhui/ / (h n ) strain key: cord- -kcatqzgs authors: zhang, xinyu; ren, dan; li, tuofan; zhou, huayan; liu, xiaoyu; wang, xiaobo; lu, hao; gao, wei; wang, yajuan; zou, xiaoyan; sun, huaichang; ye, jianqiang title: an emerging novel goose astrovirus associated with gosling gout disease, china date: - - journal: emerg microbes infect doi: . /s - - - sha: doc_id: cord_uid: kcatqzgs since the first isolation from human, astroviruses have been detected in many species. wide host range and occasional cross-transmission of astrovirus pose a risk for zoonotic infection. here, novel astroviruses were identified from goslings with recent epidemic gout disease in china. a virus, designated as gd, was efficiently isolated from a diseased gosling using lmh cells. genome of gd amplified using ′ and ′ race was nt in full length. sequence analysis revealed the genome of gd was < . % homology with others deposited in genbank. moreover, gd could be neutralized by goose convalescent sera, and the gout associated symptom in goslings could be reproduced by gd infection. our data demonstrated the goose astrovirus could be one of the causative agents of the ongoing gosling gout disease in china. the identification of the goose astrovirus not only diversified the astrovirus species, but also broadened the disease patterns caused by astroviruses. astroviruses are currently classified into two genera, mamastroviruses (mastvs) and avastroviruses (aastvs) . mastvs mainly infect mammals including human, ovine, bovine, porcine, feline, canine, mink, bat, deer, mouse, sea lion, dolphin etc., whereas aastvs generally infect aviansuch as turkey, chicken, duck, pigeon, and goose , . notably, genetic variation and crossspecies transmission of astrovirusespose the risk for zoonotic infection , . infection with astroviruses mainly cause enteric diseases such as gastroenteritis and diarrhea in human and animals as reported initially, then nephritis in chicken and pigeon, hepatitis in ducklings, and encephalitis in human, cattle, and sheep recently, which broadens the disease pattern of astroviruses and highlights its significance , - . in , a gout disease emerged in week-old goslings in anhui province, which had spread to most provinces of china by with high morbidity ( - %) and mortality ( - %) , and no pathogenic bacteria could be isolated from the diseased goslings. however, little is known about the pathogen for the goose gout disease endemic in goose flocks in china. during - , the outbreaks of gout disease were reported in broilers in india. through virus isolation and infection study, bulbule et al. demonstrated that a novel chicken astrovirus (castv) could be as one of the causative agents for the gout disease in chicken flocks in india . to control the spread of the goose gout disease, we investigated the pathogenic agent of the disease. through in vitro and in vivo experiments, we identified and isolated a novel goose astrovirus different from the castvas a causative agent of the gout disease recently circulating in gosling flocks in china. in , a gout disease emerged in -week-old goslings in anhui province, which has spread to most provinces of china by . the outbreak of the gosling gout disease has caused significant economic loss in goose industry. the clinical signs of the disease were characterized by white feces, leg joint enlargement with urate deposits and paralysis. at necropsy, kidney enlargement and intensive urate deposits were found in the gallbladder, knees, and ureters, and on the surfaces of cardiac, heart, liver, air sacs, trachea, and proventriculus ( fig. a-f ). the disease lasted for - days with high morbidity and mortality. the survival goslings grew slowly and were susceptible to bacterial infection. to investigate the causative agent for the ongoing gosling gout in china, diseased goslings with gout symptom were collected from goose flocks from jiangsu, anhui, shandong, guangdong, and fujian provinces. pcr/rt-pcr was performed using specific primers listed in table to detect viral nucleic acids. except for a bp band amplified from all samples (n = ) by a pair of degenerate primers to orf b gene of avian astrovirus, negative results for the other viruses were demonstrated. however, the samples (n = ) detected from the healthy goslings without gout disease, provided by national water fowl gene bank, taizhou, china, were negative by rt-pcr in avian astrovirus detection. the amplicons from the flocks respectively designated as ahla, ahdy, fjza, gdhz, jsxz, jsgy, jsdy, jsyz, sdjm, and sdlj, were sequenced directly for homology analysis. results indicated the sequences of the pcr products were highly consistent (å %), but had < % homology with those deposited in genbank. furthermore, the sequences detected shared only . - % homology with the astrovirus flx (nc_ ) recently identified in goose with enteritis from china . to further determine the molecular characteristics of the goose astrovirus, the viral rna genome was amplified by race-pcr from the gdhz sample. sequencing data showed that the viral rna genome was nt in length (genbank accession no. mg ) with a typical genome structure of astroviruses , . between the -nt ′ untranslated region (utr) and -nt ′ utr, three open reading frames (orfs) were predicted, including -nt orf a, -nt orf b, and -nt orf . a potential ribosomal frameshift signal (aaaaaac) was identified downstream of orf a, followed by a uag stop codon. as expected, the conserved ccgaa motif of astroviruses was found between orf b and orf . sequence alignment of the rna genome showed < . % homology with other aastvs. to gain further insight into the evolutionary relationship of the virus with other aastvs, phylogenetic tree was constructed based on the sequences of orf . the novel astrovirus formed a distinct clade in avastroviruses including tastv- (aaf ), tastv- ak/ (abx ), and dastv- c-ngb (acn ) as described in fig. . recombinant analysis was conducted among all the genomes of astroviruses deposited in genbank, and the results demonstrated no recombinant event happened in the viral genomic sequence. to isolate the novel goose astrovirus, the homogenates of the pooled liver and kidney from the diseased gosling were inoculated into chicken liver cell line lmh. after five passages, an astrovirus was isolated and identified by rt-pcr using the above primers, named as gd. to further confirm the isolation of the virus, the infected lmh cells were analyzed through indirect fluorescent assay (ifa) using the convalescent sera from the survival geese with gout symptom and the mouse sera against the capsid p of gd (generated in our laboratory) respectively. as described in fig. , the specific fluorescent signals could be found in the cytoplasm of the lmh cells infected with gd, but not in the control lmh cells. notably, there was no cytopathic effect found in infected lmh cells. moreover, gd virus could be efficiently neutralized by the convalescent sera from the survival goose with gout disease, and the neutralization titer of the goose convalescent sera against gd reached : , however, the sera from healthy geese did not show any neutralization activity, which highlighted the association between the novel goose astrovirus and the gosling gout disease. all these clearly demonstrate that a novel goose astrovirus gd has been efficiently isolated from the diseased goslings with severe gout symptoms. to investigate whether the gd isolatewould be the causative agent for the ongoing goslings gout in china, day-old goslings were infected with gd. all the infected goslings showed clinical signs with depression and started to excrete white feces at day post infection, and grew slower than the non-infected control goslings as described in fig. a . at day post infection, three inoculated goslings were killed and the tissues were collected. at necropsy, the typical gout pathological changes were found, including kidney with swelling and white substance-filled ureters. the histopathological assay also demonstrated the similar pathological signs to that of naturally infected goslings, including necrosis and abscission of renal tubular epithelial cells, presence of protein cast in renal tubules (fig. c) . moreover, the virus could be efficiently isolated from the liver, spleen and kidney of the infected goslings at day post infection, but not from the non-infected control goslings as described in fig. b . although all the infected goslings had clinical signs and histopathological changes associated with gout, all these infected goslings were survival during weeks observation post infection. in addition, the neutralizing antibody titer against gd from the infected goslings could reach : at day post infection. all these data demonstrated that the gd isolate could efficiently infect the goslings and cause diseases associated with gosling gout. it has been reported that avastrovirus infections can cause enteritis, diarrhea, hepatitis of ducklings, nephritis of chickens, and decrease of egg-hatching rate of different fowls , - . based on the amino acid sequences of the fig. identification of the novel goose astrovirus gd isolate in lmh cells by indirect immunofluorescence assay. a, b lmh cells infected with or without gd were reacted with the convalescent sera from the survival geese with gout symptomrespectively. c, d lmh cells infected with or without gd were reacted with the mouse sera against capsid p of gd respectively fig. infection study with the novel goose astrovirus gd. a bodyweight changes of the infected goslings. b viral titer in the tissues from the infected goslings. c histopathologic changes in infected goslings. a normal kidney from the non-infected gosling, b kidney from the infected gosling with renal tubular necrosis, abscission of renal tubular epithelial cells and protein cast in renal tubules orf -encoded capsid protein, these aastvs can be divided into three species: avastroviruses , , and (the international committee on taxonomy of viruses, ictv, ). in this study, a novel goose astroviruses gd was identified and isolated from goslings with gout symptom. the infection study further confirmed that the gd belonging to avastroviruses could be the causative agent of the ongoing gosling gout disease in china. although castvs could induce gout disease in chickens , castvs have only about . - . % homology with gd on amino acid sequence of capsid, and there is no recombinant event happened between them, which indicate that the gd is an under recognized novel astrovirus. in comparison with the goose astrovirus recently identified in geese with enteritis in china , the novel goose astrovirus showed only about % identity in nucleic acid. this highlights at least two species of goose astrovirus associated with different diseases are circulating in goslings flocks in china. the infection study with gd could reproduce the clinical signs and histopathogenesis associated with the gout disease, but all the infected goslings were survival during weeks observation post infection. the high morbility and low mortality for the infection study with gd might be related with the infection route and dose, and the age. although the pathogens including fowl adenovirus, goose hemorrhagic polyomavirus, goose reovirus, goose coronavirus, goose parvovirus, and duck tembusu virus were not able to be detected in the diseased goslings, we could not exclude the potential coinfections with other pathogens, which may exacerbate the clinical symptoms of gout. in addition, whether the high protein level in the feed promotes the occurrence of gout also need to be further investigated. the high-titer neutralizing antibody to gd from the survival geese highlighted the novel goose astrovirus could efficiently induce enough adapt immunity in goslings. however, whether this adapt immunity can protect goslings completely from the novel goose astrovirus need to be further studied. the mechanism for the gout disease induced by either castv reported by bulbule et al. or gd identified in this study need to be further elucidated. moser et al. reported that astroviruses could increase the permeability of the epithelial cells . the increased permeability of the kidney epithelial cells induced by the astroviruses might contribute to the gout disease . in addition, due to lacking arginase in poultry, ammonia cannot be processed into urea instead of purine, hypoxanthine, and xanthine, then oxidized to uric acid, forming sodium urate and calcium urate, excreted through kidney finally. if the rate of urate formation is greater than the excretory capacity of the urinary organs, gout can be caused with the urate deposits on visceral surfaces . therefore, factors that cause renal and urinary tract injury or urine concentration and urinary excretion disorder can promote the formation of urate deposits. in this study, the novel astrovirus gd could cause kidney damage, which maybe the main cause of gosling gout in china. in summary, this is the first demonstration of a goutdisease associated novel goose astrovirus efficiently isolated by lmh cells. the specific sequences of the astroviruses detected in samples from the five different provinces demonstrated the widespread of the novel goose astroviruses in china. however most astrovirusesare difficult to grow in cell culture , the novel goose astrovirus gd could be isolated efficiently in lmh cells in this study, which provides an efficient proliferation system for the avastrovirus in vitro. this efficient system might provide a good tool for developing inactivated vaccine and investigating the pathogenicity mechanism of goose astrovirus in the future. the full genomic rna of the novel goose astrovirus was sequenced and publicated in genbank,which pave ways for further developing vaccines and diagnostic methodsin controlling the gout disease in goslings. during the preparation of the revised version for this manuscript, other groups in china recently also reported the novel goose astrovirus [ ] [ ] [ ] . different from those recently reported by other groups, we efficiently isolate such novel goose astrovirus in vitro using the cell culture system (lmh cells) and reproduce the associated gout disease by inoculating the cell cultured virus gd. of course, the originator, host range, variation, molecular pathogenesis, and potential zoonotic infection of the novel goose astrovirus need to be further studied. fresh tissues (such as liver, kidney, trachea, etc.) were collected from diseased goslings (n = ) aged - days with gout symptom from different goose flocks in jiangsu, anhui, shandong, guangdong, and fujian provinces during july to november . the convalescent sera were collected from survived geese (n = ) aged days suffered gout disease or survived goslings (n = ) experimentally infected with astrovirus. total dna and rna of pooled liver and kidney were extracted using qiamp dna mini kit (qiagen) and trizol reagent (invitrogen) respectively based on protocols from the manufacturers. pcr/rt-pcr for the detection of fowl adenovirus, goose hemorrhagic polyomavirus, goose reovirus, goose coronavirus, duck tembusu virus, goose parvovirus, and avian astrovirus were performed using the primers listed in table [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and pcr/rt-pcr kits from takara biotechnology co., ltd (dalian). the amplified pcr fragments were sequenced by sangon biotech (shanghai) co., ltd. to determine the full-length nucleotide sequences of virus, race-pcr was performed using rna extracted from pooled liver and kidney with a smarter® race ′/ ′ kit (takaka bio usa, inc). the ′ and ′ end fragments were amplified using genome-specific primer gsp , ngsp , and gsp , ngsp listed in table . then pcr products were cloned into the prace vector and sequenced according to the manufacturer's protocol. orfs prediction and special sequences search in the genome were conducted by lasergene . nucleic acid and amino acid sequences identity analyses was conducted by protein blast in genbank. phylogenetic tree was constructed by mega . recombinant analysis was performed by rdp . and simplot . . . the homogenate of the pooled liver and kidney samples from a diseased gosling were filtered with . μm filter, then ml filtrate mixed tpck (l- -tosylamido- -phenylethyl chloromethyl ketone)-treated trypsin at the concentration of μg/ml was inoculated into chicken liver cell line (lmh, atcc) with % confluence in wells plate. h post infection, the supernatant was discarded, and opti-mem medium containing μg/ml tpck trypsin was added. the infected cells were blindly passaged every days, and the viral antigen was detected by immunofluorescent assay. according to the viral genome sequence, a codonoptimized gene of capsid p was synthesized and cloned into pet- a (novagen). the recombinant capsid p protein was expressed in e.coli blr (de ) cells (novagen) under the inducing of isopropyl-β-d-thiogalactopyranoside (sangon biotech) at concentrate of mmol/l and purified using ni-nta agarose (qiagen). after verified using convalescent goose sera by westernblot, μg purified recombinant protein was inoculated to -week old balb/c mouse in abdomen at an interval of days. days after the rd immunization, the mouse blood was collected to prepare antisera. the infected cells were fixed with % paraformaldehyde (sangon biotech) for min, followed by rinsing with pbs. . % v/v triton x- (sigma) diluted in pbs was applied to each well for min. after washing three times with sterile pbs, the cells were overlaid with pbs containing % w/v bovine serum albumin (sangon biotech) and incubated for min at °c. the mouse antiserum anti-capsid p was used as the primary antibody with a : dilution in pbs. after min incubation at °c, the cell monolayers were rehydrated by rinsing three times with pbs. coverslips were stained using goat anti-mouse igg conjugated with fitc (kpl) and incubated for a further min at °c. the stained cells were rinsed with pbs, then dyed with hoechst (solarbio) and rinsed again. the stained cells were then examined under a confocal microscope. the virus neutralization test was performed as reported . briefly, the convalescent sera both from the clinical survived goslings (n = ) with gout disease and the experimental infected survived goslings (n = ) were serially diluted with opti-mem medium. then, µl of the diluted sera were mixed with an equal volume of tcid virus. the mixture was incubated for h at °c, then inoculated into lmh cells and the inoculated cells were washed once with pbs. at day post inoculation, the inoculated cells was analyzed by indirect immunofluorescence assay to determine the neutralization titer of the sera. to determine pathogenicity of the isolated virus, twelve -day-old goslings were inoculated with the novel goose astrovirus ( × tcid ) through muscle injection routes and twelve non-inoculated -day-old goslings were used as control. the clinical signs and the bodyweight of these goslings were observed and monitored after inoculation. three infected goslings were killed at day post infection for detecting the viral titer in tissues and the histopathological analysis. fresh tissues collected from the diseased or experimental goslings were fixed with % neutral buffered formalin. then paraffin sections were prepared and stained with haematoxylin and eosin (h&e) as described . histopathological changes in the tissues were observed under an optical microscope. construction of a reverse genetic system for porcine astrovirus astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions non-human primates harbor diverse mammalian and avian astroviruses including those 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preliminary report astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos isolation and detection of duck astrovirus cph: implications for epidemiology and pathogenicity astrovirus increases epithelial barrier permeability independently of viral replication gout: a disease of kings capsid protein sequence diversity of avian nephritis virus isolation and characterization of an astrovirus causing fatal visceral gout in domestic goslings genetic characterization of a novel group of avastroviruses in geese a novel group of avian avastrovirus in domestic geese outbreaks of serotype fowl adenovirus with novel genotype a novel polyomavirus (goose hemorrhagic polyomavirus) is the agent of hemorrhagic nephritis enteritis of geese preparation and evaluation of goose reovirus inactivated vaccine molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) duck egg-drop syndrome caused by byd virus, a new tembusurelated flavivirus development of a duplex semi-nested pcr assay for detection of classical goose parvovirus and novel goose parvovirus-related virus in sick or dead ducks with short beak and dwarfism syndrome identification of chicken enterovirus-like viruses, duck hepatitis virus type and duck hepatitis virus type as astroviruses human antibody neutralizes severe fever with thrombocytopenia syndrome virus, an emerging hemorrhagic fever virus this study was supported by key laboratory of prevention and control of biological hazard factors (animal origin) for agrifood safety and quality ( ) and the priority academic program development (papd) of jiangsu higher education institutions. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -p i m authors: wang, renxi; xiao, he; guo, renfeng; li, yan; shen, beifen title: the role of c a in acute lung injury induced by highly pathogenic viral infections date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: p i m the complement system, an important part of innate immunity, plays a critical role in pathogen clearance. unregulated complement activation is likely to play a crucial role in the pathogenesis of acute lung injury (ali) induced by highly pathogenic virus including influenza a viruses h n , h n , and severe acute respiratory syndrome (sars) coronavirus. in highly pathogenic virus-induced acute lung diseases, high levels of chemotactic and anaphylatoxic c a were produced as a result of excessive complement activaiton. overproduced c a displays powerful biological activities in activation of phagocytic cells, generation of oxidants, and inflammatory sequelae named “cytokine storm”, and so on. blockade of c a signaling have been implicated in the treatment of ali induced by highly pathogenic virus. herein, we review the literature that links c a and ali, and review our understanding of the mechanisms by which c a affects ali during highly pathogenic viral infection. in particular, we discuss the potential of the blockade of c a signaling to treat ali induced by highly pathogenic viruses. the epithelium of the lung is vulnerable to damage caused by inhaled microorganisms and other noxious particles. many studies suggested the presence of complement components at the alveolar epithelium, where inhaled airborne particles and microorganisms are deposited. [ ] [ ] [ ] in addition, the complement system has been implicated in the development of acute lung diseases induced by highly pathogenic viruses including influenza a virus h n , h n , h n , severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov). however, the specific contributions of complement to lung diseases based on innate and adaptive immunity are just beginning to emerge. elucidating the role of complement-mediated immune regulation in these diseases will help identify new targets for therapeutic interventions. complement activation leads to the formation of bioactive molecules, including the anaphylatoxins, c a and c a, and the lytic membrane attack complex (c b- ). the complement-activated product c a is a strong chemoattractant and is involved in the recruitment of inflammatory cells such as neutrophils, eosinophils, monocytes, and t lymphocytes, in activation of phagocytic cells and release of granulebased enzymes and generation of oxidants. c a also displays other powerful biological activities including inducing ''cytokine storm.'' on the other hand, blockade of c a signaling has demonstrated potential benefits in the treatment of acute lung injury (ali) induced by highly pathogenic viruses. in this article, we summarize recent developments in our understanding of the role of c a in mediating aute lung injury induced by highly pathogenic viruses. highly pathogenic virus due to high mutation rates of viruses, every several years to decades a highly pathogenic virus emerges. especially in the recent decades, there were more than five highly pathogenic viruses such as sars coronavirus in , avian influenza a/h n virus in , h n virus in , h n virus in , and mers coronavirus in . as exemplified by coronaviruses and influenza viruses, bats and birds are natural reservoirs for providing viral genes during evolution of new virus species and viruses for interspecies transmission. , this is the primary cause of an outbreak by jumping directly from bird to human. in two months, laboratory-confirmed cases and deaths have been reported globally. there is an h n vaccine for human use, but there is currently no h n , sars or mers vaccine available. current vaccination strategies are still inadequate at providing protection against epidemic outbreaks. thus, it is urgent to explore the mechanism by which highly pathogenic viruses induce diseases. acute lung injury induced by highly pathogenic viral infections although highly pathogenic virus infections have the different epidemiology, there is a similar rapid progression to acute respiratory distress syndrome (ards). for example, histopathological changes in the lung from patients infected with h n are highly similar to those of patients with sars. except for influenza a h n virus, avian influenza a h n virus in also caused severe pneumonia. postmortem biopsy of patients infected with h n in showed acute diffuse alveolar damage: patient , who died days after symptom onset, had intra-alveolar hemorrhage, whereas patients and , who died days after symptom onset, had pulmonary fibro proliferative changes. patients infected with h n develop rapidly progressive pneumonia, further resulting in ali or ards. , ali may be a critical cause of death in patients with h n infection. , like h n infection, h n also causes serious lung pathology. in addition, sars-cov infection caused ali that may progress to life-threatening ards. mers-cov infection resulted in a more severe pneumonia than sars-cov infection. respiratory distress is the most common cause of death in patients infected with highly pathogenic virus. in terms of therapy, lung protective ventilation is the cornerstone of supportive care. extracorporeal membrane oxygenation is routinely used in many centers for the treatment of severe respiratory tract infections. however, due to few effective treatment options, ali is often fatal for patients infected with highly pathogenic viruses. this suggests that serious lung pathology should be of particular concern. after a microorganism infection begins, the host quickly activates the complement system to clear infected pathogens. during the complement activation, the high levels of products such as c a are commonly involved in exacerbated inflammatory reactions that can cause direct harm to the host following infections. [ ] [ ] [ ] iav belongs to the orthomyxoviridae family with single-stranded negative-sense rna virus, and has the capacity to activate the complement system. in addition, the avian influenza hemagglutinins typically bind alpha - sialic acid receptors, whereas human influenza hemagglutinins bind alpha - sialic acid receptors. thus, h n replicates in the lower respiratory tract, then causes complement activation. this suggests that upon influenza infection, the high levels of c and c including fragments c a and c a are produced. complement activation possibly contributes to the observed tissue damage in severe viral infection. studies demonstrated that ali in h n -infected mice was caused by excessive complement activation such as release of c a. thus, complement activation plays a critical role in the pathogenesis of virus-induced acute lung injury. among the complement activation products, the anaphylatoxin c a is one of the most potent inflammatory peptides. increased levels of c a were found in bronchoalveolar lavage fluid (balf) and serum from patients infected with fatally h n pandemic virus. , c a had also been found to increase in balf of mice infected with highly pathogenic avian influenza h n but not following seasonal iav infection. on the other hand, balf from recovered patients with ards demonstrated significantly reduced c a-dependent chemotactic activity. thus, c a might play a critical role in the pathogenesis of virus-induced acute lung injury. c a-mediated inflammatory cells migrate into lung tissue compared to normal controls, sars patients had increased cellularity of balf with increased alveolar macrophages. thus, mononuclear cell infiltration might have an important role in the pathogenesis of ali induced by highly pathogenic viruses like sars. anaphylatoxin c a has been implicated in the pathogenesis of ards by mediating neutrophil attraction, aggregation, activation, and subsequent pulmonary endothelial damage. [ ] [ ] [ ] [ ] reversely, c adependent chemotactic activity is significantly decreased in recovered patients with ards. these suggest that c a-mediated mobilization and activation of immune cells might be the central events to tissue injury caused by highly pathogenic viral infections. two chemoattractants c a and interleukin (il- ) can be synthesized by cells in the lung (e.g., macrophages, epithelial cells, endothelial cells, smooth muscle cells and neutrophils). il- levels have also been found to correlate with neutrophil numbers and the degree of lung dysfunction. c a could strongly amplify il- expression from human whole blood cells induced by lipopolysaccharides and other types of toll-like receptors agonists via extracellular-signal-regulated kinases / and p , but not c-jun n-terminal kinase. the data suggest that c a might be a critical effector molecule to mediate lymphocyte attraction by itself or indirectly by enhancing the production of il- . altogether, c a-mediated lymphocyte attraction plays a critical role in the pathogenesis of ali induced by highly pathogenic viruses. neutrophil extracellular traps (nets) are primarily composed of dna from neutrophils, which bind pathogens with antimicrobial proteins. nets are beneficial in antimicrobial defense and can help fight against invading pathogens. however, an excess of nets contributes to the pathology of a number of diseases including those of the lung. nets are found in infection-related ali models of influenza virus. , in vitro studies demonstrated that c a, in association with granulocyte-macrophage colony-stimulating factor, is able to induce the release of nets. c a is also able to activate macrophages and endothelial cells and to promote vascular leakage and the release of nets. thus, nets are induced by c a during iav infection and are associated with alveolar damage in iav-induced pneumonitis. the excess of net components are potent factors in lung injury. net increases the permeability of the alveolar-capillary barrier by cleaving endothelial actin cytoskeleton, e-cadherin and vecadherin. the antimicrobial peptide ll- in net structures presents cytotoxic and proapoptotic properties towards endothelial and epithelial cells. net also induces the release of proinflammatory cytokines. the data suggest that c a-mediated neutrophil extracellular traps aggravate ali in patients infected with highly pathogenic virus. c a-mediated release of reactive oxygen species c a is a strong chemoattractant for neutrophils and monocytes; it then activates these cells to generate oxidative burst with release of a study demonstrated that ros are primary pathogenic molecules in pneumonia from mice infected with influenza virus. the amount and duration of exposure of generated ros, released from respiratory, immune, and inflammatory cells, determined the extent of lung damage. in lung fibroses, inflammatory cells produce a significantly greater amount of ros. critically, antioxidant treatment significantly reduces lung damage and mortality in influenza-infected mice. these studies demonstrated a critical role of reactive oxygen intermediates (rois) in virus-induced epithelial damage. c a-c ar interaction plays a critical role in oxidative burst. interception of c a/c ar signaling with a c ar antagonist significantly inhibited oxidative burst in neutrophils induced with e. coli. similarly, anti-c a blocked the oxidative burst in whole blood induced with neisseria meningitides. phosphorylation of p phox is essential for assembly of nadph oxidase and the subsequent production of o and h o . c a is a strong activator of mitogen-activated protein kinase (including p /p ), which is an important kinase for p phox phosphorylation. except for directly affecting tissue damage, oxidant production might also be involved in signal transduction pathways. il- expression is enhanced by the oxidant sensitive transcription factor nuclear factor-kb activated in the lungs of influenza-infected mice. this means that oxygen-derived free radicals might exert much greater effects on the pathogenesis of the disease by indirectly inducing other proinflammatory mediators. thus, c a-mediated oxygen-derived free radicals are thought to be important events in the pathogenesis of the disease. c a-mediated release of histones histones are essential regulators of genome function in eukaryotic cells. the ns protein of influenza a h n subtype possesses a histone-like sequence (histone mimic), and could target the human rna polymerase-associated factor transcription elongation complex which has a crucial role in the antiviral response. thus, the virus used ns histone mimic to suppress human rna polymerase-associated factor transcription elongation complex-mediated antiviral response. diversely modified histone regulates gene replication, repair and transcription. after activation with influenza, h k me reduced association of interferon i (ifn-i) and ifn-iii promoters in dendritic cells (dcs) to suppress antiviral gene expression. in contrast to ifns, the association of tumor necrosis factor-a (tnf-a) promoter was not disturbed. histone can be excreted into cells to reduce intracellular histone to suppress antiviral gene expression. in the setting of ali both in humans and in mice, histone presence has been found in balf. in addition, when polymorphonuclear leukocytes are incubated in vitro or in vivo with c a, neutrophil extracellular histones-contained extracellular traps (nets) develop. these results suggest that engagement of c a with its receptors led to the appearance of extracellular histones in balf. extracellular histones significantly enhance inflammatory response by inducing nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain containing (nlrp ) inflammasome. furthermore, airway instillation of histones resulted in intense lung injury and inflammation, together with fibrin clots in pulmonary veins. c a-mediated release of histones has an important contribution to the pathogenesis of ali. the process of leukocyte adhesion to endothelial cells is the first critical step in neutrophil migration into an area of inflammation. adhesion molecules on the surface of endothelial cells have an important role in inflammatory cell migration. in fact, c a can regulate the expression of adhesion molecules. c a directly activates endothelial cells to upregulate adhesion molecules such as p-selectin. in addition, c a and tnf-a cooperate to enhance upregulation of intercellular adhesion molecule and e-selectin. thus, c a is an effective mediator in the first step in inflammatory cell migration into the lung. adhesion molecules on the surface of inflammatory cells also have an important role in inflammatory cell migration. in vitro studies demonstrated upregulation of cd lb/cd expression on neutrophils induced by c a. in addition, c a also induced the expression of b and b integrin on blood neutrophils. , thus, enhanced adhesive interactions of neutrophils to endothelial cells promote inflammatory cell migration into inflammatory sites. the adhesion molecules effectively enhanced pro-inflammatory cytokines such as tnf-a production by pulmonary macrophages, which, in turn, promotes the inflammatory response. blockade of cdllb, cd , intercellular adhesion molecule , or p-selectin significantly reduced ali damage by neutrophil content of the lungs. anti-c a might protect tissue injury in various organs by limiting neutrophil sequestration through downregulating the expression of adhesion molecules. these studies suggest that c a-mediated upregulation of adhesion molecules promotes the inflammatory response. c a-mediated adaptive immune response c a induces innate immune cells including mast cells, neutrophils, and macrophages to release cytokines such as il- , tnf-a and macrophage inflammatory proteins- a. il- is a strong activator of cd t cells, whereas tnf-a promotes transendothelial migration of t cells by up-regulating vascular adhesion molecules and induces ifn-c expression in t cells. these data demonstrate that c a indirectly induces adaptive immune response by activating innate immune cells. apart from innate immune cells, human dcs , and t cells also express the c a receptor (c ar, cd ). thus, c a is also a potent chemoattractant for human t cells, , b cells, and dcs. , , , in addition, during the early inflammatory stage of a pathogen infection, dcs used c a as a homing signal to take up ag, and then were primed for helping t-cell function. thus, c a induces adaptive immune response by recruiting for dcs. cd and cd l on t cells are important signaling for t-cell proliferation and differentiation induced by interaction of locallyproduced c a with c ar on antigen-presenting cells (apcs). accordingly, c a could not activate cd / cd / and cd / apcs to induce t cell activation. the data suggest that the local interaction of c a and c ar on apcs is critical to cd t cell proliferation and differentiation. the binding of the c a to the c ar also plays an important role in cd t cell responses. cd t cell activation during influenza infection requires c a, which acts as a chemoattractant for t lymphocytes. , thus, it is conceivable that c a might elicit cd t cell response upon the input stimuli. accordingly, c ar antagonist reduced the frequency and absolute numbers of flu-specific cd t cells. in patients infected with influenza a virus like h n , inflammatory cytokines such as il- b, il- , and il- play a major role in mediating and amplifying ali and ards by stimulating by chemotaxis c a. c a induces innate immune cells including mast cells, neutrophils, and monocytes/macrophages to release proinflammatory cytokines such as il- , tnf-a and macrophage inflammatory proteins- a. in addition, c a also stimulates adaptive immune cells such as t and b cells to release cytokines such as tnf-a, il- b, il- , and il- . , many cytokines, triggered by highly pathogenic viruses like h n , has been called a ''cytokine storm''. cytokines were rapidly induced at h post infection with h n . the pro-inflammatory cytokines including il- b and tnf-a might contribute to the severity of disease by promoting maximal lung inflammation caused by h n viral infection. compared to healthy volunteers, h n -infected patients have significantly higher levels of cytokines such as il- , ifn-c-inducible protein , il- , ifn-c, and tnf-a. a dangerous cytokine storm also occurs in sars. the representative sars-cov ssrnas had powerful immunostimulatory activities in inducing pro-inflammatory cytokines tnf-a, il- and il- . elevated levels of some proinflammatory cytokines including moncyte chemoattractant protein- , transforming growth factor-beta , tnf-a, il- , and il- , produced by cells infected by sars-cov, might cause ali. in addition, a cytokine could induce other cytokines to further enhance the proinflammatory response. take for example, elevated levels of tnf-a induced other cytokines like il- . thus, cytokine storm plays an important role in ali. anti-tnf-a (etanercept) significantly reduced the damage of ali. the inhibition of macrophage migration inhibitory factor alleviated h n influenza virus pneumonia in murine model by causing a significant reduction in pulmonary inflammatory cytokines il- b, il- and tnf-a and ifn-c-inducible protein a widely used antiviral agent arbidol hydrochloride efficiently inhibits both h n strains and diminishes both viral replication and acute inflammation through suppression of inflammatory cytokines such as il- b, il- , il- , and tnf-a. these studies indicate that blockade of cytokine storm is effective in treatment of infections with highly pathogenic virus. the severe h n patients were in a state of immune paralysis with general leukopenia, low antigen-presenting capacity and impaired t cell response. those suffering fatal infections with h n have particularly low proportions of peripheral blood t lymphocyte subgroups. previous studies have demonstrated that c a induces thymocyte apoptosis, which in turn results in decreased number of t cells in circulation and attendant immunosuppression. , this suggests that in a striking contrast to neutrophils, thymocytes apparently receive pro-apoptotic signals from c a. during sars-cov infection, il- and il- induced by c a inhibits the t-cell-priming ability of dcs. compared to significant up-regulation of inflammatory chemokines, the sars-cov-infected dcs showed low expression of antiviral cytokines (ifn-a, ifn-b, ifn-c, and il- p ) . these studies are in accordance with the conclusion that the n-protein of sars-cov induced ali by resulting in imbalance of pro-inflammatory and anti-inflammatory cytokines. many inflammatory and anti-viral genes were differentially expressed in sars patients. plenty of pro-inflammatory cytokines such as il- , tnf-a, and il- significantly increased, whereas a number of ifnstimulated genes like double-stranded rna-dependent protein kinase, interferon-induced guanylate-binding protein- and , c-x-c motif chemokine decreased in the acute severe case. like sars-cov, mers-cov viruses were unable to significantly stimulate the expression of antiviral cytokines (ifn-a and ifn-b) but induced comparable levels of tnf-a and il- . c a-c ar interaction might potentiate the mitochondrial apoptotic pathway and/or enhance the expression of proapoptotic factors, such as tnf-a, which has been linked to thymocyte apoptosis, in turn reducing the expression of antiviral cytokines. this suggests that c a-mediated immune paralysis plays a critical role in mediating pathogenic damage in severe patients infected with highly pathogenic virus like h n . to evaluate the effect of c a blockade, omci, a potent arthropodderived inhibitor of c activation that binds to c and prevents release of c a by complement activation, was used to treat mice infected with h n pandemic virus. omci significantly inhibited neutrophil and macrophage infiltration in the airways, nets formation, death of leukocytes, lung epithelial injury and overall lung damage. the study suggests that targeting c a could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of iav infections. c ar was found to be expressed on upper (bronchial) and lower (alveolar) airway epithelial cells. an adenovirus construct (sirna) was used to silence mrna for c ar in the lung and resulted in buildup of polymorphonuclear leukocytes, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluid. antagonism of c a receptors also significantly inhibited the development of ards induced by intravenous infusion of cobra venom factor, including neutrophil migration and bronchoalveolar vascular leakage, blood pressure alterations, pro-inflammatory cytokines including tnf-a levels in bronchoalveolar lavage fluid. the study indicates that c a signaling greatly contributes to inflammation and injury in the lung and was targeted to treat highly pathogenic virus infection. in addition, interception of c a signaling has recently shown promising beneficial effects in small animal models of ali/ards by reducing pro-inflammatory cytokines. polyclonal anti-c a antibody led to significantly reduced inflammation in lungs, alleviating ali in h n -infected mice. the study indicates that inhibition of c a might be an effective clinical intervention for h n -induced ali. however, studies in knockout mice demonstrated that c was required for protection from influenza infection, proper viral clearance, and associated with changes in cellular infiltration. the data are in accordance with the fact that complement c a is the leading mediator of the over-inflammatory response which induced ali, whereas the lytic membrane attack complex (c b- ) provide a protective role in controlling viral infection. thus, we developed a neutralized humanized anti-human c a antibody which only blocked c a effects but did not affect the formation of c b- membrane attack complex. in vitro experiments demonstrated that a novel, neutralizing, humanized anti-human c a antibody blocked the ability of c a to induce granulocytes to express cd b while not affecting the ability of c b to form the membrane attack complex. african green monkeys were inoculated with h n virus and then treated intravenously with anti-human c a antibody. anti-c a treatment in h n -infected monkeys substantially attenuated ali by reducing the lung infiltration of macrophages and neutrophils, and the levels of inflammatory mediators. the data suggest that humanized anti-human c a antibody might provide a potential therapeutic reagent for h n -infected patients. the role of c a in the different viral infections and the effect of c a blockade on acute lung injury were described in table that the neutralized humanized anti-human c a antibody would be a potential therapeutic option for h n -infected patients. the complement system, a part of innate immunity, plays a critical role in host defense against pathogens. unregulated complement activation is likely to play a crucial role in the pathogenesis of lung diseases. the complement-activated product c a displays powerful biological activities in the activation of phagocytic cells, generation of oxidants, release of histones and cytokine storm, and so on. in particular, cytokine storm is believed to be responsible for many of the deaths during the influenza pandemic, during the sars epidemic in , mers-cov in , and the human deaths from h n , h n and h n . there is growing awareness that there are key similarities in the contribution to the cytokine storm and the manifestation of lung pathology among the chronic respiratory diseases, and the cause of death such as bleeding from ebola virus. c a, as a key trigger to induce cytokine storm, could be an ideal target for many lung inflammatory diseases, and it would be important to assess the therapeutic potentials of c a blockade in human clinical trials. we have evidence that humanized anti-c a antibody greatly reduced lung histopathologic injury, as well as decreased lung infiltration of macrophages and neutrophils and the levels of pro-inflammatory cytokines including tnf-a in a monkey model of ali induced by h n and herbicide, paraquat (shihui sun et al, unpublished data). thus, it is reasonable to speculate that blockade of c a with a humanized anti-human c a antibody would be a potential therapeutic target for highly pathogenic viral infection-induced acute lung injury. anti-c a ab treatment also reduced lung injury and neutrophil infiltration especially on day after h n virus infection. also, anti-c a ab treatment increased survival rate, with % mortality in the c a ab group compared with % mortality in the control group on day after h n virus challenge. hpai h n virus infected murine model h n influenza virus infected mice had increased levels of c a 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preparing for the next pandemic confronting potential influenza a (h n ) pandemic with better vaccines the cytokine network in asthma and chronic obstructive pulmonary disease clinical features and pathobiology of ebolavirus infection this study was supported by national basic research program grants ( cb ), national nature and science fund ( , and ) and beijing natural science foundation ( , and ). key: cord- -narpqgmj authors: zhan, siyi; yang, ying ying; fu, chuanxi title: public’s early response to the novel coronavirus–infected pneumonia date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: narpqgmj nan to the editor: originated from wuhan city in central china and widely spread due to the mass migration for lunar new year holiday, , + confirmed novel coronavirus ( -ncov)-infected pneumonia (ncip) cases with + deaths have been identified in the country since december [ ] [ ] . another + exported cases have been confirmed in other countries worldwide [ ] . during : am to : pm, january th, (utc + ), we initiated an online survey on people's knowledge on ncip by snowball sampling via wechat invitation. invitees of - years old ( / females and / with bachelor's degree or higher) from all provinces in mainland china were enrolled, three quarters of which were from the six most developed provinces in china (zhejiang, guangdong, shandong, beijing, shanghai, jiangsu). worried on the highly contagious virus that currently lacks effective treatments, . % ( . - . %) subjects ( ) felt "extremely" or "very" nervous about ncip, and . % ( . - . %) believed that it was at least as terrible as the severe acute respiratory syndromes (sars) or avian influenza. adults living in urban areas had a better awareness of the knowledge on ncip than those in rural areas ( . % vs. . %, p < . protective measures such as washing hands, wearing masks and exercising can effectively prevent people from getting infected [ ] [ ] . . % (ci: . %- . %) subjects chose five or six items among all six measures provided. however, . % ( . %- . %) of subjects who would wear mask cannot wear it correctly, with common mistakes of nose exposure to the air and long-time mask wear without replacement. males (or: . , % ci: . - . ), younger adults ( . , . - . ), and subjects with higher education ( . , . - . ) were associated with better behaviours. our study indicates that general public in china has high concerns on the ncip in its rapid development phrase of the outbreak. among all subjects, . % ( . - . %) believed that the plague could be controlled within months due to the stringent measures advocated by government. as the ncip outbreak continues, further understanding of the infection will help to adjust strategies for public' health education. no potential conflict of interest was reported by the author(s). update on pneumonia of -ncov infections as of : on feb early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia world health organization. novel coronavirus ( -ncov) situation report - handwashing practice and the use of personal protective equipment among medical students after the sars epidemic in hong kong the efficacy of medical masks and respirators against respiratory infection in healthcare workers. influenza other respir viruses key: cord- -aqc zrrm authors: liang, guodong; gao, xiaoyan; gould, ernest a title: factors responsible for the emergence of arboviruses; strategies, challenges and limitations for their control date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: aqc zrrm slave trading of africans to the americas, during the th to the th century was responsible for the first recorded emergence in the new world of two arthropod-borne viruses (arboviruses), yellow fever virus and dengue virus. many other arboviruses have since emerged from their sylvatic reservoirs and dispersed globally due to evolving factors that include anthropological behaviour, commercial transportation and land-remediation. here, we outline some characteristics of these highly divergent arboviruses, including the variety of life cycles they have developed and the mechanisms by which they have adapted to evolving changes in habitat and host availability. we cite recent examples of virus emergence that exemplify how arboviruses have exploited the consequences of the modern human lifestyle. using our current understanding of these viruses, we also attempt to demonstrate some of the limitations encountered in developing control strategies to reduce the impact of future emerging arbovirus diseases. finally, we present recommendations for development by an international panel of experts reporting directly to world health organization, with the intention of providing internationally acceptable guidelines for improving emerging arbovirus disease control strategies. success in these aims should alleviate the suffering and costs encountered during recent decades when arboviruses have emerged from their sylvatic environment. despite the announcement of the successful eradication of smallpox in , the last case of rinderpest in and the current campaigns to eradicate poliomyelitis and measles through mass-immunization programmes, we still face the prospect of emerging or reemerging viral pathogens that exploit changing anthropological behavioural patterns. these include intravenous drug abuse, unregulated marketing of domestic and wild animals, expanding human population densities, increasing human mobility, and dispersion of livestock, arthropods and commercial goods via expanding transportation systems. consequently, the world health organization concluded that acquired immune deficiency syndrome, tuberculosis, malaria, and neglected tropical diseases will remain challenges for the foreseeable future. understandably, the high human fatality rates reported during the recent epidemics of ebola, severe acute respiratory syndrome and middle east respiratory syndrome have attracted high levels of publicity. however, many other rna viruses have emerged or reemerged and dispersed globally despite being considered to be neglected diseases. [ ] [ ] chikungunya virus (chikv), west nile virus (wnv) and dengue virus (denv) are three of a large number of neglected human pathogenic arthropod-borne viruses (arboviruses) whose combined figures for morbidity and mortality far exceed those for ebola, severe acute respiratory syndrome and middle east respiratory syndrome viruses. for instance, for denv, the number of cases of dengue fever/hemorrhagic fever is between - million annually, of which an estimated humans die. moreover, in the new world, within months of its introduction, chikv caused more than a million cases of chikungunya fever according to pan american health organization/world health organization, with sequelae that include persistent arthralgia, rheumatoid arthritis and lifelong chronic pain. likewise, within two months of its introduction, to polynesia, the number of reported cases exceeded and is currently believed to be approaching cases. alarmingly, this rapid dispersion and epidemicity of chikv (and denv or zika virus in oceania) is now threatening europe and parts of asia through infected individuals returning from these newly endemic regions. this is an increasingly worrying trend. for example, in france, from may to november, , suspected cases of dengue or chikungunya fever were reported. accordingly, this review focuses on the emergence or reemergence of arboviruses and their requirements and limitations for controlling these viruses in the future. arboviruses are transmitted between arthropods (mosquitoes, ticks, sandflies, midges, bugs…) and vertebrates during the life cycle of the virus. many arboviruses are zoonotic, i.e., transmissible from animals to humans. [ ] [ ] as far as we are aware, there are no confirmed examples of anthroponosis, i.e., transmission of arboviruses from humans to animals. - the term arbovirus is not a taxonomic indicator; it describes their requirement for a vector in their transmission cycle. [ ] [ ] humans and animals infected by arboviruses, may suffer diseases ranging from sub-clinical or mild through febrile to encephalitic or hemorrhagic with a significant proportion of fatalities. in contrast, arthropods infected by arboviruses do not show detectable signs of sickness, even though the virus may remain in the arthropod for life. as of , species belonging to virus families were registered in the international catalogue of arboviruses. however, this estimate is continuously increasing as advances in virus isolation procedures and sequencing methods impact on virus studies. whilst many current arboviruses do not appear to be human or animal pathogens, this large number of widely different and highly adaptable arboviruses provides an immense resource for the emergence of new pathogens in the future. in addition to evolving strategies for long-term survival, arboviruses have an enormous choice of arthropod species potentially capable of being infected the predominant species of which appear to be mosquitoes and ticks. approximately types of mosquito can transmit arboviruses. aedes and culex mosquitoes are the species most frequently associated with arbovirus transmission ( and types of arbovirus, respectively). ticks are also prevalent vectors, different species are currently known to transmit arboviruses. in addition, midge species have been shown to transmit arboviruses, mainly culicoides ( types) and lasiohelea. sandflies, blackflies, stinkbugs, lice, mites, gadfly, and bedbugs can also transmit arboviruses. this diversity of species and