key: cord- - qioku authors: rehman, zaib ur.; meng, chunchun; sun, yingjie; mahrose, khalid m.; umar, sajid; ding, chan; munir, muhammad title: pathobiology of avian avulavirus : special focus on waterfowl date: - - journal: vet res doi: . /s - - -x sha: doc_id: cord_uid: qioku avian avulaviruses serotype (abbreviated as apmv- for the historical name avian paramyxovirus ) are capable of infecting a wide spectrum of avian species with variable clinical symptoms and outcomes. ease of transmission has allowed the virus to spread worldwide with varying degrees of virulence depending upon the virus strain and host species. the emergence of new virulent genotypes from global epizootics, and the year-to-year genomic changes in low and high virulence apmv- imply that distinct genotypes of apmv- are simultaneously evolving at different geographic locations across the globe. this vast genomic diversity may be favoured by large variety of avian species susceptibility to apmv- infection, and by the availability of highly mobile wild birds. it has long been considered that waterfowls are not sensitive to apmv- and are unable to show any clinical signs, however, outbreaks from the ′s contradict these concepts. the apmv- isolates are increasingly reported from the waterfowl. waterfowl have strong innate immune responses, which minimize the impact of virus infection, however, are unable to prevent the viral shedding. numerous apmv- are carried by domestic waterfowl intermingling with terrestrial poultry. therefore, commercial ducks and geese should be vaccinated against apmv- to minimize the virus shedding and for the prevention the transmission. genetic diversity within apmv- demonstrates the need for continual monitoring of viral evolution and periodic updates of vaccine seed-strains to achieve efficient control and eradication of apmv- in waterfowls. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. newcastle disease (nd) is one of the most devastating and commonly prevalent diseases in the poultry industry, around the world. owing to immense economic losses, world organization for animal health has categorized the disease as "notifiable" [ ] . the disease outbreaks are enormous and the host spectrum is broad, thus making nd as one of the primary limiting factor in the development of the poultry industry, especially in the developing countries [ , ] . it is caused by the avian avulavirus (apmv- ), which belongs to the avulavirus genus within paramyxoviridae family. all apmv- strains can be classified into velogenic (highly virulent), mesogenic (intermediate virulent) and lentogenic (non-virulent) based on the intracerebral pathogenicity index (icpi) in day old specific pathogen free (spf) chickens [ , ] additional file references un-cleaved fusion protein cleavage site. apmv- is considered to be virulent, if these amino acids are basic in nature with a phenylalanine at position , and having icpi value of . in day old chicks [ , ] . apmv- is a single-stranded, negative-sense, non-segmented and enveloped rna virus with genome length of . kb. apmv- genome encodes for six co-linear genes that translate into six proteins and two non-structural proteins. structural proteins include nucleoprotein (np), matrix (m), fusion (f), hemagglutinin-neuraminidase (hn), phosphoprotein (p) and large rna-dependent rna-polymerase (l). the rna editing of the p gene can result in the expression of v and w only in the virus infected cells [ ] [ ] [ ] [ ] . virulence of apmv- varies and depends upon the host species, and chicken and turkey are more susceptible than ducks and geese. generally, waterfowls were considered to be the natural reservoir for apmv- [ ] ; commonly for lentogenic apmv- [ ] . traditional view on the resistance of waterfowl against apmv- has been challenged since the report of continuous outbreaks in different provinces of china in goose ( ) [ , ] and ducks ( ) [ ] . number of clinical nd outbreaks are increasing in the waterfowls [ ] [ ] [ ] [ ] . outbreaks of nd in ducks and geese indicate that these are not only the carrier, but also show clinical outcome of the disease. factors that led to the change in the pathogenic spectrum of apmv- in waterfowl remain elusive. investigation of the molecular mechanisms of increased pathogenicity of apmv- in waterfowls would provide foundations in designing any control strategies for the disease, as well as to improve health and welfare standards of the waterfowl. the purpose of this review is to analyze our current understanding on the host-spectrum, molecular pathobiology of apmv- in waterfowls, and host immune responses that may play crucial roles in the disease prevention and control. the class i apmv- isolate was reported in , however, currently these isolates are frequently being reported. recent epidemiological studies direct that class i apmv- are common in domestic waterfowl. all viruses belong to class i are avirulent except js -a and a b strains [ , ] which were generated by experimental consecutive passages through chicken. amongst all genotypes of class ii, genotype i and ii are the most prevalent genotypes in the waterfowl and have been isolated from many countries (summarized in table ). waterfowl-origin isolates belonging to genotype vii of apmv- are constantly increasing especially in china, republic of korea, and taiwan [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these emerging outbreaks are increasing the global burden of apmv- and causes heavy economic losses [ ] . genotype iv and v have not been isolated from the waterfowl (table ) . collectively, these epidemiological studies clearly demonstrate the susceptibility of waterfowls for apmv- and their possible roles in the epizootiology of viruses. most of our understanding on the surveillance of apmv- in wild birds came from epidemiological studies on avian influenza viruses. thus, it is required to design apmv- dedicated studies to effectively assess the true prevalence of the virus in wild birds. it is essential to understand and establish the foundations to devise control strategies, especially in wild-birds populated and highly vulnerable commercial poultry areas. waterfowls are less susceptible to apmv- compared to chickens, such as ducks and geese. a key reason for less susceptibility of waterfowl to apmv- is the presence of retinoic acid-inducible gene i (rig-i). rig-i is absent in the chicken [ ] whereas it is present in ducks and goose ( figure ) [ , ] . rig-i and melanoma differentiationassociated gene (mda ) are the foremost part of retinoic acid inducible gene-like receptors (rlrs) (figure ), which senses the cytoplasmic rna [ ] . these sensors can detect the nucleic acids of negative sense rna such as apmv- and influenza viruses [ , ] , resulting in the production of the ifn type i and iii, cytokines, chemokines and expression of the antiviral genes [ ] . there is a vital role of the rlrs in the recognition of the viruses and antiviral immune responses in macrophages, fibroblast, and dendritic cells [ , ] . the v protein of apmv- blocks the "downstream" signaling pathway by interacting with mda resulting in blockage of strong antiviral response of ifn-β in chicken, which provide the benefit to waterfowl via rig-i pathway [ , ] . positive correlation exist between the resistance to apmv- infection and expression of the antiviral genes including rig-i, irf , irf and ifn-β [ ] which was further confirmed in a study demonstrating increased expression of rig-i ultimately leading to decreased apmv- load in vitro as well as in vivo [ ] . apmv- infection increases the expression of interleukin (il) il- β, tumor necrosis factor-α-like factor and interferon (ifn)-β in duck embryo fibroblast (def) cells. distinct innate immune responses of the waterfowl against apmv- may reason the resistance of waterfowls to these infections [ ] . a higher level of innate immune genes expression has been observed in chicken embryo fibroblast (cef) compared to the def cells [ ] . an experimental study reveals that apmv- stimulate a strong and intense expression of the ifn-β in ducks compared to chickens [ ] , and also up-regulate the ifn-β, ifn-regulatory factor (irf- ), and decreases the virus titer in goose lung and air-sac postinfection [ ] . these observations dictate that the strong innate immune responses is a plausible reasons for less susceptibility of duck [ ] and geese because its strong protective effects have been revealed to decrease the virus titer in goose-transfected cell [ ] , which may not be compensated by the ifn-α [ ] . recently, yang et al. [ ] have demonstrated that overexpression of ′- ′-oligoadenylate synthetase-like gene lessens the replication of apmv- in goose cells. in conclusion, waterfowls have diverse innate immunity components, which possibly increase their resistance to the apmv- [ ] . besides the strong innate immune responses, waterfowl are generally considered long-term carrier of apmv- and disease outbreaks have been reported since [ ] [ ] [ ] , and were confirmed by follow up experimental studies. clinically and naturally infected ducks and geese with apmv- show clinical signs such as elevated body temperature, excessively excreted oral mucus, dried cloaca, watery, greenish-white diarrhea, vain attempts of eating and drinking, listlessness, anorexia, crouch, eyelid edema and emaciation [ , , , ] . ducks may show up to % decrease in egg production, % morbidity and % mortality [ , ] however the mortality in ducks varies with the different breeds, virus strain and dose of virus [ ] . some birds also show weakness of legs and wing along with unilateral or bilateral incomplete paralysis and the effects of this paralysis increases with progression of the disease [ ] . duck and geese also show the neurological signs such as muscular trembles, muscular dis-coordination, circling, and twisting of head and neck [ , , ] . these clinical signs disappear according to infection status; mildly affected recover sooner and severely affected birds may recover after days of infection [ , ] . apmv- infected ducks and geese show the gross lesions on the immune organs such as bursa, spleen, thymus, mild to severe tracheitis, kidney enlargement, necrosis of pancreas, congestion on the meninx and in the brain and diffuse brain edema, focal hemorrhages in the mucosa of the proventriculus and intestine (especially duodenum and upper part of jejunum) [ , , , ] . bursal atrophy, hemorrhagic thymus and splenomegaly with white necrotic spots were found in the apmv- infected geese and duck [ , ] . these lesions and histopathological changes may be due to higher viral loads, multi-systemic distribution of the virus in these immune organs [ ] . as these immune organs are the reservoir of immune cells, and their destruction may lead to low antibody titer and other infections. experimental infection studies are necessary to determine the virulence and pathogenesis, in different bird types, age, species, intervention strategies, evaluation, comparison of vaccines etc. different scientists propose diverse reasons for experimental infection studies and the design of the experimental infection studies varies greatly with the above-mentioned factors. experimental infection studies on the pathogenesis, infection route, most susceptible age, bird line and immune responses are limited in waterfowl and available information is summarized in additional file and are briefly discussed below. kang et al. [ ] have studied the immune related gene expression of chicken and duck embryonic fibroblast (cef, def), by infecting them with apmv- of moderate virulent strain nh- and highly virulent strain ss- . upregulated expression of the toll like receptor (tlr) , tlr , il- β, il- , ifn-α, ifn-γ, mhc-i and mhc-ii were observed both in cef and def, however, these expression levels were higher in cef (mechanism is described briefly in figure ). peking duck infection at week of age with nh- and ss- via intranasal route showed the systemic replication of the virus into small intestine, cecal tonsils, brain, lung, bursa of fabricius, thymus, and spleen. this study also demonstrated the increased expression of the tlr , tlr , rig-i, mda , il- β, il- , il- , il- , ifn-α, ifn-β, ifn-γ in lungs compared to thymus. furthermore, the higher expression level of tlr , tlr , il- β, il- , il- , ifn-α, ifn-γ and mhc ii were induced by nh- than ss- in the lungs. whereas, the expression of the il- and ifn-β in lung as well as thymus was higher for ss- group [ ] . similarly, a study of the zhong et al. [ ] has demonstrated the upregulation of the viperin (an ifn stimulated gene) in the defs and also in the spleen, kidneys, liver, brain, and blood of changbai ducks infected with g apmv- through intranasal or intramuscular route. experimental infection of geese with genotype vii apmv- upregulate the expression of tlr - , , , and , avian β-defensin - , , , and , il- , il- , il- β, and ifn-γ, and mhc class i in different tissues [ ] . intranasal inoculation of the japanese commercial ducks and chicken males with artificially made apmv- class i virus a b results in the higher ifn-β in the duck compared to chicken. this study also demonstrated that replication, distribution, tissue damage and apoptosis were more in the immune organs of the chicken compared to duck [ ] . intramuscular infection of different ducks (mallard, gaoyou, shaoxing, jinding, shanma, and peking ducks) with jsd strain showed that different strains vary in the susceptibility to the disease [ ] . mallard are the most susceptible and peking ducks are the most resistant species of birds. infection of the gaoyou duck at , , , , and with different routes indicate that their susceptibility to disease and virus shedding decreases with the age and birds seldom die after infection through the natural route [ ] . experimental co-infection of ducks with apmv- , and low or high pathogenic avian influenza virus (lpaiv and hpaiv) indicate that it decreases the virus shedding and transmission to the naïve ducks by contact [ ] . duck after immunization with inactivated vaccine of apmv- , and challenge with the same live virulent kenyan apmv- resulted in the development of more antibody titer than the unchallenged birds [ ] . experimental infection of geese with virulent apmv- genotype viid and goose origin apmv- showed the extensive replication of geese in the immune organs which correlated with the clinical signs and lesions [ , ] and also it transmission to spf chickens [ ] . geese and chicken were vaccinated, then infected with goose-origin apmv- /na- and chickenorigin apmv- /f e , and f e viruses (additional file ). geese are more resistant to f e virus after vaccination. results indicate that na- vaccine provides a better protection in the form of less morbidity, less mortality and less virus shedding after challenge [ ] . although several natural outbreaks and experimental infections of apmv- in waterfowl had been reported. however, it remain to be clarified whether it cause the disease in waterfowl [ ] . inconsistent results in the infection of apmv- in waterfowl are due to apmv- strain, dose and rout of inoculation, breed, and maternal antibody titer (additional file ). ducks are more resistant to infection through natural route than the geese [ ] . in conclusion, these studies indicate that different viruses affect the immune organs and innate immune genes in diverse mechanisms. waterfowls are more susceptible to apmv- infection at an early age, and through natural route results in less damage to immune organs. more comprehensive and detailed studies are warranted for the control of nd in waterfowl. waterfowl are naturally infected with large number of the viruses which are avirulent and do not cause the diseases in domesticated poultry. waterfowl are commonly considered to be the natural host as well as carrier of apmv- [ ] . apmv- isolates from the waterfowl are generally lentogenic or potentially pathogenic [ ] , and may be transmitted to the avian species, leading to increase attention for their role in the transmission/spread and emergence of nd [ ] [ ] [ ] . there are increasing concerns about the increased virulence from the lentogenic to mesogenic to virulent pathotypes upon cyclic replication in poultry. these lentogenic isolates may converted to pathogenic viruses through serial passage in susceptible birds [ , , ] and one such isolate have already been documented to be the causative agent of the outbreak in the ireland [ ] , but disappeared quickly. all the class i apmv- ′s had been isolated from the waterfowl, indicating them the natural carrier of these viruses (table ) . live bird market (lbm) epidemiological study in united states indicated that from avirulent viruses, % belong to class i and % belongs to class ii [ ] . there is close phylogenetic relationship between the avirulent viruses isolated from the lbm and waterfowl, indicating that apmv- ′s may be transmitted from the waterfowl to the domestic poultry [ , ] . transmission of apmv- may occur through different routes like, ingestion of the contaminated feed, water air, contaminated feces, animals, humans, contaminated eggs etc. [ ] . mostly domestic waterfowl are reared in semi-closed areas, where they may have the contact with the wild birds and domestic poultry. therefore, it provides the best natural environment for the spread of apmv- ′s [ , ] . waterfowl and shorebirds are not only the host, but are also infected by the apmv- and these viruses can also cause the disease in domesticated poultry [ , , ] . many virulent isolates from the domestic poultry cannot cause disease in waterfowl [ , ] . most prevalent virulent genotype vii causing the endemics in china, japan, korea [ , , ] are co circulating into the ducks and chicken [ ] . this coevolution was further confirmed when the results of huang et al. [ ] declared that some of the circulating apmv- had the multiple homologous genomes from chicken, ducks and geese. so, there is dire need to modernize the housing system of waterfowl according to biosecurity point of view to prevent their contact with terrestrial poultry and wild birds. otherwise, number of virulent [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and avirulent [ , , , ] isolates from the waterfowl may easily transmit to commercial poultry farm. apmv- infected waterfowls shed the virus for an extended period of time [ ] whereas, the infected chickens clears themselves rapidly and shed virus for short duration [ ] . this prolonged virus shedding may facilitate the transmission, persistence and evolved to get some point mutation in the virus [ ] . major issue that should be concerned for virologists is the high evolution rate of some class i apmv- isolates from waterfowls. pathogenicity of some class i apmv- isolates are constantly increasing and they are naturally converted to low virulent from avirulent viruses (unpublished findings from our lab). this was confirmed by the results of the meng et al. [ ] and shengqing et al. [ ] in which they artificially develop the virulent viruses through serial passage in the air sac and brain of chicken, from avirulent viruses of waterfowl origin. although the conditions provided in these studies are not naturally existing but recovery of the virus from the air sacs after challenged via nasal or ocular route indicate the possibility of this mechanism. in conclusion waterfowl plays a vital role in the transmission and re-emergence of nd in terrestrial poultry. it is recommended that rearing facilities of the waterfowl should be separated from commercial poultry. cumulating evidences indicate that genetic resistance to apmv- exists in various breeds of waterfowl [ ] . comprehensive studies are needed to determine the genetic variability against apmv- in different breeds of waterfowls. genetic resistance of more susceptible breeds can be improved by including the resistant birds in the breeding programs of the commercial waterfowl [ ] . apmv- resistant breeds should be used to produce highly efficient transgenic poultry birds [ ] . although constant outbreaks of nd have been reported in waterfowl from china and other east asian countries, however, vaccination against nd in waterfowl is still a matter of debate. there are two consortia of scientists; one favors the vaccination and, others think that domestic waterfowl should not be vaccinated. these veterinarians have their own views, scientist, in the favors of vaccination argue, that it will decrease the chances of outbreaks and lessons the virus shedding. but, scientists, which are against vaccination argue that it will increase the virus burden on the birds, consequently increase the variation rate. the annual rate of change of virulent viruses could be as high as ten times the rate of change of low virulence apmv- , suggesting that other selective pressures such as vaccination may accelerate the rate of evolution of virulent viruses [ , ] . they claim that the rearing conditions of waterfowl could not be changed in future because they require water for breeding. therefore, it is not plausible to prevent their contact with wild birds, resulting in transfer of the wild bird viruses to domestic waterfowls. secondly, they claim vaccine will develop the antibody titers that will interact with the virus in the future exposures. these antibody responses may force the viruses for evolution. our viewpoint supports the group of scientists, which claims that birds should be vaccinated. because, it had already been established that apmv- cause the disease in waterfowl [ , , , ] and vaccination prevent the chances of the outbreak. second most important reason is the prolonged virus shedding by non-vaccinated birds, which may transfer to other poultry species and cause heavy economic losses. theory, that vaccination will increase the virus burden is not very interesting in case of waterfowl because, without vaccination we have to face the same consequences of the high evolution rate. waterfowls are naturally infected with large and diverse groups of viruses [ ] . development of the vaccines for waterfowl may require strains of waterfowl origin [ ] because some lentogenic vaccines phylogenetically are far away than the infectious virus thus may provide partial protection to waterfowl [ ] . vaccine should also be killed because live vaccine virus replicates in the body and leads to shedding of the virus. virus shedding of live vaccine may interact with the other apmv- 's present in the environment or birds and results in recombination and future evolution. waterfowls are not only the carrier but also susceptible to apmv- . strong innate immune responses may attribute to the less susceptibility of nd to waterfowl. viral shedding by waterfowl for prolonged duration may increase the transmission, evolution and emergence of new viral strains. a number of apmv- isolates from the waterfowl are reported with high mutation rate, which is an alarming matter and may cause the endemics in future. pathogenicity of nd is affected by different factors such as type, dose, and inoculation route of the virus, and species and age of birds. further studies are needed to explore the mechanism, and its intervention to prevent the virus shedding by the waterfowl for a long period. continuous studies are also required to monitor the apmv- in the waterfowl, which may be the future threat to commercial poultry industry. studies on the humoral immune responses of waterfowl are crucial to develop better intervention strategies. it is recommended that rearing facilities of the waterfowl including ducks and geese should be separated from chicken and turkey flocks to prevent the virus transmission. additional file . experimental infection studies of ducks and geese with avian avulavirus . table summarizes temporal, geographic, and host distribution of avian paramyxovirus (newcastle disease virus) infectivity and pathogenicity of newcastle disease virus strains of different 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disease virus strain na- isolated from geese in china genetic diversity of newcastle disease viruses isolated from domestic poultry species in eastern china during complete genome sequences of new emerging newcastle disease virus strains isolated from china the authors declare that they have no competing interests.authors' contributions zur, cm and cd developed the idea; zur and cm collected the data, wrote and revised the manuscript; cm, ys, kmm and su helped in collection of data and contributed to writing the manuscript; mm helped in the illustrations; mm and cd critically revised the manuscript. all authors read and approved the final manuscript. key: cord- -lgutt r authors: lauterbach, sarah e.; nelson, sarah w.; robinson, meghann e.; lorbach, josh n.; nolting, jacqueline m.; bowman, andrew s. title: assessing exhibition swine as potential disseminators of infectious disease through the detection of five respiratory pathogens at agricultural exhibitions date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: lgutt r widespread geographic movement and extensive comingling of exhibition swine facilitates the spread and transmission of infectious pathogens. nasal samples were collected from pigs at exhibitions and tested for five pathogens. at least one pathogen was molecularly detected in pigs at ( . %) exhibitions. influenza a virus was most prevalent and was detected in ( . %) samples. influenza d virus was detected in two ( . %) samples. more than one pathogen was detected in ( . %) samples. influenza a virus remains a top threat to animal and human health, but other pathogens may be disseminated through the exhibition swine population. agricultural swine exhibitions showcase a variety of swine related educational programs, livestock auctions, and competitive shows for youth throughout the united states. swine exhibitions are unique agricultural settings where large numbers of pigs from various geographic locales that would otherwise not come in contact with one another are co-housed and comingled for the length of the exhibition. long exhibition durations ( - days), direct contact of swine, and relaxed biosecurity strategies have all been recognized as risk factors for the rapid transmission of infectious pathogens infecting swine during exhibitions [ , ] . influenza a virus (iav) has been routinely detected in swine at agricultural exhibitions, where subclinical infection is common and can make recognition difficult [ ] [ ] [ ] [ ] . swine are important mixing vessels of iav, where genetic reassortment between multiple iav strains may produce novel, potentially more virulent strains [ ] . the comingling of large numbers of iav infected swine at exhibitions may increase the chance for the emergence of these reassortant viruses. in addition to the extensive interaction of swine, exhibitions also create a unique human-animal interface, where exhibiting families and the general public are permitted to interact with swine. this unique swine-human interface facilitates zoonotic transmission of iavs during swine exhibitions [ ] . continued detection of variant iavs, iavs infecting humans that normally circulate in swine, in individuals who have association with swine exhibitions has begun to heighten disease awareness and surveillance in exhibition swine [ , ] . combined with the ability of iavs to reassort in swine, the increased swine-human interface of exhibitions creates opportunities for novel iav strains to enter the human population, potentially leading to an influenza pandemic. continued disease surveillance is vital to understanding the epidemiology of iav and other pathogens infecting swine at agricultural exhibitions in order to protect animal and public health. commonly raised on small farms with varying management practices, the exhibition swine population is unlike commercial swine, which are managed in large herds under strict biosecurity protocols [ , ] . coupled with extensive movement over a large geographic area in order to attend multiple exhibitions, exhibition swine facilitate the widespread dissemination of infectious pathogens throughout the country [ , ] . therefore, exhibition swine represent a unique niche in the total swine population that may play an important role in the ecology and epidemiology of all swine infectious diseases. with continued detection of iav and increasing awareness of disease in exhibition swine, the current study aims to identify pathogens in addition to iav that may be circulating in this swine population. other swine respiratory viruses such as porcine hemagglutinating encephalomyelitis virus (phev), porcine reproductive and respiratory syndrome virus (prrsv), porcine parainfluenza virus (ppiv ), and influenza d virus (idv) have only briefly been described in exhibition swine and their prevalence and overall impact in exhibition swine remains unknown [ , [ ] [ ] [ ] . with varying degrees of severity and impact on swine and human health, understanding the overall disease ecology and epidemiology of the exhibition swine population is vital to the development and implementation of disease mitigation strategies designed to protect animal and public health. here, we describe the overall estimated prevalence of five infectious respiratory viruses in swine at agricultural exhibitions and assess the potential epidemiological role of exhibition swine. in , as part of an ongoing iav surveillance program, pigs at exhibitions across six states were sampled by nasal swab or nasal wipe [ , ] . all samples underwent nucleic acid extraction and were tested individually for iav using real-time reverse transcription polymerase chain reaction (rrt-pcr) as previously described [ ] . five microliters of extracted nucleic acid of no more than seven individual pigs from the same exhibition were pooled and screened for ppiv , phev, and idv using rrt-pcr as previously described [ , , ] and for prrsv using the vetmax prrsv reagents and manufacturer's protocol (thermofisher scientific, waltham, ma, usa). nucleic acid of samples within positive pools was subsequently tested individually using the same methods. during field sample collection, exhibitions with ≥ pig showing clinical signs consistent with influenza like illness (ili) were recorded as having pigs with ili [ ] . five of the exhibitions were excluded from statistical analysis due to lack of clinical sign data. logistic regression (stata special edition . , college station, tx, usa) was performed to determine any association between ili noted at the exhibition and the detection of a pathogen in the pigs. at least one pathogen was detected in the pigs at ( . %) of the swine exhibitions tested. influenza a virus was detected among the pigs at ( . %) and was detected at more exhibitions than any of the other viruses. in contrast, influenza d virus was detected in the pigs at the fewest exhibitions; only pigs at two ( . %) of the exhibitions tested positive for idv (table ). more than one pathogen was detected among the pigs at ( . %) exhibitions; four pathogens were detected at three ( . %), representing the highest number of pathogens detected among the pigs at any of the exhibitions. exhibitions with pigs testing positive for at least one pathogen had . times the odds of having noted ili compared to exhibitions where no pathogens were detected (p < . ). detection of iav in swine at exhibitions was the only pathogen with a significant positive association to noted ili at exhibitions (or = . , p < . ). however, with fewer positives, this association may be hidden for the other pathogens. out of the pigs sampled, ( . %) tested positive for at least one pathogen. the most commonly detected pathogen was iav which was detected in ( . %) pigs. influenza a virus was detected in more pigs than the next two most prevalent pathogens combined, phev and ppiv , which were detected in ( . %) and ( . %) pigs, respectively. influenza d virus was the least prevalent and was only detected in two ( . %) pigs (table ) . co-infections were common with ( . %) pigs testing positive for more than one pathogen, including four ( . %) pigs testing positive for three pathogens, which were the most pathogens found in co-infected, individual pigs. due to widespread movement and prolonged intermingling, exhibition swine and the variety of pathogens they carry should be considered significant for both swine and human health. there has been increased attention on iav in swine at exhibitions from veterinarians, health officials, and exhibition attendees due to recent iav zoonotic transmission events [ , , ] . previous active surveillance has described the epidemiology of iav in swine at exhibitions in the u.s.; at exhibitions where ≥ pig tests positive for iav, over % of the pigs will have active iav infection by the conclusion of the exhibition [ ] . there were cases of variant influenza infections associated with the iavs detected in this population of exhibition swine, and iav should remain a priority for infectious disease surveillance and mitigation in exhibition swine [ ] . several mitigation strategies have been suggested and implemented at exhibitions as the concern for animal and public health rises. many of these strategies are aimed at reducing the risk of iav transmission between pigs and humans before, during, and after agricultural swine exhibitions [ , ] . still, iav continues to impact exhibition swine and was detected at almost double the prevalence of phev, the next most prevalent virus of those included in this study. despite the persistent spread of iav through exhibition swine and its implications for both animal and human health, other infectious pathogens can be detected in exhibition swine and should not be ignored. identification of active infections in exhibition swine is vital to protecting animal and human health during agricultural exhibitions. pigs at exhibitions with at least one pathogen detected in the pigs were more likely to exhibit ili. however, subclinical infections are common and there is overlap in clinical signs associated with various respiratory pathogens in swine; because of this, clinical signs should not be solely relied upon for the detection and subsequent diagnosis of infectious pathogens in swine at exhibitions [ , ] . proper biosecurity should be used at all times, even in the absence of clinical signs, in order to reduce the risk of intra-and interspecies transmission of all pathogens during exhibitions. focus on animal and public health at swine exhibitions is a priority, but the potential for introduction of infectious pathogens from exhibition swine into commercial swine facilities should also be considered. an estimated % of exhibition pigs are raised near commercial swine, with many of those returning home after attending an exhibition [ ] . detection of pathogens such as phev and prrsv in exhibition swine raises concern for commercial swine due to the potential economic implications should they enter the production system. prrsv alone causes an estimated $ million in economic loses annually in the united states swine production system [ ] . detection of ppiv and idv also raises concern, though they are more recently described in swine and their overall consequences to swine health are still unknown [ , ] . in addition to the five pathogens assessed in the present study, there is potential for other pathogens to travel with and spread through exhibition swine that may also present a threat to commercial swine, including foreign animal-diseases. exhibition swine should not be disregarded as potential hosts and disseminators of diseases such as african swine fever virus (asfv); movement and comingling of pigs have been recognized as risk factors for the spread of asfv in endemic regions [ ] . if overlooked, exhibition pigs may create a niche for devastating pathogens, with extensive animal movement resulting in rapid spread across the country. the findings of this study highlight the potential role of exhibition swine in the disease ecology and epidemiology of the united states swine population. lack of sustained disease surveillance and application of mitigation strategies for swine at agricultural exhibitions will allow for continued variant iav infections that pose considerable risk to public health and could ultimately lead to a devastating disease epizootic within the u.s. swine herd. exploration of risk factors contributing to the presence of influenza a virus in swine at agricultural fairs national association of state public health veterinarians. measures to minimize influenza transmission at swine exhibitions subclinical influenza virus a infections in pigs exhibited at agricultural fairs characterization of an influenza a virus isolated from pigs during an outbreak of respiratory disease in swine and people during a county fair in the united states simultaneous infection of pigs and people with triple-reassortant swine influenza virus h n at a u.s. county fair active surveillance for influenza a virus among swine, midwestern united states the pig as a mixing vessel for influenza viruses: human and veterinary implications evolutionary dynamics of influenza a viruses in us exhibition swine outbreak of influenza a(h n ) variant virus infections among persons attending agricultural fairs housing infected swine-michigan and ohio outbreak of influenza a (h n ) variant virus infection among attendees of an agricultural fair movement patterns of exhibition swine and associations of influenza a virus infection with swine management practices potential role of noncommercial swine populations in the epidemiology and control of porcine reproductive and respiratory syndrome virus introduction, evolution, and dissemination of influenza a viruses in exhibition swine in the united states during to porcine hemagglutinating encephalomyelitis virus and respiratory disease in exhibition swine complete genome sequence of an influenza d virus strain identified in a pig with subclinical infection in the united states widespread detection and characterization of porcine parainfluenza virus in pigs in the usa nasal wipes for influenza a virus detection and isolation from swine utility of snout wipe samples for influenza a virus surveillance in exhibition swine populations prevalence of influenza a virus in exhibition swine during arrival at agricultural fairs isolation of a novel swine influenza virus from oklahoma in which is distantly related to human influenza c viruses two cases of flu linked to contact with swine at fowlerville fair. the detroit news influenza a(h n ) virus in swine at lauterbach et al. vet res ( ) : • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc, research is always in progress. learn more biomedcentral.com/submissions ready to submit your research ? choose bmc and benefit from: agricultural fairs and transmission to humans assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers small-scale pig farmers' behavior, silent release of african swine fever virus and consequences for disease spread publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank field staff michele zentkovich, alison martin, courtney wright, lauren smith, and alex tweedy for collection of the samples as well as research assistants rachel patton, libby mcgarry and dillon mcbride for help in the laboratory. we also thank the exhibition officials and participants for partaking in the study. funding was provided by the centers of excellence for influenza research and surveillance, national institute of allergy and infectious diseases, national institutes of health, department of health and human services, under contract nos. hhsn c and hhsn c. iav: influenza a virus; phev: porcine hemagglutinating encephalomyelitis virus; ppiv : porcine parainfluenza virus ; prrsv: porcine reproductive and respiratory syndrome virus; idv: influenza d virus; rrt-pcr: real-time reverse transcription polymerase chain reaction; ili: influenza like illness.authors' contributions sel: data analysis, data interpretation, manuscript preparation. swn: laboratory sample testing, data analysis, manuscript preparation. mer: study design, laboratory sample testing, data analysis, manuscript revision. jnl: study design, data interpretation, manuscript revision. jmn: study design, data interpretation, manuscript revision. asb: study conception and design, data interpretation, manuscript revision. all authors read and approved the final manuscript. funding was provided by the centers of excellence for influenza research and surveillance, national institute of allergy and infectious diseases, national institutes of health, department of health and human services, under contract nos. hhsn c and hhsn c. all data generated or analyzed during this study are included in this published article. ethics approval for animal use in this research was provided by the institutional animal care and use committee at the ohio state university under approval number a -r . key: cord- -jfawhnsq authors: caron, alexandre; cappelle, julien; cumming, graeme s; de garine-wichatitsky, michel; gaidet, nicolas title: bridge hosts, a missing link for disease ecology in multi-host systems date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: jfawhnsq in ecology, the grouping of species into functional groups has played a valuable role in simplifying ecological complexity. in epidemiology, further clarifications of epidemiological functions are needed: while host roles may be defined, they are often used loosely, partly because of a lack of clarity on the relationships between a host’s function and its epidemiological role. here we focus on the definition of bridge hosts and their epidemiological consequences. bridge hosts provide a link through which pathogens can be transmitted from maintenance host populations or communities to receptive populations that people want to protect (i.e., target hosts). a bridge host should ( ) be competent for the pathogen or able to mechanically transmit it; and ( ) come into direct contact or share habitat with both maintenance and target populations. demonstration of bridging requires an operational framework that integrates ecological and epidemiological approaches. we illustrate this framework using the example of the transmission of avian influenza viruses across wild bird/poultry interfaces in africa and discuss a range of other examples that demonstrate the usefulness of our definition for other multi-host systems. bridge hosts can be particularly important for understanding and managing infectious disease dynamics in multi-host systems at wildlife/domestic/human interfaces, including emerging infections. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. ecological functional approaches classify organisms according to what they do, and/or what they eat. they offer an alternative perspective to taxonomic classifications for identifying trends within and making sense of ecological complexity. applications of functional group concepts, which date back to fundamental ideas about biomass distributions across different trophic levels [ ] , have been crucial in advancing ecological understanding. more recently, ecological functional analyses have achieved prominence as a way of linking taxonomic survey data and the provision of ecosystem services [ ] . functional analyses thus remain an important research area in ecology. in epidemiology, functional concepts have clear potential utility but are still in a relatively early stage of development. classical epidemiology relies heavily on single-species studies, particularly those of people (e.g., analyses of measles and smallpox in human populations [ ] ). in contemporary epidemiological studies, in the last fifteen years, under the influence of ecology, the scope of epidemiology is being broadened to include plant and animal communities in which multiple different species can contribute to the maintenance and spread of pathogens in host populations [ ] . in multihost systems, the role played by each host population in pathogen dynamics is determined by the species' competence for the pathogen (i.e., its receptivity to infection and its capacity to replicate and transmit the pathogen [ ] ), its exposure to the pathogen determined by the host ecology/behaviour and its interactions with other host populations (including vectors for vectorborne infections) leading to infectious contacts, and finally, the composition of the host community that will determine the range of inter-host interactions [ ] . one of the central questions in disease ecology is that of how the community composition of potential host species relates to the dynamics of pathogen transmission within the host community, as opposed to within a population of a single species. the complexity of this problem can be simplified by assigning epidemiological functions to relevant traits that define an organism's role in the epidemiology of a given pathogen. for example, animals that undertake long movements (a trait) may contribute to the epidemiological function (pathogen disperser) of spreading pathogens over large distances (a role). grouping organisms by epidemiological functions facilitates the development of eco-epidemiological models for a given pathogen in relation to an entire animal community [ ] . this approach could potentially play an important role in guiding research, as well as in the surveillance and control of animal and zoonotic diseases [ ] . although some progress has been made in the characterization of epidemiological functional groups, (e.g., clear definition of the maintenance function [ , ] ), other epidemiological functions remain incompletely defined, especially those relating to the transmission of pathogens between groups of hosts. in this paper we first define the transmission function in relation to the maintenance function. we then focus on the concept of "bridge hosts" and demonstrate their potential importance in the ecology of disease transmission in multi-host systems. though closely related concepts have been used previously [ ] [ ] [ ] , we believe that a refined definition embedded in a clear functional framework is still lacking. lastly, we present an operational framework to identify potential bridge host populations, using as a case study the ecology of avian influenza viruses at the wild/domestic bird interface in africa and also giving other multi-host systems examples. we use "host" to refer to a host population, a host species, or a host community. the smallest epidemiological unit to which we will refer is a host population, acknowledging the fact that individual variability can also substantially impact pathogen transmission (e.g. "superspreader", [ ] ). as defined by haydon et al. [ ] and more recently revised by viana et al. [ ] , a conceptual framework for the role of hosts in epidemiology requires the definition of the target host: "the population of concern to the observer" in the geographic area under study (table ) . a maintenance host will only be relevant to a target population if it can be in contact with and able to transmit the infection to it. the maintenance function represents the capacity to maintain the pathogen within the ecosystem. a maintenance host is a host population (single population) or community/host complex (several sympatric host populations) "in which the pathogen persists even in the complete absence of transmission from other hosts" [ ] . the maintenance function depends on host density, and on intraand inter-host infectious contacts (i.e., a contact leading to infection amongst other intra-host factors; [ ] ). in multihost systems, the notion of a maintenance community in which several populations from different species play a role in the maintenance of the pathogen seems more appropriate than the "reservoir" concept [ , ] for understanding pathogen dynamics. the reservoir concept is still being used in contradictory ways, as discussed by several authors [ ] [ ] [ ] . haydon et al. [ ] extended the definition of reservoir by adding "source populations" that may not be involved in the maintenance of the pathogen but rather in the transmission of the pathogen to the target population. ashford [ ] defined a "liaison host" as linking the reservoir to another host population, with no explicit reference to target populations. we agree with ashford [ ] that source population should not be included in the definition of the reservoir, as this term is strongly linked to the concept of maintenance and because control of infection in the reservoir would be different if targeted at the maintenance or source populations. for example, aiming at controlling the infection in a maintenance vs. a source population might have different outcomes, since the maintenance host could still re-infect the source population in the latter case. to add to the confusion, suzán et al. [ ] presented a new framework to understand patterns in space and time of meta-communities of hosts and parasites. in their first figure they display in red "reservoir species" and in orange "alternative hosts", together "maintaining higher infection of prevalence". clearly, their concept of "reservoir" differs from that of haydon et al. [ ] , who argued that any host involved in the maintenance of the pathogen should be part of the reservoir. the difference in definitions is identical with plowright et al. [ ] : they present domestic horses as potential source populations (defined in the article as "recipient" and "intermediate hosts") of hendra viruses for human populations without considering them as part of the reservoir (presented as the bat community). the extensive use of the "reservoir" concept under multiple definitions and the lack of consensus around the liaison host and source population concepts (revealed by the scarcity of use of these two last terms in the literature) requires a refined conceptual framework and definitions. agreeing with others [ , ] , we thus prefer to use only maintenance host or community, a term that refers better to the dynamic aspect of the functional role than the static notion of a reservoir [ , ] . although the maintenance-target host relationship and its link with the maintenance function have been properly defined, the function of pathogen transmission to the target host needs a clearer definition. interspecific pathogen transmission is of crucial importance for infectious disease management. disease control can target the maintenance host to stop pathogen maintenance and circulation in the ecosystem (i.e. targeting the maintenance function); however, as this option is often unfeasible (for practical or ethical reasons, notably concerning wildlife populations), one could also try to break the transmission pathway that brings the pathogen to the target host. we therefore define the transmission function as the capacity to transmit the pathogen to the target host. this function must be separated from the maintenance function, as the maintenance host does not always have infectious contact with the target host. when it has direct contact with the target host, then the maintenance host is implicated in the maintenance and transmission functions. when it does not, a bridge host (table ) can connect (i.e., have infectious contact with) both maintenance and target hosts, "bridging" the gap between them. using this functional definition, the concept of the reservoir as revisited by haydon et al. [ ] and more recently by viana et al. [ ] , does not refer clearly to a single epidemiological function, because it includes maintenance host(s) involved in the maintenance function and potentially in the transmission function as well as non-maintenance population(s) only involved in the transmission function. allocating hosts belonging to the reservoir to specific functional groups that surveillance and/or control can target is therefore difficult and provides an additional reason to focus solely on the maintenance-target hosts. bridge host is therefore used, since (i) the group is distinct from the source population, as bridge hosts do not belong to the maintenance host/community, and the liaison host as a bridge host is always in reference to a maintenance-target population system; and (ii) the word "bridge" is relevant to the definition proposed (e.g. [ ] ). -hosts in which the pathogen persists even in the complete absence of transmission from other hosts [ ] x (x) -brush-tailed possums for bovine tuberculosis in new zealand [ ] -population larger than the critical community size (i.e. size under which the pathogen cannot be maintained in the community) in which the pathogen persists [ ] -white-footed mouse (peromyscus leucopus) for lyme disease in the united states [ ] maintenance host community/ maintenance host complex -one or more epidemiologically connected populations or environments in which the pathogen can be permanently maintained [ ] x (x) -anatids for avian influenza viruses worldwide [ ] -any host complex in which disease persists indefinitely is a reservoir [ ] -amphibian sp. for the trematode ribeiroia ondatrae [ ] -host for which cross species transmission and inter-species transmission are high [ ] bridge host -non-maintenance host population able to transmit a pathogen from a maintenance host/complex to the target population, otherwise not or loosely connected to the maintenance complex (this manuscript) x previous related definitions: -little studied so far -source population: any population that transmits infection directly to the target population [ ] -red deer and domestic pigs for bovine tuberculosis in new zealand [ ] ? -liaison host: incidental hosts that transmit pathogens from a reservoir to another incidental host [ , ] -peri-domestic birds such as swallow sp., sparrow sp., etc. [ ] -spatial vector: host that transport the pathogen to target populations in new locations [ ] -temporal vector: host that can transmit the pathogen to target species across temporal scale [ ] crosses in brackets indicate that maintenance host can participate in the transmission function although this is not a necessary condition. bridge hosts refer therefore to a group of hosts that perform the same epidemiological function for a pathogen that can be targeted by specific surveillance and control interventions. in suzán et al. [ ] , information about whether alternative hosts function as bridge hosts would add an important layer of information to their framework and contribute to the understanding of the spatial spread of parasites. our bridge host definition is closely related to the "spatial and temporal vector" concepts presented by nugent [ ] but unifies them with previous definitions (see above) and broadens them. a bridge host can be defined at the level of a population or a community. bridge hosts may be frequent in disease ecology, but this term has not been explicitly defined and its usage is not common when referring to the transmission function without any role in maintenance function. for example, it would be incorrect to use the term "bridge species" as the role of a bridge host would refer to a specific host population in interaction with other hosts in a given ecosystem (e.g. contact with maintenance or target populations) and at a specific density [ ] ; the host density and the network of interaction between these hosts in another ecosystem would likely be different and would make it unlikely that a species can play the same epidemiological functional role across its range. a clearer conceptual framework is thus needed to guide the identification of bridge hosts and the characterisation of their roles in disease ecology. this framework must also be operationalised if it is to guide the design of hypotheses that can be tested through field protocols to characterise the role(s) of hosts in disease ecology. using the different target-maintenance systems proposed by haydon et al. [ ] , bridge hosts can be included in target-bridge-maintenance systems in several ways ( figure ). according to our definition, a bridge host is involved in the transmission function while not involved in the maintenance function. two main prerequisites must be fulfilled for a host to qualify as a bridge host. the first prerequisite is that the host must be competent for the pathogen (i.e., must be receptive to infection, permit pathogen replication, and be able to excrete it) without being able to maintain it alone, in which case the host would be considered as a maintenance host; or alternatively, the host should be able to mechanically transport the pathogen [ , ] . its competence will influence the capacity of a bridge host to achieve the transmission function: if the bridge host has a short pathogen excretion period, it will be able to transmit the pathogen to a target population only if the time lag between contact with a maintenance and then a target host is shorter than the excretion period, or if the distance between target and maintenance is shorter than the maximum distance that the bridge host can travel during its excretion phase. similarly, for mechanical transmission, the survival of the pathogen on/in the host body part (e.g. skin, hair, mouth, feathers) exposed to the external environment will determine for how long the host can play the bridge role. the second prerequisite is that infectious contacts must occur along the maintenance-bridge-target transmission chain. these will depend on direct and indirect (e.g. environmental transmission) contacts, the mode of transmission of the pathogen, and the site of infection. the basic reproductive number r for the bridge host (not considering here mechanical transmission) should be < as it cannot maintain the infection but its force of infection, dependent on the number and extent of infectious contacts with the target host, can be high. a bridge host that compensates for a lack of infectious contacts between maintenance and target hosts can operate across different dimensions: spatial, temporal, and behavioural. the spatial dimension arises when the bridge host creates a spatial link between the separate areas in which the maintenance and the target host populations occur. this dimension typically refers to the situations developed below for wild birds and avian influenza. it has been defined as a "spatial vector" by nugent [ ] when considering the role of feral pigs (sus scrofa) in the epidemiology of bovine tuberculosis (btb) in new zealand. the temporal dimension arises when the pathogen can persist (but not be maintained indefinitely) in the bridge host for a period of time longer than in the maintenance host or during a distinct season; this has been well described by nugent [ ] as a "temporal vector", for example when red deer (cervus elaphus) transmit mycobacterium bovis to possum populations that are controlled to levels that are well under the critical community size for btb maintenance. the behavioural dimension exists when the absence of contact between sympatric maintenance and target hosts is compensated for by another host that has infectious contacts with both. situations may occur in which the microhabitat preferences and behaviours of maintenance and target hosts mean that they do not come into direct contact despite using the same locations on a daily basis. bats, for example, are believed to be the maintenance host for ebola, and can be sympatric with people; but ebola transmission from bats to humans is enhanced by the great apes (whose susceptibility to ebola seems to indicate that they are not maintenance hosts) which feed with bats and are fed upon by humans [ ] . it is interesting to note that in all cases, even a r close to zero (approximating a dead-end host) could still be important for the transmission function: the capacity to excrete the pathogen for a few hours, associated with some form of dispersal, may be sufficient for a bridge host to come into contact with the target host and infect it. for pathogens like ebola, the range of hosts that is classically considered to be important in disease ecology may have to be broadened by including hosts that are able to transmit the pathogen over short time-and space-scales. these hosts are commonly considered as playing no role in pathogen ecology and are called dead-end hosts (e.g., most wild avian hosts for avian influenza virus -aiv -apart from anseriformes and charadriiformes). amongst the multitude of those dead-end hosts, the bridge host perspective can identify some that do play a role in disease ecology. with this framework in place, we next turn to the question of how bridge hosts can be identified in the multihost context of aiv epidemiology and suggest an operational framework (partially implemented in [ ] ) that can figure definition of different target-bridge-maintenance systems (adapted from haydon et al. [ ] ). a represents the simplest maintenance-bridge-target system. in a', the maintenance and target populations are less connected (frequency/intensity of infectious contacts) than between the maintenance-bridge-target populations. in b, mitigation strategies aimed at one bridge host cannot fully control pathogen transmission to the target host because of the alternative bridge host's pathway. if both maintenance populations were in contact with both bridge hosts (i.e. if dashed arrows exist), controlling contacts between the target population and bridge hosts should be simpler than other control options. in b', according to our definition, z is not considered as a bridge population as it belongs to the maintenance community. in c, stopping contacts between the maintenance population and the target population by acting on one of the two bridge hosts would not be enough to stop transmission, which can still occur through the second bridge host. d is a special case of b, understanding the complexity of the maintenance community is not necessary to control the pathogen transmission risk to the target population, which can be achieved through the control of arrows connecting the bridge host. in e, none of the host populations can sustain the infection by itself and according to our definition, u is not considered as a bridge population as it belongs to the maintenance community. in f, the bridge host connects the target population with another maintenance host creating a system with a maintenance meta-population, which could change the epidemiological dynamics of the system and the probability of success of intervention strategies (e.g. vaccination coverage to achieve control of the infection in the target population). g is a special case where two bridge hosts are necessary to achieve the transmission function. good knowledge of the ecological interactions in the ecosystem will be necessary to identify such complex interactions between bridge hosts. enhance disease ecology as well as pathogen surveillance and control. waterfowl (defined here as ducks, geese, waders, gulls, and terns) constitute the maintenance hosts for low pathogenic avian influenza viruses (lpaiv) [ ] . aiv represent major threats to poultry production when strains originating from wild birds evolve from low to high pathogenicity in the poultry (target) populations [ ] . the transmission of lpaiv between the wild bird maintenance community and domestic populations is therefore crucial to managing the sanitary and economic impacts of the disease. in this section, the risk of lpaiv spillover to poultry populations from the maintenance populations will be used as an example. when poultry are confined in farms or buildings, their direct contacts with the maintenance waterfowl community, which mainly lives in wetlands and on coastal shorelines, are believed to be limited due to spatial segregation between populations. many outbreaks of highly pathogenic aiv outbreaks have nonetheless occurred in domestic poultry production systems. it is therefore suspected that bridge hosts play a role in transmitting waterfowl-derived strains of aiv to poultry populations. the ability of wild birds to travel long distances, and their ubiquity in most habitats, facilitate the potential for wild bird species to act as bridge hosts. several constraints limit a better understanding of aiv ecology in bird communities: ) high host diversity, that can include several hundred species in a given ecosystem; ) the costs of diagnostic techniques that limit the number and type of samples (e.g. cloacal/tracheal swabs, blood) that can be analysed; and ) the impossibility of randomly sampling from bird communities because of bias in capture techniques (e.g. walk-in traps, mist-nets). as a consequence, the information available on most wild bird species is scarce and has been obtained mostly from by-catch (i.e. captured non-targeted species) of studies investigating aiv in maintenance waterfowl, resulting in small sample sizes that are inadequate to provide epidemiological understanding of the host roles in aiv ecology in africa [ ] . the following framework used in a recent study [ ] and here developed in detail, aims at first gathering/collecting available ecological and epidemiological information; second, at synthesizing this information to provide a priority list of species that act as potential bridge hosts; and finally, at undertaking targeted sampling that can determine the competence of the high priority species and revisit the framed hypotheses. the range of methods available to characterize host competence for aiv and contact patterns between maintenance, potential bridge and target host populations is drawn from the fields of epidemiology and avian ecology ( table ) . none of these methods alone is sufficient to identify a bridge host in a given ecosystem [ ] . molecular epidemiology (e.g. gene sequencing after virus isolation) could in principle be used to identify bridge species but it is very unlikely that related strains from three different host populations (i.e., maintenance, bridge and target hosts) are concurrently isolated except perhaps during a localised aiv outbreak. virological surveillance (e.g. polymerase chain reaction -pcr techniques) can provide information about host contacts between potential bridge and maintenance hosts if data are collected close to wetlands where waterfowl communities are known to occur. serological investigation (e.g. elisa tests) can be cheaper than virological testing but provide less information on the timing of the infection [ , ] . however, a combination of epidemiological and ecological methods could provide the necessary information to infer the bridge role of a given host population. taking into account these constraints, the proposed framework aims, first, to narrow the large number of species by ranking the most probable potential bridge hosts based on proxies of host competence and/or contacts between maintenance, target and potential bridge hosts. this step can be achieved using (or combining) available published field (e.g. [ , [ ] [ ] [ ] ) and experimental epidemiological studies (e.g. [ , ] ). however, most aiv experimental studies have concentrated so far on a very limited set of species (e.g. for lpai [ , ] and for hpai [ , ] ). reviewing available pcr viral data within a given area or region can provide information on the range of host species with a competence for aiv. for example, in sub-saharan africa, the available databases are poor representations of existing avian diversity (only . % of all african species have been sampled, additional files and , and only a few species were tested with a sample size that would be sufficient to detect % aiv prevalence). this exercise can help with ranking the species or groups of species based on the rate of infection, which provides an initial prioritization list for future investigation (additional file ). however, one shortcoming of pcr data is to link detection of genetic material and state of infectiousness of the sampled individual [ ] , an issue that is often overlooked but particularly important for the identification of bridge hosts. the first step of the proposed framework must also incorporate ecological data that provide information about the presence/abundance of potential bridge hosts in the ecosystem and their potential contacts with maintenance and target hosts. however, it is a challenge to provide evidence that contacts ( ) occur; and ( ) result in successful virus transmission. different types of data can be used or collected, each with its own strengths and weaknesses: life history traits (e.g., abundance, gregarism, foraging and migratory behaviour) obtained from the literature can be used as risk factors for contacts between wild and domestic birds or exposure to infection [ , ] ; contacts between wild and domestic birds can be estimated using satellite telemetry [ ] ; capture-recapture techniques indicate population size (e.g. using colour rings at a local scale) [ ] ; and observations at focal points that are at wild/ domestic bird interfaces (e.g. around poultry production building) can be used to quantify interactions [ ] . the second step is to synthesize the ecological and/or epidemiological data to rank bird species according to the likelihood that they play a bridge role in the ecosystem under study. risk analysis can provide such a tool [ , [ ] [ ] [ ] and may be particularly important when no information is available for an ecosystem, or prior to a field survey, by highlighting the populations that could be targeted preferentially. once the bridging potential of different species has been evaluated, the third step of the framework consists in testing the host competence of the most likely bridge hosts in the ecosystem through targeted sampling. for example, caron et al. [ ] applied this framework in a southern african ecosystem and identified bridge hosts by combining bird counts with selected sampling and pcr testing. targeted sampling facilitates the concentration of resources to obtain adequate sample sizes and relevant epidemiological information and comes in place of the practice of blind sampling from wild bird communities, which is usually biased by capture techniques. hypotheses can be revisited iteratively as more is learned about the potential of highly ranked species to act as bridge hosts. this approach can also lead to the detection of inconsistencies in the initial model (e.g., the definition of the maintenance community) and the necessity to revisit it [ ] . avian influenza provides a good example of a case in which paying conceptual and practical attention to bridge hosts can enhance our understanding of pathogen dynamics in multi-host systems. although the use of the bridge host concept may not be relevant for all multi-host systems, it has the potential to contribute to structuring investigations on the ecology of emerging pathogens shared at wildlife/livestock interfaces. to illustrate this point we present two additional examples of multi-host systems. in the first, ebola in west africa, understanding could be improved by the use of the conceptual framework developed here. in the second and better-known system, bovine tuberculosis in new zealand, bridge hosts have been identified and are an important component of the problem. ebola virus spilled over in early in west africa from an unknown animal to the human index case. knowledge of ebola ecology is still limited, despite the first outbreak having being reported in . current understanding points at bats (mammalia: chiroptera) as potential maintenance hosts, and contact between humans and some bat species occurs through the bushmeat industry [ ] as well as via bat droppings and occasional cases of sick bats that are handled by humans [ , ] . however, embracing the functional approach presented here makes sense to look for potential bridge hosts that could link maintenance bats and humans. a priori, scavenging pigs, dogs, other non-maintenance bat species and wild antelope can have direct or indirect (e.g. consumption/hunting) contact with humans [ ] . targeted surveillance of such species will provide information on their competence for the virus; and host interaction protocols that identify contact networks with maintenance and target populations can provide information on the potential for viral spread (e.g. [ ] ). once the multi-host system is better understood (case b, c or d in figure ), it may be simpler to try to block transmission pathways from bridge hosts to human populations (e.g. through changes in behavior related to bridge host consumption by people) than to control the pathogen in the maintenance hosts. [ ] [ ] [ ] [ ] risk analysis x x x [ ] [ ] [ ] serological investigation x x x xx [ , , ] virological investigation xx xx xx xx xx [ , , , , , , ] telemetry study xxx xxx xxx [ ] bird ringing and monitoring xx x x [ ] bird counts xx xx x [ , , , ] molecular epidemiology xx xx xx xx xx xxx [ , ] as the number of crosses increases in the first columns the methods provide better ecological or epidemiological information; in the last columns, cost increases as the number of crosses increases. a similar yet less complex example was recently developed [ ] , indicating that domestic horses could be "bridge hosts" for hendra viruses between bats (maintenance host) and humans (target host) even if it is not yet known if horses could maintain the virus or just act as a bridge between bats and humans [ ] . as a second example of the utility of the bridge host framework, nugent [ ] offers a comprehensive description of the btb multi-host system in new zealand. the cattle industry in new zealand suffers from continuous spillover of the btb mycobacterium from the maintenance host, the brush-tailed possum (trichosurus vulpecula). the control of possum populations by depopulation is mainly implemented in areas around farms that are at high-risk of transmission to cattle, leaving high densities of possums in more distant forest and providing a gradient of btb prevalence. this apparently efficient strategy is, however, thwarted by three potential bridge hosts (feral pigs sus scrofa, red deer cervus elaphus, and feral ferrets mustela furo) that are involved in transmission (case g in figure , called "link-host" in the article but lacking a more conceptual definition). infected pigs and deer with large home ranges may leave the forest to die (or be hunted) around farms, providing an opportunity for ferrets to become infected when feeding on carcasses and subsequently infecting cattle or possums. this study is particularly interesting for reasons: ( ) the complexity and low probability of the chain of events leading to infection of the target population do not prevent btb occurrence and the failure of disease control; ( ) disease control targeted at the maintenance population prevents the transmission link between the maintenance and target hosts but the transmission pathways built by bridge hosts (case a' in figure ) reduce the effectiveness of control, proving the importance of considering this epidemiological function and host role; and ( ) the plasticity of the roles of host populations in disease epidemiology, which is heavily influenced by the environmental, ecological and anthropological context. the concepts of transmission function and bridge host contribute to a better understanding of disease ecology in multi-host systems by clarifying the epidemiological processes that are relevant for disease transmission and maintenance. this perspective fits better with the way that people operationalize the complexity theory and makes it easier to develop models of these systems. when maintenance and target hosts are not in direct contact, pathogen transmission relies on successive infectious contacts along the chain of maintenance, bridge and target hosts. bridge hosts can play a pertinent and legitimate role in disease ecology and could become the targets for surveillance and control for some multi-host systems. for example, in some ecosystems, domestic bird populations are rarely in direct contact with wild waterfowl populations but phylogenetic analyses have indicated that most precursors of hpaiv in gallinaceous poultry have originated from wild waterfowl [ ] , suggesting that bridge hosts play a role in aiv transmission at the wild/domestic bird interface. more recently, evidence supporting a role for some passerines (finches, sparrows) in the transmission of the avian-origin human influenza a (h n ) to human and poultry in china [ ] suggests a potential role for passerines as bridge hosts between poultry and humans. the functional approach emphasizes the need to focus on transmission pathways between hosts (and their directionality) instead of relying solely on intrinsic host properties (e.g. density, shedding capacity) [ , ] . the presence of a target host defines directionality in the transmission processes and implies a network of interconnected hosts with different epidemiological roles. our framework thus provides a better empirical approach to some kinds of epidemiological problems, such as the risk of spread of a specific pathogen towards a target population or the potential for disease emergence in emerging disease hotspots. the maintenance and transmission function concepts can be related to the roles of vectors in vector-borne disease ecology. blood-feeding arthropod vectors that transmit a pathogen between hosts [ ] may be involved in distinct epidemiological functions, including the transmission function. the term "bridge vector" has already been used (e.g. [ , ] ) to group mosquitoes that transmit west nile virus to humans (here the target population). however, so far, the distinction between the maintenance and transmission function has not been properly defined. this distinction could be important if maintenance and bridge vectors are different species, opening different control strategy options (i.e. on the maintenance or on the bridge hosts). the identification of bridge hosts for a given pathogen in a given ecosystem has consequences for disease management, surveillance and control. once bridge hosts are known, managers can adopt mitigation strategies specifically aimed at reducing contact between the target and the bridge populations. in the case of aiv, this mitigation can be achieved through strengthening biosecurity measures or decreasing the quantity of attractors on the farm (e.g. water sources or open feedlots) [ ] . the adoption of adequate management measures targeting contacts between maintenance, bridge and target hosts is also more environmentally acceptable than controlling (wild) host populations. the distinction between maintenance and bridge hosts may under some circumstances be difficult. in the case of aiv, for example, our current level of knowledge about the maintenance hosts and the apparent lack of contact in some ecosystems between the maintenance community and the target populations suggest a role for bridge hosts. the identification of hosts that do not fit into either maintenance or target host groups, as in [ ] , raises two possibilities: either these susceptible hosts act as bridge hosts, or they may act as previously unknown maintenance hosts for aiv epidemiology. to differentiate between these two hypotheses may require focused experimental research, for example by using infection of captive animals to determine their capacity to maintain the virus. other approaches using meta-analysis of existing data sets have also been proposed [ ] . in both cases, our conceptual framework helps with framing hypotheses based on current knowledge and using empirical tests to either confirm these hypotheses or call for a revision of our understanding of the epidemiological system (e.g. this host is not a bridge host and therefore has no (or another) role in the local context). our framework does have some weaknesses. in particular, proving that a bridge host in a complex multi-host system where maintenance communities are composed of numerous interacting populations does not take part in the maintenance function (i.e. that removing the bridge host will not drive the pathogen to extinction, according to haydon et al. [ ] ) may necessitate an experimental design that would be difficult to achieve in practice [ ] . in addition, only cases in which maintenance and target populations are not in contact have been considered so far. if they are loosely in contact (case a' in figure ), the frequency and efficacy of contacts between different pairs (maintenance-target, maintenance-bridge and bridgetarget) would need to be weighted against each other. decreasing the maintenance-target contacts through management will reveal the relative importance of bridge-target contacts and could require interventions in order to efficiently stop pathogen transmission (as in the case of control of possums for btb in new zealand mentioned earlier). finally, we have assumed that a bridge host must be competent for the pathogen but in some cases simple mechanical transmission (e.g., a bird carrying the virus on its feathers [ ] ) may be possible, relaxing the prerequisite on host competence for the bridge host. the development of complex human/livestock/wildlife interfaces, due to the encroachment of human activities within natural ecosystems, triggers new epidemiological dynamics that may permit a range of wild or domestic bridge hosts to link maintenance communities with new target hosts [ ] . we would expect that domestic species and newly farmed or traded wildlife species will increasingly play bridge host roles in the emergence of new zoonoses. the epidemiology of ebola, sars, lyme disease, and h n aiv, for example, are not yet fully understood but are known to involve multiple hosts. we believe that introducing our definitions and operational framework into research and surveillance could contribute to more efficient use of resources to fill some knowledge gaps. our approach builds on that of haydon et al. [ ] and refines it to take into account potential circumstances under which an extra conceptual development is necessary. whether this extra development will be necessary in many multi-host systems or will be used only under exceptional circumstances will be answered by studies to come. the examples given here indicate that they could be used for at least a few important diseases. the recent appearance in the epidemiological literature of similar concepts [ , , ] that are not always placed soundly within a conceptual framework and/or ignore previous definitions suggests also the need for a consolidated review and refinement of these concepts and definitions. while no individual element of our proposed framework is new, it is clear from our discussion above that approaching the problem of understanding multi-host disease systems from a more integrated, functional perspective has the potential to offer a wide range of valuable insights into both epidemiology and its applications to pathogen control. our approach, which requires both epidemiological and ecological approaches (and also social science approaches when the human host is considered) fits well within current initiatives that call for more transdisciplinary integration between the health sciences and other fields of research. finally, the global fight against emerging infectious diseases is increasingly focused on identifying potential emerging pathogens from high-risk maintenance hosts (e.g., bats and rodents, [ , ] ). recent advances in genetics and genomics have increased drastically the pace at which new micro-organisms are discovered and identified [ ] . but adding new names to the list of parasites and pathogens does not provide information on which of these microorganisms might present a significant threat to animal or human health. a maintenance population hosting a large range of potentially new emerging pathogens does not constitute a threat for target populations if no transmission route exists between the maintenance and target populations. focusing on pathogen transmission pathways, including potential hosts bridging the gap between maintenance and target populations, will help to guide "pathogen hunting" approaches as functional ecology complements taxonomy. such an approach will help to guide high-throughput sequencing tools towards key hosts within a given epidemiological context, increasing the efficiency of surveillance and control efforts. epidemiology and public health unit contrasting spatial patterns of taxonomic and functional richness offer insights into potential loss of ecosystem services infectious diseases of humans: dynamics and control causal inference in disease ecology: investigating ecological drivers of disease emergence experimental infection of north american birds with the new york strain of west nile virus the ecology of infectious disease: effects of host diversity and community composition on lyme disease risk epidemiological interaction at the wildlife/livestock/human interface: can we anticipate emerging infectious diseases in their hotspots? 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[ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] .additional file : aiv rt-pcr results for wild birds sampled in africa until . this table displays detailed results of wild bird species and families sampled for aiv (rt-pcr) in africa until following the gathering of data as described in additional file . the authors declare that they have no competing interests. ac drafted the first version of the manuscript after in-depth discussions with the various authors about the concept and framework developed in this manuscript. jc, gsc, mdgw and ng commented on various versions of the manuscript. all authors read and approved the final manuscript. this article was made possible by the involvment of authors in three projects: the gripavi project sponsored by grants from the french ministry of foreign affairs, the tcp of fao through additional grants from the government of france, and the usaid-sponsored (through the wildlife conservation society) gains project. we are grateful to the numerous epidemiologists, veterinarians and field assistants who participated in field operations. this work was conducted within the framework of the research platform "production and conservation in partnership", rp-pcp and the ahead initiative.author details key: cord- -qsaewje authors: wang, pengcheng; bai, juan; liu, xuewei; wang, mi; wang, xianwei; jiang, ping title: tomatidine inhibits porcine epidemic diarrhea virus replication by targeting cl protease date: - - journal: vet res doi: . /s - - -y sha: doc_id: cord_uid: qsaewje porcine epidemic diarrhea virus (pedv) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. there is an urgent need to find new therapeutic methods to prevent and control pedv. not only is there a shortage of commercial anti-pedv drugs, but available commercial vaccines fail to protect against highly virulent pedv variants. we screened an fda-approved library of natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of pedv replication in vero and ipec-j cells in vitro. molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of pedv cl protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (itc). the inhibiting effect of tomatidine on cl protease was determined using cleavage visualization and fret assay. tomatidine-mediated blocking of cl protease activity in pedv-infected cells was examined by western blot detection of the viral polyprotein in pedv-infected cells. it indicates that tomatidine inhibits pedv replication mainly by targeting cl protease. in addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (tgev), porcine reproductive and respiratory syndrome virus (prrsv), encephalo myocarditis virus (emcv) and seneca virus a (sva) in vitro. these results may be helpful in developing a new prophylactic and therapeutic strategy against pedv and other swine disease infections. pedv, an enveloped, positive-sense, single-stranded rna virus, is a member of the coronavirinae subfamily [ , ] , which comprises viruses that cause a variety of diseases in mammals and birds, ranging from enteritis in cows and pigs, to upper respiratory disease in chickens, and potentially lethal human respiratory infections, such as severe acute respiratory syndrome (sars) [ ] , middle east respiratory syndrome (mers) [ ] , and the novel coronavirus disease (covid- ) [ ] . fecal-oral transmission is believed to be the main mode of pedv transmission [ ] . the latest research indicates that airborne transmission may also contribute to a pedv outbreak [ ] , similar to sars-cov- and mers-cov. owing to antigenic, genetic (> % amino acid variation between respective s-proteins) and phylogenetic (g vs g ) differences between vaccine and field epidemic strains, current pedv vaccines appear to have low to moderate efficacy [ ] . there is a need for alternative approaches to control this disease, such as effective antiviral drugs for pedv treatment. the interaction between pedv and pigs has been well studied. several host antiviral factors, including the bone marrow stromal cell antigen (bst ) [ ] , interleukin- (il- ) [ ] , interferon-lambda (ifn-lambda) [ ] , gtpase-activating protein-binding protein (g bp ) [ ] , and cholesterol -hydroxylase (ch h) [ ] , have been reported to show antiviral activity against pedv infection. some natural compounds and compositions have also been reported to show anti-pedv activity in vitro, such as griffithsin [ ] , coumarin [ ] , and prenylated phenolic compounds [ ] . however, the mechanism of antiviral activity is not well understood, and there are no commercial anti-pedv drugs available to the pig breeding industry. in this study, we screened a library of natural products and found that tomatidine, a steroidal alkaloid that can be extracted from the skin and leaves of tomatoes [ ] , significantly inhibited the replication of pedv by directly inhibiting clpro activity, and showed antiviral activity against other swine disease viruses as well, demonstrating excellent potential as a natural broad-spectrum antiviral product. vero cells, st cells, marc- cells, bhk- cells, and ipec-j cells were maintained in dulbecco's modified eagle's medium (dmem) (gibco, usa) with % fetal bovine serum (lonsera, uruguay), penicillin ( u/ml), and streptomycin ( ug/ml). the cells were incubated at °c in a humidified incubator with % co . pedv ms (genbank accession no. mt ), yz (genbank accession no. mk . ), sh (genbank accession no. mk . ) [ ] , and cv (genbank accession no. af . ) strains were maintained in our laboratory and passaged in vero cells with . % trypsin. the ms, yz, and sh strains belong to variant strains; cv belongs to a classical strain. the ms strain was used for all experiments and is represented by "pedv" in this article. tgev js (genbank accession no. kt . ) passaged in st cells, the highly pathogenic prrsv strain bb (genbank accession no. hq . ) passaged in marc- cells, emcv nj (genbank accession no. hm ), and sva ch-sd (genbank accession no. mh . ) passaged in bhk- cells, were maintained in our laboratory. tomatidine with purity > % was used for in vitro experiments (selleck chemicals, usa). a library (fda approved) of natural products was purchased from selleck chemicals. these compounds were stored as mm stock solutions in dmso at − °c until use. the workflow for high-throughput screening (hts) of the library was carried out as shown in figure a and b. vero cells were seeded in -well plates at × cells per well. when approximately % confluent, the cells were treated with μm compound or dmso ( μl, . % v/v) for h and then infected with pedv ( . moi) or mock infected for h. the cells were then washed with pbs, then culture medium containing μm compound was added back to each well. at hpi, cpe and ifa were observed under a microscope. the fluorescence intensity of ifa was measured by imagej software. the percentage of inhibition of fluorescence intensity is calculated by drug treatment relative to dmso treatment. during primary screening, compounds were ruled out if they resulted in any visible cytotoxicity or demonstrated less than % reduction of cpe compared with the dmso control group. for the second round of screening, cell viability had to be % or greater, and the inhibition of pedv had to be % or greater as determined by ifa. the ic (concentration of compounds was . , . , . , . , . , and . μm) and cc (concentration of compounds was . , . , . , . , . , . , and . μm) of each remaining candidate compound were calculated using log (inhibitor) vs. response-variable slope (four parameters) method by graphpad prism . software (graphpad software, ca, usa), and those that displayed dose-dependent inhibition of pedv and selectivity index (si, si = cc /ic ) over were considered for further study. in addition, cell viability was tested using an enhanced cell counting kit- (cck- ) (beyotime, china) following the manufacturer's instructions. the cc was calculated using graphpad prism . software. dmso was used as the negative control. figure screening protocol for pedv inhibitors. a screening procedure. vero cells were treated with μm compound for h, then infected with pedv ( . moi) for h. the cells were washed with pbs, then incubated in medium containing μm compound for another h. b screening process flowchart. the criteria for passing the primary screening were that the compound must have no apparent cytotoxicity and must reduce cpe by at least % compared with the dmso treatment. the criteria for passing the secondary screening were that the compound must leave cells at least % viable and inhibit pedv by more than %. compounds that passed the third screen inhibited pedv in a dose-dependent manner and had a selective index (si) higher than . c each dot represents the percent inhibition of each compound. the dots located above the dotted line indicate % or greater inhibition. d ifa of infected cells treated with one of the four designated compounds. pedv n-protein is colored green; a bright field shows cpe. e ic and cc curves of the four designated compounds. cell viability is calculated as a percentage of the viability in the cells treated with the compounds divided by that in the dmso-treated cells. the structure of each compound is inset. f sis of the four designated compounds. (see figure on next page.) wang et al. vet res ( ) the cells were lysed with μl of ripa lysis buffer (beyotime, china) on ice for min, then resolved by sds-page and transferred to a nitrocellulose membrane. after transfer, the membrane was incubated in blocking buffer ( % non-fat milk in pbst w/v) for h at room temperature, washed three times with pbst, then probed with the following antibodies: anti-pedv n-protein ( the detection of mrna levels of tgev, prrsv, and sva was performed as described previously [ , , ] . quantitative rt-pcr was performed using aceq ® qpcr sybr ® green master mix (vazyme, china). each reaction was performed in triplicate and results are expressed as mean ± standard deviation (sd). vero cells grown in -well plates were infected with tenfold serial dilutions of pedv samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. st cells grown in -well plates were infected with tenfold serial dilutions of tgev samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. marc- cells grown in -well plates were infected with tenfold serial dilutions of prrsv samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. bhk cells grown in -well plates were infected with tenfold serial dilutions of emcv/sva samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. virus titers are expressed as tcid , calculated using the reed-muench method. tomatidine ( μm) or dmso was incubated with pedv ( . moi) at °c for h and h. a mixture of tomatidine ( μm) or dmso and pedv was placed into vero cells seeded in -well plates. after incubation at °c for an additional h, the culture supernatants were replaced with fresh dmem and incubated for an additional h ( figure a ). the cells were then washed with pbs, and the mrna levels of pedv n and gapdh in the cells were measured using qrt-pcr. vero cells were pretreated with tomatidine ( μm) or dmso for h at °c, and then infected with pedv ( . moi) for the time indicated ( min, min, h) at °c ( figure b ). the cells were then washed with icecold pbs, and the mrna levels of pedv n and gapdh in the cells were measured using qrt-pcr. vero cells were infected with pedv ( . moi) at °c for h. the supernatant was replaced with dmem containing tomatidine ( μm) or dmso, and then incubated at °c for the time indicated ( min, h, h) ( figure c ). the cells were washed with citrate buffer (ph ) to remove non-internalized virus. the mrna levels of pedv n and gapdh in the cells were then measured using qrt-pcr. vero cells were incubated with pedv ( . moi) at °c for h and washed three times with pbs to remove free virus. at hpi, the culture medium was replaced with fresh dmem containing tomatidine ( μm) or dmso, and the cultures were incubated at °c ( figure d ). the mrna levels of pedv n and gapdh in the samples collected at , , hpi were measured using qrt-pcr. vero cells were infected with pedv ( . moi) for h at °c. the culture medium was then replaced with fresh dmem. at hpi, the cells were washed three times with pbs and the culture medium was replaced with fresh dmem containing tomatidine ( μm) or dmso. the cultures were incubated at °c for . , , and h, at which time the supernatants were harvested ( figure e ). the mrna levels of pedv n in the released virus was quantified by absolute fluorescence quantification. the crystal structure of pedv nsp was obtained from the protein data bank ( clpro, pdb: xfq). due to lack of crystal structure, the protein sequences of nsp (plp ), nsp (rdrp), nsp (ntp), nsp (exon), nsp (nendou), and nsp ( ′-o-methyltransferase) were subjected to comparative homology modeling using swiss model, which has been widely used and cited by more than articles, to generate a putative d model. swiss model performs the sequence alignments and searches the putative template protein to generate a d model for a query sequence. all the modeling parameters were set to default. the three-dimensional structure of tomatidine was obtained from pubchem (compound cid: ). the autodock . program was used for the docking of tomatidine to the active pocket of potential replication-relevant proteins. we generated a grid map around selected active site residues with grid point spacing of . Å. the potential tomatidine-protein complex was generated by genetic algorithm using the default parameters. the estimated free energy of binding was ranked, and the top two complexes were employed for further md analysis. the docking results were visualized using pymol . . . the ligand bound to the gromos /gromos force field generated by prodrg . . simulations were conducted with the gromacs package using the gro-mos a force field. the simple point charge (spc ) model of water was used to solvate the protein in a periodic dodecahedron box extending Å from the nearest protein atom. the solvated system was then neutralized with na+ and cl− ions, minimized by the steepest descent method ( steps), and equilibrated with a -ps constant volume (nvt) simulation. the production runs were conducted in a constant pressure ensemble (npt). the temperature was set to k and controlled with v-rescale. long-range electrostatic interactions were treated with the particle-mesh ewald (pme) method. the pressure was coupled to bar using the parrinello-rahman method. all bond lengths were constrained with the linear constraint solver (lincs) algorithm. a cut-off of Å was used to calculate short-range van der waals and electrostatic interactions. the time step was fs and the total simulation time was ns. rmsd, distance, and the number of hydrogen bonds between tomatidine and active pockets of the proteins were analyzed to judge binding stability and convergence. the pet- a- clpro was transformed into e. coli strain bl , and the cells were cultured at °c in lb medium. when optical density at nm (od ) reached . , the culture was cooled to °c and supplemented with mm iptg. the cells were harvested after incubation at °c for h, resuspended in pbs and disrupted by ultrasonication. the supernatant was filtered and loaded onto ni-sepharose (ge healthcare, usa). finally, the histagged protein was eluted using a linear gradient between the binding buffer and elution buffer a ( mm tris, ph . , mm nacl, and mm imidazole). low concentration imidazole ( mm) was used to wash impurities, and high concentration imidazole ( mm) was used to elute targeted protein. the target protein was condensed and desalinated using amicon ultra- ( kda, ge healthcare, usa). the proteins were analyzed by sds-page. all of the purification procedures were performed at °c to avoid unexpected degradation. the fluorescence quenching assay was measured by a perkinelmer enspire multimode plate reader. the reaction medium ( μl) contained μl of clpro solution at the concentration of μm and μl of tomatidine with different final concentrations ( , , , , , and μm). following incubation at room temperature for min, the fluorescence spectra of clpro with the different concentrations of tomatidine were recorded in the wavelength range of - nm upon excitation at nm. the stern-volmer equation was used to describe fluorescence quenching as follows: . in this equation, f and f represent the fluorescence intensities in the absence and presence of tomatidine. τ ( − s) indicates the lifetime of the fluorophore without quencher. k q is the bimolecular quenching constant. [q] refers to the concentration of the quencher, and k sv is the stern-volmer quenching constant. hence, the above equation may be applied to determine k sv by linear regression of a plot of f /f versus [q] . each measurement was repeated three times. all measurements were performed using the microcal itc calorimeter in an itc buffer ( mm tris, ph . , mm nacl, ph . ) while stirring at rpm. the stock solution of compound and clpro protein were diluted with the itc buffer to a compound concentration of μm and a protein concentration of μm before titrations. the final concentration of dmso in the reaction buffer was less than % of the total volume. protein solution was titrated using the compound. all titrations were performed using an initial injection of . μl followed by identical injections of μl with a duration of s and an interval of s between injections. the data were analyzed by microcal itc software. the last three data points were averaged and subtracted from each titration to account for the heat of dilution. each measurement was repeated three times. for pedv clpro eukaryotic and prokaryotic expression plasmids, the coding sequence for clpro was reverse transcribed and amplified from pedv ms strains. the resulting pcr products were assembled into pcaggs-flag and pet- a. mutagenesis of pedv clpro constructs were carried out by overlap extension pcr using specific mutagenic primers. the plasmids were verified by sequencing and double enzyme digestion. for functional substrates of pedv clpro, a nucleotide sequence encoding for an -amino-acid stretch corresponding to the cleavage site of pedv clpro derived from the junction of the nsp and nsp genes (ygvnlq^sg) in the pedv genome was inserted into the coding sequence of gfp between amino acids g and d . the first pcr product (n-gfp) was amplified with a reverse primer harboring the n-terminal of the cleavage site coding sequence (ygvnlq). the second pcr product (c-gfp) was amplified with a forward primer harboring the c-terminal of the cleavage site coding sequence (vnlqsg). the first and second pcr products were assembled using overlap pcr and cloned into pcaggs. vero cells cultured in -well plates were co-transfected with ng/well of gfp nsp / encoding plasmid, ng of clpro expressing plasmid, or an empty vector. at hpi, the culture supernatants were replaced with fresh dmem containing the indicated concentrations ( , , μm) of tomatidine or dmso. at hpi, the cells were harvested and the cleaved fragment of gfp nsp / was visualized using western blot. the polypeptide substrate dabsyl-ynstlq↓aglrkm-e-edans was chemically synthesized by the genscript corporation. pedv cl protease was used at final concentrations of . , , and . μm; and fret peptide was added to the protein in a black -well plate at final concentrations of μm. prrsv gp protein expressed and purified at the same time with cl protease was used as a negative control at final concentrations of μm. the mixtures were then further incubated at °c for min, and fluorescence was monitored at nm excitation and nm emissions every minute. obacunone was used as a control drug. it is an fdaapproved compound with molecular weight similar to tomatidine, but it has no antiviral activity on pedv (according the data from hts). tomatidine ( , μm), obacunone ( μm, negative control) or dmso were pre-incubated with μm clpro for min at °c, and μm fret peptide was added to the mixture in a black -well plate [ ] . the mixtures were then further incubated at °c for min, and fluorescence was monitored at nm excitation and nm emissions every minute. the mock represents fret peptides with no protease. the relative fluorescence units (rfu) of the experimental group were obtained after subtracting fluorescence readings of the mock. the percentage of inhibition was calculated as follows: percentage of inhibition (%) = × [ − rlu of the tomatidine group ( - min)/rlu of the negative control group ( - min)] ( ) . all procedures involving animals for this study were approved by and performed in accordance with the animal ethics committee and nanjing agricultural university animal experiment central guidelines, respectively. female - week-old balb/c mice were immunized with μg of purified clpro protein in . ml, emulsified in the same amount of freund's complete adjuvant. two booster injections were administered with equal amounts of immunogen and freund's incomplete adjuvant at -week intervals. ten days after the last injection, antiserum was collected. pedv was cultured and passaged by passages in ipec-j cells in the presence of tomatidine at an increasing concentration of - - μm or in dmso. the total rna was extracted from the virus cultures, tomatidine-f , dmso-f , and f . the clpro gene was amplified and sequenced by using the primers located at ~ bp upstream and downstream of clpro. to assess the sensitivity of f viruses to tomatidine, ipec-j cells were inoculated with the virus (tomatidine-f , dmso-f , and f ) at . moi, and the culture medium was replaced with fresh dmem containing tomatidine ( μm) or dmso at hpi. the mrna levels of pedv n and gapdh in the samples collected at hpi were measured using qrt-pcr. western blot, qpcr, and tcid were used to examine tomatidine's inhibition of other swine disease viruses. three designated compound concentrations were added to the culture medium (final concentrations were . , , and μm). dmso was used as the negative control. statistical analysis was performed using graphpad prism software. results are expressed as mean ± standard deviation (sd). differences between groups were examined for statistical significance using one-way or two-way analysis of variance (anova). the asterisks in the figures indicate significant differences (*p < . ; **p < . ; ***p < . ; ns = not significant). vero cells were treated with µm natural product compounds, and infected with pedv as detailed in the timeline ( figure a) . the results indicated that ( . %) compounds showed no apparent cytotoxicity, and reduced cytopathic effect (cpe) by % compared with dmso alone. these compounds were then subjected to a second round of screening ( figure b) . four of the compounds produced negligible cytotoxicity and inhibited pedv infection by over % as determined by indirect immunofluorescence assay (ifa) ( figure c and d). in addition, they inhibited pedv in a dose-dependent manner and had a selectivity index (si) greater than (figure e and f) . tomatidine was chosen for further study as it had the highest si and the lowest price. to determine the dose range of tomatidine having anti-pedv activity, vero cells were treated with . , , and μm tomatidine for h and then infected with pedv. median tissue culture infectious dose (tcid ), western blot and qrt-pcr analysis showed that the virus titers, n-protein, and mrna levels decreased in a dosedependent manner (figure a-c) . at h post-infection (hpi), ifa showed that the number of infected cells in the tomatidine-treated groups were obviously lower than those in the negative control ( figure d ). in addition, tomatidine had similar inhibitory effects on the different pedv variant strains tested (yz, ms, and sh), as well as on the classic cv strain ( figure e ). to understand the anti-viral activity of tomatidine in porcine cells, ipec-j cells were treated with . , , and μm tomatidine for h and then infected with pedv ( . moi). tcid and qrt-pcr analysis showed that the virus titers and orf mrna levels significantly decreased in a dose-dependent manner ( figures f, g) . meanwhile, the ipec-j cells indicated that the tomatidine displayed no cytotoxicity ( figure h ). to further explore the mechanism by which tomatidine inhibits pedv infection, we first tested whether tomatidine is directly virucidal and kills pedv particles. as shown in figure a , tomatidine treatment failed to directly inactivate pedv. next, the relative amount of pedv n mrna to gapdh was detected by qrt-pcr to determine the effect of tomatidine on pedv attachment, internalization, and replication. the results showed that tomatidine treatment prior to pedv infection did not significantly block virus attachment to vero cells, indicating that tomatidine does not inhibit pedv attachment to cells ( figure b ). in evaluating the effect of tomatidine on pedv internalization, as shown in figure c , we determined that tomatidine treatment accompanied by virus internalization did not significantly impede virus replication compared to treatment with dmso. we then examined the effect of tomatidine on pedv replication by adding tomatidine during the replication stage. as shown in figure d , tomatidine treatment decreased pedv n mrna levels by approximately tenfold compared to treatment with dmso, suggesting that tomatidine inhibits pedv replication. in addition, the virus release assay showed that there was no significant difference in pedv rna levels in the supernatants between the treatment with tomatidine and dmso ( figure e ). taken together, these results indicate that tomatidine inhibits pedv infection primarily by affecting viral replication. several replicative enzymes regulate virus replication [ ] . considering that tomatidine significantly inhibited infection at the virus replication stage, we speculated that tomatidine may work directly with important viral replicative enzymes. to ascertain which replicative enzymes were targeted by tomatidine, potential binding sites were analyzed in detail using autodock to dock tomatidine into the pedv replicative enzyme structures ( figure a ). the estimated binding free energies were ranked as shown in figure b , and the top two complexes (binding energy = − . kj/mol, − . kj/mol) nsp and nsp were selected for further molecular dynamics (md) analysis. md utilizes newtonian physics to simulate atomic movements in a solvated system and is an accurate computational method for simulating protein-drug interactions [ ] . to judge the findings of our docking model, a ns molecular dynamic simulation was carried out for the assessment of receptor and ligand stability via root mean square deviation (rmsd), the distance and the number of hydrogen bonds between tomatidine and the active pockets of the proteins. in tomatidine-nsp ( clpro) simulation, the rmsd values of the backbone converged at less than . nm and remained stable starting at ns. the higher and more volatility rmsd in tomatidine-nsp implied structure instability (figure c(i) ). distances between tomatidine and the active pocket of nsp are highly conserved and tight during simulation calculations, whereas those between tomatidine and nsp fluctuated wildly ( figure c(ii) ). we measured the number of hydrogen bonds during the md simulations to better capture the intermolecular polar interactions. no hydrogen bonds were detected between tomatidine and nsp . however, tomatidine did form hydrogen bonds with the active pocket of clpro, which may contribute to the stability of the tomatidine- clpro complex ( figure c(iii) ). this means that clpro is a more likely target of tomatidine than nsp . in addition, the low binding energy of tomatidine to inactive cl (see figure on next page.) figure in silico tomatidine targeted the active pocket of pedv cl protease. a docked conformations of tomatidine with pedv nsp plp , nsp clpro, nsp rdrp, nsp ntp, nsp exon, nsp nendo u, and nsp ′-o-methyltransferase. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. b the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. c overall dynamic behaviors in the md simulations. (i) rmsd of backbones of nsp (red) and nsp (blue); (ii) distance between tomatidine and active pocket of nsp (red) and nsp (blue); (iii) number of hydrogen bond interactions of tomatidine with nsp (red) and nsp (blue). d docked conformations of tomatidine with pedv clpro or inactive clpro. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. e the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. wang et al. vet res ( ) : protease in silico strengthens the possibility that clpro is indeed the target for tomatidine ( figure d and e) . to verify the binding of clpro and tomatidine, pedv clpro fused with his tag was expressed in soluble form using an e. coli expression system ( figure a(i) ). after ni-sepharose purification, a single clear target band indicated that the clpro obtained could be used for further experiments ( figure a (ii), (iii)). the purified recombinant clpro was then used for a fluorescence quenching assay, itc and fret. a fluorescence quenching assay was carried out to determine the interaction between clpro and tomatidine. the results showed that the fluorescence emission intensity of clpro decreased distinctly in a dose-dependent manner with increasing concentration of tomatidine ( figure b upper) . the stern-volmer plot is shown in figure b lower; the kq value was calculated to be . × l·mol − s − . the maximum scatter collision quenching constant of the various quenchers is × l·mol − s − . this indicates that the quenching mechanism was static and the interaction between clpro and tomatidine was strong. the binding affinities of tomatidine with pedv clpro were measured by itc, which is widely used to determine thermodynamic parameters of protein-ligand interactions such as the dissociation constant (kd) and binding stoichiometry (n). the results showed that tomatidine directly interacted with pedv clpro with a kd of . μm and n of . sites. this result further supports the premise that pedv clpro is the target protein of tomatidine. these results allowed us to speculate that tomatidine may block the activity of the clpro, thereby inhibiting pedv replication. to explore the impact of tomatidine on clpro activity, we constructed plasmids expressing nsp ( clpro), a series of inactive clpro mutants (h a, c a, h / c a), and gfp nsp / containing the nsp /nsp junction (ygvnlq^sg) of pedv. after the vero cells were co-transfected with these plasmids, western blot assay showed that cleaved fragments of gfp nsp / were detected in the presence of clpro, but not in the presence of clpro mutants h a, c a, and h /c a, indicating that pedv clpro was active ( figure a ). when vero cells were treated with increasing concentrations of tomatidine, the cleaved fragments clearly decreased in a dose-dependent manner ( figure b ), indicating that tomatidine inhibited the activity of pedv cl protease. we also used fret to confirm the effect of tomatidine on clpro activity. the dose-dependent increase of fluorescence intensity shows that the clpro obtained possessed catalytic activity ( figure c ). moreover, compared with obacunone, tomatidine significantly inhibited the activity of pedv clpro ( figure d ). the percentage of inhibition of μm and μm tomatidine was . % ± . % and . % ± . % respectively, at min incubation. each reaction was performed in triplicate and the results are expressed as mean ± standard deviation (sd). in order to explore the inhibition of viral clpro activity of tomatidine in pedv-infected cells, anti- clpro serum antibody was prepared by vaccinating mice with the purified recombinant clpro ( figure e, lines - ) . vero cells were inoculated with pedv ( . and . moi) and μm tomatidine. western blot showed that the partial virus polyprotein (nsp - complex, rather than clpro) was detected with the anti- clpro serum antibody at hpi, as previously described [ ] . the ratios of nsp - complex/n in pedv not treated with tomatidine were similar to each other between the two doses of . and . moi. but the ratios of nsp - complex/n in pedv treated with tomatidine were significantly higher than those treated with dmso ( figure e , line - ), indicating that tomatidine may block the cl protease activity in a natural situation. tomatidine strongly inhibits virus particle production of dengue virus and chikungunya virus [ , ] . to investigate whether tomatidine has an antiviral effect against other swine disease viruses, we selected several other viruses including a coronavirus, tgev, [ ], an arterivirus, prrsv [ ] , and picornaviruses emcv and sva [ , , ] . st cells were treated with . , , and μm tomatidine for h and then infected with tgev for h. the results indicated that tgev proliferation was significantly suppressed by tomatidine and almost completely cut off at the concentration of μm ( figure a-c) . marc- cells were treated with the indicated concentrations of tomatidine for h and infected with prrsv for h. these results indicated that tomatidine exhibits antiviral activity against prrsv ( figure d -f). bhk- cells were treated with the indicated concentrations of tomatidine for h and then infected with emcv or sva for h. western blot, qpcr, and tcid analysis revealed substantial inhibition activity of tomatidine against emcv and sva (figures g-i, and j-l). none of the various cells treated with the indicated concentrations of tomatidine showed any cytotoxicity ( figure m ). outbreaks of porcine epidemic diarrhea cause significant lethality rates in neonatal piglets, creating a heavy economic burden in the global swine industry. unfortunately, available commercial vaccines fail to protect against the high virulence of pedv variants [ , ] . because there is no antiviral drug available to treat this disease, we need to develop new therapeutic methods to prevent and control pedv. natural compounds and compositions have been a rich source of drugs against many viral infections. for example, griffithsin, a highmannose-specific lectin, has been shown to reduce pedv infection in vero cells by preventing viral attachment and disrupting cell-to-cell transmission [ ] . prenylated phenolic compounds from the leaves of sabia limoniacea figure tomatidine directly inhibited the activity of pedv cl protease. a vero cells cultured in -well plates were co-transfected with ng of gfp nsp / encoding plasmid, ng of clpro or inactive mutants expressing plasmid or empty vector. at hpi, the cells were harvested and the cleaved fragment of gfp nsp / was visualized using western blot. b vero cells cultured in -well plates were co-transfected with ng of gfp nsp / encoding plasmid, ng of clpro expressing plasmid or empty vector. at hpi, the culture supernatants were replaced with fresh dmem containing the indicated concentrations ( , , and μm) of tomatidine or dmso. at hpi, the cells were harvested and the cleaved fragment of gfp nsp / was visualized using western blot. c pedv clpro was used at final concentrations of . , , and . μm; and μm ( clpro substrate) was added to the protein in a black -well plate. prrsv gp protein was used as a negative control. the mixtures were then further incubated at °c for min, and fluorescence was monitored at nm excitation and nm emissions every minute. the rfu were calculated by subtracting the mock from the fluorescence readings to eliminate the effect of background signals. d tomatidine ( , μm) or obacunone ( μm, negative control) or dmso was pre-incubated with μm protease for min at °c, and μm ( clpro substrate) was added to the mixture in a black -well plate. the mixtures were then further incubated at °c for min, and fluorescence intensity was monitored at nm excitation and nm emissions every minute. rfu were calculated as above. e vero cells were inoculated with pedv ( . and . moi) and μm tomatidine. cells were transfected with ng pcaggs- clpro as clpro positive control. after incubation for h, the cellular proteins were collected and the virus polyprotein was detected with western blot using anti- clpro mouse antibody, as previously described ( ) . meanwhile, pedv n-protein, gapdh, and clpro-flag were detected with western blot using molecular antibodies against pedv n-protein, gapdh, and flag. the ratio of nsp - complex/n protein levels was depicted by integrated density analysis. the arrow indicates the location of nsp - complex. all results are mean ± sd from three independent experiments performed in triplicate. exhibit promising antiviral activities against pedv replication [ ] . but the mechanisms involved in these studies are not well understood. in this study, tomatidine, screened from a library of natural products, was shown to inhibit pedv proliferation in vitro. tomatidine displayed remarkable inhibition of pedv replication by targeting cl protease, which we investigated using molecular docking and md analysis, fluorescence spectroscopy, itc, cl protease activity, and fret assays. the pedv life cycle is composed of four stages: attachment, entry, replication, and release [ ] . our results indicate that tomatidine significantly inhibits viral infection at the replication stage. several nonstructure proteins are key to viral replication, hence we speculated that tomatidine, which is shown to inhibit viral replication, may target a non-structural protein. making use of molecular docking and molecular dynamics, we speculated that tomatidine may inhibit pedv proliferation by directly targeting cl protease. fluorescence quenching is an important technique for measuring binding affinity between ligands and proteins. itc is a technique used in a wide variety of quantitative studies of biomolecular interactions. itc works by directly measuring the heat that is either released or absorbed during a biomolecular binding event. in our study, clpro-tomatidine binding was indicated by fluorescence quenching and itc. in order to confirm the effect of tomatidine on clpro activity, we constructed a plasmid expressing gfp nsp / protein containing the nsp /nsp junction (ygvnlq^sg) of pedv. our results show that gfp nsp / was cleaved into two fragments by clpro, indicating that pedv clpro was active. tomatidine inhibited the activity of pedv cl protease in vero cells in a dose-dependent manner. the inhibition ratio of tomatidine was also shown in a fret assay. the nm fluorescence intensity remained stable until the labeled substrate was fractured by the protease, which indicates that the purified protein possesses cl protease activity. compared with the control drug obacunone, tomatidine significantly reduced the fluorescence intensity. the ratio of nsp - complex/n in virus treated with tomatidine was significantly higher than in the dmso treated virus, indicating that tomatidine blocked the cl protease activity in a natural situation. however, anti- clpro serum raised in mice detected nsp - complex rather than clpro (nsp ) only. this phenomenon underlines the possibility that there is a quantitative difference between nsp - complex and nsp of products in pedv-infected cells at hpi. the c-like protease, which is responsible for processing polyproteins of nidoviruses and picornaviruses, is an attractive target for drug therapy. it has been reported that a series of compounds such as flavonoids, chalcones, and benzothiazolium display significant antivirus activity by inhibiting clpro activity. we found that tomatidine strongly inhibits pedv replication by targeting cl protease. tomatidine has many cell biological properties. it enhances expression of nuclear factor erythroid -related factor (nrf ) [ ] , which when knocked out benefits viral propagation [ ] . ′-amp-activated protein kinase catalytic subunit alpha- (ampk) promotes innate immunity and antiviral defense through modulation of sting signaling [ ] . tomatidine also stimulated ampk phosphorylation by activating the camkkβ pathway [ ] . tomatidine inhibits dengue virus particle production partly by inhibiting cyclic amp-dependent transcription factor atf- (atf ) [ ] . in this study, we noted that the ic ( % inhibitory concentration) of tomatidine against pedv in cells was low micromolar ( . μm); the cc ( % cytotoxic concentration) was about . μm; while the inhibition of pedv clpro by tomatidine in the fret assay at μm was only %. in addition, the binding energy of tomatidine with the viral nsp was close to that of the clpro. these results suggest that there may be other antiviral mechanisms involved. clpro of pedv, as well as of other coronaviruses (porcine deltacoronavirus), picornaviruses (hepatitis a virus, foot and mouth disease virus), and arterivirus (prrsv), has been shown to inhibit production of infβ, hijacking the host's innate antiviral immune response [ ] [ ] [ ] [ ] . this effect has been attributed to the direct cleavage of nf-kappa-b essential modulator (nemo) figure tomatidine shows broad-spectrum antiviral activity against other swine disease viruses. tcid , western blot, and qpcr were used to examine the inhibition activity of tomatidine against other swine disease viruses. three designated concentrations of compounds were added to the culture medium (final concentrations were . , , μm, with no-observable cytotoxicity). dmso was used as the negative control. tgev ( . moi), prrsv (e), and emcv (h)/sva (k) were then used to infect st, marc- , and bhk- cells, respectively, and samples were harvested at h, h, and h. the tcid of tgev (a), prrsv (d), emcv (g), and sva (j) treated with μm tomatidine or dmso were calculated using the reed-muench method. the n-protein level of tgev (b), prrsv (e), emcv (h), and sva (k) were determined by western blot. relative tgev n (c), prrsv n (f), emcv vp (i), and sva vp (l) mrna levels, was determined by qrt-pcr, and expressed relative to that in dmso-treated cells. m viability of st, marc- , and bhk- cells pretreated with the indicated concentrations of tomatidine and incubated for h, h, and h, respectively in medium containing tomatidine. the results are from one of three independent experiments. the internal loading control was β-actin. error bars represent the sd. the asterisks in the figures indicate significant differences (*p < . ; **p < . ; ***p < . ; ns = not significant). (see figure on next page.) (an important substrate in the rlr cascade) [ , ] . by blocking clpro, tomatidine may also prevent clpro cleaving nemo, thereby enhancing the innate antiviral immunity, which could in turn help to inhibit virus replication. tgev, prrsv, emcv, and sva contain c or cl protease. our results showed that tomatidine effectively inhibits tgev, prrsv, emcv, and sva replication in vitro. but the antiviral activity of tomatidine against picornaviridae looks more significant than tgev and prrsv. it may be related to the different characterization of the virus replication. the molecular docking analysis demonstrated that the binding energy between the c or cl protease of the viruses with tomatidine were weak and no hydrogen bonds were detected, which being different from that of pedv clpro (additional file ). it indicates that the antiviral activity of tomatidine is likely to rely on other pathways rather than only targeting c proteases, which is worthy being further explored. tomatidine has been proved to be safe within a certain dosage in mice. unfortunately, as a dietary supplement approved by the fda, tomatidine lacks detailed safety and pharmacokinetics evaluation in pigs. the safety, pharmacokinetics, and efficacy of tomatidine for prevention, treatment, and control of pedv and perhaps other porcine nidoviruses need to be further evaluated in pigs. it would be interesting to test whether its clpro blocking activity also holds for nidoviruses infecting humans. we conclude that tomatidine effectively inhibits pedv proliferation via inhibition of clpro activity. in addition, tomatidine displays antiviral activity against tgev, prrsv, emcv, and sva. these findings offer novel and promising therapeutic possibilities for fighting infections caused by these viruses. supplementary information accompanies this paper at https ://doi. org/ . /s - - -y. additional file . the binding energy of tomatidine to cl or c protease of tgev, prrsv, emcv, and sva in silico. a docked conformations of tomatidine with cl or c protease of tgev, prrsv, emcv, and sva in silico. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. b the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains characterization of a novel coronavirus associated with severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study structural basis for the recognition of sars-cov- by full-length human ace evaluation of 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porcine epidemic diarrhea virus (pedv) inhibitory activity prenylated phenolic compounds from the leaves of sabia limoniacea and their antiviral activities against porcine epidemic diarrhea virus therapeutic potential of steroidal alkaloids in cancer and other diseases pathogenicity and immunogenicity of a new strain of porcine epidemic diarrhea virus containing a novel deletion in the n gene xanthohumol inhibits prrsv proliferation and alleviates oxidative stress induced by prrsv via the nrf -hmox axis seneca valley virus c and c inhibit type i interferon production by inducing the degradation of rig-i identification of two antiviral inhibitors targeting c-like serine/ c-like protease of porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus functional and genetic analysis of coronavirus replicase-transcriptase proteins molecular dynamics simulation for all alternative proteolytic processing of the arterivirus replicase orf a polyprotein: evidence that nsp acts as a cofactor for the nsp serine protease tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro efffects of electrostatic and hydrophobic interaction on the stability of the tgev main proteinase dimer structure and cleavage specificity of the chymotrypsinlike serine protease ( clsp/nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) encephalomyocarditis virus c protease attenuates type i interferon production through disrupting the tank-tbk -ikkepsilon-irf complex seneca valley virus c(pro) abrogates the irf -and irf -mediated innate immune response by degrading irf and irf two novel porcine epidemic diarrhea virus (pedv) recombinants from a natural recombinant and distinct subtypes of pedv variants genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china coronaviruses: an overview of their replication and pathogenesis tomatidine enhances lifespan and healthspan in c elegans through mitophagy induction via the skn- /nrf pathway amp-activated kinase (ampk) promotes innate immunity and antiviral defense through modulation of stimulator of interferon genes (sting) signaling tomatidine reduces palmitate-induced lipid accumulation by activating ampk via vitamin d receptor-mediated signaling in human hepg hepatocytes aspartic acid at residue modulates the capacity of hp-prrsv nsp to antagonize ifn-i expression porcine deltacoronavirus nsp antagonizes type i interferon signaling by cleaving stat hepatitis a virus c protease cleaves nemo to impair induction of beta interferon foot-andmouth disease virus antagonizes nod -mediated antiviral effects by inhibiting nod protein expression arterivirus nsp antagonizes interferon beta production by proteolytically cleaving nemo at multiple sites porcine epidemic diarrhea virus c-like protease regulates its interferon antagonism by cleaving nemo publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank miss. huixin zhu for her generous help with the animal experiment. in addition, the critical and helpful comments from the reviewers are highly appreciated. conception of the work: pj and pw; cellular and animal experiment: pw, jb, xl and mw; analysis and interpretation of data: pj, xw and pw; preparation of the manuscript: pj and pw. all authors read and approved the final manuscript. not applicable. all animals were housed in the animal facility of nanjing agricultural university (nau), nanjing, jiangsu, china. all experimental protocols for animals were conducted following the national guidelines for housing and care of laboratory animals (china) and performed in accordance with nau institutional regulations after approval by the institutional animal care and ethics committee of nau (syxk(su) - ). not applicable. none of the authors have any possible conflicts of interest. key: cord- -txfuuu d authors: lim, byeonghwi; kim, sangwook; lim, kyu-sang; jeong, chang-gi; kim, seung-chai; lee, sang-myeong; park, choi-kyu; te pas, marinus f. w.; gho, haesu; kim, tae-hun; lee, kyung-tai; kim, won-il; kim, jun-mo title: integrated time-serial transcriptome networks reveal common innate and tissue-specific adaptive immune responses to prrsv infection date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: txfuuu d porcine reproductive and respiratory syndrome virus (prrsv) infection is the most important viral disease causing severe economic losses in the swine industry. however, mechanisms underlying gene expression control in immunity-responsible tissues at different time points during prrsv infection are poorly understood. we constructed an integrated gene co-expression network and identified tissue- and time-dependent biological mechanisms of prrsv infection through bioinformatics analysis using three tissues (lungs, bronchial lymph nodes [blns], and tonsils) via rna-seq. three groups with specific expression patterns (i.e., the -dpi, lung, and bln groups) were discovered. the dpi-specific group showed antiviral and innate-immune signalling similar to the case for influenza a infection. moreover, we observed adaptive immune responses in the lung-specific group based on various cytokines, while the bln-specific group showed down-regulated ampk signalling related to viral replication. our study may provide comprehensive insights into prrsv infection, as well as useful information for vaccine development. porcine reproductive and respiratory syndrome (prrs) is one of the most important diseases affecting commercial pig productivity in the swine industry worldwide [ ] . prrs is caused by the prrs virus (prrsv), a single-stranded rna virus [ , ] , resulting in severe reproductive losses for breeding pigs and respiratory problems for growing pigs [ ] . vaccination-a solution for this problem-is still limited because of the high mutation rate in the viral proteins and the intrinsic characteristics of prrsv that impede innate immune responses [ ] [ ] [ ] . therefore, to date, several studies have been performed to identify host factors that confer resistance to prrsv infection. some mutations in the guanylate-binding protein and cluster of differentiation (cd ) genes were reported to be associated with prrsv susceptibility [ , ] , and also cd knockout was proved to show full resistance for prrsv infection [ ] . porcine alveolar macrophages (pams) represent the main cellular target for prrsv infection [ ] . prrsv replication is initiated in the cytosol in infected cells following receptor-mediated endocytosis and disassembly, and replication primarily occurs in the lung and lymphoid organs, but not the spleen [ ] . prrsv can modulate host immune responses by down-regulating interferon-β production and suppressing the activity of antigen-presenting cells [ , ] . in addition, serum viral loads have been found to increase for approximately week after infection and then gradually decrease over the course of a month [ ] . in contrast, viral loads in lymphoid organs are reported to reflect high viral replication for a few months [ ] . several recent studies have involved the use of rna sequencing (rna-seq) to identify the functional basis of host responses to prrsv infection. serial blood transcriptomes observed following prrsv infection in commercial pigs showed three main clusters related to immune signalling, dna repair, and cell signalling [ ] . additionally, the blood transcriptomes in prrsvinfected gilts indicated the development of innate immunity at days post infection (dpi) and t cell signalling at dpi [ ] . relatively few prrsv studies in tissues based on rna-seq have been conducted thus far. the lung transcriptomes of prrsv-infected pigs revealed genes that were potentially related to early innate immune responses [ ] . analysis of tracheobronchial-lymph node transcriptomic responses to highly pathogenic prrsv infection revealed that they were mainly associated with cell death [ ] , and the tonsil transcriptomes of pigs revealed that high viral levels activated polarisation of blood cell functions [ ] . however, in most studies, analysis was performed at one or two time points in a single tissue after prrsv infection. therefore, an examination of the dynamic regulatory changes in gene expression levels at serial time points in multiple tissues is needed to gain comprehensive insights into prrsv infection. in this study, we investigated the molecular mechanisms of prrsv infection by integrating the differences in the expression of various genes in tissues responsible for respiration (lungs) and immunity (bronchial lymph nodes [blns] , and tonsils) using rna-seq data at serial time points. differentially expressed genes (degs) were identified at each time point and in each tissue after prrsv infection. then, dynamic molecular networks were constructed to identify tissue-and time-dependent gene expression levels and patterns. marc- cells, which are highly permissive to prrsv infection, were used for virus propagation and functional assays. marc- cells were maintained in rpmi- medium (gibco ® rpmi- , life technologies, carlsbad, ca, usa) supplemented with heat-inactivated % foetal bovine serum (life technologies), mm l-glutamine, and antibiotic-antimycotic (anti-anti, life technologies) containing iu/ml penicillin, µg/ ml streptomycin, and . µg/ml amphotericin b in a humidified chamber at °c under % co conditions. the prrsv- strain ja (genbank: ay . ) was used in this study. weeks-old piglets (n = ) were obtained from a prrsv-negative farm and housed in animal rooms at our facility. after days of acclimation, pigs were intramuscularly challenged with ml of prrsv (ja strain; × tissue culture infectious dose (tcid) /ml), diluted in sterile pbs. all infected pigs were humanely euthanised at , , , , and dpi, respectively. the remaining pigs were humanely euthanised without virus infection as an uninfected control ( dpi) group. a schematic overview of the animal study is shown in figure a . blood was collected at , , , , , and dpi from the euthanised pigs, and serum was separated for viral load detection and serological assays. the lungs, blns, and tonsils were aseptically extracted after euthanasia. these tissues were collected in tubes, snap-frozen using liquid nitrogen, and stored immediately at − °c for rna extraction. all animal experiments were approved by the jeonbuk national university institutional animal care and use committee, republic of korea (approval number - ). viral rna was extracted from µl of each serum sample and g of each tissue sample, using a mag-max ™ viral rna isolation kit (ambion, applied biosystems, life technologies) and a total rna extraction kit (hybrid-rtm, geneall, seoul, republic of korea), respectively, per the manufacturers' instructions. serum and lung viral loads were measured using a prime-q pcv , prrsv detection kit (genet bio, inc., daejeon, republic of korea) with a fast real-time pcr system (applied biosystems, foster city, ca, usa). a standard curve was generated from known titres of prrsv and used to calculate the quantity of prrsv in each sample by converting each cycle threshold value to the tcid /ml-equivalent values. the prrsv titres in lung tissues were measured with marc- cells, using a microtitration-infectivity assay. briefly, tissue homogenates [ % (weight/ volume)] from the extracted lungs were prepared in dulbecco's modified eagle's medium with antibiotics, vortexed for - min, and centrifuged at ~ × g for h at ℃. after centrifugation, each collected supernatant was filtered through a sterile . μm syringe filter and incubated with marc- cells to measure the viral titre. prrsv titres were calculated at to dpi, based on the observed cytopathic effects, and were expressed as tcid /ml. prrsv-specific immunoglobulin g (igg)-type antibodies were detected in the serum using a commercially available elisa kit (bionote prrs ab . , hwaseong, republic of korea) based on the detection of the nucleocapsid protein, according to the manufacturer's instructions. the sample to positive (s/p) ratio of each serum sample was ≥ . , which was considered to be indicative of the presence of prrsv antibodies. total rna was extracted from the lung, bln, and tonsil tissues using the trizol reagent (invitrogen, life technologies) according to the manufacturer's recommendations. total rna concentrations were calculated using quant-it ribogreen (invitrogen, life technologies, carlsbad, ca, usa). to assess the rna-integrity number, samples were run on the tapestation rna screentape system (agilent technologies, santa clara, ca, usa) (additional file ). a cdna library was independently prepared with µg of total rna for each sample using the illumina truseq stranded mrna sample prep kit (illumina, inc., san diego, ca, usa). the first step in the workflow involved removing the rrna from the total rna, using the ribo-zero rrna removal kit (human/mouse/rat; illumina, inc.). subsequently, the figure overview of the study design and the measured phenotypes. a schematic representation of the experimental design in terms of the sample types, target tissues (lungs, blns, and tonsils), and time points ( dpi, dpi, dpi, dpi, dpi, and dpi) after prrsv infection. b serum and lung viral loads and serum antibody levels in prrsv-infected pigs. remaining mrna was fragmented into small pieces using divalent cations under elevated temperature conditions. the cleaved rna fragments were copied into first-strand cdna using superscript ii reverse transcriptase (invitrogen, life technologies) and random primers. this step was followed by second-strand cdna synthesis using dna polymerase i, rnase h, and dutps. the cdna fragments were subjected to an end-repair process, involving the addition of a single ' a' base, after which adapters were ligated. the products were then purified and enriched by pcr to create the final cdna library. the libraries were quantified using kapa library quantification kits for illumina sequencing platforms, according to the qpcr quantification protocol guide (roche, basel, switzerland), and the libraries were validated using the tapestation d screentape system (agilent technologies, santa clara, ca, usa). the indexed libraries were then analysed on an illumina hiseq instrument (illumina, inc.), and paired-end ( × base pair) sequencing was performed. all raw rna-seq data generated in this study were deposited in the ncbi sequence read archive database under the accession number prjna . to select the quality-filtering strategy, a quality check of raw read data was performed for each sample using the fastqc software v . . , and the reads were trimmed with adaptors using the trimmomatic software v . based on the quality results. then, the trimmed reads were re-checked with fastqc and mapped to the reference genome (sus scrofa . , gca_ . ) of the ensembl genome browser (https ://www.ensem bl.org/ sus_scrof a/) as the default option of the hisat v . . programme. raw counts corresponding to the genes in each library were calculated based on the exons in sus scrofa gtf v (ensembl) as the genomic-annotation reference file, using the featurecounts of subread package, v . . . all deg analyses for the obtained raw counts were performed using the edger software package v . . of bioconductor. to reduce statistical bias in the deg analyses, genes were excluded when all samples had raw counts of ≤ . normalisation of the raw counts was performed using the trimmed mean of m-value (tmm) method, and dispersion parameters were estimated and applied using the cox-reid profile-adjusted likelihood method in edger. degs were identified for each time point ( , , , , and dpi; versus gene expression at dpi) for each tissue (lungs, blns, and tonsils) using a negative binomial-generalised linear model, and p-values were corrected for multiple comparisons based on the false discovery rate (fdr). degs were determined based on an fdr of < . and an absolute log fold-change (fc) of ≥ . multidimensional scaling (mds) was performed using the limma function of the r package to identify the similarities among samples. gcn analysis was conducted by filtering out: (i) degs with no significant fdr (fdr < . ) observed at any of the time points in tissues and (ii) degs with a not stringent significant value (absolute log fc ≥ . ), in order to increase the efficiency of network construction. before gcn analysis, significant associations between the filtered genes were calculated using the partial correlation coefficient with information theory (pcit) algorithm [ ] . correlations were estimated to assess coexpression, and the network was constructed using genes with absolute co-expression correlations of ≥ . . gcn visualisation was performed using the cytoscape v . . software, and the resulting network consisted of genes (nodes) and connections (edges). clustering analysis was performed using the log fc values of genes in the constructed network. after determining the optimal number of clusters, the genes were analysed using the k-means clustering algorithm with iterations, using the multi experiment viewer (mev) software. the genes in the constructed gcn were classified as up-and down-regulated genes based on the time point for tissue that showed the maximum fc, and were annotated to the kyoto encyclopaedia of genes and genomes (kegg) using database for annotation, visualization and integrated discovery (david) v . . in addition, enrichment analyses were performed with bps, using gene ontology (go) terms and kegg pathways for the genes in each gcn. go annotations were filtered with the direct option and applied to enrichment analyses with the following cut-offs: p value < . and counts ≥ . next, treemaps for the enriched go terms were visualised using the revigo tool. kegg annotations were also enriched using the same cut-off criteria and are represented by the -log p value and fold enrichment. all data used in the enrichment analyses were annotated in sus scrofa. gsea for gcn group-specific genes were conducted using the gene-ranking method based on gene sets in the kegg database to determine the enrichment scores and statistically significant differences, using the gsea v . . software. all analyses were performed using the log -normalised tmm counts of the selected tissues and time points that showed the largest expression changes in each group. counts corresponding to: (i) dpi in all tissues were used for the dpi-specific group, (ii) dpi in lung tissues were used for the lung-specific group, and (iii) and dpi in bln tissues were used for the bln-specific group. gsea results were visualised as enrichment maps with significant pathways (fdr < . ) following the benjamini-hochberg correction using cytoscape, and their connections indicated the similarities between gene sets. additionally, the core enriched genes of pathways showing the highest normalised enrichment score (nes) were expressed as heatmaps. then, the modulations of responsible gene products (proteins) in the selected representative kegg pathways (determined through gcn and gsea) were confirmed using the clusterprofiler package in the r software. among the genes corresponding to each protein, genes showing the maximum changes were used as the representative values. the ppi network was investigated for the top interactions (identified in this study) with extremely high gene expression levels using the homo sapiens database of string v . . . the viral loads decreased markedly beyond dpi, and lung samples without detectable viral loads were observed during these time points. serum antibodies (igg) were first detected at dpi and their levels increased up to dpi, but slightly decreased at dpi. a total of . billion paired-end sequence reads were produced from tissue samples (from tissues of individuals), and the average of the number of reads produced per sample was . million (additional file ). the reads that passed the trimming process were mapped to pig reference genome . (~ . % identity), and the average unique mapping rate was . %, ranging from . to . %. transcriptome read data were produced using tissues (lungs, blns, and tonsils) at time points ( , , , , , and dpi), as shown in figure a . the transcriptomes produced under prrsv infection showed clear clustering for each tissue type, as determined by mds analysis (figure a ). degs were confirmed by comparing the gene expression levels at each time point ( , , , , and dpi), relative to those at dpi, and overlapping degs among tissues and different time points are shown in a venn diagram ( figure b ). we observed dynamic changes in the gene expression levels in lung and bln tissues at each time point. lung tissues demonstrated a large proportion of up-regulated genes at all time points ( dpi: %, dpi: %, dpi: %, dpi: %, and dpi: %) against dpi, whereas bln tissues mostly displayed a large proportion of down-regulated genes ( dpi: %, dpi: %, dpi: %, dpi: %, and dpi: %). interestingly, we observed a tendency towards gene up-regulation at dpi in all tissues. moreover, the numbers of degs in lung and bln tissues increased markedly at dpi, then sharply decreased, and slightly increased at dpi. tonsil tissues showed relatively subtle changes; therefore, we had difficulties in analysing them at all time points, except at dpi ( genes). gcn construction using genes and significant connections (selected using the pcit algorithm) was performed to integrate the transcriptomes for one respiratory and two immunity-related tissues at all time points (figure ). the locations of genes were marked close to one another when many common neighbours were found. the resulting network was precisely concentrated for three groups ( dpi, lung tissue, and bln tissue), and the groups were linked by some genes. five clusters were identified via clustering analysis using the log fc values of genes in the gcn and were specifically matched to each group. cluster , representing the dpi-specific group, was composed of genes that were up-regulated in all tissues with prrsv at dpi. clusters and , which represented the lung-specific group, contained down-regulated genes at dpi and up-regulated genes at - dpi in the lungs, respectively. the blnspecific group comprised two clusters of strongly downregulated genes ( genes in cluster and genes in cluster ) at and dpi in bln tissues. analysis of cluster showed that the genes were slightly up-regulated at and dpi in blns. additionally, the bln-specific group contained some genes that showed maximum changes in the tonsils. we selected representative up-and down-regulated genes at different time points in different tissues with relatively high absolute fc values in the constructed gcn and categorised them as follows: up-regulated genes and down-regulated genes; significant pathways were identified by performing kegg enrichment analyses using david ( figure a ). the bubble plot of the enriched biological pathways revealed up-regulated genes in kegg pathways that are mainly related to immune responses (i.e., cytokine-cytokine receptor interaction, influenza a, cytosolic dna-sensing pathway, and rig-i like receptor signalling pathways) and down-regulated genes in pathways mainly related to energy metabolism (i.e., nitrogen metabolism, ppar signalling pathway, and ampk signalling pathway). these pathways provided important insights into the specific immune responses of host cells to prrsv infection. functional-enrichment analyses based on the kegg ( figure b -d) and go ( figure e -g) databases were performed to investigate the biological processes (bps) associated with each group following prrsv infection. the kegg pathways of the dpi-specific group were enriched for terms related to viral infection, including influenza a, the rig-i-like receptor signalling pathway, herpes simplex infection, and the cytosolic dna-sensing pathway ( figure b ). in the lung-specific group, kegg terms associated with immune responses, such as cytokine-cytokine receptor interaction, rheumatoid arthritis, and the chemokine signalling pathway, were identified ( figure c) . interestingly, the bln-specific group showed kegg terms related to lipid metabolism such as the ampk signalling pathway, the ppar signalling pathway, glycerolipid metabolism, and the adipocytokine signalling pathway ( figure d ). the illustrated treemaps revealed bps for significant go terms such as defence response to virus and negative regulation of viral genome replication in the dpi-specific group (figure e ); immune response in the lung-specific group ( figure f ); and regulation of epithelial cell proliferation in the bln-specific group ( figure g ). the enrichment results were consistent between the kegg and go terms. to validate the results of specific groups, gsea based on the kegg database were performed using log -normalised tmm counts at each time point for each tissue to identify the maximum gene expression changes for each group, and the gsea results were consistent with the kegg enrichment analyses of degs. gsea using the -dpi data for all tissues revealed many significant pathways related to viral infection, among which influenza a showed the highest nes ( figure a ). the expression levels of core enriched genes in influenza a were visualised by generating a heatmap, and (rsad , ddx , cxcl , mx , rnasel, ifnb , ifih , oas , ifn-alphaomega, il figure b ). gsea using the -dpi data for lung tissues revealed many significant pathways related to immune responses, among which the cytokine-cytokine receptor interaction pathway showed the highest ness (figure a) . the expression levels of core enriched genes involved in cytokine-cytokine receptor interactions were visualised by generating a heatmap, and (tnfrsf , tnfrsf , tnfrsf b, ccl , cd , cxcr , il ra figure b ). the gsea results obtained with the -and -dpi data for the bln tissues could not be used to construct an enrichment map because only a few statistically significant pathways and low similarity were observed among the gene sets. in the ppi network, the top proteins (i.e., those with the strongest interactions with ifnb (ifnb ), and the largest differences in expression levels) were all immunerelated proteins (ifnar , irf , irf , ifnar , stat , irf , socs , irf , stat , irf , ifih , tyk , irf prrsv infection in pigs can cause a complicated disease when functioning as a primary respiratory infectious agent or as a cofactor in porcine respiratory disease complex (prdc), and prrsv was reported to be the most common virus associated with prdc [ ] [ ] [ ] [ ] . in addition, prrsv was reported to inhibit the host immune defence system, which can lead to further infections (secondary/opportunistic pathogens), resulting in more serious and chronic diseases [ , ] . therefore, understanding the functional and regulatory mechanisms of respiratory and immunity-responsible tissues in the host during prrsv infection is essential for preventing and controlling diseases directly linked to animal productivity. in this study, we compared and integrated serial whole transcriptomes for three tissues (lungs, blns, and tonsils) during prrsv infection. interestingly, the lungs had lower viral loads than the serum ( figure b) , even though the lungs showed major symptoms during prrsv infection [ ] . based on mds analysis, a well-defined (see figure on next page.) ; b and e) , lung-specific network (clusters and ; c and f), and bln-specific network (clusters and ; d and g) were used for the analyses. kegg-enriched pathways for the whole gcn network (a) and each specific network (b-d) were visualised by generating bubble and bar plots. a bubble plot corresponding to the whole network was generated for each analysis for the up-regulated (red) and down-regulated genes (blue) (a). data information: significantly enriched pathways represented in the plots met the following cut-off criterion: − log p value > . . go treemaps were created based on the p values associated with the bp terms for each specific network (e-g). lim et al. vet res ( ) : trajectory of transcriptomes related to prrsv infection was identified in each tissue (figure a ). the number of degs increased until dpi, then decreased until dpi, and finally increased slightly at dpi ( figure b ). the drift curves for the degs were very similar to serum and lung viral load curves, confirming that the overall host responses according to prrsv infection may be most active at approximately dpi, although they were not comparable to the serum antibody levels ( figure b) . previous reports showed that viral loads were detectable in lymphoid organs (excluding the spleen) until immediately before viral extinction [ , ] , although no similarity prrsv can infect cells of the macrophage and monocyte lineages in pigs, and thus, it may affect many aspects of tissue remodelling, development, immunity, and pathology [ ] . prrsv can delay innate and adaptive immune responses by inhibiting the production of type-i interferons (ifns), especially ifn-α, which is important for intracellular signal transduction [ , ] . because the lungs are known as the main target of prrsv infection and lymphoid organs can serve as viral reservoirs [ ] , the observation of common degs probably indicates identical host responses among these tissues, whereas unique degs for each tissue may serve specific functions in the respective tissues during prrsv infection. weighted gcns, which indicate specific gene expression changes, have been used as a powerful approach for identifying specific molecular mechanisms at the system level [ ] . we integrated the transcriptomes of multiple tissues and time points by constructing a gcn with stringent degs and a pcit algorithm (figure ). the constructed network revealed a clear separation of one time point group ( dpi), one respiratory responsible tissue group (lungs), and one immunity-responsible tissue group (blns), and each group showed dynamic changes in the host-response system after prrsv infection. in addition, the genes included in the lung and bln groups showed specific differential expression in each tissue, whereas the genes included in the -dpi group were commonly expressed in all tissues. these features suggest that host responses to prrsv infection are mainly regulated through the expression of different genes in the lungs and lymphoid organs (blns and tonsils). furthermore, gene expression levels may be similarly regulated at dpi in three tissues. dynamic changes in these significant gene subsets at dpi may represent biological signals associated with general and early immunological mechanisms in response to virus infection. significant kegg terms related to viral infections and immune signalling were identified through kegg enrichment analyses performed for each up-and down-regulated genes ( and genes, respectively), based on the time point for each tissue with a maximum fc value in the gcn, which was constructed using serial degs ( figure a ). in particular, the enriched terms related to rna viral infection (influenza a, the rig-i-like receptor signalling pathway, measles, hepatitis c virus (hcv), and the toll-like receptor signalling pathway) and innate immune signalling (cytokine-cytokine receptor interaction, the jak-stat signalling pathway, haematopoietic cell lineage, and the chemokine signalling pathway) were found among the highly significant pathways in the dpi-specific group ( figure b ). in addition, gsea results based on the expression levels at dpi in all tissues revealed clustering with kegg terms related to viral infection, and influenza a showed the highest nes (figure a) . previous reports showed that rig-i-like receptors (rlrs), consisting of three proteins (rig-i, mda , and lgp ), are one type of pattern-recognition receptors (prrs) that activate innate immune-signalling pathways by detecting viral rna in the cytosol [ , ] . influenza a viruses are rna viruses that express non-structural protein (ns ), which is important for evading toxic innate immune responses. ns can suppress mrna processing and transport, which inhibits the binding of the viruses to double-stranded rna molecules and restrains rlr activation [ ] [ ] [ ] . these functions are known to interfere with, and delay, both the expression of the cytokine ifnα/β and the initiation of an ifn-induced antiviral state [ ] . it has been reported that both the structural and non-structural proteins of prrsv have polygenic toxicity in hosts [ ] , particularly, nsp tf and nsp n generated by a ribosomal frameshift mechanism affect the suppression of cellular innate immune responses [ ] . these features of prrsv can be very susceptible to ifn-α/β, although they do not exhibit typical innate immune-signalling activation, including type-i ifn responses [ ] . in addition, signalling associated with the rig-i-like receptor and jak-stat pathways, which play important roles in ifn production, can become disrupted by prrsv during acute infection [ ] . additionally, analysing the lung transcriptomes of prrsv (north american strain ch a)-infected pigs (landrace × yorkshire) showed degs associated with inflammatory signalling at dpi [ ] , which is similar to our results. thus, the enriched biological terms for the dpi-specific group found in this study suggest that both prrsv and influenza a virus, both of which cause