the wide distribution of these transmission vectors explain why arboviruses are so successful in dispersing globally via the mechanisms highlighted earlier. [ ] [ ] [ ] arboviral diseases are primarily associated with specific vectors. however, many other arthropod species, in which viruses have been identified, may be involved in perpetuating the virus life cycle without having been associated with overt disease in humans or animals. for example, wnv is typically mosquito-borne but can be vectored by many different mosquito species and also by ticks and other arthropods. - , moreover, japanese encephalitis virus appears to be transmissible by culex, anopheles, and other mosquito species, as well as midges, sandflies and ticks. as a general rule, a specific arthropod species, is likely to predominate during an epidemic. however, if the availability of the vertebrate host, for example birds, becomes limited towards the end of the summer as the birds migrate to warmer countries, the vector might switch its preference to a different vertebrate host. this is consistent with the observation that outbreaks of west nile fever/encephalitis in north america often appear to occur in the late summer, following peak incidence in birds, after they commence their migration to warmer regions. alternatively, aedes aegypti has adapted to the urban environment whereas aedes albopictus occurs more commonly in semi-urban or rural areas. however, these are not hard and fast rules. for example, on the french island of la reunion, where aedes aegypti was not recognizably present, chikv was simultaneously epidemic in both rural and urban environments, and was primarily associated with aedes albopictus. a high proportion of arboviruses associated with human and animal disease circulate in tropical, and subtropical regions, where mosquitoes, and other flying insects, tend to be abundant. however, many arboviruses also circulate amongst wildlife species in temperate regions of the world. despite the global distribution of viruses such as wnv, dengue virus, bluetongue virus and now chikv, most other arboviruses are generally endemic to specific regions of the world. for example, mosquito-borne japanese encephalitis virus is prevalent in india, central and southeast asia, largely due to the prevalence of highly competent culex tritaeniorhynchus mosquito species and intensely farmed pigs which act as amplifying hosts for the virus and, importantly, the mosquitoes. [ ] [ ] in southeast asia, rice cultivation with enormous areas of paddy fields also attracts migratory birds and provides ideal breeding areas for the mosquitoes which transmit the virus to the birds, ensuring virus dispersion over large areas of asia. in contrast, tick-borne encephalitis virus occurs mainly in northern temperate regions which are the primary habitats of ixodes species ticks (forests etc.). nevertheless, even within this relatively localized distribution of arboviruses, dispersion to distant locations occurs via animal or vector migration. as an example, powassan virus, a close relative of tick-borne encephalitis virus, is found in far east asia and also in canada. in contrast, rift valley fever virus (rvfv) was localized in africa but in , it was introduced to the middle east, where it caused thousands of human infections, with an estimated deaths, during a single epidemic. more than species of arbovirus that cause human/animal or zoonotic diseases have been identified. four virus families, togaviridae, flaviviridae, bunyaviridae and reoviridae, contain most of the arboviruses that cause human/animal diseases. arbovirus infections are not always clinically obvious and often resolve spontaneously after - weeks. however, some arboviral infections result in high fever, hemorrhage, meningitis, encephalitis, other serious clinical symptoms, and even death. therefore, they cause a large social and economic burden. a summary listing the arboviruses associated with human diseases and their geographic distributions was published previously. arboviruses have evolved a wide variety of strategies to ensure their long-term success, dispersal and survival. they associate with specific arthropods and exhibit distribution characteristics that reflect the environmental preferences of these particular species. however, once an epidemic has run its course, the virus survives via its sylvatic life cycle which may involve a wide variety of species currently not identified. alternatively, arboviruses can be maintained for months or maybe even years in mosquito eggs which remain dormant until the rainy season triggers hatching of the new, healthy but infected mosquito larvae. eggs can also provide a long term reservoir for tickborne arboviruses but in many cases the viruses exploit the extended life cycle of some tick species, surviving for years through the transstadial stages, reproducing at low levels. this long-term survival strategy is also enhanced by non-viraemic transmission during which infected and non-infected ticks co-feed on small animals in the forests. non-viraemic transmission provides an efficient mechanism for transmission of the virus directly between ticks, without necessarily infecting the vertebrate host. whilst co-feeding is taking place, virus infectivity in the tick salivary glands may increase by orders of magnitude presumably increasing virus transmission efficiency between the ticks. a similar non-viraemic co-feeding transmission process involving mosquitoes and blackflies, has also been described for wnv and vesicular stomatitis virus. alternatively some insect-specific flaviviruses exist in the form of dna when they infect insect cells. diverse sequences have been discovered that appear to be related to insect-specific flaviviruses, amplified from mosquitoes, belonging to factors, strategies and challenges for arbovirus control g liang et al the culicine genera culex, aedimorphus, ochlerotatus and/or stegomyia. many of these sequences may represent dna integrations into mosquito genomes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] moreover, srnas related to wnv in apparently uninfected culex mosquitoes have also been identified. whilst there is no current evidence that such forms of dna, provide a long-term arbovirus survival strategy, the similarity with other viruses that use dna intermediates and episomal dna should not be ignored. these and other diverse survival strategies provide safe havens for their longterm survival from which they can reemerge to cause epidemics amongst human and animal populations. changing anthropological behaviour, climate change and high mutation frequency are important determinants of arbovirus emergence. arboviruses adapt readily to new susceptible hosts by alteration of receptor specificity, transmission efficiency, antigenicity, and ecological and environmental conditions. humans, livestock and/or domestic animals are not an essential part of this arbovirus life cycle. therefore, unlike, smallpox virus, measles virus or poliovirus, arbovirus disease control based on humans, livestock and/or domestic animals cannot eradicate the arbovirus. consequently, the reservoir for arboviruses in wild species places a limitation on our ability to control disease emergence. for instance, chikv was a zoonotic arbovirus that cycled harmlessly between simians and mosquitoes in the african tropical forests causing localized outbreaks of polyarthralgia in humans. it also occasionally ''escaped'' to asia gradually becoming zoonotic. however, prior to , chikv was rarely an epidemic arbovirus until a mutation occurred in the gene encoding the surface protein of the african strain which increased its capacity to infect, reproduce and be transmitted by the striped asian ''tiger'' mosquito aedes albopictus. coincidentally this mosquito species has gradually dispersed westwards and chikv is now a major global human epidemic pathogen throughout asia. moreover, on december , it was reported to have crossed the atlantic ocean, reaching the french caribbean island of saint martin from where it dispersed to the americas. it also dispersed eastwards from southern asia, reaching polynesia by october . worryingly, chikv is now frequently being introduced into non-endemic europe and northern asia by incoming humans infected in the americas and polynesia. in the americas, wnv, was first reported in new york in august , following a hot and humid summer and many publications describe its emergence and dispersal. , [ ] [ ] [ ] in contrast with chikv, the major determinant for the dispersal of wnv, throughout north america, over a period of five years was primarily birds and their associated culex mosquito species. the dispersal and increasing epidemicity of dengue fever/dengue hemorrhagic fever which is confined to humans in the tropics, sub-tropics and southern temperate regions, can generally be attributed to human and aedes aegypti population density increase during the past century, resulting from intensive urbanization and the influence of increased transportation of humans, commercial goods, livestock and major military movements across the oceans. on the other hand, rvfv is enzootic and confined to a wide range of animals, mosquitoes and sandflies throughout africa and the arabian peninsula. generally, the virus circulates without causing major disease outbreaks which usually arise following periods of rainfall when herded ruminants are introduced to rvfv-endemic areas. two important factors are believed to have influenced the epidemiology of rvfv, firstly, major irrigation projects and secondly, the el niño effect. in each case, cited above, a combination of two or more of the factors identified earlier have had an important influence on the appearance of these emerging arboviruses in new territories and/or old territories. other recently emerging arboviruses, not included in this brief outline, include, zika virus in oceania, bluetongue virus and schmallenberg virus in northern europe and bagaza virus in spain. [ ] [ ] [ ] [ ] strategies for arbovirus control the concept of arthropod-borne disease transmission was born out of the studies of a physician, josiah clark, and years later developed by carlos finlay who proposed mosquitoes as the agents for transmission of yellow fever. subsequently, empirical methods, such as mosquito eradication, which was used very successfully in cuba, and the development of a yellow fever vaccine has protected millions of humans from potentially fatal infection by yellow fever virus. [ ] [ ] [ ] now, in the st century genetically engineered live attenuated vaccines can be manufactured within months, to protect humans against the ravages of pandemic influenza and other virus diseases. moreover, a spectrum of antiviral molecules has been developed to treat humans dying from infection with human immunodeficiency virus and several antiviral drugs have also been developed against other viruses. does this mean that if we develop vaccines and antiviral drugs to prevent or treat humans against infection by pathogenic arboviruses we will resolve the challenges associated with emerging arboviruses? regrettably it is not that simple! it is a remarkable fact that in the future, because of their high mutation rates, many new pathogenic arboviruses will emerge even though they do not currently exist as epidemic strains in the sylvatic environment. it is also becoming clear, from early results of genome sequencing, that mosquitoes carry large numbers of known and unknown viruses that infect humans, primates, mammals, birds, insects, and plants. [ ] [ ] therefore, should we attempt global eradication of arthropods? the answer is a definite no. this would have a catastrophic impact on the survival of many wildlife species, as first became apparent when dichlorodiphenyltrichloroethane was used widely as a general insect control agent rather than being precisely targeted to relevant mosquito species. however, implementation of temporary localized arthropod control measures during epidemics, for example in high density urbanized areas, can still play an important but transient role in reducing the impact on humans and animals of emerging arboviruses. the breadth and depth of arbovirus surveillance differs regionally, and several areas lack surveillance altogether. there is also a lack of interdisciplinary expertise on arbovirus diseases and understanding their vectors, and epidemiology. in addition, only a small number of arboviral diseases can be prevented using vaccines or specific antiviral drugs, and there are few validated diagnostic reagents, with which to monitor disease progress and control. until such disease control reagents become available, the most effective alternative is to focus on practical procedures to reduce risks of exposure to arthropods. it is clear that we are now entering a period in virological discovery that will reveal a ''pandora's box'' of new viruses with unique characteristics, which circulate in the ecosystem and will potentially lead to the evolution of novel pathogenic arboviruses. future disease control strategies must therefore recognize this and be planned accordingly. what are the major objectives that need to be addressed if we are to win the war against these versatile viruses that appear to have an infinitesimal variety factors, strategies and challenges for arbovirus control g liang et al of strategies to confuse and disorganize our ability to bring them under control? (i) develop vaccines to reduce the incidence of disease caused by known viruses; (ii) develop therapeutic drugs to treat clinical diseases caused by known viruses; (iii) develop unified vector-controlled strategies that will not unduly threaten the survival of wildlife species but locally will reduce the risk of disease in humans and animals; (iv) develop universal teaching/training courses to be taught worldwide, to provide cores of expertise to implement these policies; (v) encourage the strengthening of levels of cooperation between academia and drug and vaccine development companies; (vi) encourage the development of research programmes to understand the underlying mechanisms of arboviral pathogenicity, evolution, emergence and dispersal; (vii) develop, at the international level, public health measures to inform and educate citizens in local arboviral disease control measures, including monitoring and reporting; (viii) implement measures to improve monitoring procedures at borders, harbours, airports to reduce the influx of arthropods to new countries; (ix) develop unified public health strategies for arboviral disease control; (x) simplify the procedures for establishing safety and efficacy of antiviral drugs; (xi) establish an international committee of experts charged with the objective of reviewing global arthropod control strategies; (xii) develop and implement internationally acceptable and user-friendly guidelines for avoiding exposure to the different types of arthropod likely to carry human pathogens. this is undoubtedly an incomplete list of recommendations and probably some recommendations cannot easily be implemented. nevertheless, the list provides the building blocks for a unified strategy on which to develop methods that will reduce the high morbidity and mortality rates due to human or animal arbovirus infections. finally, we also have to recognise that arboviruses and arbovirus-related viruses infect more than just humans, invertebrates and land-based animals. they also infect plants, fish and marine animals. these relatively new discoveries also need to be accommodated if arboviral disease strategies are to be developed in a rational and unified manner. the lesson of the past is that human endeavour is at its best in the face of adversity. let this be the mantra for arbovirus disease control in the future! who and the future of disease control programmes the global burden of disease study : interpretation and implications for the neglected tropical diseases global trends in emerging infectious diseases chikungunya virus imported into french polynesia outbreak news today. france reports cases of chikungunya local transmission medically important arboviruses of the united states and canada biological transmission of arboviruses: new insights into components mechanisms, unique traits and their evolutionary trends present and future arboviral threats virus taxonomy: ninth report of the international committee on taxonomy 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programmes, silver and predemics (grant agreement nos and , respectively). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. this work is licensed under a creative commons attribution . unported license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / factors, strategies and challenges for arbovirus control g liang et al key: cord- -teplpmi authors: horm, srey viseth; tarantola, arnaud; rith, sareth; ly, sowath; gambaretti, juliette; duong, veasna; y, phalla; sorn, san; holl, davun; allal, lotfi; kalpravidh, wantanee; dussart, philippe; horwood, paul f; buchy, philippe title: intense circulation of a/h n and other avian influenza viruses in cambodian live-bird markets with serological evidence of sub-clinical human infections date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: teplpmi surveillance for avian influenza viruses (aivs) in poultry and environmental samples was conducted in four live-bird markets in cambodia from january through november . through real-time rt-pcr testing, aivs were detected in % of samples collected throughout the year. detection rates ranged from % and % in duck and chicken swabs, respectively, to % in carcass wash water samples. influenza a/h n virus was detected in % of samples positive for influenza a virus and % of all samples collected. sequence analysis of full-length haemagglutinin (ha) and neuraminidase (na) genes from a/h n viruses, and full-genome analysis of six representative isolates, revealed that the clade . . reassortant virus associated with cambodian human cases during was the only a/h n virus detected during the year. however, multiplex reverse transcriptase-polymerase chain reaction (rt-pcr) analysis of ha and na genes revealed co-circulation of at least nine low pathogenic aivs from ha , ha , ha , ha , ha , ha , ha , ha and ha subtypes. four repeated serological surveys were conducted throughout the year in a cohort of poultry workers. serological testing found an overall prevalence of . % and . % for antibodies to a/h n and a/h n , respectively. seroconversion rates of . and . cases per person-months participation were detected for a/h n and a/h n , respectively. peak aiv circulation was associated with the lunar new year festival. knowledge of periods of increased circulation of avian influenza in markets should inform intervention measures such as market cleaning and closures to reduce risk of human infections and emergence of novel aivs. avian influenza viruses (aivs) naturally infect the gastrointestinal tracts of wild birds from the orders anseriformes (ducks, geese and swans) and charadriformes (waders and gulls). within these hosts, haemagglutinin (ha) and nine neuraminidase (na) surface glycoprotein types have been described. the ha and na types carried by the virus are used for classification and can be found in various combinations, such as a/h n and a/h n . wild birds have been implicated in the global dissemination of aivs and, in some instances, the introduction of aivs into domestic poultry populations. although low pathogenic avian influenza (lpai) viruses (which have limited impact on poultry mortalities) constitute the majority of these viruses, highly pathogenic avian influenza (hpai) viruses such as a/h n can result in mortalities approaching % when introduced into domestic flocks. influenza a viruses, which constitute all of the known aivs, have a segmented genome with eight separate rna strands enclosed in the virus. when an animal is co-infected with two different influenza viruses, reassortment of these rna segments can occur, resulting in a new virus with characteristics distinct from the parent viruses. the emergence and resulting pandemic from a/h n pdm virus was caused by a virus that underwent multiple reassortment events with strains from pigs, birds and humans before suddenly gaining the ability to efficiently transmit between humans. pandemic events resulting from reassortment of influenza viruses have occurred at least three times in the last years, , resulting in high human morbidity and mortality worldwide. the most severe influenza pandemic in recorded history, the 'spanish flu', is thought to have arose from an avian influenza strain that directly adapted to the human host. clearly it is imperative that close monitoring of reassortments and mammalian-adapted mutations in avian influenza strains is needed. since the emergence of hpai a/h n in southern china in and , descendants of this virus have caused considerable economic losses in poultry populations, primarily in east and southeast asia, but also in africa and europe. influenza a/h n has been identified as a significant threat for pandemic emergence. to date, at least people have been infected worldwide, resulting in deaths ( % case fatality rate). the virus was first detected in cambodian poultry in early and the first human cases were detected in . as of december , human cases (including deaths) and poultry outbreaks of influenza a/h n have been recorded in the country. , live-bird markets (lbms) have been implicated as an important source of human sporadic cases and dissemination of avian influenza since the emergence of influenza a/h n . previous studies have established that lbms serve as hubs for the circulation and persistence of aivs through the presence of multiple avian species, the constant introduction of immunologically naive hosts and the frequent lack of biosecurity measures. , , recent surveillance studies have documented that a/h n and a/h n commonly co-circulate with other subtypes of avian influenza in lbms, [ ] [ ] [ ] thus increasing the likelihood of reassortment events. in the present study we initiated poultry and environmental surveillance for a/h n and other aivs in four lbms in cambodia to better ascertain virus circulation in this setting. a longitudinal human serosurvey was also conducted to investigate the risk of human avian influenza infections in exposed lbm workers. commencing in january , through november , environmental and poultry samples were collected at four lbms in cambodia: an lbm in central phnom penh (m ), a wholesaling farm/slaughter house in phnom penh (m ), an lbm in kampong cham province (m ) and an lbm in takeo province (m ) (figure ). these lbms were selected for the study as they represent the largest poultry collection sites in the most densely populated region of the country. samples were collected from each market every - weeks (figure ). the lbms in cambodia typically have poor biosecurity, with slaughtering of animals onsite occurring for a range of domestic animals ( figure ). birds are typically sourced from backyard flocks by middlemen who transport the animals through a convoluted system of semi-commercial farms and stock houses before being transported to the main lbms. thus, tracking the original source of poultry is usually not possible. during each market investigation, tracheal and cloacal swabs were collected (both samples pooled in one tube of viral transport medium for each animal) from four randomly selected poultry (three ducks and one chicken). environmental samples were also collected in the same cage/site where the poultry swabs were collected to investigate contamination of the lbms with aivs. during each mission, ml of carcass wash water (water used to wash the carcasses once the poultry has been slaughtered and defeathered), ml of poultry drinking water (small bowl of water placed in cages), soil/mud from an area within cm around the poultry cages/poultry resting area and samples of discarded feathers were collected. the water, soil/mud and feather samples were processed and nucleic acids extracted following techniques described previously. , extracts were then tested by quantitative real-time reverse-transcriptase polymerase chain reaction (qrt-pcr) for the detection of m, h and n genes. m-gene-positive samples, for which there was sufficient sample and high virus concentration (cto ; n = ), were inoculated into specific pathogen-free embryonated chicken eggs for virus isolation. isolates were then tested using influenza universal multiplex rt-pcr assays to test for all known subtypes of influenza. universal multiplex rt-pcr typing assays were also applied to the original samples where isolation could not be achieved but discordance between m-gene and h qrt-pcr testing suggested the presence of a non-h aiv. the amplified rt-pcr products from isolates and original material were submitted to a commercial sequencing facility (macrogen, seoul, south korea) for sequencing by the sanger method. full-genome sequences were generated from representative a/h n isolates using methods previously described. , contiguous sequences were assembled using clc workbench (clc bio) and compared to representative influenza virus sequences downloaded from the ncbi genbank database. neighbour-joining trees were constructed with mega and bootstrap values were calculated and expressed as a percentage from replicates. the longitudinal human serological study was approved by the national ethics committee for human research (approval no. , december ). serum samples were collected, after obtaining informed consent, from lbm workers at the start of the study (january ) to form a baseline, and weeks after the three major national festivals shown by previous work to be associated with increased a/h n circulation in markets: lunar new year, week ; khmer new year, week ; pchum ben, week . all adult-age lbm sellers or workers were exhaustively recruited in the four targeted livepoultry markets. the sample size could not be calculated as transmission to humans was unlikely and its probability in cambodia is unknown. participants were informed to report any acute febrile, respiratory or digestive signs, and were provided with a toll-free phone number. serum samples were tested for avian influenza a/h n , a/h n and a/h n antibodies using the haemagglutination inhibition assay (hia) and microneutralization assay (mn). the hia and mn testing were performed using influenza a/h n clade . . reassortant viruses (a/cambodia/x / and a/cambodia/ x -/ ), which were isolated from human cases during , and influenza a/h n virus (a/environment/cambodia/e / ), which was isolated from the lbms during . the hia and mn testing for a/h n were performed using the strain a/anhui/ / , supplied through the world health organization (who) global influenza surveillance and response system (dr sylvie van der werf, department of virology, institut pasteur, paris, france). exposure to these viruses was considered 'confirmed' with a hia titre of ≥ and a mn titre of ≥ . a seroconversion was defined as the detection of antibodies equal to or above the thresholds defined above following no detection of antibodies in the serum sample from the previous period. laboratory data were entered using an excel spreadsheet (microsoft excel, microsoft, redmond, wa, usa). a baseline assessment was documented using point prevalence for influenza antibodies, and incidence rates during follow-up were then computed using data on laboratory-confirmed seroconversions in lbm workers (numerator), and the number of days elapsed during the last serosurvey (denominator). poisson confidence intervals for the incidence rates were computed using stata (stata corp., college station, tx, usa) with the function cii for the binomial ci, and the functions cii and the option 'poisson' for the poisson ci. during the study, a total of samples were collected, with % of all samples positive for influenza a rna by qrt-pcr (table ) . influenza a viruses were detected in at least one sample during % of collection missions, with influenza virus detected in three of the markets (m , m and m ) on all but one sampling mission and detected in % of sampling missions from the remaining market (m ). influenza a (m-gene) ribonucleic acid (rna) was detected most frequently in carcass wash water samples ( %, n = ), followed by feathers ( %, n = ), poultry drinking water ( %, n = ), soil/mud ( %, n = ), duck swabs ( %, n = ) and chicken swabs ( %, n = ). h and n genes were detected in % (n = ) and % (n = ), respectively, of the samples that tested positive for influenza a (m-gene), which accounted for % and % of all samples collected, respectively. peak aiv circulation was detected from january through march, with particularly high detection rates during the lunar new year festival period (figure ) . influenza a/h n virus was isolated from % ( / ) of samples from which h was detected and isolation was attempted. isolation was only attempted on high viral load samples (m-gene qrt-pcr ct full-gene sequences were generated for h (n = ) and n (n = ) for all viruses detected in the study where sufficient virus concentration allowed for successful sequencing (figure ) . , fullgenome sequences (eight fragments) were generated for six influenza a/h n viruses detected during the study (supplementary figure s ) . for other viruses full-gene sequences could not be generated for all fragments (supplementary table s ). all a/h n viruses detected in the study clustered with the clade . . reassortant strains associated with human cases and poultry outbreaks of a/h n during . non-h avian influenza detection a large number and variety of non-h avian influenza strains were detected during the study. full-ha sequences were generated from two h , two h , three h , nine h , h , one h , h , one h and twelve h viruses (supplementary figure s ) . full na sequences from twenty-two n , one n , one n , two n , one n and two n subtypes were also generated from the same samples (supplementary figure s ) . however, as many of the environmental samples contained evidence of multiple avian influenza strains, it was difficult to ascertain that ha and na sequences derived from the same sample actually belonged to the same virus strain. co-infections between aivs were not detected in any poultry samples. many other partial ha and na sequences were also generated, but not included in these results due to difficulties in determining close phylogenetic relationships from incomplete gene sequences. aivs were detected throughout the year ( figure ) , with a distinct peak of activity during january − march, perhaps mostly due to the increased circulation of a/h n during major cambodian festivals, as previously reported. longitudinal human serosurvey successive serological surveys in the poultry worker cohort provided evidence of seroconversions and some prior exposure (table ) . at baseline sampling (january ), participants were enrolled in the study, with one person testing positive to a/h n antibodies and another testing positive to a/h n antibodies. participant retention was high throughout the study, with , and people resampled at the second (march ), third (june ) and fourth (november ) sampling missions, respectively. seroconversions to a/h n were detected for two participants at the third sampling and two participants at the fourth sampling. seroconversions to a/h n were detected for one participant at the third sampling. overall seroprevalence was . % for a/h n and . % for a/h n . rates of seroconversion were . infections per person-months for a/h n and . infections per person-months for a/h n . there was no serological evidence of exposure or molecular detection of a/h n before or during the study. there were no calls to the toll-free number reporting an incident with clinical signs or symptoms compatible with influenza infection. lbms have been established as important foci for the transmission of aivs and the potential emergence of reassortant strains. the presence of multiple host species and the continued introduction of naive birds create an ideal environment for the persistence and emergence of aivs. exposure to live poultry has been associated with fatal human a/h n infections, which for instance led to the government of hong kong rapidly closing lbms in and slaughtering large numbers of poultry. the lbms surrounding phnom penh have been established as foci for poultry movement in the country , and a previous market surveillance study in revealed a high rate of influenza a/h n circulation in this setting. to our knowledge, aivs were detected in cambodian lbms during at a higher frequency than any other study published previously, avian influenza viruses in cambodian live-bird markets sv horm et al chicken swabs positive for influenza a rna. the majority of the aivs detected were likely a/h n , with % of samples positive for h qrt-pcr. although there was clearly high co-circulation of lpai viruses, much of the difference between the detection rates of m-gene, h and n was probably due to the differing qrt-pcr assay sensitivities (m h n ). we previously detected high circulation of a/h n in these same markets in . however, in there was a . -fold increase in the frequency with which the virus was detected despite the same sample processing, nucleic acid extraction and rt-qpcr methods being used. in , cambodia had the highest confirmed human a/h n caseload per capita in the world. during alone, human a/h n cases ( deaths) were detected in cambodia, a dramatic increase in the total cases that had been detected in the preceding years, - . this rise in reported human cases of a/h n coincided with the emergence of a reassortant virus that contained the ha and na genes from the previously circulating clade . . genotype z virus, and the matrix and internal genes from a clade . . . virus previously circulating in southern vietnam. sequence and phylogenetic analyses of the ha and na genes ( figure ) and the matrix and internal genes (supplementary figure s ) from the a/h n market strains revealed that all of the viruses clustered closely with other clade . . reassortant strains associated with poultry outbreaks and human cases in . questions remain regarding the causes of the dramatic increase in human cases in and whether the reassortant strain is more transmissible in poultry, resulting in the intense circulation that we observed in this report. alternatively, increased surveillance and education of clinicians may have resulted in improved detection of human a/h n cases. presently we do not know the exact role played by lbms in the infection of poultry workers and market clients. similarly to our previous lbm surveillance in , increased circulation of aivs was detected before and during the major cambodian festivals (figure ). in particular, increased circulation of influenza a viruses was detected during the period between the lunar new year and khmer new year festivals. a previous study on the poultry trade links in cambodia established that there was a significant increase in the trade volume of poultry prior to these festivals, with an assumed rise in cross-border poultry trade in response to increased demand. the greater volume of poultry trade and the expansion of poultry trading networks during these festival periods present opportunities for virus spread throughout domestic flocks and the introduction of new strains of aivs. peaks in aiv levels were also observed during times that were not associated with known festivals (e.g. week and ). climatic factors, which were not analysed in this study, are also likely to have an influence on levels of aiv circulation. knowledge of these periods of intense circulation should inform future control policies such as targeted poultry vaccination, quarantining and improved market cleaning. such measures have proven effective in reducing avian influenza viral isolation rates in lbms [ ] [ ] [ ] and may reduce the spread of aivs back to farms through fomites and personnel. environmental sample testing showed that the lbm environment is highly contaminated with aivs (table ) . influenza a detection rates were highest in carcass wash water samples, which serve as 'pooled' samples for multiple slaughtered birds. during the slaughtering process at the markets, birds are defeathered and eviscerated before carcasses are washed in a large bucket of water. we observed that - carcasses were often washed in the same container of water before it was refreshed. these samples were positive for influenza a rna in % of samples from all four markets (ranging from to %). poultry drinking water was also a useful surveillance sample, with % of samples testing positive for influenza a rna; a/h n isolation rates were higher when compared to carcass wash water. detection rates and isolation rates were also high with soil/mud and discarded feather samples, but these specimens required more complex processing before testing. high contamination of the lbm environment, including relatively high rates of virus isolation, is evidence that the risk of human exposure is very high. carcass wash water and poultry drinking water samples proved a useful adjunct to poultry swabs for monitoring purposes in the lbms. based solely on the ha typing, analysis of lbm samples collected in revealed the presence of at least nine other subtypes of aivs co-circulating with a/h n ( figure ). this situation increases the likelihood of reassortment events occurring, which may result in the emergence of new influenza subtypes. the recent emergence of reassortant hpai subtypes of h , such as a/h n , a/h n , a/h n and a/h n , [ ] [ ] [ ] [ ] [ ] is evidence of the risk of new viruses arising through reassortment events with a/h n . the sudden emergence of multiple h subtypes has not been adequately explained but could be related to the diversity of aivs currently circulating in domestic poultry populations. the emergence of a/h n virus in china in early has also prompted increased concerns about the possibility of a pandemic virus emerging in the asia-pacific region. although most of the non-h aivs detected in this study likely pose little or no risk to humans, the high prevalence of multiple avian influenza strains is an indictment on the poor biosecurity associated with poultry rearing and selling in the region. previous studies and surveillance have also established that there is intense circulation exposure to these viruses was considered positive with a haemagglutination inhibition assay titre of ≥ and a microneutralization assay titre of ≥ . c only participants who provided samples for at least two time points were included in these analyses. d participants were removed from further calculations once they were recorded as 'positive' in the assumption that antibodies are protective against further infections. e one-sided, . % ci. of poultry between regions and across borders, which facilitates the regional spread of aivs. , in our study, the detection of a/h n was achieved using qrt-pcr, whereas the detection of other aivs was done using universal multiplex rt-pcr assays with comparatively reduced sensitivity. in addition, multiplex testing was only conducted where there was disparity between m-gene and h qrt-pcr assays. furthermore, due to the difficulties with classifying influenza viruses using incomplete gene sequences we have only reported lpai detection where we have been able to generate full ha or na sequences. it is therefore likely that the true prevalence of non-h aivs was far greater than what we have reported in these market samples. the potential for reassortment between aivs, including a/h n , could result in the emergence of viral strains with considerable impacts on poultry and/or human health. this study, coupled with other recent studies in lbms from other asian countries, [ ] [ ] [ ] confirms that the environment of lbms provides a pool of genes for potential emergence of new pandemic viruses. the silent circulation of a multitude of lpai viruses, which remain undetected and unmonitored in most asian countries, heightens the threat when they co-circulate with pandemic candidates such as a/h n and a/h n . as no poultry workers reported any symptoms in relation to acute avian influenza infection, seroconversions in this study would most likely be related to sub-clinical or very mild cases. it has been reported that asymptomatic and mild avian influenza infections lead to seroconversions with low antibody titres that quickly decrease below the threshold recommended by who for a confirmed a/h n case (hia ≥ ; mn ≥ ). for this reason, we considered as positive all individuals with a hia titre of ≥ and a mn titre of ≥ , which is consistent or more stringent - than the cut-off levels suggested by other authors. however, it is difficult to directly compare the results between studies as there is no consensus on the antibody titre that results from a mild or asymptomatic infection, and consistent cut-off levels have not been used in past studies. according to our classification, . % of workers who participated in the study had antibodies against a/h n . this prevalence is much higher than that reported among lbm workers in egypt, thailand or china, and much higher than in villagers in thailand or cambodia. the incidence was . per person-months in our study, a figure twice that described in bangladesh in - . there were no reported clinical signs in our cohort, confirming that a large proportion of human a/h n infections may go undetected. poultry workers in cambodia and other asian countries, where there is endemic circulation of a/h n , are constantly exposed to high levels of virus. although this may lead to mild or sub-clinical infections with seroconversions, it seems that transmission to humans resulting in acute infections is still rare. influenza a/h viruses, predicted to be a/h n based on the phylogenetic analysis of the ha and na sequences repeatedly detected in the same samples, were detected in . % of samples analysed in this study. although a/h n viruses have low pathogenicity for poultry, novel hpai and lpai viruses affecting humans, such as a/h n , a/h n and a/h n , contain internal genes originating from influenza a/h n . [ ] [ ] [ ] this may have facilitated the ability of these viruses to cause infection and deaths in humans. human infections with a/h n have been reported in the literature, and seroprevalence studies have suggested that asymptomatic or mild infections commonly occur in high-risk locations such as lbms and slaughterhouses. , , , the detection of a/h n antibodies in . % of lbm workers participating in our study and an incidence of . infections per person-months confirm other data on poultry-tohuman transmission of a/h n , , including among rural villagers in cambodia. in addition, a/h n infections have also been detected in other mammals such as guinea pigs, dogs, horses and pigs. , the ability of a/h n to cross the species barrier and the relatively high frequency in which it has been implicated in reassortment events suggest that this virus significantly contributes to the emergence of viruses that pose an important public health risk and should therefore be very closely monitored. in this study we document intense co-circulation of influenza a/h n and lpai viruses in cambodian lbms during . in addition, serological surveys provided evidence of sub-clinical a/h n and a/h n infections. interventions such as regular cleaning/disinfection, bans on overnight poultry storage, targeted closure during periods of peak circulation and segregation of poultry slaughtering areas should be considered in lbms to reduce the threat of the emergence of aivs with public health or animal health impacts. further monitoring of the circulation of influenza a/h n in cambodian lbms and research into the mechanisms associated with human cases is warranted. ); and the world health organization country office in cambodia (grant no ecology of avian influenza viruses in a changing world ecology of avian influenza virus in birds a new avian influenza virus from feral birds in the ussr: recombination in nature? 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clinical and epidemiological characteristics of a fatal case of avian influenza a h n virus infection: a descriptive study evolution of the h n influenza genotype that facilitated the genesis of the novel h n virus avian influenza h n seroprevalence among poultry workers in pune a systematic review and meta-analysis of the seroprevalence of influenza a (h n ) infection among humans a global phylogenetic analysis in order to determine the host species and geography dependent features present in the evolution of avian h n influenza hemagglutinin the authors would like to thank poultry workers at the live-bird markets for their cooperation with the study team. we thank the technical staff at the virology unit and investigation team at the epidemiology and public health unit, institut pasteur in cambodia. the authors would also like to thank the investigation team from the national veterinary research institute, ministry of agriculture, forestry and fisheries. this work was funded by the key: cord- -c w bw u authors: lui, pak-yin; wong, lok-yin roy; fung, cheuk-lai; siu, kam-leung; yeung, man-lung; yuen, kit-san; chan, chi-ping; woo, patrick chiu-yat; yuen, kwok-yung; jin, dong-yan title: middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk -dependent phosphorylation of irf date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: c w bw u middle east respiratory syndrome coronavirus (mers-cov) infection has claimed hundreds of lives and has become a global threat since its emergence in saudi arabia in . the ability of mers-cov to evade the host innate antiviral response may contribute to its severe pathogenesis. many mers-cov-encoded proteins were identified to have interferon (ifn)-antagonizing properties, which correlates well with the reduced ifn levels observed in infected patients and ex vivo models. in this study, we fully characterized the ifn-antagonizing property of the mers-cov m protein. expression of mers-cov m protein suppressed type i ifn expression in response to sendai virus infection or poly(i:c) induction. this suppressive effect was found to be specific for the activation of ifn regulatory factor (irf ) but not nuclear factor-κb. mers-cov m protein interacted with traf and disrupted traf –tbk association leading to reduced irf activation. m proteins from mers-cov and sars-cov have three highly similar conserved n-terminal transmembrane domains and a c-terminal region. using chimeric and truncation mutants, the n-terminal transmembrane domains of the mers-cov m protein were found to be sufficient for its inhibitory effect on ifn expression, whereas the c-terminal domain was unable to induce this suppression. collectively, our findings suggest a common and conserved mechanism through which highly pathogenic mers-cov and sars-cov harness their m proteins to suppress type i ifn expression at the level of tbk -dependent phosphorylation and activation of irf resulting in evasion of the host innate antiviral response. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in september as a novel coronavirus that causes severe acute respiratory disease. since then, this virus has caused recurrent outbreaks in the arabian peninsula and has spread, occasionally, to other parts of the world. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] according to the world health organization, laboratory-confirmed cases were reported between september and january with related deaths in countries. in particular, people were infected and were killed in one recent outbreak in south korea. mers-cov is classified into lineage c of betacoronavirus and is most phylogenetically related to two bat coronaviruses, hku and hku , providing insight on its evolutionary origin. , mers-cov is a polycistronic positive-sense single-stranded rna virus with a genome of~ kb in size. the ′ most two-thirds of mers-cov genome encodes polyproteins a and ab, which are further cleaved to yield non-structural proteins, whereas the ′ end of the genome encodes several structural or lineage-specific proteins. upon infection, these proteins are expressed to facilitate viral replication and propagation in the host. mers-cov infection has been widely reported to mildly induce type i interferons (ifns), including ifn-α and -β, in patients as well as in animal and cellular infection models. [ ] [ ] [ ] [ ] [ ] [ ] [ ] this has been attributed to the ifnantagonizing property of some mers-cov-encoded proteins, which directly perturb the host ifn production mechanisms, [ ] [ ] [ ] [ ] [ ] lending support to the notion that mers-cov uses multiple strategies to evade the innate immune response. in non-specialized epithelial cells as well as a subset of specialized immune cells that are susceptible to mers-cov infection, , , type i ifn production is an important part of the host innate immune response and is initiated by ubiquitously expressed cytoplasmic viral sensors in the retinoic acid-inducible gene-i (rig-i)-like receptor (rlr) family in response to the detection of viral pathogen-associated molecular patterns such as double-stranded rna (dsrna). , stimulated rlrs mobilize downstream signal transducers that lead to the activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-κb (nf-κb) that drive ifn-β expression. the transduction events within this signaling cascade are prone to negative regulation by many mers-cov proteins. in a comparative analysis of mers-cov structural and accessory proteins, it has been shown that m, orf a, orf b and orf possess ifn-antagonizing properties. we, and others, have characterized the orf a protein as a dsrna-binding protein that interferes with the activation of rlr by either a dsrna ligand or the protein co-activator pact. , however, the molecular mechanisms through which other mers-cov proteins manipulate the rlr signaling pathway to disrupt ifn-β expression have not been elucidated. in this study, we focused on the characterization of the mers-cov m protein in ifn antagonism. coronavirus m protein is a transmembrane glycoprotein localized predominantly to the golgi complex and is required for virion assembly. [ ] [ ] [ ] mers-cov m protein is of particular interest because sars-cov m protein also inhibits ifn production through a mechanism by which the formation of traf ·tank·tbk /ikk-ε complex is impeded to ablate the activation of irf transcription factor. in contrast, m protein encoded by human coronavirus hku associated with common cold has no influence on ifn production. here we reported that the mers-cov m protein also specifically inhibited irf activation but not nf-κb signaling. mers-cov m protein was capable of interacting with traf adapter protein and hampered traf -tbk interaction leading to diminished irf activation. using a chimeric protein containing the mers-cov m protein n-terminal transmembrane domains and a dormant sars-cov m protein c-terminal domain, we confirmed that the n-terminal transmembrane domains of mers-cov m protein sufficiently account for its inhibitory effect. although another chimera containing sars-cov m protein n-terminal transmembrane domains and a mers-cov m protein c-terminal domain was fully competent in ifn antagonism, a truncation mutant lacking the functional first transmembrane domain of sars-cov m was not, suggesting that the c-terminal domain of the mers-cov m protein is largely dispensable for its immunosuppressive activity. the ifnβ-luc reporter plasmid and rig-i expression plasmid are gifts from professor takashi fujita (kyoto university, kyoto, japan). the expression plasmids for tbk , irf and traf were generous gifts from dr genhong cheng (university of california, los angeles, ca, usa), , whereas those for rig-i n, ikk-ε and mavs and iκb-α as well as irf -luc and κb-luc reporter plasmids have been described elsewhere. , [ ] [ ] [ ] viral rna was extracted from mers-cov-infected vero-e cells. the m gene was pcr-amplified from complementary dna and cloned into the ecori/xhoi sites of pcagen plasmid with the addition of a c-terminal v -tag with the following primers: ′-atg tct aat atg acg caa ctc act ga- ′ (forward) and ′-agc tcg aag caa tgc aag ttc- ′ (reverse). the sars-cov m protein expression plasmid has been described elsewhere. , the expression plasmids for the sn and mn chimeras were constructed by assembly pcr with the following forward primers covering the breakpoints: ′-agg ctg ttt gct cgt acc cgc tca tgg tgg tca ttc aat cct gag- ′ (sn) and ′-ccg gct gtt tat gag aac tgg atc aat gtg gtc att caa ccc a- ′ (mn). the reverse primers were complementary to their respective forward primers. the truncation mutant of the sn chimera lacking the first transmembrane domain was constructed using the forward primer: ′-atg gta aca ctt gct tgt ttt gtg ct- ′. mouse anti-flag (m ) and anti-β-actin antibodies were purchased from sigma-aldrich (st. louis, mo, usa). mouse anti-v and anti-ha (y ) antibodies were purchased from life technologies (grand island, ny, usa) and santa cruz biotechnology (dallas, tx, usa), respectively. rabbit anti-irf and anti-phospho-irf (ser ) antibodies were purchased from ibl-america (minneapolis, mn, usa). cell culture and sendai virus hek- human embryonic kidney cells were maintained in dulbecco's modified eagle's medium with % fetal bovine serum (life technologies) at °c in a humidified chamber supplemented with % carbon dioxide. plasmid transfection was performed with genejuice (merck millipore; billerica, ma, usa). poly(i:c) was purchased from sigma-aldrich and was transfected with lipofectamine (life technologies). sendai virus (cantell strain) was purchased from american type culture collection (manassas, va, usa). dual-luciferase reporter assay, co-immunoprecipitation and western blotting were performed as previously described. , particularly, relative luciferase activity in arbitrary units was calculated by normalizing firefly luciferase activity to renilla luciferase activity recovered from cell lysate. non-denaturing native polyacrylamide gel electrophoresis (page) was performed as previously described. , , bioinformatic analysis sequence alignment was performed using cluster omega, an online tool based on the hidden markov model, and hosted by the embl-ebi server (http://www.ebi.ac.uk/tools/msa/clustalo/). transmembrane domain prediction was performed using tmfinder, which considers hydrophobicity and helicity of the amino-acid sequence (http://tmfinder.research.sickkids.ca/). to characterize the mers-cov m protein in terms of its ifn antagonism, m protein was ectopically expressed in cultured cells for functional assays ( figure a) . a luciferase reporter construct driven by ifn-β promoter was used to reflect ifn-β promoter activity stimulated during infection. sendai virus was used as a model virus to potently induce ifn expression in transfected cells. when increasing doses of m proteins were expressed in advance, a dose-dependent inhibition of ifn-β promoter activity was observed ( figure b ; bars - compared with bar ). a similar observation was also noted when synthetic dsrna analog poly(i:c) was used as an alternative inducer that specifically stimulates the rlr pathway of ifn production ( figure c ; bars - compared with bar ). these two pieces of data are generally consistent with a previous report, and they further strengthen the current model of the ifn antagonism of mers-cov m protein. cellular ifn-β expression is under the control of multiple transcription factors, which work cooperatively to form a large enhanceosome complex. in particular, irf and nf-κb are two transcription factors that are primarily activated by rlr signaling. , , mers-cov m protein has previously been shown to have no influence on nf-κb activation induced by sendai virus infection. however, it remains to be seen whether the mers-cov m protein could preferentially inhibit irf and nf-κb signaling after rig-i activation. to address this issue, two different luciferase reporter constructs, in which tandem copies of either irf -or nf-κb-binding elements were inserted into their promoter region, were used. the truncation mutant of rig-i known as rig-i n that contains only the n-terminal card domain was chosen to be the inducer in these assays because it is constitutively active and highly competent to induce these two pathways. the mers-cov m protein was able to suppress the promoter activity of the irf -driven luciferase construct in a dose-dependent manner (figure a ; bars - compared with bar ), but no inhibitory effect was observed with the nf-κb-driven construct ( figure b ; bars - compared with bar ) although the canonical inhibitor iκb-α could efficiently blunt its activation as a positive control ( figure b ; bar compared with bar ). hence, the suppressive effect of the mers-cov m protein is specific for irf signaling but not nf-κb activation. to delineate the action point of the mers-cov m protein in ifn antagonism, we tested the ability of the m protein to inhibit the activation signal induced by different signal transducers of the rlr pathway individually. the activation signal will be mostly unaffected if the activation event mediated by that transducer is downstream of the action point where m protein exerts its inhibitory effect. as described above, rig-i n is a constitutively active mutant that resembles ( μl/ml) in c for h