respiratory illnesses, exhibit similar infection mechanisms, immune evasion, and innate immune signalling. additionally, the rig-i-like receptor is thought to function as the main prr for prrsv during early immune responses. kegg enrichment analysis of the lung-specific group (containing clusters and of the gcn) implicated cytokine-cytokine receptor interactions, cell-adhesion molecules, rheumatoid arthritis, and chemokine signalling pathways in prrsv infection, all of which are related to immune signalling ( figure c ). cluster included several genes related to immune responses, which were especially up-regulated at - dpi in the lungs (figure ). gsea at dpi revealed clustering of kegg terms related to immune signalling (which showed the largest changes in expression levels), among which cytokine-cytokine receptor interactions showed the highest nes ( figure a ). in contrast to the common early immune responses, terms in the lung-specific group were not enriched for innate immune signalling. moreover, lung viral loads were not detected after dpi, and the levels of anti-prrsv antibodies (igg) started to increase after approximately dpi ( figure b ). therefore, based on the viral loads and antibody levels found in this study, we proposed that the immune signalling pathway terms identified in the lung-specific group represent the adaptive immune-response mechanism occurring in the respiratory system. many studies regarding the adaptive immune responses that occur during prrsv were mainly focused on humoral responses associated with various cytokines and the development of cell-mediated immunity (cmi). with regard to humoral responses, it has been reported that non-neutralising antibodies against prrsv proteins (i.e., the n protein and non-structural proteins) are produced beginning at approximately dpi, whereas neutralising antibodies (nas) were detected later after approximately dpi [ , ] . although prrsv-specific t cells were previously observed early in lymphoid tissues (beginning at approximately dpi), the t cell responses did not last long and did not correlate with viral loads [ ] . in addition, another study showed that the activity of ifn-γ-secreting cd + t cells against prrsv was weak and delayed [ , ] . as previous studies have shown, nas (humoral responses) and cellular responses (cmi) involved in adaptive immune signalling against prrsv were detected at abnormally low levels and were delayed, which may reflect the sequential triggering of interference and delayed innate-immune signalling. lymphoid organs (blns and tonsils) were not highlighted in the lung-specific group due to weaker immune responses in these tissues compared to those in the lungs. significant go terms related to immune responses and apoptosis were identified in each tissue at - dpi in the bln in this study (additional file ), but the tonsils tissues were difficult to study in terms of associated mechanisms because of the small number of degs. in agreement with the results of this study, a previous study demonstrated that changes in the expression of proinflammatory cytokines (il- α, tnf-α, and il- ) at to dpi in prrsv (european strain )-infected lymphoid organs (retropharyngeal lymph nodes, mediastinal lymph nodes, and tonsils) were found only in the lymph nodes [ ] . consequently, we discovered that adaptive host immune signalling in response to prrsv infection was relatively active in the lungs, although such signalling was significantly lower in lymphoid organs (blns and tonsils). the bln-specific group contained clusters and (showing down-regulated genes) and was highly enriched for the ampk signalling pathway, the ppar signalling pathway, glycerolipid metabolism, and the adipocytokine signalling pathway, which are related to lipid metabolism ( figure d) . during viral infection, lipid metabolism can be regulated by viruses; it promotes membrane fusions during viral entry and efficient replication [ , ] . among these pathways, several viruses have been reported to inhibit ampk signalling, which plays a major role in cellular-energy homeostasis including lipid production. human immunodeficiency virus (hiv) is known to encode a trans-activating regulatory protein (tat), which has been reported to inhibit phosphorylation of the ampk α-subunit at thr and to concomitantly reduce the phosphorylation of the ampk substrate, acetyl-coa carboxylase [ ] . it was also reported that the hcv proteins ns b and ns a can activate protein kinase b (pkb/akt), which inhibits ampk by phosphorylating ser and thr [ ] [ ] [ ] [ ] . moreover, previous reports showed that hiv, hcv, and influenza a virus replication could be inhibited by -aminoimidazole- -carboxamide ribonucleotide (aicar), which is an ampk activator [ , , ] . marc- and porcine monocyte-derived dendritic cells (mdcs) infected with prrsv (north american strain p -gfp) showed significant suppression by ampk activators (sodium salicylate and u a) [ ] . recently, it was confirmed that ampk activity increased at up to . dpi in pk- cd cells, that pams were infected with prrsv (north american strain wuh ), that replication was inhibited through acetyl-coa carboxylase (acc ), and that fatty acid biosynthesis was reduced by the ampk activator a [ ] . in addition, proteins nsp , nsp , and nsp of prrsv are also well known to induce cell membrane rearrangement for efficient replication [ ] , and it may interact with lipid metabolism such as ampk signalling. in this study, we performed enrichment analyses for the bln-specific group in the gcn, which confirmed that genes related to ampk signalling were only downregulated in bln tissues (figure ) . these results suggest that prrsv regulates ampk signalling to create a suitable environment for viral replication in lymphoid tissues including blns, which function as a reservoir, thereby establishing persistent infection. we examined highly significant pathways including influenza a and cytokine-cytokine receptor interactions through enrichment analyses for each group in the gcn construction and gsea, based on all genes. we also intensively investigated the expression levels of significant genes identified through both analyses. firstly figure b ). all selected genes were associated with antiviral and immune signalling. in particular, rsad (viperin), ddx (rig-i), mx (mxa), and oas ( ′- ′oas and oas) were highly expressed in all tissues at dpi (log fc ≥ . ), and ifnb (ifnβ) and ifn-alphaomega (ifnα) were extremely highly expressed at dpi in the lymphoid organs (blns and tonsils) (log fc ≥ . ). rsad (viperin) encodes a cellular protein that was found to inhibit the replication of various dna and rna viruses, including influenza a [ ] . during influenza a infection, viperin decreased lipid raft formation by reducing the activity of farnesyl diphosphate synthase, which inhibited viral budding and release [ ] . in addition, viperin up-regulation in prrsv (north american strain bb )-infected marc- cells was reported to inhibit viral replication [ ] . rig-i, a prr responsible for type-i interferon responses, is an essential molecule for innate immune signalling that recognises virus-infected cells in mammals, including pigs [ ] . the human mxa protein has been shown to exert antiviral activity against a wide range of rna viruses (including influenza a) and some dna viruses [ ] . moreover, expression of the porcine mx protein was reported to increase at up to dpi in prrsv (american strain snuvr )-infected marc- cells [ ] . oas is known to encode a member of the - a synthetase family, which comprises essential proteins involved in the innate immune responses to viral infection; it promotes viral rna degradation and inhibition of viral replication [ ] . furthermore, it was reported that oas overexpression in pams inhibited prrsv (north american strain bj- ) replication [ ] . ifn-alphaomega encodes a unique isoform found only in a few species such as pigs and cows [ ] , and its antiviral activity against prrsv (north american strain sdsu- -p ) infection was found to differ depending on the target cell [ ] . in addition, ifnb , one of the representative signalling genes involved in antiviral innate immunity in mammals including pigs, showed high antiviral activity in pams, but not in marc- cells, indicating an opposite effect compared to that of ifn-alphaomega [ ] . in this study, we also focused on the top proteins showing high interactions with ifnb through the ppi network (figure ) , some of which (ifnar , ifnar , irf , irf , irf , stat , and stat ) were associated with ifn-mediated immune responses (autocrine and paracrine signalling) and promoted ifn secretion through intracellular pathogen recognition [ ] . generally, pathogens are recognised by prrs, and ifn expression can be induced in the nucleus through the activation and phosphorylation of irf and irf . ifns secreted from cells can bind to the receptors ifnar and ifnar , which initiates signal transduction pathways; subsequently, ifn-stimulated genes are expressed after the formation of the isgf complex (irf , p-stat , and p-stat ). collectively, these findings suggest that the host responses at dpi in all tissues were associated with up-regulated genes related to antiviral signalling that were common to the case of influenza a infection, and immune-related genes specifically expressed in lymphoid organs (blns and tonsils) were identified. in particular, the ifn-alphaomega and ifnb genes, which encode type-i interferons (which were expressed at extremely high levels in lymphoid organs), need to be further studied, so as to clarify their molecular functions in prrsv infection. secondly, cytokine-cytokine receptor interactions (confirmed by gcn-enrichment analysis and gsea) included up-regulated expression of tnfrsf (ox ), tnfrsf ( - bb), tnfrsf b (dcr ), ccl (ccl ), cd (cd ), cxcr (cxcr ), il ra (il ra), tnfsf (rankl), ccr (ccr ), ccl (ccl ), il (il ), and il rb (il rb), which include adaptive immune-related genes ( figure a ). tnfsf and tnfrsf are known to control innate and adaptive immune cells by regulating various mechanisms, inducing the costimulation or co-inhibition of immune responses [ ] . ox is a co-stimulatory receptor expressed in activated t cells after antigen recognition, and interaction with its ligand can promote t cell proliferation and survival, as well as cytokine production. - bb is an inducible co-stimulatory receptor that is mainly expressed in t cells, and interaction with its ligand is essential for t cell development, survival, proliferation, effector function, and memory t cell formation [ ] . rankl is a strongly up-regulated ligand in t cells after antigen receptor stimulation, and it affects bone homeostasis, lymphoid organ development, and t cell-dendritic cell interactions [ ] . in summary, we identified genes affecting t cell maturation, proliferation, and survival with respect to adaptive immune signalling at dpi; however, further studies should be performed to clarify the roles of these genes during prrsv infection. a possible concern regarding the generation of serial data for these respiratory and immune tissues is the use of tissue samples obtained from different animals at each time point. however, the trends observed for serum viral loads and antibody titres ( figure b ) corresponded with those of previous studies [ , , ] that used blood collected from genetically identical pigs from multiple time points, indicating that the pigs used in this study could have had similar responses to prrsv at any given time point. we performed deg profiling at six serial time points with one respiratory and two immunity-responsible tissues during prrsv infection, based on rna-seq data. additionally, three groups with specific expression patterns (i.e., the -dpi, lung, and bln groups) were discovered by integrating the data via gcn construction. our findings suggested the involvement of key signalling pathways through functional-enrichment analyses. at dpi, all three tissues showed antiviral and innateimmune signalling similar to the case for influenza a infection, with the lymphoid organs (blns and tonsils) showing relatively stronger expression levels in response to infection than the lungs. moreover, we observed the adaptive immune responses that were most active in the lung tissues, based on high expression levels of various cytokines, whereas the responses were relatively weak in the lymphoid organs. independently, ampk signalling appeared to be down-regulated specifically in bln tissues, 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virus challenge in mice the ancient drug salicylate directly activates amp-activated protein kinase fatty acids regulate porcine reproductive and respiratory syndrome virus infection via the ampk-acc signaling pathway viperin, a key player in the antiviral response the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year ready to submit your research ? choose bmc and benefit from monkey viperin restricts porcine reproductive and respiratory syndrome virus replication rig-i in rna virus recognition human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity expression of interferon-α and mx protein in pigs acutely infected with porcine reproductive and respiratory syndrome virus (prrsv) the oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities porcine ′, ′-oligoadenylate synthetase inhibits porcine reproductive and respiratory syndrome virus replication in vitro evolution of the class cytokines and receptors, and discovery of new friends and relatives differential expression and activity of the porcine type i interferon family differential regulation of type i and type iii interferon signaling the tnf family of ligands and receptors: communication modules in the immune system and beyond agedependent resistance to porcine reproductive and respiratory syndrome virus replication in swine emergence of two different recombinant prrsv strains with low neutralizing antibody susceptibility in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. all data generated or analysed during this study are included in this published article. all animal experiments were approved by the jeonbuk national university institutional animal care and use committee, republic of korea (approval number - ). not applicable. the authors declare that they have no conflict of interest. key: cord- - iift zy authors: zhang, xiaorong; liao, kai; chen, shuqin; yan, kun; du, xubin; zhang, chengcheng; guo, mengjiao; wu, yantao title: evaluation of the reproductive system development and egg-laying performance of hens infected with tw i-type infectious bronchitis virus date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: iift zy the prevalence of tw i-type infectious bronchitis virus (ibv) has been increasing rapidly, and it has become the second most common genotype of ibv in china threatening the poultry industry. in this study, -day-old specific-pathogen-free (spf) chickens infected with tw i-type ibv were continuously observed for days. tw i-type ibv affected the respiratory, urinary, and female reproductive systems, resulting in a mortality rate of % as well as a decrease in egg quantity and an increase in inferior eggs. during the monitoring period, serious lesions occurred in the female reproductive system, such as yolk peritonitis, a shortened oviduct, and cysts of different sizes with effusion in the degenerated right oviduct. the infective viruses persisted in vivo for a long time, and due to the stress of laying, virus shedding was detected again after the onset of egg production. our findings suggest that tw i-type ibv is deadly to chickens and could cause permanent damage to the oviduct, resulting in the poor laying performance of female survivors and decreasing the breeding value and welfare of the infected flock. avian infectious bronchitis is a highly contagious acute viral disease in chickens caused by infectious bronchitis virus (ibv) [ ] . as a member of the genus gammacoronavirus, ibv has a remarkably high mutation and recombination rate, leading to numerous types and variants that differ from each other in pathogenicity [ , ] . although the site of entry of ibv is the upper respiratory tract, where the initial infection occurs, the virus can spread systemically, replicating in the epithelial cells of many organs and causing injuries of the kidneys and female reproductive tract [ ] . the kidney, trachea, caecal tonsil, and cloaca have been demonstrated to be tissues in which the long-term persistence of ibv is observed [ ] . it has been reported that the virus can even be re-excreted from the faeces of h -infected and h -infected chickens at days post-infection [ ] . the mechanisms involved in the long-term persistence of virulent ibv in convalescent chickens are assumed to be related to viral pathogenicity, and further study is needed [ ] . tw i-type ibv was first discovered in in taiwan and subsequently identified as a new genotype. since the first report in chinese mainland in , the prevalence of this ibv type has increased rapidly nationwide [ ] . it [ ] . due to differences in antigenicity, the protection provided by commercial vaccines in chickens infected with tw i-type ibv is not complete, and the incidence of immune failure caused by this strain type has increased in recent years [ ] . the majority of tw i-type ibv strains exhibit widespread tissue tropism, and they can affect the respiratory, urinary and reproductive systems, and lead to the death of young chickens [ ] . there is evidence suggesting that tw i-type viruses have undergone extensive evolution, with diverse strains circulating in chicken flocks, and the need to comprehensively evaluate the pathogenicity of this type of strain has become even more urgent [ , ] . in this study, the pathogenicity of tw i-type ibv was evaluated by examining clinical symptoms, mortality rates, virus shedding, lesions, and laying performance in terms of egg quantity and quality in infected chickens. the aim of the study was to comprehensively reveal the pathogenicity of tw i-type ibv, particularly regarding the long-term impact on egg production. strain ck/ch/ah/ / (abbreviation: ah ) of tw i-type ibv used in this study was isolated in from the trachea and kidney of chickens in a broiler flock exhibiting respiratory signs and death [ , ] . ah was serially diluted, and five replicate samples of − , − , − , − , and − dilutions were inoculated into -day-old embryonated spf chicken eggs, the % egg infections dose (eid ) of this strain was calculated by the reed-muench method [ ] . spf chicken eggs were purchased from the beijing boehringer ingelheim merial vital laboratory animal technology co., ltd, china. one-day-old spf white leghorn chickens were purchased from the jinan sipai furui livestock technology co., ltd. the operation and treatment of the animals were approved by the institutional animal care and use committee of yangzhou university (yzudwll- - ). one-day-old spf chickens (n = ) were randomly divided into two groups, each group contained chickens. at days of age, all cockerels were picked out and discarded. the chickens were housed in separate negative-pressure isolators in biosafety level facilities and supplied with feed and water ad libitum. at day of age, the challenge group was infected via oculo-nasal route, with μl of pbs diluent containing . eid of the ah ; and the control group was administered pbs instead. after challenge, the chickens were observed daily. the full trial period lasted days. the pathogenicity of tw i-type ibv in the early stage post-infection was evaluated via the clinical symptoms, pathological lesions, and virus shedding from trachea and cloaca. dyspnea, gasping, tracheal rales, diarrhoea, depression, anorexia, and death in the infected chickens were recorded as clinical symptoms. at days post-infection (dpi), five chickens in each group were euthanized by cervical dislocation for microscopic lesion examination. at dpi and dpi, the oral and cloaca swabs were collected from chickens in each group for the detection of virus shedding by reverse transcription-quantitative polymerase chain reaction (rt-qpcr). at days of age, the effects of tw i-type ibv infection on the female reproductive system were evaluated. nine hens of the challenge group and three hens of the control group were euthanized by cervical dislocation and necropsied. the developmental status of oviducts and ovaries were checked and documented. then, fourteen chickens in each group were randomly chosen for comparison of egg production performance and egg quality after the onset of laying. in the laying period, the eggs were collected daily, and quality traits were evaluated for each egg in the next morning [ ] . eggs were weighed individually, and their egg weight recorded to the nearest . g. egg length and width was measured to the nearest . cm using calipers. the width was divided by the length and multiplied by to obtain the shape index. eggs were broken onto a flat surface and the height of the albumen was measured in millimeters using a tripod micrometer halfway between the edge of the yolk and thick albumen. the thickness of the eggshell was measured in places around the midline to the nearest . mm, and averaged. at days of age, the oral and cloaca swabs were collected from hens in each group for the detection of virus re-excretion by virus isolation and rt-qpcr. at days of age, all of the hens were euthanized by cervical dislocation and necropsied. the samples of trachea, lungs, kidneys, oviduct, heart, liver, spleen, cecal tonsils, duodenum, and glandular stomach of chickens were collected, and tissues from every two chickens were pooled for viral rna detection by rt-qpcr. the oral and cloaca swabs were washed in pbs, and suspensions of tissues were also prepared in pbs ( % w/v). samples of -day-old hens were blind passaged three times in the allantoic cavity of -day-old embryonated spf chicken eggs. total rna of the samples were extracted using the ultrapure rna kit (cowinbio, beijing, china), and reverse transcription was performed with the easyscript ® reverse transcriptase [m-mlv, rnaseh-] kit (transgen biotech, beijing, china). the primers and probe used for rt-qpcr were described in a previous study [ ] . the reaction mixture and the thermal profile employed for rt-qpcr were as specified in the aceq qpcr probe master mix kit (vazyme biotech, nanjing, china). for haematoxylin-eosin staining, the collected samples of the trachea, lungs, kidneys, and oviduct were fixed in % neutral formalin for h at room temperature. the fixed samples were processed, embedded in paraffin wax, and cut into μm sections and examined by light microscopy for the presence of lesions. for the examination of ultrastructural alterations, the cylindrical trachea were cut crosswise with two sharp blades into small strips of mm wide and - mm long, and then the strips were cut into mm small squares. the squares were then fixed in . % glutaraldehyde in . m pbs for h at °c. the samples were then washed with pbs and dehydrated via an alcohol gradient, followed by drying (cpd- d) and conductive treatment (scd ). finally, the ultrastructure of the tracheal cilia was observed by scanning electron microscopy (gemin-isem ) with a magnification of ×. all statistical analyses were performed with spss . software. non-parametric mann-whitney u test was used to analyze the significant difference, and p < . was considered as significant. the clinical manifestations in the early infection stage caused by tw i-type ibv were identified as respiratory symptoms. nearly half of the chickens in the challenge group exhibited gasping, tracheal rales or dyspnea. seven chickens died from to dpi, accounting for % of the infected flock. gross lesions such as light hyperaemia and serious catarrhal exudation in the tracheal mucosa and urate deposition in the tubules of the pale kidney, appeared in the infected flock from to dpi. the development of oviducts and ovaries was assessed at days of age. the reproductive system of chickens in the control group was well developed. however, there were / chickens in the challenge group that showed delayed development of oviducts and ovaries ( table ). the right oviducts of all chickens in both control and challenge groups were completely degenerated. right cystic dilatations attached to the cloaca with a watery content were present in / of the chickens from the challenge group, and another / chicken had a cyst on the left immature oviduct wall (table , figure ). the pathogenicity of tw i-type ibv in the laying period was characterized by a decrease in the quantity of eggs and an increase in the quantity of inferior eggs (table , figure ). during the . -to . -week-old in which laying was monitored, egg production in the challenge group was lower than that in the control group; the decrease of egg production was . % in the challenge group; and the peak egg production per week ( . %) was lower than that in the control group ( . %). the mean albumen height in the challenge group was . mm thinner than that in the control group, representing a decrease of . % (p < . ). these results indicate that tw i-type ibv has a significant effect on subsequent laying performance when the infection occurs at a very young age in chickens. at the age of days, various lesions were observed in the reproductive systems of the hens (table , figure ). in the challenge group, lesions of yolk peritonitis were found in infected females, which were assumed to have been caused by the mature follicles or eggs falling into the abdomen and failing to be captured (table ) . almost all of the females exhibited a large or small oviduct cyst, three of which presented a diameter greater than cm (figure ) . a fibrin clot (yolk-like) in the lumen of the oviduct was observed in females (figure ). none of these lesions were found in the control group. the rt-qpcr results for the oral and cloaca swabs collected at dpi and dpi indicated virus shedding via the respiratory tract and cloaca at dpi (figure ). at days of age, the viruses were re-excreted from the cloaca ( / in the challenge group), about . copies/μl, and two positive samples were blind passaged three times in spf chicken eggs, . and . copies/ μl were detected. at dpi, one of the pooled caecal tonsil samples was detected containing . copies/μl ( figure ). the ultrastructural examination showed the presence of catarrhal exudates and the adhesion, lodging, and shedding of cilia on the tracheal surface of the infected flock at dpi (see additional file ). the tracheae were collected at dpi; the lesions of the blurred boundary between the cilia, congestion, inflammatory cell infiltration, and necrosis of ciliated epithelial cells were widespread. in the laying period, the lesions of the congestion, inflammatory cell infiltration, broadening of the interstitial region, and desquamation of epithelial cells in the oviducts were common in the infected flock ( figure ). no significant lesions were observed in the control group. the prevalence of tw i-type ibv has increased greatly in china in recent years, which has caused tremendous economic losses to the poultry industry [ ] . some reports have illustrated that the majority of tw i-type ibv show tissue tropism to the kidneys and trachea and result in a moderate mortality rate [ , ] . however, there are no long-term reports on the effects of tw i-type ibv on the quantity and quality of eggs when infection occurs at a very young age in chickens, although the poor laying performance and occurrence of "false layers" caused by qx-type ibv have attracted widespread attention [ , ] . in this study, a -day pathogenicity study of the tw i-type ibv strain in chickens was performed. the results suggest that this strain can result in death and has consecutive adverse effects on the female reproductive system, which decrease the breeding value and welfare of the infected flock. in the early infection stage, the pathogenicity of tw i-type ibv in chickens is similar to that of other prevalent types of strains regarding, for example, the symptoms of tracheal rales, and dyspnea or lesions of urate deposition in the kidneys [ , ] . in terms of the ultrastructural and microstructural examination of the trachea, the lesions of adhesion and lodging and the shedding of tracheal cilia caused by tw i-type ibv are assumed to result directly from inflammatory cell infiltration and the presence of serious catarrhal exudates [ ] . respiratory lesions, which are the most common pathogenic characteristic of ibv, subsequently recover and do not lead to the death of infected chickens [ , ] . similar to qx-type strains, the renal lesions of urate deposition may be responsible for the death of chickens, although the mortality rate caused by tw i-type ibv was relatively low [ , ] . during the laying period, the impacts caused by tw i-type ibv in females can be summarized into four categories: a high incidence of oviduct cysts, consecutive lesions in the female reproductive system, decreases in egg production, and poor quality of eggs. the mechanisms involved may contribute to the lesions in the reproductive system that emerged throughout the monitoring period and were induced by the virus reserves in the caecal tonsil. some previous studies have illustrated that ibv exhibits reproductive tissue tropism and that the virus induces an immune response involving an influx of cytotoxic cells and upregulation of inflammatory cytokines in the oviduct and ovary [ , ] . according to a previous report, the two main candidate sites for ibv persistence are cecal tonsils and the kidneys [ ] . however, in this study, we found that the cecal tonsil was the only site of virus replication at days of age. in addition, the exacerbated inflammation and physiological disorders in the oviduct caused by reinfection would be further worsened due to the effect of oestrogen [ ] . as a result, during the laying period, the congestion and inflammatory cell infiltration in the oviducts were more severe than at any earlier time. these adverse impacts were subsequently reflected in a series of lesions in the female reproductive system, such as shortening of the length of the oviduct and a decrease in the number of hierarchical follicles in the ovary, which were directly responsible for a portion of the decrease in egg production. however, the pathogenicity of ibv in the female reproductive system is irregular, and the factors involved include the strains of the virus, the age of the infected chickens, and the protection afforded by antibodies [ , ] . some qx-type strains can also induce oviduct cysts with fluid accumulation in chickens of different ages, but some / -type strains fail to do so [ , ] . not all mass-type strains can induce cystic oviducts or the occurrence of false layers, which differ in different strains [ , ] . some recombinant strains can also induce oviduct cysts even when chickens have been inoculated with commercial vaccines [ ] . the long-term decrease in egg production and the shortened oviduct observed at dpi suggest that the damage to the reproductive system caused by tw i-type ibv might be a permanent impairment rather than just a developmental delay [ ] . in conclusion, tw i-type ibv is characterized by respiratory symptoms, urate deposition lesions in the kidneys, and the continuous disturbance of the female reproductive system, resulting in a high incidence of oviduct cysts and a decline in the quantity and quality of eggs. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . abbreviations ibv: infectious bronchitis virus; spf: specific-pathogen-free; dpi: days post infection; rt-qpcr: reverse transcription-quantitative polymerase chain reaction. coronavirus avian infectious bronchitis virus evolution of infectious bronchitis virus in china over the past two decades recombination in avian gamma-coronavirus infectious bronchitis virus induction of ibv strain-specific neutralizing antibodies and broad spectrum protection in layer pullets primed with ibv massachusetts (mass) and b vaccines prior to injection of inactivated vaccine containing mass antigen the long view: years of infectious bronchitis research a long-term study of the pathogenesis of infection of fowls with three strains of avian infectious bronchitis virus re-excretion of infectious bronchitis virus in chickens induced by cyclosporin phylogenetic analysis of the s glycoprotein gene of infectious bronchitis viruses isolated in china during epidemiology and characterization of avian infectious bronchitis virus strains circulating in southern china during the period from serotype, antigenicity, and pathogenicity of a naturally recombinant tw i genotype infectious bronchitis coronavirus in china altered pathogenicity of a tl/ch/ldt / genotype infectious bronchitis coronavirus due to natural recombination in the ′- kb region of the genome genetics, antigenicity and virulence properties of three infectious bronchitis viruses isolated from a single tracheal sample in a chicken with respiratory problems characterization and analysis of an infectious bronchitis virus strain isolated from southern china in molecular epidemiology of infectious bronchitis virus in china between and , and development of recombinant vaccine using marek's disease virus as vector development and efficacy of an attenuated tw-i like infectious bronchitis virus strain as a candidate live vaccine for chickens in china coronaviruses: methods and protocols. in: walker jm (ed) methods in molecular biology egg quality traits differ in hens selected for high as compared with low antibody response to sheep red blood cells development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks characterization of the complete genome, antigenicity, pathogenicity, tissue tropism, and shedding of a recombinant avian infectious bronchitis virus with a ck/ch/ljl/ -like backbone and an s fragment from a / -like virus induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype d (genotype qx) by maternally derived antibodies and by early vaccination comparative histopathology and immunohistochemistry of qx-like, massachusetts and /b serotypes of infectious bronchitis virus infection in chickens pathogenicity differences between a newly emerged tw-like strain and a prevalent qx-like strain of infectious bronchitis virus safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine genetic and pathologic characterization of a novel recombinant tc - -type avian infectious bronchitis virus pathogenicity differences between qx-like and mass-type infectious bronchitis viruses preparation and protective efficacy of a chicken embryo kidney cell-attenuation gi- /qx-like avian infectious bronchitis virus vaccine a multi-omics study of chicken infected by nephropathogenic infectious bronchitis virus effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct effects of the routine multiple vaccinations on the expression of innate immune molecules and induction of histone modification in ovarian cells of layer chicks the effects of oestrogen and progesterone on re-excretion of infectious bronchitis virus strain g (correction of straing) in spf chickens the effect of estrogen on the early cytotoxic response to ib virus infection in hen oviduct new insights on infectious bronchitis virus pathogenesis: characterization of italy serotype in chicks and adult hens comparative pathogenicity of malaysian qx-like and variant infectious bronchitis virus strains in chickens at different age of exposure to the viruses comparison of the pathogenicity of qx-like, m and • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc /b infectious bronchitis strains from different pathological conditions pathogenicity of virulent infectious bronchitis virus isolate yn on hen ovary and oviduct publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank dr. xiaobo wang of yangzhou university for providing help during histopathologic examination. the datasets analyzed during the current study are available upon request from the corresponding authors. the authors declare that they have no competing interests.received: february accepted: july key: cord- -ztqjo e authors: feng, min; xie, tingting; li, yuanfang; zhang, nan; lu, qiuyuan; zhou, yaohong; shi, meiqing; sun, jingchen; zhang, xiquan title: a balanced game: chicken macrophage response to alv-j infection date: - - journal: vet res doi: . /s - - -y sha: doc_id: cord_uid: ztqjo e avian leukosis virus subgroup j (alv-j) infection can cause tumors and immunosuppression in infected chickens. macrophages play a central role in host defense against invading pathogens. in this study, we discovered an interesting phenomenon: alv-j replication is weakened from hours post-infection (hpi) to hpi, which was verified using western blotting and rt-pcr. to further investigate the interaction between alv-j and macrophages, transcriptome analysis was performed to analyze the host genes’ function in chicken primary monocyte-derived macrophages (mdm). compared to the uninfected control, up-regulated differentially expressed genes (deg) and down-regulated deg at hpi, and up-regulated deg and down-regulated deg at hpi were identified in chicken mdm, respectively. alv-j infection induced strong innate immune responses in chicken mdm at hpi, instead of hpi, according to the analysis results of gene ontology and kegg pathway. importantly, the host factors, such as up-regulated mip- α, il- β, inos, k , irg , ch h, nfkbiz, lysozyme and oasl were involved in the host defense response during the course of alv-j infection. on the contrary, up-regulated ex-fabp, il i , cox- , nfkbia, tnfaip and the jak stat pathway inhibitors including cish, socs and socs are beneficial to alv-j survival in chicken macrophages. we speculated that alv-j tropism for macrophages helps to establish a latent infection in chicken mdm from to hpi. the present study provides a comprehensive view of the interactions between macrophages and alv-j. it suggests the mechanisms of defense of chicken macrophages against alv-j invasion and how alv-j escape the host innate immune responses. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. avian leukosis virus subgroup j (alv-j) is an oncogenic retrovirus, primarily inducing neoplastic diseases and reproduction problems in infected chickens. it is well known that alv-j causes enormous economic loss in the global poultry industries. to date, there are no vaccines or treatments to protect against alv-j infection. since little is known about the interaction between alv-j and the host, current strategies are focused on alv-j eradication. rna viruses are prone to mutations. in contrast to the virus, the host does not change quickly. it is therefore an enticing strategy to try to overpower alv-j by finding ways to make chickens less permissive to viral replication. studies concerning host innate and adaptive immune responses to alv-j are in their infancy [ ] . macrophages are found in all tissues and have a well-defined role in host responses against viral infection [ ] . however, macrophages are susceptible to infection for human immunodeficiency virus (hiv) [ ] , dengue virus [ ] , and porcine reproductive and respiratory syndrome viruses [ ] . in particular, macrophages serve as a reservoir throughout hiv infection [ ] . importantly, macrophages also play a key role in avian viral infections including infectious bursal disease virus (ibdv) [ ] , avian influenza virus (aiv) [ ] , newcastle disease virus (ndv) [ ] and infectious bronchitis virus (ibv) [ ] . however, the role of macrophages in alv-j infection remains unclear. in our previous study, we found that primary chicken monocyte-derived macrophages (mdm) were susceptible to alv-j and infection resulted in expression of immune-related genes [ ] . however, the number of genes we examined was too low to comprehensively map the involvement of immune host factors in an alv-j infection. the goal of the current study was to examine host gene expression profile to improve our understanding of the relationship between macrophages and alv-j during infection. in this study, rna-seq analysis platform and gene overexpression verification were employed to analyze chicken mdm gene expression after alv-j infection. our findings provide a comprehensive view of alv-j immune escape and the host defense response to alv-j infection in chicken macrophages. a total of six-week-old specific-pathogen-free (spf) white leghorn chickens, half females and half males, were purchased from guangdong dahuanong animal health products co., ltd (guangzhou, china) and housed under pathogen free conditions. laboratory alv-j strain scau-hn was kindly provided by prof. weisheng cao, south china agricultural university. all animal experiments were performed with approval and guidance from south china agricultural university institutional animal care and use committee. chicken primary mdm were cultured and identified according to previous studies [ , ] . briefly, peripheral blood mononuclear cells (pbmc) were isolated from blood obtained from spf chickens using chicken lymphocyte separation medium (solarbio, beijing, china) according to the manufacturer's instructions. the supernatant was removed and adherent cells were washed twice with pbs to remove thrombocytes, non-adherent lymphocytes and other semi-adherent cells after h of incubation. these adherent cells were primarily chicken monocytes. subsequently, fresh rpmi- medium with % chicken serum, u/ml penicillin and mg/ml streptomycin were added to the remaining monocytes. chicken monocytes were then cultured for days to generate mature macrophage differentiation. the culture medium was changed every days in order to ensure stable and consistent conditions. chicken mdm were infected with a tcid /ml of alv-j strain scau-hn . dna, rna and total proteins were extracted from the alv-j infected mdm at , , , and h post-infection (hpi). rt-pcr was employed to detect the alv-j replication using specific pcr primers h /h [ ] . western blotting was performed with alv-j envelope protein specific mouse antibody je (kindly provided by dr aijian qin, yangzhou university, yangzhou, china) and rabbit anti-β-actin antibody (bioworld, louis park, usa) according to the method described previously [ ] . irdye dx-conjugated antirabbit igg and irdye -conjugated anti-mouse igg (rockland immunochemicals, limerick, pa, usa) was used as the secondary antibody. membranes were visualized and analyzed with an odyssey infrared imaging system (li-cor biosciences, lincoln, ne, usa). alv-j provirus was detected by pcr with primers h /h using dna template. total rna for rna sequencing (rna-seq) was isolated from pooled mdm (isolated and cultured from spf chickens) infected with alv-j ( tcid /ml) at and hpi using trizol reagent (invitrogen, ca, usa). samples were collected from two independent experiments. non-infected mdm were used as a control group. purity and quantity of total rna were assessed using the nanophotometer ® spectrophotometer (implen, ca, usa) and the bioanalyzer system (agilent technologies, ca, usa). rna degradation and contamination were further monitored using agarose gel electrophoresis. after quality inspection, approximately μg rna per sample was used as input material for the rna sample preparations. briefly, ribosomal rna was first removed using the ribo-zero ™ rrna removal kit (epicentre, wi, usa), and rrna free residue was cleaned up by ethanol precipitation. subsequently, sequencing libraries were generated using the rrna-depleted rna by nebnext ® ultra ™ directional rna library prep kit for illumina ® (neb, ma, usa) according to the manufacturer's recommendations. first strand cdna was synthesized with random hexamers and m-mulv reverse transcriptase. second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. in the reaction buffer, dntp containing dttp were replaced with dutp. remaining overhangs were converted into blunt ends via the exonuclease and polymerase activities. after adenylation of ′ ends of dna fragments, neb-next adaptor with hairpin loop structure were ligated to prepare for hybridization activities. in order to select cdna fragments of preferentially - bp in length, the library fragments were purified with ampure xp system (beckman coulter, beverly, usa). then μl user enzyme (neb, ipswich, ma, usa) was used with size-selected, adaptor-ligated cdna at °c for min followed by min at °c before pcr. then, pcr was performed with phusion high-fidelity dna polymerase, universal pcr primers, and index (x) primers. at last, products were purified (ampure xp system) and library quality was assessed on the agilent bioanalyzer system. the mrna libraries were sequenced at the novogene (beijing, china) on an illumina hiseq platform. raw data (raw reads) of fastq format were first processed through in-house perl scripts. in this step, clean data (clean reads) were obtained by removing adapter sequences as well as reads containing poly-n and low quality reads. therefore, only high quality data were analyzed and quality scores (q and q ) and gc content were subsequently calculated. all the following analyses were based on the clean data with high quality. reads were mapped to the chicken genome assembly [ ] using tophat (v . . ). the mapped reads of each sample were assembled by both scripture (beta ) and cufflinks (v . . ) in a reference-based approach. the fpkm (fragments per kilo-base of exon per million fragments mapped) was calculated based on the length of the fragments and reads count mapped to this fragment. cuffdiff (v . . ) was used to calculate fpkm of coding genes in each sample. gene fpkm were computed by summing the fpkm of transcripts in each gene group. cuffdiff software was used to provide statistical routines for determining differential expression in digital transcripts or gene expression data using a model based on the negative binomial distribution. in the present study, for differentially expressed genes (deg), the threshold was q value < . , log |(fold change)| ≥ with an fpkm value no less than in infected or uninfected samples. deg were subjected to gene ontology (go) categorization and kyoto encyclopedia of genes and genomes (kegg) pathway analysis using the database for annotation, visualization, and integrated discovery (david) version . [ ] . total rna was extracted from alv-j-infected ( tcid /ml) and uninfected mdm at hpi and hpi using rnaiso reagent (takara, japan). for gene expression analysis, cdna synthesis of mrna was performed using a primescript rt reagent kit (perfect real time) (takara, japan) according to the manufacturer's protocol. the qpcr primers were designed using the ncbi primer blast program [ ] and were based on published target sequences (additional file a) [ ] [ ] [ ] [ ] . the gapdh gene was used as an internal control. qpcr was performed on a bio-rad cfx real-time detection system using itaqtm universal sybr ® green supermix kit reagents (bio-rad, ca, usa) according to the manufacturer's specifications. data analyses were performed using the −ΔΔct method. mdm were cultured in -well plates and transfected with μg plasmids including k , irg , oasl, ch h, cish, ex-fabp, il i and socs using lipofectamine reagent, respectively. egfp was used as a control. after incubation for h, lipofectamine transfection reagent was removed, and the cells were replenished with rpmi- medium with % chicken serum. h later, the transfected mdm were infected with tcid /ml of alv-j strain scau-hn . at hpi, alv-j replication level was analyzed by western blot and qpcr. the primers used in the construction of these plasmids are summarized in additional file b. statistical comparisons were performed using graphpad prism (graphpad software inc., san diego, ca, usa). the results are presented as the mean ± sem. two-way anova analysis was used to analyze the statistical significance among multiple groups and unpaired student's t-test between two groups. statistical significance is indicated by p values of > . (non-significant, ns), < . (*), . (**) or . (***). the sequencing data obtained from rna-seq were released to the geo database under the accession numbers gse . alv-j infections of mdm resulted in either genome integration or reverse transcription into cdna during - hpi ( figure a ). the rate of alv-j replication was very high at hpi and gradually decreased from to hpi ( figure b) . furthermore, the viral envelope protein was detectable by western blotting at and hpi but not at later times. similarly, env gene expression at hpi was greater than that at hpi ( figure c ). these results demonstrate that the replication rate of alv-j was extremely high at hpi but low after hpi, alv-j replication might be inhibited in chicken mdm. the illumina hiseq platform produced raw reads. after discarding adaptor and low-quality sequences, we obtained clean reads ( . gb). the clean reads were mapped onto the chicken reference genome (gallus_gallus- . ), and the mapping rate of each library ranged from . to . % (additional file ). we found that at and hpi, and deg were uniquely up-regulated, and and were uniquely down-regulated in alv-j infected mdm, respectively. there were up-regulated and downregulated deg in common at the two time points (figures a and b ). in addition, three deg, oasl, fkbp and mcf , were up-regulated at hpi and down-regulated at hpi ( figure c ). nine deg were down-regulated at hpi but up-regulated at hpi ( figure d ). more details of the deg are shown in additional file . the go biological process analysis shows that the up-regulated deg were mainly enriched for immunerelated terms but down-regulated deg were not (figure ) . for the up-regulated deg at hpi, the top three significant go terms were inflammatory response, response to lipopolysaccharide and regulation of apoptotic process ( figure a ). the top three significant go terms for up-regulated deg at hpi were inflammatory response, innate immune response and toll-like receptor signaling pathway ( figure b ). down-regulated deg at hpi were significantly enriched for transmembrane transport, endocytic recycling and positive regulation of interleukin- production ( figure c ). the five down-regulated deg at hpi included hmox , slc a , slc a , gab and slc a were significantly enriched on four go terms ( figure d ). more details of the deg involved in go enrichment analysis can be found in additional file . kegg analysis illustrated that up-regulated deg induced by alv-j in mdm at hpi were involved in immune-related pathways including mapk signaling, toll-like, nod-like, rig-i-like and jak-stat signaling pathway, and etc. (figure a ). up-regulated deg identified at hpi were significantly enriched in cell adhesion molecules, influenza a, toll-like receptor and adipocytokine signaling pathway ( figure b ). however, just two pathways were significantly enriched by the down-regulated deg at hpi ( figure c ). moreover, down-regulated deg at hpi did not enrich any pathway. more details of the deg involved in kegg enrichment analysis can be found in additional file . the immune-related genes were selected according to gene function annotation. a greater number of immune-related deg were found at hpi than at hpi, and most of these immune-related deg were up-regulated by alv-j infection at hpi ( figure a ). according to published studies [ ] [ ] [ ] , and differentially expressed interferon-stimulated genes (isg) were identified in alv-j-infected mdm at hpi and hpi, respectively (additional file ). similarly, the expression of most isg ( ) was significantly increased at hpi, especially irg , ripk , ch h, irf and etc. ( figure b ). to validate the rna-seq results, we chose eight immunerelated deg for qpcr analysis. these included ch h, pkr, socs , nod , tlr , il- , isg - and oasl. the qpcr data matched the rna-seq results and both methods indicate similar trends for these eight genes ( figure ). overexpression of k , oasl, ch h and irg significantly decreased alv-j replication at the protein (figures a and b) and mrna ( figure c ) levels in chicken mdm cells at hpi when compared to the control group (egfp). overexpression of cish, ex-fabp and socs significantly increased the expression of alv-j env gene at protein levels ( figures a and b) and mrna levels ( figure c ) in chicken mdm cells at hpi. however, overexpression of il i only significantly increased the expression of the alv-j env gene at the mrna level, but there was no difference at the protein level ( figure ). as an avian retrovirus, alv-j has been studied for many years although many interesting scientific problems such as tumorigenesis, immunosuppression and immune responses induced by alv-j infection are still not understood [ ] . in our previous studies, we found that chicken mdm are susceptible to alv-j infection [ ] . in the present study, we observed that alv-j replication in mdm was active at hpi, but inhibited from to hpi. it is reported that recombinant chicken ifn-α as well as the isg, ccch type zinc finger antiviral protein (zap), could inhibit alv-j replication in df cells [ , ] . so, we speculated that isg may also resist alv-j replication in chicken mdm. alv-j infection induced most immune-related deg in mdm at hpi (tables and ) . strikingly, the expression of isg including ch h, pkr, oasl, mx, and etc. were significantly increased in alv-j-infected mdm at hpi. isg exert numerous antiviral effector functions by targeting almost any step in the virus life cycle [ ] . for example, ch h broadly inhibited growth of enveloped viruses including vsv, hsv, hiv and the acutely pathogenic viruses ebov, rvfv and rssev by converting cholesterol to -hydroxycholesterol ( hc) [ ] . additionally, up-regulated isg were identified in alv-j mdm at hpi. all of these up-regulated isg in alv-j-infected mdm at and hpi might serve as candidates resisting alv-j infection. in addition to the above isg, some deg have significant changes in expression at hpi and hpi (table ) . mip- α, macrophage inflammatory protein- alpha, is responsible for the chemo-attraction of dendritic cells, and effector and memory b and t cells [ ] . moreover, mip- α exhibited anti-microbial and anti-hiv activities [ ] [ ] [ ] . il- β is produced primarily by activated