before harvest for dual-luciferase reporter assay. bars represent the mean of three biological replicates (n = ) and error bars indicate their s.d. the statistical significance between selected samples was evaluated using a two-tailed student's t-test for unpaired samples with equal variance and p-value (p) was indicated. figure a ; bars - compared with bar ). a similar result was also obtained using mavs as an activator ( figure b ; bars - compared with bar ), which is a mitochondrial adapter that diverts the activation signal from rig-i to the irf and nf-κb pathways. [ ] [ ] [ ] [ ] when activators committed to the irf pathway were used, greater inhibitory effects were observed, as in the cases of tbk ( figure c ; bars - compared with bar ) and ikk-ε ( figure d ; bars - compared with bar ), which are kinases which recognize and phosphorylate irf as direct substrate. [ ] [ ] [ ] [ ] surprisingly, when a constitutively active mutant of irf transcription factor (irf d), with five inducible phosphorylation sites at ser/thr residues mutated to asp, was employed, the expression of the m protein no longer quenched the irf -induced activation of ifn-β promoter ( figure e ), suggesting that the inhibitory effect of m protein occurs upstream of irf activation. to further analyze the molecular mechanism and consequences through which mers-cov m protein exerts its inhibitory effect, we first investigated what signal transducer molecule might interact with the m protein. several rlr transducers were ectopically expressed with mers-cov m protein in cultured cells for a coimmunoprecipitation experiment. when the transducers were precipitated with an anti-flag antibody, the m protein was only detected in traf -containing precipitate ( figure a ; lane compared with lanes - ) even though m protein was abundantly expressed in all samples with other transducers (figure a ; lower panel for input), traf functions as an adapter that bridges the mitochondrial transducer mavs with the downstream signaling complex containing tbk and ikk-ε kinases that are essential for irf activation. , the physical association of the mers-cov m protein with traf ( figure a ) prompted us to test whether the adapter function of traf would be particularly affected by m protein. we performed another co-immunoprecipitation experiment to explore the possibility that m protein could perturb the interaction of traf with the downstream transducer complex. when traf and tbk were ectopically expressed in cultured cells, the detection of tbk in traf -immunoprecipitate confirmed the specific recruitment of tbk to traf in the absence of m protein ( figure b ; lane compared with lane ). however, when m protein was added to the system, the interaction between traf and tbk was significantly disrupted ( figure b ; lane compared with lane ), demonstrating that the physical association of mers-cov m protein with traf perturbs traf -tbk interaction. it was observed that mers-cov m protein disrupted traf -tbk interaction ( figure b ), which is required for irf activation. we then evaluated whether irf activation would be affected by the expression of m protein. irf dimerization visualized by non-denaturing native page is a sensitive assay for evaluating direct irf activation. therefore, we ectopically expressed irf and m protein with the inducer rig-i n in cultured cells and subjected cell lysates directly to native page to check for irf dimerization. when the inducer rig-i n was exclusively co-expressed with irf , the detection of an additional slow-migrating band indicated the activation and dimerization of irf , which would otherwise be entirely in its monomeric form in the absence of any activator ( figure c ; lane compared with lane ). interestingly, when m protein was expressed, the signal reflecting the dimeric form of irf molecules was significantly diminished, even though the total irf level expressed in all samples was highly comparable as detected by conventional denaturing sds-page ( figure c ; lower panel for sds-page), suggesting that irf activation was greatly inhibited by the mers-cov m protein. irf phosphorylation was also suppressed with the expression of mers m protein in a similar experimental setup ( figure d ; lane compared with lane ). together with other results, mers-cov m protein was thought to interact with traf to perturb traf -tbk interaction, which, in turn, affects irf phosphorylation and activation. given that the m proteins of both sars-cov and mers-cov were capable of antagonizing ifn production through highly similar innate immunosuppression by mers-cov m protein p-y lui et al mechanisms, it will be of interest to analyze the sequence and domain architecture of the two proteins. sequence alignment of sars-cov and mers-cov m proteins revealed a strikingly high sequence similarity ( %) and the presence of three transmembrane domains at the n-termini ( figure a ). according to the prediction results, we have initially constructed two truncation mutants for mers-cov m protein, an n-terminal transmembrane domain-containing mutant and a c-terminal mutant, and tested their inhibitory capacity in suppressing ifn-β expression using a luciferase reporter assay. however, neither exhibited an inhibitory effect (data not shown), possibly due to unstable expression or aberrant localization. to overcome the inactivity of truncation mutants and to define the inhibitory activity of different domains, we decided to create chimeric proteins using domain swapping between mers-cov and sars-cov m proteins. particularly, the sn chimera contains the n-terminal transmembrane domains from sars-cov m protein and the c-terminal domain from mers-cov m protein, whereas the mn chimera contains the n-terminal transmembrane domains from the mers-cov m protein and the c-terminal domain from the sars-cov m protein ( figure b ). the breakpoint was designed to occur immediately after the third predicted transmembrane domain at residue and before the conserved ser residue in both proteins at residue ( figure b ). we next compared the inhibitory effect of these two chimeras and the full-length m proteins on ifn-β expression using the luciferase reporter assay. our previous study showed that the ifn-antagonizing activity of the sars-cov m protein is mediated by its n-terminal transmembrane domains, but the c-terminal domain has no effect. when we swapped the c-terminal domain of the sars-cov m protein with that of the mers-cov m protein in the sn chimera, a similar suppressive effect on ifn-β promoter activity was observed ( figure c ; bar compared with bar ), consistent with our previous results. likewise, when we swapped the c-terminal domain of mers-cov m with that of sars-cov m protein in mn, the chimera was capable of suppressing ifn-β promoter activity to comparable level ( figure c ; bar compared with bar ). given that the c-terminal domain of sars-cov m protein possesses no suppressive effect, the inhibitory activity of the mn chimera would be predominantly due to the n-terminal domains of mers-cov m protein. a biochemical assay also confirmed that the mn chimera maintained the ability to interact with the traf adapter protein in a co-immunoprecipitation experiment ( figure d ; lane compared with lane ). therefore, we concluded that the n-terminal transmembrane domains of the mers-cov m protein are required and sufficient for its innate immunosuppressive activity. to further determine whether the c-terminal domain of the mers m protein also possesses ifn-antagonizing activity, we utilized the knowledge that the first transmembrane domain of sars-cov m protein is fully responsible for its suppression effect in this study, we reported that the mers-cov m protein inhibited irf activation, hence ifn expression, by disrupting traf -tbk interaction. this innate immunosuppressive activity of the mers-cov m protein was due to its conserved n-terminal transmembrane domains. our mechanistic study complemented the previous work that showed that mers-cov m protein had ifn-antagonizing activity. both studies demonstrated that mers-cov m protein suppressed irf activity but not nf-κb signaling. it is known that the activation of rig-i and mavs results in the activation of both irf and nf-κb. , [ ] [ ] [ ] our results indicated that the mers-cov m protein was capable of differentially suppressing the rig-i-induced activation of irf (figure ). this provides further support to the bifurcation of irf and nf-κb signaling subsequent to rig-i and mavs activation. further investigations should elucidate the mechanism by which the mers-cov m protein preferentially modulates irf activators such as tbk , while sparing nf-κb activators such as card . we provided evidence that the traf -tbk interaction as well as irf phosphorylation and dimerization were affected by the mers-cov m protein (figure ). our findings fill the knowledge gap by providing novel mechanistic insight into the innate immunosuppressive activity of mers-cov m protein. in our study, traf was also shown to interact with the mers-cov m protein ( figure a ). in line with this, the adapter function of traf in tbk recruitment and subsequent irf activation was perturbed by mers-cov m protein ( figures b-d) , which plausibly contributed to the ifn antagonism of the mers-cov m protein (figures - ) . the mers-cov m protein is a transmembrane protein that was shown to co-localize with markers of the golgi apparatus and endoplasmic reticulum (er)-golgi intermediate compartments in the perinuclear area. although traf is known to adapt the mitochondrial transducer mavs, it is not associated with mitochondria but rather with the golgi apparatus and er-golgi intermediate compartments in unstimulated conditions, , rendering it susceptible to interaction with the mers-cov m protein. upon stimulation with ligands or viral infection, traf appears on membrane-bound fragments originating from golgi. retention of traf in er-to-golgi compartments and inability to form golgi fragments rendered ifn-β expression inefficient. therefore, whether traf -containing golgi fragment formation is affected by mers-cov m protein warrants further analysis. this may serve as a novel mechanism by which virus-encoded proteins counteract host ifn production. mers-cov and sars-cov are two highly pathogenic coronaviruses that have caused hundreds of deaths. on one hand, the development of relevant prophylactic and therapeutic agents has been well under way. [ ] [ ] [ ] on the other hand, the identification of the pathogenic factors in these viruses is also in full swing. the m protein is a pathogenic factor by virtue of its ifn-antagonizing property. both the mers-cov and sars-cov m proteins were found to suppress ifn production with a highly similar mechanism in which the irf phosphorylating complex of traf ·tank·tbk /ikk-ε was affected by their n-terminal transmembrane domains. , interestingly, in the case of community-acquired human coronavirus hku , which normally causes common cold in infected individuals, its m protein showed no ifn antagonistic property, further supporting the importance of the m protein in sars-cov and mers-cov pathogenesis. using a side-by-side comparison of sars-cov and mers-cov m proteins, we discovered that the extent by which the mers-cov m protein suppressed ifn-β promoter activity was lower than that by sars-cov m protein. this difference might be explained by the strengths of the interaction of mers-cov m protein with other transducers. whereas the mers-cov m protein was found to be strongly associated with traf , its interaction with tbk or ikk-ε was undetectable (data not shown). this distinguished mers-cov m protein from the sars-cov m protein, which interacts potently with every component of the traf ·tank·tbk /ikk-ε complex. further investigations are required to shed light on how the interaction of the m protein with traf complex might influence mers-cov pathogenesis. coronaviruses encode multiple proteins to counteract the host innate antiviral response. - mers-cov is no exception. several mers-cov-encoded proteins have been identified to be ifn antagonists. we, and others, have characterized at least three ifn-antagonizing proteins encoded by mers-cov. in addition to the m protein reported in this study, orf a is a dsrna-binding protein, which directly inhibits rlr activation induced by dsrna and/or the protein co-activator pact. , in addition, our unpublished data also revealed that orf b is a potent ifn antagonist. this is in line with findings by other groups although the mechanistic details of its action have not well documented. , one recent report suggested that orf b might not only interact directly with tbk / ikkε in the cytoplasm but also perturb ifn production in the nucleus through an as yet unknown mechanism. nevertheless, how m, orf a, orf b and the other ifn-antagonizing proteins of mers-cov coordinate with each other to modulate the host innate antiviral response to facilitate viral replication and propagation remains elusive. severe respiratory illness associated with a novel coronavirus-saudi arabia and qatar severe respiratory illness caused by a novel coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in a 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structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon to sense or not to sense viral rna-essentials of coronavirus innate immune evasion a molecular arms race between host innate antiviral response and emerging human coronaviruses middle east respiratory syndrome coronavirus orf b protein inhibits type i interferon production through both cytoplasmic and nuclear targets this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- -iex lr authors: niu, xuefeng; zhao, lingzhai; qu, linbing; yao, zhipeng; zhang, fan; yan, qihong; zhang, shengnan; liang, renshan; chen, peihai; luo, jia; xu, wei; lv, huibin; liu, xinglong; lei, hui; yi, changhua; li, pingchao; wang, qian; wang, yang; yu, lei; zhang, xiaoyan; bryan, l. aubrey; davidson, edgar; doranz, j. benjamin; feng, liqiang; pan, weiqi; zhang, fuchun; chen, ling title: convalescent patient-derived monoclonal antibodies targeting different epitopes of e protein confer protection against zika virus in a neonatal mouse model date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: iex lr the zika virus (zikv) outbreak and its link to microcephaly triggered a public health concern. to examine antibody response in a patient infected with zikv, we used single-cell pcr to clone heavy and light chain-paired monoclonal antibodies (mabs) that bind to zikv envelope (e) proteins isolated from memory b cells of a zikv-infected patient. three mabs ( b , c , and a ) that showed the most potent and broad neutralization activities against the african, asian, and american strains were selected for further analysis. mab b showed an ic value of . ng/ml against the circulating american strain gz . epitope mapping revealed that mabs b and c targeted residue k of the lateral ridge (lr) epitope of the ediii domain, but b has a broader lr epitope footprint and recognizes residues t , g , e , and n as well. mab a recognized residues d , k , and k of the edii domain. interestingly, although the patient was seronegative for denv infection, mab c , originating from the vh - and vk - germline pair, neutralized both zikv and denv . administration of the mabs b , c , and a protected neonatal scid mice infected with a lethal dose of zikv. this study provides potential therapeutic antibody candidates and insights into the antibody response after zikv infection. zika virus (zikv) is a member of the flaviviridae family which includes dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), west nile virus (wnv), and tick-borne encephalitis virus (tbev) [ , ] . zikv is mainly transmitted by aedes mosquitoes but can also spread through sexual contact, blood transfusions, or via mother-to-child transmission during pregnancy [ , ] . zikv was first discovered in africa in [ ] and was confined within the equatorial zone of africa and asia until the outbreak in yap island, which was then transmitted to french polynesia and other southern pacific islands in [ , ] . it is believed that the adaptation and infectivity of zikv in mosquito-vectors contributed to the spread of the virus from asia to the americas [ ] . the zikv outbreak and associated increase in microcephaly cases in brazil increased global awareness [ ] ; to date, more than countries have reported zikv infections [ ] . it is known that zikv can cross the placental barrier, leading to fetal microcephaly, and can cause neurological complications in adults, such as guillain-barré syndrome [ ] [ ] [ ] . currently, there are no approved drugs or vaccines to mitigate the risk of zikv infection. the zikv surface is formed by copies of each envelope (e) glycoprotein and associated membrane (m) protein [ , ] . e proteins are arranged as dimers, with three parallel dimers connected to form a raft, and with rafts covering the viral surface [ ] . the e protein mediates viral entry into host cells and membrane fusion and is the major target for neutralizing antibodies and vaccine immunogens [ ] . the flavivirus e ectodomain consists of three distinct domains, edi, a -stranded beta-barrel that acts as a bridge between edii and ediii [ ] ; edii, a finger-like structure that is responsible for the dimerization of soluble e protein monomers and viral fusion [ ] ; and ediii, an immunoglobulin-like segment that is involved in host cell receptor recognition and viral fusion [ , ] . in recent years, a number of neutralizing antibodies (nabs) have been isolated from individuals infected with zikv [ ] [ ] [ ] [ ] [ ] . these nabs mainly recognize edii, ediii, and tertiary or quaternary epitopes that constitute e ectodomains. although ediii-targeted antibodies represent a relatively small population of e protein-binding antibodies, their presence is associated with serum neutralizing activity against zikv [ , ] . among these nabs, ediii-targeted antibodies and edii/e-dimer epitope (ede)-targeted antibodies showed the most potent neutralization activities. in this study, we cloned and characterized e-targeted monoclonal antibodies (mabs) from a chinese patient who returned to china from a visit to venezuela. selected mabs were evaluated for their neutralizing activities in vitro and in vivo via a zikv-infected neonatal severe combined immunodeficiency (scid) mouse model. the patient was a -year-old male who returned from venezuela in february . he was hospitalized in guangzhou th people's hospital (guangzhou, china). zikv rna was detected in serum, saliva, and urine samples by rt-pcr. the patient manifested relatively mild symptoms including fever, rash, sore throat, and fatigue, and recovered and was discharged approximately weeks after the onset of symptoms with no detectable zikv. the patient tested serologically negative for denv - infection using an ns based elisa kit (euroimmun, lubeck, germany), indicating that the patient had no previous exposure to denv - before infection with zikv [ , ] . single b cell sorting, rt-pcr, sequencing, and cloning freshly isolated peripheral blood mononuclear cells (pbmcs) were stained with a cocktail of antibodies including anti-human cd -fitc (invitrogen, carlsbad, ca), igg-apc-h /cd -pacific blue/cd -percp-cy . (bd biosciences, franklin lakes, nj), and anti-his-pe (miltenyi biotec, bergisch gladbach, germany). for antigen-specific memory b cells, we used zikv e protein (cat. no. -v b ; sino biological inc., beijing, china) as a probe. after washing, cd − cd + cd + igg + his + memory b cells were sorted using a multi-laser ariaii sorter. individual b cells were sorted into -well pcr plates containing µl lysis buffer per cell. the lysis buffer contained . µl rnasin inhibitor (promega, madison, wi), µl x first-strand buffer, . µl . m dtt, and . µl igepal (sigma-aldrich, st. louis, mo). pcr plates with sorted cells were frozen on dry ice and then stored at − °c or subjected to reverse transcription. rt-pcr and cloning into expression vectors was performed as previously described [ ] . briefly, µl random hexamers ( ng/µl; promega), µl dntps (each at mm), and . µl superscript iii (invitrogen) were added to each well, followed by incubation at °c for h. the igg heavy and light chain variable regions were amplified independently by nested pcr [ ] . first round pcr was performed using µl cdna directly following reverse transcription, with hotstart taq plus dna polymerase (qiagen, hilden, germany) and the primer mix. the pcr programme was initiated by min incubation at °c, followed by cycles of °c for s, °c (first round) or °c (second round) for s, and °c for min, followed by °c for min before cooling to °c. using % agarose gels, the pcr products were evaluated, excised from the gel (approximately bp for the heavy chain and bp for kappa and lambda chains), and sent for sanger sequencing after purification. human antibody sequences were analysed using imgt/v-quest (http://www.imgt.org/) [ ] . fulllength igg was expressed by co-transfecting hek- t cells with equal amounts of paired heavy and light chain plasmids based on the backbone of the pci-neo vector [ ] . culture media were harvested four days after transfection and purified using protein a agarose (ge healthcare, chicago, il). zikv particles were successfully isolated from the infected patient's blood plasma or urine. the virus was passaged once in suckling mouse brains and cultured in vero cells to prepare stocks, which were stored at − °c before use. denv (hawaii strain), denv (new guinea-c strain), denv (h strain), and denv (h strain) were prepared in vero cells. recombinant zikv e and ediii protein (cat. no. -v b and -v h, respectively) were purchased from sino biological inc. the e protein was derived from zikv strain sph (ku ), isolated from brazil in . denv ediii protein (cat. no. -v b), denv ediii protein (cat. no. -v y ), and denv e protein (cat. no. -v b ) were also purchased from sino biological inc. nunc maxisorp plates (thermo fisher scientific, waltham, ma) were coated with zikv e or e domain iii protein ( µg/ml) and incubated at °c overnight. after blocking for h, the plates were washed six times with phosphate-buffered saline (pbs) and transfected with antibody supernatants at a : dilution with blocking buffer or a serial dilution of purified mabs. secondary antibody (goat anti-human igg; ab ; abcam, cambridge, uk) was applied at a : dilution in blocking solution and then the plates were incubated at room temperature for h, and tmb substrate. absorbance values were determined at nm using a biotek plate reader (biotek instruments, winooski, vt). neutralization activity of purified antibodies was measured using a flow cytometry-based neutralization assay with vero cells as previously reported [ , ] with minor modifications. briefly, × cells were plated in a -well plate h before the experiment. purified human mabs were serially diluted in dmem (gibco; thermo fisher scientific) supplemented with % feta bovine serum (fbs; gibco) and incubated with zikv ( × pfu) for h at °c under % co . the cells were then incubated with µl of the mixture for h at °c under % co , after which ml/well mem medium containing % fbs was added and incubated for another h. zikv-infected and uninfected cells were used as positive and negative control, respectively. cells were then trypsinized, fixed, and permeabilised with fixation and permeabilization solution (bd biosciences) on ice for min, followed by staining with g antibody ( µg/ml) diluted with x perm/wash buffer and staining with anti-mouse igg fitc ( : in x perm/wash buffer) on ice for min. after washing, the percentage of e-positive cells was measured using bd facscanto ii (bd biosciences). the antibody dilution that neutralized % of the viruses (half-maximal neutralizing inhibitory concentration; ic ) was calculated by nonlinear, dose-response regression analysis with graphpad prism version . (graphpad software inc., la jolla, ca). epitope mapping was performed by shotgun mutagenesis as previously described [ ] . zikv prm-e protein expression constructs (based on zikv strain sph ) were subjected to high-throughput alanine scanning mutagenesis to generate a comprehensive mutation library [ , ] . each residue within prm-e was changed to alanine, with alanine codons mutated to serine. in total, zikv prm-e mutants were generated ( % coverage)which were confirmed by sequencingand arrayed into -well plates. each zikv prm-e mutant was transfected into hek- t cells and incubated for h. cells were then fixed in % (v/v) paraformaldehyde (electron microscopy sciences, hatfield, pa) and permeabilised with . % (w/v) saponin (sigma-aldrich) in pbs plus calcium and magnesium (pbs++). for mapping, cells were sequentially incubated with . µg/ml mabs and . µg/ml alexafluor -conjugated secondary antibody (jackson immunoresearch laboratories, west grove, pa) diluted in pbs++, % normal goat serum (sigma-aldrich), and . % saponin. cells were washed three times with pbs++/ . % saponin, twice with pbs, and then mean cellular fluorescence was recorded using a high-throughput flow cytometer (htfc; intellicyt, albuquerque, nm). antibody reactivity against each prm-e mutant relative to the wildtype (wt) protein was calculated by subtracting the signal from mock-transfected controls and normalizing to the signal from wt prm-e-transfected controls. binding competition between mabs b , c , and a and five other ediii-targeted antibodies was determined using a real-time, label-free, bio-layer interferometry assay on an octet red biosensor (fortebio, fremont, ca) as previously described [ ] . the experiment was performed at °c in pbs buffer with shaking at , rpm. ni-nta biosensors (forte-bio) were first loaded with μg/ml his-tagged-e protein for s and then associated with the first mab ( b , f , d , c , a , b , d , or f ) for s. an irrelevant mab, d , was used as negative control and pbs was used as blank solution. the biosensors were then dipped into the second mab and incubated in the presence of the first mab. the capacity of additional binding was monitored by measuring further shifts for s. all mabs were evaluated at concentration of nm, except for b ( nm), d ( nm), and f ( nm), for saturation measurement. the ni-nta biosensors were regenerated with mm glycine-hcl (ph . ; ge healthcare) and re-charged with mm nicl . the response of mab binding to the e protein was compared and the data were processed using biaevaluation software (biacore, uppsala, sweden). the use of pregnant scid and suckling mice in this study was carried out in strict compliance with the association for the assessment and accreditation of laboratory animal care. the experimental protocol was approved by the guangzhou institute of biomedicine and health (gibh) institutional animal care and use committee. scid beige mice were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china). one-day-old suckling mice (n = - ) were intraperitoneally inoculated with zikv at . × pfu. zikv mabs were administered as a single dose h after virus infection. mouse brain, spleen, and serum samples were then collected days after virus infection for rna extraction ( ; qiagen). zikv rna detection was determined by one-step qrt-pcr ( ; qiagen) on a cfx real-time pcr system (bio-rad laboratories, hercules, ca) using published primers and conditions. briefly, µg rna together with a mixture of . µl sybr green, . µl rt-mix, and . µl each of forward and reverse primers were placed in -well pcr plates in µl reaction volumes. amplification was performed at °c for min, °c for min, followed by cycles of °c for s, °c for s, and °c for s. viral load was expressed on a log scale as viral rna copies per µg after comparison with a standard curve. three replicates were conducted for each sample. flow cytometric data were analysed using flowjo version . (tree star inc., ashland, or). the ec (half-maximal effective binding concentration) and ic values obtained from the elisa assay of zikv e/ediii binding activity to mabs and the neutralization assay, respectively, were calculated using a dose-response inhibition model and sigmoidal curves were generated using graphpad prism version . (graphpad software inc.). statistical significance of the difference in viral loads between groups was determined using one-way anova. p values < . were considered statistically significant. to clone e-targeted mabs from a zikv-infected patient, we used recombinant zikv e protein as a probe to isolate e-specific memory b cells from a chinese patient that had returned from venezuela and was identified as infected with zikv during border entry. thirty-one mabs that bind to e protein were successfully cloned days after the onset of symptoms. we first used a facs-based neutralization assay to screen neutralizing activities in the cultured media of hek- cells transfected with expression plasmids for each heavy and light chain of the mabs. eight mabs with the best neutralizing activities and binding to e protein were selected for further analysis. the binding activities of these eight mabs to zikv e, ediii, and denvi ediii proteins were assessed ( figure and table ). mabs b , c , d , and b bound both zikv e and ediii, while mabs a , a , e , and a bound e protein but not ediii ( figure (a) ). mabs b and c showed strong binding activities to zikv e and ediii protein in the nanomolar range. we then measured the neutralization activities of purified mabs against the zikv gz strain. zikv gz has the same genomic sequence as the current circulating zikv strain in south america and was the same as the zikv strain isolated from the patient [ ] . three mabs, b , c , and a , showed the best neutralization activities with ic values of . , . , and ng/ml, respectively; thus, we further analysed these mabs for epitope identification and in an animal model (figure (b) ). we also tested the neutralizing activities of these three mabs against two other zikv isolates, mr (the first zikv isolated from the zika forest in uganda) [ ] and prvabc (a zikv isolated from puerto rico). all three antibodies cross-neutralized mr and prvabc (table ) . intriguingly, one of the mabs, c , showed binding to denv ediii protein but not to other denv ediii proteins. the ed of c with denv ediii was . ng/ml, which was comparable to zikv ediii binding ( . ng/ml). we then measured the neutralization activity of these three representative mabs against denv and found that mabs b and a showed no denvi ediii cross-reactivity and failed to neutralize denvi virus at µg/ml, whereas c showed potent neutralizing activity at an ic of . µg/ml (table ) . neutralizing mabs recognized either the ediii or edii lateral ridge (lr) ediii has been recognized as a major target site for nabs that bind to the same or different epitope region. we used a bio-layer interferometry (bli) competition assay to probe whether our e-targeted mabs bind to the same epitope region. a his-tagged e protein, captured to a ni-nta biosensor, was first saturated with one mab, after which competitive binding of a second mab was assessed. the binding competition between mabs b , c , and a and five other zikv e-targeted mabs was measured (figure (a) ). mabs c , d , d , f , and f but not a could block the binding of mab b to e protein, indicating that b and these five mabs share the same or overlapping epitopes on zikv ediii. interestingly, mab c binding to e protein was blocked by mab b and no other etargeted mab, indicating that b may bind to a broader epitope footprint on ediii, whereas c binds to a smaller epitope region that overlaps with the binding region of b . we next mapped the detailed epitope residues for mabs b , c , and a using a shotgun alaninescanning mutagenesis library of zikv prm and e protein variants [ , ] . mab b recognized an epitope region that includes residues t , g , e , n , and k along the lr region of ediii. notably, mab c only recognized k on the lr region as a key residue (figure (b and c) ). amino acid sequence alignment between e proteins of zikv and denv revealed that residue k on zikv e protein corresponds to k on denv , which is not present on the e proteins of denv , denv , or denv ( figure (d) ). mab a is an edii-targeted antibody that recognized residues d , k , and k on the edii. we confirmed that an individual mutation of these key residues reduced binding activity by more than % when compared with that of wt prm-e protein (figure (b) ). analysis of the immunoglobulin heavy chains of b , c , and a revealed that they were derived from germlines hv - * , hv - * , and hv - - * , respectively (figure (a) ). the shm rates of these heavy chains compared with their predicted germline sequences were relatively low, at . % for b h, . % for c h, and . % for a h, which is lower than that of antibodies isolated from annual trivalent inactivated influenza vaccine (tiv) donors [ ] and chronic human immunodeficiency virus (hiv)- patients (> %) [ , ] . compared with their germline sequences, the shms of a h were in the cdr , cdr , and cdr regions, whereas shms of c h and b were found sporadically in the cdr , fr , cdr , fr , and cdr regions ( table ). we paired the heavy chain predicted germline (hgl) of c h ( c hgl) with the light chain (kappa) of c ( c k) and similarly paired a hgl with a k and expressed these heavy chain germline-reverted antibodies in hek- cells. these germline-reverted antibodies showed reduced binding activities to e protein at . µg/ml for a gl, which is about times lower than that of mature a ( . µg/ml), and . µg/ml for c gl, which almost completely lost binding ability compared with that of mature c ( . µg/ml; table ). therefore, shms are necessary for strong binding to e protein, but only a low level of shms is needed to improve binding. to evaluate the protective effects of e-targeted mabs, we developed a zikv lethal infection mouse model. intraperitoneal infection of -day-old scid neonates with . × pfu zikv gz caused % lethality within days; neonatal mice that received no treatment showed disease symptoms, including ruffled fur, trembling and shaking, and body weight loss ( figure (a) ). neonatal mice treated with mab b at either , µg, or µg h after infection survived the lethal infection (figure (b) ). titration of viral load from brains and spleens collected days after infection revealed a dose-dependent decrease in viral rna in mice treated with mab b , whereas mice that received no treatment had much higher viral rna levels in the brain and spleen (figure (c and d) ). treatment with µg mab b resulted in the best protection, with no detectable viral rna in the brain and spleen. we also demonstrated in subsequent experiments that zikvinfected neonatal scid mice survived and did not exhibit weight loss after treatment with µg of either mab c or a (figure (e and f) ). in a separate experiment, an unrelated mab, g , which is specific for h n influenza virus, showed no protective effects on zikv-infected neonatal scid mice (data not shown). therefore, both ediii-targeted and edii-targeted mabs can effectively protect neonatal scid mice from lethal zikv infections. the emergence of zikv in south america has raised a global health concern due to the link between zikv infection and microcephaly in infants and guillain-barré syndrome in adults. the search for and development of vaccines and therapeutics to prevent and control zikv infection are thus necessary. for example, nabs have been found effective in combating emerging viruses, such as the middle east respiratory syndrome coronavirus (mers-cov), ebola virus, and influenza virus [ , [ ] [ ] [ ] . in the present study, we cloned zikv e proteinbinding mabs from the memory b cells of a zikvinfected chinese patient and selected three mabs for further study. we characterized the zikv e protein epitopes recognized by these mabs and analysed the shm pattern. two of the most potent mabs, b and c , are ediii-targeted and showed neutralizing activities against three different zikv strains, including the south american circulating strain (gz ), african strain (mr ), and american strain (prvabc ). mab b recognized several residues in the lr region of ediii, and an individual mutation in these residues led to a > % decrease in binding activity compared with that of wt prm-e protein. a previous study also demonstrated that an e k mutation alone abolished the neutralizing activity of the lr-targeted mab zka [ ] . meanwhile, the heavy and light chains of mab c were derived from the vh - and vk - germlines, respectively. it has been reported that zikv mabs with vh - /vk - paired antibodies are present in five of six people that were sequentially infected with denv and zikv; thus, these mabs were determined as recurrent antibodies that recognize both zikv and denv viruses [ ] . our findings, along with observations by other studies [ ] , suggested that vh - may be preferentially enlisted in response to zikv and denv infections. it is interesting to note that both mab b and c recognized k in the lr region of zikv ediii. mabs reported by others, such as z and z , also recognized k on zikv ediii or k on denv ediii [ ] . although mab c neutralized the african zikv strain mr , another reported mab, zikv- , that also recognized k of the zikv e protein and neutralized the h/pf/ strain, failed to neutralize the mr strain [ ] . this difference may because the current zikv strains gz and prvabc possess e in the ediii region, whereas african strain mr has d in the ediii region. therefore, k in lr region of ediii appears to be a key target residue for ediiitargeted antibodies to exert neutralizing activities. mabs that recognized k showed potent in vitro neutralizing activity, supporting the notion that residue k is a hotspot for effective neutralizing activity of ediii-targeted mabs [ , , , , ] . it is important to note that most reported e-targeted neutralizing mabs are ediii-targeted, whereas only a few neutralizing mabs are edii-targeted. mabs showing broad neutralization activities against flaviviruses have been reported to recognize the edii targeting either the fusion loop epitope (fle) or ede regions [ , ] . antibodies targeting the fle are conserved among flaviviruses and can bind to zikv e protein. it has been reported that fle-targeted antibodies show poor neutralizing activities but have strong infection enhancement in vitro [ ] . meanwhile, antibodies targeting the ede show potent neutralizing activities against zikv and can protect against zikv infection in mouse and rhesus macaque models [ , ] . notably, mab a in our study showed potent neutralizing activities against all three tested zikv strains and is likely an edii-targeted mab. it also recognized residues d , k , and k and is similar to the reported ede-targeted antibody zikv- , which recognizes d , k , and q [ ] . both mabs recognize the same two residues in the edii region. structural analysis indicated that mab zikv- is cross-linked with e monomers within dimers and neighbouring dimers in the zikv particle [ , ] . therefore, mab a may not exhibit the same interaction with the virus particle as zikv- . recently, another ede-targeted mab, zikv- , was reported to neutralize multiple zikv strains and recognize residues d , m , r , and k in the edii region [ ] ; both mab a and zikv- recognize residues d and k . notably, mab a , zikv- , and zikv- were all isolated from memory b cells of zikv table . cdr , fr , cdr , and fr amino acid sequence of b h, c h, and a h with their corresponding vh germline sequences and predicted cdr sequences. convalescent patients. it is possible that residues d , k , and k are hotspots for ede-targeted neutralizing antibodies. future structural biology analysis is required to confirm if mab a is indeed an edetargeted antibody. antigen-specific b cells undergo a process termed shm to increase antigen affinity. interestingly, the neutralizing mabs b , c , and a had relatively low shm rates in their vh genes, which is much lower than the antibodies isolated from annual tiv vaccine donors [ ] and chronic hiv- patients [ , ] . it is possible that when a person is exposed to zikv infection, e-targeted antibodies that require low shm rates to bind and neutralize zikv were generated. even with relatively low shm rates, these limited mutations appeared essential for conferring binding activities. when the mabs c and a heavy chains were reverted to their predicted germline sequence and paired with their respective mature c and a light chains, binding activity to zikv e protein was found almost completely impaired and times lower than that of mature a , respectively. therefore, (d) viral load in the spleen (n = - ). total rna was extracted from the homogenates of the brain and spleen. zikv genomic rna was evaluated using one-step qpcr. viral loads are expressed as the genome copy number per microgram tissue. (e) body weight changes in zikv-infected mice treated with mab a or c at µg/mouse. (f) survival curve of zikv-infected mice treated with mab a or c . data are representative of two independent experiments and presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significance (one-way anova). a few mutations are sufficient but necessary to confer neutralizing activities against zikv infection. in addition to evaluating the neutralizing activities of representative mabs in inhibiting zikv infection in cultured cells, we also tested the protective efficacy of these mabs in a neonatal scid mouse model. zikv infection in wt mice does not result in disease, whereas suckling wt mice are susceptible to infection [ , ] . mice lacking interferon signalling, such as a (type i ifnar ko) [ ] , interferon regulatory factor (irf) / / triple ko [ ] , and ag (type and type ifn ko) [ ] , are susceptible to zikv infection with detectable zikv in the brain, spinal cord, and testes; these mice died within - days post-infection. in this study, we developed a scid beige suckling mouse model for evaluating the protective ability of mabs against zikv infection. scid beige mice are deficient in t, b, and nk cells and are thus more suitable for evaluating the net effect of anti-zikv mabs. zikv infection of scid beige suckling mice led to neurological symptoms and high viral loads in the brain and spleen, which was eventually lethal. all three mabs tested (ediii-targeted mabs b and c and edii-targeted mab a ) showed protective effects in zikv-infected scid mice. therefore, nabs against zikv cloned from convalescent patients have the potential to be further developed for treating zikv infection. there is an opinion that e-targeted mabs may mediate antibody-dependent enhancement (ade) of virus infection. we found that mab b enhanced zikv infection in k cells at a very low concentration of ng/ml. however, we believe that ade is not a critical concern because the concentration of mabs used for treatment are much higher. nevertheless, engineering of the mab fc fragment to minimize fc gamma receptor-mediated infection would be beneficial in improving the practical usage of these neutralizing mabs. overall, our study findings provided insights into the antibody response after zikv infection and demonstrated the potential of mabs in zikv treatment. zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus: a previously slow pandemic spreads rapidly through the americas effect of acute zika virus infection on sperm and virus clearance in body fluids: a prospective observational study the emergence of zika virus and its new clinical syndromes isolations and serological specificity zika virus outside africa evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes assessing the global threat from zika virus zika virus: recent advances towards the development of vaccines and therapeutics guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study possible association between zika virus infection and microcephaly -brazil zika virus associated with microcephaly the . 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infection a human antibody against zika virus crosslinks the e protein to prevent infection structural basis of a potent human monoclonal antibody against zika virus targeting a quaternary epitope a mouse model of zika virus pathogenesis zika (prvabc ) infection is associated with t cell infiltration and neurodegeneration in cns of immunocompetent neonatal c bl/ mice characterization of lethal zika virus infection in ag mice key: cord- -mar zt t authors: su, liang; ma, xiang; yu, huafeng; zhang, zhaohua; bian, pengfei; han, yuling; sun, jing; liu, yanqin; yang, chun; geng, jin; zhang, zhongfa; gai, zhongtao title: the different clinical characteristics of corona virus disease cases between children and their families in china – the character of children with covid- date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: mar zt t this study aims to analyze the different clinical characteristics between children and their families infected with severe acute respiratory syndrome coronavirus . clinical data from nine children and their families were collected, including general status, clinical, laboratory test, and imaging characteristics. all the children were detected positive result after their families onset. three children had fever ( . %) or cough ( . %) symptoms and six ( . %) children had no symptom. among the adult patients, the major symptoms included fever ( . %), cough ( . %), chest tightness/pain ( . %), fatigue ( . %) and sore throat ( . %). nearly % of the patients had normal ( . %) or decreased ( . %) white blood cell counts, and % ( / ) had lymphocytopenia. there were adults ( . %) showed abnormal imaging. the main manifestations were pulmonary consolidation ( %), nodular shadow ( %), and ground glass opacity ( %). five discharged children were admitted again because their stool showed positive result in sars-cov- pcr. covid- in children is mainly caused by family transmission, and their symptoms are mild and prognosis is better than adult. however, their pcr result in stool showed longer time than their families. because of the mild or asymptomatic clinical process, it is difficult to recognize early for pediatrician and public health staff. in late , an outbreak of pneumonia with unknown etiology was found in wuhan, hubei province, china. then the pathogen was isolated soon and named the novel coronavirus ( -ncov) on january [ ] and on february, the international committee on taxonomy of viruses announced that its official classification is severe acute respiratory syndrome coronavirus (sars-cov- ). the virus spread very fast in wuhan. even more unfortunate, as the chinese spring festival is approaching, aggregation of large numbers of people flow caused it to spread quickly across the country and even spread to more than countries [ ] . the current case reports are mainly concentrated in hubei province and adults, but cases of children outside hubei province are rare. meanwhile, the clinical characteristics of cases in hubei province and other provinces were significantly different. here, we report the clinical manifestations, laboratory test results, imaging characteristics, and treatment regimen of nine sars-cov- infected children and their families in jinan, shandong province to increase awareness of this disease, especially in children. a retrospective review was conducted of the clinical, lab tests, and radiologic findings for nine children and their families admitted to the jinan infectious diseases hospital identified to be nucleic acid-positive for sars-cov- from january to february . sample collection and pathogen identification after admission to the hospital, respiratory tract samples including sputum and nasopharyngeal swabs were collected from the patients, which were tested for influenza, avian influenza, respiratory syncytial virus, adenovirus, parainfluenza virus, mycoplasma pneumoniae and chlamydia, along with routine bacterial, fungal, and pathogenic microorganism tests. real-time pcr used the sars-cov- (orf ab/n) nucleic acid detection kit (bio-germ, shanghai, china) and performed refer to previous literature [ ] . all the patients were recorded with basic information and epidemiological histories [ ] including ( ) history of travel or residence in wuhan and surrounding areas or other reported cases within days of onset; ( ) history of contact with new coronavirus infection (nucleic acid-positive) days before onset; ( ) history of contact with patients with fever or respiratory symptoms from wuhan and surrounding areas, or from communities with case reports within days before onset; ( ) cluster onset, along with disease condition changes. laboratory test results were compiled, including standard blood counts, blood biochemistry, c-reactive protein (crp), procalcitonin (pct), erythrocyte sedimentation rate(esr), interleukin- (il- ) and myocardial enzyme spectrum. additional data collected included medical imaging, treatment regimens, and prognosis (any severe complications, including death), and recover or discharge date (table ) . this study was conducted in accordance with the declaration of helsinki. informed consent was waived because of the retrospective nature of the study and the analysis used anonymous clinical data. continuous data are expressed as medians and ranges, and categorical data are presented as counts and percentages. there were three boys, six girls and their families admitted to jinan infectious disease hospital of shandong university were investigated in this study. the youngest of the nine children was a pair of elevenmonth-old twins and the oldest is nine years and months old (mean age was . years, median age . years, table ). there were families were infected by sars-cov- , and adults were enrolled in this study (two patients hospitalized in another hospital). the patients consisted of males and females with a mean age of . years (median age, years [range, - years]). all nine pediatric patients came from eight families. as shown in table , six children had no information from the epidemiological data, / ( %) of the adults were infected through household contact, ( . %) was found to be infected after returning from wuhan or hubei in late january and ( . %) patients couldn't find the exact source of infection. as shown in table , / ( . %) children had normal or decreased white blood cell counts, consistent with the main characteristic of viral infection. six children ( . %) showed increased ck-mb. alt, ast and the other index of liver and kidney were all normal. all inflammation indicators, including crp, pct, esr and il- were all within the normal range. two children ( . %) showed bronchitis and one ( . %) showed bronchial pneumonia. one ( . %) boy (the older of the twins) showed pulmonary consolidation and ground glass opacity on the first day ( figure (a)) admitted in the hospital, and disappeared after five days (figure (b) ). five other ( . %) children showed no abnormal chest radiograph. all the adult patients had normal ( / , . %) or decreased ( / , . %) white blood cell counts and ( . %) have lymphopenia. there were ( . %) patients had increased crp, pct, serum amyloid a (saa), d-dimer and il- , meanwhile, their ct-scan showed larger lung consolidation. compared to children, there were only two ( . %) patients showed increased ck-mb. ferritin in the adult patients were higher than the children but most of them were normal ( / , . %). the imaging of adult chest was mix and the most common characters of imaging were pulmonary consolidation ( %), nodular shadow ( . %), and ground glass opacity (ggo, . %) (figure ) . four ( . %) adults showed normal chest imaging. at present, there are no drugs available that can target sars-cov- . therefore, treatment was focused on symptomatic and respiratory support. all the children inhaled interferon and one of the twins was prescribed ribavirin ( - mg/kg.d) in addition. ten ( . %) adults with pneumonia were treated lopinaviritonavir ( / mg, tablets, bid), interferon and chinese medicine. the patients with higher infection index (such as crp, pct, esr, saa, il- ) were prescript antibiotics for - days in addition. all the nine children and adult patients recovered in - weeks and were discharged after two negative nucleic acid tests. unfortunately, our follow up found that there were five discharged children were admitted again before we submit this article because their stool showed positive result in sars-cov- pcr. meanwhile, all their families were negative in all the specimen. coronaviruses are a large family of viruses that are known to cause illness ranging from the common cold to more severe diseases. aa an enveloped rna virus, cov is ubiquitous in humans, other mammals, and birds, which can cause respiratory, digestive, liver and nervous system disorders [ , ] . to date, six covs have been known to cause human infection [ ] . among them, two zoonotic viruses, sars-cov and mers-cov, were responsible for serious outbreaks: in china in - [ , ] of particular concern, our observations found that all the children were diagnosed after their families, which indicated that they were infected by the household contact. however, after an epidemiological investigation, we found that six adults ( . %) had a definite or suspicious contact history and six families ( . %) contacted them were infected, while the other two patients ( . %) denied any epidemiological history. among them, the father of case did not contact anyone who came back from wuhan or hubei, but also denied contact with any person with respiratory symptoms. at the same time, through official investigations, they did not find that someone was diagnosed with sars-cov- infection on the vehicle he was travelling on, prompting the virus to spread. in addition, from the official information, more and more patients can't find the clue of infection and more and more cluster outbreak showed that no contact, no close communication and even never go out the door. so, we think that these phenomena maybe suggest that: ( ) the virus spreads very strongly and the transmission of the virus may not be limited to contact, droplets and airborne transmission, and aerosol transmission may also exist, which was similar to sars [ ] . ( ) the virus may be carried asymptomatically after infecting the human body but can infect other people. in china, the sars outbreak of is still impressive, because the - sars outbreak infected individuals leading to deaths in eight affected areas [ ] . during the sars outbreak, there were less children patients and the symptoms are significantly milder in children than in adults [ ] [ ] [ ] [ ] . similarly, the official data to date suggest that children infected with the sars-cov- are relatively rare too [ ] , and their overall symptoms are significantly mild. the main reasons for this phenomenon may be: ( ) the range of activities for children is relatively small, they are mainly infected by their adult families. and, as an rna virus, the sars-cov- virus maybe also is prone to mistakes in replication, mutating, and surviving without recognition by the immune system, but can also cause a decline in virulence. so, children are infected with second or third generation or even fourth generation virus and they get milder symptoms; ( ) it may be because of differences in the immune responses of children compared to adults. one hypothesis is that the innate immune response, that is the early response that is aimed broadly at groups of pathogens, tends to be more active in children. the innate immune system is the first line of defense against pathogens. cells in that system respond immediately to foreign invaders. the adaptive immune system, by contrast, learns to recognize specific pathogens, but takes longer to join the battle. if the innate immune response is stronger in children exposed to sars-cov- , they may fight off infection more readily than adults, suffering only mild symptoms. other coronaviruses, including sars and mers, also show this pattern [ ] . ( ) the number or function of ace receptors in children is not as good as in adults. recently, one studies had investigated the role of the ace receptor and found that the sars-cov- uses the sars-coronavirus receptor ace and the cellular protease tmprss for entry into target cells [ ] . as we know, the distribution of ace receptors in different organs and populations is different. therefore, it may be that different receptor levels or functions in children and adults lead to different severity of illness. ( ) other reasons: such as children have fewer basic diseases, children smoke less, and children have strong self-healing capabilities and so on. ck-mb is an indicator of myocardial injury. in the present study, we found six children and two adults had high ck-mb, which means that sars-cov- can cause heart injury. it is reported that the main mechanisms of sars-cov- -induced myocardial injury may be the direct injury of virus, the inflammatory storm and the distribution of ace receptor [ ] . as human lifestyles change, more and more viruses are spreading across species. current research confirms that sars-cov- are transmitted from animals to humans. like other viruses, the relationship between sars-covs and humans has the following possibilities: ( ) the virus disappears for some unknown reasons, such as sars-cov. ( ) viruses coexist with humans and have seasonal onsets, such as flu influenza viruses. the first is the best outcome of the current situation, but the second possibility is very large. if, as we analyzed above, many people, especially children with mild or no clinical symptoms carry the virus but do not develop the disease, however, the virus spread very strongly, it may lead to the silent spread of the disease and leading to major losses. therefore, the chinese government will face greater risks after school starts and work resumes. and, clinicians, especially pediatricians, need to be vigilant to prevent widespread spread of the disease. children who have infected family members should be monitored or evaluated and family clustering should be reported to ensure a timely diagnosis. in addition, just before we submit, we found that five of six discharged children returned to the hospital because of positive pcr in their stool, however, their families were all negative. one girl (case ) didn't return to the hospital but isolated in home because she had mild mental symptoms after discharge. although positive results cannot confirm there were live virus in the stool or not. however, for insurance of public health, they were admitted to the hospital again to get clinical observation. interestingly, their onset was later than their families, but the period of positive pcr was longer than adults. we should pay more attention to this phenomenon and study the possible mechanism. several important limitations of this study should be noted. first, the size was small. second, the retrospective study included only of children who were hospitalized in one hospital. but as one of the rare reports in children out hubei province, it's helpful to improve the ability to recognize patients with mild illness. further studies with large multi-center samples are needed. in conclusion, by analyzing confirmed cases of covid- in jinan, shandong province, this study's findings indicate that new control measures should include rapid medical assessment and removal of the case from the home, as well as increased awareness of the importance of protective measures after symptom onset. public health measures such as home isolation should be aimed at minimizing such risk factors when addressing household transmission of serious infections spread through droplet transmission. geneva: world health organization who. coronavirus disease (covid- ) situation reports clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province national health commission of people's republic of china e e faf b ecd df .pdf?ich_args = - _ eae af a e d e ae b b _ _ c c fdec f d f a _c eab a ddd cb f f daf coronavirus pathogenesis fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin epidemiology, genetic recombination, and pathogenesis of coronaviruses infectious diseases. battling sars on the frontlines epidemiology and cause of severe acute respiratory syndrome (sars) in people's republic of china isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and short-term outcomes of patients with sars in the greater toronto area summary of probable sars cases with onset of illness from severe acute respiratory syndrome in children: experience in a regional hospital in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children new and emerging infectious diseases the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china the novel coronavirus ( -ncov) uses the sarscoronavirus receptor ace and the cellular protease tmprss for entry into target cells heart injury signs are associated with higher and earlier mortality in coronavirus disease (covid- ) we thank all patients involved in the study. dr zhang and gai had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. no potential conflict of interest was reported by the author(s). this study was funded by the jinan science and technology bureau [grant number ]. the funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. xiang ma http://orcid.org/ - - - key: cord- - xetsc c authors: drosten, christian title: virus ecology: a gap between detection and prediction date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: xetsc c nan virologists have been surprised by a recent report that has changed our long-standing conception of the ecology of influenza viruses. potentially, we can no longer rely on waterfowl to be the only source of new flu variants, as bats have now been found to harbor influenza viruses whose internal genes share common ancestry with all known influenza a viruses. other genome portions share even older ancestry, while the main surface protein lies within the known diversity of 'usual' influenza a viruses. the appearance of such a vast mixture of genes suggests that more undiscovered flu strains are lurking in bats. for any virus, the identification of a mammalian reservoir is highly relevant because the 'fitness valley' that viruses need to cross for the conquest of new hosts is shallow if the hosts are genetically related. our knowledge of mammalian viruses is fairly opportunistic, focusing on agents of obvious disease in livestock and pets. the range of viruses carried unnoticed by our phylogenetic next of kin may be huge. for instance, wild small mammals including bats and rodents have now been shown to harbor a tremendous spectrum of relatives of human paramyxoviruses-a family that contains the mumps virus, several different respiratory agents and the measles virus. not all of these have yet been proven to have their cognates in bats, but the sample studied so far is just a tiny fraction of the tremendous bat diversity. some of these agents have already been suggested to cross-infect humans. that is a worrying perspective because the concept of liberating humankind from some of its most notorious viruses by mass vaccination is essentially dependent on the absence of animal sources from which eradicated viruses could be replenished. the implications of recent findings might even reach into the future agenda of virus eradication: the hepatitis c virus, one of the most important human viruses and a prime candidate for eradication pending vaccine availability, has relatives in companion animals including dogs and horses. , these and other recent findings remind us of an important issue in viral reservoir ecology: non-persisting viruses are maintained on a social level, requiring large, dense and interconnected host groups for their perpetual transmission. human immunodeficiency virus and its ape reservoir with a rather small group size might have been a decoy rather than a paradigm for this field of research because the virus is able to persist in individuals and depends less on efficient transmission for maintenance. on the contrary, candidates for the next pandemic would be agents that are transmitted efficiently and cause acute disease-such as severe acute respiratory syndrome and flu. the novel human coronavirus emc/ with its connection to bats might establish another recent case. , within the class of mammals, bats form the largest contiguous social groups. their association with pathogenic viruses has been proposed to be due to specific immune functions, but these remain to be proven. large social group sizes and a migratory lifestyle may suffice to make certain bat species become breeders of viruses. the reliance of bat-borne viruses on transmissibility rather than persistence could explain their high onward transmissibility after host-switching. there are prominent examples of bat-borne viruses that can be passed between humans, including ebola virus, marburg virus, nipah virus and the severe acute respiratory syndrome agent. for comparison, we have few examples of rodent-derived viruses that are routinely passed from human to human. lassa virus may be the only relevant exception, and even there, transmission seems to be possible only under conditions of very close contact. apart from certain bat species, there is only one other mammalian species that forms interconnected social groups of more than one million individuals-humans. we may thus provide a familiar environment for bat-borne viruses that are optimized for transmission in large social groups. in the virus-hunting scene, there is now a rush to study bat-borne viruses, doubtlessly triggered by the finding of severe acute respiratory syndrome-related viruses and the conjecture that bat-borne viruses might spark the next pandemic. however, there remains a large gap between the many studies describing novel reservoir-borne viruses and our capabilities to use this knowledge to predict or prevent future human disease outbreaks. this is not to say there is no progress. there are reports emerging of longitudinal and quantitative studies of reservoir-borne viruses showing potential utility for prevention. for instance, very recent work has identified adolescent bats as pronounced carriers of marburg virus in a crowded bat cave in uganda where at least two well-documented human infections have occurred. interestingly, these adolescents are forced to roost in less preferred places close to the cave's entrance-areas preferentially touched and passed by humans visiting the cave. other studies have convincingly shown that the breeding season is a time when several bat-borne viruses are amplified-a situation that is highly similar to a kindergarten where runny noses are commonplace. however, beyond such practical insight, we still know little about the fundamental ecological mechanisms driving virus emergence. the idea that reservoir-borne viruses should exist peacefully with their hosts is most likely not widely valid. as we dig deeper into viral reservoir ecology, including its man-made modifications, we may find that changes in host populations affect the transmission and maintenance of viruses with possible consequences for their potential to infect humans (figure ). for example, analogously to the dilution effect theory, one could expect that either the reduction or expansion of the host group density would allow more virulent virus variants. obviously, the investigations necessary to probe such effects need to be led by ecologists rather than virus hunters. as for virologists, we will contribute little to the prevention of the next pandemic by piling up virus sequences-we need to generate functional insight to further triage among reservoir-borne viruses with regard to their epidemic risks. for example, we can identify bona-fide interferon antagonists in reservoir-borne viruses using sequence homology and systematically test how potent these proteins are at breaking our innate immunity barrier. beyond innate immunity and receptor-mediated cell entry, there are exciting new results from comparative studies of virushost interactions across the family tree of viruses that identify new cellular pathways that can be hijacked by viruses or that suppress their replication. not only can these additional targets be used as tests for viral cross-host compatibility, but their comparison between mammals may also yield targets for cross-host antiviral drugs. such drugs could confer practical pandemic preparedness. attribution-noncommercial-noderivative works . unported license. to view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/ . habitat fragmentation resource abundance change of social structure risk virus replication / transmission duration of excretion / infectivity figure modification of viral maintenance optimum. the maintenance of non-persisting viruses requires a sufficient rate of transmission (x-axis) within the host population or group. a longer duration of excretion or infectivity (y-axis) allows for a lower virus transmission rate while still successfully maintaining the virus. increased transmission correlates with increased replication and virulence. as virulence kills or incapacitates hosts, it limits the duration of infectivity, leaving a limited space in which maintenance can be optimized (triangle). the optimum is virus-specific (gray curve). changes in host ecology can move the maintenance optimum within the limits of the optimization space (dashed curve). those viruses whose maintenance optimum is moved into the red area of the optimization space may pose increased pandemic risks. emerging microbes and infections a distinct lineage of influenza a virus from bats the neuraminidase of bat influenza viruses is not a neuraminidase host species barriers to influenza virus infections bats host major mammalian paramyxoviruses novel potentially-zoonotic paramyxoviruses from the african straw-colored fruit bat, eidolon helvum measles eradication: past is prologue characterization of a canine homolog of hepatitis c virus serology-enabled discovery of genetically diverse hepaciviruses in a new host infectious diseases in primitive societies isolation of a novel coronavirus from a man with pneumonia in saudi arabia human betacoronavirus c emc/ -related viruses in bats, ghana and europe what links bats to emerging infectious diseases? crossing the line: selection and evolution of virulence traits seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection amplification of emerging viruses in a bat colony canine hepacivirus ns serine protease can cleave the human adaptor proteins mavs and trif viral immune modulators perturb the human molecular network by common and unique strategies the deutsche forschungsgemeinschaft (germany's national research council) has established a priority program on ecology and species barriers in emerging viral diseases (coordinator's grant to christian drosten, dr / - ). key: cord- -xx imhzb authors: lambour, jennifer; naranjo-gomez, mar; piechaczyk, marc; pelegrin, mireia title: converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: xx imhzb monoclonal antibodies (mabs), which currently constitute the main class of biotherapeutics, are now recognized as major medical tools that are increasingly being considered to fight severe viral infections. indeed, the number of antiviral mabs developed in recent years has grown exponentially. although their direct effects on viral blunting have been studied in detail, their potential immunomodulatory actions have been overlooked until recently. the ability of antiviral mabs to modulate antiviral immune responses in infected organisms has recently been revealed. more specifically, upon recognition of their cognate antigens, mabs form immune complexes (ics) that can be recognized by the fc receptors expressed on different immune cells of infected individuals. this binding may be followed by the modulation of the host immune responses. harnessing this immunomodulatory property may facilitate improvements in the therapeutic potential of antiviral mabs. this review focuses on the role of ics formed with different viral determinants and mabs in the induction of antiviral immune responses in the context of both passive immunotherapies and vaccination strategies. potential deleterious effects of ics on the host immune response are also discussed. therapeutic potential of antiviral monoclonal antibodies monoclonal antibodies (mabs) have gained an important place in the therapeutic arsenal against severe human diseases. more than mabs have been approved or are under review for human use, and several hundred are currently being tested in the clinic, , most of them to treat patients suffering from a variety of cancers or inflammatory diseases. concerning antiviral mabs, only one, directed against respiratory syncytial virus (rsv), has been approved for the prophylactic treatment of pediatric infections. however, employing mabs as antiviral drugs is under consideration for the treatment of several chronic and acute severe viral infections, especially to address the public health emergencies such as the recent ebola virus and middle east respiratory syndrome coronavirus outbreaks. [ ] [ ] [ ] [ ] [ ] illustrating this trend, the number of antiviral mabs developed and tested in preclinical and clinical trials has grown exponentially in the past years and includes mabs directed against life-threatening agents, such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv), influenza virus, dengue virus, ebola virus and severe acute respiratory syndrome virus coronavirus, among others. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] importantly, recent clinical data have also demonstrated the efficacy of anti-hiv mabs in controlling viremia, when administered to hiv-infected patients, lending strong support to the idea that mabs could broaden the therapeutic arsenal against severe viral infections. , their use as antiviral agents is all the more likely to be considered given that multiple biological activities may account for their therapeutic effects. although a few mabs have been developed to inhibit the recognition of viral receptors or co-receptors on the surface of target cells, most antiviral mabs have been selected for their ability to neutralize virions through the binding of their antigen-binding (fab) fragment to viral surface antigens essential for entry into host cells. however, the biological activity of antibodies is also mediated by the fragment crystallizable region (fc) moiety. thus, it is interesting to note that most antiviral mabs in use are immunoglobulin (ig)-gs displaying a variety of effector functions, including binding to both complement and fcγ receptors (fcγrs). different types of fcγrs are expressed in a regulated manner by many cells of the immune system, including b cells, natural killer cells, dendritic cells (dcs), monocytes/macrophages, granulocytes and mast cells, and their engagement by the fc antibody moiety is essential for regulating the antibody effector functions. , upon recognition of their target antigens, antiviral mabs can facilitate virus elimination via two types of complementmediated mechanisms: (i) inactivation of viral particles and/or phagocytosis of opsonized virus mediated by cells of the innate immune system ( figure ) and (ii) opsonization and subsequent lysis of infected cells, when viral antigens are also expressed on the cell surface (for example, envelope (env) glycoprotein of lentiviruses such as hiv) via complement-dependent cytotoxicity. in addition to complementmediated actions, recognition of fcγrs can entail antibody-dependent cellular phagocytosis and antibody-dependent cell-mediated cytotoxicity ( figure ). , [ ] [ ] [ ] [ ] finally, antiviral mabs also have a role in viral blunting by inhibiting cell-to-cell viral transmission. in addition to controlling the viral propagation by these mechanisms, the opsonization of viral particles and/or infected cells by therapeutic antiviral mabs of the igg type leads to the formation of immune complexes (ics) recognizable by the fcγrs expressed on antigen-presenting cells (apcs) such as dcs. this can potentially affect the endogenous antiviral adaptive immune response of passive immunotherapy-treated individuals. despite the fact that the immunoregulatory functions of antibodies (as well as ics) have been known for a long time, and have been reported in different experimental settings and physiopathological situations, , - the immunomodulatory role of mabs with clinical potential as antiviral drugs has only recently been considered. this review mainly focuses on the induction of antiviral immune responses by ics in both passive immunotherapies and vaccination strategies. the potential deleterious effects of antiviral antibodies on the host immune dysfunction and/or viral propagation are also discussed. only recently have studies addressed whether and how passive immunotherapies utilizing antiviral mabs are able to enhance the antiviral immunity in infected individuals. this is largely due to the limited availability of suitable immunocompetent animal models of viral infection that allow in-depth investigations of the endogenous immune response. the concept that passive immunotherapies utilizing antiviral mabs can induce long-term protective immunity has recently been established using an immunocompetent mouse model, consisting of short immunotherapies given to young animals infected with the frcase murine leukemia virus. the induction of such 'vaccine-like' effects by antiviral mabs, as well as some of the mechanisms involved, are reviewed in detail elsewhere. in brief, the inoculation of mouse pups with frcase is fatal because the antiviral immune response is too weak to control the viral propagation. in contrast, treatment with a neutralizing mab for several days shortly after infection blunts viral propagation and induces a lifelong protective antiviral immunity composed of both a highly neutralizing humoral response and a cytotoxic cd + t-cell response. [ ] [ ] [ ] [ ] [ ] [ ] this induction of protective immunity strictly depends on the fc fragment of the neutralizing mabs. , moreover, the formation of ics composed of the administered mabs and infected cells rather than virions is crucial for the enhanced antiviral immune response. such ics are recognized by the fcγrs expressed by dcs, which facilitate ics internalization and lead to stronger activation and more efficient antigen presentation by dcs, eventually leading to stronger cytotoxic t-lymphocyte (ctl) responses. an fc-mediated effect that occurs concurrently is the inhibition of regulatory t-cell (treg) expansion. this depends on the mab effector functions and occurs rapidly. moreover, it is necessary for the development of the protective humoral and cellular responses, as treg-mediated immunosuppression is observed in all cases of chronic viral infections, where it dampens antiviral immune responses, thereby permitting the establishment of chronicity. finally, breastfeeding and placental transfer of maternal anti-frcase igs induced by mab immunotherapy not only led to the viral propagation blunting in infected pups, but also to the induction of long-lasting protective humoral immunity in these animals. this is a particularly interesting observation when one considers that the frcase model is reminiscent of perinatal infection by hiv, including breastfeeding-mediated mother-to-child virus transmission. other evidence for the induction of 'vaccine-like' effects by antiviral mabs comes from studies in several preclinical models of human viral infections and from hiv-infected patients. in a mouse model of rsv infection, the administration of a neutralizing mab directed against the virus attachment protein g induced a shift in the adaptive immune response from th -to th -type, leading to sustained and enhanced humoral and cd + t-cell responses. however, this effect was not fc-dependent, but rather due to the ability of the therapeutic mab to counteract the intrinsic immunosuppressive activity of the rsv g protein. mab-driven enhancement of the humoral response has also been reported in two preclinical models of henipavirus infection in african green monkeys. , recovery from both hendra and nipah virusinduced disease correlated with the development of host antibody responses consequent to the administration of the highly neutralizing . mab. this hendra and nipah virus cross-reactive mab is currently being considered for human use. finally, anti-hiv antibodies can modulate immune responses in infected organisms. such effects were initially reported in several nonhuman primate models of hiv infection and then observed in infected humans. macaques were infected with different strains of simian immunodeficiency virus (siv) or simian hiv (shiv, a chimeric virus in which hiv env substitutes for that of siv and allows for the assessment of the antiviral effects of anti-hiv antibodies) following different protocols. these experiments showed that the administration of highly neutralizing antibodies (either mabs or polyclonal igs) enhanced both the humoral and cellular antiviral immune responses of treated animals. , [ ] [ ] [ ] interestingly, recent clinical data describe the elicitation of host humoral responses in viremic subjects upon single injection of the potent bnc anti-hiv mab. however, the mechanisms leading to the stimulation of antiviral immune responses in these preclinical models of hiv infection or in infected patients remain uncharacterized. moreover, it is unknown whether these antiviral responses have genuine protective vaccine-like effects. in any case, these important observations open new avenues for the improvement of mab-based antiviral hiv therapies. moreover, as the in vivo activity of anti-hiv- bnabs, including viral load control, was recently shown to crucially depend on fc effector functions, , an important issue is identifying that fc-fcγrs interactions are involved in the induction of vaccinelike effects by antiviral mabs. to understand the mechanisms underlying the enhancement of antiviral responses by ics, several in vitro studies have addressed whether antibody-mediated viral uptake by dcs could lead to stronger activation of these cells and the development of stronger virus-specific cd + and cd + t-cell responses in an fc-dependent manner. such an increase in the cellular immune response has been reported in different infectious settings using ics made with different types of antigens, including recombinant viral proteins and whole virions, as well as infected cells (table ) . concerning ics made with viral proteins, several reports have shown that ics made up of anti-hbv mabs and the hepatitis b surface antigen (hbsag) can affect dc function and enhance t-cell responses. hbsag/anti-hbv ics significantly increased the uptake of the immunocomplexed hbsag antigen, and augmented the in vitro proliferation of virus-specific t cells and their production of interferon (ifn)-γ. moreover, dcs from hbv-infected patients incubated with hbsag/anti-hbv ics showed higher expression of major histocompatibility complex (mhc)-ii molecules and higher production of interleukin (il)- . ic-loaded dcs also enhanced production of il- and ifn-γ by co-cultured t cells. interestingly, the therapeutic efficacy of hbsag/anti-hbv ics has been tested in clinical trials (see below) in hbv-infected patients with encouraging results. [ ] [ ] [ ] more recently, in experiments aimed at visualizing immunopotentialization by hbsag/anti-hbv ics (see below), live-cell imaging revealed that ics were internalized via the fcγrs of apcs and were subsequently transported through early and late endosomes into lysosomes, where they co-localized with mhc-i and mhc-ii molecules. consistent with the latter observation, the administration of dcs loaded with hbsag/anti-hbv ics to mice increased the number of ifn-γ-and tumor necrosis factor-α-producing cd + and cd + t cells. similarly, in an siv infection setting, the incubation of apcs with ics made with a recombinant full-length gag p protein and an anti-p igg increased siv capsid cross-presentation. capsid cross-presentation was dependent on fcγr-mediated uptake of the immunocomplexed siv capsid protein, and required its proteasomal and endosomal degradation to generate stronger gag-specific cd + t-cell responses. from a mechanistic standpoint, these studies indicate that antiviral antibodies might enhance the priming and expansion of virus-specific cd + and cd + t cells by both promoting the secretion of key cytokines and facilitating the uptake and crosspresentation of viral ags by fcγr-expressing dcs. immune-complexed whole virions have also been shown to affect the functional activation of dcs. the stimulation of dcs with ics composed of siv virions and highly neutralizing siv-hyperimmune sera (svig) led to the increased virus-specific cd + t-cell responses in an fc-dependent manner. in contrast, dcs stimulated with ics made of hiv- and a polyclonal igg pool from hiv-infected subjects showed only weak hiv-specific ctl-stimulating activity. this suggested that opsonization of hiv- by iggs might be associated with decreased ctl-stimulatory dc activity. however, not all igg isotypes display equivalent effector functions. therefore, the undefined nature of the antibodies (both in terms of predominant isotypes and neutralization potential) used to form the hiv-ics in these experiments might explain these observations. whether hiv-ics made with highly neutralizing anti-hiv mabs of a specific igg isotype might induce stronger cd + t-cell responses is an important issue deserving further investigation (figure ) . moreover, the nature of the viral determinant present in ics might also be crucial in the stimulation of antiviral responses. interestingly, as mentioned above, in the mouse frcase infection model, ics made up of a neutralizing mab and infected cells, but not those made with virions, efficiently induce strong gag-specific cd + t-cell responses with high cytotoxic activity. this observation shows that the viral and cellular ics can trigger different immunologic outcomes. in the case of frcase, this is explained by the fact the frcase-gagl ctl immunodominant epitope is, at best, poorly incorporated into virions. taken together, these data indicate that the uptake of cellular ics might allow the presentation of a broader viral antigenic repertoire, leading to stronger effects on immunity ( figure ). ics formed with endogenous antibodies generated in virally infected mice have been shown to influence antiviral cellular immune responses in several models (table ) . notably, the highly neutralizing humoral response generated against the frcase retrovirus in mab-treated-infected mice was demonstrated to limit the viral propagation and to enhance memory cellular responses long after the disappearance of the therapeutic mab (which occurs within two weeks post administration, reflecting the natural igg lifespan in vivo). ic-mediated activation of dcs upon binding to fcγr was key for this effect. similarly, in an influenza virus infection model, ics formed with endogenous antiviral antibodies promoted more sustained antigen presentation by dcs, resulting in stronger cd + t-cell proliferation. interestingly, such prolonged antigen presentation by dcs was dependent on virus-specific, isotype-switched antibodies that facilitated the capture and cross-presentation of viral antigens by figure parameters to consider for achieving optimal ic-mediated modulation of antiviral immune responses. the optimization of vaccine-likeeffect-inducing protocols will require the consideration of several parameters such as the nature of the antigen (that is, purified viral proteins, whole virions and infected cells) and the antibody (that is, monoclonal vs polyclonal, nature of the isotype, engineered fc domain with improved effector functions and so on) used to form the immunogenic ics, as differences in these parameters might impact immunological outcomes. in addition, whether the optimized ics are used alone or in combination with immunostimulatory molecules might also be of paramount importance. several other parameters, including the ic dosage, the route of administration, the choice of adjuvant and the immunological status of patients, will also have to be considered. fcγr-expressing dcs. in addition, serum antibodies can affect the virus-specific cd + /cd + t-cell balance in an fc-dependent manner during rsv infection. an enhanced ratio of rsv-neutralizing to -non-neutralizing antibodies profoundly enhanced the cd + t-cell response. in a murine lymphocytic choriomeningitis virus (lcmv) infection model, endogenous virus-specific antibodies could stimulate innate immunity and thereby positively affect both the induction and the maintenance of the virus-specific cd + t-cell response. notably, anti-lcmv antibodies limited viral replication in peripheral organs, but allowed replication of the virus in the marginal zone of the spleen, promoting cd + t-cell priming. interestingly, anti-lcmv antibodies were also reported to be essential for long-term maintenance of the memory ctl response in infected mice. , these observations, together with the in vitro studies described above, demonstrate that virus-specific antibodies can promote the acquisition, processing and presentation of antigens that are subsequently instrumental for priming t-cell responses and programming functional cd + memory in an fc-dependent manner. they strengthen the concept that antiviral antibodies can regulate the quality and function of antiviral t-cell responses through the formation of ics. moreover, they also provide a rationale for developing novel ic-based therapeutic vaccination strategies. in , terres and wolins demonstrated the ability of ics to induce higher antibody titers than antigens alone. since then, the immunogenic potential of ics, alone or in combination with different types of adjuvants, has been tested in several viral infection systems, including animal models of human infections, for example, those involving hbv, hiv, rsv or flaviviruses. the immunostimulatory effects of ics are principally attributed to the ability of fc antibody fragments to recruit the host immune system. however, evidence also implicates fab fragments in modulation of the antiviral immune response, although the outcomes are less documented and were proposed to occur via alterations in antigen conformation and/or in the exposure of specific epitopes. we describe the enhancement in antiviral immune responses observed in ic-based vaccination experiments below (table ) . ics have been tested as vaccines to augment protective immune responses in different animal models of hbv infection. in ducks, ics made with duck hbsag and rabbit anti-duck hbsag (dhbsag/ anti-dhb) were used as immunogens in the form of solid matrixantibody-antigen complexes (smaa). such smaas contained killed staphylococcus aureus as a solid matrix and mab-opsonized viruses. they were initially shown to induce both humoral and ctl responses against the paramyxovirus simian virus in immunized mice. immunization of hbv-infected ducks with smaa-based dhbsag/anti-dhb ics led to the viral clearance in % of infected ducks. notably, the administration of dhbsag/anti-dhb ics lacking staphylococcus aureus showed decreased immunization efficiency, suggesting that the bacteria-based solid matrix functions as an adjuvant. ics have also been tested as a therapeutic vaccine against hbv infection in mice , , and woodchucks. balb/c mice immunized with hbsag/anti-hbv ics produced the increased levels of virusspecific antibodies. moreover, administering hbsag/anti-hbv ics to balb/c mice via intranasal inhalation induced both mucosal and systemic th -polarized immune responses, when administered with adjuvants such as cholera toxin or oligodeoxynucleotides containing modulation of antiviral immune responses by immune complexes j lambour et al immunostimulatory cpg motifs (cpg). this was not observed using hbsag alone. in addition, the administration of hbsag/anti-hbv ics to hbsag-positive transgenic mice decreased the serum hbsag levels and induced stronger ctl responses than hbsag alone. notably, the co-administration of ics and a plasmid coding for hbsag increased the antiviral immune response induced by ics, indicating the adjuvant effect of dna in this setting. a similar effect was also reported in a woodchuck model of hbv infection: immunization of woodchuck hepatitis virus (whv)-infected animals with whv surface antigen/anti-whv antibody ics combined with a dna vaccine resulted in a higher reduction of both viral load and antigenemia relative to ics alone. interestingly, the whv-infected animals were pretreated with lamivudine (a potent hbv antiviral drug able to enhance t-cell responses in chronically hbv-infected patients) before ic/dna immunization, suggesting that combination strategies should be considered in treating chronic hbv infections (figure ). the enhancement of antiviral immune responses by ics in vitro and in preclinical models of hbv infection paved the way for the development of ic-based therapeutic vaccination strategies against chronic viral hepatitis b infection. a therapeutic vaccine composed of yeast-derived recombinant hbsag/anti-hbv immunogenic complexes (yics) has been tested in a series of clinical trials. this vaccine approach was initially shown to be safe and to induce higher titers of hbsag antibodies, as well as to increase serum ifn-γ and il- levels in a phase i trial. importantly, a subpopulation of chronic viral hepatitis b patients showed a decrease in serum hbv viral load and hbsag levels together with an increase in anti-hbsag antibody titers in subsequent phase ii trials. , from a mechanistic standpoint, recent data showing that the administration of yic-loaded dcs to mice increased both cd + and cd + t-cell responses suggest that the improved immune responses induced by yics might account for the antiviral effect observed in a fraction of patients. in an attempt to enhance the immunogenic potential of yic-based vaccines, a phase iii trial tested the therapeutic effect of a higher number of ic doses. unfortunately, overstimulation with yic decreased the vaccine efficiency due to host immune fatigue. this suggests that vaccination protocols must be optimized and must take into account both the nature and the dose of ics, as well as other parameters such as the route of administration, the type of adjuvant and the immunological status of patients to achieve efficient protective immunization ( figure ). in , a study immunized healthy volunteers with hiv peptides. the authors found that compared with free antigen, recall immunization with ics induced stronger t-cell responses through uncharacterized mechanisms. other reports also describe alteration of the anti-hiv response by ics. [ ] [ ] [ ] in particular, the immunization of immunocompetent mice with ics containing a recombinant hiv- gp env glycoprotein and a mab ( -d mab) directed to the cd -binding site induced a higher virus-neutralizing antibody response relative to free antigen. as described above, humoral responses were further increased upon the co-administration of ics and monophosphoryl lipid-a/dimethyldioctadecylammonium adjuvants. notably, the interaction of the anti-cd -binding site mab with hiv- gp induced conformational changes in the latter, leading to the enhanced antigenicity and immunogenicity of neutralizing epitopes localized in the hiv- v loop. these observations highlight the ability of anti-hiv- antibodies to induce antigenic alterations in specific hiv- gp epitopes upon ic formation. interestingly, further improvement in the immunogenicity of the v loop was obtained in ics generated with gp mutants lacking site-specific n-linked glycans. , , taken together, these observations suggest that the ability of ics to stimulate the induction of neutralizing antibodies is dictated by the nature of the antigen, as well as the specificity and affinity of the mabs utilized. these results also indicate the potential contribution of fab-mediated activities in the enhancement of antiviral humoral responses by ics. tsouchnikas et al. investigated the influence of immunization with ics on the specificity of antibody responses using the e protein of the tick-borne encephalitis virus as an immunogen. mice were immunized with a dimeric soluble form of e (se) alone or in complex with mabs specific for each of the three domains of e. the antibody response induced by these ics was compared with that observed after immunization with se alone. unexpectedly, immunization with ics did not change the extent of the overall antibody response in immunized mice. however, substantially different antibody responses were observed between the different ics. these differences most likely reflected an epitope-shielding phenomenon and antibody-mediated structural changes that led to the dissociation of the se dimer. thus, such phenomena can profoundly influence the fine specificity of antibody responses to the same immunogen and must be considered in ic-based vaccination strategies. as mentioned above, serum anti-rsv antibodies can affect virusspecific t-cell responses. on the basis of this, kruijsen et al. tested whether ics made with the commercial rsv-neutralizing mab palivizumab could influence adaptive immune response priming after intranasal administration. substantial anti-rsv t-cell priming and b-cell responses were observed in mice receiving rsv-ics, resulting in predominant th -type cd + t-cell response and igg c antibody responses. importantly, the ics also primed anti-rsv cd + t cells. these data have important implications for the prophylaxis and treatment of pediatric rsv infections. nevertheless, interactions between ics and neonatal versus adult innate and adaptive immune systems still need to be investigated because mouse studies have revealed potential antibody-induced neonatal autoimmunity in certain settings. , ics and immune dysfunction in the course of viral infections, the formation of ics composed of viral determinants and the resulting host humoral responses can potentially produce deleterious effects. persistent ics are formed in a variety of chronic viral infections and may lead to unregulated and protracted fcγr signaling. this may lead to immune dysfunction instead of stimulating antiviral immune responses. in this regard, the high levels of ics formed during lcmv infection interfere with fcγr-mediated activities. , these endogenously formed ics were shown to outcompete the effector functions of exogenously administered therapeutic mabs, in particular binding to fcγrs expressed by immune cells. persistent endogenous ics are also linked to dysfunctional b-cell responses in hiv infection, including the suppression of antiviral iga responses and impaired production of neutralizing antibodies (reviewed in moir et al. ). the composition of ics might also negatively affect the efficiency of the antiviral immune response. for instance, the composition of ics has been shown to be dynamic throughout the course of hiv infections due to changes in both antibody specificities and virion levels. notably, circulating ics are initially comprised of antibodies that opsonize both infectious and non-infectious virions. this results in a decrease in the availability of antibodies able to blunt viral propagation. this phenomenon probably contributes to the reduced efficiency of the antibodies generated during acute infection. changes in circulating ics have also been reported in hcv infections. the level of circulating ics is low in acutely infected patients, whereas chronically infected individuals show a high proportion of immunocomplexed hcv, raising the possibility that ics may have a role in the pathogenesis of hcv, namely liver damage. moreover, the formation of ics with non-neutralizing antibodies may also lead to the antibody-dependent enhancement of viral infection of fcγr-expressing cells. this happens in several viral infections, including those by the dengue virus. [ ] [ ] [ ] [ ] along this line, the binding of ics to fcγrs on monocytes/macrophages can paradoxically suppress innate immunity, induce il- production and bias responses from th toward th . this in turn leads to the increased infectious outputs by infected cells via intrinsic antibodydependent enhancement. , finally, ics have also been reported to have a role in increasing viral loads in the context of gene transfer-based vaccination strategies. in the step hiv- vaccine trial, which evaluated a replication-defective adenovirus type vector vaccine, the ics formed with pre-existing anti-adv antibodies improved the environment for hiv- replication in t cells. this may have been due to the ic-driven activation of a dc-t-cell axis that induces the activation of cd + t cells and leads to a permissive environment for hiv- infection. this environment probably explains the increased propagation of hiv- infection among adenovirus type -seropositive vaccine recipients. several approaches can be considered to enhance the immunomodulatory potential of antiviral mabs, both alone and in the form of ics, in particular through combining neutralizing mabs and ic-based vaccination strategies with other therapies. a first possibility would consist of inhibiting immunosuppressive mechanisms in infected individuals by either depleting the treg response, as suggested by nasser et al. or targeting immune checkpoints, the latter strategy having already led to improved immune responses against both viral infections and cancer. [ ] [ ] [ ] [ ] in addition, the combination of antiviral mabs with different immunostimulatory agents can also be envisaged. because the primary structure and glycosylation pattern of the fc fragment are both essential for antibody effector functions due to their impact on the engagement of type i and type ii fcr family members, , fc engineering might also represent another approach, not only to improve direct antiviral effects, but also to induce stronger vaccine-like effects. in this regard, identification of the main fcrs and fcr-mediated mechanisms involved in enhancing the antiviral immune response will be of utmost importance. taking into account that the various igg isotypes display different effector functions and interact differently with fcγrs, the careful selection of antiviral mab subclasses is also crucial for enhancing antiviral immune responses. finally, as fcγr polymorphisms have already been associated with differences in viral disease progression and the therapeutic efficiency of anticancer mabs, it will be important to evaluate the extent to which such polymorphisms can affect the vaccine-like effects induced by mab-based antiviral immunotherapies. the therapeutic potential of antiviral mabs is now widely accepted, and their use as antiviral drugs is increasingly under consideration. the diverse biological activities of these mabs lead to the direct control of viral propagation and the modulation of antiviral immunity. this provides a novel rationale for their use in diverse prophylactic and therapeutic approaches. the improvement in both humoral and cellular responses achieved through the administration of mabs, either free or in the form of immunogenic ics, offers new therapeutic options. the challenge now is to improve our understanding of how ics convert mab-based immunotherapies from 'passive' to 'active' and to exploit the underlying mechanisms. this conversion will be crucial in reaching the goal of using antiviral mabs to induce 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production in monocytes activation of a dendritic cell-t cell axis by ad immune complexes creates an improved environment for replication of hiv in t cells restoration of hbv-specific cd + t cell function by pd- blockade in inactive carrier patients is linked to t cell differentiation a randomized, double-blind, placebo-controlled assessment of bms- , a fully human monoclonal antibody to programmed death- (pd- ), in patients with chronic hepatitis c virus infection immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential augmentation of hepatitis b virusspecific cellular immunity with programmed death receptor- /programmed death receptor-l blockade in hepatitis b virus and hiv/hepatitis b virus coinfected patients treated with adefovir key: cord- -vfq ft authors: ai, jing-wen; zhang, yi; zhang, hao-cheng; xu, teng; zhang, wen-hong title: era of molecular diagnosis for pathogen identification of unexplained pneumonia, lessons to be learned date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: vfq ft unexplained pneumonia (up) caused by a novel coronavirus sars-cov- (severe acute respiratory syndrome coronavirus ) emerged in china in late december and has infected more than cases by january . shanghai reported the first imported case of covid- (coronavirus disease ) in january . a combinative approach of real-time rt–pcr, crispr-based assay and metagenomic next-generation sequencing (mngs) were used to diagnose this unexplained pneumonia patient. real-time rt–pcr and crispr-based assay both reported positive. this sample belonged to betacoronavirus and shared a more than % nucleotide (nt) identity with the wuhan sars-cov- isolates. we further compared pros and cons of common molecular diagnostics in up. in this study, we illustrated the importance of combining molecular diagnostics to rule out common pathogens and performed mngs to obtain unbiased potential pathogen result for the diagnosis of up. unexplained pneumonia (up) was defined as pneumonia without confirmed diagnosis. in late december , chinese social media first released news concerning the possible severe acute respiratory syndrome (sars) virus infected cases in wuhan. on december , the health commission of hubei province, china announced a cluster of unexplained pneumonia (up) and later a novel coronavirus sars-cov- as named by the who was declared to be the causative agent [ , ] . during the following month, multiple sars-cov- cases were gradually reported all over china and around definite cases were declared by january [ ] . as of today, case reports are still escalating on a daily basis. the reasons behind this outbreak are numerous, the highly infectious nature of sars-cov- , limited pathogen detection method for unexplained pneumonia, the high population density of the hubei province and across china, etc. various diagnostic methods were used during the initial search of this novel virus, yet their pros and cons have not been discussed thoroughly. on january, shanghai reported the first imported case of sars-cov- infection, and through this case, we seek a combinative approach of selected techniques to improve pathogen identification of unexplained pneumonia in the future. the patient was a years old woman and came to shanghai from wuhan on january. she went to the fever clinic of a local hospital due to fever and fatigue. the patient had relatively lower lymphocyte count. under the close collaboration with the shanghai cdc, we first performed filmarray multiplex pcr respiratory panel . (biomerieux, utah, usa) with negative results. we then performed real-time rt-pcr, crispr-based assay and metagenomic next-generation sequencing (mngs) on her respiratory sample and finally diagnosed her with covid- . total rna was extracted from the patient's pharyngeal swab using qiaamp viral rna mini kit (qia-gen, germany). we performed real-time pcr for this patient as described [ ] . crispr-ncov was performed using crispr-ncov kit from vision medicals, china. the rna sample was also prepared for highthroughput mngs. the qualified library was sequenced on an illumina nextseq using a single-end mode ( × bp). low-quality reads and reads derived from human genome sequences were removed. after that, de novo assembly was done using spades. genomes of coronaviruses and sars-cov- genomes released by china were downloaded from ncbi for phylogenetic analysis. phylogenetic tree was built according to the n gene using multiple sequence alignment algorithm clustalx [ ] . phylogenetic tree was then built through mega x [ ] . meanwhile, we downloaded a total of β family coronavirus genomes from ncbi and used pyani (https://pypi.org/project/pyani/) for homology analysis. a positive result of sars-cov- was reported by rt-pcr. as illustrated from figure (a), ct value was . while that of the positive control was . . crispr-ncov was also positive with a foldchange value of compared to the negative control. as shown in figure (b), the sequence homology among the sample and other coronaviruses were assessed. classified as subgenus sarbecovirus of the genus betacoronavirus, this sample shared a great nucleotide (nt) identity (> %) with the two sars-cov- isolates and was % identical with that of sars-cov- betacov/wuhan/ipbcams-wh- / (genbank mt . ). by contrast, a substantially lower nt identity of % was found with sars-cov (genbank nc . ). the genome mapping coverage with sars-cov- wuhan-hu- (genbank mn . ) was . % and . x in depth ( figure (c) ). in analysis of complete genome similarity in figure (d), we revealed a high similarity between this sample and two sars-cov- isolates. ever since the diagnosis of pathogen reached the era of molecular approaches, a variety of methods have showed to increase diagnostic efficacy. in this particular case, rt-pcr, crispr, and metagenomics sequencing have all exhibited their potential in the rapid diagnosis of newly emerging microorganism. however, lessons should be learned from this outbreak. acute viral respiratory tract infection is one of the major causes of unexplained pneumonia, especially in winter. culture, as the widely recognized gold standard, is time-consuming and labour intensive. conventional diagnostic methods such as direct/indirect immunofluorescence assay (ifa) and rapid antigen detection also have their own significant limitations. what's more, various culture-independent nucleic acid amplification tests (naats) and point-of-care tests (poct) could aid the diagnosis of unexplained pneumonia, including polymerase chain reaction (pcr), loop-mediated isothermal amplification (lamp), clustered regularly interspaced short palindromic repeats (crispr), etc. [ ] . however, these methods were not able to detect novel pathogens as in the case of hubei province. the latest multiplex respiratory virus infections assay includes xpert xpress flu/rsv assay (genexpert system, cepheid, sunnyvale, ca, usa), qiagen resplex ii v . kit and fil-marray multiplex pcr system [ ] . these methods offered better diagnostic rates for up by broadening pathogen coverage. despite the lack of capability when it comes to identifying novel pathogens. these syndromic tests are still valuable for ruling out the common pathogens in the first-line diagnosis of unexplained pneumonia, as reported in the articles revolving around the sars-cov- outbreaks. at present, several companies have developed and produced virus assays (supplementary material) for sars-cov- and its typical betacoronavirus organization consists of a ′ untranslated region (utr), replicase complex (orf ab), s gene, e gene, m gene, n gene, ′ utr, and several unidentified nonstructural open reading frames [ ] . the current nucleic acid amplification tests methods mainly targeted the open reading frames of the replicase complex (orf ab), s, e, m and n genes. besides various naats, a new rapid antigen detection assay has also been developed. in order to put diagnostic assays into use as soon as possible, hubei province has adopted a trial-and-request approach, which allows trials at medical institutions and review procedures after epidemic situation is resolved. shotgun metagenomics sequencing including shortread and long-read sequencing could obtain genomic data from both known and novel pathogens. mngs has been used in investigations of covid- outbreaks after exclusion of common pathogens by naats [ ] [ ] [ ] . current bioinformatic pipeline for metagenomics works by mapping the sequenced reads to an existing database of previously known microbes. this approach may result in partial alignment of a novel pathogen genome to that of some previously identified microbes and lead to incorrect taxonomic classification [ ] . in fact, during the sars-cov- outbreak, one of the first news releases came from a report of commercial sequencing company stating their identification of sars virus, causing wide panic among society. and only later after careful analysis on more sequencing data, the causative agent was found to be a novel species. the advantage of metagenomic sequencing lies in its unbiased nature which enables it to play a crucial role in the future diagnosis of the unexplained pneumonia, especially when encountering untypical causative pathogen. the alert should be raised that results from metagenomic sequencing should always be interpreted with the consultant of medical professionals, especially when dealing with a potentially novel pathogen or clustered cases. in the present case, the combination of rt-pcr, crispr and mngs confirmed our clinical diagnosis for covid- . this cross-validation was crucial at the early stage of the outbreak when our understandings on the novel pathogen were limited. for instance, binding of certain primers might be abrupted by genetic variants in the viral genome, resulting in false negative results. moreover, by harnessing an unbiased metagenomic assay, the clinicians were also granted with information on potential concomitant infections to guide more appropriate treatment. looking forward, as the crispr-based techniques further mature, we envisage its broader clinical uses for targeted, rapid pathogen detection especially in under-resourced settings where complicated pcr instruments may not be available. despite its relatively longer turn-around time, metagenomics holds great promises as a unique diagnostic, especially for patients with acute or severe infections. all these three methods consist of an identical rna extraction procedure, which takes about an hour. the residual workflow of rt-pcr and crispr is library preparation and amplification, while the residual workflow of mngs breaking into several part: library preparation ( h), mngs sequencing ( h) and data analysis ( h). thus, turn-around time (tat) of rt-pcr, crispr and mngs is about , and h, respectively. rna extraction is a necessity, the experimenters need to expose to the samples directly in the biohazard safety equipment with tertiary protection. we suppose there is no obvious difference in safety among these approaches. we further compared tat, diagnostic efficiency and detected pathogens of updated diagnostic methods in up in figure (e) [ , ] . in the future, pros and cons of each molecular diagnostics should be thoroughly taken into consideration by the physicians to reach a more informational and accurate clinical diagnosis. here we reported the first definite case of covid- in shanghai, in which the virus was detected by employing various methods. this case illustrated the importance of combining targeted naats to rule out common pathogens and metagenomic assay to obtain unbiased genomic information on the pathogen for diagnosis of up. a novel coronavirus from patients with pneumonia in china wuhan wet market closes amid pneumonia outbreak. china daily clinical features of patients infected with novel coronavirus in wuhan multiple sequence alignment with the clustal series of programs mega x: molecular evolutionary genetics analysis across computing platforms recent advances in the detection of respiratory virus infection in humans a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china rna based mngs approach identifies a novel human coronavirus from two individual pneumonia cases in wuhan outbreak clinical metagenomic sequencing for diagnosis of meningitis and encephalitis comparison of the filmarray respiratory panel and prodesse realtime pcr assays for detection of respiratory pathogens we acknowledge shanghai cdc for their efforts and hard works during this case. no potential conflict of interest was reported by the author(s). we have uploaded the assembled sequence to gisaid as epi_isl_ . key: cord- -elartlro authors: sun, jing; zhu, airu; li, heying; zheng, kui; zhuang, zhen; chen, zhao; shi, yongxia; zhang, zhaoyong; chen, si-bei; liu, xuesong; dai, jun; li, xiaobo; huang, shuxiang; huang, xiaofang; luo, ling; wen, liyan; zhuo, jianfen; li, yuming; wang, yanqun; zhang, lu; zhang, yanjun; li, fang; feng, liqiang; chen, xinwen; zhong, nanshan; yang, zifeng; huang, jicheng; zhao, jincun; li, yi-min title: isolation of infectious sars-cov- from urine of a covid- patient date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: elartlro sars-cov- caused a major outbreak of severe pneumonia (covid- ) in humans. viral rna was detected in multiple organs in covid- patients. however, infectious sars-cov- was only isolated from respiratory specimens. here, infectious sars-cov- was successfully isolated from urine of a covid- patient. the virus isolated could infect new susceptible cells and was recognized by its’ own patient sera. appropriate precautions should be taken to avoid transmission from urine. a novel coronavirus sars-cov- emerged to cause a major outbreak of severe pneumonia in humans in china and has spread to over other countries [ ] . as of april th , , , cases with , deaths ( . % case fatality rate) had been reported to the world health organization (who). the major transmission routes are believed to be exposure to virus-containing droplets or contaminated fomites [ ] . although viral rna can be detected in multiple organs in covid- patients, infectious sars-cov- has only been isolated from respiratory specimens [ , ] . since the viral receptor, angiotensin-convertase- (ace ) protein, is expressed in the kidney, the testis and the bladder [ , ] and viral rna has been detected in the sink and toilet of covid- patients, the virus may be secreted through the urinary system [ ] . however, it is currently unclear if infectious virus is secreted in urine. on january , a -year-old man who had a history of recent travel to wuhan was admitted to the eighth people's hospital of guangzhou medical university with cough and fever. he was then transferred to the first affiliated hospital of guangzhou medical university ( figure a ). oropharyngeal swabs were positive for sars-cov- rna by rt-pcr on january th. computed tomography (ct) scan of the chest revealed multiple ground-glass opacities in both lungs as shown in figure s . on february rd, the patient's condition deteriorated and he was intubated and put on a ventilator. the urine sample tested positive for sars-cov- rna on day post infection (p.i.) (february th) for the first time and had periodically showed positive results in rt-pcr test until march th. rt-pcr positive urine specimens (ct ) from day p.i. was serially diluted in infection media and inoculated onto vero e cells. cytopathic effects (cpe) were clearly observed after days ( figure b ). full-length viral genome sequence was acquired by next-generation sequencing (accession number mt ) with nucleotide mutations as compared to the original wuhan viral stain (accession number nc . ) (table s ). cell culture supernatant was negatively stained and visualized by transmission electron microscopy (tem). spherical-shaped particles with distinct surface projections, resembling spikes, that were consistent with sars-cov- virions as published previously ( figure c ) were observed [ ] . viral load in respiratory specimens and urine were quantitated by rt-pcr. viral loads decreased over time in oropharyngeal swabs. viral load in urine was low but detectable at day and day p.i., but not at day ( figure d) . the patient had a high level of sars-cov- -specific igm at early time points and declined over time while the igg antibodies remained relatively stable ( figure e ). in order to prove that the isolated virus was infectious to susceptible cells, fresh vero e cells was inoculated with the virus and stained in an immunofluorescent assay (ifa) using serum obtained from the patient and from a healthy donor. results showed positive fluorescence with patient's serum but not with healthy control serum ( figure f ). although it is hard to determine whether the kidney, the testis or the bladder were infected and produced infectious virus from current study, isolation of infectious sars-cov- in urine raises the possibility of fecal/urine-respiratory transmission. in the severe acute respiratory syndrome (sars) epidemic, patients were infected resulting in deaths in a community called amoy gardens in hong kong [ ] . investigation of the building structure showed that faulty sewage pipelines led to aerosolization of feces and urine contaminated with sars-cov, which infected almost people in the apartment complex. these findings raise the importance of using appropriate precautions to avoid transmission from urine. a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical characteristics of coronavirus disease in china viral load of sars-cov- in clinical samples single-cell rna-seq data analysis on the receptor ace expression reveals the potential risk of different human organs vulnerable to -ncov infection. front med ace expression in kidney and testis may cause kidney and testis damage after -ncov infection characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding evidence of airborne transmission of the severe acute respiratory syndrome virus we thank the patient who took part in this study. no potential conflict of interest was reported by the author(s). this study was approved by the health commission of guangdong province to use patient's specimen for this study. this study was funded by grants from the national key research and development program of china ( yfc ), national science and technology major project ( zx ), the emergency grants for prevention and control of sars-cov- of ministry of science and technology ( yfc ) and guangdong province ( b , b ). key: cord- - e u aza authors: tian, xiaolong; li, cheng; huang, ailing; xia, shuai; lu, sicong; shi, zhengli; lu, lu; jiang, shibo; yang, zhenlin; wu, yanling; ying, tianlei title: potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: e u aza the newly identified novel coronavirus ( -ncov) has caused more than , laboratory-confirmed human infections, including deaths, posing a serious threat to human health. currently, however, there is no specific antiviral treatment or vaccine. considering the relatively high identity of receptor-binding domain (rbd) in -ncov and sars-cov, it is urgent to assess the cross-reactivity of anti-sars cov antibodies with -ncov spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against -ncov. here, we report for the first time that a sars-cov-specific human monoclonal antibody, cr , could bind potently with -ncov rbd (kd of . nm). the epitope of cr does not overlap with the ace binding site within -ncov rbd. these results suggest that cr may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of -ncov infections. interestingly, some of the most potent sars-cov-specific neutralizing antibodies (e.g. m , cr ) that target the ace binding site of sars-cov failed to bind -ncov spike protein, implying that the difference in the rbd of sars-cov and -ncov has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to -ncov rbd. very recently, a novel coronavirus which was temporarily named " novel coronavirus ( -ncov)" emerged in wuhan, china [ ] . as of february , -ncov has resulted in a total of , laboratory-confirmed human infections in china, including deaths, and exported cases in countries outside of china (https://www.who.int/emergencies/ diseases/novel-coronavirus- /situation-reports). currently, there is no vaccine or effective antiviral treatment against -ncov infection. based on the phylogenetic analysis (gisaid accession no. epi_isl_ ) [ ] , -ncov belongs to lineage b betacoronavirus and shares high sequence identity with that of bat or human severe acute respiratory syndrome coronavirus-related coronavirus (sarsr-cov) and bat sars-like coronavirus (sl-cov) (figure (a) ). in previous studies, a number of potent monoclonal antibodies against sars coronavirus (sars-cov) have been identified [ ] [ ] [ ] [ ] [ ] . these antibodies target the spike protein (s) of sars-cov and sl-covs, which is a type i transmembrane glycoprotein and mediates the entrance to human respiratory epithelial cells by interacting with cell surface receptor angiotensin-converting enzyme (ace ) [ ] . more specifically, the amino acid length (n -v ) receptor binding domain (rbd) within the s protein is the critical target for neutralizing antibodies [ ] . some of the antibodies recognize different epitopes on rbd; e.g. the sars-cov neutralizing antibodies cr and cr bound noncompetitively to the sars-cov rbd and neutralized the virus in a synergistic fashion [ ] . we predicted the conformation of -ncov rbd as well as its complex structures with several neutralizing antibodies, and found that the modelling results support the interactions between -ncov rbd and certain sars-cov antibodies (figure (b) ). this could be due to the relatively high identity ( %) of rbd in -ncov and sars-cov (figure (c) ). for instance, residues in rbd of sars-cov that make polar interactions with a neutralizing antibody m as indicated by the complex crystal structure [ ] are invariably conserved in -ncov rbd (figure (d) ). in the structure of sars-cov-rbd-m , r in rbd formed a salt bridge with d of m -vl. concordantly, the electrostatic interaction was also observed in the model of -ncov-rbd-m , forming by r (rbd) and d (m -vl). this analysis suggests that some sars-cov-specific monoclonal antibodies may be effective in neutralizing -ncov. in contrast, the interactions between antibody f g [ ] or r [ ] and the rbd in -ncov decreased significantly due to the lack of salt bridges formed by r -d in sars-cov-rbd-f g or d -r in sars-cov-rbd- r, respectively. furthermore, while most of the r-binding residues on the rbd of sars-cov are not conserved on rbd of -ncov ( figure (c) ), it is unlikely that the antibody r could effectively recognize -ncov. therefore, it is urgent to experimentally determine the cross-reactivity of anti-sars-cov antibodies with -ncov spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against -ncov. in this study, we first expressed and purified -ncov rbd protein. we also predicted the conformations of -ncov rbd and its complex with the putative receptor, human ace . comparison of the interaction between the complex of ace [ ] and sars-cov rbd and homology model of ace and -ncov rbd revealed similar binding modes (data not shown). in both complexes, β -β loop and β -β loop form extensive contact, including at least seven pairs of hydrogen bonds, with the receptor. notably, r on the forth α helix in sars-cov rbd builds a salt bridge with e and a hydrogen bond with q on ace . however, the arginine (r in sars-cov rbd) to asparagine (n ) mutation in -ncov rbd abolished the strong polar interactions, which may induce a decrease in the binding affinity between rbd and the receptor. interestingly, a lysine (k in -ncov rbd) replacement of valine (v in sars-cov rbd) on β formed an extra salt bridge with d on ace , which may recover the binding ability. these data indicate that the rbd in s protein of -ncov may bind to ace with a similar affinity as sars-cov rbd does. indeed, we measured the binding of -ncov rbd to human ace by the biolayer interferometry binding (bli) assay, and found that -ncov rbd bound potently to ace . the calculated affinity (k d ) of -ncov rbd with human ace was . nm (figure (f) ), which is comparable to that of sars-cov spike protein with human ace ( nm) [ ] . these results indicate that ace could be the potential receptor for the new coronavirus, and that the expressed -ncov rbd protein is functional [ ] . next, we expressed and purified several representative sars-cov-specific antibodies which have been reported to target rbd and possess potent neutralizing activities, including m [ ] , cr [ ] , cr [ ] , as well as a mers-cov-specific human monoclonal antibody m developed by our laboratory [ ] , and measured their binding ability to -ncov rbd by elisa (figure (e)) . surprisingly, we found that most of these antibodies did not show evident binding to -ncov rbd. to confirm this result, we further measured the binding kinetics using bli. an irrelevant anti-cd antibody was used as a negative control. similarly, the antibody m , which was predicted to bind -ncov rbd (figure (d) ), only showed slight binding at the highest measured concentration ( . µm). further studies are needed to solve the high-resolution structure of -ncov rbd and understand why it could not be recognized by these antibodies. notably, one sars-cov-specific antibody, cr , was found to bind potently with -ncov rbd as determined by elisa and bli (figure (e,f) ). it followed a fast-on (k on of . × ms − ) and slow-off (k off of . × − s − ) binding kinetics, resulting in a k d of . nm (figure (f) ). this antibody was isolated from blood of a convalescent sars patient and did not compete with the antibody cr for binding to recombinant s protein [ ] . to further elucidate the binding epitopes of cr , we measured the competition of cr and human ace for the binding to -ncov rbd. the streptavidin biosensors labelled with biotinylated -ncov rbd were saturated with human ace in solution, followed by the addition of the test antibodies in the presence of ace . as shown in figure (g), the antibody cr did not show any competition with ace for the binding to -ncov rbd. these results suggest that cr , distinct from the other two sars-cov antibodies, recognizes an epitope that does not overlap with the ace binding site of -ncov rbd. the rbd of -ncov differs largely from the sars-cov at the c-terminus residues (figure (c) ). our results implied that such a difference did not result in drastic changes in the capability to engage the ace receptor, but had a critical impact on the cross-reactivity of neutralizing antibodies. some of the most potent sars-cov-specific neutralizing antibodies (e.g. m , cr ) that target the receptor binding site of sars-cov failed to bind -ncov spike protein, indicating that it is necessary to develop novel monoclonal antibodies that could bind specifically to -ncov rbd. interestingly, it was reported that the antibody cr completely neutralized both the wild-type sars-cov and the cr escape viruses at a concentration of . μg/ml, and that no escape variants could be generated with cr [ ] . furthermore, the mixture of cr and cr neutralized sars-cov in a synergistic fashion by recognizing different epitopes on rbd [ ] . these results suggest that cr has the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of -ncov infections. we expect more cross-reactive antibodies against -ncov and sars-cov or other coronaviruses to be identified soon, facilitating the development of effective antiviral therapeutics and vaccines. no potential conflict of interest was reported by the author (s). genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets. the lancet human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus coronavirus spike proteins in viral entry and pathogenesis a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme structure of severe acute respiratory syndrome coronavirus receptorbinding domain complexed with neutralizing antibody structural insights into immune recognition of the severe acute respiratory syndrome coronavirus s protein receptor binding domain structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, r structure of sars coronavirus spike receptor-binding domain complexed with receptor unexpected receptor functional mimicry elucidates activation of coronavirus fusion junctional and allelespecific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody key: cord- -tu bn hu authors: pérez-sautu, unai; wiley, michael ross; iglesias-caballero, maría; pozo, francisco; prieto, karla; chitty, joseph alex; garcía-garcía, maría luz; calvo, cristina; casas, inmaculada; palacios, gustavo title: target-independent high-throughput sequencing methods provide evidence that already known human viral pathogens play a main role in respiratory infections with unexplained etiology date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: tu bn hu despite the advanced pcr-based assays available, a fraction of the pediatric respiratory infections remain unexplained every epidemic season, and there is a perception that novel viruses might be present in these specimens. we systematically collected samples from a prospective cohort of pediatric patients with respiratory infections, that returned negative results by validated molecular rt–pcr assays, and studied them with a target-independent, high-throughput sequencing-based approach. we also included a matched cohort of children with no symptoms of respiratory infection, as a contrast study population. more than fifty percent of the specimens from the group of patients with unexplained respiratory infections were resolved. however, the higher rate of detection was not due to the presence of novel viruses, but to the identification of well-known viral respiratory pathogens. our results show that already known viral pathogens are responsible for the majority of cases that remain unexplained after the epidemic season. high-throughput sequencing approaches that use pathogen-specific probes are easier to standardize because they ensure reproducible library enrichment and sequencing. in consequence, these techniques might be desirable from a regulatory standpoint for diagnostic laboratories seeking to benefit from the many advantages of these sequencing technologies. the rate of discovery of new microbes and of new associations of microbes with health and disease has accelerated significantly in the last decade [ ] [ ] [ ] [ ] . many factors are contributing to this phenomenon including those that favour the true emergence of new pathogens as well as new technologies and paradigms that enable their detection and characterization [ ] [ ] [ ] . popular media have focused attention on biodefense and emerging infectious diseases, providing a foundation for unprecedented support of basic and translational research in host, vector, and microbe biology, as well as diagnostics and surveillance. new molecular technologies based on high-throughput sequencing (hts) have facilitated discovery [ ] [ ] [ ] [ ] . appreciation that more than % of emerging infectious diseases represent zoonoses has also had an impact [ ] . moreover, the databases needed to recognize microbial sequences improved dramatically with the development of more sophisticated algorithms for searching and identification [ , ] . thus, the current accepted vision is that we are in a position to tackle effectively the problem of the detection of the "unknown known" (e.g. the unexpected rare pathogen) while true reliable agnostic pathogen discovery for detection of the "unknown unknown" (e.g. a true novel pathogen) is still an art. according to the latest global health estimates from the world health organization, respiratory infections are among the five leading causes of death worldwide, causing near million deaths in [ ] . acute respiratory infections (aris) account for - % of deaths in children less than years of age in industrialized countries and - % of deaths in developing countries [ ] . aris of viral origin are one of the main causes of hospitalization among those patients, who typically suffer more than one episode during the season [ ] . despite advances, a significant proportion of the pediatric aris remain without a causative agent every epidemic season [ ] . this fact is not exclusive of aris, more than % of the cases of viral encephalitis, and near % of all deaths from gastroenteritis and from foodborne disease, remain unassigned to a specific pathogen even after syndromic laboratory testing [ ] [ ] [ ] . although there are multiple reasons that explain these results that are not related with diagnostic technology failure (e.g. non-infectious causes, inadequate, late or "investigational" sampling), the perception that there are pathogens lurking undetected in those specimens is widespread. unfortunately, no comprehensive risk assessments of the threat have been performed yet. in this report, we performed a systematic study of respiratory specimens collected from a carefully characterized and highly representative, prospective cohort of pediatric cases suffering unexplained ari, and we compared the rate of detection of pathogens by utilizing validated molecular assays, and a comprehensive sequence-independent, high-throughput sequencing-based analysis. in order to assess for the clinical relevance of the viral identifications made by hts in the specimens collected from the unexplained cases of respiratory infections, a second cohort of age-matched healthy individuals from the same epidemiologic environment was also studied with the same methodology. between september and june , , children < years of age with a respiratory tract disease were admitted to the severo ochoa hospital (madrid, spain), and evaluated by an attending physician. clinical data recorded included age, gender, gestational age (when less than weeks), clinical diagnosis, need and length for oxygen therapy, axillary temperature, duration of fever, total white blood cell count, c-reactive protein serum levels, presence of infiltrate/atelectasis in chest x-rays, administration of antibiotic therapy, hospital stay, and inclusion in pediatric intensive care unit. upper respiratory tract infection (urti) was diagnosed when rhinorrhea and/or cough were found with or without fever in the absence of wheezing, dyspnea, crackling rales, or bronchodilator use. asthma was diagnosed on the basis of the national asthma education and prevention program guidelines [naepp, retrieved from https://www.nhlbi.nih. gov/files/docs/guidelines/asthmafullrpt_archive.pdf]. acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children < years. all other episodes of acute expiratory wheezing were considered wheezing episodes. cases were considered pneumonia when both focal infiltrates and consolidation in chest x-rays were detected. cases of apparent life-threatening event (alte) were defined as an episode that is frightening to the observer and is characterized by some combination of apnea, colour change, marked change in muscle tone, choking or gagging [ ] . fever without source (fws) was diagnosed when otherwise healthy child to months of age presented with fever of less than days in duration and in whom alternative infectious etiologies were ruled out. nasopharyngeal aspirates (npas) were systematically taken from these patients and analysed by multiplex real-time rt-pcr based on previously published methods [ ] [ ] [ ] . these assays allow for the detection of the main viral respiratory pathogens including: influenza virus a, b and c, human rhinovirus, enterovirus, human orthopneumovirus (human respiratory syncytial virus), human bocavirus, human metapneumovirus, adenovirus, human rubulavirus - (human parainfluenza virus) and human coronavirus ( e, oc , hku , and nl ). fiftyseven of these npas (age range < month to years old; . % female and . % male) gave negative results in all the assays and were included in the present study. the clinical presentation of these patients (referred in the text as the "case" group) is described in table . a second group of npas taken from a prospective cohort of age-matched healthy donors was included as a control (referred in the text as the "control" group). the individuals included in the control group were children visiting the hospital for other causes (e.g. food allergy testing) with no history of respiratory infection days before to days after their visit. these patients came from the same geographic area as the patients with respiratory tract disease and the npas were collected during the same period of time covering the epidemic season of virus circulation, and analysed with the molecular assays as described above, always resulting negative. the study was approved by the medical ethics committee of the instituto de salud carlos iii (cei pi _ ) and informed written consent was obtained from all participants. virus identification by target-agnostic highthroughput sequencing analysis all respiratory specimens included in the study were processed and sequenced individually. from each npa, a μl-aliquot was homogenized by passing the sample through ml sterile syringes with g needles (becton dickinson, new jersey, usa) and by vortexing, centrifuged at , ×g for min at room temperature, and filtered through a . μm pore-size filter (ultrafree-mc, millipore, massachusetts, usa) [ , ] . each filtrate was processed with the rneasy micro kit (qiagen, maryland, usa) performing an on-column dna digestion following the manufacturer's protocol. rna extracts were then amplified by sispa rt-pcr as described previously [ , ] . amplification products were purified with the minelute pcr purification kit (qiagen), sheared using the covaris s instrument ( table ). data analysis was performed as follows: cutadapt v . [ ] , prinseq-lite v . . [ ] and picard (http:// broadinstitute.github.io/picard), were used for removal of adaptors, primers, pcr duplicates, and for quality filtering of the index (< phred) and reads (< phred). removal of reads belonging to the host was performed by aligning the quality-trimmed reads to the human genome reference grch (https://www. ncbi.nlm.nih.gov/genome/guide/human) with bowtie v . . [ ] . after the host removal step, reads were subjected to de novo assembly using ray v . [ ] . assembled contigs ( - , bp) were taxonomically identified through sequence similarity to the nucleotide collection (nr/nt) database in genbank using ncbi blast v . . + (megablast and dc-megablast; e-value threshold of × e− ). those contigs with no match were analysed with orffinder (https://www.ncbi.nlm.nih.gov/orffinder/) and the identified orfs were compared to the non-redundant protein sequences (nr) database in genbank by blastx analysis (e-value threshold of ). unmapped reads were aligned to the nr database in genbank with diamond, using sensitive mode [ ] . negative controls consisting on sterile rnasefree water were processed in parallel with the respiratory specimens. hits identified in the negative controls were considered as laboratory contaminants and not reported (suplemmentary methods). the results from the blastn and blastx analysis were manually curated and those contigs backed up by reads with no paired mate, as well as those that matched repeatedly to low complexity genomic regions or to endogenous and integrated viral sequences were discarded. all sequencing data generated in this study have been deposited in the ncbi sra database under the bioproject number prjna . all those npa specimens that contained reads of any respiratory virus were analysed by rt-pcr in order to confirm the results of the hts analysis. primer pairs were designed with the viral nucleotide sequence information obtained in the hts analysis (supplementary table ). rt-pcr was performed in µl of the same rna extracts that were initially used in the hts analysis. assays were performed with the agpath-id one-step rt-pcr kit (thermofisher scientific, massachusetts, usa) following the manufacturer's protocol. amplification conditions were as follows: min at °c , min at °c, cycles of s at °c, s at °c and min at °c, followed by a final extension step of min at °c. rt-pcr products were analysed in % agarose gel electrophoresis, purified with the minelute pcr product purification kit (qiagen), and sequenced with the bigdye terminator v . cycle sequencing kit (life technologies, california, usa). target-agnostic hts analysis of the nasopharyngeal aspirates taken from the "case" group produced a total of . million reads and , contigs that were taxonomically identified by blastn analysis (figure (a)). a total of , contigs (with . million reads) were identified by blastn analysis from the samples taken from the "control" group ( figure (b) ). in the "case" group, contigs assigned to viruses represented . % ( , contigs), while viruses represented . % ( , contigs) in the "control" group (figures (a,b) ). among the , contigs assigned to viruses in the "case" group, % ( contigs with . million reads mapped) corresponded to viruses that infect eukaryotic organisms, while % ( , contigs with . million reads mapped) corresponded to bacteriophages. among the contigs assigned to viruses that infect eukaryotic organisms different families were identified namely, papillomaviridae, pneumoviridae, picornaviridae, paramyxoviridae, anelloviridae, coronaviridae, circoviridae, orthomyxoviridae, and polyomaviridae (figure (c) ). altogether, the number of contigs assigned to any respiratory viral pathogen accounted for % ( contigs) ( table ) . when taking into account the number of reads, the vast majority ( . %; . million reads) mapped to any of the contigs assigned to respiratory viral pathogens (table ) . among these, human orthopneumovirus (human respiratory syncytial virus; hrsv) and human rhinovirus (hrv) were the most frequently table ) . altogether, at least one respiratory viral pathogen could be identified by target-agnostic hts analysis in out of the npa specimens. other viruses identified in these samples included: sapelovirus a, human parechovirus (hpev- ), human papillomavirus (hpv), human poscv -like circular virus, torque teno virus, torque teno mini virus, and human polyomavirus (wu polyomavirus) ( table ) . from the respiratory specimens taken from the "control" group, . % of the contigs ( contigs with , reads mapped) corresponded to viruses that infect eukaryotic organisms, while . % ( , contigs with . million reads mapped) corresponded to bacteriophages. among the contigs assigned to viruses that infect eukaryotic organisms, members of the papillomaviridae, circoviridae, pneumoviridae, anelloviridae, picornaviridae, herpesviridae, totiviridae, virgaviridae, and reoviridae were identified (figure (d) ). viral respiratory pathogens were identified only in two specimens. one produced contigs that were identified as hmpv, with the vast majority of the reads mapping to them ( . %; , reads) ( table ). in a second specimen, hrv was identified, with contigs to which reads mapped. the remaining , reads mapped to a total of contigs identified as the following viruses: hpv, human poscv -like circular virus, torque teno virus, red clover powdery mildew associated totivirus, tobacco mosaic virus, rotavirus a, and human betaherpesvirus ( table ) . results of the contig-specific rt-pcr assays confirmed those of the target-agnostic hts analysis. in the "case" group, hrsv and hrv were the most prevalent viruses, followed by ev and hpiv, hcov, influenza b virus, and hmpv (table ). in the "control" group, the two npa where hmpv and hrv had been identified by hts analysis were confirmed to be positive for these viruses (table ) . several specimens among the "case" group contained more than one virus (table ). among these co-infections, hrsv was detected with either hpiv, ev or hrv, and hcov was detected along with hrv (table ) . no co-infections were detected in the "control" group. in out of the ( . %) npas taken from the cases of respiratory infection the target-agnostic hts analysis did not identify any known respiratory viral pathogen (table ). in these samples, only bacteriophages, torque teno virus, hpv, human poscv -like circular virus, and human polyomavirus (wu polyomavirus) were identified (table ). further analysis of the unidentified contigs assembled from these samples by looking for sequence homologies to known viral proteins (blastx) returned no significative results. although some hits to viral proteins were identified, all of these contigs mapped also to other protein sequences in the gen-bank database with the same e-values, and with comparable ranges of coverage and identity percentage values, and thus could not be considered viral-specific hits. representative examples of such kind of identifications are shown in table . reads that did not form contigs were mapped against the nr genbank database with diamond [ ] . the analysis showed that those reads matched to proteins of bacterial or human origin or belonging to bacteriophages, and no putative novel virus was identified (results not shown). we compared the bacterial hits identified by targetagnostic hts analysis in these samples with those identified in the specimens taken from the control group. several contigs were identified by blastn analysis as wellrecognized bacterial respiratory pathogens, such as streptococcus pneumoniae, haemophilus influenzae, and klebsiella pneumoniae. however, all these hits to bacterial respiratory pathogens were not exclusive of the specimens taken from the cases of respiratory infection and could be identified also in the specimens taken from the control group (results not shown). since our analysis is not designed to be quantitative, no conclusions regarding frequency could be made. between and , we tested by multiplex realtime rt-pcr [ ] [ ] [ ] a total of , specimens from pediatric respiratory infections. out of these, remained negative after the analysis with different molecular assays. in order to shed light on the etiology of these infections, these specimens were subjected to target-independent hts analysis, along with age- table . respiratory viral pathogens identified by target-agnostic hts analysis and confirmed by contig-specific molecular assays in the respiratory specimens from the cases of respiratory infection and from the control group. matched specimens from a control group. similarly to the specimens from the respiratory infections, the specimens from the control group resulted negative in the aforementioned molecular assays. upon hts analysis we further identified known viral respiratory pathogens (hrsv, hrv, ev, hpiv, hcov, hmpv and influenza b virus). altogether, at least one respiratory viral pathogen could be identified in out of the npa specimens from the group of respiratory infection. by contrast, in the control group, only samples contained contigs attributed to viral respiratory pathogens (hmpv and hrv). moreover, the hmpv contigs detected in one of these samples, hoarded the vast majority of the reads assigned to any eukaryotic virus ( . %). the remaining viral entities were anelloviruses and papillomaviruses common to both groups. identification of respiratory viruses by hts was confirmed by contig-specific rt-pcr analysis. the hmpv and hrv identified in the control group occurred in two cases that showed no symptoms of respiratory disease. asymptomatic infections by hrv have been previously reported and are more frequent in young children [ ] . although asymptomatic infections by hmpv can occur at the pediatric stage, they are more frequent in immunocompetent adult individuals [ ] [ ] [ ] . apart from common respiratory viral pathogens, human parechovirus (hpev- ) and human polyomavirus (wu polyomavirus) were identified by hts analysis. both viruses were identified in specimens from the group of respiratory infection ( sample each); none in the control group. the detection of hpev- is not surprising as this virus is a frequent cause of infection in childhood, where it causes mild gastrointestinal and respiratory disease [ ] . human polyomavirus (wu polyomavirus) was originally detected in respiratory secretions of a pediatric patient diagnosed with pneumonia of unknown origin, and from patients with acute respiratory co-infections [ ] . nonetheless, a subsequent larger study found no link between wu polyomavirus and acute respiratory disease [ ] . in our study, wu polyomavirus was identified in one case of respiratory infection. although no other respiratory virus was identified in this sample, wu polyomavirus presents low prevalence in cases of respiratory infection and high rates of co-infection with other common respiratory viral pathogens, and further studies are needed to ascertain its clinical significance [ ] [ ] [ ] . thus, out of , ( . %) remain unexplained from a virological standpoint, as no known respiratory or novel virus was identified. analysis by blastn only returned matches to anelloviruses, papillomaviruses, table . representative examples of matches in genbank identified by blastx analysis of the contigs that remained unassigned to any taxon by blastn analysis. and human poscv -like circular virus. in spite of thorough, in-depth analysis of those contigs with no match as well as of the unmapped reads, no viral match in the genbank database was obtained. anelloviruses are frequently detected in most tissues and organs, including the respiratory tract of healthy individuals, and there is no association to any disease in humans [ ] . papillomaviruses are very common worldwide, and most infections are asymptomatic and resolve spontaneously. apart from the common warts and the various types of cancer (including an oropharyngeal form) associated to infection by hpv, low-risk types and are the predominant cause of respiratory papillomatosis, a disease in which noncancerous tumours grow in the air passages of the respiratory tract [ ] . however, other than oropharyngeal cancer and respiratory papillomatosis, there is no evidence of an association between papillomavirus infection and respiratory disease. in addition to the lack of evidence for an association with respiratory disease, in our study anelloviruses and hpv were also identified in the control group, strengthening the conclusion that their presence was not related to the respiratory disease. human poscv -like circular virus was recently identified in respiratory secretions from an unexplained human case of febrile illness, although its association with disease was not determined [ ] . small, circular, single stranded, rep-encoding, dna (cress-dna) viruses have been increasingly identified by metagenomic hts techniques [ ] in environmental samples and in a variety of vertebrates as well as various invertebrates [ ] [ ] [ ] [ ] . in humans, they have been reported in feces of healthy individuals [ ] , and in samples from unexplained cases of encephalitis and diarrhea [ ] , pericarditis [ ] , acute central nervous system infections [ ] , as well as in npas from children with respiratory infections [ ] . however, neither of these studies could establish a direct association with disease. thus, it is not clear what is the value of finding contigs that match this virus in the specimens from the group of respiratory infection. further studies are needed to determine their role (if any) in human respiratory disease. our findings agree with previous studies with similar design and tools [ , , ] . xu et al. employed hts to analyse a set of respiratory specimens taken from children with community-acquired pneumonia that had returned negative results in a commercial respiratory viral panel detection assay [ ] . they also identified hpiv, torque teno virus, torque teno minivirus, and wu polyomavirus [ ] . zhou et al. studied cell-cultured supernatants with apparent cytopathic effect that had been prepared from undiagnosed respiratory specimens and identified a high prevalence of ev accompanied by hrv, hrsv, hpiv, adv, influenza c virus, herpesvirus and dengue virus [ ] . taboada et al. studied nasal washings from children with respiratory infections previously found negative for common bacterial and viral respiratory pathogens by pcr, where they identified at least one known respiratory virus (including hrsv, hcov, and hrv) in the vast majority of the specimens [ ] . neither of these studies revealed the presence of any putative novel virus on the undiagnosed respiratory infections subject of study, and the large majority of the cases could be attributed to known viral respiratory pathogens [ , , ] . these studies suggest that all respiratory viral pathogens of clinical relevance during the pediatric stage have already been identified. in our study, we analysed cases that, out of a total of , , were negative in all pathogen-specific pcr assays. in consequence, only . % ( / , ) of all respiratory specimens collected during the entire epidemic season remained without a known etiology of infection after using pathogen-targeted diagnostic techniques. while target-independent hts analysis allowed us to come to a specific diagnostic in more than half of the undiagnosed infections, all the additionally resolved cases were produced by known respiratory pathogens. from these results, we can conclude that the current pathogen-specific techniques should be able to diagnose the vast majority of the respiratory infections. in consequence, we can conclude that the risk of overlooking a novel unknown viral respiratory pathogen in the pediatric population is very low when using target-specific diagnostic methods and that there is very little value in using target-independent assays. routine virological surveillance based on target-specific techniques, such as real-time (rt)pcr or target enrichment hts approaches (which use probes specifically designed against the viral genomic sequence of interest in order to enrich specifically for sequencing libraries derived from said virus), is appropriate and constitute a first-line diagnostic tool. the question about the etiology behind those cases of respiratory infection that remain negative for all routine diagnostic assays have been previously addressed by several groups [ , , [ ] [ ] [ ] . although in our study we used an overall approach which was similar to such previous studies, we introduced several improvements to our specific study design in order to address different limitations of the previous works. the identification of viral reads in clinical specimens remains controversial because it does not necessary imply that such viruses are responsible for the symptoms observed. many viruses can cause asymptomatic or subclinical infections, or simply be present among the normal, healthy microbiota and replicate without any pathogenic consequences. this hinders the interpretation and understanding of the results unveiled by virus discovery studies based on hts in regard to their clinical significance [ , , ] . in our study, we included a contrast study population (control group) formed by a prospective cohort of healthy individuals. such healthy control group was matched at all the critical levels with the cohort of patients with unexplained infection of the respiratory tract: (i) the individuals of the control group were age-matched; (ii) they came from the same geographic area; (iii) their samples were also taken systematically during the same time window covering the epidemic season of virus circulation. by including the control group, we were able to determine that the viruses detected in the clinical specimens taken from the patients with an infection of the respiratory tract were not circulating among the healthy population, providing evidence that such viruses were responsible for the respiratory disease observed in those cases. this would acquire special importance if a novel viral entity for which there is no previous information available is detected. in summary, the inclusion of the healthy control group allowed us to assess the clinical relevance of the viruses identified in the samples taken from the cases of unexplained respiratory infections. whenever possible, any future viral discovery studies should include a matched healthy control group to which the viruses identified by hts in the patients with clinical disease can be contrasted. in addition to the inclusion of a matched healthy control group, we processed and sequenced in parallel negative controls consisting on sterile nuclease-free water (see the materials and methods section) and we confirmed the viral identifications made by hts with specific rt-pcr assays. the objective of these procedures was to minimize the chances of reporting any false viral identification. it is worth highlighting also, that we performed a systematic sampling on our study groups: samples were collected from all the children arriving consecutively at the hospital during the period of the study, whose parents or legal guardians gave explicit written consent, and according to preestablished clinical and medical criteria (see the materials and methods section) with no further selection. by combining all the procedures discussed above, our study design constitutes a novel integrated approach that ensures a robust representativeness of the results, minimizes any possible bias, and provides a better understanding about the clinical implications of the viral identifications made by hts analysis. while hts was superior in the overall rate of detection of pathogens, that was not due to the presence in the cohort of unknown pathogens, but mostly on the underperformance of molecular methods (real-time rt-pcr) against targeted pathogens. all the viruses additionally identified by hts in our study were well-known respiratory pathogens, and no novel viruses were detected. altogether, our results show that already known viral respiratory pathogens play a main etiologic role behind the unexplained cases of respiratory infection in the pediatric population. this is a very significant finding, because if extrapolated to other clinical syndromes and specimens, it might allow us to quantitatively assess the risk. under that new paradigm, "agnostic" technologies would still have a role in pathogen detection under outbreak and event situations where a true "unknown unknown" is suspected, but "targeted" approaches would become desirable for next-generation sequencing-based microbial diagnostic. this paradigm might be desirable from a regulatory standpoint for those diagnostic laboratories seeking to incorporate these technologies, since "targeted" approaches allow for specific and reproducible library enrichment and thus they are easier to assess and validate. genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa discovery of an ebolavirus-like filovirus in europe prolonged shedding of zika virus associated with congenital infection redefining the invertebrate rna virosphere a metagenomic survey of microbes in honey bee colony collapse disorder panmicrobial oligonucleotide array for diagnosis of infectious diseases using metagenomics to characterize an expanding 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undiagnosed respiratory specimens and identification of human enteroviruses at a thailand hospital retrospective use of next-generation sequencing reveals the presence of enteroviruses in acute influenza-like illness respiratory samples collected in south/south-east asia during - no potential conflict of interest was reported by the authors. key: cord- - ye tfvz authors: pfaender, stephanie; brown, richard jp; pietschmann, thomas; steinmann, eike title: natural reservoirs for homologs of hepatitis c virus date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: ye tfvz hepatitis c virus is considered a major public health problem, infecting %– % of the human population. hepatitis c virus infection causes acute and chronic liver disease, including chronic hepatitis, cirrhosis and hepatocellular carcinoma. in fact, hepatitis c virus infection is the most frequent indication for liver transplantation and a vaccine is not available. hepatitis c virus displays a narrow host species tropism, naturally infecting only humans, although chimpanzees are also susceptible to experimental infection. to date, there is no evidence for an animal reservoir of viruses closely related to hepatitis c virus which may have crossed the species barrier to cause disease in humans and resulted in the current pandemic. in fact, due to this restricted host range, a robust immunocompetent small animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control and prophylactic vaccine development. recently, several studies discovered new viruses related to hepatitis c virus, belonging to the hepaci- and pegivirus genera, in small wild mammals (rodents and bats) and domesticated animals which live in close contact with humans (dogs and horses). genetic and biological characterization of these newly discovered hepatitis c virus-like viruses infecting different mammals will contribute to our understanding of the origins of hepatitis c virus in humans and enhance our ability to study pathogenesis and immune responses using tractable animal models. in this review article, we start with an introduction on the genetic diversity of hepatitis c virus and then focus on the newly discovered viruses closely related to hepatitis c virus. finally, we discuss possible theories about the origin of this important viral human pathogen. hepatitis c virus (hcv) infection is a major cause of chronic liver disease. currently, about million individuals are persistently infected with hcv. acute hcv infection is asymptomatic in many cases, but %- % of infected individuals are unable to clear the virus and a state of persistent viral replication and hepatic inflammation, i.e., chronic hepatitis c, follows. some patients remain asymptomatic, but over years or decades others develop cirrhosis, portal hypertension, deteriorating liver function and hepatocellular carcinoma. it is due to these late complications that chronic hepatitis c is a leading cause of liver-related death and the prime indication for liver transplantation worldwide. combination treatment with pegylated interferon-a and ribavirin has been the standard of care for more than a decade. recently, the first directly acting antivirals targeting the viral ns - a protease were licensed and triple therapy has further improved treatment options for genotype . however, resistance development, side effects and viral genotype-specific efficacy of these drugs require alternative antiviral treatment options. the hcv genome is . kb in size, encodes for a single polyprotein that is cleaved by cellular and viral proteases into at least different proteins: the structural proteins core, e , e , the ion channel p and the non-structural proteins ns , ns , ns a, ns b, ns a and ns b. these viral factors act in concert with host proteins to mediate virus entry and to coordinate rna replication and virus production which is coupled to lipoprotein biogenesis. ongoing transmission of productive hcv infection is limited to human populations, although higher primates are susceptible to experimental infection. due to this narrow host range, a robust immunocompetent small animal model is still lacking. however, a recent study demonstrated infection, replication and de novo virus production in an inbred mouse strain engineered to express human hcv entry factors in the liver and carrying a targeted lesion within innate immune signalling genes (stat , irf , irf , ifn-abr). although not fully immunocompetent, this landmark study shows that hcv can be propagated in mice and raises the hope that more robust models ultimately with fully functional innate and adaptive immune system can be developed. despite the rapid advancements in hcv research over the past decade, the origin of hcv still remains elusive, with no evidence of an animal population that might have transmitted the virus to humans. nevertheless, the idea that there might be a nonhuman primate source for hcv infections, as described for hiv, has engaged researchers since the discovery of hcv in . the recent discovery of novel hepaciviruses in different animals could now reveal new insights in the origin of hcv. several reports identified new homologs of hcv in mammals that cluster in the genus hepacivirus or the newly proposed genus pegivirus. non-primate hepaciviruses (nphv) were initially discovered in domestic dogs and subsequently in horses , and other diverse and widespread hcv-like viruses have been reported in wild populations of rodents and bats. [ ] [ ] [ ] tentatively assigned to this genus is gb virus b (gbv-b), a virus distantly related to hcv identified in a laboratory-housed tamarin, a new world monkey and causing hepatitis. however, gbv-b infection has not been reported in any other tamarin or other new world monkey species and its origin, as well as its natural host, remains unknown. besides hepaciviruses, the flavivridae family consists of three other genera: flaviviruses including yellow fever and dengue virus; pestiviruses including bovine viral diarrhea virus and classical swine fever virus and the recently assigned pegivirus genus. pegiviruses encompass the previously unclassified gb virus a (gbv-a) which was found in primates, gb virus c (gbv-c) or hepatitis g virus (hgv) which infects humans and chimpanzees , and gb virus d (gbv-d) identified in bats. of note is that in contrast to the hepaciviruses, gbv-a and some gbv-c/hgv isolates do not appear to encode a core protein. hcv is genetically highly variable and, based on phylogenetic analyses, viral isolates are grouped into seven genotypes ( - ) (figure ), which are associated with specific global regions and modes of transmission. the high observed genetic diversity is a result of the error-prone replication of the virally encoded rna dependent rna polymerase, coupled with the high replication rate in vivo. this genetic variability exhibited by hcv facilitates immune evasion and contributes to viral persistence. hcv genotypes differ from each other by %- % at the nucleotide level. however, this genetic heterogeneity is not evenly distributed throughout the viral genome, and is concentrated in regional hot-spots. the greatest levels of diversity are observed in the e e genes encoding the envelope glycoproteins e and e , while other genome regions, such as those encoding the core protein or the helicase domain of the ns protein, exhibit higher levels of conservation. despite substantial sequence variation, all genotypes share the same genome structure, encoding a single polyprotein which is postranslationally cleaved by viral and host proteases into mature proteins. the single open reading frame (orf) is flanked by two highly conserved untranslated regions (utrs). hcv genotypes can be further divided into multiple distinct subtypes (a, b, c, etc.) differing by %- % at the nucleotide level. even within an infected patient, hcv exists as a heterogeneous population of genetically distinct yet related variants. the prevalence of hcv genotypes differs significantly in different parts of the world. seventy percent of patients in north america and europe are infected with genotype , while hcv genotypes and account for about % of cases, but are overrepresented among patients who inject drugs. in contrast, hcv genotype can be detected in about % of patients in south east asia and genotype is the major genotype in the middle east and northern africa. genotypes and have been detected mainly in africa, while genotype has been found in china and southeast asia. phylogenetic analyses, coupled with global distribution patterns of hcv genotypes, indicate that genotypes and emerged ca - years ago. the worldwide dissemination of genotype began later some - years ago, with the restricted diversity of sequences among genotype b indicating emergence around - years ago. one has to keep in mind that this molecular clock analysis might be under-or overestimated due to mutational masking and substitutional saturation. utilizing both deep sequencing and serology-based approaches, a number of novel hepaciviruses have been identified in different mammalian host species. kapoor and colleagues were the first to discover a canine homolog of hcv which was phylogenetically closely related to hcv. initially termed canine hepacivirus (chv), the virus was independently sequenced from dogs with a respiratory illness from two different outbreaks of respiratory disease in the united states, whereas no healthy animals were found to be infected. high viral loads (. viral copies/ nasal swab) were detected in nasal swabs of the infected animals. testing liver and lung samples from unrelated dogs which had died of unexplained gastrointestinal illness, chv rna was found at low levels (, copies/ ng of total rna) in liver samples, although lung tissue contained no detectable viral rna. furthermore, using in situ hybridization, kapoor et al. revealed focal and dispersed infection of canine liver and the presence of viral rna mainly in the cytoplasm of hepatocytes. strikingly, there was a high degree of genetic relatedness between viral sequences from different animals which is rather unexpected for a rna virus. the viral genome of chv encodes a amino acid (aa) polyprotein, which is predicted to be cleaved into mature viral proteins (figure ), in a similar fashion to that described for hcv (table ) . comparative phylogenetic analyses of conserved regions of the predicted helicase (ns ) and rna-dependent polymerase (ns b), revealed chv to be the closest relative of hcv discovered to date, and equidistant from all seven hcv genotypes (figure ) . the virus displayed approximately % nucleotide sequence divergence from hcv with a maximum aa identity to hcv in the non-structural proteins ns and ns b (. %- %), whereas e , the n-terminal half of e , ns and the c terminus of ns a showed the lowest aa identity (, %- %). surprisingly, the glycoprotein regions of chv were readily aligned with hcv, with marked similarity in the c-terminal half of e . analyses of the e /e glycosylation sites revealed and potential potential n-linked glycosylation (png) sites, respectively, comparable to those predicted for hcv (e or and e up to ). png sites are essential for immune shielding, efficient entry and correct folding of the hcv envelope glycoproteins incorporated into infectious virions. intriguingly, the binding site for mir- , which is important for hcv replication, in the utr could not be identified in the chv sequence. together with the absence of microrna sequences in the dog genome capable of binding to the equivalent site in chv, this finding argues that this virus might not depend on mir- for its replication. the mean time to the most recent common ancestor between the hcv genotypes and chv was estimated to be between and years before present (ybp). in an effort to further investigate the host range of chv, the same group utilized a serology-based approach to screen for the presence of the virus in other mammalian species. burbelo and colleagues used a recombinant protein expressed from the helicase domain of chv ns as antigen to detect chv antibodies in sera from different animals. surprisingly, they detected immunoreactivity against chv ns in samples of horses ( %), with eight horse sera also carrying viral rna, whereas dogs, rabbits, deer and cows were seronegative, with one intermediate positive sample from a cow. initial sequencing of the eight viral rna positive horse samples identified a series of genetically diverse viruses. based on these findings in a different host, the authors tentatively termed these viruses nphv. complete genomic sequences from these eight nphv variants were obtained ( table ). the original chv variants showed a very high similarity (maximum of . % divergence) to one of the eight nphv variants, whereas the eight horse-derived nphv sequences themselves had moderate genome sequence diversity from each other ( . %- . %). this high degree of homology between nphv and chv combined with the lack of other hepaciviruses in dogs suggests that the chv isolate may in fact be a horse virus that was transmitted to a dog. if that was a true transmission event or simply a contamination, possibly by feeding on horse meat or veterinary products which utilized horse based components is unclear. at the aa level, the structural region showed a greater divergence than the non-structural region. of note, most sequence diversity between nphv variants was attributable to synonymous substitution (d s ), with low levels of nonsynonymous substitution (d n ) variation observed. indeed, low d n /d s ratios of between . and . indicate the action of strong purifying selection on nphv genomes when compared to hcv, which displays a higher frequency of d n variation and thus higher d n /d s ratios. in general, sequence divergence among nphv isolates was greater than subtype diversity within hcv. even though the originally predicted chv secondary structure of the utr showed (table ) . unlike hcv, which chronically infects %- % of exposed humans, only % of horses showed co-presence of igg antibodies and viral genomes. this could be indicative for either an acute infection or would suggest that possibly the majority of equine hosts are able to clear nphv infection. to further investigate the species specificity of nphv and to address questions regarding tissue tropism or pathogenesis of this newly identified hepacivirus, the group of simmonds and colleagues analyzed nphv in domestic horses in the united kingdom. screening for the presence of nphv in other mammals, the authors performed large-scale polymerase chain reaction (pcr)-based investigation of samples from dogs, cats, pigs, rodents, donkeys and horses. from this survey, three plasma samples from horses ( %) were found positive for nphv rna. sequence comparison demonstrated that each of the positive horses was infected with nphv variants distinct from the eight previously identified in horses. the newly identified nphv variants displayed similar branching orders in each genome region, indicating a lack of recombination as observed previously. clinical records from the time of sampling displayed no evidence for a hepatic or systemic disease. furthermore, liver function analyses revealed no indication for hepatic inflammation as c-glutamyl transferase and glutamate dehydrogenase values were within reference range, with the exception of a mildly elevated c-glutamyl transferase new hcv-like viruses in different mammalian hosts pfaender et al level in one horse. moreover, with the bile acid levels within reference range, there was no sign of hepatic damage. repeated samplings from one horse four and five months after the initial sampling revealed a persistent infection. the horse remained viremic, but the viral load decreased between the fourth and fifth months from the initial . copies/ml to . and . copies/ml, respectively. during the sampling period, the horse remained clinically unremarkable and the liver indices stayed mainly within the reference ranges, although frequently at the upper end of the reference ranges. this data indicate that hepaciviral infections in horses can be persistent. the organ tropism of nphv has yet to be elucidated and further studies on viral associated pathogenesis, the course of clinical disease and likely mode(s) of transmission are required to fully understand the nature of nphv infection in horses. the discovery of closely related hepaciviral homologs of hcv, which naturally infect equine hosts, raised the possibility that additional table ). based on these sequences, the rhv genome is predicted to encode a polyprotein of aa flanked by and utrs (table ). in one of the sequenced variants (rhv- ), a putative mir- seed site was apparent in the utr, suggesting a possible dependence on mir- of these novel viruses. the polyprotein is predicted to encode proteins which were similar in predicted size to those of hcv and other hepaciviruses ( figure ). the rhv glycoproteins e and e contained two and four png sites, respectively, considerably less than those observed for hcv and nphv. the computed genetic distance between rhv- and hcv in the structural regions ranged from % to % and % to % in the non-structural regions, and was therefore substantially greater than that between hcv and nphv. additionally, another group independently described novel hepaciviruses infecting rodent hosts. drexler and colleagues screened for bloodborne viruses in sera and organs from rodents and sera from bats. even though bat serum showed cross-reactivity with hcv antigens, no hepacivirus rna was detected in these samples. nevertheless, the group identified three highly divergent novel rhv clades in european bank voles (myodes glareolus) and in south african four-stripped mice (rhabdomys pumilio). to determine the genome organization and structural features of rhv, near full genomes of five representative hepaciviruses from all rodent clades were determined ( table ). the five sequenced rhv genomes were predicted to encode a typical hepacivirus polyprotein comprising three structural and seven non-structural proteins of comparable size to those previously described for other hepaciviruses (figure : rhv- , and ). again, png sites in the envelope proteins, in particular in the putative e protein, were predicted to be fewer when compared to hcv (table ) . the minimum aa identity of the novel rodent viruses to hcv averaged in the structural proteins between . % and . % and in the nonstructural proteins from . % to . %. based on the high degree of nucleotide sequence homology of the ns b genes between all members of the flaviviridae family, comparative analysis was possible and revealed the grouping of rhv as a monophyletic sister-clade to hcv. interestingly, all rhv clades were slightly more related to gbv-b than to hcv (figure ). further genomic analyses revealed the presence of one mir- binding site, again pointing to a possible hepatotropism for these viruses. indeed, the authors were able to find high viral loads in bank vole tissues infected with rhv, with the highest rna concentrations found in liver tissue: . copies/g compared to significantly lower concentrations in lung, kidney, serum, spleen, heart, brain and intestine. in situ hybridization revealed foci of viral rna in the cytoplasm of m. glareolus hepatocytes, whereas no evidence of virus infection by in situ hybridization could be found in the other organs. evidence for liver inflammation was observed by histopathological examinations, which revealed low-grade focal lymphatic invasion in the rna-positive animals compared to the rna-negative animals. serological investigations of the myodes hepaciviruses revealed the presence of antibodies against the ns antigens of these viruses in only some of the animals ( . % and . %), and no cross-reactivity of the sera with hcv could be observed. this indicates a specific immune reaction against rhv. rna and antibodies were found in of ( . %) pcr-positive bank vole sera and this low co-occurrence could indicate that bank voles might be able to clear hepacivirus infections in the majority of cases. the identification of rodent homologs of hcv could pave the way for novel surrogate animal models of hcv which may ultimately facilitate vaccine design and treatment approaches. although the group of drexler and colleagues failed to directly recover hepacivirus genomes in bats, they were able to show serological evidence for these viruses in bats, suggesting the existence of bat hepaciviruses (bhvs). indeed, the group of quan and colleagues identified bats as a major natural reservoir for hepaciviruses and pegiviruses. the group utilized an unbiased high-throughput sequencing approach to enhance the knowledge of viral diversity in bats and encountered a highly diverse group of bat-derived viruses which were related to hepaciviruses and pegiviruses. the group was able to detect viral genomes in six of the eight bat families tested and in a total of sera/plasma samples, one lung specimen as well as one rectal swab. the viral load was determined by quantitative pcr and ranged from to rna copies/ml in the sera or plasma of infected bats. taken together, the group was able to identify bat-derived viruses, which potentially represent novel viral species. the bhvs fell into three highly divergent clades exclusively composed of viruses from two species of african bats (hipposideros vittatus, otomops martiensseni). clade a viruses were most closely related to gbv-b whereas clade c and d viruses fell into a basal position relative to the clades containing nphv and hcv. bhv serum levels ranged from . to rna copies/ml. five near full-length genome sequences were obtained representing all three clades of bhv (table ) . bhv genomes encode a single positive-stranded rna genome containing a single orf, flanked at the and end by non-translated regions, encoding a polyprotein precursor of about to aa ( figure ). consistent with other viruses in the family of flaviviridae, conserved protein domains were recognized in the predicted polyprotein. comparable to other hepaciviruses, the most variable regions of the genome encode the envelope glycoproteins, the non-structural proteins ns and ns a with less than . %, . % and . % aa sequence identity, respectively. the most conserved regions were the ns and ns b proteins, with aa sequence identities of . %- . % in the ns gene and . %- % in the ns b gene. again, the translated bhv e and e protein sequence showed fewer png ( - ) sites compared to hcv (table ) . regarding pathogenesis of these new viruses, further investigations are required. even though there were high levels of viremia detected, all bats collected were apparently healthy, suggesting that bhvs may not be pathogenic to their host. bats are probably the most abundant, diverse and geographically dispersed vertebrates worldwide. the discovery of bhv and bat pegivirus (bpgv) in bats adds new members to the ever expanding list of viruses that can infect this animal reservoir. many different zoonotic viruses, including rabies virus and related lyssaviruses, nipah and hendra viruses as well as ebola and severe acute respiratory syndrome coronavirus and recently also hepadnaviruses have been found to originate in bats. , further research might shed new light on the role of these mammals as carriers of members of the flaviviridae and may provide new insights into the evolutionary origins of hcv. pfaender et al initially, novel hepaciviruses were all identified in non-primate species. recently, the group of lauck and colleagues expanded this host range further with the discovery and characterization of the first hepacivirus infecting a wild non-human primate, the black-and-white colobus (colobus guereza), an old world monkey from uganda. notably, all infected animals appeared healthy at the time of sampling with no observed overt clinical symptoms. deep sequencing of rna from plasma samples of nine of these animals revealed the presence of viral rna in three animals, with the genomic architecture matching other viruses from the hepacivirus genus. the virus, named gnereza hepacivirus (ghv), was shown to share a common ancestry with gbv-b, the recently identified rhvs and one of the three recently discovered bhv clades (clade a). viral sequences covering the entire coding region as well as partial and utrs were obtained from all three animals. the new viruses share limited nucleotide identity across the coding region with other known hepaciviruses (hcv %, nphv %, rhv %, gbv-b % and bhv %). based on sequence comparison, two highly similar variants were classified as subtype ghv- , whereas the third variant represents subtype ghv- . interestingly, a canonical mir- binding site was present in both ghv subtypes. the predicted cleavage sites of the ghv polyprotein are similar to other hepaciviruses, with ten mature viral proteins predicted. the glycoproteins e and e contain four png sites each (table ) . a striking feature of theses hepaciviruses is their unusually long ns a gene ( - aa), approximately twice the length of any other known ns a within the flaviviridae, rendering the genomic sequence over kb longer than that of hcv ( figure ). slidingwindow analyses of aa similarities between ghv and other members of the hepaciviruses revealed that across the region from core to ns b ghv is most similar to bhv, whereas ghv ns b shares the highest sequence similarity with rhv. this suggests that cross-species transmission and ancient recombination might have contributed to the evolution of ghv. phylogenetic analyses support the grouping of ghv within the hepacivirus genus (figure ) and the shared common ancestry of ghv, gbv-b, rhv and bhv- . analyses to determine the most recent common ancestor for ghv, hcv and nphv suggests an early divergence of ghv from the other hepacivirus lineages, at least - ybp. , taken together, the detection of ghv represents the first documented natural infection of a non-human primate with a hepacivirus and further expands the known host range of this viral genus. as a newly proposed genus of the family of the flaviviridae, the genus pegivirus has been proposed to encompass the so far unclassified viruses gbv-a, gbv-c/hgv and gbv-d. based on this new classification, different groups also identified multiple viruses falling into the pegivirus genus, which are emerging in new hosts. chandriani and colleagues identified a highly divergent member of the flaviviridae family, which is likely the causative agent for an outbreak of acute hepatic disease occurring on a horse farm. the virus was present in equine serum from two overtly clinical index cases of hepatitis and from an equine plasma product which was administered to the horses before the outbreak. this new pathogen was designated theiler's disease-associated virus (tdav), presumably the causative agent of theiler's disease, an acute hepatitis in horses. viral titers in most of the infected horses ranged from to genomes/ml. the viral genome contained a single orf encoding a polyprotein of aa flanked by a and utr. based on comparison with related members of the flaviviridae, the virus is predicted to encode three structural proteins (core, e and e ) and seven putative non-structural proteins (a protein between e and ns as well as ns , ns , ns a, ns b, ns a and ns b). phylogenetic analyses grouped tdav as belonging to the newly proposed pegivirus genus (figure ) . over the length of the entire polyprotein, the virus shares . % aa identity with gbv-d, . % identity with hcv (genotype ) and . % identity with nphv. the most conserved regions can be found in ns and ns b, with regions exceeding % aa identity with gbv-d. interestingly, even though tdav is linked to an acute hepatitis, no mir- binding site could be identified, suggesting that, unlike hcv, the virus can replicate independently of mir- . transmission between horses appears to be low as the virus was undetectable in horses which had contact with other infected animals. the tdav-positive horses were further ranked as clinical or subclinical, with the clinical cases displaying significantly elevated liver enzymes in serum. the subclinical cases displayed varying degrees of elevated liver enzymes in the serum, but without overt clinical manifestation of liver disease in most cases. in general, a slightly higher proportion of asymptomatic tdav-positive animals were observed. furthermore, a clear relationship between viral load and clinical hepatitis could not be observed, indicating that the viral load was not predictive of the extent of hepatic injury. further analyses of the infected horses year after the outbreak provided evidence that tdav can establish a chronic infection. to further investigate the route of transmission for tdav infections, the authors performed an inoculation study in which they experimentally injected four healthy animals with a tdav-positive plasma product. even though the viral genome could be detected in all inoculated horses, only one horse showed a clear elevation of liver enzymes over the course of the study. in summary, this study provides evidence for the association of tdav with theiler's disease, identifying tdav as the sole member of the newly described genus pegivirus for which disease association has been demonstrated. kapoor and colleagues independently identified another equine pegivirus (epgv) which is genetically distinct to tdav. the group screened serum samples from horses with elevated liver enzyme levels and detected viral rna in two samples, which were highly divergent from all previously known pegiviruses ( table ) . the virus encodes a utr, a single orf encoding a putative polyprotein of aa and a utr. sequence alignments revealed the divergence of epgv from other pegiviruses, ranging from % to % in the structural genes and from % to % in the non-structural region. the most conserved regions were the ns and ns b genes, as described for the hepaciviruses genus, whereas the highest sequence diversity can be found in the glycoproteins e /e and ns b. to analyze the prevalence and persistence of epgv, two horse cohorts were studied. in one cohort, the viremia frequencies were % ( / ) among horses with elevated liver enzymes and . % ( / ) among healthy animals. in another herd analyzed at different time points over four years, %- % of the horses were found to be positive, with two horses remaining viremic for at least three and a half years and two horses being able to clear the infection. viral genome copy numbers ranged from . to . genome equivalents/ml in the serum and viral rna could also be detected in liver and lymph node biopsy samples, as well as in peripheral blood mononuclear cells, with no major differences in viral rna between the tissues, suggesting that the virus is not strictly hepatotropic. further studies are necessary to determine the prevalence, tissue tropism and possible disease association of this newly identified virus. the same group additionally identified two new species of pegiviruses in rodents (see the section on 'rodents/bats'), one from white-throated wood rats (rpgv-cc ) from which the whole genome was acquired (table ) and the other from deer mice. the viral genome displayed the typical flaviviridae genome organisation including a utr, a polyprotein coding region ( aa) as well as a utr. genetic analyses revealed that rpgv was substantially divergent from gbv-a, gbv-c, gbv-d and from the epgv (figure ). amino-acid divergence ranged from % to % in the structural proteins and % to % in the non-structural region. apart from rodents, bats comprise the most diverse group of mammals. the pteropus giganteus bats have already been identified as carriers of gbv-d. the group of quan and colleagues further enhanced our understanding of the viral diversity in bats, with the identification of bat-derived hepaciviruses and pegiviruses (see the section on 'rodents/bats'). among the total of bat-derived viruses uncovered, potential novel viral species within the pegivirus genus were identified, which formed three distinct lineages. clade h viruses clustered with the previously identified gbv-d, whereas the clade g and k viruses formed distinct phylogenetic clusters within the genus pegivirus. interestingly, in some of the animals, coinfections of clade g and k viruses and in one case even coinfection with a hepacivirus (clade c) and a pegivirus (clade k) were observed. the genomic organization of these pegiviruses is similar to that observed for other flaviviruses. amino acid sequence identities were greatest in non-structural proteins ns and ns b, whereas the envelope proteins as well as ns and ns a displayed the highest variability. in summary, these studies show that also pegiviruses are widely distributed among different mammalian species and future studies are necessary to investigate disease association, transmission and tissue tropism. viruses are usually well adapted to their hosts, resulting in multiple barriers to cross-species transmission. nonetheless, the majority of recent emerging infections in human populations represent zoonoses from wild animal species. a recent survey, using fruit bats as a model organism, uncovered novel viruses from seven viral families. extrapolating this number to all mammalian species, the authors estimate a minimum of . mammalian viruses awaiting discovery. although this number is likely to be an overestimation due to the tendency of bats to act as reservoirs for a broad spectrum of viruses, it is clear that many as yet undiscovered viruses circulate in wild animals, all with the potential to jump the species barrier to humans. cumulatively, these findings would support a scenario where hcv in humans is likely the result of a cross-species transmission, probably from an as yet unidentified source. current patterns of global hcv diversity could be due to a single ancestral zoonotic transmission to archaic humans prior to their global dispersal, with subsequent viral diversification in isolated populations. identification of a more closely related hepaciviral progenitor closer to the root of extant circulating hcv isolates than nphv, the current closest relative, would provide support for this scenario. however, the geographical associations of different hcv genotypes, which appear to have been evolving and diverging in geographically discrete human populations over an extended time scale, could also be the result of multiple, more recent, independent cross-species transmissions to geographically separated human populations. identification of novel hepaciviruses, isolated from animal hosts, which fall within the global hcv radiation and are positioned basally to distinct hcv clades, would ultimately provide strong evidence for this alternative. the . bat species are known to harbour a diverse array of viruses from multiple families, many of which have crossed the species barrier to cause a variety of diseases in humans, often via an intermediate host. for example, the hendra virus, an rna virus from the family paramyxoviridae, has caused multiple outbreaks in horses and four reported human fatalities. interestingly, hendra virus was subsequently demonstrated to originate in fruit bats and was transmitted to humans via close contact with horses. of note, the most closely related animal hepaciviruses to hcv described to date are found in horses (nphv) , and kenyan fruit bats (bhv) (figure ) . thus, if continued wild animal sampling fails to identify more likely candidates for the ancestor(s) of hcv, one could envisage an historical scenario whereby the progenitor of hcv was transmitted to humans from bats via horses, as all three species are susceptible to hepaciviral infection. however, viruses are dependent on a multitude of host factors to complete their life cycle in susceptible cells. thus, species-specific barriers to viral cross-species transmissions are likely to be proportional to the genetic relatedness of host species: one would expect viruses to jump the species barrier more easily between closely related host species. in addition to hcv infection of humans, gbv-b infection of new world monkeys and the recent discovery of distantly related hepaciviral homologs to hcv in old world monkeys indicates the susceptibility of a broad array of primates to hepaciviral infection. two of the biggest infectious disease burdens afflicting humanity presently are hiv and malaria, zoonoses from chimpanzees and gorillas, respectively, our closest great-ape relatives. thus, it might be also possible that great apes, or other primate species, harbor as yet undescribed hepaciviruses, which may ultimately have given rise to the current hcv pandemic. despite successful development of cell culture systems to study the complete hcv replication cycle, analysis of mechanisms of virus pathogenesis and immune control as well as vaccine development are severely hampered by the lack of robust immunocompetent small animal models. furthermore, the origin of hcv has remained elusive. recently, studies combining powerful next-generation sequencing technologies with coordinated sample collection from multiple geographical localities have uncovered related hepaciviral and pegiviral homologs in diverse animal species. these research efforts have resulted in a rapid expansion of the known viral diversity in hepacivirus and pegivirus genera, and identified an increasing number of animal host species susceptible to hepacivirus and pegivirus infection. differences and similarities between these newly discovered viruses pdb- a only viruses listed whose near full-length genomes were generated. new hcv-like viruses in different mammalian hosts pfaender et al and hcv may ultimately advance our understanding of hepacivirus biology with respect to mechanisms of hepaciviral replication, permissiveness of small animal models to productive infection, epidemiology and vaccine development, in addition to further elucidating hcv origins. future studies should address transmission and ecology of these new viruses in their natural hosts to evaluate any potential risk of trans-species transmission to humans. the generation of functional cdna clones will further advance our knowledge on hepaciviral replication strategies and could be used for the development of recombinant hcv vaccines. in conclusion, the recent discoveries of multiple novel hepaciviruses in diverse mammalian species have undoubtedly intensified research efforts to identify the true origins of hcv. ultimately, these studies will expand our knowledge of circulating mammalian viruses, in addition to further illuminating barriers/pathways to viral cross-species adaptation. evolving epidemiology of hepatitis c virus hepatitis c and liver transplantation novel therapies for hepatitis c-one pill fits all? hepatitis c virus therapy update replication of hepatitis c virus assembly of infectious hepatitis c virus particles transmission of hepatitis c by intrahepatic inoculation with transcribed rna completion of the entire hepatitis c virus life cycle in genetically humanized mice hepatitis viruses in non-human primates origin of hiv- in the chimpanzee pan troglodytes troglodytes isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome 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friend or foe? emerging infectious diseases. link to mers virus underscores bats' puzzling threat evolution. great apes and zoonoses cell culture systems for hepatitis c virus this work is licensed under a creative commons attribution . unported license stephanie pfaender was funded by a stipend from the international research training group (irtg ) provided by the deutsche forschungsgemeinschaft. eike steinmann was supported by the deutsche forschungsgemeinschaft (ste / - ) and an intramural young investigator award of the helmholtz centre for infection research. thomas pietschmann is supported by grants from the deutsche forschungsgemeinschaft (sfb , project a ) and (pi / - ) by grants from the helmholtz association (so- , hai-idr) and by a grant from the european research council (erc- -stg_ -virafront). we thank gisa gerold (centre of experimental and clinical infection research, hannover, germany) for critically reading the manuscript. key: cord- -h i t cr authors: hoang, van-thuan; dao, thi-loi; ly, tran duc anh; belhouchat, khadidja; chaht, kamel larbi; gaudart, jean; mrenda, bakridine mmadi; drali, tassadit; yezli, saber; alotaibi, badriah; fournier, pierre-edouard; raoult, didier; parola, philippe; de santi, vincent pommier; gautret, philippe title: the dynamics and interactions of respiratory pathogen carriage among french pilgrims during the hajj date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: h i t cr we conducted this study to describe the dynamics of the acquisition of respiratory pathogens, their potential interactions and risk factors for possible lower respiratory tract infection symptoms (lrti) among french pilgrims during the hajj. each participant underwent four successive systematic nasopharyngeal swabs before and during their stay in saudi arabia. carriage of the main respiratory pathogens was assessed by pcr. pilgrims were included and . % reported respiratory symptoms during the study period. the acquisition of rhinovirus, coronaviruses and staphylococcus aureus occurred soon after arrival in saudi arabia and rates decreased gradually after days and . in contrast, streptococcus pneumoniae and klebsiella pneumoniae carriage increased progressively until the end of the stay in saudi arabia. haemophilus influenzae and moraxella catarrhalis carriage increased starting around days and , following an initial clearance. influenza viruses were rarely isolated. we observed an independent positive mutual association between s. aureus and rhinovirus carriage and between h. influenzae and m. catarrhalis carriage. dual carriage of h. influenzae and m. catarrhalis was strongly associated with s. pneumoniae carriage (or = . ). finally, our model showed that m. catarrhalis carriage was negatively associated with k. pneumoniae carriage. chronic respiratory disease was associated with symptoms of lrti. k. pneumoniae, m. catarrhalis-s. aureus and h. influenzae-rhinovirus dual carriage was associated with lrti symptoms. our data suggest that rtis at the hajj are a result of complex interactions between a number of respiratory viruses and bacteria. each year, an increasing number of people travel to the kingdom of saudi arabia (ksa) for the hajj and umrah pilgrimages, which attract around million pilgrims annually from more than countries. more than two million pilgrims from outside saudi arabia participated in the hajj pilgrimages in [ ] . each year, about , pilgrims from marseille, france, participate in the hajj [ ] . the event presents major challenges for public health, including inter-human transmission of infectious diseases, notably respiratory tract infections (rtis), due the crowded conditions experienced by pilgrims [ ] . in a recent study on morbidity and mortality among indian hajj pilgrims, infectious diseases represented % of outpatient diagnoses, with rtis and gastroenteritis being the most common [ ] . between . % and . % of french pilgrims presented rti symptoms during the hajj [ ] . a recent literature review suggested that etiology of rtis at the hajj is complex; several studies showed a significant acquisition of respiratory pathogens by pilgrims following participation in the hajj in both symptomatic and asymptomatic individuals [ ] . in a systematic review of studies, al-tawfiq et al. showed that human rhinovirus (hrv) and influenza viruses were the most common viral respiratory pathogens isolated from ill hajj pilgrims [ ] . in addition, human non-mers coronaviruses (hcov) were also a common cause of rtis during the event [ ] . on the other hand, streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus were shown to be the most commonly acquired respiratory bacteria at the hajj [ ] . rtis are caused by the antagonistic and synergistic interactions between upper respiratory tract viruses and predominant bacterial pathogens [ ] . pathogens are usually studied individually, although in their natural environment they often compete or coexist with multiple microbial species. similarly, the diagnosis of infections often proceeds via an approach which assumes a single agent etiology [ ] . nevertheless, complex interactions occur between the different infectious microorganisms living in the same ecological niche and mixed infections are frequent [ ] . a better understanding of polymicrobial interactions in the nasopharynx among hajj pilgrims is important for many reasons. carriage of more than one pathogen is common among hajj pilgrims, whether or not they present with respiratory symptoms [ ] . colonization is the initial step in the disease process [ ] . nasopharyngeal colonization is likely to be a reservoir for respiratory pathogens resulting in interhuman transmission between pilgrims during close contact experienced during the hajj ritual. furthermore, antibiotic use or vaccines, which target specific pathogen species, may alter polymicrobial interactions in the nasopharynx and have unanticipated consequences [ , ] . to our knowledge, the dynamics and interaction between the main respiratory pathogens acquired during the hajj pilgrimage have not been specifically investigated, to date. risk factors for possible lower respiratory tract infection (lrti) symptoms at the hajj have not been studied. we conducted this study among french pilgrims during the hajj, to describe the dynamics of the acquisition of respiratory pathogens and their potential interactions. in addition, we investigated risk factors for possible lrti symptoms. participants and study design ( figure ) pilgrims travelling to mecca, saudi arabia during the hajj from marseille, france, were recruited through a private specialized travel agency. potential adult participants were invited to participate in the study. they were included and followed-up by two bilingual (arabic and french) medical doctors who travelled with the group. all participants departed to ksa on the same date, were housed in the same accommodation during their stay and performed the rituals together. upon inclusion, before departing from france, pilgrims were interviewed using a standardized pre-hajj questionnaire that collected information about demographic characteristics, medical history and immunization status. pilgrims were considered to have been immunized against influenza when they had been vaccinated within the past year and until before days of the date of travel. pilgrims were considered to be immune to invasive pneumococcal disease (ipd) when they had been vaccinated with the -valent conjugate pneumococcal vaccine (pcv- ) in the past five years [ , ] . a post-hajj questionnaire was completed two days before the pilgrims' return to france. clinical data, antibiotic intake and information on compliance with face masks use as well as hand washing, the use of hand gel disinfectant and disposable handkerchiefs was collected. to evaluate the dynamic and interaction of respiratory pathogens during the hajj, all pilgrims underwent four successive systematic nasopharyngeal swabs at different times: pre-travel, five to six days post arrival, - days post arrival and just prior to leaving ksa (post-hajj). the hajj rituals took place from - august. influenza-like illness (ili) was defined as the presence of cough, sore throat and subjective fever [ ] . possible lrti was defined by presence of productive cough without nasal or throat symptoms; febrile productive cough; dyspnea or febrile dyspnea [ ] . based on the who classification, "underweight" was defined as having a body mass index (bmi) below . , "normal" corresponded to a bmi between . and , "overweight" corresponded to a bmi ≥ , and "obese" referred to those with a bmi ≥ [ ]. nasopharyngeal swabs were obtained from each pilgrim, transferred to sigma-virocult® medium and stored at − °c until processing. the sampling was done by the doctors accompanying the group, in a standardized way ( cm in the nostril, turns; post wall of the pharynx, streaks). the rna and dna were extracted from the samples using the ez advanced xl (qiagen, hilden, german) with the virus mini kit v . (qiagen) according to the manufacturer's recommendations. all quantitative real-time pcr were performed using a c touch™ thermal cycle (bio-rad, hercules, ca, usa). one-step simplex real-time quantitative rt-pcr amplifications were performed using multiplex rna virus master kit (roche diagnostics, france) for influenza a, influenza b, hrv and internal controls ms phage [ ] . hcov was detected by one-step duplex quantitative rt-pcr amplifications of hcov/ hpiv-r gene kit (ref: - , biomérieux, marcy l'etoile, france), according to the manufacturer's recommendations. real-time pcr amplifications were carried out using lightcycler® probes master kit (roche diagnostics, france) according to the manufacturer's recommendations. the shd gene of h. influenzae, phoe gene of klebsiella pneumoniae, nuca gene of s. aureus, lyta cdc gene of s. pneumoniae and copb gene of moraxella catarrhalis were amplified with internal dna extraction controls tiss, as previously described [ , ] . human adenovirus, human metapneumovirus, respiratory syncytial virus, bordetella pertussis and mycoplasma pneumoniae were not tested because a low proportion (< %) of returning french pilgrims or international pilgrims were found positive for these pathogens in previous works [ , , , ] . negative controls (pcr mix) and positive controls (dna from bacterial strain or rna from viral strain) were included in each run. positive results of bacteria or virus amplification were defined as those with a cycle threshold (ct) value ≤ . a threshold value of was used in each experimental run and we calculated the rfu cut off value recommend by cfx manager software version . (bio-rad) [ ] in order to verify the positive cases. results were considered positive when the cycle threshold value of real-time pcr was greater than the cut off value. pilgrims were considered to be positive for respiratory pathogens during the hajj if they were positive at the days and and/or days and sample. stata software version . (copyright - statacorp llc, http://www.stata.com) was used for statistical analysis. the main outcomes of interest were the relationships between respiratory pathogens among pilgrims during the hajj. we evaluated the carriage of hrv, hcov, s. aureus, s. pneumoniae, h. influenzae, k. pneumoniae and m. catarrhalis using logistic mixed models. because each pilgrim provided four successive samples, we used a repeated measures design to take into account the variability of series samples from each pilgrim. to evaluate the effect of covariates on each respiratory pathogen carriage, we modelled carriage of hrv, hcov, s. aureus, s. pneumoniae, h. influenzae, k. pneumoniae and m. catarrhalis separately. we did not separately model the outcome of carriage of influenza a and b viruses because of the low prevalence of these viruses. only the variables with a prevalence ≥ . % were considered for statistical analysis. unadjusted associations between respiratory pathogen carriage with multiples factors: sociodemographic characteristics (gender, ≥ years), chronic respiratory disease, bmi classification, smoking status; individual preventive measures (vaccination against influenza, vaccination against ipd, use of a face mask, hand washing, disinfectant gel and disposable handkerchiefs); antibiotic intake days before each sample; respiratory virus or bacteria and dual carriage were analysed by univariable analysis. variables with p values < . in the univariable analysis were included in the multivariable analysis. a mixed model with the subject being random effect was used to estimate the relationships between respiratory pathogens and to take into account the repeated measures for pathogen carriage for each subject. regarding risk factors for lrti, the outcome was possible lrti symptoms reported during the hajj. the independent factors were sociodemographic characteristics (gender, ≥ years), chronic respiratory disease, smoking status, bmi classification; vaccination against influenza, vaccination against ipd, respiratory virus or bacteria and dual carriage during the hajj. unadjusted associations between multiple factors and possible lrti symptoms were examined using univariable analysis. variables with p values < . in the univariable analysis were included in the multivariable analysis. a logistical regression model was used to estimate factors' adjusted odds ratios regarding possible lrti. the results were presented by odds ratio (or) with a % confidence interval ( %ci). results with a p value ≤ . was considered to be statistically significant. the protocol was approved by the aix-marseille university institutional review board ( july ; reference no. -a - ). the study was performed according to the good clinical practices recommended by the declaration of helsinki and its amendments. all participants provided their written informed consent. the study included pilgrims. the sex ratio of the population was : . and the median age was years with . % of pilgrims aged years and over. most pilgrims were born in north africa ( . %) and sub-saharan africa ( . %). there was a high prevalence of overweight ( . %), obesity ( . %), diabetes mellitus ( . %) and hypertension ( . %) and . % participants reported that they suffered from chronic respiratory disease. in line with french recommendation, / pilgrims ( . %) had an indication for vaccination against ipd [ , ] ( table ) . a total of / ( . %) pilgrims reported that they had been vaccinated against influenza in the past year. only / ( . %) pilgrims with an indication for ipd had been vaccinated against pneumococcal disease (pcv- ) in the past five years. regarding non-pharmaceutical preventive measures, / ( . %) pilgrims reported using face masks during the pilgrimage. also, / ( . %) and / ( . %) pilgrims reported washing their hands more often than usual and using hand gel, respectively during the pilgrimage. finally, / ( . %) reported using disposable handkerchiefs during the hajj. a total of / ( . %) pilgrims presented at least one respiratory symptom during their stay in ksa. a cough and rhinitis were the most frequent symptoms affecting . % and . % of participants. over half of the pilgrims ( . %) reported expectoration and . % reported a dry cough. voice failure was reported by . %, fever by . % and ili by . % of participants. antibiotic use for rtis was reported by . % pilgrims. only one ( . %) pilgrim was hospitalized in ksa. regarding possible lrti symptoms, / ( . %) participants reported a productive cough without nasal or throat symptoms. in addition, / ( . %), / ( . %) and / ( . %) pilgrims presented febrile dyspnoea, dyspnea and a febrile productive cough, respectively. at total of / ( . %) pilgrims with respiratory symptoms were still symptomatic at return. the mean time between arrival in ksa and the onset of symptoms was . ± . days (range = - days) (data not shown). most ill pilgrims presented the onset of respiratory symptoms when stationed at mecca with a second minor wave in mina (figure ). table shows the prevalence of the carriage of respiratory pathogens according to sampling time and figure show the dynamics of most prevalent pathogens over the study period. overall, / ( . %) of all samples tested positive for at least one pathogen. s. aureus was the pathogen most frequently isolated with . % of all samples testing positive. high positivity rates were also observed for h. influenzae ( . %), k. pneumoniae ( . %), hrv ( . %) and m. catarrhalis ( . %). only . % of the samples were positive for coronaviruses and . % for s. pneumoniae. very few samples tested positive for influenza viruses. no case was positive for hpiv. of the positive samples, the proportion that was positive for more than one pathogen was . % ( / ). a total of / ( . %) samples were positive for two pathogens, / ( . %) for three pathogens, / ( . %) for four pathogens and / ( . %) for five pathogens (data not shown). in pre-travel samples, virus carriage was very low with only a few participants testing positive for hrv and hcov (< %). bacterial carriage was higher, notably for h. influenzae ( . %) m. catarrhalis ( . %) and s. aureus ( . %). k. pneumoniae and s. pneumoniae carriage were relatively low ( . % and . %, respectively). a dramatic increase in hrv carriage was observed on days and of the pilgrimage with prevalence times higher than that of pre-travel. hrv carriage decreased progressively in subsequent samples but was still eight times higher in post-hajj samples compared to pre-hajj. a seven-fold increase of hcov carriage was observed on days and that persisted on into days and of the pilgrimage and tended to slightly decrease in post-hajj samples. regarding bacteria, carriage of s. aureus increased by a factor of three on days and and decreased progressively in subsequent samples but was still double in post-hajj samples compared to pre-hajj. interestingly, the carriage curves of hrv and s. aureus were strictly parallel. m. catarrhalis carriage was about - % in pre-travel, days and and and samples and increased to % in post-hajj samples. k. pneumoniae carriage increased three-fold between pre-hajj and days and samples and slightly increased in subsequent samples. s. pneumoniae carriage increased constantly overtime with a seven-fold increase in post-hajj samples compared to pre-hajj. finally, h. influenzae carriage first decreased on days and and and by a factor . and then increased in post-hajj samples to a carriage rate which was higher than that of pre-hajj samples. table shows the factors that were independently associated with the carriage of respiratory pathogens on swabs from pilgrims. a positive association was observed between males and carriage of hrv and s. pneumoniae. chronic respiratory disease was also associated with s. pneumoniae carriage. finally, the use of disposable handkerchiefs was associated with a decreased carriage of h. influenzae. antibiotic intake ten days before each sampling was positively associated with hcov and k. pneumoniae carriage. regarding interactions between pathogens, we observed that hrv carriage and s. aureus carriage were mutually positively associated. the same applied to h. influenzae and m. catarrhalis carriage. pilgrims carrying s. pneumoniae were more likely also to carry m. catarrhalis. patients with a dual carriage of h. influenzae and s. pneumoniae were six times more likely also to be carrying s. pneumoniae. by contrast, m. catarrhalis carriage was associated with a reduced carriage of k. pneumoniae. table shows the results of multivariable risk factor analysis for possible lrti symptoms. chronic respiratory disease was associated with all possible lrti symptoms. obesity was associated with dyspnea. carriage of k. pneumoniae or m. catarrhalis-s. aureus or h. influenzae-rhinovirus combination was associated with a four-fold, -fold and eight-fold increase of dyspnoea prevalence, respectively. finally, m. catarrhalis-s. aureus dual carriage was associated with a five-fold increase in the prevalence of febrile dyspnea (table ). despite the recommendation to take individual preventive measures to prevent rtis [ , ] , these infections remain common among hajj pilgrims. overcrowding during the event is thought to increase the risk of the transmission of infectious diseases, but interaction between respiratory pathogens is probably one factor contributing towards the development of rtis. to our knowledge, no study on respiratory microbiota alteration among pilgrims during the hajj has been conducted. our results about the occurrence of rti symptoms are in line with previous results obtained regarding french pilgrims [ ] and others [ , ] . notably, we observed that rti symptoms occur soon after the pilgrims' arrival in mecca, with most symptoms starting between and days after arrival, corresponding to the period when pilgrims are stationed in mecca hotels and are visiting the grand mosque daily, where highly crowded conditions are common [ ] . we also confirmed that an overall increase in the carriage of respiratory viruses and bacteria can be seen when comparing pre-travel samples and post-hajj samples, as previously documented [ , , , [ ] [ ] [ ] . higher acquisition rates were observed for rhinovirus with a nine-fold increase when comparing pre-travel to post-hajj carriage and for s. pneumoniae with a seven-fold increase, but an increase was observed for all pathogens tested in this study. the unique design of our study with sequential systematic sampling at regular intervals allows for a better understanding of the dynamics of pathogen carriage during the pilgrimage. carriage rates of bacteria and viruses in this study are in line with those observed during recent studies conducted on french pilgrims and in pilgrims of other nationalities, using the same methods of detection [ , , [ ] [ ] [ ] . the acquisition of respiratory viruses and s. aureus occurred soon after arrival in saudi arabia and decreased gradually after days and . by contrast, s. pneumoniae and k. pneumoniae carriage increased progressively until the end of the visit, h. influenzae and m. catarrhalis carriage increased later, after an initial clearance. we hypothesize that the brutal acquisition of respiratory viruses upon arrival was the initial step that triggered subsequent changes in the relative abundance of resident bacteria [ ] that were already present in the nasopharynx of pilgrims. the apparent simultaneity of viruses and s. aureus carriage increase and the initial wave of respiratory symptoms, suggests that this pathogen association was responsible for the rtis that affected most pilgrims soon after arriving in mecca. the subsequent increase in resident bacteria that occurred during the second half of pilgrims' stays in saudi arabia appears to be contemporaneous with a second wave of respiratory symptoms, suggesting that these rtis were of bacterial origin. regarding interaction between respiratory pathogens, we observed a very clear pattern of positive association between the carriage of s. aureus and rhinovirus with acquisition curves which were strictly parallel. furthermore, an independent positive mutual association between the carriage of the two pathogens was evidenced in our multivariate model. several studies revealed a positive interaction between natural or experimental rhinovirus infection and s. aureus nasal carriers [ ] [ ] [ ] [ ] [ ] . these studies also underlined that rhinovirus infection may facilitate the propagation of s. aureus from staphylococcal carriers to the environment and the transmission of the bacterium between humans. among healthy persons who were experimentally infected by rhinovirus, the relative abundance of s. aureus first increased and then returned to its baseline level after the rhinovirus infection was cleared [ ] . these results suggest that changes in the composition of the respiratory microbiota following rhinovirus infection may play a role in the development of bacterial superinfection. morgene et al. proposed several potential mechanisms through which rhinovirus may increase bacterial infection [ ] . rhinovirus infection promotes pro-inflammatory cytokines and ifn production mainly through the activation of nfκb. in rhinovirus infected cells, the adherence of s. aureus was significantly higher compared to uninfected cells. the inflammation due to rhinovirus infection also increased cellular patterns that facilitate the adhesion and internalization of s. aureus within host cells [ ] . we also observed a parallel increase of h. influenzae and m. catarrhalis carriage in days and and post-hajj samples. an independent positive mutual association between the carriage of the two pathogens was evidenced in our multivariate model. dual carriage of h. influenzae and m. catarrhalis strongly associated with s. pneumoniae carriage which in turns associated with m catarrhalis carriage. these results are consistent with those of several studies conducted among children with upper rtis [ ] [ ] [ ] . in these studies, the competitive interaction between s. pneumoniae and h. influenzae was dependent on neutrophils and complement. the additional carriage of m. catarrhalis might alter the competitive balance between h. influenzae and s. pneumoniae [ ] . co-colonization of s. pneumoniae or h. influenzae with m. catarrhalis associating with increased risk of otitis media has been documented [ ] . using in vivo models, mixed species biofilms play a role in increasing the persistence of ear disease [ ] . other proposed mechanisms for positive associations between bacterial species include interspecies quorum sensing and passive antimicrobial resistance, which have been observed in experimental models of otitis media [ ] . finally, our model showed that m. catarrhalis carriage was negatively associated with k. pneumoniae carriage which, to our knowledge, has not previously been published. additionally, we found that the male gender was independently associated with an increase in rhinovirus and s. pneumoniae carriage. we have no explanation for this unexpected observation. the carriage of s. pneumoniae was higher among pilgrims with chronic respiratory disease which support the current french recommendations that vaccination against ipd be proposed to at-risk pilgrims [ ] . in one of our recent studies, pilgrims who were vaccinated against ipd were seven time less likely to harbour s. pneumoniae after the hajj compared to unvaccinated pilgrims [ ] . in this study, the use of disposable handkerchiefs was associated with a significant decrease in h. influenzae carriage. non-pharmaceutical individual preventive measures such as cough etiquette, hand hygiene, use of a face mask, disinfectant gel and disposable handkerchiefs are recommended for hajj pilgrims [ ] . nevertheless, the effectiveness of these measures has been poorly investigated and available results are contradictory [ ] . the apparent association between antibiotic use and hcov and k. pneumoniae carriage warrants further investigation to better explore this unexpected observation. we also confirm that chronic respiratory disease is a risk factor for lrti. we also evidenced the role of respiratory bacteria including k. pneumoniae and m. catarrhalis-s. aureus association and h. influenzaerhinovirus association in the occurrence of possible lrti symptoms. this reinforces the need for antibiotic use in case of lrti symptoms [ ] . our study has some limitations. the study was conducted among french pilgrims only with a relatively small sample size and cannot be generalized to all pilgrims. qpcr used to detect respiratory pathogens does not distinguish between dead and living micro-organisms. only a small number of respiratory pathogens were investigated. respiratory bacteria serotypes were not investigated. influenza viruses were not included in the model due to low carriage rates. in addition, we did not recruit a control group of individuals that did not participate to the hajj. a study addressing interactions between respiratory pathogens in the general french adult population could be of interest. nevertheless, our study is the first study on the dynamics of and interaction between the respiratory pathogens that are most frequently isolated among hajj pilgrims. our data suggest that rtis at the hajj are a result of complex interactions between a number of respiratory viruses and bacteria. further studies aimed at studying the respiratory microbiota with tools allowing the identification of larger numbers of pathogens will be necessary to better elucidate these ecological changes and their potential role in the occurrence of respiratory symptoms. mass gatherings medicine: public health issues arising from mass gathering religious and sporting events travel reported by pilgrims from marseille, france before and after the hajj morbidity and mortality amongst indian hajj pilgrims: a -year experience of indian hajj medical mission in massgathering medicine the inevitable hajj cough: surveillance data in french pilgrims infectious diseases and mass gatherings a systematic review of emerging respiratory viruses at the hajj and possible coinfection with streptococcus pneumoniae acquisition of respiratory viruses and presence of respiratory symptoms in french pilgrims during the hajj: a prospective cohort study associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections medically important bacterial-fungal interactions nasal carriage as a source of staphylococcus aureus bacteremia. study group impact of the hajj on pneumococcal carriage and the effect of various pneumococcal vaccines effect of conjugate pneumococcal vaccine followed by polysaccharide pneumococcal vaccine on recurrent acute otitis media: a randomised study haut conseil de la santé publique. bull epidemiol hebdo. recommandations sanitaires pour les voyageurs ministère des affaires sociales, de la santé et des droits des femmes influenza and the hajj: defining influenza-like illness clinically antibiotic use for respiratory infections among hajj pilgrims: a cohort survey and review of the literature rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than , routine pcr tests mass gathering and globalization of respiratory pathogens during the hajj quantitative detection of moraxella catarrhalis in nasopharyngeal secretions by real-time pcr circulation of respiratory viruses among pilgrims during the hajj pilgrimage respiratory viruses and bacteria among pilgrims during the miniopticon system instruction manual for miniopticon real-time pcr detection system with cfx manager spfware expected immunizations and health protection for hajj and umrah -an overview non-pharmaceutical interventions for the prevention of rtis during hajj pilgrimage bacterial respiratory carriage in french hajj pilgrims and the effect of pneumococcal vaccine and other individual preventive measures: a prospective cohort survey a cohort study of the impact and acquisition of nasopharyngeal carriage of streptococcus pneumoniae during the hajj impact of the hajj on pneumococcal transmission respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia airborne dispersal as a novel transmission route of coagulase-negative staphylococci: interaction between coagulase-negative staphylococci and rhinovirus infection gesundheit!" sneezing, common colds, allergies, and staphylococcus aureus dispersion preventing the airborne spread of staphylococcus aureus by persons with the common cold: effect of surgical scrubs, gowns, and masks dispersal of staphylococcus aureus into the air associated with a rhinovirus infection coinfection can trigger multiple pandemic waves changes in microbiota during experimental human rhinovirus infection staphylococcus aureus colonization and non-influenza respiratory viruses: interactions and synergism mechanisms carriage of streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, and staphylococcus aureus in indonesian children: a cross-sectional study microbial interactions during upper respiratory tract infections nasopharyngeal bacterial interactions in children bacterial and viral interactions within the nasopharynx contribute to the risk of acute otitis media residence of streptococcus pneumoniae and moraxella catarrhalis within polymicrobial biofilm promotes antibiotic resistance and bacterial persistence in vivo divergent mechanisms for passive pneumococcal resistance to β-lactam antibiotics in the presence of haemophilus influenzae ministère des affaires sociales, de la santé et des droits des femmes no potential conflict of interest was reported by the authors. this study was supported by the institut hospitalo-universitaire (ihu) méditerranée infection, the french national research agency under the "investissements d'avenir" programme, reference anr- -iahu- , the région provence alpes côte d'azur and european feder primi funding. key: cord- - zhmmvdg authors: to, kelvin kw; lu, lu; yip, cyril cy; poon, rosana ws; fung, ami my; cheng, andrew; lui, daniel hk; ho, deborah ty; hung, ivan fn; chan, kwok-hung; yuen, kwok-yung title: additional molecular testing of saliva specimens improves the detection of respiratory viruses date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: zhmmvdg emerging infectious diseases in humans are often caused by respiratory viruses such as pandemic or avian influenza viruses and novel coronaviruses. microbiological testing for respiratory viruses is important for patient management, infection control and epidemiological studies. nasopharyngeal specimens are frequently tested, but their sensitivity is suboptimal. this study evaluated the incremental benefit of testing respiratory viruses in expectorated saliva using molecular assays. a total of hospitalized adult patients with suspected respiratory infections were included. their expectorated saliva was collected without the use of any special devices. in the first cohort of patients whose nasopharyngeal aspirates (npas) tested positive for respiratory viruses during routine testing, the viral load was measured using quantitative reverse transcription pcr. seventeen percent of the patients ( / ) had higher viral loads in the saliva than in the npa. the second cohort consisted of patients whose npas tested negative for respiratory viruses using a direct immunofluorescence assay. their npa and saliva specimens were additionally tested using multiplex pcr. in these patients, the concordance rate by multiplex pcr between npa and saliva was . %. multiplex pcr detected viruses in saliva samples from patients, of which nine ( . %) had at least one virus that was not detected in the npa. decisions on antiviral or isolation precautions would be affected by salivary testing in six patients. although npas have high viral loads and remain the specimen of choice for most patients with respiratory virus infections, supplementary molecular testing of saliva can improve the clinical management of these patients. viruses play important roles in respiratory tract infections. [ ] [ ] [ ] [ ] recently, several novel respiratory viruses have emerged, including the pandemic influenza virus a(h n )pdm , the avian influenza viruses a(h n ) and a(h n ), [ ] [ ] [ ] and the middle east respiratory syndrome (mers) coronavirus. the novel avian influenza viruses and the mers coronavirus are associated with high mortality rates of over %. prompt and accurate detection of respiratory viruses is important for guiding antiviral treatment. for influenza virus infection, earlier administration of neuraminidase inhibitors is associated with faster resolution of symptoms in randomized clinical trials and improved survival in retrospective studies. , hyperimmune intravenous immunoglobulin confers survival benefit for severe influenza only if administered within days after symptom onset. antibiotic usage can be reduced if a respiratory virus is detected without evidence of bacterial infection. early detection is also important for infection control and public health measures. nasopharyngeal aspirate (npa), nasopharyngeal swabs (nps) (including flocked swabs), and nasal or throat swabs/washes are the recommended upper respiratory tract specimen types for diagnostic testing of respiratory viruses. , nasopharyngeal specimens are usually considered to have the highest detection rate for respiratory viruses. nasopharyngeal specimens are often used as the only specimen type in routine clinical practice and in many surveillance studies for the detection of respiratory viruses. however, in studies that have tested multiple specimen types, nasopharyngeal specimens have been found to be negative in some patients with respiratory virus infections. [ ] [ ] [ ] [ ] [ ] sputum or other lower respiratory tract specimens may contain a higher viral load for some patients, which will improve the detection of viruses. jeong et al showed that nasopharyngeal swabs were negative in % of adult patients with sputum positive for respiratory viruses. however, many patients do not have sputum production or cannot expectorate good quality sputum. additionally, the collection of tracheal or bronchial specimens involves invasive procedures that are associated with significant discomfort and risk to the patient and pose a risk to healthcare workers. , saliva can be easily provided by patients without any invasive procedures. however, saliva is rarely used for the detection of respiratory viruses, because it is generally considered to have inferior sensitivity compared with other respiratory tract specimens. muc b, salivary gp- , histatins, and human neutrophil defensins in saliva have been shown to neutralize influenza a virus. moreover, saliva is not a suitable specimen type for direct immunofluorescence assay (dfa) because it does not contain a sufficient amount of infected respiratory epithelial cells. recently, there has been renewed interest in using saliva for the detection of respiratory viruses with molecular assays. , [ ] [ ] [ ] [ ] some of these studies showed that the detection rate of respiratory viruses was actually higher for saliva than for nasopharyngeal specimens. however, these studies were conducted either in children or among adult patients with mild symptoms who did not require hospitalization. , some studies only evaluated the detection of influenza virus. , furthermore, many of these studies used special collection devices that are not usually available in most hospitals. the utility of saliva for the detection of respiratory viruses among hospitalized adult patients has not been comprehensively studied. the current study sought to evaluate the benefit of testing expectorated saliva in addition to npa among adult patients hospitalized for a suspected respiratory tract infection. the aim of the first part of the study was to assess the difference in the viral load between npa and saliva using quantitative pcr with reverse transcription (rt-pcr). the aim of the second part of the study was to evaluate the incremental benefit of testing saliva in addition to npa using a commercially available molecular assay. as the current study was designed to enable the easy application of our saliva specimen collection method to any clinical setting, we asked the study participants to provide saliva specimens by simple expectoration into a sterile bottle without using any special collection devices. this study was a -year prospective study conducted between march , and february , in queen mary hospital, which is a teaching hospital in hong kong with beds. this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster. written informed consent was obtained from all study participants. patients were eligible for recruitment if they were hospitalized adult patients aged years or above with a suspected respiratory tract infection and had npa samples obtained for respiratory virus testing. saliva specimens were collected from the enrolled patients when the result of the routine clinical testing for respiratory viruses in the clinical microbiology laboratory of queen mary hospital was known. therefore, a patient's saliva specimen was always collected after the npa specimen. patients were excluded from the study if they were discharged from the hospital before enrollment, were unable to provide a saliva specimen, or refused or were unable to provide written informed consent. the first part of the study consisted of patients whose npa samples tested positive for respiratory viruses by dfa or the influenza a virus m gene by real-time rt-pcr during routine respiratory virus testing in our clinical microbiology laboratory ( figure ). the viral load was determined in both the npa and the saliva samples using quantitative rt-pcr (qrt-pcr). during the first phase ( march to june ), only patients who tested positive for influenza a virus were included. during the second phase ( june to february ), patients who tested positive for any respiratory viruses were included. the second part of the study consisted of patients whose npa specimens either tested negative for respiratory viruses by dfa or had insufficient nasopharyngeal columnar epithelial cells (npcs) in the npa sample for dfa during routine clinical testing ( figure ). insufficient npcs was defined as o npcs in the entire well. multiplex pcr was used to test for respiratory viruses in both the npa and the saliva samples. patient data concerning demographics, underlying diseases, clinical findings and radiological changes were recorded in a predesigned database. the charlson comorbidity score was calculated. pneumonia was defined by radiological evidence of new or increased pulmonary infiltrate in a chest radiograph and at least one of the following symptoms (cough with or without sputum production, dyspnea, tachypnea, or pleuritic chest pain) plus one auscultatory finding or one sign of infection (core body temperature °c, shivers, leukocyte count cells/μl or o cells/μl) as described previously. routine respiratory virus testing in the clinical microbiology laboratory npa samples were collected in viral transport medium (vtm) as described previously. routine testing for respiratory viruses was performed using antigen detection by dfa (d ultra dfa respiratory virus screening and identification kit, diagnostic hybrids, inc., quidel, san diego, ca, usa), which included influenza a virus, and influenza b virus, parainfluenza viruses - , respiratory syncytial virus (rsv), human metapneumovirus (hmpv) and adenovirus. from march to april (during the peak influenza a virus season in hong kong), real-time rt-pcr for the influenza a virus m gene was performed for patients admitted to the general medical ward as described previously. during this period, dfa was not performed if the npa samples tested positive for influenza a virus using rt-pcr. patients were instructed to expectorate saliva into a sterile container and ml of vtm was added to the container in the microbiology laboratory. the volume of saliva ranged between~ . and ml. figure study design. nasopharyngeal aspirate, npa; quantitative pcr with reverse transcription, qrt-pcr. a routine clinical testing was performed using antigen detection by the dfa, which included the influenza a and b viruses, parainfluenza virus types - , respiratory syncytial virus, human metapneumovirus and adenovirus. from march to april (during the peak influenza a virus season), monoplex real-time rt-pcr for the influenza a m gene was performed for patients admitted to the general medical ward. b patients whose npa specimens either tested negative for respiratory viruses by dfa or had insufficient npcs for dfa during routine clinical testing. insufficient npcs is defined as o npcs in the entire well. quantitative rt-pcr for respiratory viruses total nucleic acid (tna) extraction was performed using the easy-mag instrument (biomerieux, boxtel, netherlands) as described previously. , npa or saliva specimens in vtm ( μl) were mixed with lysing buffer. after extraction, the nucleic acids were recovered using μl of elution buffer. qrt-pcr for influenza a virus detection was carried out using superscript iii platinum one-step rt-pcr reagents (invitrogen, carlsbad, ca, usa) as described previously with modifications. , , the reagent mixture ( μl) contained the × reaction mix, superscript iii rt/platinum taq mix, rox reagent, . μm of the forward and reverse primers, . μm of the probe and μl of tna as the template. the thermal cycling conditions were min at °c for reverse transcription, min at °c for rt inactivation/initial denaturation, and cycles of s at °c and s at °c. all reactions were performed using the steponeplus real-time pcr system (applied biosystems, foster city, ca, usa). the qrt-pcr for the detection of influenza b virus, parainfluenza viruses - , hmpv and respiratory syncytial virus was carried out using agpath-id one-step rt-pcr reagents (applied biosystems). the reagent mixture ( μl) contained × rt-pcr buffer, × rt-pcr enzyme mix, . μm of the forward and reverse primers, . μm of the probe and μl of tna as the template. the thermal cycling conditions were min at °c for reverse transcription, min at °c for rt inactivation/initial denaturation, and cycles of s at °c and s at °c. all reactions were performed using the lightcycler real-time pcr system (roche, basel, switzerland). the primer and probe sequences are shown in table . the multiplex pcr for respiratory viruses was performed using the nxtag respiratory pathogen panel (ivd) (luminex, austin, tx, usa) according to the manufacturer's instructions. respiratory viruses detected by this assay included influenza a virus, influenza b virus, rsv a and b, enterovirus/rhinovirus (ev/rv), parainfluenza viruses - , hmpv, adenovirus, coronaviruses hku , nl , e and oc , and human bocavirus. extracted tna was added to pre-plated lyophilized bead reagents. the reaction was amplified via rt-pcr, and the reaction product underwent bead hybridization within the sealed reaction well. the hybridized and tagged beads were sorted and read on the magpix instrument, and the signals were analyzed using the nxtag respiratory pathogen panel assay file for the synct software (luminex, austin, tx, usa). the mann-whitney u-test and fisher's exact test were used for comparisons of continuous and categorical variables, respectively. the wilcoxon matched-pairs signed rank test was used for the comparison between the npa and saliva viral loads. correlations between the viral loads in the npa and saliva specimens were determined using spearman's correlation coefficient. mcnemar's test was used to compare the positivity rates between the npa and the saliva specimens. log-transformed data were used for statistical calculations of the viral load. the statistical analysis was performed using spss . (ibm corp., armonk, ny, usa) or graphpad prism . (graphpad software, la jolla, ca, usa). first cohort: comparison of the viral loads between the npa and the saliva samples the first cohort consisted of patients with known respiratory virus infection (table and supplementary table s ). the median age was years, with a range from to years. the median charlson comorbidity score was . forty ( . %) patients had pneumonia, two patients ( . %) required admission to the intensive care unit, and two patients ( . %) died. most of the saliva specimens were collected o days after the collection of the npa sample ( . %, / ). among this first cohort of patients with a respiratory virus detected in the npa sample during routine clinical testing, the qrt-pcr for the same virus was positive in the npa specimens of all patients and in the saliva specimens of . % ( / ) of the patients ( table ) . among the patients who tested positive for influenza a virus, the npa specimens of patients were tested by influenza a virus m gene rt-pcr only during the routine clinical service. the saliva sample was positive by qrt-pcr in out of these patients ( . %). although the median viral load was significantly greater in the npa than in the saliva samples ( . vs . log copies per ml, po . ), . % ( / ) of the patients had a higher viral load in the saliva than in their npa sample (figure a) . no significant correlation was found between the viral load in the npa and that in the saliva (p = . ). among the patients with influenza a or influenza b virus infection, no significant difference was found in the median salivary viral load detection of respiratory viruses in saliva kkw to et al between the patients with saliva collected before and those with saliva collected after oseltamivir treatment ( . vs . log copies per ml, p = . ; figure b ). no significant difference in the median viral load was detected in the saliva specimens between patients with pneumonia and those without pneumonia ( . vs . log copies per ml, p = . ; figure c ). respiratory viruses were detected in the saliva specimens of ( . %) of the patients whose npa specimens tested negative by nxtag respiratory pathogen panel, including three patients with influenza a virus, two patients with hmpv, two patients with ev/rv and one patient with coronavirus oc (table ) . for the patients whose npa specimens tested positive by the nxtag respiratory pathogen panel, one patient had an additional virus species detected in her saliva (patient in table ). potential changes in the antiviral treatment or infection control practice would be possible for six of the nine patients with additional viruses detected in their saliva. the three patients with influenza a virus infection could have been given neuraminidase inhibitor, whereas the three patients with hmpv should have been placed under contact precaution. table detection of viruses using quantitative real-time reverse transcription pcr in the first cohort of patients [ ] [ ] [ ] [ ] [ ] which have greatly enhanced the sensitivity of the detection of respiratory viruses. however, very few studies have reported an evaluation of specimen types to improve the detection rate. this study evaluated the use of saliva in addition to npa for the diagnosis of respiratory viral infections in two parts. in the first part of this study, saliva had a higher viral load than npa in . % of the patients who tested positive for respiratory viruses by dfa or influenza a virus by rt-pcr in their npa samples. in the second part of this study, saliva specimens tested positive in eight ( . %) of the patients whose npa specimens tested negative by multiplex pcr. among the patients whose npa specimens were unsuitable for dfa or tested negative by dfa but positive by multiplex pcr, one patient ( . %) had an additional respiratory virus detected in her saliva specimen using multiplex pcr. importantly, a change in antiviral treatment or isolation precaution could have occurred in six out of these patients with additional respiratory viruses detected in their saliva. in some previous studies, saliva was obtained using a dropper or a special sponge on a stick. the advantage of using these devices is that the amount of saliva is standardized, and the saliva is less likely to be contaminated by sputum. however, these collection devices are not usually available in a general medical ward setting. furthermore, the collection of saliva with these devices requires help from healthcare workers. in the present study, we simply asked the patients to expectorate saliva into the standard sterile sputum containers used routinely in our hospital. as no special collection device is required, the use of expectorated saliva can be implemented easily in daily clinical practice. one possible concern is that the expectorated saliva may be mixed with sputum, which may contain a higher viral load than saliva. however, because the aim of using saliva is to increase the detection rate, this perceived limitation is actually favorable. in real-life clinical practice, testing both npa and saliva simultaneously is too costly. a more cost-effective approach is to test saliva only if the npa result is negative. hence, in the current study, we mimic this situation by collecting saliva only when the npa result is available. our previous studies on influenza viruses showed that patients usually had higher viral loads in their npa upon hospital admission. , , therefore, saliva collected later may have a lower viral load and may result in a lower sensitivity of detection. although this study was primarily designed to evaluate the incremental benefit of testing saliva in addition to npa, the results provided some insights into the potential use of testing saliva alone. among npa-positive specimens, the detection rate in saliva was . % ( / ) in the first part of the study and . % ( / ) in the second part of the study. notably, the detection rate of respiratory viruses by multiplex pcr in the second part of the study was slightly higher in saliva ( . %) than in npa ( . %). kim et al also showed that multiplex pcr of saliva and nps specimens had similar detection rates. therefore, the use of saliva alone may not be inferior to the use of npa if only a single type of specimen is tested. testing saliva over npa has many advantages. first, collecting saliva rather than npa avoids patient discomfort. second, the collection of saliva is suitable for patients for whom the collection of nasopharyngeal specimens is contraindicated, such as patients with severe bleeding tendency. third, patients can provide saliva specimens after simple instruction, whereas the collection of nasopharyngeal specimens must be performed by healthcare personnel. this approach would reduce the delay in specimen collection. finally, the collection of saliva does not require any special infection control precautions and can be performed in any clinical setting with standard precautions. by contrast, the procedures for the collection of nasopharyngeal specimens are potentially aerosolgenerating and therefore pose significant risks to healthcare workers and other patients. some health authorities have recommended that nasopharyngeal specimens should be collected in a negative pressure isolation room for patients with suspected mers coronavirus or novel influenza viruses. sputum is also a non-invasive specimen that has been used for the detection of respiratory viruses, especially in patients with pneumonia. , , however, many patients with respiratory virus infection do not have sputum production or cannot expectorate good quality sputum. by contrast, saliva specimens can be obtained much more easily than sputum specimens. dfa is routinely used in our clinical setting for the diagnosis of respiratory virus infections. the advantages of dfa include a relatively low cost, rapid results and simultaneous detection of multiple viral pathogens. however, the sensitivity of dfa is low compared with molecular assays. during the influenza pandemic, the sensitivity of dfa ranged from % to %. dfa also has a low sensitivity for influenza a h n virus infection. another disadvantage of dfa is that it is not suitable for saliva specimens, because dfa can only detect viral antigens that are present inside infected cells, which are not present in the saliva. therefore, to avoid bias between different tests, we used a multiplex pcr panel to test both the npa and the saliva specimens. although the multiplex pcr panel used in the second cohort (the nxtag respiratory pathogen panel) is more sensitive than dfa, it has lower sensitivity than real-time rt-pcr assays. the sensitivity of the nxtag respiratory pathogen panel ranged from . % to % compared with real-time pcr or rt-pcr. therefore, some patients with respiratory virus infection may be missed even when npa and saliva specimens are tested with this multiplex pcr panel. this study has several limitations. first, our saliva collection method is not suitable for patients who cannot expectorate saliva, such as patients who are unconscious. for these patients, suction aspiration of saliva is required. second, in the first cohort, we only evaluated viruses included in the routine dfa or influenza a virus rt-pcr. other important respiratory viruses, such as rhinoviruses and coronaviruses, need to be further evaluated. third, the number of specimens of each virus species is too small to compare the differences in sensitivity for specific virus species. although the viral load in the saliva is lower than the viral load in the npa for most patients, we have shown that testing both expectorated saliva and npa can significantly improve the detection of respiratory viruses compared with testing of npa alone. saliva should be obtained from patients with a suspected respiratory virus infection but a negative test for respiratory viruses. furthermore, saliva should be evaluated as the specimen type in a diagnostic testing for novel respiratory 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development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses the natural viral load profile of patients with pandemic influenza a(h n ) and the effect of oseltamivir treatment flocked nasal swab versus nasopharyngeal aspirate in adult emergency room patients: similar multiplex pcr respiratory pathogen results and patient discomfort recommended personal protective equipment (ppe) in hospitals/clinics for suspected or confirmed cases with middle east respiratory syndrome (mers) under different response levels partial comparison of the nxtag respiratory pathogen panel assay with the luminex xtag respiratory panel fast assay v and singleplex real-time polymerase chain reaction for detection of respiratory pathogens key: cord- - k jucae authors: zhang, qingshui; cao, yanxin; wang, jun; fu, guanghua; sun, mengxu; zhang, lijiao; meng, li; cui, guolin; huang, yu; hu, xueying; su, jingliang title: isolation and characterization of an astrovirus causing fatal visceral gout in domestic goslings date: - - journal: emerg microbes infect doi: . /s - - - sha: doc_id: cord_uid: k jucae astroviruses are recognized as a leading cause of gastroenteritis in humans and animals. they are also associated with extra-intestinal diseases, such as hepatitis in ducklings, nephritis in chickens, and encephalitis in cattle. in february , a fatal infection of goslings characterized by visceral urate deposition was reported in the shandong province, china. our systematic investigation led to the isolation of an astrovirus, designated aastv/goose/chn/ /sd , and similar disease was reproduced by experimental infection of healthy goslings, fulfilling koch’s postulates. the isolated astrovirus replicated well and resulted in % mortality of goose embryos. complete genome sequence analysis revealed that the isolate was genetically distinct from known astroviruses and closely related to members of the avastrovirus genogroup ii. experimental infection showed that the isolate was highly pathogenic in goslings, causing clinical signs, growth repression and in many cases mortality. histopathological examination indicated that lesions occurred mainly in the kidneys of infected birds. however, virus-specific genomic rna was detected in all representative tissues, and virus shedding was detected up to days after inoculation, suggesting that the isolate was able to spread systemically and replicate efficiently in vivo. collectively, our study demonstrates, for the first time, the etiological role of a genetically distinct astrovirus in the fatal infection of goslings. astroviruses (astvs) are non-enveloped, positive-sense, single-stranded rna viruses belonging to the astroviridae family. currently, two genera: namely mamastrovirus and avastrovirus are distinguished within this family. the genus mamastrovirus includes astrovirus species isolated from humans and a number of mammals. isolates originated from avian species, such as turkey, chickens, ducks, and other birds are classified into the genus avastrovirus , . astvs have been detected in humans and a variety of animal species, including nonhuman primates, other mammals and avian species [ ] [ ] [ ] . their genomes are . - . kb in length, consisting of a ′untranslated region (utr), three open reading frames (orfs), a ′-utr and a poly (a) tail . the high degree of genetic diversity among astvs and their recombination potential signify their capacity to cause a broad spectrum of diseases in multiple host species , , . human classical astvs are a frequent cause of acute gastroenteritis in young children and the elderly, occasionally with encephalitis . in poultry, astv infections have been found to be associated with multiple diseases, such as poult enteritis mortality syndrome, runting-stunting syndrome of broilers, white chick syndrome, kidney and visceral gout in broilers and fatal hepatitis of ducklings, leading to substantial economic losses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . increasing evidence indicates that there is a high degree of cross species transmission of astvs between domestic birds, and even the potential to infect humans . by comparison, fewer astv infection cases have been described in domestic goose flocks. bidin et al. reported the detection of avian nephritis virus infection in croatian goose flocks and provided evidence that this astv was associated with stunting and prehatching mortality of goose embryos. studies to detect astv genomes from the clinical samples of geese suggested that these viruses might distribute widely among goose flocks, as seen in other poultry flocks , . in february , an outbreak of disease was reported in a goose farm in weifang, shandong province, china. affected flocks (containing - goslings) experienced continuous mortality rates ranging from to % during the first weeks of the outbreak despite antibiotic and supportive treatment. we conducted a systematic investigation to identify the causative agent of this disease and report here the isolation and characterization of a genetically distinct avian astv. the pathogenicity of this virus was evaluated by experimental infection of goslings. in the field, affected goslings displayed signs of depression and were observed sitting alone (fig. a) . the palpebra tertia of some of the goslings showed an obvious gray-white cloudy appearance (fig. b) . death occurred from when the goslings were - days old, and peaked at - days old; then the mortality rate decreased gradually to the end of the third week. a common feature at postmortem was visceral urate deposition on the serous surfaces of the heart, liver and kidney (fig. c, d) . distended bile sacs with abundant urate particles were also observed (fig. e) . virulent bacteria were not isolated and tissue samples were negative by pcr for goose parvovirus, goose hemorrhagic polyomavirus, reovirus, or tembusu virus. however, a dna fragment was amplified from the rna sample extracted from the pooled spleen, liver and kidney tissues using pan-astv rt-pcr targeting the astv rnadependent rna polymerase (rdrp) gene . sequence and phylogenetic analysis of the amplified rdrp gene with other known astv sequences retrieved in the genbank database showed that the detected virus could be assigned to the subgroup . within the avastrovius group , with the closest relationship to the astrovirus detected from dropping samples of northern shovelers (anas clypeata) in hong kong (fig. ) . however, the nucleotide sequence of the rdrp gene had ≤ . % similarity to the sequences of other astroviruses within avastrovirus group , suggesting that the virus was genetically distinct from known avastroviruses. therefore, the isolation of astv was initiated by inoculating tissue samples into goose embryos. for the first inoculation, significant thickening of the embryo's chorioallantoic membrane was noted although no death occurred by days post inoculation (dpi). the subsequent passage of the isolate caused - % mortality of the embryos by dpi. the dead embryos exhibited severe subcutaneous edema and hemorrhages with necrotic foci in the liver (fig. ). using the gene specific rt-pcr, the astv was consistently detected in the allantoic fluids. quantal assays showed that the infectious virus titers of the embryo allantoic fluid increased from × eld /ml for the fourth passage to × . eld /ml for the th passage, indicating that the isolates adapted to the goose embryo culture system. therefore, the isolate was designated aastv/goose/ chn/ /sd (sd hereafter) as proposed by martella et al. . the complete genome of the sd was identified by sequencing of the rt-pcr products and was submitted to the genbank database under accession number mf . the genome was nucleotides (nt) in length with similar gene organization to other known avastroviruses, consisting of a ′-utr of nt, three sequential orfs (orf a, orf b and orf ), a ′-utr of nt and a poly (a) tail stretching nt (fig. a) . orf a of the isolate was nt long, encoding a polypeptide of amino acids (aa) with . - . % identity to corresponding regions of other known avian astvs as determined by blast analysis ( table ). the predicted nonstructural protein contained a trypsin-like peptidase domain as revealed by pfam analysis with a serine protease motif at position (gnsg), a nuclear localization signal motif at position (kkkgktk), and four predicted transmembrane domains. as is the case with other known avian astvs, there was an overlapping region between orf a and orf b ( - nt), which contains the highly conserved ribosome frameshift sequence ( ′-aaaaaac- ′) and a downstream hairpin structure ( - nt) as predicted by rna folding analysis. orf b was nt long and was predicted to encode a rna-dependent rna polymerase. there was an nt spacer between the stop codon of orf b and the start codon of orf . orf was nt long encoding a capsid protein of aa. a stem-loop-ii-like (s m) motif consisting of nt was revealed adjacent to nt of orf in the ′-utr by rfam analysis. to determine the potential genetic mutation(s) that might occur during the goose embryo passage, the initial virus genome was sequenced using the total rna extracted from the clinical case tissue homogenate. nucleotide differences between the initial virus genome and that of the fourth embryo-passaged isolate was shown in table s . a single-mutation exhibited in the orf gene of the adapted isolate, leading to the amino acid change from r to q . the potential effect of the mutation on the virus adaptation need to be further evaluated. the complete genome sequence of astv sd had the highest similarity to those of turkey astv (tastv- ) strains, at the level of . - . % (representative tastv- va/ in table ). the next was the duck astrovirus- (dastv- ) sl , with . % nucleotide identity. phylogenetic analysis of the full-length sequences showed that the sd formed a sister clade neighboring dastv- and tastv- in the avastrovirus genogroup ii (fig. b) . further analysis with the complete amino acid sequence of rdrp (fig. c ) and the capsid protein ( fig. d ) revealed close alignment and closely matched phylogenetic trees. the pairwise comparison of nucleotide and amino acid identities of the three orfs among the representative avastrovirus isolates was shown in table . based on the available complete sequences of avian astv strains, the amino acid of orf a, orf b, and orf of sd shared the highest identities of . - . %, . - . %, and (table s ). according to the species demarcation criteria in the genus avastroviruses (p-dist range between genotypes range between . and . ), the sd was grouped within the genotype consisting of tastv- , tastv- , and dastv- . however, the p-dist values between tastv- , tastv- , and dastv- included in this genotype was much lower, ranging between . and . (table s ). these results suggested that sd has a higher variability than those previously detected avastroviruses in the genotype. seven out of the infected goslings displayed signs of depression from to dpi. one bird died at , , and dpi, respectively, resulting in a mortality rate of % ( / ) during the experimental period. at necropsy, slight to moderately swollen kidneys were noted for the deceased birds (fig. a) . histologic examination revealed degeneration and necrosis of the epithelial cells of the tubules of the kidneys (fig. c ). neuronophagia and microgliosis was detected in the cortex of the cerebrum and the dying neuron was surrounded by satellite microglia (fig. e) . following embryo inoculation, the inoculated virus was re-isolated from the liver and kidney tissues and confirmed by rt-pcr. infected goslings exhibited signs of depression from dpi and this symptom persisted for - days. one bird died at dpi and severe urate deposition, similar to that seen in the field cases, was evident on the surface of the heart, liver, and kidney (fig. b) . for the three infected goslings killed at dpi, no evident gross lesion was noted in the visceral organs at postmortem. however, histologic examination revealed the presence of an eosinophilic proteinaceous substance in the renal tubules, and mild interstitial lymphocyte infiltration was noted in sample of two goslings ( / ) (fig. s ). astv rna were detected in the collected tissues of three goslings (fig. s ) , indicating that the isolate has a wide tissue tropism after infection. all tissues from the uninfected birds were normal. when the samples were tested by rt-pcr for virus shedding evaluation, the aastv specific rna was sequentially detected from the cloacal swabs of infected goslings from to dpi (fig. ). viral rna could still be detected in the liver and spleen when the infected birds were killed at dpi. neither viral shedding nor positivity in the tissue samples was detected in the uninfected control goslings during the experiment. infected goslings showed decreased body weight gain and the average body weight of infected birds was statistically significantly lower than that in the uninfected group from dpi to the end of the experiment (fig. ) . the average body weight in the infected group was ± g versus ± g in the control group at dpi, and ± g versus ± g at dpi, respectively. orally inoculation of goslings with the isolate resulted in depression of birds from to dpi. one gosling died at dpi with evident urate deposition on the surfaces of heart and liver at necropsy. another severely ill bird was killed humanely at dpi for animal welfare reasons. viral rna was detected by rt-pcr in the cloacal swabs from dpi (fig. s ) and growth depression was noticed in this group (fig. s ) . for the goslings infected by intranasal inoculation, no death occurred in the group, but virus shedding and growth depress was observed (fig. s & s ) . these results indicated that the isolate might infect goslings via oral and nasal routes, further demonstrating the infectivity of the isolate. several studies have reported the existence of astv in goose flocks , , but the prevalence and pathogenicity of astv among domestic geese remains poorly understood due to the lack of efficient in vitro culture techniques and diagnostic assays. in this study, aastv sd was astv infection occurred within the first days or week of life usually resulted in a worse outcome, as the agedependent pathogenicity of aastv has been reported . in this study, the mortality of young goslings caused by subcutaneous or oral inoculation indicated that aastv sd was highly pathogenic. the experiment was fairly a represent of the situation in the field, where susceptible goslings are exposed to astv soon after they are placed in contaminated houses. apart from mortality, avian astv infection can decrease feed intake and alter feed conversion efficiency, leading to growth repression. the decrease in body weight of infected goslings is a major concern as a % lighter body weight at dpi has a considerable economic impact. histologic examination revealed the presence of a proteinaceous substance in the renal tubules, indicating that aastv sd infection caused increased permeability of the kidney epithelia barrier. degeneration and necrosis of tubular epithelial cells found in the deceased goslings provided further evidence of kidney function damage. these results could explain the development of visceral urate deposition in infected goslings. increased epithelium permeability due to astv infection has been reported in both human and avian species , . extra-intestinal infection with nephritis has been reported in birds infected with chicken astrovirus and avian nephritis virus , . viral rna was found in all of the tissues sampled from the infected goslings killed on dpi, indicating that the goose astv has a wide tissue tropism and spread systemically after inoculation. virus shedding was detected by rt-pcr and persisted in the infected goslings for about days, further indicating that the virus replicated efficiently in vivo. it is interesting that encephalitis lesions were observed in the deceased goslings (data not shown), along with the detection of aastv sd rna in the brain tissue (fig. s ) . however, no neurological symptoms were noted in either the field cases or the experimentally infected goslings. the neurologic infection of aastv sd is worthy of further investigation since there are numerous reported cases of astv-associated encephalitis and meningitis in humans and mammals , . nonetheless, based on the limited number of goslings infected in present study, it is not likely to get accurate evaluation for the virulence of the isolate. the present work describes the isolation of the astrovirus aastv/goose/chn/ /sd from tissue samples of goslings dying from a disease characterized by visceral urate deposition. the successful reproduction of the disease by experimental infection demonstrates the etiological role of this aastv. based on the genetic analysis of the complete capsid region at amino acid level, the isolate should be assigned as a member within the genotype consisting of tastv- and dastv- strains. the high variability of the genomic sequence to other known astroviruses suggest more detailed antigenic investigations should be performed. for bacteriological diagnosis, liver, and kidney samples from dead goslings were first inoculated onto tryptic soy agar plates (bd science, md, usa) containing % fetal calf serum, and incubated at °c under an atmosphere with % co for h. then the spleen, liver, and kidney tissue were pooled and tested for the presence of goose parvovirus , goose hemorrhagic polyomavirus , astv , reovirus , and tembusu virus , respectively. to isolate astv, the kidney, spleen, and liver samples were homogenized with sterile phosphate buffered saline (pbs, ph . ) to a % suspension (w/v) and centrifuged at ×g, at °c for min. the supernatant was filtered using a syringe-driven filter unit with a pore size of . μm and the filtrate was inoculated into five -day-old goose embryos ( . ml/egg) via the chorioallantoic membrane route. embryos were incubated at °c and candled daily. embryos that died beyond h and those that survived until day after inoculation were chilled to °c overnight. the allantoic fluids were collected for a hemagglutination (ha) activity test performed by a standard method using % chicken red blood cells as an indicator, and then were subjected to additional passage in goose embryos. to determine the infectious titers of the th and th passage, the virus suspension was -fold serially diluted with pbs and inoculated into -day-old goose embryos via the chorioallantoic membrane route. the embryos were incubated for days at °c and the mean embryo lethal dose (eld ) of infectious virus was calculated using the reed-muench method . to sequence the complete genome of the isolate, total rna was extracted from the goose embryo allantoic fluids of the fourth passage using a viral rna kit (omega, ga, usa) and the cdna was synthesized using a reverse transcription system (promega, wi, usa) with random primers following the manufacturer's instructions. viral genomic fragments were amplified by pcr with primer sets designed against conserved regions of the astv sequences retrieved from the genbank database (table ) . pcr products were purified using gel extraction kit (omega, ga, usa) and ligated into peasy-blunt simple cloning vector (transgen biotech, beijing, china). the recombinant vector was transformed into competent escherichia coli trans-t (transgen biotech, beijing, china) and transformants containing the pcr amplified fragment were selected by pcr following the manufacturer's instruction. at least two representative transformants were subjected to bidirectional dna sequencing using applied biosystems abi (shanghai meiji biological medicine technology co., ltd. shanghai, china). the ′ and ′ ends of the viral genome were amplified using the ′/ ′ race kit (clontech, ca, usa) following the guidelines of the manufacturer. the initial complete genome was assembled and manually edited using the software contigexpress. based on the initial genome sequence, additional primer pairs were designed (table s ) , and pcr amplicons were sequenced to determine the genome. to evaluate the potential adaptive mutation (s) of the virus that might occur during the process of goose embryo passage, we sequenced the complete genome of initial virus using the total rna extracted from the clinical case tissue homogenate of kidney, spleen, and liver using the method described above. the genome sequence was compared with that of the isolate of fourth passage. nucleotide sequences of the virus genome and the deduced amino acids of the orfs were compared with known astv orf sequences retrieved from the genbank database. neighbor-joining trees of the complete genome nucleotide sequences, orf b and orf amino acid sequences were constructed using mega . software, with bootstrap values calculated from replicates. the mean amino acid genetic distance (p-dist) of the viral rdrp and capsid were calculated using mega . with a bootstrap test obtained from replicates. one-day-old goslings (anser anser domesticus) were obtained from a local hatchery. birds were raised in negative pressured isolators with ad libitum access to feed and water. three experiments were conducted to investigate the pathogenicity of the isolate. animal infection experiments were approved by the china agricultural university animal ethics committee. gosling experiment the aim of this experiment was to evaluate whether the isolate was pathogenic in goslings. thirteen -day-old goslings were infected by subcutaneous inoculation with . ml of the virus suspension prepared from the infected goose embryos at the fourth passage (containing approximately . × eld ). goslings inoculated with sterile pbs (uninfected control) were kept in a separate isolator. clinical signs and mortality were recorded for days. dead birds were necropsied immediately, and tissue samples of the heart, liver, spleen, kidney, lung, thymus, bursa, and brain were collected. a portion of these tissues were fixed in % neutral buffered formalin, embedded in paraffin, and - μm sections were cut and stained with hematoxylin and eosin (h&e). pieces of liver and kidney tissue were frozen and subjected to virus isolation. this experiment was designed to investigate the in vivo replication of the virus and its impact on the growth of infected birds. fourteen -day-old goslings were infected as in experiment . birds inoculated with sterile pbs were used as the uninfected control. the tissue distribution of the virus was analyzed in three infected goslings dpi. representative tissues samples were collected and subjected to histopathological examination and virus rna detection. for virus shedding detection, cloacal swabs were collected from both infected and control birds on , , , , , , and dpi. swabs were immersed in ml of × dulbecco's modified eagle's medium (gibco, ny, usa) and frozen at − °c. simultaneously, individual birds were weighed. the difference between the mean body weights of infected and uninfected control birds was tested by student's t-test. significant differences were defined by p-values < . (*), < . (**). to investigate the possible infection route, three groups of -day-old goslings were kept at separate isolator. birds were inoculated orally (n = ) or intranasally (n = ) at day old with the astrovirus isolate at the dose as experiment . five goslings were kept as uninfected control. mortality were checked daily. cloacal swabs were collected for virus shedding detection as described in experiment . tissue samples were prepared as a % suspension (w/ v) in pbs and were homogenized using beads in a highthroughput tissuelyser (nibo scientz biotechnology co., ltd., zhejiang, china). then, the homogenate was centrifuged at ×g for min at °c and μl of the supernatant was used for rna extraction. total rna was extracted and converted to cdna using the kits described above. pcr amplification was conducted using a set of specific primers (gastvf /gastvr , table ), targeting the orf gene of the isolate. for virus shedding detection, swab samples were vortexed and centrifuged. after centrifugation, μl of the supernatant was processed for rt-pcr detection in the same manner. astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions virus taxonomy th edn, family astroviridae the broad host range and genetic diversity of mammalian and avian astroviruses non-human primates harbor diverse mammalian and avian astroviruses including those associated with human infections molecular characterization of avian astroviruses genetic heterogeneity and recombination in human type astroviruses ecological drivers of virus evolution: astrovirus as a case study epidemiology of classic and novel human astrovirus: gastroenteritis and beyond viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus the role of type- turkey astrovirus in poult enteritis syndrome the isolation and characterisation of astroviruses from chickens detection and characterization of a new astrovirus in chicken and turkeys with enteric and locomotion disorders a seroprevalence investigation of chicken astrovirus infections chicken astrovirus detected in hatchability problems associated with 'white chicks role of chicken astrovirus as a causative agent of gout in commercial broilers in india detection of antibodies against turkey astrovirus in humans astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos complete genome sequence of a novel avastrovirus in goose circulation and phylogenetic relationship of chicken and turkeyorigin astroviruses detected in domestic ducks (anas platyrhynchos domesticus) novel astroviruses in insectivorous bats a novel group of avian astroviruses in wild aquatic birds analysis of the orf of human astroviruses reveals lineage diversification, recombination and rearrangement and provides the basis for a novel sub-classification system pathogenesis of astrovirus infection oral administration of astrovirus capsid protein is sufficient to induce acute diarrhea in vivo detection and characterization of avian nephritis virus in ducklings neurologic cinical signs in cattle with astrovirus-associated encephalitis isolation and molecular characterization of a new muscovy duck parvovirus from muscovy ducks in the usa pathological and epidemiological significance of goose haemorrhagic polyomavirus infection in ducks isolation and characterization of a reovirus causing spleen necrosis in pekin ducklings duck egg-drop syndrome caused by byd virus, a new tembusurelated flavivirus a simple method for estimating fifty percent end points key: cord- - rg n fr authors: he, biao; fu, yuhong; xia, shuai; yu, fei; wang, qian; lu, lu; jiang, shibo title: intranasal application of polyethyleneimine suppresses influenza virus infection in mice date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: rg n fr nan emerging and reemerging viruses that cause respiratory infectious diseases, such as severe acute respiratory syndromes coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and influenza viruses, are a significant threat to public health worldwide. although vaccines are the most effective strategy to prevent viral infection, vaccine development is a long process and may be effective only against the corresponding virus. therefore, it is essential to develop antiviral agents for intranasal administration as nonspecific prophylaxis against infection by emerging or reemerging respiratory viruses with epidemic or pandemic potential. cholera toxin (ct), which acts as a mucosal adjuvant to stimulate mucosal and systemic immune responses, has the potential for intranasal application as an immunotherapeutic against infection by respiratory viruses. however, ct can exacerbate lung pathology after influenza virus infection through the induction of il- , a potent proinflammatory cytokine, because co-administration of an il- ra neutralizing antibody with ct attenuates lung pathology and increases protection against influenza virus infection. therefore, we hypothesized that intranasal application of a mucosal stimulant that induces antiviral cytokines, except il- , may be effective against influenza virus infection. in this regard, polyethyleneimine (pei), a mucosal adjuvant, exhibits stronger mucosal adjuvanticity but induces much lower il- expression than cholera toxin subunit b (ctb). therefore, for the first time, we tested pei for its potential protective effects against influenza virus infection. ten-week-old specific-pathogen-free (spf) female balb/c mice were anesthetized with pelltobarbitalum natricum. then, μl of pbs containing μg of kd linear pei (polysciences, warrington, pa) or . μg of ctb (sigma-aldrich, st louis, mo, usa) or pbs alone as a control was intranasally administered twice at and h before challenge with ld influenza virus h n (a/pr/ / ). mouse body weights were monitored every day, and those with greater than % loss of their initial body weight were euthanized as described. as shown in figure a , the body weight of the mice in the pei-pretreated group remained stable from day to day after h n challenge and then gradually decreased until day , for a total loss of %, before recovering. by contrast, the body weights of the mice in the ctb-and pbs-pretreated groups decreased significantly beginning on day and reached losses of more than % by day and , respectively, after h n challenge. the final survival rate of the mice in the pei-pretreated group was %, whereas that of the mice in the ctb-and pbs-pretreated groups was % ( figure b) . we then examined the viral titers in mouse lungs as previously described and found that pei significantly reduced lung viral titers on day after h n challenge, whereas the viral titers in the lungs of mice in the ctb-and pbs-pretreated groups showed no significant differences ( figure c ). subsequently, we examined lung sections stained with hematoxylin and eosin as previously described. on day after h n infection, the pulmonary alveoli were relatively intact, and only a few inflammatory cells were observed in the lung tissues of mice in the pei-pretreated group. however, the lungs of mice in the ctb-and pbs-pretreated groups were filled with abundant inflammatory cells ( figure d ). these results suggest that intranasal application of pei has efficacy in protecting mice from challenge by influenza virus h n . to determine the efficacy of pei against another subtype of influenza virus, we pretreated mice with pbs containing pei or pbs alone and challenged them with ld influenza virus h n (a/guizhou/ / ). mice in the pei-pretreated group were fully protected against h n challenge, showing no significant body weight loss ( figure e ) and a % survival rate, whereas those in the pbs group lost more than % of their body weight and showed a % survival rate on day post-challenge ( figure f ). therefore, pei-mediated protection against influenza virus infection is not subtype-specific. to elucidate the mechanism of action of pei, we examined the rna levels of ifn-α , ifn-β, ifn-γ, gm-csf, ifitm and il- in the lungs of mice pretreated with pei, ctb, and pbs, respectively, before viral challenge using quantitative reverse transcription-pcr (qrt-pcr). as previously reported, some of these cytokines, such as interferon, gm-csf and ifitm , were effective in protecting against influenza infection. [ ] [ ] [ ] as shown in supplementary figure s , pei induced a significantly higher rna level of ifn-α than ctb or pbs. furthermore, the rna levels of gm-csf and ifitm elicited by pei were similar to those induced by ctb but much higher than those induced by pbs. the rna level of il- in mice pretreated with ctb, an il- -inducing adjuvant, was approximately -and -fold higher than that in mice pretreated with pei and pbs, respectively. these results suggest that ifn-α , gm-csf, and ifitm are 'good cytokines' because they act as protective mediators against influenza virus infection, whereas il- is a 'bad cytokine' that exacerbates pathology, primarily in the lung, after influenza infection. in summary, we demonstrated that pei, a mucosal stimulant for topical intranasal administration, is highly effective in preventing influenza virus infection. compared to the bacteria-produced toxin ctb, the chemically synthesized polymer pei is safer for mucosal application in humans. pei has been tested in several clinical trials for gene delivery in vivo, demonstrating a good safety profile. moreover, its low cost of production and abundance makes pei more suitable for urgent and widespread use during a time of influenza epidemic or pandemic. bacteria and their toxins tamed for immunotherapy mucosal pre-exposure to th -inducing adjuvants exacerbates pathology after influenza infection polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens putative suppressing effect of igg fc-conjugated haemagglutinin (ha) stalk of influenza virus h n on the neutralizing immunogenicity of fc-conjugated ha head: implication for rational design of ha-based influenza vaccines infection of influenza virus neuraminidase-vaccinated mice with homologous influenza virus leads to strong protection against heterologous influenza viruses protection from pulmonary tissue damage associated with infection of cynomolgus macaques by highly pathogenic avian influenza virus (h n ) by low dose natural human ifn-alpha administered to the buccal mucosa gm-csf in the lung protects against lethal influenza infection ifitm restricts the morbidity and mortality associated with influenza recent developments in nucleic acid delivery with polyethylenimines we thank dr ze chen at the shanghai institute of biological products in china for providing influenza virus h n (a/guizhou/ / ). this work was supported by grants from the national nature science foundation of china ( to shibo jiang, to lu lu) and shanghai kai star project ( qa ) to lu lu. for panels e and f, *a significant difference (*po . ) and **very significant difference (**po . ) were observed between the pei-and pbs-pretreated groups. data are expressed as means ± sd. all results were repeated and verified at least twice. key: cord- -vlwzzrgc authors: kiambi, stella; corman, victor m.; sitawa, rina; githinji, jane; ngoci, james; ozomata, abdullahi s.; gardner, emma; von dobschuetz, sophie; morzaria, subhash; kimutai, joshua; schroeder, simon; njagi, obadiah; simpkin, piers; rugalema, gabriel; tadesse, zelalem; lubroth, juan; makonnen, yilma; drosten, christian; müller, marcel a.; fasina, folorunso o. title: detection of distinct mers-coronavirus strains in dromedary camels from kenya, date: - - journal: emerg microbes infect doi: . /s - - -z sha: doc_id: cord_uid: vlwzzrgc nan between july and october , nasal swabs were randomly taken from n = dromedaries in five counties, namely, turkana (n = ), marsabit (n = ), isiolo (n = ), laikipia (n = ), and nakuru (n = ). in addition, monthly repeated sampling was performed on dromedaries from four herds in two different countries (isiolo and nakuru) for a period of months (from april to october ). in total, n = nasal swab samples were collected. all samples were stored frozen in trizol buffer at − °c. rna extraction (direct-zol™ rna kit, zymo research) and mers-cov nucleic acid detection were performed following the manufacturer's instructions and according to previously established protocols . in seven of ( . %) tested nasal swabs, mers-cov rnas were detected by the upe mers-cov rt-pcr screening assay (supplementary table) . for / samples, which had very low mers-cov rna concentrations (< x copies/ml), confirmatory rt-pcr testing and sequencing were unsuccessful. the mean viral load for / samples was . x (range . x - . x ) rna copies per ml buffer. four of the five mers-cov rna-positive animals were female and < year old, consistent with previous observations that juvenile dromedaries and possibly females may be the main sources for mers-cov excretion . the mers-cov rna-positive animals belonged to two different dromedary camel herds in dabel and lombolio, which are both located within isiolo country. however, the herds neighbor each other and share common pastures and water sources. introduced into the two herds. the dromedaries were sampled on the same day, suggesting simultaneous cocirculation of two different mers-cov strains and perhaps an unexplored infection dynamic in isiolo, which is a camel congregation location. to experimentally confirm the presence of two independently circulating mers-cov strains and to rule out sample cross-contamination, we generated complete mers-cov genome sequences using a previously established protocol . full genome sequences were generated for one specimen of each of the two positive herds using the samples with the highest mers-cov rna concentrations ( . x and . x copies/ml). other confirmed mers-covpositive samples were assigned to two different mers-cov isolates ("dabel" or "lombolio") by amplifying and sequencing single-nucleotide polymorphisms in the spike gene and the open reading frame (supplementary table) . all three viruses from dabel and both viruses from lombolio shared the same polymorphism patterns. for phylogenetic analysis, we included two representatives of mers-cov lineages representing mers-cov clades a and b, as defined earlier , along with all published clade c (non-a, non-b) mers-cov complete genomes (genbank accessed april ). a phylogenetic tree was constructed using the maximum-likelihood method based on the general time reversible model and bootstrap replicates using the phyml plugin in geneious r (www.geneious.com, biomatters ltd, new zealand). as shown in fig. , both kenyan dromedary mers-cov isolates clustered with the proposed clade c viruses from ethiopia and egypt in a sister relationship to all arabian mers-covs (clades a and b). the two kenyan mers-cov isolates diverged by . % at the nucleotide level, confirming the circulation of at least two different mers-cov strains in kenya. the next closest mers-cov relative was obtained from a dromedary sampled in egypt in (nrce-nc / ; acc no. ku , clade c) and showed . - . % nucleotide distance. recombination analysis by rdp v . indicated that none of the two kenyan mers-cov strains had recombined with any of the known clade a, b, or c strains. the previously described clade c african mers-cov strains , had several mutations in the spike protein, which is responsible for cellular receptor interaction, virus entry, and antibody-directed virus neutralization . an alignment of the amino-acid sequences of all known mers-cov spikes showed that the kenyan mers-cov strains had one unique amino-acid change (s p) within the core part of the receptor-binding domain (supplementary figure) . as the mutation was not among the amino-acid residues that directly interact with the dipeptidyl peptidase- receptor , phenotypic traits of these new clade c mers-cov strains may be comparable to epidemic mers-cov strains as described previously . however, without further extensive experimental assessment, we cannot rule out the possibility that the observed mutation in the spike protein causes differences in the receptor interaction or receptor binding affinity, which may influence virus transmission or host tropism. recently described mers-cov strains from western africa had genomic deletions within open reading frame (orf) a/b that were not seen in eastern african mers-cov strains . both of the encoded proteins, proteins a and b, have anti-immune functions , and may represent important virulence factors in vivo. we provide independent evidence that mers-cov from eastern african dromedaries encode a complete orf a/b. the observation that mers-cov strains in different parts of eastern africa have a complete orf a/b suggests the predominance of these strains on the african continent and emphasizes that the orf a/b deletion is most likely geographically restricted to western africa. taken together, differential spike-receptor interactions and anti-immune activity may influence virus replication and transmission. the limited number of human mers cases in africa would certainly favor the idea that mers-cov strains differ in virulence and transmissibility. further experimental confirmation, preferably by animal transmission experiments in combination with coronavirus reverse genetics , are warranted. the phylogenetic relationship of mers-cov strains from the african continent (clade c) with the strains circulating on the arabian peninsula (clades a and b) hints at the divergence of these clades some time ago. the putative absence of clades a and b mers-covs on the african continent may be explained by a lack of surveillance and testing and/or by the genetic drift of mers-cov on the arabian peninsula. the unidirectional export routes from africa to the arabian peninsula may prevent the reintroduction opportunities of clades a and b mers-covs into african dromedary herds. interestingly, to date, no clade c mers-cov strains from africa have been detected on the arabian peninsula, which is rather surprising, given the continuous and extensive export of african dromedaries to the arabian peninsula. an explanation for this observation may again be a lack of testing of imported animals and/or the fact that previous clade a/b mers-cov infections may have established herd immunity in the arabian dromedary populations. as cov infections do not elicit long-lasting (mucosal) immunity, the introduction of clade cmers-cov strains on the arabian peninsula may be possible in the future and should therefore be monitored. to shed light on possible reasons for the restricted geographic circulation of different mers-cov strains, enhanced virological surveillance of mers-cov is urgently needed in dromedary populations of the affected regions. putative underlying evolutionary and molecular mechanisms that influence the geographic distribution of differentially virulent mers-cov strains should be assessed through phenotypic characterizations of different mers-cov strains. the early detection and characterization of emerging mers-cov strains with new phenotypic features will be highly relevant for future vaccination strategies and the prediction of epidemics in humans. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers and the dromedary camel trade between africa and the middle east mers coronaviruses from camels in africa exhibit regiondependent genetic diversity cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt antibodies against mers coronavirus in dromedary camels mers-cov antibodies in humans detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction time course of mers-cov infection and immunity in dromedary camels rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia structure of mers-cov spike receptor-binding domain complexed with human receptor dpp mers-cov b protein interferes with the nf-kappabdependent innate immune response during infection middle east respiratory syndrome coronavirus accessory protein a is a type i interferon antagonist transgene expression in the genome of middle east respiratory syndrome coronavirus based on a novel reverse genetics system utilizing redmediated recombination cloning key: cord- - aps kvp authors: zhang, wei; du, rong-hui; li, bei; zheng, xiao-shuang; yang, xing-lou; hu, ben; wang, yan-yi; xiao, geng-fu; yan, bing; shi, zheng-li; zhou, peng title: molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: aps kvp in december , a novel coronavirus ( -ncov) caused an outbreak in wuhan, china, and soon spread to other parts of the world. it was believed that -ncov was transmitted through respiratory tract and then induced pneumonia, thus molecular diagnosis based on oral swabs was used for confirmation of this disease. likewise, patient will be released upon two times of negative detection from oral swabs. however, many coronaviruses can also be transmitted through oral–fecal route by infecting intestines. whether -ncov infected patients also carry virus in other organs like intestine need to be tested. we conducted investigation on patients in a local hospital who were infected with this virus. we found the presence of -ncov in anal swabs and blood as well, and more anal swab positives than oral swab positives in a later stage of infection, suggesting shedding and thereby transmitted through oral–fecal route. we also showed serology test can improve detection positive rate thus should be used in future epidemiology. our report provides a cautionary warning that -ncov may be shed through multiple routes. coronaviruses (covs) belong to the subfamily orthocoronavirinae in the family coronaviridae and the order nidovirales. a human coronavirus (sars-cov) caused the severe acute respiratory syndrome coronavirus (sars) outbreak in . most recently, an sars-related cov was implicated as the etiological agent responsible for the outbreak in wuhan, central china. this outbreak is estimated to have started on th december and , laboratory confirmed cases with deaths as of rd february in china [ ] . the virus has spread to other countries by travellers from wuhan [ ] . typical symptoms are fever, malaise, shortness of breath and in severe cases, pneumonia [ ] [ ] [ ] . the disease was first called unidentified viral pneumonia. we quickly identified the etiological agent, termed -ncov (virus name designated by the world health organization). the newly identified virus is an sars-related virus (sarsr-cov) but shares only . % genome identity to sars-cov [ ] . we developed molecular detection tools based on viral spike genes. our previous studies indicate that qpcr method can be used for the detection of -ncov in oral swabs or in bronchoalveolar lavage fluid (balf) [ ] . additionally, we developed igm and igg detection methods using a cross-reactive nucleocapsid protein (np) from another sarsr-cov rp [ ] , which is % identical to -ncov np. using these serological tools, we demonstrate viral antibody titres increase in patients infected with -ncov [ ] . like sars-cov, -ncov induced pneumonia through respiratory tract by clinical observation. therefore, the presence of viral antigen in oral swabs was used as detection standard for -ncov. similarly, two times of oral swabs negative in a -h interval was considered as viral clearance by patients officially. here we launched an investigation of -ncov in a wuhan hospital, aiming to investigate the other possible transmission route of this virus. human samples, including oral swabs, anal swabs and blood samples were collected by wuhan pulmonary hospital with the consent from all patients and approved by the ethics committee of the designated hospital for emerging infectious diseases. two investigations were performed. in the first investigation, we collected samples from patients, of which were in severe conditions. in the second investigation, we collected samples from patients, yet their clinical records were not available. we only showed patients who were viral nucleotide detection positive. patients were sampled without gender or age preference unless where indicated. for swabs, . ml dmem+ % fbs medium was added in each tube. supernatant was collected after rpm, s vortex and - min standing. supernatant from swabs were added to lysis buffer for rna extraction. serum was separated by centrifugation at g for min within h of collection, followed by °c min inactivation, and then stored at °c until use. whenever commercial kits were used, manufacturer's instructions were followed without modification. rna was extracted from μl of samples with the high pure viral rna kit (roche). rna was eluted in μl of elution buffer and used as the template for rt-pcr. qpcr detection method based on -ncov s gene can be found in the previous study [ ] . in brief, rna extracted from above used in qpcr by hiscript® ii one step qrt-pcr sybr® green kit (vazyme biotech co., ltd). the μl qpcr reaction mix contained μl × one step sybr green mix, μl one step sybr green enzyme mix, . μl × rox reference dye , . μl of each primer ( μm) and μl template rna. amplification was performed as follows: °c for min, °c for s followed by cycles consisting of °c for s, °c for s, and a default melting curve step in an abi machine. in-house anti-sarsr-cov igg and igm elisa kits were developed using sarsr-cov rp np as antigen, which shared above % amino acid identity to all sarsr-covs, as reported previously [ ] . for igg test, maxisorp nunc-immuno well elisa plates were coated ( ng/well) overnight with recombinant np. human sera were used at : dilution for h at °c. an anti-human igg-hrp conjugated monoclonal antibody (kyab biotech co., ltd, wuhan, china) was used at a dilution of : , . the od value ( - ) was calculated. for igm test, maxi-sorp nunc-immuno wellelisa plates were coated ( ng/well) overnight with anti-human igm (µ chain). human sera were used at : dilution for min at °c, followed by anti-rp np-hrp conjugated (kyab biotech co., ltd, wuhan, china) at a dilution of : . the od value ( - ) was calculated. in the first investigation, we aimed to test whether viral positive can be found in anal swab and blood as well as oral swabs. we conducted a molecular investigation to patients in wuhan pulmonary hospital, who were detected as oral swabs positive for -ncov upon admission. we collected blood, oral swabs and anal swabs for -ncov qpcr test using previously established method [ ] . we found patients who still carry virus following days of medical treatments. of these patients, were oral swabs positive ( . %), were anal swabs positive ( . %), blood positives ( %) and serum positives ( %). two patients were positive by both oral swab and anal swab, yet none of the blood positive was also swabs positive. not surprisingly, all serum positives were also whole serum positive (table ). in summary, viral nucleotide can be found in anal swab or blood even if it cannot be detected in oral swabs. it should be noted that although swabs may be negative, the patient might still be viremic. we then did another investigation to find out the dynamic changes of viral presence in two consecutive studies in both oral and anal swabs in another group of patients. the target patients were those who received around days of medical treatments upon admission. we tested for both viral antibody and viral nucleotide levels by previously established method [ ] . we showed that both igm and igg titres were relatively low or undetectable in day (the day of first sampling). on day , an increase of viral antibodies can be seen in nearly all patients, which was normally considered as a transition from earlier to later period of infection ( figure and supplementary table ). igm positive rate increased from % ( / ) to % ( / ), whereas igg positive rate increased from % ( / ) to % ( / ). this is in contrast to a relatively low detection positive rate from molecular test (below). for molecular detection, we found oral swabs positive ( %) and anal swabs ( %) in these people on day . on day , we were only able to find oral swabs positive ( %). in contrast, we found anal swabs positive ( . %). when counting all swab positives together, we found most of the positives came from oral swab ( / , %) on day . however, this trend appears to change on day . we found more ( / , %) anal swab positive than oral swab positive ( / , %). another observation is the reoccurrence of virus in patients who were detected negative on day . of note, of these viral positives were from anal swabs ( table ) . these data suggested a shift from more oral positive during early period (as indicated by antibody titres) to more anal positive during later period might happen. within month of the -ncov disease outbreak, we rapidly developed molecular and serological detection tools. this is the first molecular and serological study on this virus after the initial identification of -ncov from patients diagnosed with unidentified viral pneumonia [ ] . we detected the virus in oral swabs, anal swabs and blood, thus infected patients can potentially shed this pathogen through respiratory, fecal-oral or body fluid routes. in addition, we successfully applied serology test a large population and showed which could greatly improved detection positive rate. we show that the current strategy for the detection of viral rna in oral swabs used for -ncov diagnosis is not perfect. the virus may be present in anal swabs or blood of patients when oral swabs detection negative. in sars-cov and mers-cov infected patients, intestinal infection was observed at later stages of infection [ ] [ ] [ ] . however, patients infected with -ncov may harbour the virus in the intestine at the early or late stage of disease. it is also worth to note none of the patients with viremia blood had positive swabs. these patients would likely be considered as -ncov negative through routine surveillance, and thus pose a threat to other people. in contrast, we found viral antibodies in near all patients, indicating serology should be considered for -ncov epidemiology. a possible shift from oral positive during early infection to anal swab positive during late infection can be observed. this observation implied that we cannot discharge a patient purely based on oral swabs negative, who may still shed the virus by oral-fecal route. above all, we strongly suggest using viral igm and igg serological test to confirm an infection, considering the unreliable results from oral swabs detection. in summary, we provide a cautionary warning that -ncov may be transmitted through multiple routes. both molecular and serological tests are needed to definitively confirm a virus carrier. who press statement related to the novel coronavirus situation clinical features of patients infected with a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin serological evidence of bat sars-related coronavirus infection in humans severe acute respiratory syndrome associated coronavirus is detected in intestinal tissues of fatal cases organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus we thank dr. danielle anderson of duke-nus medical school for critical review of this report. we thank national virus resource center (ncrc) in wuhan institute of virology. key: cord- -a ba dz authors: chen, qi; wang, leyi; yang, chenghuai; zheng, ying; gauger, phillip c.; anderson, tavis; harmon, karen m.; zhang, jianqiang; yoon, kyoung-jin; main, rodger g.; li, ganwu title: the emergence of novel sparrow deltacoronaviruses in the united states more closely related to porcine deltacoronaviruses than sparrow deltacoronavirus hku date: - - journal: emerg microbes infect doi: . /s - - -z sha: doc_id: cord_uid: a ba dz nan porcine deltacoronavirus (pdcov), has been identified as one of the major enteric pathogens causing diarrhea in pigs since , . both clinical and experimental evidence demonstrated that pdcov is pathogenic to swine and causes similar lesions in the pig intestine as porcine epidemic diarrhea virus (pedv) , . pdcov strains reported from the united states and other countries had . - . % nucleotide identity at the whole-genome level , suggesting that a single genotype is circulating worldwide. regarding sparrow deltacoronavirus (spdcov), one sparrow cov strain, hku - , has been reported thus far . the genomic diversity of sparrow deltacoronaviruses remains unclear. we report the detection and genetic characterization of novel spdcov in fecal samples collected from sparrows found on swine farms in the midwestern united states. in january , multiple batches of porcine oral fluid (pen-based) and floor-picked fecal samples were submitted from a grow-finish stage pig farm in illinois for routine health monitoring. unexpectedly, multiple oral fluid samples and one fecal sample were tested positive for pdcov (the ct value ranged between and ) by duplex pedv and pdcov real-time rt-pcr (see technical appendix). however, no clinical diarrhea or other signs were observed among pigs on the index farm, which prompted us to further investigate the source of positive pcr results. since the pigs were housed in hoop-style buildings with uncontrolled access for wild bird entry and congregation, fecal samples were also collected from sparrows found inside the pig barn (isu - to − ) and were submitted to our laboratory for testing in january , along with feed samples (isu - to − ). surprisingly, / fecal samples were positive using pcr targeting the pdcov n gene, with a ct of . - . , while all feed samples were negative. four additional sparrow fecal samples (isu - to − ) collected in june from the same farm were tested, and one of the samples (isu - ) was positive by pdcov n gene-based pcr, with a ct of . . similarly, in an unrelated grow-finish swine farm in minnesota, two oral fluid samples collected from clinically healthy pigs at the end of october were tested positive (ct of . and . ), and one sparrow fecal sample (isu - ) collected in early november from that barn were tested positive (ct of . ) using a pdcov n gene-based pcr. the pdcov pcr-positive sparrow samples then underwent next-generation sequencing (ngs) using an illumina miseq platform (see supplementary methods). after using an in-house bioinformatics analysis pipeline (supplementary methods), four nearly complete genomic sequences of spdcov (isu - , isu - , isu , and isu ; genbank accession numbers mg , mg , mg , and mg , respectively) were obtained. the genome organizations of all four spdcov strains are similar to those of sparrow cov hku and pdcov with the characteristic gene order ′replicase orf ab, spike (s), envelope (e), membrane (m), and nucleocapsid (n)- ′ (fig. a) . similar to hku , the four spdcov strains contain three orfs downstream of n encoding three nonstructural proteins (ns a, ns b, and ns c), as illustrated in fig. a . whole-genome nucleotide sequence comparison showed that spdcov isu shares a higher identity with isu ( . %) and isu - and − ( . %) than with pdcov ( . %) and hku ( . %), and it has an even lower identity with other avian deltacoronaviruses, such as hku ( . %), hku ( . %), and hku ( . %) (fig. b, supplementary table s ). spdcov isu - and isu - share . % wholegenome nucleotide identity with each other, whereas both strains share . %, . %, . %, and . % identity with isu , isu , pdcov, and hku , respectively, and they share an even lower identity with other avian deltacoronaviruses, such as hku ( . %), hku ( . %), and hku ( . %). these data suggest that the newly identified spdcov isu - /isu - , isu , and isu represent novel spdcov strains. to exclude the possible issue of contamination of sparrow fecal samples with porcine samples, all fecal samples directly collected from swine were tested and were negative for both sdcov and spdcov. in addition, sanger sequencing of four rt-pcr products of the orf ab, m, and n genes of spdcov showed that the sequences had % identity to those from ngs. the four spdcov strains identified in this study share a slightly higher identity ( . - . % and . - . %) to a sparrow cov strain hku - than to pdcov strains ( . - . % and . - . %) in their orf ab and n proteins, respectively ( fig. b and supplementary table s ). in contrast, the four spdcov strains share a significantly lower identity to sparrow cov hku ( . - . %) than to three pdcov strains ( . - . %) in their s protein (fig. b and supplementary table s ). in addition, they share a relatively lower identity ( . - . % and . - . %) to sparrow cov hku than the pdcov strains ( . - . % and . - . %) in their e and m proteins, respectively (supplementary table s ). these findings were also confirmed by phylogenetic tree analysis of amino-acid sequences in which the four spdcov strains clustered together and were closely related to pdcov and sparrow cov hku strains in orf ab, e, m, and n trees ( fig. c and supplementary figure s ). however, the four spdcov strains were close to pdcov but distant from sparrow cov hku in the s tree (fig. d) . in the case of non-structural proteins (ns , ns a, and ns b), the spdcov strains share a high amino-acid identity with each other. however, spdcov isu - / has . % and % identity to isu and isu , respectively, in ns c, which is much lower than the . % identity shared between isu and isu (supplementary table s ). an -nt deletion in the ns c gene of isu - and - resulted in a protein that was amino acids shorter than that of isu and isu in length (supplementary figure s ) . overall, these data further support that the sparrow covs identified in our study are novel spdcov. recombination analysis revealed that pdcov and sparrow cov hku are highly variable from spdcovs in two regions. the most prominent variable region is located in the s-e-m gene region, and another region is located within the orf ab gene (~ nt to nt) (supplementary figure s ) . however, there is no obvious pattern showing that the emergence of spdcov strains identified in the study was through recombination between pdcov and sparrow cov hku . comparison of pdcov pcr primer and probe sequences (targeting the n gene) with spdcov sequences revealed that the probe sequence was exactly identical to the sequence observed in spdcov, and only and / nucleotide mismatches were observed in the forward and reverse primers, respectively (supplementary figure s ) . therefore, pdcov real-time rt-pcr can cross-react with spdcov, which complicates the molecular detection of pdcov. it is plausible that contamination with sparrow feces containing spdcovs caused non-specific positive results for pdcov when the oral fluid and fecal samples collected from clinical healthy pigs were tested using pedv/pdcov duplex rt-pcr. this also raises a concern regarding the specificity of detecting pdcov from environmental and environmentally contaminated samples. to avoid the cross-reaction issue, further modification of the primers and probe for pdcov real-time rt-pcr is needed. pdcov, as a causative agent for pig diarrhea, has been reported in many countries, including the united states, canada, japan, south korea, thailand, vietnam, mainland china, and laos, indicating that pdcov is widespread in pig populations. however, all known pdcovs to date have been relatively conserved and shared . - . % nucleotide identity at the whole genome level. in contrast, the spdcov strains identified in the present study (isu - / , isu , and isu ) as well as one previously identified sparrow cov strain, hku - , exhibited higher genetic diversity ( . - . % nucleotide identity at the whole-genome level). further studies to understand the genetic diversity of spdcov by sequencing more spdcov strains are warranted. another important finding is that, due to the relative conservation between pdcov and spdcov n gene sequences in some regions, the pdcov n gene-based pcr used in this study can cross-react with spdcov. thus, pdcov pcr results should be interpreted carefully when pig samples that can be contaminated by bird feces or other excretions are tested. the spdcov sequence data reported in this study may help determine the evolutionary relationship of various dcovs and help to understand the interspecies transmission of dcovs in future studies. virus taxonomy, classification and nomenclature of viruses coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus full-length genome sequence of porcine deltacoronavirus strain usa/ia/ / detection and genetic characterization of deltacoronavirus in pigs porcine deltacoronavirus: histological lesions and genetic characterization pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -day-old neonatal piglets analysis was completed using cgview comparison tool software. the corresponding strains/samples for rings are detailed on the bottom. orf open reading frame, s spike, e envelope, m membrane, n nucleocapsid, ns , ns a, ns b, and ns c represent nonstructural proteins , a, b, and c, respectively. c phylogeny of orf ab amino-acid sequences. d phylogeny of spike protein sequences. the sparrow deltacoronavirus strains identified in this study are indicated with red squares, hku - is marked with a blue square, and three hku strains are marked with a pink triangle. the phylogeny was inferred using the neighbor-joining method in mega version . . statistical support was obtained using bootstrap resampling ( replications) and is drawn on the inferred tree. reference sequences representing coronavirus diversity were obtained from ncbi genbank (pedv, porcine epidemic diarrhea virus, nc_ ; sc-batcov- , scotophilus bat coronavirus , nc_ , tgev transmissible gastroenteritis virus, aj , fipv feline infectious peritonitis virus, nc_ , prcv porcine respiratory coronavirus, dq , phev porcine hemagglutinating encephalomyelitis virus, dq , bcov bovine coronavirus, nc_ , ibv infectious bronchitis virus, nc_ , ibv-partridge partridge coronavirus, ay , tcov turkey coronavirus, nc_ ; bulbul, cov strain hku - , fj ; bulbul cov strain hku - , fj ; thrush cov strain hku - , nc_ ; munia cov strain hku - , nc_ supplementary information accompanies this paper at https://doi.org/ . /s - - -z. key: cord- -y vhh oq authors: zhang, yimin; liu, jimin; yu, liang; zhou, ning; ding, wei; zheng, shufa; shi, ding; li, lanjuan title: prevalence and characteristics of hypoxic hepatitis in the largest single-centre cohort of avian influenza a(h n ) virus-infected patients with severe liver impairment in the intensive care unit date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: y vhh oq avian influenza a(h n ) virus (a(h n )) emerged in february . liver impairment of unknown cause is present in % of patients with a(h n ) infection, some of whom experience severe liver injury. hypoxic hepatitis (hh) is a type of acute severe liver injury characterized by an abrupt, massive increase in serum aminotransferases resulting from anoxic centrilobular necrosis of liver cells. in the intensive care unit (icu), the prevalence of hh is ∼ %– %. here, we report a . % ( / ) incidence of hh in the largest single-centre cohort of icu patients with a(h n ) infection. both hh patients presented with multiple organ failure (mof) involving respiratory, cardiac, circulatory and renal failure and had a history of chronic heart disease. on admission, severe liver impairment was found. peak alanine aminotransferase (alt) and aspartate aminotransferase (ast) values were and u/l, and and u/l, respectively, in the two patients. unfortunately, both patients died due to deterioration of mof. a post-mortem biopsy in case confirmed the presence of centrilobular necrosis of the liver, and real-time reverse transcription polymerase chain reaction of a(h n )-specific genes was negative, which excluded a(h n )-related hepatitis. the incidence of hh in a(h n ) patients is similar to that in icu patients with other aetiologies. it seems that patients with a(h n ) infection and a history of chronic heart disease with a low left ventricular ejection fraction on admission are susceptible to hh, which presents as a marked elevation in alt at the time of admission. avian influenza a(h n ) virus (a(h n )) emerged in february and infected patients according to the latest world health organization (who) report, updated on february . - the mortality rate of a(h n ) infection is . % ( / ). liver derangement commonly occurs in a(h n )-infected patients. [ ] [ ] [ ] indeed, these patients can suffer from severe liver injury; however, the underlying reason remains unclear. the causes of severe acute liver impairment include hepatotropic virus infection, drug-induced liver injury (dili), ischaemic liver injury and liver trauma. thus, whether the novel a(h n ) virus is hepatotropic and plays an important role in the dramatic liver injury sometimes seen in patients with severe liver impairment warrants investigation. furthermore, it is also necessary to identify other causes of severe liver impairment if a(h n )-related hepatitis is excluded in these patients. hypoxic hepatitis (hh) is characterized as a massive but transient increase in serum alanine aminotransferase (alt) activity secondary to anoxic necrosis of centrilobular liver cells. the prevalence of hh in intensive care unit (icu) patients is %- %. hh is one cause of acute liver injury in patients with respiratory failure. , severe a(h n ) infection always leads to respiratory failure, which reduces oxygen supply to the liver. hence, hh is likely one possible cause of severe liver impairment in a(h n )-infected patients with respiratory failure. hh patients are not always on hepatology wards, which may complicate its recognition. to the best of our knowledge, there has been no report of hh in patients infected with a(h n ) or any other type of avian influenza virus. however, the overall in-hospital mortality rate of hh can be as high as %- % due to accompanying multiple organ failure (mof). [ ] [ ] [ ] [ ] because of this high mortality rate, early diagnosis and treatment of hh are necessary to ensure a good outcome. from march to february , patients with a(h n ) infection were admitted to the icu of the first affiliated hospital, zhejiang university school of medicine which represents the largest single-centre cohort of patients with a(h n ) infection worldwide. we report here the prevalence of hh in this single-centre cohort. the clinical characteristics of a(h n )-infected patients with hh are described. post-mortem biopsies were obtained from one fatal case of hh with a(h n ) infection. the pathological features were reported, and a(h n )-related hepatitis was excluded based on a negative viral real-time reverse transcription polymerase chain reaction (rt-pcr) assay of liver tissue. a total of patients with avian flu h n infection admitted to the icu at the first affiliated hospital, zhejiang university school of medicine from march to february were screened. this study was approved by the ethics committee of the first affiliated hospital. liver function was tested daily for the first week and at least twice during the remainder of the hospital stay. the presence of respiratory, cardiac, renal or circulatory failure was recorded. serologic testing for viral hepatitis a, b, c, d, e, epstein-barr virus and cytomegalovirus was performed. serum autoantibodies were also assayed. liver ultrasound, echocardiogram and chest radiography were performed. all relevant medical information, such as past history, recent contact with live poultry, coagulation function and medications taken during the hospital stay, was recorded. patients who met all of the following criteria were diagnosed as having hh according to previous reports , , : (i) a massive but transient elevated alt level (more than -fold the upper limit of normal (uln)), (ii) the presence of respiratory, cardiac or circulatory failure and (iii) exclusion of other causes of liver injury. post-mortem biopsy, histological examination and rt-pcr a limited post-mortem biopsy that included the liver, lung and kidney was performed in one confirmed fatal case of hh with a(h n ) infection (case ). written consent was obtained from the relatives of the patient prior to the biopsy. two post-mortem biopsy cores from the liver and one biopsy from each lung and kidney were taken according to institutional protocols. one biopsy core from each organ was fixed in % formalin for histopathological examination by haematoxylin and eosin staining. the second liver biopsy core was directly homogenized and subjected to real-time rt-pcr for a(h n )-specific genes as reported previously. the primers and probes used are listed in table . statistical analysis was performed using the spss software package (version . , chicago, il, usa). non-parametric variables are presented as n (%). parametric variables are presented as means sd and were compared by t-test. a p-value , . (two-tailed) was considered to indicate significance. the patients in the icu with a(h n ) infection were predominantly male (n , . %), aged - years (n , . %) ( table ) . their underlying medical conditions, described in table , included hypertension (n , . %), coronary heart disease (n , . %), chronic obstructive pulmonary disease (n , . %), cerebrovascular disease (n , . %), chronic liver disease (n , . %), chronic renal disease (n , . %), diabetes mellitus (n , . %), rheumatoid arthritis (n , . %) and cancer (n , . %). the proportion of patients with liver impairment was lower on admission than during the hospital stay (n ( . %) vs. n ( . %), p , . ). antiviral therapy was performed in all patients, while antibiotic therapy was performed in ( . %) and glucocorticoid therapy in ( . %) ( table ). an alt level of . -fold the uln was found in only two patients, not only on admission but also during the hospital stay. the extent of hypoxic hepatitis in a(h n )-infected patients y zhang et al alt elevation was considerably higher in hh patients than in non-hh patients with liver injury (on admission, . . u/l vs. . . u/l, p , . ; hospital stay, . . u/l vs. . . u/l, p , . ) ( table ). these two cases were diagnosed as hh. the incidence of hh in our single-centre cohort of icu patients with a(h n ) infection was . % ( / ). both cases exhibited respiratory, renal, circulatory and cardiac failure. in addition, viral hepatitis and autoimmune diseases were excluded based on the negative results of serum marker tests. dili was excluded based on no history of drug intake (such as acetaminophen, chinese herbs, etc.) suspected to induce dili prior to admission. liver ultrasound excluded surgical biliary tract diseases and space-occupying lesions. details of the two patients are reported below. an -year-old male was admitted to the icu with a -day history of shortness of breath and sudden deterioration with a fever of . c prior to admission. on admission, the patient exhibited ventricular tachycardia and was in atrial fibrillation ( bpm), and had tachypnoea ( /min), oliguria and a low oxygen index (pao /fio ) (, mmhg). his mean arterial pressure was maintained at mmhg by norepinephrine administration at a dose of . mg/kg?min. the patient had a history of contact with live poultry seven days before admission. chest radiography showed bilateral pulmonary infiltrates with consolidation at admission ( figure a ). respiratory secretions were positive for a(h n ) h , n and m genes by realtime rt-pcr. on admission, severe liver injury was identified. serum alt, aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) activities were elevated to , and u/l, respectively. total bilirubin level was mmol/l. the international normalized ratio (inr) was prolonged to . with a d-dimer value of mg/l. the dynamic changes in liver function are shown in figure . peak alt, ast and ldh activities ( , and u/l, respectively) occurred on the second day of admission, while the inr peaked at . on the sixth day. renal failure was identified as the creatinine level reached mmol/l with anuria. serologic test results were negative figure d) . the patient had a -year history of hypertension and coronary artery disease status and had twice undergone placement of intracoronary stents at six and two years previously. concomitant acute myocardial infarction was excluded according to the echocardiogram, electrocardiogram (ecg), myocardial enzyme spectrum and troponin (tni) results. the patient was identified as having a severe a(h n ) infection with mof and hh, placed on a mechanical ventilator and given fluid resuscitation. antiviral therapy with oral osetalmavir ( mg) twice daily was administered through a feeding tube. plasma exchange and haemofiltration were performed in the patient as liver and renal replacement therapies. unfortunately, the patient died on day of hospitalization. the liver biopsy showed well-demarcated multifocal centrilobular coagulative necrosis without accompanying inflammation. the necrotic hepatocytes were partially replaced by red blood cells, outlined by sinusoidal endothelial cells. sinusoid congestion and mild hepatocellular atrophy were identified in adjacent tissue ( figure a and b). the lung showed interstitial pneumonitis with hyaline membranes, congestion, intrapulmonary haemorrhage and anthracosis ( figure c ). acute tubular necrosis and generalized renal tubule atrophy were found in the kidney ( figure d ). a -year-old male was admitted to the icu with a two-week history of cough with sputum and sudden deterioration with a fever of . c prior to admission. on admission, the patient had paroxysmal ventricular tachycardia, and his mean arterial pressure was maintained at mmhg by administration of norepinephrine at a dose of . mg/kg?min. oxygen inhalation through a nasal tube was administered to maintain oxygen saturation at . %. the patient had a recent history of contact with live poultry days prior to admission. respiratory secretions were positive for a(h n ) h , n and m genes. chest radiography on admission showed bilateral pulmonary infiltrates ( figure a ). on admission, an echocardiogram showed a dilated left ventricle with a low lvef of . % ( figure d ), together with weakened cardiac wall motion. the serum creatine phosphokinase (ck) level was u/l, and tni was negative. ecg did not show the evidence of acute myocardial infarction on admission. a second echocardiogram showed recovery of lvef to % without accompanying weakened cardiac wall motion at day . the patient had a -year history of bronchiectasia that had been progressing to pulmonary heart disease for years. on admission, severe liver injury was identified. alt, ast and ldh activities were elevated to , , and u/l, respectively, the highest levels seen during the patient's hospital stay ( figure ). total bilirubin level was mmol/l. inr was prolonged to . with a d-dimer value of mg/l. the dynamic changes in liver function and inr are described in figure . the patient was identified as having a severe a(h n ) infection with mof and hh. antiviral therapy of oral oseltamavir ( mg) twice daily was administered. due to the progression of the infiltrates and consolidation ( figure b and c), the patient was placed on a kidney, heart, etc. , , , according to early reports, the occurrence of liver injury in a(h n ) patients was relatively frequent. , our study is not only in agreement with these reports, but it also indicated a higher frequency of liver impairment during the hospital day (n , . %) than on admission (n , . %) (p , . ). however, severe liver impairment (alt . -fold the uln) was rare (n , . %). the distinct difference between the extent of elevated alt between hh patients and non-hh patients with liver impairment indicated that hh could be differentiated from other types of liver injury by means of a sharp elevation in the alt level. the majority of a(h n )-infected patients are treated with antiviral medications, antibiotics and steroids, which are potentially hepatotoxic. [ ] [ ] [ ] [ ] there are also reports of liver impairment induced by respiratory viruses, such as severe adult respiratory syndrome coronary virus (sars-cov). hence, it is meaningful to identify the cause of liver injury, especially in cases of severe liver impairment. hh is a common cause of acute liver injury in the icu setting and is characterized by an abrupt and massive increase in aminotransferase activity secondary to anoxic centrilobular liver necrosis. , in other words, it is the clinical syndrome underlying hepatic necrosis homogeneously distributed around the central veins. centrilobular liver necrosis without inflammation was found in this study, which is consistent with the typical histological changes of hh ( figure a and b) . , the pathologic features differed from those of sars-cov-associated viral hepatitis, in which hepatocyte ballooning and lobular lymphocytic infiltration are commonly found. the negative results of rt-pcr testing of post-mortem liver tissue excluded a(h n )-related viral hepatitis. in addition, the peak ldh activities of and u/l in these patients were notable (figures a and a ). these may be useful for differentiation of hh from viral hepatitis. according to previous reports, the occurrence of hh requires a pre-existing condition that chronically compromises oxygen supply to the liver, together with an acute event that further decreases hepatic oxygen supply. , severe a(h n ) infection will lead to respiratory failure, and respiratory failure will in turn induce a sudden decrease in oxygen supply to the liver. the presence of a chronic disease that reduces baseline oxygen supply to the liver is an indicator of hh risk in a(h n ) patients. in case , coronary artery disease that chronically reduced blood flow, and hence oxygen supply, to the liver was found in both hh cases. in case , chronic pulmonary dysfunction decreased blood oxygen saturation, which led to an insufficient baseline oxygen supply to the liver. in addition, temporary left ventricular failure and respiratory failure decreased the oxygen supply to the liver. temporary left ventricular failure was suspected due to myocarditis. the ck, tni and ecg results supported this speculation. in another temporary left ventricular failure patient with a(h n ) infection, typical myocarditis histological changes, such as cluster lymphocyte infiltration, were found in post-mortem heart tissue. cardiac dysfunction was confirmed on cardiogram by the decrease in lvef to % and . % in the two patients on admission. in case , the detection of dilated intrahepatic veins by abdominal ultrasound ( figure b) and a prolonged history of coronary disease with two intracoronary stent implantation procedures suggested chronic right ventricular failure. during hospitalization, these two patients required persistent intravenous norepinephrine transfusion to maintain diastolic blood pressure. this clearly indicated circulatory failure, which would also reduce oxygen supply to the liver. the reduction in oxygen supply caused by respiratory failure was ameliorated in the icu. the peak alt and ast levels occurred on the day of admission or the following day in this study, which is in accordance with the report of raurich et al. ours is the largest centre for the treatment of a(h n ) patients in the world. a(h n ) patients in our icu represent , % ( / ) of the total number of patients confirmed infected with a(h n ) according to the latest report by the who. the prevalence of hh in icu patients with a(h n ) infection in our centre is . %. the incidence of hh in icu patients varies from . % to . %. most studies have reported a prevalence of %- %. this is in agreement with our findings, which suggest that patients with a(h n ) infection in the icu setting have a similar prevalence of hh to that of those with other aetiologies. the in-hospital mortality rate of hh is o %. according to raurich et al., risk factors for mortality include prolonged inr and need for renal replacement. , both of these risk factors were present in the two patients reported herein, which was indicative of a poor prognosis. both patients died despite treatment of the accompanying mof (such as by fluid resuscitation and blood purification). according to previous reports, a cytokine storm can occur in severe a(h n ) or a(h n ) patients. , , moreover, inflammatory cytokines were found to contribute to hypoxic hepatopathy in animal models. a cytokine storm was also detected in two hh patients with a(h n ) infection. however, the relatively low prevalence of hh ( hh cases vs. non-hh cases) makes it difficult to reach a conclusion regarding the contribution of a cytokine storm to the pathogenesis of a(h n ) infection in hh. a large-scale study of a(h n )infected patients should be performed to determine the contribution of a cytokine storm to hh and the correlation between them. in conclusion, we report here a . % prevalence of hh in icu patients with a(h n ) infection. the typical pathological change of hh, centrilobular necrosis of the liver, was confirmed. a(h n )related viral hepatitis was excluded based on negative real-time rt-pcr for viral genes in liver tissue. a lower lvef accompanying underlying chronic heart disease and abrupt elevation of serum alt levels at the time of admission in patients with a(h n ) infection suggest the need for identification and treatment of hh. h n influenza-infected patients with chronic heart disease accompanying acute heart failure are at elevated risk of severe liver damage. lan-juan li and colleagues at zhejiang university in hangzhou studied patients admitted to intensive care with h n influenza over years, two of whom had hypoxic hepatitis, a type of acute severe liver injury caused by reduced oxygen supply to the liver. the results indicated a combination of pre-existing heart conditions and sudden decreasing of left ventricular ejection fraction in the two patients, combined with the respiratory failure caused by h n , reduced oxygen supply to the liver, which caused hereditary hemochromatosis. the researchers suggest that h n patients with a history of heart disease and acute left heart failure on admission should be carefully checked for liver damage. this research summary is based on your manuscript that was recently accepted for publication in emerging microbes & infections (emi). it provides a non-specialist audience with a synopsis of your key research outcomes and conclusions. this value-added service provided by npg is designed to raise interest in your research across the broader community. npg will publish the summary on the journal's website, and it will be freely available under a creative commons ''by-nd-nc . unported'' license (see the journal website for details). we encourage you to re-use the summary to bring attention to your research; for example, you can host it on your own website and share it via social-networking platforms. please attribute 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a(h n ) virus infection a recombinant viruslike particle influenza a (h n ) vaccine sars-associated viral hepatitis caused by a novel coronavirus: report of three cases hepatic histological findings in suspected drug-induced liver injury: systematic evaluation and clinical associations evaluation of prognostic markers in severe drug-induced liver disease hypoxic hepatitis -epidemiology, pathophysiology and clinical management extreme serum elevations of aspartate aminotransferase surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia cytokine and chemokine levels in patients infected with the novel avian influenza a (h n ) virus in china comparison of the cytotoxic actions of hypoxia and endotoxin in the perfused cat liver this work was supported by the grants zx and zx from the china national science and technology major project and zb from the zhejiang ctm science and technology project. we thank prof. rajiv jalan from university college london for his constructive suggestions in the revision of the manuscript.