macrophages and possess multiple and diverse properties in their response to infection [ , ] . thus, host damage following infection induces macrophage secretion of a variety of inflammatory mediators including il- and no that activate anti-pathogenic microorganism defense mechanisms [ ] . no production is primarily catalyzed by inos and is a part of innate host defenses [ ] . alv-j infection in mdm at hpi increased expression of the two orthologues of chicken il- , k (il l ) and il (il l ) [ , ] . il- is a potent chemo-attractant and activator of macrophages [ ] . furthermore, il- has been shown to attract and activate t lymphocytes [ , ] , which would aid in raising an immunologically specific response against alv-j. immune response gene (irg ) was originally identified as a highly inducible gene in murine macrophages following lps stimulation [ ] . the role of irg in the course of virus infection has not been extensively reported. irg was identified as an isg with antiviral effects against different neurotropic viruses [ ] . oasl has been found to broadly inhibit the replication of viruses such as swine fever virus, rsv and hcv through a variety of mechanisms [ ] [ ] [ ] . lysozyme is a differentiation marker for macrophage, and is activated during macrophage differentiation [ ] . we found that lysozyme expression increased incrementally from hpi to hpi in alv-j-infected mdm. this result reminded us that alv-j infection could stimulate chicken macrophage maturation. lysozyme is a cornerstone of innate immunity due to its direct antimicrobial activity through peptidoglycan hydrolysis and immune regulatory functions [ ] . interestingly, lysozyme also possesses antiviral properties [ , ] . nfkbiz encodes the protein iκbz and is known as a partner of nfκb that regulates innate host defense factors [ ] . accordingly, the host genes mip- α, il- β, inos, k , irg , ch h, oasl, lysozyme and nfkbiz served as restriction factor candidates against alv-j infection in chicken macrophages. we further selected several of the anti-alv-j candidates for verification. exactly, the experiments in vitro show that overexpression of k , irg , ch h, and oasl could significantly decrease alv-j replication in mdm at hpi (figure ) . initially, we found that alv-j infection activated many pattern recognition receptors (prr) pathways including toll-like receptors (tlr), rig-i-like receptors (rlr), nod-like receptors (nlr) and cytosolic dna-sensing pathway at hpi (figure ). up to now, the specific innate sensors responding to alv were unknown [ ] . we speculated that alv-j should theoretically be recognized by prr such as tlr, rlr, ifi , and cgas, similar to hiv [ ] . however, no functional prr such as tlr , tlr , tlr or mda were induced in alv-j-infected mdm at hpi. only tlr was up-regulated by alv-j at hpi (table ) . therefore, we speculate that chicken macrophages lack functional prr for alv-j to escape host immune attack at the early stage of infection. indeed, it has been reported that other retroviruses use this strategy to achieve immune escape in macrophages [ ] . interestingly, the expression of tlr , tlr and tlr was significantly increased in alv-j-infected mdm at hpi (table ) . as a result, alv-j replication was inhibited and weak innate immune responses were induced at hpi. at this infection stage, alv-j may be recognized by tlr , tlr and tlr . the jak-stat pathway is a major signaling pathway in the function of immune cells and is activated by cytokines and growth factors [ ] . in this study, many negative feedback regulators of cytokine signaling mediated by this pathway were identified. cish, socs and socs were significantly induced in alv-j-infected mdm at hpi and enriched on the jak-stat signaling (table ) . cish and suppressor of cytokine signaling (socs) family proteins are jak-stat inhibitors, including members, cish, socs , socs , socs , socs , socs , socs and socs [ ] . cish, socs , socs and socs are the best characterized socs family members [ ] . cish is induced by cytokines that activate stat and block the stat binding to cytokine receptors. socs binds to the jaks and inhibits catalytic activity, while socs binds to jak-proximal sites on cytokine receptors and inhibits jak activity [ ] . it has been reported that socs enhances hiv- replication in macrophages by inhibiting antiviral ifn-β signaling [ ] . the socs expression was also significantly increased in alv-j-infected mdm (table ). further verification found that overexpression of cish and socs promoted alv-j replication in mdm at hpi (figure ). we speculated that alv-j infection inhibited the jak-stat pathway via inducing expression of cish, socs and socs . indeed, the key factors such as jak and stat in jak-stat pathway were not remarkably induced by alv-j infection in mdm. the role of socs has not been well identified during viral infection. however, a novel role for socs has been found to restrain the early phase of influenza a infection by inhibiting egfr activity [ ] . coincidently, socs expression was inhibited by alv-j in mdm at hpi (table ). in addition, the expression of nfkbia and tnfaip (a ) were significantly increased in alv-j-infected mdm at hpi (table ). the iκbα protein is encoded by nfkbia and is an important negative regulatory factor in the nf-κb pathway [ ] . the protein tnfaip is known as a powerful suppressor of cytokine signaling and innate antiviral pathways, and it can inhibit the activity of nf-κb and nf-κb-mediated inflammatory responses [ , ] . tnfaip deficiency in myeloid cells and lung epithelial cells could protect against influenza a virus infection [ , ] . we speculated that nfkbia and tnfaip were significantly induced by alv-j infection in mdm to down-regulate cytokines in macrophages, resulting in viral persistence in the host. altogether, nfkbia, tnfaip , cish, socs and socs were considered as a counterbalance to the antiviral immune responses induced by alv-j infection in chicken mdm at hpi. and ex-fabp significantly changed expression at hpi and hpi (table ). we also found that overexpression of il i and ex-fabp could enhance alv-j replication in chicken mdm at hpi (figure ). il i is an immunosuppressive enzyme and primarily expressed in professional antigen-presenting cells and it inhibits t-cell proliferation and activation [ , ] . cox- is one of the important mediators of inflammation in response to viral infection, which contributes to viral replication and this has been shown for hcv [ ] , hbv [ ] , dengue virus [ ] and cytomegalovirus [ ] . the ex-fabp gene encodes an extracellular fatty acid binding protein and it is significantly induced by salmonella enteritidis infections in chickens [ ] . this protein may provide fatty acids for mitochondrial respiration during infection. ex-fabp expression is enhanced after treatments with inflammatory stimuli and is repressed by anti-inflammatory agents, behaving as an acute phase and constitutively expressed survival protein [ ] . ex-fabp was robustly induced in alv-j-infected mdm at hpi and therefore may provide protection for mdm after the chemokine and cytokine production induced by alv-j at hpi. there is a rule that the virus is lost when the cells die. consequently, induction of ex-fabp at hpi may be a strategy of alv-j to live in harmony with chicken macrophages at the late stages of infection. consequently, ex-fabp, il i and cox- together with nfkbia, tnfaip , cish, socs and socs enable alv-j survival in chicken macrophages. immunity to avian leukosis virus: where are we now and what should we do? macrophage biology in development, homeostasis and disease the importance of monocytes and macrophages in hiv pathogenesis, treatment, and cure activation 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viruses interferon-inducible oligoadenylate synthetase-like protein acts as an antiviral effector against classical swine fever virus via the mda -mediated type i interferon-signaling pathway ′- ′-oligoadenylate synthetase-like protein inhibits respiratory syncytial virus replication and is targeted by the viral nonstructural protein ′-oligoadenylate synthetaselike gene highly induced by hepatitis c virus infection in human liver is inhibitory to viral replication in vitro lysozyme gene activity in chicken macrophages is controlled by positive and negative regulatory elements from bacterial killing to immune modulation: recent insights into the functions of lysozyme lysozyme and rnases as anti-hiv components in beta-core preparations of human chorionic gonadotropin structural and functional modeling of human lysozyme reveals a unique nonapeptide, hl , with anti-hiv activity iκbζ regulates human monocyte pro-inflammatory responses induced by streptococcus pneumoniae hiv- infection of macrophages is dependent on evasion of innate immune cellular activation the jak-stat pathway: specific signal transduction from the cell membrane to the nucleus socs proteins, cytokine signalling and immune regulation suppressors of cytokine signaling and immunity suppressor of cytokine signaling inhibits antiviral ifn-beta signaling to enhance hiv- replication in macrophages suppressor of cytokine signaling (socs) ameliorates influenza infection via inhibition of egfr signaling role of nuclear iκbs in inflammation regulation regulation of inflammatory and antiviral signaling by a convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year ready to submit your research ? choose bmc and benefit from inhibition of nf-kappab signaling by a through disruption of ubiquitin enzyme complexes a (tnfaip ) deficiency in myeloid cells protects against influenza a virus infection a deficiency in lung epithelial cells protects against influenza a virus infection the novel immunosuppressive enzyme il i is expressed by neoplastic cells of several b-cell lymphomas and by tumorassociated macrophages il i is a novel regulator of m macrophage polarization that can inhibit t cell activation via l-tryptophan and arginine depletion and il- production green tea phenolic epicatechins inhibit hepatitis c virus replication via cycloxygenase- and attenuate virus-induced inflammation induction of cyclooxygenase- expression by hepatitis b virus depends on demethylation-associated recruitment of transcription factors to the promoter cyclooxygenase- facilitates dengue virus replication and serves as a potential target for developing antiviral agents inhibition of cyclooxygenase blocks human cytomegalovirus replication characterization of chicken spleen transcriptome after infection with salmonella enterica serovar enteritidis inhibition of cell proliferation and induction of apoptosis by exfabp gene targeting host genes associated with hiv- replication in lymphatic tissue hiv- latency in monocytes/macrophages the low levels of alv-j replication in mdm at hpi was accompanied with fewer immune-related deg involved in host defense responses. superficially, we could conclude that alv-j replication was inhibited at - hpi due to robust host immune responses induced at hpi. however, in hiv-related studies, the conventional host immune response does not contain hiv- replication and even contributes by increasing virus replication through immune activation [ ] . moreover, viruses can proactively hide in the host to evade the host immune elimination and hiv typically establishes latency within the macrophage [ ] . based on our findings, it is also possible that alv-j is capable of escaping from host immune responses and establishing latency in chicken mdm after h of viral infection. according to the above analyses, we should take a balanced view to consider the interactions between alv-j and host immune response. additional studies are needed to elucidate the mechanisms of alv-j immune evasion and host defense responses in chicken macrophages.in summary, gene expression profiling analysis in chicken mdm infected with alv-j provides insights into the mechanisms underlying the host immune responses and alv-j immune escape. strong immune responses were induced by alv-j infection in mdm at hpi. we found that numerous differentially expressed genes such as mip- α, il- β, inos, k , irg , ch h, nfkbiz, lysozyme and oasl were involved in host defense of alv-j infection. alv-j countered host immune attacks by inhibiting the expression of functional prr and facilitating expression of jak-stat pathway inhibitors. these results provide valuable insights into the antagonism between host antiviral immune responses and alv-j infection. additional file . rna-seq data statistics. the authors declare that they have no competing interests. mf participated in the design of the study, performed the experiments, collected and analyzed data, and drafted the manuscript. tx and yl performed western blot assay and qpcr. nz, ql and yz helped with the animal experiment and data analysis. ms, js and xz participated in the design and coordination of the study. all authors read and approved the final manuscript. key: cord- -eseo lyh authors: li, chong; culhane, marie r.; cheeran, maxim; galina pantoja, lucina; jansen, micah l.; amodie, deborah; mellencamp, martha a.; torremorell, montserrat title: exploring heterologous prime-boost vaccination approaches to enhance influenza control in pigs date: - - journal: vet res doi: . /s - - -z sha: doc_id: cord_uid: eseo lyh influenza a viruses evolve rapidly to escape host immunity. in swine, this viral evolution has resulted in the emergence of multiple h and h influenza a virus (iav) lineages in the united states (us) pig populations. the heterologous prime-boost vaccination strategy is a promising way to deal with diverse iav infection in multiple animal models. however, whether or not this vaccination strategy is applicable to us swine to impart immunity against infection from north american strains of iav is still unknown. we performed a vaccination-challenge study to evaluate the protective efficacy of using multivalent inactivated vaccine and/or a live attenuated iav vaccine (laiv) in pigs following multiple prime-boost vaccination protocols against a simultaneous h n and h n iav infection. our data show that pigs in the heterologous prime-boost vaccination group had more favorable outcomes consistent with a better response against virus challenge than non-vaccinated pigs. additionally, delivering a multivalent heterologous inactivated vaccine boost to pigs following a single laiv administration was also beneficial. we concluded the heterologous prime boost vaccination strategy may potentiate responses to suboptimal immunogens and holds the potential applicability to control iav in the north american swine industry. however, more studies are needed to validate the application of this vaccination approach under field conditions. influenza a viruses are important zoonotic pathogens and one of the most prevalent causes of respiratory disease. swine influenza a virus (iav) causes respiratory disease in pigs worldwide and is considered a significant player in the porcine respiratory disease complex together with other viruses and bacteria such as porcine reproductive and respiratory syndrome virus (prrsv) and mycoplasma hyopneumoniae (m. hyopneumoniae) [ ] . influenza causes important economic losses in the swine industry with estimates ranging between $ and $ /pig [ ] . h n , h n , and h n are the major subtypes of iav found in pigs around the world and there is significant genetic diversity within those subtypes [ ] . influenza a virus is an enveloped rna virus with a genome consisting of negative-sense rna segments. its genetic diversity is driven mostly by two mechanisms: antigenic drift which is the result of mutations in antigenic sites due to the poor proofreading ability of the rna polymerase, and antigenic shift, or reassortment, which is the exchange of gene segments between distinct viruses resulting in new strains with a gene combination distinct from the parental strains. these viral evolution mechanisms are responsible for the emergence of multiple novel distinct h and h iav lineages in pigs open access *correspondence: torr @umn.edu college of veterinary medicine, university of minnesota, st. paul, mn , usa full list of author information is available at the end of the article during the last years [ ] . between and , genome patterns were documented for the h subtype from us swine alone [ ] . the high diversity of iav makes control of the disease using vaccination difficult. the differences of prevailing lineages between different continents and regions require the iav vaccines for swine to be produced locally and contain distinct strains for each region. currently, vaccination is the primary measure to control iav infection in pigs. the success of vaccination control measures is largely attributed to the ability of the vaccine to stimulate the host immune system and elicit high levels of humoral and cell meditated immune response. the protection imparted by the humoral immune response comes not only through the secretion of anti-hemagglutinin antibodies that neutralize the virus but also from the production of antibodies that stimulate complement system and subsequent antibodydependent cellular cytotoxicity (adcc) which further inhibits virus replication [ ] . cellular immunity also plays a vital role in virus elimination by activating cytotoxic t cells and macrophages to lyse infected cells and destroy ingested microbes [ ] . even though much progress has been made to improve the efficacy of recombinant subunit, vector and dna vaccines against iav and these have promising results as demonstrated in experimental studies, whole-cell inactivated vaccines (wiv) remain the most commonly used licensed vaccines to control iav in pigs. wiv are widely used in sows and gilts to induce serum antibodies against the viral hemagglutinin and enhance transfer of passive immunity to newborn piglets. more than percent of large breeding herds in the u.s vaccinated gilts against iav before or at entry into the herds by using commercial or autogenous wiv [ ] . furthermore, over one-half of large breeding herds vaccinated sows during the last weeks of gestation [ ] . in addition, a live attenuated influenza vaccine (laiv) became commercially available in the us in [ ] . this vaccine, a multivalent laiv with an ns truncated protein, has been purported to elicit both humoral and cell-mediated immune responses in pigs day of age and older [ ] . however, there is less overall information regarding the use of laiv. laiv are usually administrated intranasally to piglets to bypass or minimize the limiting effect of maternal antibodies. due to antigenic drift and shift, vaccines for iav need to be updated on a regular basis to provide sufficient protection. however, updating the vaccine requires significant time to determine the vaccine composition, testing, distribution, and administration [ ] . the delayed time, almost months for human vaccines, is enough for new strains to emerge and undergo antigenic drift and shift, which renders the immunization too late to have substantial impact on the next influenza outbreak [ ] . in the case of vaccines for animals, this time may be even longer [ ] . moreover, there is no systematic worldwide surveillance program for iav in pigs and iav vaccines for swine are updated far less frequently than humans [ ] . therefore, it is much harder to develop vaccines that incorporate strains that are appropriately matched with the circulating strains in a farm or region and provide complete protection against iav for pigs. for these reasons, broadening the immune response is key to providing more complete protection and one way to do this is to adopt a heterologous prime-boost vaccination strategy. heterologous prime-boost vaccination refers to the delivery of antigens with overlapping nucleotides or same antigenic inserts expressed by different vectors or delivery systems for primary and boost vaccination [ , ] . previous research with h n influenza virus in poultry, h n in mice, and h n in ferrets has resulted in the desired broad and long-lasting immune response using the prime-boost approach [ ] [ ] [ ] . the benefit of heterologous prime-boost vaccination has also been evaluated in controlled experiments with pigs and the desired broader protection against h n iav was demonstrated [ ] . whether the heterologous primeboost strategy is applicable to us swine infected with north american strains of iav using currently available multivalent iav vaccines for pigs is still unknown. therefore, we evaluated the protective efficacy of heterologous prime-boost vaccination with wiv and laiv vaccines in a swine model against h and h iav co-infection. the outcomes of this study may be used to develop better vaccination protocols and new control strategies for iav in pigs. this project consisted of two separate studies ( table ). the first study included only pigs vaccinated with commercial (com) or autogenous (aut) wiv and had five different treatment groups: com/com, aut/aut, aut/com, com/aut and no vaccination but challenged (no vac/cha). the second study included pigs vaccinated with the laiv and two treatment groups: laiv/com and laiv/none (none = no boost vaccination). a combined total of ninety, -week-old pigs were enrolled in the project. pigs originated from a farm seronegative to iav, prrsv and m. hyopneumoniae and the farm was monitored for these pathogens regularly. all pigs tested negative for antibodies against the iav nucleoprotein (herdchek, idexx elisa) and negative for iav rna in nasal swabs using a matrix gene real-time rt-pcr (rrt-pcr) [ ] upon arrival to the university of minnesota bsl- animal isolation units. pigs were randomly allocated into treatment groups as depicted in table and figure a . a microchip was implanted intramuscularly in the neck of each pig (lifechip ® , destron fearing, south saint paul, mn) to monitor body temperature. sixty of the pigs were vaccinated against influenza using different prime-boost vaccination protocols at and/or weeks of age. the vaccines were administered according to their labels. the com and aut vaccines were administrated intramuscularly (im) with ml per dose, while, the laiv was administrated as a single ml dose intranasally (in) for each pig. both, sixteen control pigs (include no vac/cha and no vac/no cha groups) and fourteen seeder pigs received two administrations of a saline solution intramuscularly at and weeks of age. the seeder pigs were challenged with either an h or h iav at weeks of age and served as infection sources to the vaccinated pigs and no vac/ cha pigs. each seeder pig was inoculated intratracheally and intranasally with a ml dose of × ^ tcid /ml table description of vaccination protocols applied to pigs by treatment groups in the two separate studies. i.m: intramuscular; i.n: intranasal; iav: influenza a virus. a three pigs from the no vac/no cha group were necropsied prior to challenge and the remaining pigs were necropsied at the termination of the study ( dpc challenge virus ( ml intratracheally and ml intranasally). when seeder pigs were confirmed iav positive in their nasal secretions by rrt-pcr, two seeder pigs (one h seeder and one h seeder) were commingled with the pigs in each room. there were five rooms in total for the study with each room containing ten contact pigs (two from each wiv treatment group), and two seeder pigs for a total of pigs per room ( figure b ). similarly, the two rooms in the second study had twelve total pigs per room with five pigs from laiv/com, five pigs from laiv/none and two seeder pigs ( figure c ). six pigs from no vac/no cha group served as unvaccinated negative controls and were kept in a separate room. three of the no vac/no cha pigs were euthanized at weeks of age ( days post-contact (dpc)) for histopathological evaluation and the remaining of the pigs were euthanized at weeks of age ( dpc). all the vaccines used in this study were licensed vaccines. antibiotic-antimycotic (gibco, life technologies, grand island, ny, usa)) and following a common laboratory virus isolation protocol and incubated at °c, % co for h [ ] . after that, fresh medium was added and the cells were further incubated for at least h until at least % cytopathogenic effect was observed. the media containing virus were harvested and centrifuged ( rpm, min) and viruses were titrated on mdck cells based on established protocols [ ] . the virus titer for h n challenge strain was × ^ . tcid /ml and the titer for h n challenge strain was × ^ . tcid / ml. the harvested viruses were aliquoted and stored at − °c. blood samples were collected on arrival to the animal isolation units, week after boost vaccination and at challenge. the pigs were manually restrained and to ml of blood was collected from the cranial vena cava using to gauge needles and blood collection tubes (bd vacutainer ® sst ™ , franklin lakes, nj, usa). nasal swabs were collected from pigs on arrival, before challenge and at days , , and after contact challenge with the seeder pigs using swabs (bd bbl ™ culture swabs ™ , sparks, md, usa) inserted - cm into the pig's nostrils and gently rotated. the nasal swabs were stored refrigerated until processing which took place within h of collection. all pigs were euthanized and necropsied at days postcontact (dpc) with the seeder pigs by an overdose of intravenously injected pentobarbital (fatal-plus, vortech pharmaceuticals ltd, dearborn, mi, usa). the percentage of gross lung lesions for each pig was analyzed and scored by a pathologist blinded to the treatments based on previously published protocols [ , ] . bronchoalveolar lavage fluid (balf) was collected from each pig using a saline solution as described previously [ ] (gibco, life technologies, grand island, ny, usa). an iav matrix gene real-time rt-pcr (rrt-pcr) was used to detect iav shedding in all the nasal swabs and balf samples [ ] . iav nucleic acid was extracted from the nasal swabs and balf using the magmax- viral isolation kit (applied biosystems, life technologies, carlsbad, ca, usa) based on the methods previously described [ ] . the iav rrt-pcr was performed using the agpath-id one-step rt-pcr kit (applied biosystems, life technologies, carlsbad, ca, usa) following the established protocols [ ] . a sample was considered positive by rrt-pcr if the cycle threshold (ct) value was or lower and, due to the semiquantitative nature of the rrt-pcr test, the lower the ct value, the higher the amount of viral rna detected in the sample. virus isolation was attempted on all positive nasal swabs and balf samples on mdck cells and titrated by calculating the tcid per ml [ ] . a sample was considered positive by virus titration if the virus titer was × ^ . or higher. to evaluate which strain the infected pigs were shedding, the nasal swabs and balf samples with ct value less than or equal to (based on the matrix rt-pcr described above) were selected for next generation sequencing. one step reverse transcription-pcr amplification was performed on extracted rna from selected samples by using superscript iii one-step rt-pcr system with high fidelity platinum taq dna polymerase (invitrogen, life technologies, usa) with degenerate primers ( um mbtuni- m and mbtuni- ) [ ] . the pcr product was visually verified by gel electrophoresis, the quality and quantity of rt-pcr product was checked by nanodrop (thermo fisher scientific). the pcr product was then cleaned up by qiagen qiaquick pcr purification kit (qiagen, usa). the sequencing library was prepared by using the nextera dna xt sample preparation kit (illumina, san diego, ca, usa) and quantified by using the quant-it ™ picogreen ™ dsdna assay kit (invitrogen). the barcoded libraries were pooled in equimolar concentrations and sequenced in multiplex for bp paired-end on illumina nextseq mid-output mode ( m) at the university of minnesota genomics center (umgc). the raw contigs released from umgc underwent quality assessment by fast-qc [ ] and then trimmed by trimmomatic [ ] to remove the adapter, barcode and low-quality sequences. the trimmed contigs were de novo assembled by shovill [ ] and the consensus sequences were annotated by flan [ ] which is a web based influenza annotation tool available at ncbi influenza virus resource. blood samples collected prior to challenge were tested using the hemagglutination inhibition (hi) assay according to methods previously described [ ] . before the hi assay, the serum was separated from the blood cells and the serum was treated with three parts receptor destroying enzyme (rde; denka seiken, tokyo, japan) for h at °c, followed by heat inactivation at °c for min. after the sera cooled down to room temperature, % of turkey red blood cells were added and hemadsorbed for at least min to remove nonspecific hemagglutinin inhibitors and natural serum agglutinins. the hi assay was performed using turkey red blood cells and the challenge strains (a/swine/minnesota/pah- / h n and a/swine/minnesota/ / h n ) as antigens. hi antibody titer was calculated as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. the elispot analysis of ifn-γ secreting cells was performed on peripheral blood mononuclear cells (pbmc) collected week after boost vaccination on pigs and on lymph nodes collected at necropsy. pbmcs were isolated from heparinized whole blood collected from pigs in treatment groups com/com, aut/aut, laiv/com, laiv/none and no vac/no cha prior to challenge [ ] . lymph nodes were collected during necropsy from all vaccinated pigs. the lymph nodes were crushed to separate the cells from the tissue layers and passed through a -µm mesh nylon membrane (bd falcon ). after treating them with ack lysis buffer (thermo fisher scientific, grand island, ny, usa) and washing with hank's balanced salt solution (hbss; bd falcon, franklin lakes, nj, usa), the lymph node cells were ready to proceed for elispot assay. an elispot assay to determine ifn-γ secreting cells specific for h and h challenge strains was performed as described [ ] . briefly, -well elispot filter plates (maips , millipore corp., ma, usa) were coated overnight with porcine ifn-γ capture antibody (r&d systems, minneapolis, mn, usa). freshly isolated pbmc cells ( × ^ cells per well) or lymph node cells ( × ^ cells per well) were loaded into duplicate wells and the stimulants (heat-inactivated h and h challenge strains) were also added into corresponding wells. after h incubation ( °c, % co ), the porcine ifn-γ detection antibody (r&d systems, minneapolis, mn, usa) was added to each well and the ifn-γ secretion cells were visualized using the streptavidin-ap and nbt/bcip for membranes (r&d systems, minneapolis, mn, usa) and counted with an elispot plate reader. non-specific spots detected in wells coated with mockinfected rpmi (gibco, life technologies, grand island, ny, usa) medium were subtracted from the counts of influenza-specific ifn-γ secretion cells. statistical analyses were performed by comparing results of treatment groups receiving wiv (study ) and separate analyses for comparing results from laiv groups (study ). the data from no vac/no cha pigs was summarized but not statistically analyzed. quantitative rrt-pcr, virus titers from nasal swabs, weight, body temperature and influenza-specific antibody responses were analyzed by a generalized linear mixed model (glmm) approach for repeated measures. virus titers, hi titers, and the elispot reads were log-transformed while percent lung lesions were transformed using arcsine square-roots prior to analysis. using the r software (version . . ), transformed and non-transformed data were analyzed with a model that considered the fixed effects of treatment, day, the interaction of treatmentby-day, the random effects of room and the residual error. day was considered the repeated factor. treatment and treatment-by-day were assessed at the % level. if the treatment-by-day interaction was significant then treatment comparisons were assessed for each study day separately. percent lung lesions, elispot counts and balf were analyzed using a glmm approach with a model that considered the fixed effects of treatment and the random effects of room and the residual error. binary data were analyzed with r software (version . . ) by fisher's exact test. comparisons of lsmeans for all continuous variables were performed by the tukey's multiple method at the % level of significance. lsmeans from transformed data were back-transformed (geometric means) after analyses. no pigs displayed any clinical signs of respiratory disease such as coughing or nasal discharge from the start of the study to necropsy. the average daily weight gain (adg) from challenge to necropsy for the different treatment groups was measured in grams and is summarized in figure a . no statistically significant differences in adg were observed among pigs receiving the wiv combinations (p = . ). the adg for com/com pigs was . ± . g, . ± . g for aut/aut pigs, . ± . g for aut/com pigs, . ± . g for com/aut pigs, and . ± . g for no vac/cha pigs. also, there was no statistical difference in adg for pigs receiving the live-attenuated vaccines. the adg for laiv/com pigs was . ± . g and ± . g for laiv/none pigs (p = . ). the no vac/no cha pigs had an adg of . ± . g. the mean body temperatures for each treatment group shown as mean (± sem) are summarized in figure b . we considered fever present if body temperature was greater than °c with no pigs having fever prior to the challenge. three of com/com pigs, / aut/ aut pigs, / aut/com pigs, / com/aut pigs and / no vac/cha pigs had fever after challenge. in contrast, none of the pigs receiving the laiv vaccine (laiv/com and laiv/none) and none of the pigs from no vac/no cha had fever during the study. there were no significant differences in body temperatures between any of the groups at any time-point (additional file ). there were mild gross lung lesions in all challenged pigs in all treatment groups. the range of gross lung lesions for all vaccinated pigs ranged from to %, the average gross lung lesions of pigs from each group were less than % and no significant differences were detected between treatment groups ( figure c ). the nasal mucosa and lungs are major targets for virus replication in the upper and lower respiratory tract of pigs. before commingling with vaccinated pigs, the seeder pigs were confirmed iav positive by rrt-pcr on nasal swabs at day post-challenge (ct value range . to . ) . to determine the presence and amount of virus in the respiratory tract, we collected nasal swabs at , , and days post-contact with the seeder pigs and balf samples at necropsy from all pigs. the detailed infection kinetics of the individual pigs is shown in additional file . no iav rna was detected in any of the no vac/ no cha pigs from either balf samples or nasal swabs. based on the balf samples collected from pigs receiving wiv, the least amount of virus post-contact was detected in the heterologous treatment group aut/ com (table ) . only a small quantity of iav rna was detected in the balf sample from a single pig in this treatment group (ct value = . ). the amount of viral rna detected and the number of iav rrt-pcr positive pigs in homologous treatment group aut/aut was similar to the heterologous treatment group com/aut. the wiv group with the highest amount of viral rna detected was the homologous treatment group, com/ com. nevertheless, com/com pigs still had fewer positive pigs and less virus detected in balf samples compared with the pigs from no vac/cha group. for the pigs receiving laiv, all pigs in laiv/none treatment group were positive by rrt-pcr and only half of the pigs in the laiv/com treatment group were rrt-pcr-positive, with the average ct value for balf samples from laiv/none pigs significantly lower, e.g. more iav viral rna detected, than the laiv/com pigs (p < . ). there was less viral rna detectable by rrt-pcr in the nasal cavities of pigs receiving wiv combinations and, similarly, the number of pigs positive (positive rate, pr), was also lower. for the laiv treatment groups, the laiv/com treatment had less infected pigs and significantly higher ct values in both balf samples and nasal swabs at necropsy (table ) . we performed next-generation sequencing on all nasal swabs and balf samples that tested positive by rrt-pcr to determine the h and/or h gene segment in each sample (table ) . among the balf samples collected from wiv pigs, no h or h genes were detected in the heterologous aut/com pigs. there were only h sequences detected in the balf of out of pigs from the heterologous com/aut group. for the homologous wiv pigs, both ha sequences were detected in a balf sample of out of aut/aut pigs. based on the nasal swabs collected from wiv pigs, the ha sequences detected from heterologous aut/com, com/aut and homologous com/com pigs belonged to the h subtype. in contrast, only h sequences were detected in nasal swabs from homologous aut/aut pigs. in addition, both h and h sequences were found in nasal swabs of no vac/cha pigs. among laiv administrated pigs, both h and h gene sequences were detected in pigs from laiv/none and laiv/com group no matter on nasal swabs or balf samples. furthermore, we observed the co-infection of both challenge strains happened on multiple pigs from no vac/cha and laiv/none group either based on results from nasal swabs or balf samples (additional file ). to better understand the shedding and transmission of the challenge viruses in the vaccinated pigs' lungs and nasal cavities, we performed virus titration on all the balf samples and nasal swabs collected at , and days post-contact with the seeder pigs to determine the extent of virus shedding. among the wiv treatment groups, no virus was isolated from any balf samples from heterologous prime-boost treatment pig groups com/aut and aut/com ( figure a ). only one pig in the homologous aut/aut treatment group was shedding a low quantity of virus ( . tcid /ml). however, in the homologous com/com, virus was isolated in the balf of / pigs and at higher virus concentrations than in other inactivated vaccinated groups. for the laiv treatment groups, virus was isolated from balf samples of / pigs in laiv/none group and only figure b ). pigs in no vac/cha group had significantly higher amounts of nasal virus shedding than all the wiv treatment groups on day and day post-contact. for the pigs from laiv groups, the heterologous laiv/ com treatment had significantly lower virus shedding from the nasal cavities at all selected time-points compared to that from pigs in laiv/none group. to quantify the antibody titers against h and h challenge strains induced by different vaccine primeboost combinations, the hi test was performed on the sera collected from each pig prior to challenge. consistent with the virus shedding results, the heterologous aut/com group had the highest antibody titers against both the h and h challenge strains (figures a and b) . also, pigs in all four wiv groups had significantly higher hi antibody levels against both h and h challenge strains than pigs from the no vac/ cha group. anti-h titers were significantly higher in heterologous aut/com than in the homologous prime-boost groups (e.g. com/com and aut/aut). pigs from aut/com groups had significantly higher antibody levels against the h challenge strain than pigs from com/com and com/aut groups. the laiv only group had no serum antibody responses detected against either h or h challenge strains. in contrast, pigs in the laiv/com treatment group had significantly higher antibody responses against both h and h challenge strains. we compared the frequency of ifn-γ secreting cells between groups and assessed the antigen-specific cd t cell response in pigs. the cells of lymph nodes that were collected from pigs during the necropsy were stimulated with the challenge h or h viruses and the numbers of virus-specific ifn-γ secreting cells were determined by elispot plate reader (figures c and d) . the h and h specific ifn-γ secreting cells were undetectable in no vac/no cha pigs. for the h response in the pigs in the wiv treatment groups, the ifn-γ secreting cell counts in no vac/ cha pigs were significantly higher than homologous prime-boost wiv treatment groups (com/com and aut/aut) and pigs in all four of the wiv treatment groups had similar counts of h ifn-γ secreting cells. the h ifn-γ secreting cell response was at comparable levels for all pigs in the wiv treatment groups and did not differ significantly from the no vac/ cha group. for pigs in the laiv treatment groups, the com boost vaccination preceded by laiv prime increased the ifn-γ secreting cells against h and h challenge strains, but it did not differ significantly from pigs in the treatment group receiving a single laiv vaccination. we also tested the frequency of virus-specific ifn-γ secreting cells in pbmc samples collected at week after boost vaccination from pigs in selected groups (com/com, aut/aut, laiv/ com and laiv/none) and obtained similar results between the wiv administration groups (com/com and aut/aut). however, we observed significantly increased number of h and h specific ifn-γ secreting cells in pigs from laiv/com group than pigs in laiv/none group (additional file ). the advantage of heterologous prime-boost vaccination in protecting pigs and reducing influenza infections has been illustrated in a previous study [ ] . to test whether this vaccination approach is applicable to commercial u.s. pig farms, we mimicked field conditions by using a seeder pig infection model where seeder pigs were infected with either an h or h virus and commingled with the vaccinated pigs to serve as challenge. the vaccinated pigs received different licensed multivalent vaccine combinations as prime and boost doses. our results suggested that the heterologous aut/com prime/boost vaccine combination resulted in lower numbers of infected pigs than a wiv homologous prime/boost vaccine combination when compared to no vac/cha pigs. among the laiv groups, lower infection levels were also observed in pigs receiving heterologous laiv/com prime/boost vaccination compared to a single administration of laiv. since the ability of vaccination to induce an immune response in pigs is an important indicator for vaccine efficacy, we also examined the humoral and cell-mediated immune responses for all vaccinated pigs receiving the different vaccine administrations. we found the heterologous prime-boost vaccination protocols may have expanded the antibody response to both h and h challenge strains as demonstrated by the higher hi titers especially in pigs from aut/com and laiv/com treatment groups. however, as expected we did not observe a significantly enhanced cell-meditated immunity in the wiv groups [ ] . overall the pigs in the treatment group that received the heterologous aut/com vaccination had more favorable outcomes consistent with a better response against the virus challenge than non-vaccinated pigs and compared to all the other wiv groups. the responses seen in the heterologous laiv/com treatment group were also more favorable than those responses observed compared to the treatment group receiving the single dose of laiv vaccine. compared with traditional vaccination approaches, the goal of a heterologous prime-boost vaccination approach is two-fold in that there is not only an increase in the titer and longevity of the immune responses but also an expansion of the scope of immune responses elicited through humoral and cell-mediated immune responses [ ] . as a result, heterologous prime-boost approaches have been applied against a wide range of pathogens and even complex diseases such as malaria or tuberculosis [ , ] . the heterologous prime-boost vaccination approach was first employed in a macaque model against the simian immunodeficiency virus in the s and displayed one of the most promising protection results in the early human immunodeficiency virus vaccine development effort [ ] . similar strategies to prevent influenza infections have been documented [ ] . applying heterologous prime-boost influenza regimens are thought to elicit higher levels of anti-hemagglutinin stalk antibodies which typically exhibit much broader and neutralizing activity than antibodies that bind to conventional antigenic sites on the hemagglutinin head [ ] . therefore, the heterologous prime-boost strategy may potentiate responses to suboptimal immunogens and may elicit broadly cross-reactive responses that could eliminate the need for additional vaccinations. in our study, the heterologous aut/com and laiv/com groups appeared to have the best immune responses among their comparison groups. although we did not measure antibodies against the stalk part of the ha protein, hi titers were highest for aut/com in wiv groups and laiv/com in laiv groups against both h and h challenge viruses, which resulted in less virus detected in pigs within these groups. we also quantified the t cell immune response for the vaccinated pigs using elispot to evaluate the antigen-specific cell-mediated immune responses. even though we did not observe a significant difference between different treatment groups, there were increased h -specific ifn-gamma secreting cells in lymph nodes examined from pigs in the no vac/cha group. this may reflect differences in iav exposure of pigs in the different treatment groups to the seeder pigs or individual differences in the ability of the pigs to respond to nonspecific challenges as exemplified by the higher counts of ifn-γ secretion cells in pigs in the no vac/cha group. it is also plausible that the higher counts are due to the intense reactivity of pigs' natural defenses against severe influenza infection [ ] . the amount of virus shedding is one of the key factors used to demonstrate the extent of protection against the virus challenge afforded by vaccination. since the iav can replicate in both the upper and lower respiratory tract and the lung is the target organ that correlates with disease, we performed virus detection and sequencing on nasal swabs and balf samples to evaluate not only the virus shed in nasal secretions and the amount of virus detected in the lower respiratory tract but also assessed lung pathology. diverse infection patterns of each subtype viral population were observed in pigs from homologous and heterologous wiv groups, and in multiple pigs from no vac/cha and laiv/none groups shedding both challenge viruses through the lungs and nasal cavities. in our study, we used a seeder pig model that intended to mimic transmission via nose-to-nose contact instead of direct intranasal or intratracheal inoculation of pigs with high quantities of challenge virus as these direct inoculation methods are not an accurate reflection of what occurs naturally [ , ] . furthermore, direct intratracheal challenge is a consistent factor in vaccination/challenge studies wherein enhanced respiratory tract pathology is observed [ , ] . meanwhile, high dose intranasal challenge could cause high virus nasal shedding at the next day after challenge which represents a problem for vaccine evaluation, especially when evaluating t cell-inducing vaccines which usually have poor ability to prevent the virus entry into cells [ ] . pigs from the laiv vaccine treatment groups were not housed in the same rooms as pigs in the wiv treatment groups because the laiv vaccine is shed for a short period of time after administration (https ://www.ingel vacpr ovenz a.com/addit ional -data). as a result, direct comparisons between pigs in the wiv treatment groups to pigs in the laiv treatment groups could not be made. all pigs receiving laiv vaccine were confirmed iav rrt-pcr negative on nasal swabs collected prior to challenge and the hemagglutination sequences obtained from the iav positive samples all belonged to the challenge viruses. thus, we can conclude that the virus shed after challenge is the challenge strain rather than the vaccine strain. we found the heterologous laiv/com pigs shed reduced levels of virus in both the upper and lower respiratory tract compared with laiv/none pigs. among the wiv groups, the heterologous aut/com vaccination may be said to offer the best protection since no virus was isolated from any balf samples or nasal swabs from pigs in this group. even though we did not detect significant differences in virus shedding with the com/ aut group, this group had lower hi titers compared to the aut/com pigs. although the limited number of pigs in each treatment group may have reduced the sensitivity of our study, thereby impairing our ability to detect differences between vaccination protocols, there remain questions regarding the order of the vaccine used in heterologous prime-boost vaccinations and whether the benefits imparted by vaccination are dependent on which vaccine is used as the prime and which vaccine is used as the boost. which vaccine is first and which is second may be irrelevant. if, perhaps, order does affect heterologous prime-boost vaccination performance, then this order "phenomenon" seen in our study and also present in other influenza vaccination studies [ ] may be explained by the assumption that the heterologous prime-boost favors the antibody response against the first encountered antigen(s) without impairing the immune response to the second encountered antigen(s), since in our study, the ha protein of the aut vaccine strains shared the highest amino acid identity with the ha of the challenge viruses. moreover, this is also supported by the concept of "back-boosting" which refers to the tendency of the immune system during second influenza exposures to boost the titers of antibodies against the previous encountered vaccine strains [ , ] . the variability of ha homology between the different vaccines and the challenge strains could also lead to differences on results between homologous wiv groups. however, it was not the main aim of the study to compare the differences between the homologous vaccinated groups but rather to compare as a whole the differences between homologous and heterologous vaccinated groups. nevertheless, the key question remains whether this assumption applies to any heterologous prime-boost scenario; thus, the order and delivery of multivalent vaccine combinations is still an area that needs further investigation. the seeder pig model, while closely mimicking on-farm exposure to influenza, is often challenging to perform. one difficulty was the variability encountered in virus shedding from seeder pigs for a duration of sufficient length to uniformly expose in-contact pigs. however, the effects on the overall study results due to this were mitigated given that each room contained animals from all wiv vaccination treatment groups, thereby minimizing the impact of the seeder and/or room effect. the distribution of pigs from all treatments in each of the rooms was a strength of the study and, though increasing the study complexity, was helpful in minimizing the seeder and/or room effect. control of iav in pigs is complicated, and thorough evaluation of available vaccines for pigs in a controlled vaccine/challenge experiment that mimics field conditions is necessary. to accomplish this, we chose virus challenge strains that represent the iav subtypes and clades most commonly detected in u.s. swine iav surveillance [ ] , used a seeder-pig model to simultaneously exposed pigs to both iav challenge strains, and vaccinated pigs with vaccines that are commercially licensed and ready-to-use in u.s. pig populations. in summary, we demonstrated the advantage of applying a heterologous vaccine combination using inactivated vaccines against simultaneous exposure to two iav subtypes. meanwhile, delivering a whole inactivated vaccine boost to pigs following a single priming administration of laiv also significantly improved protection that may be imparted by vaccines, as was evident in decreased virus detected and increased immune response. the potential to apply this vaccination strategy to pigs in the united states is appealing. more studies are still needed to validate the concept of 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the following people at the college of veterinary medicine, university of minnesota: jorge garrido mantilla, gustavo lopez, carlos vilalta and erika vasquez for their help in sampling the pigs. the authors also thank my yang for her laboratory assistance and data organization, and venkatramana d krishna for his help on the elispot methodology. additional file . virus detection via rrt-pcr on nasal swabs and balf samples from each individual pig before and after contact.additional file . hemagglutinin sequences detected by next generation sequencing from nasal swabs and balf from individual pigs after contact with infected seeder pigs.additional file . the number of ifn-γ specific h (a) and h (b) secreting cells in pigs from peripheral blood mononuclear cells (pbmc) collected at necropsy by treatment group. the number of ifn-γ secreting cells in each treatment group are summarized as mean ± sd. the spot counts for individual pigs are displayed as data points. the asterisks denote the significant difference (p < . ) of the number of ifn-γ secreting cell spots between groups. the dark blue, green and dark red bars and/or data points represent the pigs from the whole inactivate vaccine comparisons, negative control and live attenuate vaccine comparisons, respectively. authors' contributions mt, mc, mam and lgp conceived the study. mc and mt designed and supervised the experiments. cl performed the experiments. da and cl performed the statistical analysis. all the authors reviewed and interpreted the data. cl wrote the first draft of the manuscript. all authors contributed to the paper revision, had full access to the data. all authors read and approved the final manuscript. the authors gratefully acknowledged the funding from zoetis. all data generated or analyzed during this study are included in this published article. all protocols and procedures were approved by the university of minnesota institutional animal care and use committee (protocol id: - a) and the institutional biosafety committee (protocol id: - h). not applicable. lg., m.j., d.a. and m.m. are employed by zoetis at the time of study. all other authors declare no competing interests. key: cord- - wqtu ey authors: lee, ji eun; kye, yoon-chul; park, sung-moo; shim, byoung-shik; yoo, sungsik; hwang, eunmi; kim, hyungkuen; kim, sung-jo; han, seung hyun; park, tae sub; park, byung-chul; yun, cheol-heui title: bacillus subtilis spores as adjuvants against avian influenza h n induce antigen-specific antibody and t cell responses in white leghorn chickens date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: wqtu ey low-pathogenicity avian influenza h n remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. bacillus subtilis (b. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. the objective of the present study was to elucidate the effect of b. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus h n . herein, the adjuvanticity of b. subtilis spores in chickens was demonstrated by enhancement of h n virus-specific igg responses. b. subtilis spores enhanced the proportion of b cells and the innate cell population in splenocytes from chickens administered both inactivated h n and b. subtilis spores (spore + h n ). furthermore, the h n and spore administration induced significantly increased expression of the pro-inflammatory cytokines il- β and il- compared to that in the h n only group. additionally, total splenocytes from chickens immunized with inactivated h n in the presence or absence of b. subtilis spores were re-stimulated with inactivated h n . the subsequent results showed that the extent of antigen-specific cd (+) and cd (+) t cell proliferation was higher in the spore + h n group than in the group administered only h n . taken together, these data demonstrate that b. subtilis spores, as adjuvants, enhance not only h n virus-specific igg but also cd (+) and cd (+) t cell responses, with an increase in pro-inflammatory cytokine production. this approach to vaccination with inactivated h n together with a b. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and t cell responses against avian influenza virus. avian influenza has been a global problem not only because it infects wild and domestic birds but also because it can be transmitted to humans. one of the low-pathogenicity avian influenza viruses, h n , does not induce severe pathology in birds or humans compared to that induced by highly pathogenic viruses; however, it has been focused on for decades because of its economic damage in the poultry industry. since it was first identified in [ ] , h n has become endemic worldwide, especially in asia and africa. some countries, including china, republic of korea, and egypt, have adopted a vaccination scheme against h n in their poultry farms [ ] . however, h n outbreaks have been continuously reported even in farm animals immunized against avian influenza [ ] , implying that the current vaccination strategy is in need of advancement for improved performance. this could be due to the antigenic shift and drift of viruses, weak antigenicity of current vaccines and/or inappropriate vaccination strategy in poultry farms [ ] . with growing interest in the importance of the gut microbiota, probiotics that contain beneficial bacteria or yeast have also been tried in the domestic animal industry. a large number of field studies have shown the positive effect of probiotics on growth performance or the immune system [ , ] . in particular, bacillus subtilis, a gram-positive bacterium, is a non-pathogenic species that has also been used as a probiotic for both animals and humans as feed [ ] or health food [ ] , respectively. it is indeed classified as a generally regarded as safe microorganism by the food and drug administration. b. subtilis is an endospore-forming bacterium that can differentiate into a form of dormant spores under harsh environmental conditions, including nutrient starvation and extreme thermal changes [ ] . sporulation initiates when dna segregation is completed and concurrently with the asymmetric invagination of the membrane by forming a polar septum near one pole of the cell [ , ] . then, the immature spore stage (i.e., the forespore) is surrounded by a double membrane of the mother cell and develops into the mature spore [ ] . in previous studies, b. subtilis spores showed potential for use as an adjuvant in mice. b. subtilis spores not only enhance innate immunity that protects against respiratory infections [ ] [ ] [ ] but also induce an increase in antigen-specific antibody and t cell responses when co-administered with a soluble antigen [ ] [ ] [ ] . b. subtilis spore-induced cross-presentation in response to a co-administered antigen suggests that the spore instructs diverse antigen-specific adaptive immune responses [ , ] . other reports also suggested that genetically modified b. subtilis spores displaying antigens on their surface can enhance antibacterial or antiviral immunity [ , [ ] [ ] [ ] [ ] [ ] ]. an additional advantage of b. subtilis spores as adjuvants in influenza vaccines includes the enhanced effect of the vaccine and the reduced frequency of immunization required for the optimal immune response for full protection [ , ] . a previous study demonstrated that b. subtilis spores could be a viable vaccine adjuvant against influenza in mice [ ] , with a reservation for safety and efficacy issues for further empirical investigation. thus, we explored the ability of b. subtilis spores to influence the diversity of immune responses induced by inactivated h n avian influenza virus in chickens. specifically, we attempted to elucidate the mechanism for intrinsic induction of humoral and cell-mediated immune responses in chickens immunized with inactivated h n and b. subtilis spores as adjuvants. b. subtilis spores have been suggested as probiotics against enteric pathogens in chickens [ , ] . however, it is important to note that very few studies using b. subtilis spores as vaccine adjuvants have been performed in the poultry field. therefore, in the present study, we examined whether the b. subtilis spores work as adjuvants against influenza based on the induction of b cell and t cell responses in chickens. fertile eggs from white leghorn chickens were provided by university animal farm, college of agriculture and life sciences, seoul national university (pyeongchang, republic of korea). the eggs were incubated in a . - °c incubator (rcom, gimhae, republic of korea) for days. five chickens were allotted to each group. the care room was maintained at - °c, with % humidity under positive pressure. hatched chickens were raised under conventional conditions and were allowed free access to feed and water. the experiment was approved by the institutional animal care and use committee of seoul national university (snu- - - ). bacillus subtilis strain hb (national culture collection for pathogen, republic of korea) was spread on an agar plate containing % trypticase soy broth (tsb), . % yeast extract (ye) and . % bacto agar (all from bd biosciences, san jose, usa) and incubated at °c for h. one colony was randomly picked and inoculated in ml of % tsb and . % ye liquid medium. then, the culture was incubated for h in a shaking incubator (biofree, seoul, republic of korea) at °c until the od value reached between . and . . for sporulation, the culture was transferred to ml of autoclaved % tsb and . % ye medium containing ml of % kcl, ml of . % mgso . (h o) (ph . ), and . ml of m ca(no ) , . m mncl , and mm feso . the culture was incubated at °c for h in a shaking incubator. the cells were collected by centrifugation at × g for min, re-suspended in distilled water, and incubated at °c for h on a rocker. then, the cells were sonicated at % amplitude ( watt) for s with a . s pulse. spores, loaded on an optiprep density gradient (sigma-aldrich, st. louis, usa) with layers of %, %, and %, were centrifuged at × g for min at °c without disruption for purification. the b. subtilis spores were washed three times and re-suspended in pbs. h n influenza virus (a/chicken/korea/ / , strain ce , from prof. jae hong kim, college of veterinary medicine, seoul national university) was inactivated with formalin at °c for h at a final concentration of . % [ ] . the formalin was neutralized by the addition of nahso solution to the inactivated virus as previously described [ ] . one-week-old white leghorn chickens were immunized with phosphate-buffered saline (pbs), b. subtilis spores, inactivated h n , or both b. subtilis spores and inactivated h n in a volume of μl. for vaccination, × eid of h n viruses/ μl per animal was administered intramuscularly. for comparison with a commercial vaccine, the same dose and strain of commercial h n inactivated oil vaccine (kbnp, anyang, republic of korea) was used in the oil-vaccine group. randomly selected white leghorn chickens were allotted into five different groups (four to five animals per group) as follows: pbs control (con) as a negative control, × cfu of b. subtilis spores alone (spore), inactivated h n alone (h n ), inactivated h n together with b. subtilis spores (spore + h n ) or commercial h n oil vaccine (oil vaccine). the immunization regimen comprised two injections via the intramuscular (i.m.) route at and days of age. blood samples were collected at and days (i.e., and days old) after the last immunization from the wing vein, and serum was collected with centrifugation at × g for min at °c to analyse the antigenspecific antibody responses. spleens were collected and used for in vitro culture and flow cytometric analysis, as explained below. antigen-specific igg in serum was analysed by elisa. for h n -specific igg, μl of formalin-inactivated h n influenza a virus per well was used for coating onto a -well microplate (thermo, waltham, usa) overnight at °c. serially diluted ( -fold) sera along with controls were incubated for h at room temperature, followed by a h incubation with μl of rabbit anti-chicken igg conjugated with hrp at a : dilution (bethyl laboratories, montgomery, usa). after incubation for h at room temperature, tmb (merck, darmstadt, germany) was added until colour developed, and then the reaction was stopped by the addition of μl of n h so . absorbance was measured at nm using an elisa microplate reader (molecular devices, san jose, usa). the haemagglutination inhibition (hi) titre was determined by using chicken erythrocytes collected with alsever's solution (sigma-aldrich). serially -fold diluted sera ( μl/well) from each group of chickens (four to five animals per group) at weeks old were incubated with μl of h n virus ( hau)/well in a u-bottom -well plate (thermo) for min at room temperature. chicken erythrocytes ( μl/well) were added and incubated for min at room temperature. then, the plate was analysed to distinguish agglutinated from non-agglutinated wells, of which the highest dilution showing clear red dots was determined as the hi titre. splenocytes from -week-old chickens were stained with a mouse anti-chicken cd antibody (southern biotechnology, birmingham, usa) followed by incubation with anti-mouse igg beads (miltenyi biotec, bergisch gladbach, germany) for min. cd + t cells were isolated by macs magnetic bead sorting (miltenyi biotec) and stained with μm celltrace ™ violet (ctv) dye (invitrogen, waltham, usa) for min at °c. then, the cells were washed with pre-warmed complete medium. cd + t cells stained with ctv were cultured in anti-chicken cd and cd antibody-coated flat-bottom plates (thermo) for days. for some experiments, chicken monocyte/macrophage cells were isolated by using mouse anti-chicken monocyte/macrophage (kul ) antibodies followed by anti-mouse microbead (miltenyi biotec) and macs separators. then, the cells were stimulated with inactivated h n and/or b. subtilis spores, and the supernatants were collected after centrifugation at × g for min. the supernatant was treated with t cells together with anti-cd /cd antibodies. the cells were stained with anti-chicken cd and cd antibodies, and proliferative activity was determined by flow cytometry (facs canto ii, bd biosciences) and analysed using flowjo software (tree star, ashland, usa). division index scores were calculated manually according to the following equation suggested by flowjo software (tree star), as adopted from the previous report [ ] : where i is the generation number and n i is the number of cells in generation i. the spleen was collected, minced and filtered through a -μm nylon cell strainer (corning, new york, usa) to obtain a single cell suspension. the splenocytes were then suspended in ml of rpmi containing heat-inactivated % (vol/vol) fbs and % (vol/vol) antibiotics/antimycotic solution (all from invitrogen) and centrifuged at × g for min at °c. then, the pellet was treated with ml of ack lysing buffer (thermo) incubated for min at room temperature and centrifuged at × g for min at °c. the pellet was washed and re-suspended in medium and filtered through a -μm strainer. a single-cell suspension of total splenocytes was stained for min at °c in the dark with a combination of the following fluorochrome-conjugated monoclonal antibodies: anti-chicken cd -pacblu (ct- ), anti-cd -fitc (ct- ), anti-cd α-pe (ct- ), anti-bu- -alexa fluor ® (av ), and anti-monocyte/macrophage-pe (kul ) (southern biotechnology). an anti- aad-percp-cy . antibody was purchased from bd biosciences. after staining, the cells were washed, and the expression of surface markers was measured by flow cytometry (facs canto ii). all the flow cytometric data were analysed using flowjo software (tree star). to analyse the antigen-specific t cells in the spleen, splenocytes were collected from vaccinated chickens days after the last immunization. splenocytes were re-stimulated with hau of inactivated h n [ ] for h. then, splenocytes were stained with an anti-chicken cd (ct ) antibody (southern biotechnology). after washing with macs buffer (pbs containing . % bsa and mm edta), the cells were incubated with anti-mouse igg microbeads (miltenyi biotec) for min in the dark and centrifuged at × g for min at °c. then, the cell suspension was separated on a macs ls column that was placed in the magnetic field of a macs separator (miltenyi biotec). the magnetic fraction of positively selected cd + cells was used in the mrna experiments for ifn-γ, il- and il- . total rna was extracted from splenocytes or purified t cells using nucleozol (machery-nagel, duren, germany) according to the manufacturer's instructions. briefly, single cells of the splenocytes or t cells were treated with ml of nucleozol per × cells. total rna was isolated by the addition of μl of rnasefree water (sigma-aldrich) followed by centrifugation at × g for min. then, μl of aqueous phase was transferred into a new tube, and the same volume of isopropanol was added. next, the samples were incubated for min at room temperature for rna precipitation and centrifuged at × g for min. the rna pellet was obtained at the bottom of the tube after washing with % ethanol followed by air drying for - min and resuspension with rnase-free water. rna concentration was quantified with a nanodrop (amersham biosciences, buckinghamshire, uk) at a . subsequently, ng of purified rna was reverse transcribed to cdna using m-mlv reverse transcriptase (invitrogen) according to the manufacturer's instructions. real-time quantitative pcr was performed on cdna using a stepone plus real-time pcr system (applied biosystems, waltham, usa). sybr ® green pcr master mix was used according to the manufacturer's specification (applied biosystems). pcr was carried out in a -well reaction plate with μl of sybr ® green pcr master mix, . μl of primers, - μl of cdna template and - μl of nuclease-free h o. each reaction involved a pre-incubation at °c for min, followed by thermal cycles at °c for s, °c for s and elongation at °c for s. relative quantification of target genes was calculated using the −ΔΔct method. target gene expression was normalized to the β-actin mrna level. primer sequences used for real-time quantitative pcr (table ) were designed using ncbi primer-blast and synthesized by bioneer inc. (daejeon, republic of korea). all data are expressed as the mean values ± standard deviations (sds). for comparison of means between two groups, the data were analysed using two-tailed, paired student's t test and considered statistically significant when the p-value was less than . . for multiple group comparisons, one-way anova followed by a friedman test was applied. all statistical analyses were performed using graphpad prism (version . , graphpad software, inc., san diego, usa). to determine the adjuvant effect of b. subtilis spores on antigen-specific antibody responses, chickens were intramuscularly immunized with inactivated h n with or without b. subtilis spores according to the immunization schedule ( figure a) . none of the chickens at days after hatching showed abnormalities, signs of illness, body weight loss or death during the experimental period (data not shown). serum h n virus-specific igg production in chickens immunized with inactivated h n and b. subtilis spores (spore + h n ) was significantly higher than that of other groups at both day and day ( figure b) . to investigate the virus-specific inhibition of serum antibodies, we conducted a hi assay against h n virus with serum from day . the results showed that the hi titre of spore + h n group was significantly higher than those of the other groups ( figure c ). collectively, these results suggested that b. subtilis spores act as adjuvants in chickens, enhancing antigen-specific immune responses when immunized with the inactivated h n avian influenza virus. we next investigated the changes in immune cells in the spleens of chickens immunized with inactivated h n and/or b. subtilis spores. the total cell number of splenocytes seemed to increase slightly, but there were no significant changes among the groups (figure a) . however, administration of b. subtilis spores led to an increase in the percentage ( figure b ) and absolute number ( figure c ) of kul + monocyte/macrophage populations compared to those of these populations in the h n group. we next analysed the changes in b cells, which are major adaptive immune effectors producing antigenspecific antibodies. the percentage ( figure d ) and absolute number ( figure e ) of the bu- + b cell population were significantly increased only in the spore + h n group but not in the h n group or spore group after the second immunization. these results suggested that b. subtilis works as an adjuvant to enhance the proliferation of b cells when co-administered with the h n antigen. collectively, b. subtilis spores efficiently activated innate immune cells by themself and increased the proportion and number of b cells when administered with antigens. next, we sought to determine what kind of immunerelated genes were upregulated in response to b. subtilis spores. to examine the gene expression pattern for proinflammatory cytokines, quantitative rt-pcr analysis was conducted in splenocytes treated with h n and/ or b. subtilis spores in vitro. the expression of il- β and il- , major pro-inflammatory cytokines produced in innate immune cells, was significantly increased in splenocytes stimulated with the b. subtilis spore adjuvant ( figure a ). the mrna expression levels of baff, baff receptor (baff-r), transmembrane activator and calciummodulating cytophilin ligand interactor (taci), cd and cd l, which are responsible for the proliferation and survival signals in b cells, were higher in the spore + h n group than in the other groups ( figure b ). in addition, the mrna expression of il- and il- , which are prosurvival factors for b cells or t cells, was significantly upregulated in the spore + h n group ( figure c ). these results indicated that the expression of pro-inflammatory cytokines and key regulators of b cells was enhanced in splenocytes treated with the b. subtilis spore adjuvant. for the best t cell immunity, the combination of three components is necessary: tcr stimulation, co-stimulatory signalling and cytokines. among them, we first examined the role of cytokines induced by b. subtilis spores. to test the role of cytokines from innate immune cells, we collected the supernatant from splenocytes or monocytes/ macrophages stimulated with b. subtilis spores in vitro. cd + t cells from unimmunized chickens and labelled with ctv were treated with the supernatant and examined for proliferation [ ] . tcr and co-stimulatory signalling was induced by using anti-cd and anti-cd antibodies together with the supernatant, as shown in the experimental scheme ( figure a ). flow cytometric analyses showed a strong cd + t cell proliferation when t cells treated with the supernatant from the b. subtilis spore-treated group ( figures b and c) . similar to total splenocytes, the supernatant from kul + monocytes/macrophages stimulated with spores promoted cd + t cell proliferation (figures b and c). in addition, cd + t cells cultured in the supernatant from b. subtilis spore-treated total splenocytes or monocytes/macrophages also showed high proliferative capacity regardless of the presence of h n (figures d and e) . these results demonstrated that cytokines from innate cells induced by b. subtilis spores could further enhance the proliferation of cd + and cd + t cells. generally, oil adjuvant vaccines are used against h n in poultry farms, but their limitations still exist. since the treatment of b. subtilis spores together with inactivated h n showed an increase in kul + monocytes/ macrophages ( figure b) and b cells ( figure d ) in chickens, we compared the major immune cell changes with those of commercial h n oil vaccine-immunized chickens. chickens immunized with the commercial h n oil vaccine or spore + h n chickens showed a significantly increased percentage of the kul + monocyte/ macrophage population compared to that in the control chickens ( figure a ), yet the absolute number was significantly higher only in chickens immunized with spore + h n treatment ( figure a ). both the proportion and the absolute number of the b cell populations were significantly higher in the spore + h n group than in the oil vaccine groups (figure b ). there were no significant differences in cd + t cells among the treated groups ( figure c ), while the percentage and absolute number of the cd + t cell population were significantly higher in chickens immunized with spore + h n than in the oil vaccine group chickens ( figure d ). these findings suggested that chickens administered the spore + h n vaccine showed a higher antigen-specific immune cell population than the chickens administered the commercial oil vaccine. next, we investigated antigen-specific t cell responses in chickens immunized with inactivated h n with/without b. subtilis spores in vivo compared to those in oil vaccine-administered chickens. to investigate h n virus antigen-specific t cell activities, the expression levels of the ifn-γ, il- , and il- genes were analysed in cd + t cells isolated from immunized chickens after re-stimulation with h n . the expression levels of ifn-γ (figure a ) and il- ( figure b ) were strongly induced in t cells from chickens immunized with spore + h n treatment, while minor changes were found in other groups ( figure b ). the expression levels of il- were relatively seven-day-old chickens (n = ) were immunized twice with inactivated h n and/or b. subtilis spores at one-week intervals. single cells from splenocytes were stained with the proper combination of anti-chicken cd , bu- , and kul antibodies, and flow cytometric analysis was performed. a total splenocytes, b the percentage and c absolute number of kul + monocytes/macrophages cells, and d the percentage and e absolute number of bu- + cells were examined by using flow cytometry. to determine the significance, one-way anova followed by a friedman test corrected by dunn's multiple comparison test was performed. data are expressed as the mean values ± sds. different letters on each group denote a significant difference at p ≤ . . low in all groups ( figure c ). these results demonstrated that b. subtilis spores as adjuvants could promote stronger antigen-specific th -and th -driven immune responses against h n than a commercial oil vaccine. adjuvants are now considered as an essential component of most, if not all, inactivated virus vaccines in the poultry industry [ ] . the elucidation of the action mechanism of adjuvants has contributed greatly to the development of the appropriate adjuvants in a rational approach to modern vaccine formulation [ , ] . although adjuvants have diverse activities [ ] , the exact role of each adjuvant has not yet been well clarified, especially in chickens. oil adjuvant, one of the most commonly used adjuvants in domestic animal vaccines, is known to have safety and robust efficacy profiles, with antigen-specific antibodies induced against influenza viruses. it has also been applied to vaccines for low-pathogenicity avian influenza h n vaccines for decades [ , ] . however, h n has not been conquered worldwide, and moreover, outbreaks have been reported in animals vaccinated against h n infection. it has also been reported that oil adjuvants are not appropriate for inducing t cell responses, which are essential for memory t or b cell generation [ ] ; therefore, novel adjuvants and formulations of vaccine adjuvants are essential. in the present study, we demonstrated that b. subtilis spores could act as potential vaccine adjuvants that synergistically provide antigen-specific immune responses against the avian influenza virus h n in white leghorn chickens. our results showed that b. subtilis spores could induce the expression of the pro-inflammatory cytokines il- β and il- in innate immune cells such as monocytes and macrophages. these cytokines play a critical role not only in controlling and eliminating invading pathogens but also in provoking the activation of adaptive immune cells such as t cells, especially th and th differentiation [ , ] . it has been suggested that high expression of pro-inflammatory cytokines upon b. subtilis spore treatment is associated with improved resistance to viral infection [ ] . we showed that soluble factors such as cytokines from b. subtilis spore-treated innate immune cells can enhance the proliferation of t cells, suggesting the importance of cytokines from innate cells treated with spores that subsequently enhance antigen-specific adaptive immune responses. in the present study, upregulation of gene expression of b cell-related genes such as baff or cd l in total splenocytes after b. subtilis spore treatment showed a positive correlation with increased b cell numbers. we also showed that the proliferation of cd + and cd + t cells was increased by b. subtilis spores, which deserves further research to validate and identify the cross-presentation of exogenous antigens in antigen-presenting cells followed by activation of antigen-specific cd + t cells. on the other hand, since there is no suitable model to examine antigen-specific immune responses in chickens, we need more sophisticated in vitro and in vivo models to evaluate antigen-specific b and t cell responses. furthermore, antigen-specific t or b cell responses should also be analysed at the protein level or single-cell level using elisa or intracellular staining and flow cytometry in subsequent studies. one of the most important factors for vaccine development is probably whether the vaccine induces a longterm immunological memory response. in particular, the figure cd + and cd + t cell proliferation after tcr stimulation co-treatment with the supernatant from splenocytes or monocytes/ macrophages stimulated with inactivated h n and b. subtilis spores. a ctv-labelled purified cd + t cells were cultured in anti-cd and anti-cd antibody-coated plates with supernatants from splenocytes or monocytes/macrophages stimulated with inactivated h n and/ or b. subtilis and analysed by using flow cytometry. b-e the proliferative populations of cd + t cells (b and c) and cd + t cells (d and e) were measured by ctv histograms and division index scores. to determine the significance, one-way anova followed by a friedman test corrected by dunn's multiple comparison test was performed. data are expressed as the mean values ± sds. different letters on each group denote a significant difference at p ≤ . . induction of proper memory responses should be more important in laying hens and great grandparent stocks that generally stay alive for a longer period of time. the generation of memory responses is largely dependent on the activation of t cells, especially cd + helper t cells. most vaccine studies performed in chickens have focused only on humoral responses, including antigen-specific or neutralizing antibody responses; however, the evaluation of t cell quality has been less studied. our results showed that the commercial oil adjuvant vaccine could not induce a strong t cell response compared to that induced by h n with the b. subtilis spore adjuvant. this result is in line with a previous study that demonstrating the ability of b. subtilis spores to increase the level of t cell responses [ ] . as b. subtilis spores induced stronger recall responses than a commercial oil adjuvant-based vaccine, they may have advantages as adjuvants in terms of the generation of antigen-specific memory responses, which should be further investigated in chickens in the industrial field. as we have demonstrated in the current study, b. subtilis spores instruct th and th immune responses rather than th responses when re-stimulated with h n , suggesting that antigen-specific t cell responses could be optimal for anti-viral or anti-bacterial responses. it is important to note that the spore + h n group had much higher levels of th and th responses than the commercial oil vaccine group. th immune responses are considered to be essentially required against viral infection. il- is known to trigger autoimmune disease or anti-bacterial immune responses [ ] but is also reported to have a role in anti-viral immune responses [ ] . in the same context as our results, il- and il- β produced from innate immune cells, such as macrophages, have been reported to be required for il- production. in addition, some studies have suggested that il- β can induce both th and th responses [ , ] . we focused on systemic immune responses, but tissuespecific immune responses have also been considered a primary target for protection against influenza infection. according to mouse studies [ ] , spore adjuvants could induce lung-specific immune responses when delivered as intranasal vaccines. therefore, further studies with b. seven-day-old chickens (n = ) were immunized twice with inactivated h n and/or b. subtilis spores at one-week intervals. at one week post second immunization, single cells from splenocytes were stained with anti-kul , anti-bu- , anti-cd and anti-cd antibodies, and flow cytometry analysis was performed. the percentages and absolute numbers of a kul + monocytes/macrophage cells, b bu- + b cells, and c cd + and d cd + t cells were examined. to determine the significance, one-way anova followed by a friedman test corrected by dunn's multiple comparison test was performed. data are expressed as the mean values ± sds. different letters on each group denote a significant difference at p ≤ . . subtilis spore adjuvants should be performed not only as a mucosal adjuvant but also with challenge studies against highly pathogenic influenza while comparing with commercially available vaccine adjuvants. taken together, the current study results demonstrated that a b. subtilis spore adjuvant can effectively induce antigen-specific antibody and ifn-γ-and il- -producing t cell immune responses more potently than a traditional oil adjuvant. therefore, b. subtilis spores could be novel and potential vaccine adjuvants against h n avian influenza virus in chickens. 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synergize to enhance nitric oxide production and mrna expression of inducible nitric oxide synthase, proinflammatory cytokines and chemokines in chicken monocytes il- β, but not programed death- and programed death ligand pathway, is critical for the human th response to mycobacterium tuberculosis tc , a unique subset of cd t cells that can protect against lethal influenza challenge reciprocal developmental pathways for the generation of pathogenic effector th and regulatory t cells regulating human th cells via differential expression of il- receptor publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors gratefully acknowledge the nicem, seoul national university for technical support. the data sets used and analysed during the current study are available from the corresponding author upon reasonable request. the experiment was approved by the institutional animal care and use committee of seoul national university (snu- - - ). the authors declare that they have no competing interests. key: cord- -li sc z authors: ma, jingjiao; wu, rujuan; xu, guanlong; cheng, yuqiang; wang, zhaofei; wang, heng’an; yan, yaxian; li, jinxiang; sun, jianhe title: acetylation at k of the ns protein is important for the replication and virulence of influenza virus date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: li sc z non-structural protein (ns ) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. in this study, an acetylation modification was identified at the k residue of the ns protein of h n influenza virus. to further explore the function of the k acetylation modification of the ns protein, a deacetylation-mimic mutation (k r) and a constant acetylation-mimic mutation (k q) were introduced into the ns protein in the background of a/wsn/ h n (wsn), resulting in two mutant viruses (wsn-ns - r and wsn-ns - q). in vitro and mouse studies showed that the deacetylation-mimic mutation k r in the ns protein attenuated the replication and virulence of wsn-ns - r, while the constant acetylation-mimic mutant virus wsn-ns - q showed similar replication and pathogenicity as the wild-type wsn virus (wsn-wt). the results indicated that acetylation at k of the ns protein has an important role in the replication and virulence of influenza virus. to further explore the potential mechanism, the type i interferon (ifn-i) antagonistic activity of the three ns proteins (ns - q, ns - r, and ns -wt) was compared in cells, which showed that the k r mutation significantly attenuated the ifn-β antagonistic activity of the ns protein compared with ns -wt and ns - q. both ns -wt and ns - q inhibited the ifn-β response activated by rig-i card domain, mavs, tbk , and irf more efficiently than the ns - r protein in cells. taken together, the results indicated that acetylation at ns k is important for the ifn antagonistic activity of the ns protein and virulence of the influenza virus. influenza virus non-structural protein (ns ) is a multifunctional protein that is responsible for interacting with cellular factors to antagonize the host antiviral response during viral infection [ ] . the major role of the ns protein is inhibition of both interferon (ifn) and ifn-stimulated proteins by different mechanisms. ns inhibits the transcription of type i ifn by binding the ′ triphosphate viral double-stranded rnas generated during viral replication to prevent the recognition of viral genomic material by host pattern recognition receptors (prrs), including rig-i, dsrna-dependent protein kinase r (pkr), and ′ ′-oligoadenylate synthetase (oas)/rnase l [ ] [ ] [ ] . ns can also interact directly with rig-i in the absence of rna binding to inhibit the conformational change of rig-i required for mavs activation [ ] . moreover, ns is able to interrupt mrna maturation by inhibiting the nuclear export of host mrnas by binding to host poly(a)-binding protein ii (pabpii) and cleavage and polyadenylation specific factor (cpsf ), which are required for host mrna processing, resulting in the accumulation of ifn pre-mrnas in the nucleus of infected cells [ ] . in addition, ns also antagonizes the open access *correspondence: lijinxiang@caas.cn; sunjhe@sjtu.edu.cn shanghai key laboratory of veterinary biotechnology, key laboratory of urban agriculture (south), ministry of agriculture, school of agriculture and biology, shanghai jiao tong university, shanghai , china chengdu national agricultural science and technology center, sichuan, china full list of author information is available at the end of the article ifn signalling response by regulating other host factors, such as phosphoinositide -kinase (pi k) activity, crklike protein (crkl), and the jak-stat signalling pathway [ ] [ ] [ ] [ ] [ ] . the ns protein, typically - aa in length depending on the strain, contains four functional regions: an rna binding domain (rbd, - aa), linker region (lr, - aa), effector domain (ed, - aa), and c-terminal "tail" (ctt, - aa) [ ] . multiple basic amino acids (e.g., r, r, k, and r) in the rbd are important for rna binding activity and suppressing the activation of pkr [ , ] . the ed plays an important role by targeting multiple host factors, such as pkr, cpsf , and p β (pi k), to inhibit antiviral responses and enhance viral replication [ ] . the residue w in the ed domain is important for the dimerization of the ns protein, and the w r substitution impaired ns dimerization and attenuated the virus in vivo [ ] . in addition, residues e, d, and v play important roles in the binding of ns to cleavage and polyadenylation specificity factor (cpsf ), and mutations in those residues weaken the binding of ns to cpsf and impair the ability of the ns protein to shut off host gene expression [ , ] . post-translational modifications, such as phosphorylation, sumoylation, and acetylation, are important for protein function. phosphorylation at t, t, and t of the ns protein are important for interferon antagonistic activity and replication of human influenza virus [ , ] . sumoylation at positions and of ns are crucial for host protein expression shutoff and replication of h n influenza virus [ ] . acetylation is an important post-translational modification that occurs in two forms [ ] . one is the co/post-translational acetylation at the n α -termini of the nascent polypeptide chains [ ] . the other form is acetylation of the ε-amino group of lysine, which was first recognized in histones regulating gene translation [ ] and later was found in non-histone proteins [ ] . the acetylation status is reversible and well balanced by lysine (k) acetyltransferases (kats) and lysine deacetylases (kdacs), which are tightly regulated to perform many cellular functions [ ] . dysfunction of the acetylation machinery can inhibit protein functions and consequently lead to severe diseases [ , ] . acetylation has been found in multiple proteins of influenza viruses. acetylation was identified in the np protein of influenza virus, and deacetylation of the np protein prevented the virus from assembling functional virus particles [ ] . a histone-like sequence (histone mimic) was identified in the ns protein of influenza a h n , which contributes to suppression of the antiviral response [ ] . the n-terminal acetylation of pa-x is required for the host shutoff activity of pa-x and for viral polymerase activity [ , ] . acetylation of pa has been reported to be crucial for polymerase activity, and deacetylated pa protein restricts iav rna transcription and replication. the influenza virus haemagglutinin (ha) has three conserved cysteine residues ( , , and ) at its c terminus serving as acylation sites that are essential for the formation of fusion pores and infectivity [ ] . in the present study, an acetylation modification at k of the ns protein was identified and characterized. the results showed that the deacetylation-mimic mutation k r in the ns protein attenuated the replication and virulence of the virus in vitro and in vivo. ifn-β antagonist assays indicated that the k r mutation attenuated ifn antagonistic ability compared with the ns -wt or ns - q (constant acetylation-mimic) proteins. overall, this study indicated that acetylation at k of the ns protein plays an important role in the replication and virulence of influenza virus. madin-darby canine kidney (mdck) cells were maintained in eagle's minimal essential medium (emem, hyclone, grand island, usa) with % foetal bovine serum (fbs, gibco, grand island, usa), l-glutamine (gibco), and % antibiotic (gibco). human embryonic kidney (hek) t cells and adenocarcinomic human alveolar basal epithelial cells (a cells) were maintained in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with % fbs (gibco), l-glutamine (gibco), and % antibiotic (gibco). the virus strain a/wsn/ h n (wsn), a mouse-adapted human influenza virus, was propagated and titrated in mdck cells. to identify the putative acetylation sites in influenza viral proteins, mass spectrometry was conducted with concentrated influenza virus. briefly, the h n virus was propagated in mdck cells, and then a total of ml of virus stock was prepared. to concentrate the virus, the collected virus was pelleted by centrifugation at g for min to remove the cell debris. clarified virus supernatants were layered on a % (w/v) sucrose cushion and centrifuged at g for h. the virus pellet was suspended in water and subjected to mass spectrometry analysis performed by ptm biolabs llc (hangzhou, china). to generate mutant viruses, the site mutations k r (deacetylation-mimic mutation) and k q (constant acetylation-mimic) were introduced into the reverse genetic plasmid phw -wsn-ns by a commercial site-directed mutagenesis kit (invitrogen, grand island, usa). the mutant viruses were rescued in the background of wsn-h n virus as described previously [ ] , resulting in wsn-ns - r and wsn-ns - q viruses, and all the mutant viruses were verified by sequencing. then, the ns -wt, ns - r, and ns - q genes were amplified and cloned into the pcdna . -flag expression vector (flag-ns -wt, flag-ns - r, and flag-ns - q). the three viruses were inoculated on monolayer mdck and a cells cultured in -well plates with multiplicities of infection (mois) of . and . for each virus, respectively. each time point was set up in triplicate, and then the samples were collected at , , , and hours post-inoculation (hpi). the supernatants were titrated on mdck cells cultured in -well plates following the reed and muench method to calculate tcid /ml. the cells were collected and subjected to western blotting. briefly, the cell lysates were separated on a sodium dodecyl sulfate (sds)-polyacrylamide gel and transferred to a pvdf membrane. the membrane was blocked in pbs containing % skim milk and then incubated with rabbit anti-ns polyclonal antibody (genscript, piscataway, usa) and then with horseradish peroxidase-conjugated secondary anti-mouse antibody (thermo fisher, grand island, usa). the proteins were visualized by using an ecl kit (yeasen, shanghai, china). additionally, to determine the protein expression levels of the three ns expression plasmids, the flag-ns -wt, flag-ns - q, and flag-ns - r plasmids were transfected into t cells. forty-eight hours post-transfection, the cells were collected and subjected to western blotting. forty-eight -week-old female balb/c mice were randomly allocated into four groups, and each group contained mice. three groups were challenged with the indicated viruses, and one group was challenged with pbs as a control. the mice were inoculated with virus intranasally with . tcid of virus in µl solution under slight anaesthesia with co . the mice were monitored for body weight, clinical signs, and survival rate each day until days post-infection (dpi), and they were euthanized if they lost more than % of their original body weight. three mice from each group were euthanized at and dpi. the mouse lungs were collected for viral titration and cytokine analysis. to quantify the cytokine levels of il- β, ifn-β, and tnf-α in mouse lungs, total rna was extracted from lung tissues, reverse-transcribed and subjected to quantitative realtime polymerase chain reaction as described previously [ ] . to detect the ifn-β antagonistic ability of ns proteins, t cells were transfected with the indicated ns expression plasmids ( . μg/well) together with a plasmid expressing firefly luciferase under the control of the ifn-β promoter (pgl-ifn-β-luc, . μg/well), the renilla luciferase expressing plasmid prl-tk ( . μg/ well), and the ifn-β stimulator poly(i:c) ( . μg/well) or a plasmid expressing the active caspase recruitment domain (card) of rig-i (pcdna-rig-i . μg/well), pcdna-mavs ( . μg/well), pcdna-tbk ( . μg/well) or pcdna-irf ( . μg/well) as described previously [ ] . twenty-four hours post-transfection, the cells were lysed and subjected to a dual-luciferase reporter assay kit (promega, madison, usa). mdck cells cultured on glass slides were infected with wsn-ns -k r, wsn-ns -k q, or wsn-wt at an moi of . all cells were fixed with % paraformaldehyde (pfa) and permeabilized with . % triton x- in pbs at , , and hpi. to detect the ns proteins, the fixed cells were incubated with rabbit anti-ns polyclonal antibody (genscript), followed by fitc-conjugated antirabbit igg antibody (yeasen), and then the cells were stained with dapi. all images were obtained on a leica tcs sp confocal microscope (leica microsystems inc., buffalo grove, usa). the animal study was conducted in accordance with the guidelines of the animal care and use committee of shanghai jiao tong university, and the animal study protocols were approved by shanghai jiao tong university (approval no. ). all data were analysed using analysis of variance (two-way anova) in graph-pad prism version . (graphpad software inc., la jolla, usa); a p-value of . or less was considered significant. the mass spectrometry results showed that one acetylation modification was identified at position k of the ns protein of the wsn-wt virus (figure ). to further explore whether k is subtype-specific, we compared the ns amino acid sequences of randomly selected influenza virus strains of each endemic subtype in birds and humans from genbank. most avian h n ( %), h n ( . %), h n ( %), human h n ( . %), and human h n ( . %, isolated before ) viruses contained k in the ns genes, whereas . % of pandemic h n viruses contained r in the ns protein. these data demonstrated that the k residue is relatively conserved in influenza viruses except the pandemic h n . since the ns protein is an important virulence marker and antagonist of host innate immunity, we chose to further explore the influence of acetylated k on virus replication and virulence in this study. to mimic deacetylated lysine, a k r substitution was introduced into the ns protein, since an r substitution prevents acetylation but preserves the positive charge, and a mutant virus containing the ns -k r substitution was generated in the background of wsn virus (wsn-ns - r). moreover, to mimic constantly acetylated lysine at k of the ns protein, a k q substitution that is a known acetylation mimic was introduced into ns , resulting in a mutant virus containing ns -k q (wsn-ns - q). to determine the effect of acetylated k on virus replication, mdck and a cells were infected with wsn-wt or the two mutant virus wsn-ns - r (deacetylation mimic) or wsn-ns - q (constant acetylation mimic) at the indicated mois to obtain multicycle growth curves. all three viruses replicated efficiently in mdck and a cells, and wsn-ns - q and wsn-wt replicated to similar levels at each time point. however, the growth of the deacetylated mutant virus wsn-ns - r was significantly impaired compared with that of the other two viruses in mdck and a cells at and hpi, which indicated that the acetylated k of ns is important for virus replication in vitro at the late stage of infection (figures a and b) . similarly, ns protein levels were detected by western blotting. the results showed that the ns expression levels of wsn-ns - r were lower than those of wsn-wt and wsn-ns - q at different time points in mdck and a cells (figures c and d) . all the mice infected with viruses showed clinical signs such as ruffled fur, depression, and inappetence. the mice infected with constantly acetylated wsn-ns - q displayed more severe clinical signs and started to show mortality earlier than wsn-wt-infected mice. both wsn-ns - q and wsn-wt caused % mortality in infected mice. however, the deacetylated wsn-ns - r virus infection resulted in % mortality, and the mortality was delayed by days compared with other viruses, which indicated that the deacetylation-mimic k r substitution attenuated the wsn-ns - r virus in mice ( figures a and b) . virus titers were slightly lower in the lungs of mice infected with wsn-ns - r than in the other two groups ( figure c) . notably, significantly higher levels of ifn-β, il- β, and tnf-α mrna were detected in wsn-ns - r-infected mice than in the other two groups at dpi but not at dpi ( figures d-f) , which indicated that wsn-ns - r was less efficient at inhibiting the innate immune response than wsn-ns - q and wsn-wt at dpi in mice. to determine whether the k r and k q mutations affect the expression of the ns protein, the expression levels of flag-ns -wt, flag-ns - q, and flag-ns - r in t cells were compared. the three proteins were expressed at similar levels in transfected t cells, which indicated that the k r and k q mutations did not influence protein expression ( figure e) . the major function of the ns protein is inhibition of type i ifn induction, and the acetylated k residue is located in the effector domain of ns , which is important for its ifn antagonistic ability. to determine the effect of the acetylated k residue on ifn suppression by the ns protein, the inhibition of ifn-β promoter activity by flag-ns -wt, flag-ns - q, and flag-ns - r was evaluated. the results showed that flag-ns -wt and acetylation-mimic flag-ns - q suppressed the ifn-β promoter activity stimulated by poly(i:c) ( figure a) ; however, the deacetylation-mimic flag-ns - r protein was significantly less capable of inhibiting the activation of the ifn-β promoter compared with the acetylated ns proteins ( figure a ). this result indicated that the impaired ifn-β antagonistic ability might be responsible for the attenuation of the wsn-ns - q virus in vitro and in vivo. influenza virus infection stimulates type i ifn production by signal transduction from rig-i to tbk to irf . to further explore how the k r mutation attenuated the ifn-β antagonistic ability of the ns protein, we co-transfected ns expression plasmids, an ifn-β reporter plasmid and different type i interferon pathway components, including rig-i card, tbk , and the active form of irf , into t cells. the results showed that ns - q and ns -wt inhibited the ifn-β response stimulated by each component more efficiently than ns - r, which suggested that the acetylated k residue is important for inhibiting the ifn-β response of ns that targets factors downstream of irf or other proteins (figures b-d) . two nuclear localization signals ( - and - ) have been identified in the ns protein, which drive ns to the nucleus during the early stage of infection. to determine whether k r changes the subcellular localization of ns during infection, mdck cells were infected with the three viruses. the ns protein of wsnwt was located in the nucleus and cytoplasm of infected cells at and hpi ( figures a and b) . at the late stage of infection ( hpi), the ns protein was mainly located in the nucleus and perinuclear area of infected cells (figure c ). the ns - q protein was located in both the nucleus and cytoplasm at hpi and hpi, while it was mainly located in the perinuclear area of infected cells at hpi. in contrast, the ns - r protein accumulated mostly in the cytoplasm during the whole infection course. this result indicated that the deacetylation-mimic k r substitution retained ns protein in the cytoplasm of infected cells, suggesting that the acetylated k residue is important for the nuclear localization of the ns protein ( figures a-c ). post-translational modification is important for protein function, stability, cellular localization, and protein-protein interactions. recent studies have shown that posttranslational modifications of viral proteins modulate the virus life cycle, e.g., phosphorylation of influenza viral proteins (ns , m , and np) plays important roles in virus replication [ ] [ ] [ ] [ ] . the ubiquitination of np and m proteins is crucial for viral rna replication and the production of infectious virus particles [ ] . acetylation is an important post-translational modification in eukaryotes, but the occurrence and function of acetylation in influenza viral proteins remain largely unclear. giese et al. reported that acetylation of k, k, and k of np proteins is important for virus polymerase activity and replication [ ] . in the present study, we identified and characterized the acetylation of k in the ns protein, and the deacetylation of k q affected viral replication in cells at and hpi. the expression levels of ns -k q and ns -k r in transfected t cells were similar ( figure e ), while the ns levels in infected mdck and a cells were different ( figures c and d) . this result could be attributed to the deacetylation affecting virus replication, which resulted in low expression of ns in infected cells. moreover, the acetylation of q contributes to the ifn antagonistic ability of the ns protein. the mrna levels of ifn-β, il- β, and tnf-α in mouse lungs of the wsn-ns -k r-infected group were significantly higher than those in the other two groups at dpi, which indicated that ns - r was less efficient at inhibiting the production of innate antiviral cytokines at dpi in mice. the ns protein of influenza virus is a virulence factor that inhibits the antiviral immunity of the infected host, and c-terminal truncation has been widely used as a strategy to generate attenuated virus vaccine candidates [ ] . one mechanism used by ns to inhibit the ifn response is through direct binding and sequestration of rna as well as direct interaction with trim and complex formation with the rna sensor rig-i, resulting in inhibition of the activation of the rig-i card and hence inhibition of irf activation [ ] . the rna binding, rig-i and trim interacting domains are located in the n-terminus ( - aa) of the ns protein. however, the acetylated k is located in the ed domain, and acetylation of k may not affect the rna binding capability of the ns protein. the ns -k r substitution impaired the suppression of ifn promoter activation by poly(i:c), rig- card, tbk- , and irf , which suggested that the ns -k r substitution affected the ifn antagonism of ns either through targeting downstream of irf or a general mechanism that ns uses to inhibit ifn, such as interaction with cpsf , resulting in inhibition of the processing of mrna, including ifn mrna [ ] . notably, the cpsf protein is mainly located in the nucleus and is required for the ′ end processing of all host pre-mrnas. interestingly, the deacetylation-mimic k r substitution retained ns protein in the cytoplasm of infected cells, resulting in a possible impaired interaction between cpsf and the ns protein, subsequently leading to attenuated ifn antagonism. benjamin hale and colleagues found that the a/california/ / (h n ) virus has r in ns , and ns was unable to suppress general host gene expression. nevertheless, the rk substitution in the ns / protein restored its ability to block general gene expression and bind cpsf [ ] . this could explain why attenuation of the ifn antagonistic ability of ns -k r is independent of rig-i card, tbk- , and irf activation. in addition, anastasina et al. [ ] reported that the ns protein binds to cellular dna to block the cellular transcription of ifns and isgs; thus, ns proteins retained in the cytoplasm lose their cellular dna binding function, resulting in impaired ifn-β antagonistic ability. two known nuclear localization sequences (nlss) of ns proteins are located at the - and - positions. the - nls of the ns protein is highly conserved among influenza a virus strains [ ] . the second nls ( - ) is virus strain specific, and the pandemic h n lacks the second nls in the ns protein [ ] . single point mutations, either r a, r a, or k a, completely eliminated importin protein binding, which transports target proteins to the nucleus [ ] . notably, in this study, acetylation of k located outside of the nls affected the cellular localization of ns protein, and the deacetylation-mimic k r substitution blocked the nuclear localization of the ns protein in infected cells; however, the underlying mechanism remains unknown. interestingly, the ns -k residue is relatively conserved in most influenza viruses, except for the pandemic h n . the pandemic h n has r in ns , which causes inefficient general host gene expression shutoff, while r k restores its ability to block general host genes and bind cpsf [ ] . potentially, the pandemic h n virus might use different strategies to overcome the ifn response compared with the other influenza viruses. overall, we identified an acetylation of k of the ns protein of influenza virus, and the acetylation of k plays an important role in the cellular localization, ifn antagonistic ability, replication, and virulence of influenza virus. conformational plasticity of the influenza a virus ns protein inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus rig-i-mediated antiviral responses to single-stranded rna bearing ′-phosphates immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns genes structural basis for a novel interaction between the ns protein derived from the influenza virus and rig-i structural basis for suppression of a host antiviral response by influenza a virus influenza virus non-structural protein (ns ) disrupts interferon signaling a site on the influenza a virus ns protein mediates both inhibition of pkr activation and temporal regulation of viral rna synthesis the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the ′- ′ oligo (a) synthetase/rnase l pathway influenza a virus inhibits type i ifn signaling via nf-kappabdependent induction of socs- expression influenza a virus abrogates ifn-gamma response in respiratory epithelial cells by disruption of the jak/stat pathway the multifunctional ns protein of influenza a viruses influenza a virus virulence depends on two amino acids in the n-terminal domain of its ns protein to facilitate inhibition of the rnadependent protein kinase pkr 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synthesis acetylation and deacetylation of non-histone proteins the world of protein acetylation genetic dissection of histone deacetylase requirement in tumor cells the many roles of histone deacetylases in development and physiology: implications for disease and therapy role of influenza a virus np acetylation on viral growth and replication suppression of the antiviral response by an influenza histone mimic n-terminal acetylation by natb is required for the shutoff activity of influenza a virus pa-x hdac restricts influenza a virus by deacetylation of the rna polymerase pa subunit acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity analysis of recombinant h n wild-type and mutant viruses in pigs shows that the q l mutation in ha is important for transmission quantification of murine cytokine mrnas using real time quantitative reverse transcriptase pcr herpes simplex virus ubiquitinspecific protease ul inhibits beta interferon production by deubiquitinating traf mapping the phosphoproteome of influenza a and b viruses by mass spectrometry effects of the s residue of the h n swine influenza virus ns protein on interferon responses and virus replication ubiquitination of the cytoplasmic domain of influenza a virus m protein is crucial for production of infectious virus particles phosphorylation and dephosphorylation of threonine in nucleoprotein is crucial for the replication of influenza a virus attenuation of the virulence of a recombinant influenza virus expressing the naturally truncated ns gene from an h n equine influenza virus in mice inefficient control of host gene expression by the pandemic h n influenza a virus ns protein influenza virus ns protein binds cellular dna to block transcription of antiviral genes nuclear and nucleolar targeting of influenza a virus ns protein: striking differences between different virus subtypes influenza a h n subtype virus ns protein targets into the nucleus and binds primarily via its c-terminal nls /nols to nucleolin and fibrillarin this study was supported by the national natural science authors' contributions jm, jl, and js designed the study; jm was involved in the acquisition of data, analysis, and figure preparation; rw, gx, yc, and zw contributed to some of the laboratory experiments and data analysis; hw and yy helped revise the manuscript; jl and js supervised the study; jm drafted the original paper. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -um ds dh authors: paul, mathilde; tavornpanich, saraya; abrial, david; gasqui, patrick; charras-garrido, myriam; thanapongtharm, weerapong; xiao, xiangming; gilbert, marius; roger, francois; ducrot, christian title: anthropogenic factors and the risk of highly pathogenic avian influenza h n : prospects from a spatial-based model date: - - journal: vet res doi: . /vetres/ sha: doc_id: cord_uid: um ds dh beginning in , highly pathogenic avian influenza (hpai) h n virus spread across southeast asia, causing unprecedented epidemics. thailand was massively infected in and and continues today to experience sporadic outbreaks. while research findings suggest that the spread of hpai h n is influenced primarily by trade patterns, identifying the anthropogenic risk factors involved remains a challenge. in this study, we investigated which anthropogenic factors played a role in the risk of hpai in thailand using outbreak data from the “second wave” of the epidemic ( july to may ) in the country. we first performed a spatial analysis of the relative risk of hpai h n at the subdistrict level based on a hierarchical bayesian model. we observed a strong spatial heterogeneity of the relative risk. we then tested a set of potential risk factors in a multivariable linear model. the results confirmed the role of free-grazing ducks and rice-cropping intensity but showed a weak association with fighting cock density. the results also revealed a set of anthropogenic factors significantly linked with the risk of hpai. high risk was associated strongly with densely populated areas, short distances to a highway junction, and short distances to large cities. these findings highlight a new explanatory pattern for the risk of hpai and indicate that, in addition to agro-environmental factors, anthropogenic factors play an important role in the spread of h n . to limit the spread of future outbreaks, efforts to control the movement of poultry products must be sustained. (received july ; accepted december ) abstract -beginning in , highly pathogenic avian influenza (hpai) h n virus spread across southeast asia, causing unprecedented epidemics. thailand was massively infected in and and continues today to experience sporadic outbreaks. while research findings suggest that the spread of hpai h n is influenced primarily by trade patterns, identifying the anthropogenic risk factors involved remains a challenge. in this study, we investigated which anthropogenic factors played a role in the risk of hpai in thailand using outbreak data from the ''second wave'' of the epidemic ( july to may in the country. we first performed a spatial analysis of the relative risk of hpai h n at the subdistrict level based on a hierarchical bayesian model. we observed a strong spatial heterogeneity of the relative risk. we then tested a set of potential risk factors in a multivariable linear model. the results confirmed the role of freegrazing ducks and rice-cropping intensity but showed a weak association with fighting cock density. the results also revealed a set of anthropogenic factors significantly linked with the risk of hpai. high risk was associated strongly with densely populated areas, short distances to a highway junction, and short distances to large cities. these findings highlight a new explanatory pattern for the risk of hpai and indicate that, in addition to agro-environmental factors, anthropogenic factors play an important role in the spread of h n . to limit the spread of future outbreaks, efforts to control the movement of poultry products must be sustained. avian influenza / epidemiology / poultry farming / spatial analysis / thailand after emerging in southern china in the mid- s, the highly pathogenic avian influenza (hpai) h n virus spread across east and southeast asia, causing unprecedented epidemics in - [ ] . as of september , the virus has caused human cases, with deaths worldwide . controlling the spread of h n disease in poultry may contribute to the reduction of risk for humans [ ] by preventing the emergence of a viral form with efficient human-to-human transmission capable of triggering a global pandemic [ ] . determining the factors involved in the spread of h n in poultry and producing risk maps are critical to disease control as they would enable control measures to be targeted and surveillance in ''high-risk'' areas to be strengthened. the hpai h n virus is now well established in the poultry population in asia, where the virus has been able to maintain itself and spread as well as periodically re-emerge. the main pathways that have been identified for the spread of h n are the migration and trade of wild birds and the transport of poultry and poultry products [ ] . however, the respective roles of these pathways at the global or national scale are still unclear [ , ] . the persistence of hpai h n virus in southeast asia has been linked to a specific agro-ecosystem [ ] that associates freegrazing ducks with rice cultivation. a separate study found that the risk of hpai outbreaks was reduced in areas with agricultural activities other than rice farming [ ] . free-grazing ducks form a reservoir of hpai h n in asia [ ] and may contribute to the spread of the virus when they are moved among rice fields which also constitute a habitat for wild waterfowl [ ] . in addition to free-grazing ducks, it is likely that backyard poultry raised in low biosecurity systems and fighting cocks are involved in the diffusion of the virus [ ] ; however, their roles are still unclear. research to date suggests that the spread of hpai h n is influenced primarily by human activities related to poultry production and poultry trading [ ] , however, little is known about the underlying processes involved. live poultry markets probably play a role in the maintenance of the virus in asia [ , ] and the movement of poultry within trade chains may have facilitated the spread of the hpai h n virus. in southeast asia, thailand was affected by hpai h n early, with the first official report of poultry and human outbreaks on january . by the end of january , provinces throughout the north and several in the south experienced outbreaks in many types of poultry. the disease caused human cases from january to june . the epidemic peaked during a ''second wave'' with outbreaks in poultry. beginning in early , thai authorities implemented a control strategy based on the prohibition of vaccination and the use of preemptive culling. approximately million poultry were culled during the first wave, with stamping-out measures applied inside a -km radius around an outbreak. from july , culling was restricted to suspected farms or villages; million poultry were destroyed during the second wave of outbreaks. the movement of poultry and poultry products was also restricted around infected areas and fighting cocks and free-grazing ducks were targeted by control measures. apart from these control strategies, the thai authorities strengthened the surveillance of hpai. in addition to the passive surveillance system and routine laboratory surveillance, the government implemented an intensive survey known as the ''x-ray campaign'' in october with volunteers conducting door-to-door surveys [ ] to check poultry in every house nationwide. from mid- , the number of outbreaks in poultry decreased substantially but the occurrence of poultry outbreaks in two provinces of thailand in late indicates that the threat of hpai in thailand remains present. the fact that surveillance was strengthened in response to the large hpai epidemic makes thailand a prime place to analyse which factors play an important role in the spread of the disease. a set of environmental risk factors was identified in thailand [ ] , but aside from the findings of tiensin et al. [ ] , little has been learned about anthropogenic risk factors in the country. some aspects of the role of human activities in hpai risk were recently reported from vietnam [ ] . the discovery of hpai h n in thai poultry markets in and [ ] suggests that the hpai virus has continued to spread among poultry through trade activities despite the presence of control measures. apart from the duck-rice agro-ecosystem which has been shown to be a source of infection, the role of humans in the spread of hpai h n has not yet been fully investigated. the risk of hpai varies spatially according to the anthropogenic characteristics of the different geographical areas of interest, each characterized by a variety of human activities such as poultry farming practices, trade activities and market rules, land use and agro-ecosystems, and veterinary services structure and control. the objective of the present work was to study, through a spatial approach, the risk factors of hpai h n linked to human activities. it complements previous work by identifying the high-risk areas of hpai h n in thailand and by determining which anthropogenic factors are associated with an increased risk. the subdistrict (an administrative unit covering an average area of . km , called ''tambon'' in thaï) was used as the spatial scale of interest for the study. out of the subdistricts in thailand, we excluded from the analysis those located on islands since we assumed that they played a minor role in disease distribution. this resulted in a geo-database of subdistricts which we considered as the statistical units for the analysis. epidemiological data relevant to hpai h n outbreaks in poultry were provided by the avian influenza control center, department of livestock development (dld, bangkok, thailand), a unit in charge of surveillance and monitoring of avian influenza (ai) in poultry. since january , dld has been recording information on all poultry outbreaks confirmed by a diagnostic test. tests were carried out by diagnostic laboratories on sick or dead poultry or cloacal samples using reverse-transcriptase polymerase chain reaction and virus isolation [ ] . we restricted the study to outbreaks in chickens and ducks which occurred during the second wave of hpai because at that point both the passive and active components of the surveillance system were fully implemented to detect the disease. the data included records of laboratory-confirmed cases (flocks of affected poultry) dating from july to may . the background susceptible population was calculated for each subdistrict based on the poultry census data that dld collected during the x-ray survey of february . this was used to model the relative risk. we computed several anthropogenic factors to investigate the role of humans in disease spread since poultry product trading activities operate at different geographic scales. to take into account these different commercial activities, we built anthropogenic indicators based on the road network and human population settlements. using the human population census database of the thailand department of provincial administration , we calculated the human population density in subdistricts to study its association with the relative risk of hpai, previous papers having found differing results regarding the effect of human population density on hpai [ , ] . in addition, we computed the distance from the subdistrict to major cities (defined as having a population of or more). we believed that major cities may have played a role in disease spread due to the intensity of poultry trade in the areas surrounding them. information on the road network (primary and secondary roads, highways) was obtained from the ministry of transport, bangkok, thailand. this information made it possible to compute the road density per subdistrict (grouping primary and secondary roads) which was taken as an indicator of the intensity of the local trade of poultry products within a subdistrict. we suspected that highways played a role not only in the long-distance spread of the virus through the dispersal of infected materials, but also in the short-distance spread to subdistricts located in their vicinity. therefore, we introduced the distance of a subdistrict to the closest highway as an explanatory variable. finally, we computed the distance to the closest highway junction, which was assumed to function as a ''dissemination node'' for the hpai virus. we assumed that if the virus was transported mainly through the road network, the subdistricts located close to a highway junction were more likely to come in contact with the virus than those located further away. to take into account environmental risk factors, we used topographic data (altitude, hydrology) which were obtained from the digital database of the thailand environment research institute (bangkok). research has shown that rice-cropping intensity is a relevant risk factor in thailand and other asian countries [ ] . therefore, we included maps of rice-cropping intensity based on modis sensor images processed from satellite-based mapping algorithms developed by xiao et al. [ ] . using data from the dld poultry census, we also estimated for each subdistrict the density of animals and households raising poultry for different types of poultry: native chickens, fighting cocks, broiler and layer chickens, free-grazing ducks, and broiler and layer ducks. all data were integrated into a geographical database and geoprocessing was carried out with the spatial analyst extension of arcgis software v. disease modelling and mapping was performed for the whole of thailand at the subdistrict level. we ran two parallel models (one for chickens, one for ducks) since we assumed that the respective spatial patterns for chickens and ducks were different. we aimed to produce disease maps based on ''relative risk'', which was taken to be a ratio of the risk of hpai in a given subdistrict to the average risk nationwide. the latter was estimated from the overall number of cases and the poultry farm population in thailand. due to the widely varying number of poultry farms in each subdistrict, and because of spatial dependency between the subdistricts [ ] , we applied the hierarchical bayesian approach described by besag et al. [ ] to the hpai h n data. this method made it possible to compute area-specific relative risk estimates [ ] while considering spatial interactions through a spatial smoothing based on a gaussian auto-regressive model [ ] . we used a first order spatial interaction neighbourhood based on the contiguity between the spatial units. the original method in besag et al. [ ] uses a poisson distribution to model the occurrence of cases, which is appropriate for rare, non-contagious diseases such as cancers in humans [ ] or bovine spongiform encephalopathy in cattle [ ] . however, due to the contagiousness of hpai within each spatial unit, applying this method to hpai would result in overdispersion compared to the poisson distribution. we handled this problem by modelling the locally observed number of cases using a negative binomial distribution [ ] , an approach that has been used to model influenza by fraser et al. [ ] . monte carlo markov chain (mcmc) simulations were used to estimate the parameters of the model, including the estimate of relative risk for each spatial unit [ ] . the estimation was performed using linbugs [ ] , with million iterations, each producing a random simulation of the relative risk for all of the statistical units (e.g. subdistricts). geweke and heidelberger-welch tests were used to assess the convergence of the models [ ] . considering a long safety burn-in period, parameters were estimated from a subset of of the random simulations (with a systematic step of over to overcome auto-correlation problems). from this subset of simulations, we computed credible intervals containing % of the values of relative risk. we tested the link between the relative risk values in subdistricts on chickens and ducks using a spearman rank order correlation test. the relative risk was mapped for chickens and ducks using arcgis software v. . (esri inc.). maps made it possible to identify groups of subdistricts with either a significantly high or a significantly low risk of hpaiinfected flocks compared to the rest of the country. we aimed to identify the factors associated with the spatial risk of hpai. to do so, we constructed a linear model with fixed effects. we used the logarithm of the relative risk estimated through the bayesian approach (which modelled the exponential of relative risk values through a gaussian distribution in each statistical unit, as mentioned by besag et al. [ ] ) as the dependent variable. separate models of hpai outbreaks were constructed for the chicken and duck populations. each of the two models contained variables which included environmental, poultry farming, and anthropogenic factors. multicollinearity was investigated by checking the standard errors of regression coefficients and the variance inflation factors (vif) [ ] . the density of native chickens and the density of farms with native chickens were found to be positively correlated; consequently, only the former was introduced into the analysis. multicollinearity was finally assumed not to cause any serious problem in the model (vif values < . ) [ ] . since we expected some non-linear relationships, continuous variables were transformed into categorical data before they were entered into the model. four categories were chosen for each variable with the exception of free-grazing ducks (density of animals and density of farms) whose distribution allowed only categories. we selected the thresholds that simultaneously fitted the non-linear relationships and had a sufficient number of statistical units per category. finally, we added the level of relative risk in the duck population as a covariate in the chicken model and vice versa for the duck model. for each species, this thereby made it possible to adjust the analysis to account for the level of disease occurrence in the other species. the multivariate analysis was carried out using a stepwise backward elimination process; the significance of each variable in the full model was assessed in turn, with the least significant variable deleted and the process repeated until all of the remaining variables were significant at p value . (fisher test) [ ] . from the effect estimates computed in the final linear model, we deduced values of risk ratios (rr) and their confidence intervals ( %) for the different variables. for each variable, the reference category was defined as the category expected to be at the lowest level of risk based on our hypothesis and findings of previous studies in thailand [ , ] . although spatial dependence already had been taken into account through the spatial contiguity in the bayesian approach, we looked for any remaining spatial autocorrelation in the linear models. the semi-variograms of model residuals showed that autocorrelation may have played a role only in a very short-distance range ( m). computing the distances between each subdistrict to the next nearest subdistrict, we found that only a small portion of subdistricts (< %) actually had a chance of being influenced by their neighbours within that range. thus the likelihood that spatial autocorrelation affected the results was assumed to be low. the statistical analysis was performed using r software v. . . . figure shows the geographical distribution of hpai outbreaks in chicken and duck flocks during this time period. figure presents the spatially smoothed relative risk maps for chickens and ducks and shows that the two maps resemble each other fairly closely (spearman rho = . , p < eÀ ). the maps visually confirm the presence of a ''hot spot'' of hpai risk in the central plain of thailand where the relative risk was significantly higher than the national average (relative risk > for both chickens and ducks). for ducks, however, the high-risk area tended to extend further across the western part of the central plain of thailand. in contrast, the extreme south of thailand appeared to be a high-risk area for chickens, with values of relative risk significantly > . on the contrary, some areas were especially low-risk for both chickens and ducks despite the occurrence of outbreaks (relative risk significantly < . ), as in northeastern thailand and in the middle part of the peninsula. northern thailand had low values of relative risk (significantly < . ) only for chickens. we focussed on the rr values and highlighted the variables with high or very low rr. the estimated effects of the environmental, poultry farming and anthropogenic risk factors are displayed in tables ia, ib and ic. the level of relative risk of hpai for ducks was the main risk factor associated with the relative risk for chickens, and vice versa. apart from this, the mean number of rice crops per year was the most relevant risk factor for the relative risk of hpai for both chickens and ducks. a low average altitude in a subdistrict ( m) was also found to be a risk factor of hpai for chickens and ducks, while medium altitude was associated to rr below . a high density of free-grazing ducks appeared to be one of the main risk factors for hpai. for chickens and ducks, the hpai risk was connected more closely to animal density than to the density of farms or households with poultry. areas with a high density of broiler and layer ducks were associated with a strong increase in the relative risk for both chickens and ducks (rr > for subdistricts with more than ducks per km ). a high density of broiler and layer chickens (> chickens per km ) was found to be associated with a high risk of hpai (rr > ) only in the duck model. our results demonstrated a relationship between a high density of native chickens and a low risk of hpai h n for both chickens and ducks. to a lesser extent, a high density of fighting cocks was found to be associated with an increase in the hpai risk for both species. the role of several anthropogenic factors related to proximity to main transportation axes and major cities (fig. ) still remained strong after adjusting for the effects of the other variables. we found that a high hpai risk was strongly associated with highly-populated areas, short distances to the highway junction (< km), and a high density of roads in a subdistrict. moreover, the hpai risk decreased when the distance radius to major cities (with a human population of over ) increased. to a lesser extent, a very short distance to the closest highway (< km) was also associated significantly with a higher hpai risk for chickens and ducks. we used bayesian spatial analysis to characterise hpai risk areas in thailand during the second wave of the hpai h n epidemic and to explore the link between anthropogenic factors and the relative risk of hpai. we focussed on risk factors that contributed to the spread of hpai h n . this analysis shows that, when adjusted for the effects of environmental and poultry variables, several anthropogenic factors were significantly associated with an increased risk of hpai in both chicken and duck populations. first, we generated maps of the relative risk of hpai h n for chicken and duck flocks, and showed that the spatial pattern for chickens and ducks was similar. this indicated that chickens and ducks either infected each other or shared the same spatial source of infection. the results of the multivariate analysis suggest that both explanations may be valid. when, for example, we considered chickens, we found that the relative risk in ducks and several other risk factors were significant when adjusted for each other in the final model. furthermore, the model for chickens and the model for ducks indicated a common set of risk factors. second, our results were consistent with previous studies on ecological risk factors of hpai in thailand [ , ] . however, adding on to previous work, our analysis made it possible to identify classes of values associated with higher risk, which provides greater detail regarding the possible role of ecological risk factors. a high hpai risk was associated with a high density of free-grazing ducks (> ducks per km ), more than one rice crop per year, and a short distance to a river ( km). altitude may be considered as an indicator of other unmeasured environmental variables related to hpai risk. subdistricts with a low average altitude ( m) were associated with a high risk of hpai. the mixture of wetlands, ponds, irrigation networks and agriculture in these areas combined with intensive land use [ ] , may have constituted a favourable environment for the hpai h n virus. in contrast, subdistricts with a medium altitude ( . - m) have higher slopes and a land cover dominated by forests and permanent vegetation [ ] . medium average altitude in subdistricts associated with low rr was found to constitute a kind of protective factor regarding hpai risk. we also provide new insight into the role of factors related to poultry farming in the spread of hpai. like tiensin et al. [ ] , we found no indication that native chickens represent an increased hpai risk despite the fact that these chickens are raised in low biosecurity systems and were affected massively by the disease ( out of the chicken flocks infected during the second wave). conversely, a high density of native chickens ( - and > native chickens/km ) was associated with rr significantly below . in thailand, wet markets have always been rare [ ] and native chickens mainly are raised for family consumption using little input and involving little trading activity. this may have resulted in a protective effect against hpai in subdistricts with a high density of native chickens. these subdistricts probably were less exposed to the virus because they were not connected to trading chains which potentially spread the disease. in addition, the pre-emptive culling, which focussed in the beginning on native chickens around an outbreak, may have contributed to containing the spread within these subdistricts. fighting cocks are believed to have worsened the hpai situation in other asian countries [ ] and in thailand [ ] . the association we found between high densities of fighting cocks and hpai risk was significant but weak, as did gilbert et al. [ ] . in thailand, fighting cocks were also targeted when control measures were implemented in , with a prohibition on cockfighting, compulsory registration, and disease monitoring. given their high monetary and cultural value, roosters receive very special attention from their owners, who may have changed their practices early to protect their poultry from the disease. together, these two elements may have resulted in a decreased effect of fighting cock abundance on the hpai risk. third, we identified a new set of significant risk factors that help to refine the current understanding of the hpai h n epidemic in thailand. previous studies have suggested that broiler and layer ducks do not constitute a risk factor for hpai risk in thailand [ , ] . in contrast, we found a significantly increased risk of hpai, both in chicken and duck flocks, in areas with a high density of broiler and layer ducks. during the period studied, only a quarter of the total number of broiler and layer ducks were raised in closed facilities with high biosecurity systems [ ] . since it has been proven that farm duck breeds can shed the h n virus with minimal clinical signs [ ] , our results suggest that farm ducks may also have played the role of silent carriers during the second wave of the epidemic, contributing to the spread of the disease. in addition, an increased risk in duck flocks was shown for subdistricts with a high density of broiler and layer chickens (> chicken/km ). in thailand, broiler and layer chicken production range from large-scale industrial farms to small, family-run operations [ ] . the latter refer to small or medium-scale businesses with links to several middlemen or companies for the transformation and transportation of both farm inputs and outputs (feed, wastes, poultry products. . .) [ ] . during the second wave of the epidemic, it is possible that biosecurity rules were not applied fully throughout these complex poultry production chains, thus resulting in the spread of h n in subdistricts with a high density of broiler and layer chickens. furthermore, we identified several statistically significant relationships between indicators of human activity and the relative risk for hpai in chicken and duck populations. human population density was the only anthropogenic risk factor thus far identified in previous research in thailand. on the contrary to tiensin et al. [ ] , but in accordance with gilbert et al. [ , ] , our study found a progressive increase of hpai risk with an increase of human population density for both chickens and ducks. in addition, we showed that areas located within a short distance radius around major cities and highway junctions constitute ''hot spots'' for hpai risk. cities are characterised by highly intense poultry trade activities involving live poultry markets, food markets, slaughterhouses, and poultry plants. this intensity may have resulted in increased possibilities of virus introduction and spread in surrounding areas. if the hpai virus was transported through the road networks, the subdistricts located a short distance from the highway junction were more likely to be in contact with the virus than those situated further away. highway junctions thus may have functioned as ''dissemination nodes'' for the hpai h n virus. a significant association was identified between a high hpai risk and proximity to the closest highway. this may underline the role of the movement of poultry and poultry products during the second wave of the epidemic, not only in the long-distance spread of the hpai virus, but also in the short distance dissemination in the areas surrounding the highways. free-grazing ducks that are moved along the central plain for hundreds of kilometers and are known to be reservoirs of hpai may have contributed to the spread of the disease [ , ] . other types of poultry also were moved from farms to slaughterhouses, markets, or fighting arenas, while farm inputs and poultry products such as eggs, meat, litter, and poultry manure were transported along collection circuits. the movement of live poultry, people, and infected material may have resulted in the spread of the virus between houses through direct or indirect transmission. subdistricts with a high road density were associated with an increased risk of hpai h n . local poultry product and by-product business activities involve frequent contacts which revolve around road networks. once the hpai virus was introduced into a subdistrict, a dense road network may have facilitated its local spread. thus, while the spatial pattern of relative risk is known to be largely associated with an abundance of free-grazing ducks and rice-cropping intensity, we found that several indicators of human activities were also associated with hpai risk in thailand during the second wave of the epidemic. this suggests that in addition to the ''rice paddy-free grazing duck system'', major cities, the highway network, and local road networks may also have played key roles in the spread of hpai in thailand. through the transportation of potentially infected live poultry or contaminated poultry products, highways may have contributed to both the long distance and the local spread of the hpai h n virus. local road networks were possibly involved in short-distance spread. in addition, major cities and highway junctions may have functioned as ''dissemination nodes'' for the hpai h n virus through the intense traffic of poultry products and poultry trading activities in their surrounding areas. moreover, our results suggest that activities related to layer and broiler ducks may have played a more significant role than previously thought, as well as to a lesser extent -layer and broiler chickens. to tackle the outbreaks, thai authorities implemented different control measures which evolved over time, but the initial plan aimed to control poultry product movement countrywide. beginning in july , in addition to pre-emptive culling, the movement of poultry and avian products was restricted within a -km radius of an infected flock; these restrictions were extended during the second wave. the whole country was also zoned into areas, and poultry movements were strictly controlled through check points between zones [ ] . this helped to contain the disease from spreading countrywide. in addition to this set of control measures, and because free-grazing ducks were suspected of being h n hpai reservoirs, the thai government encouraged duck producers to change their practices from a free-grazing to a housed system. however, farmers were not able to change their practices in a short period of time [ ] . in , ducks were still allowed to graze in paddy fields, but the dld prohibited long-distance movements.the free-ranging practice became illegal in march , obliging farmers to house every duck flock [ ] . however, by this time the epidemic was already under control: the number of outbreaks had dropped from during the second wave to during the third wave ( july to november ). thus, while the housing of all free-grazing ducks took time to achieve, restrictions on the long-distance movements of free-grazing ducks had already contributed largely to limiting hpai spread in thailand. the h n virus may now be well established in different southeast asian countries. despite the implementation of control measures, it is probable that these countries will continue to face new outbreaks in poultry. the conditions under which the virus maintains itself in the environment are not well known. it is difficult to prevent virus re-emergence in possible local persistence spots, or the periodic reintroduction of the virus [ ] . controlling the disease within the poultry population is a critical issue for both the public health and agricultural economic systems. the restructuring of poultry production from open-housed to closed systems has started in thailand but the process will take time and considerable cooperative effort. therefore, to limit the number and size of future outbreaks in the poultry population, the focus of efforts should be on controlling the movement of both live poultry and avian products. poultry, pig and the risk of bse following the feed ban in france -a spatial analysis influenza virus (h n ) in live bird markets and food markets bayesian image restoration, with two applications in spatial statistics surveillance activities and 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thank the department of livestock development (dld), bangkok, thailand for its support in this research. data acquisition and integration was performed with funding from the international emerging infections program (ieip) in thailand through a dld-ieip collaborative project. we also thank the french research agency (anr) project ecoflu and the phc program which provided us additional support. drs. xiao and gilbert were supported by the grant from the us national institutes of health (r - - ). key: cord- -vwdxr bl authors: Świętoń, edyta; tarasiuk, karolina; Śmietanka, krzysztof title: low pathogenic avian influenza virus isolates with different levels of defective genome segments vary in pathogenicity and transmission efficiency date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: vwdxr bl defective interfering particles (dips) of influenza virus are generated through incorporation of highly truncated forms of genome segments, mostly those coding polymerase complex proteins (pb , pb , pa). such particles are able to replicate only in the presence of a virus with the complete genome, thus dips may alter the infection outcome by suppressing production of standard virus particles, but also by stimulating the immune response. in the present study we compared the clinical outcome, mortality and transmission in chickens and turkeys infected with the same infectious doses of h n low pathogenic avian influenza virus containing different levels of defective gene segments ( / (dvg-high) and / (dvg-low)). no clinical signs, mortality or transmission were noted in spf chickens inoculated with neither virus stock. turkeys infected with / (dvg-high) showed only slight clinical signs with no mortality, and the virus was transmitted only to birds in direct contact. in contrast, more severe disease, mortality and transmission to direct and indirect contact birds was observed in turkeys infected with / (dvg-low). apathy, lower water and food intake, respiratory system disorders and a total mortality of % were noted. shedding patterns in contact turkeys indicated more efficient within- and between-host spread of the virus than in / (dvg-high) group. sequencing of virus genomes showed no mutations that could account for the observed differences in pathogenicity. the results suggest that the abundance of dips in the inoculum was the factor responsible for the mild course of infection and disrupted virus transmission. the genome of influenza a virus consists of eight single-stranded negative-sense rna segments numbered according to their length in a descending order [ ] . the length of segments and is nucleotides (nt) and segment contains nt. they encode proteins of the polymerase complex (polymerase basic , pb ; polymerase basic , pb ; polymerase acidic, pa, respectively). the mid-length segments - code for hemagglutinin (ha), nucleoprotein (np) and neuraminidase (na). the m (matrix) and ns (non-structural) segments are nt and nt in length, respectively, and each codes for two proteins: m and m , and ns and nep [ ] . the rna segments are associated with pb , pb , pa and np forming ribonucleoprotein complexes (vrnp), which are basic replication units [ ] . replication of influenza virus is an error-prone process but apart from point mutations introduced during the synthesis of novel rna molecules, defective viral segments are also produced and assembled into virus particles [ ] . virions containing highly deleted forms of genome segments (defective viral genes-dvgs) are able to replicate only in the presence and at the expense of fully infectious virus, hence the term "defective interfering particles" (dips) [ ] . generation of dips was first described in s by von magnus, who noted that passages of non-diluted influenza virus in embryonated eggs led to a gradual decrease of the infectious titre despite an increase in the amount of viral particles [ ] . further studies showed that this phenomenon is common for rna and dna viruses passaged at high multiplicity of infection in laboratory conditions, but dips were found also in natural infections [ ] [ ] [ ] [ ] . defective genomes of influenza virus arise through deletion of a large portion in the middle part of genome segments, mostly in pb , pb and pa, while the ′ and ′ termini with packaging signals are retained [ ] . therefore, in cells co-infected with both defective and standard virus particles, the production and packaging of shortened genome segments outcompetes those of full-length [ ] . due to the ability of in vitro derived dips to interfere with replication of full virus particles and induce immune response, their potential application as antiviral and immunostimulatory agents has been studied extensively in recent years [ ] [ ] [ ] . however, the biological role of dips emerging during in vivo infection is not clearly defined. they might be a tolerable effect of rapid, but error-prone replication [ ] . otherwise, they might mitigate the disease enabling survival of the host thus favouring virus spread [ ] . several studies on influenza in humans and animals suggest the latter possibility is more likely [ ] [ ] [ ] . experiments on the effect of influenza dips in mice showed that protection from severe disease is a result of reduction of the amount of infectious virus [ ] or modulation of host immune response [ , ] . the dip-mediated stimulation of innate immunity occurs due to a preferential recognition of short viral rnas by retinoic acid inducible gene i (rig-i), one of cellular sensor of viral rna, whose activation initiates antiviral and inflammatory response [ ] . despite numerous evidence that the dips activity may reduce the disease severity and increase host survivability in mice and ferrets, there is little data on their properties during infection with avian influenza in birds. it was demonstrated that increased generation of defective particles might had contributed to the reduced virulence of a highly pathogenic h n avian influenza virus in chickens [ , ] . to evaluate the effect of dips on the course of infection with low pathogenic avian influenza virus (lpaiv), a comparison of pathogenicity of two virus stocks of h n lpaiv with different levels of defective genomes was performed in turkeys and chickens. the low pathogenic avian influenza virus a/turkey/ poland/ / (h n ) was used. this strain represented a group of lpaiv h n causing outbreaks in meat and breeder turkeys in poland in mid- s [ ] . despite low pathogenicity of the virus in experimental chickens (intravenous pathogenicity index, ivpi = . ), clinical disease was observed in the field outbreaks. respiratory symptoms were noted in meat turkeys and breeders showed also a drop in egg production [ ] . the virus stock from the th passage in embryonated specific pathogen free (spf) chicken eggs was shown to have contained a high level of defective viral gene segments (see below), therefore it was designated as / (dvg-high). to reduce the amount of defective particles from the virus stock, additional three passages were performed in spf chicken eggs using highly diluted inoculum ( - ) and short incubation periods ( - h) . the resulting virus stock was designated as / (dvglow). both isolates were characterized by hemagglutination (ha) assay and titrated in spf chicken eggs according to standard procedures [ ] . the rna was extracted from / (dvg-high) and / (dvg-low) stocks using viral mini kit (syngen, poland) following the manufacturer's instructions. the virus genome was amplified in rt-pcr using universal primers flanking all eight genome segments [ ] with a modification to improve the yield of pb , pb and pa segments [ ] . reactions were performed with super-script iii one-step rt-pcr system with platinum taq high fidelity dna polymerase (thermofisher scientific, usa). the pcr products were purified using pcr/ dna clean-up purification kit (eurx, poland) and processed with nextera xt dna library preparation kit (illumina, usa) according to the manufacturer's manual. the obtained libraries were sequenced in miseq using paired-end bp mode (illumina. usa). raw reads were cleaned using trimmomatic [ ] and aligned using bwa [ ] and a sequence of a wild-bird origin h n aiv as the reference genome. consensus sequences were generated with samtools [ ] and reads were mapped again using these sequences as references. coverage data were obtained using samtools. consensus sequences of / (dvg-high) and / (dvg-low) obtained after the second round of mapping were compared to identify any mutations that may result in an altered pathogenicity. in addition, variant calling was performed using var-scan [ ] and variants of ≥ % were included in further analysis. to assess the differences in the proportions of defective and full genome segments, a real time rt-pcr method was developed with two sets of primers and probe targeting different regions of the pb and pa genes ( table ) . the primer and probe set binding near the ′ terminus (t) of the gene allows detection of both defective and full segments, while the set targeting the internal (i) part of segment allows detection of those of full length only. reactions were performed using quantitect probe rt-pcr kit (qiagen, germany) according to the manufacturer's recommendations in an abi fast system. three biological replicates were tested for each virus stock. based on the ct values obtained with terminal and internal assays, relative differences in the amount of defective rnas between / (dvg-high) and / (dvg-low) were evaluated as previously described [ ] . briefly, a ratio (-ctt) : (-cti) was calculated and compared for both virus stocks. welch t-test was used to assess whether the differences were statistically significant with p value of . as a threshold. the defective segments were amplified with the eightsegment protocol as described above. the pcr products were separated in % agarose and a band of - bp was excised from the gel and extracted using nucleospin gel and pcr clean-up kit (macherey nagel, germany) according to the manufacturer's protocol. the purified fragments were cloned into a plasmid using topo ta cloning kit for sequencing (thermofisher scientific, usa) as per the manufacturer's manual. colonies containing plasmids with inserts were identified by colony pcr. twenty clones were sequenced using m primers and bigdye terminator v . cycle sequencing kit (thermofisher scientific, usa) in genetic analyzer. sequences of inserts were assembled and analyzed in seqscape v . (applied biosystems, usa). the experiments were performed in -week-old spf chickens and commercial turkeys. chickens were hatched from spf eggs (valo biomedia, germany) and -day-old turkey poults were purchased from a commercial hatchery. birds were reared until the age of weeks in the animal facility of the national veterinary research institute (nvri), poland. experiments for each virus stock and each species were conducted separately in the bsl + animal facility. birds were housed in open metal grid cages with feed and water provided ad libitum. to exclude previous exposure to aiv, blood samples and swabs were collected before infection and tested by serological and molecular methods, respectively. additionally, turkeys were examined for the presence of common viruses and bacteria that may exacerbate infection with aiv: mycoplasma spp., ornithobacterium rhinotracheale, turkey coronavirus, astrovirus, rotavirus, adenovirus, parvovirus, avian metapneumovirus, and reovirus (protocols available upon request) with negative results. birds were randomly divided into three groups: inoculated, direct contact (placed in the same cage) and indirect contact (placed in a neighbouring cage at a distance of approximately cm). five birds were inoculated intranasally and intraocularly with the dose of eid of either / (dvg-high) or / (dvg-low) in a total volume of . ml. at day post infection (dpi) five direct contact birds were placed in the same cage and another five birds were placed in an adjacent cage to monitor the indirect contact transmission of the virus. birds were monitored daily for the presence of clinical signs and mortality. at , , , , and dpi oropharyngeal and cloacal swabs were collected and immersed in viral transport medium (copan, italy). at dpi blood samples were collected and birds were humanely euthanized. the rna was extracted from µl of swab sample using viral rna mini kit (syngen, poland). the viral load was examined without discrimination between defective and standard genomes using primers and a probe targeting the m gene [ ] with quantitect probe rt-pcr kit (qiagen, germany). ten-fold dilutions of the virus inoculum were used to generate standard curve and calculate the equivalents of eid (eqeid ) per . ml of swab medium. statistical analysis for the corresponding / (dvg-high) and / (dvg-low) groups was performed using mann-whitney test with p < . considered statistically significant. due to the mortality in / (dvg-low) groups, comparisons were made for , , and dpi in the case of inoculated birds, , and dpi in the case of direct contact birds, and , , , for indirect contact birds. serum samples were tested in hemagglutination inhibition (hi) test using homologous h n antigen according to a standard procedure [ ] . the aiv-positive rnas from oropharyngeal swabs collected at dpi were subjected to whole genome amplification and deep sequencing as described above. additionally, sequencing of ha cleavage site (hacs) for swabs from and dpi, and a kidney sample collected from euthanized bird at dpi was performed. briefly, a fragment encompassing the hacs was amplified in rt-pcr using gk . and gk . primers [ ] with onestep rt-pcr kit (qiagen, germany). the pcr products were sequenced using bigdye terminator v . cycle sequencing kit in genetic analyzer (applied biosystems, usa). the sequences were analyzed in seqscape v . (applied biosystems, usa). titration of / (dvg-high) and / (dvg-low) showed an increase in the amount of infectious virus (from . eid / . ml to . eid / . ml, respectively), despite similar quantity of virus particles as evidenced by identical ha titre. the presence of dvgs in / (dvg-high) was confirmed in rt-pcr by poor amplification of long genome segments and presence of short pcr products of about - bp ( figure ). coverage plots obtained from deep sequencing data revealed uneven distribution of reads mapped to the pb , pb and pa segments with high coverage in the ′ and ′ termini ( figure ). the highest disproportion between the segment ends and internal part was observed for the pb gene. in the case of / (dvg-low), efficient amplification of long segments was noted ( figure ). weak bands indicative of shortened segments were also observed, but with a different length pattern than that in / (dvghigh) (figure ). deep sequencing confirmed the low level of defective segments in / (dvg-low), noticeable only in the case of pb and pa genes ( figure ). the differences in the amount of truncated forms were also identified in real time rt-pcr targeting distinct fragments of the pb and pa genes ( table ) . higher (−ctt) : (−cti) ratios were found for / (dvg-high) (p < . for pb and pa) indicating higher levels of dvgs than in / (dvg-low). sequencing of defective segments of / (dvg-high) cloned into plasmids showed that all clones contained defective forms of polymerase complex genes. several patterns of deletions were found for each segment ( figure ). the highest number of clones contained defective forms of the pb gene which showed also the highest diversity in terms of length and position of deletions ( figure ) . full-length sequences of all gene segments obtained in deep sequencing were compared to identify mutations that may affect the virus pathogenicity. four consensuslevel nonsynonymous differences between / (dvglow) and / (dvg-high) were revealed. however, all these mutations were found in / (dvg-high) as minority variants ( . to . %) ( table ) . none of these substitutions have been reported to alter aiv pathogenicity or have any specific function. additionally, analysis of minority variants present in the virus population showed low diversity in / (dvg-low). none of the polymorphic positions found in / (dvg-high) was retained in / (dvg-low) indicating that passages of / (dvg-high) in eggs using highly diluted inoculum eliminated most of viral subpopulations (additional file ). no clinical signs or mortality were observed in chickens infected with / (dvg-high) or / (dvglow). shedding and seroconversion were noted only in directly inoculated chickens in both groups, indicating a lack of transmission to direct and indirect contact birds (table ). chickens inoculated with / (dvg-high) shed the virus until dpi and in group inoculated with / (dvg-low) shedding was observed until dpi (table , additional file ). in both groups, the viral rna was detected almost exclusively in oropharyngeal swabs (only one cloacal swab positive in / (dvg-high) group at dpi). turkeys inoculated with / (dvg-high) showed slight lethargy and no mortality was observed. respiratory and cloacal shedding was noted until dpi (table , figure ). hi titres ranged from to . the virus was transmitted effectively to direct contact birds, as evidenced by high level of shedding ( figure ) and seroconversion (hi titres - ) (additional file ). poor transmission to indirect contact turkeys was observed as positive results of rt-qpcr were found only for three oropharyngeal swabs collected at dpi showing low levels of viral rna (table , figure ) and all serum samples were negative in hi test. in contrast, a severe clinical outcome was observed in group infected with / (dvg-low). first clinical signs appeared in inoculated birds at dpi and from dpi, clinical signs were observed also in both contact groups. turkeys demonstrated lethargy, reluctance to move, ruffled feathers, reduced feed and water intake, dyspnoea, nasal discharge, conjunctivitis and oedema of infraorbital sinuses. a total of birds died between and dpi, including inoculated turkeys, direct and indirect contact birds. at necropsy, oedema and congestion of kidneys were observed in most dead turkeys. congestion of lungs, small intestine, duodenum, pancreas and spleen were also noted in some birds. respiratory and cloacal shedding was observed in all turkeys, indicating efficient transmission to both direct and indirect contact groups (table , figure ). this observation was confirmed by seroconversion in all birds that survived until the end of the experiment (hi titres ranging from to ) (additional file ). there were the consensus sequence of / (dvg-high) was used as a reference. nt nucleotide. figure ). however, differences in the patterns of cloacal shedding were observed as it was noted earlier than in / (dvg-high) direct contact group. in addition, higher loads of viral rna were found in / (dvg-low) direct contact group at dpi (p < . ) and dpi (p < . ). the most prominent differences in the duration and intensity of shedding were noted for indirect contact groups. both oral and cloacal shedding in / (dvg-low) indirect contact turkeys was observed as soon as at dpi and continued until dpi (table , figure ). the differences in the level of viral rna were observed for oropharyngeal swabs at , , and dpi (p < . ) and for cloacal swabs at , and dpi (p < . ). due to the differences in the clinical manifestation of infection with / (dvg-high) and / (dvg-low) in turkeys, a possibility of transformation into highly pathogenic form was taken into consideration. to verify this possibility, samples collected at the end of the experiment were subjected to ha cleavage site sequencing. all tested samples showed typical monobasic hacs (peipkgr*glf) indicating low pathogenic phenotype. additionally, sequences generated in deep sequencing were analysed to reveal any mutation that could have an effect on the pathobiological outcome. the presence of nonsynonymous mutations that differentiated both virus stocks and those newly emerged was investigated in swabs from dpi. sequences of viruses from / (dvglow) group reflected the dominant population in the virus the number of positive samples per the number of samples tested is indicated. op oropharyngeal swab, cl cloacal swab, nt not tested. inoculum, i.e. in most birds there were no differences at the consensus level. seven birds showed a nonsynonymous mutation at the ha protein (i v) (frequency of . - . %) which was already present as a minority variant in the virus stock (table , additional file ). the / (dvg-high) group showed high variation in the frequency of the nonsynonymous mutations between birds (table , additional file ) indicating a lack of particular selection pattern. pathogenicity of avian influenza viruses depends on host-and virus-related factors. the traditional classification into low and highly pathogenic aiv is based on the result of intravenous inoculation of the virus into chickens. however, gallinaceous poultry are considered more susceptible to aiv infection than waterfowl [ ] and clinical course of lpaiv infection can be sometimes more severe than hpaiv infection in ducks or geese [ , ] . moreover, virus-specific factors can also influence pathogenicity, a feature that has been well described in gs/gd h hpaiv lineage viruses, even closely related ones [ ] . additionally, pathogenicity experiments that are not performed in specific pathogen free birds should also take into account the subclinical presence of other pathogens that may exacerbate the infection. so far, the possible variation in clinical outcome following infection with the same virus strain in the same species of birds has gained little attention even though the potential implications caused by the interference of defective viral particles on the replication of fully infectious particles have been known for a few decades [ ] . in our study we investigated the pathogenicity and transmissibility of a turkey-origin low pathogenic aiv h n strain with a high and low load of dvgs in spf chickens and in aivnegative turkeys. since the turkeys used in the study were not obtained from a specific pathogen free flock, a number of tests were carried out prior to the experiment to exclude the presence of potential subclinical infections with the most common turkey pathogens. infected birds received the same infectious dose of the virus but with different amount of dvgs. the semiquantitative analysis of defective particles was done by a combination of rt-pcr, real time rt-pcr and whole genome sequencing and indicated significantly higher amount of truncated gene segments in / (dvg-high). consequently, the infectious titre was higher in / (dvg-low). sequencing of defective segments showed patterns similar to those described previously in influenza viruses, i.e. they were generated mostly from polymerase complex genes by deletion of a large middle fragment while retaining ′ and ′ packaging signals [ , ] . no significant differences were observed in chickens as inoculated birds remained healthy, shed moderate amounts of the virus without transmission to direct and indirect contact chickens. the longer shedding duration of / (dvg-low) than / (dvg-high) might indicate slightly better replication efficiency of / (dvglow) but requires further investigations. on the other hand, the experiment in turkeys showed striking differences in pathogenicity and transmissibility between / (dvg-high) and / (dvg-low) aivs. infection of turkeys with the / (dvg-high) virus stock induced mild clinical signs with no mortality and resulted in transmission only to birds placed in direct contact. in contrast, severe respiratory and systemic disease was noted in turkeys inoculated with / (dvglow) aiv as well as in direct and indirect contact turkeys followed by mortality in all groups (cumulative mortality of %). the possibility that the severe clinical outcome observed in / (dvg-low)-infected turkeys had been caused by the transition of the virus into highly pathogenic form was ruled out by sequencing of the postpassage virus and demonstration of the typical lpaiv cleavage site. additionally, the comparison of the wholegenome sequence of the inoculum and virus excreted by birds showed no difference at the consensus level. since the amino-acid sequences of dominant populations in both virus stocks differed at four sites, analysis of polymorphisms at these positions in virus populations from swabs was also performed. turkeys infected with / (dvg-high) showed high variability in the frequency of the analysed variants, while no polymorphisms were found in turkeys infected with / (dvg-low) which reflected the homogeneity of the virus inoculum. the maintenance of both variants at each position in turkeys infected with / (dvg-high) and high betweenhost diversity in terms of frequency suggests that none of these mutations conferred any specific advantage for the virus replication efficiency or transmissibility. this allows to draw the conclusion that the difference in the amount of defective particles was the factor responsible for the observed disparities in the pathobiology of / (dvghigh) and / (dvg-low). there are two possible explanations of the significant pathobiological differences between lpaiv with high and low dips load. firstly, the production of incomplete particles at the expense of fully infectious particles led to the attenuation of clinical outcome, decline in mortality rate and reduction in transmission efficiency. there were no statistically significant differences in the amount of viral rna between inoculated birds but the earlier onset and higher level of cloacal shedding in the / (dvg-low) direct contact turkeys and high oral and cloacal shedding in the indirect contact group suggest that higher amounts of fully infectious particles were transmitted to turkeys exposed to / (dvglow) enabling more efficient and faster dissemination of virus within the host. this hypothesis is supported also by the gross lesions and high load of viral rna found in kidneys (data not shown) indicating that higher amounts of infectious particles (or the lack of the interfering activity of dips) enabled systemic spread of the virus. the second explanation is that dips trigger innate immune response at the early stage of infection. it was shown recently that defective viral genomes of human respiratory syncytial virus stimulated the antiviral response in mice and humans [ ] . it is also possible that the observed outcome is a combined effect of early antiviral response triggered by dips accompanied by the interference with the generation of viral particles with complete infectious capacities. the protective effect of dips in influenza infection has been shown in mice and ferrets [ ] [ ] [ ] [ ] and a relationship between the severity of infection and amount of dips was also identified in humans [ ] . differences in pathogenicity that could be attributed to abundance of dips were also found for h n aiv [ , ] . however, the role of dips in natural infections of avian hosts is unknown and their presence in field samples has been rarely reported [ ] . it is possible that under field conditions there are fluctuations in the proportions between quantities of complete and defective particles at flock level, leading to the alternate phases of suppression and exacerbation of clinical outcome. this hypothesis needs further verification in an experiment with a longer chain of subsequent transmissions and analysis of viral populations derived from each passage but such phenomenon could be perceived as advantageous from the perspective of survival and subsequent spread of the virus. in conclusion, to the best of our knowledge this is the first report investigating the role of aiv containing defective genomes in the modulation of disease outcome in birds under experimental conditions. significant differences observed in turkeys can result from either the suppressive effect of dips on the production of functional viral particles capable of causing disease and/or the early stimulation of innate antiviral response. the results can also have implications for the interpretation of virulence assessment results routinely conducted for aiv field isolates. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . additional file . frequency of variants found in viral populations of virus stocks and swabs collected from infected turkeys and results of quantitative analysis of virus shedding. aiv: avian influenza virus; dip: defective interering particle; dpi: day post infection; dvg: defective viral genome; eid : % embryo infectious dose; eqeid : equivalent of eid ; ha: hemagglutination; hacs: hemagglutinin cleavage site; hi: hemagglutination inhibition; hpaiv: highly pathogenic avian influenza virus; lpaiv: low pathogenic avian influenza virus; nt: nucleotide; rt-qpcr: quantitative real time rt-pcr; spf: specific pathogen free. influenza. in: swayne de (ed) diseases of poultry influenza virus rna polymerase: insights into the mechanisms of viral rna synthesis population diversity and collective interactions during influenza virus infection the defective component of viral populations incomplete forms of influenza virus functional characterization of naturally occurring variants of human hepatitis b virus containing the core internal deletion mutation defective interfering viral particles in acute dengue infections internally deleted wnv genomes isolated from exotic birds in new mexico: function in cells, mosquitoes, and mice smrt sequencing revealed the diversity and characteristics of defective interfering rnas in influenza a (h n ) virus infection influenza defective interfering viral rna is formed by internal deletion of genomic rna segment-specific noncoding sequences of 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vaccine: a novel antiviral that converts a potentially virulent infection into one that is subclinical and immunizing protection of mice from lethal influenza: evidence that defective interfering virus modulates the immune response and not virus multiplication defective interfering influenza virus confers only short-lived protection against influenza virus disease: evidence for a role for adaptive immunity in di virus-mediated protection in vivo preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing defective interfering virus associated with a/chicken/pennsylvania/ influenza virus avian influenza in poland manual of diagnostic tests and vaccines for terrestrial animals chapter single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses viral population analysis and minority-variant detection using short read next-generation sequencing trimmomatic: a flexible trimmer for illumina sequence data fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools varscan: variant detection in massively parallel sequencing of individual and pooled samples droplet digital pcr: a novel method for detection of influenza virus defective interfering particles convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc development and evaluation of a one-step real-time rt-pcr assay for universal detection of influenza a viruses from avian and mammal species identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the european union pathogenesis and pathobiology of avian influenza virus infection in birds unexpected infection outcomes of china-origin h n low pathogenicity avian influenza virus in turkeys infectivity, transmission and pathogenicity of h highly pathogenic avian influenza clade (h n and h n ) united states index viruses in pekin ducks and chinese geese different pathogenicity of two strains of clade . . . c h n highly pathogenic avian influenza viruses bearing different pa and ns gene in domestic ducks ghedin e; insight flu study group; insight flu study group ( ) sequence analysis of in vivo defective interfering-like rna of influenza a h n pandemic virus immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans the influence of defective-interfering particles of the pr- strain of influenza a virus on the pathogenesis of pulmonary infection in mice cloned defective interfering influenza virus protects ferrets from pandemic influenza a virus and allows protective immunity to be established influenza virus protecting rna: an effective prophylactic and therapeutic antiviral defective interfering virus protects elderly mice from influenza emergence of the virulence-associated pb e k substitution in a fatal human case of highly pathogenic avian influenza virus a(h n ) infection as determined by illumina ultradeep sequencing publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank krzysztof wyrostek, urszula sadurska and elżbieta juszczuk for excellent technical assistance. we are also grateful to our colleagues from the department of omics analyses for performing high-throughput sequencing. eŚ and kŚ conceived the study. kt performed animal experiments. eŚ and kt participated in laboratory tests. eŚ, kt and kŚ analysed and interpreted the results. eŚ and kŚ prepared the manuscript. all authors read and approved the final manuscript. the study was funded by the national science centre in poland (grant no. / /b/nz / ). the datasets generated in the current study are included in the article or in additional file, or are available from the corresponding author on reasonable request. the animal experiments were carried out according to the requirements and with an approval of the local ethical committee (permissions no. / and / ). not applicable. the authors declare no competing interests.received: july accepted: august key: cord- -nnorilo authors: lu, mingmin; tian, xiaowei; yang, zhang; wang, wenjuan; tian, ai-ling; li, charles; yan, ruofeng; xu, lixin; song, xiaokai; li, xiangrui title: proteomic analysis revealed t cell hyporesponsiveness induced by haemonchus contortus excretory and secretory proteins date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: nnorilo haemonchus contortus has evolved highly integrated and sophisticated mechanisms to promote coexistence with hosts. the excretory-secretory (es) products generated by this parasite contribute to the regulation of the host immune response to facilitate immune evasion and induce chronicity, but the proteins responsible for this process and the exact cellular mechanisms have yet to be defined. in this study, we identified h. contortus es proteins (hcesps) interacting with host t cells and t cell binding receptors via co-immunoprecipitation and shotgun liquid chromatography-tandem mass spectrometry analysis. based on bioinformatics analysis, we demonstrated that hcesps could inhibit t cell viability, induce cell apoptosis, suppress t cell proliferation and cause cell cycle arrest. furthermore, the stimulation of hcesps exerted critical control effects on t cell cytokine production profiles, predominantly promoting the secretion of interleukin (il)- , il- a and transforming growth factor-β and inhibiting il- , il- and interferon-γ production. collectively, these findings may provide insights into the interaction between es proteins and key host effector cells, enhancing our understanding of the molecular mechanism underlying parasite immune evasion and providing new clues for novel vaccine development. epidemiological data suggest that more than one billion people worldwide, as well as numerous groups of livestock, are infected with at least one species of gastrointestinal (gi) nematode [ ] . these parasitic species have evolved sophisticated and highly integrated mechanisms to reside in the gi tract of the hosts [ ] . gi nematodes can release certain factors, generally termed excretorysecretory (es) products or proteins, by actively exporting or passively diffusing into the host environment to ensure survival [ , ] . to date, the investigation of nematode es proteins has been incorporated into taxonomic composition analysis, immunodiagnostic applications, and vaccine development [ ] . importantly, increasing attention has been paid to the immunomodulatory properties of es proteins, with multitudinous findings demonstrating the selective immunosuppressive or regulatory effects of certain nematode products on host immune cells [ ] . the barber's pole worm, haemonchus contortus, is a voraciously blood-feeding and highly pathogenic gi nematode inhabiting the abomasum of small ruminants (mainly sheep and goats) [ ] . given its widespread occurrence and substantial morbidity among affected animals, haemonecrosis is one of the most economically important parasite diseases, representing a major constraint on the livestock industry worldwide, especially in tropical, subtropical and warm climatic zones [ ] . h. contortus is transmitted via a complex life cycle involving three open access *correspondence: lixiangrui@njau.edu.cn moe joint international research laboratory of animal health and food safety, college of veterinary medicine, nanjing agricultural university, nanjing , jiangsu, china full list of author information is available at the end of the article free-living larval stages and two parasitic stages. after oral ingestion by the host in contaminated pastures, the infective third-stage larvae (l ) moult into the parasitic fourth-stage larvae (l ) via an exsheathment process triggered by the gastric acidic environment and then develop into adults, causing severe pathology and normally inducing chronicity [ ] . unlike the defined rapid and delayed rejection of l and iga-induced hypobiosis toward l feeding, little is known about the exact molecular basis of host protective mechanisms against adult worm-mediated damage [ ] . due to anthelmintic resistance and the increasing demands for drug-free animal production [ ] , a better understanding of the mechanisms by which adult worms regulate host immune responses to promote coexistence with hosts may contribute to the exploitation of novel control strategies against h. contortus infection. importantly, accumulating evidence has revealed that an array of adult h. contortus es proteins (hcesps), for example, hco-gal-m/f [ ] , hcstp- [ ] , miro- [ ] , and hc-ak [ ] , contribute to the facilitation of immune evasion by suppressing the proliferation of host peripheral blood mononuclear cells (pbmcs) and the production of protective cytokines. similar to other gi nematodes, host cellular immunity against h. contortus infection is associated with the establishment of a type immune response characterized by the secretion of interleukin (il)- , il- and il- , as well as the development of a th -type immune response related to chronic infections [ ] . as the regulators and the regulated at the host-parasite interface, t cells play pivotal roles against gi nematode infections. however, immunosuppressed hosts cannot generate persistent and effective anti-nematode immunity clinically due to the impairment of t cell functions. for instance, cd + th responses were notably inhibited by myeloid-derived suppressor cells induced by primary heligmosomoides polygyrus (hp) infection [ ] , and hp infection could also block t cell activation by promoting p-glycoprotein activity [ ] . moreover, recent studies demonstrated that es products derived from gi nematodes contributed to suppressing host t cell responses, as exemplified by the inhibition of cd + and cd + t cell proliferation induced by ancylostoma caninum and toxocara canis es proteins [ ] . however, the exact role of t cells as putative key effector cells in h. contortus infection is still poorly understood, and the exact molecular basis of the regulation between t cells and hcesps remains to be elucidated. based on the stage-specific binding of hcesps to host pbmcs in vivo [ ] and the immunosuppressive effects of hcesps on pbmcs in vitro [ ] , the aim of this study was to characterize the interactions between hcesps and host t cells, as well as to elucidate the immunomodulatory potential of hcesps in the t cell-mediated immune response. to our knowledge, this is the first proteomics-guided comprehensive investigation of the relevance of hcesps to host key effector cells. all experimental protocols were reviewed and approved by the science and technology agency of jiangsu province (approval no. syxk (su) - ). all animal experiments were performed in strict compliance with the guidelines of the animal welfare council of china. all efforts were made to minimize the suffering of animals, and daily health checks were performed throughout the experiments. the h. contortus strain was preserved in the laboratory of veterinary parasitology at nanjing agricultural university. worms were maintained and propagated by serial passage in nematode-free goats ( - months old). the recovery procedures for the eggs and l of h. contortus were performed as previously described [ ] . sprague-dawley (sd) rats (body weight ~ g) were purchased from the experimental animal center of jiangsu, china (quality certificate: scxk - ) and were raised in a sterilized room with free access to sterilized food and water. local crossbred and healthy goats ( - months of age) were reared in individually ventilated cages to prevent accidental infection with nematodes. they were fed with hay and whole shelled corn and provided water ad libitum in pens. peripheral venous blood samples were obtained by venipuncture from these goats, and the isolation of goat pbmcs was conducted as previously described [ ] . total t cells were sorted from goat pbmcs by a magnetic-activated cell sorting (macs) system (miltenyi biotech, auburn, ca, usa) as described elsewhere [ ] . briefly, freshly isolated pbmcs were resuspended to a density of × cells/ml in phosphate-buffered saline containing mm edta and . % bovine serum albumin (bsa, sigma, st. louis, mo, usa). then, × pbmcs in µl of staining buffer were incubated with μl of mouse anti-bovine cd primary antibody (bio-rad inc, kidlington, uk), which was cross-reacted with goat t cells at room temperature for min. after two washes, × total cells in µl of staining buffer were labelled with μl of anti-fitc microbeads (miltenyi biotec) at room temperature for min. subsequently, the cell suspension was loaded on a macs ms column (miltenyi biotec) placed in the magnetic field of the macs separator (miltenyi biotec). the magnetically labelled t cells were retained in the column, while the unlabelled cells passed through the column. after removing the column from the macs separator, t cells were eluted as the positively selected cell fraction. the t cells were then resuspended to a density of × cells/ml in rpmi (gibco, grand island, ny, usa) containing u/ml penicillin and mg/ml streptomycin (gibco) and % heat-inactivated foetal calf serum (fcs, gibco). the viability of the t cells was > %, as assessed by the trypan blue exclusion test. the purity of isolated t cells was above %, as measured by flow cytometry. three biological replicates (three goats) were used in each experiment. hcesp collection was performed following standard procedures as previously described [ ] . briefly, adult worms were obtained from the abomasum of experimentally infected goats, washed with pbs containing % penicillin/streptomycin (gibco) five times, seeded into -well culture plates ( worms per well), and cultured in % ps/rpmi- (gibco) at °c for h with worm viability assessment and medium exchange every h. the supernatant containing hcesps was harvested, pooled, centrifuged, filtered, and concentrated with an amicon ultra centrifugal tube with a -kd molecular weight cutoff (merck millipore, bedford, ma, usa). hcesp concentrations were measured by the bradford assay, and hcesp samples were stored at − °c until further analysis. five biological replicates (five goats) were used for hcesp collection. to generate polyclonal antibodies against hcesps, µg of hcesps blended with freund's complete adjuvant was injected subcutaneously into sd rats for primary immunization. sd rats were later boosted four times with the same dose of hcesps mixed with freund's incomplete adjuvant at -week intervals. one week after the last injection, rat sera containing specific anti-hcesp antibodies were collected and stored at − °c for later use. hcesp samples were resolved on % sds-page gels and then electrotransferred onto nitrocellulose membranes. subsequently, the membranes were blocked with % skim (non-fat) milk in tris-buffered saline containing . % tween- (tbst) for h at room temperature before probing with the primary antibodies (anti-hcesp antibodies or normal rat igg, : in tbst) overnight at °c. after five washes, the membranes were incubated with horseradish peroxidase (hrp)-conjugated rabbit anti-goat igg (h + l) secondary antibody (sigma) in tbst ( : ) for h at °c. immunoreactions were detected using , ′-diaminobenzidine (dab, sigma) as a chromogenic substrate after - min of colour development. immunocytochemistry assays were performed to verify the interaction of hcesps with goat t cells as previously described [ ] . briefly, freshly sorted t cells ( × cells/ml) were incubated with µg/ml hcesps at °c for h. after five washes, the cells were fixed with % paraformaldehyde for min at room temperature, permeabilized by . % triton x- in pbs for min and blocked with % bsa in pbs for min to minimize background staining. subsequently, the cells were treated with rat anti-hcesp polyclonal antibody ( : ) or normal rat igg (control) in a humidified atmosphere at °c for h, followed by staining with cy -coupled secondary antibody ( : ) at room temperature for min. -( -amidinophenyl)- -indole carbamidinedihydrochloride (dapi, sigma) was used for nuclear staining. immunofluorescence-labelled cells were imaged at × magnification using a zeiss lsm laser scanning confocal microscope (zeiss, jena, germany). digital images were analysed by zen imaging software (zeiss). each experiment was performed in triplicate. co-ip assays were performed to determine the proteins of hcesps that interacted with goat t cells using protein a/g plus-agarose (santa cruz biotechnology, texas, usa) as described elsewhere [ ] . the goat t cells were incubated with µg/ml hcesps at °c with % co for h. after three washes with pbs, the cell pellets were lysed with pierce ™ ip lysis buffer (thermo fisher inc, rockford, il, usa) containing halt protease inhibitor cocktail and phosphatase inhibitor cocktail (thermo fisher). the cell lysates were collected after centrifugation at × rpm for min at °c, followed by preclearing with . μg of normal rat igg and μl of protein a/g plus-agarose at °c for min. after pretreatment, duplicate cell lysates ( mg) were incubated overnight at °c with rat anti-hcesp igg as the ip sample and normal rat igg as the negative control sample. immune complexes were precipitated with μl of protein a/g plus-agarose at °c for h. after four washes with ip lysis buffer, the immunoprecipitates were collected and resuspended in μl of × sds loading buffer. the resulting protein samples were resolved on a % sds-page gel and then transferred onto nitrocellulose membranes. the membranes were probed with rat anti-hcesp igg as the primary antibody for western blot analysis. each experiment was performed in triplicate. identification of the immunoprecipitates was performed by in-solution trypsin digestion and lc-ms/ms analysis using a q exactive instrument (thermo finnigan, san jose, ca, usa) at shanghai applied protein technology co., ltd., as previously described [ ] . based on the corresponding uniprot database of the h. contortus genome, the raw files from the lc-ms/ms tests were analysed using mascot . search software (v. . , matrix science, london, uk), specifying carbamidomethyl (c) as the fixed modification and oxidation (m) as the dynamic modification and allowing less than two missed cleavages. meanwhile, all the identified peptides were screened by false discovery rate (fdr) ≤ . and mascot score ≥ . after the multi-analysis stated above, proteins with ≥ unique peptides in three biological replicates were thought to be the identified interacting proteins. gene ontology (go) annotation of parasite and host interacting proteins was performed for functional classification based on the categories of molecular function, cellular component and biological process using blast go based on the blastp results. the effects of the hcesps on the viability of goat t cells were determined using the cell counting kit- (cck- ) assay (dojindo, kumamoto, japan) as described elsewhere [ ] . t cells activated with concanavalin a (cona, μg/ml, sigma) were treated with serial dilutions of hcesps ( , , , and µg/ml) at °c with % co in a humidified atmosphere. following h of incubation, μl of cck- reagent was added for another h of incubation in the dark. the optical density at nm (od ) was measured using a microplate reader (bio-rad, hercules, california, usa). three independent experiments were performed, and each experiment was performed in triplicate. flow cytometry analysis of apoptosis was performed as previously described using the annexin v-pe kit (bd biosciences, san jose, california, usa) [ ] . briefly, freshly isolated t cells were incubated with different concentrations of hcesps ( , , , and µg/ml) for h followed by staining with annexin v and -aminoactinomycin d ( -aad) according to the manufacturer's protocols. unstimulated goat t cells were used as a negative control. the apoptosis rate was calculated from the percentage of early (annexinv + aad − ) and late (annexinv + aad + ) apoptotic t cells. three individual experiments, each consisting of three replicates, were carried out. cell proliferation assays were performed by directly measuring dna synthesis using an alexa fluor click-it plus edu flow cytometry kit (thermo fisher) according to the manufacturer's instructions. -ethynyl- ′-deoxyuridine (edu, μm) was added to the culture medium for a -h incubation in a humidified atmosphere at °c with % co at the end of the -h coincubation ( , , , , µg/ml hcesps with t cells) period. subsequently, t cells were fixed with % paraformaldehyde in pbs and permeabilized with click-it saponin-based permeabilization and wash reagent, followed by click-it reaction to couple edu with the alexa fluor dye. after two washes with ml of % bsa in pbs, t cells were treated with -aad staining solution (bd biosciences), and standard flow cytometry methods were used to determine the percentage of s-phase cells in the population. each experiment consisting of three replicates was performed in triplicate. cell cycle analysis was performed following the manufacturer's dna staining protocol for flow cytometry. during the -h coincubation with hcesps ( µg/ml) at °c in a humidified atmosphere with % co , t cells were collected at different time points ( , , , and h), washed and fixed with ice-cold % ethanol. after incubation at − °c for h, the cells were washed twice to remove the ethanol and resuspended in pi/rnase staining buffer ( × cells/ µl, bd biosciences) for flow cytometry analysis. three independent experiments were performed, and each experiment was performed in triplicate. t cells treated with various concentrations ( , , , and µg/ml) of hcesps for h were harvested for cell apoptosis analysis, and t cells treated with µg/ ml hcesps for different durations ( , , , and h) were collected for cell cycle analysis. total rna samples were extracted, and the resulting cdna was synthesized by reverse transcription pcr in accordance with the manufacturer's specifications. transcriptional analysis of candidate genes was conducted by real-time pcr with a standard procedure on a quantstudio real time pcr system (applied biosystems, carlsbad, ca, usa) using published specific primers for endogenous reference genes and target genes (additional file ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the amplification efficiencies and correlation coefficients were verified to be stable and similar, and the relative mrna expression levels of candidate genes were calculated by the −ΔΔct method. each experiment was performed in triplicate. detection of cytokine secretion was performed using goat il- , il- , il- , il- a, interferon (ifn)-γ and transforming growth factor (tgf)-β elisa kits (mlbio, shanghai, china). fresh isolated t cells activated by cona were incubated with the presence of hcesps ( , , , and µg/ml) for h in a humidified atmosphere with % co at °c. the supernatants were collected and assayed for cytokine production according to the manufacturer's protocols. the limits of detection were between and pg/ml depending on the analytic assay. each experiment was performed in triplicate. statistical analysis by one-way anova for significant differences was performed using graphpad premier . software (graphpad prism, san diego, ca, usa). p < . was considered to be statistically significant. data are expressed as the mean ± standard deviation (sd). to identify the highly concentrated hcesps obtained via in vitro culture, ~ µg of hcesps were separated by % sds-page gels. the comus bright blue stain showed that the molecular weights of the collected hcesps ranged from ~ kd to ~ kd ( figure a, lane ) . western blot analysis revealed that rat anti-hcesp igg ( figure a , lane ) recognized all the bands of natural hcesps distributed from ~ kd to ~ kd, but the control normal rat igg ( figure a , lane ) did not recognize any band, indicating that the rat anti-hcesp igg was specific to the hcesps. freshly sorted goat t cells with a purity of > % were obtained by positive selection and used for functional and immunological studies ( figure b) . the interactions of hcesps with goat t cells in vitro were investigated by immunocytochemistry assays. the immunostaining results showed that intense red fluorescence resulting from tagging hcesp proteins with specific rat anti-hcesp igg was detected in hcesp-pretreated t cells ( figure c, panel b) , indicating the cytomembrane and cytoplasmic localization of hcesps. however, no red fluorescence was observed in either unstimulated cells labelled with rat anti-hcesp igg ( figure c, panel a) or hcesp-pretreated cells labelled with normal rat igg ( figure c, panel c) . based on the positive results of the immunostaining assay, co-ip assays were conducted in hcesp-stimulated t cells. the immune complexes resolved on % sds-page gels were extracted from the interacting proteins using rat anti-hcesp igg ( figure d , lane ) or normal rat igg ( figure d , lane ) as the ip antibody. consistent with the observation from coomassie brilliant blue staining, western blot analysis demonstrated that the interacting proteins were present in rat anti-hcesp igg-precipitated immune complexes, as indicated by the multiple detected bands ranging from ~ kd to ~ kd ( figure d , lane ). however, no significant protein bands, except those for the heavy and light chains of normal rat igg, were detected in normal rat igg-precipitated immune complexes (figure d, lane ) . the immunoprecipitates detected by lc-ms/ms analysis were identified by extraction and quantification of the peptides obtained by digestion. a total of matched es proteins with unique peptides and t cell binding partners are given in additional file and additional file , respectively. among the interacting es proteins, a variety of enzymes, namely, hydrolase, proteases, acyltransferase, kinases, phosphatases, and lipases, were identified. meanwhile, protein transporters, ion-binding proteins, conserved regulatory proteins such as ubiquitin and ras, and conserved structural proteins such as histones, actins, collagen and actin-binding proteins were identified. furthermore, proteins with unknown functions, as well as novel proteins that are yet to be annotated, were also detected in this study (additional file ). importantly, the goat fasl protein was confirmed as being present among the t cell binding proteins (additional file ). the association of fasl with its receptor fas may trigger the transmembrane signalling of an apoptotic pathway that plays a vital role in cellular development and immune regulation [ ] . blast go was used to identify the go terms for the es proteins and t cell binding receptors. of these es proteins, were annotated into subcategories of cellular component terms. in contrast, proteins were enriched in subcategories of biological process terms, and proteins were assigned to subcategories of molecular function terms (figure a) . consistent with previous proteomic analyses [ , , ] , serine-threonine/tyrosine protein kinases and histidine/tyrosine phosphatases were enriched in protein kinase/phosphatase activity subcategories in this study. both protein kinases and phosphatases are key regulators of cellular functions and are particularly prominent in signal transduction and coordination involved in the cell cycle, cell survival, protein-protein interaction, and inflammation [ , ] . notably, . % of the identified es proteins (n = ) were enriched in binding activity terms, namely, ion binding, substrate compound binding, and nucleic acid binding. proteins involved in these functions are normally associated with energy metabolism, signalling, and transcription [ ] [ ] [ ] . as external stimuli, these exogenous proteins may bind to host t cells, interfere with the intracellular homoeostatic balance and disrupt multitudinous cellular functions. additionally, among the go terms of t cell binding proteins, cellular component terms were allocated to the extracellular space (n = ) and integral component of membrane (n = ) subcategories. biological process terms were allocated to the dna repair (n = ), apoptotic process (n = ) and immune response (n = ) subcategories, and molecular function terms were grouped into the dna binding (n = ) and atp binding (n = ) subcategories (figure b ). in particular, as go annotation revealed that t cell binding receptors were enriched in biological process terms associated with cell apoptosis and immunomodulation, which were mainly attributed to the functional annotation of fasl, it is likely that the external stimuli of hcesps played a pivotal role in the regulation of t cell survival and growth, as well as the t cell immune response. to verify the new clues from go annotation indicating that hcesp stimuli might affect t cell growth and survival, we further investigated the biological effects of hcesps on the viability of goat t cells. consistent with this hypothesis, our data showed that the viability of goat t cells was significantly inhibited by treatments with μg/ml (p < . ), μg/ml (p < . ), μg/ml (p < . ) and μg/ml (p < . ) hcesps ( figure a) . furthermore, an annexin v-pe/ -aad dual staining kit was used to test the pro-apoptotic effects of hcesps on t cells in this study. flow cytometry analysis demonstrated that treatment with hcesps dramatically promoted t cell apoptosis in a dose-dependent manner compared to the control group (p < . ) ( figure b ). concurrently, transcriptional analysis of key genes in fas-mediated apoptotic signal pathways further confirmed the induction of t cell apoptosis by hcesps. treatment with ≥ μg/ml hcesps significantly upregulated the mrna transcripts of fasl (p < . ), fas (p < . ), fadd (p < . ), bid (p < . ), caspase (p < . ), caspase (p < . ) and caspase (p < . ) ( figure c ). taken together, these data revealed that hcesp stimuli induced t cell apoptosis via the upregulation of the transcription of several essential genes in the apoptosis pathway. hcesps significantly inhibited t cell viability. cell viability tests were performed by cck- incorporation, and the cell viability index was determined by setting the od values of the control group as %. b flow cytometry analysis of t cell apoptosis in response to hcesps. apoptosis of t cells was determined by staining with annexin v-pe and -aad. the apoptosis rate was calculated from the percentage of early (annexinv + aad − ) and late (annexinv + aad + ) apoptotic t cells. c the mrna transcripts of candidate genes in t cells stimulated with hcesps. the results presented here are representative of three independent experiments. the data are presented as the mean ± sd, minimum to maximum; *p < . , **p < . , ***p < . , ****p < . vs the control group. as apoptosis, proliferation and the cell cycle are interconnected cellular movements, we next explored the influence of hcesp stimuli on the cell proliferation and cell cycle of host t cells. in a dose-dependent manner, treatment with hcesps significantly inhibited the proliferation of t cells in vitro, as indicated by the decreasing percentage of cells in s phase compared with control cells (p < . ) ( figure a ). given that the treatment with μg/ml hcesps had significant biological effects on cell viability, apoptosis and proliferation, as well as the transcription of certain key genes, we next treated t cells with µg/ml hcesps for cell cycle determination. flow cytometry analysis with pi staining revealed that hcesp stimuli induced cell cycle arrest at g phase in a time-dependent manner, as indicated by the increasing percentage of cells in g phase (p < . ) and the decreasing percentage of cells in s phase (p < . ) (figure b) . consistent with the above findings, real-time pcr analysis of key genes in g /s checkpoints and g /m checkpoints showed that the transcription of ccnd (p < . ), ccne (p < . ), cdk (p < . ), cdk (p < . ) and cdk (p < . ) was significantly downregulated, whereas no significant transcriptional changes in ccnb or cdk were observed ( figure c ). in addition, the transcription of essential genes in the akt/pkb pathway that regulate cell proliferation and cell growth was also detected in this study. the transcripts of foxo (p < . ), p (p < . ) and p (p < . ) were dramatically enhanced, while akt transcription was significantly suppressed (p < . ) ( figure c ). collectively, hcesp stimuli restrained t cell proliferation and caused t cell cycle stalling at the g phase. to investigate the modulatory effects of hcesps on t cell cytokine production, the secretion of il- , il- , il- , il- a, ifn-γ, and tgf-β was examined by elisa in this study. the results showed that goat t cells exposed to hcesps changed their cytokine production profile ( figure ). the production of il- was predominantly inhibited by stimulation with μg/ml (p < . ), μg/ml (p < . ), μg/ml (p < . ) and μg/ ml (p < . ) hcesps. il- a secretion was significantly promoted by treatment with μg/ml (p < . ), μg/ml (p < . ) and μg/ml (p < . ) hcesps. meanwhile, treatment with μg/ml and μg/ml hcesps dramatically inhibited il- (p < . and p < . , respectively) and ifn-γ production (p < . and p < . , respectively). importantly, a high dose of hcesps ( μg/ ml) promoted the secretion of il- (p < . ) and tgf-β (p < . ) ( figure ). taken together, the results show that hcesp stimuli mainly played an immunosuppressive role in the t cell immune response via alteration of cytokine secretion profiles. for decades, significant efforts have been made to identify the composition and structure of hcesps from different developmental stages to better understand the pathophysiology and develop novel controls. yatsuda et al. identified adult hcesps recognized by anti-h. contortus serum for the first time [ ] , whereas wang et al. recently characterized unique proteins in es products from different life-cycle stages [ ] . here, we identified adult es proteins that interacted with goat t cells through co-ip and lc-ms/ms analysis. notably, a cascade of proteolytic peptidases, including aspartic peptidases and metallopeptidases, was identified from these interacting es proteins and is postulated to play crucial roles in larval development, adult survival and reproduction via the digestion or degradation of host haemoglobin [ , ] . similarly, comparable expression of these peptidase family members in es products was reported in a series of blood-feeding nematodes, such as a. caninum [ ] , necator americanus [ ] , and ancylostoma ceylanicum [ ] . simultaneously, other essential molecules, e.g., c-type lectins (clecs) and venom allergen-like proteins (vals), were detected in the current study. as members of the lectin superfamily, clecs engage in a diverse set of immune regulation processes by glycan binding and are as indispensable to nematode survival as the secretome [ , ] . given their wide distribution and potential binding with the host, clecs have been speculated to be immunomodulators in parasite-host interactions or parasitic immune evasion [ ] . in addition, recent studies reported abundantly secreted vals in es products that caused severe damage to host tissue, favouring the establishment of persistent infections during parasitism, and vals could inhibit innate immune responses and remodel the extracellular matrix [ ] . in this study, our preliminary bioinformatics analysis revealed that hcesp stimuli might affect t cell growth and survival, based on the identification of fasl, a binding ligand of fas. apoptosis can be induced through the activation of death receptors, including fas, tnfαr, dr , dr , and dr , by their respective ligands [ ] . consistent with the go annotation, our data proved that hcesp stimuli dramatically inhibited t cell viability and induced cell apoptosis. generally, death receptor ligands initiate signalling via receptor oligomerization, which in turn results in the recruitment of specialized adaptor proteins and activation of caspase cascades [ ] . regarding fas-mediated signalling, fas trimerization resulting from binding to fasl leads to recruitment of the initiator caspase- via the adaptor protein fadd, inducing caspase- oligomerization and activation via autocatalysis. subsequently, activated caspase- stimulates apoptosis by directly cleaving and activating caspase- or alternatively cleaving bid, which is a pro-apoptotic bcl- family protein. then, truncated bid (tbid) translocates to mitochondria, facilitating the release of cytochrome c to activate caspase- and caspase- sequentially [ , ] . in this study, hcesps exerted modulatory effects on the transcript levels of several essential genes in the fas-mediated death receptor pathway. the mrna transcription of fasl, fas, fadd, bid, caspase- , caspase- and caspase- was notably upregulated due to hcesp stimulation, indicating a potential mechanism of hcespinduced intrinsic and extrinsic apoptosis of t cells. cell apoptosis, the cell cycle, and cell proliferation are fundamental and ultimately linked processes. analogous to its pro-apoptotic effects, hcesp stimuli repressed cell proliferation in a dose-dependent manner. in eukaryotic cells, cell cycle progression is controlled by the g /s checkpoint via cdk / -cyclin d and cdk -cyclin e kinase complexes and the g /m checkpoint via the cyclin b-cdc (cdk ) complex [ , ] . here, pi/rnase staining results revealed that hcesp stimuli induced cell cycle arrest at the g phase in a time-dependent manner. furthermore, the commitment of t cells to enter from the g /s phase into g /m phase was prevented by hcesps through the downregulation of ccnd , cdk / , ccne , and cdk transcription. the serine/ threonine kinase akt contributes to cell proliferation by phosphorylating the cdk inhibitors p and p , both of which have direct inhibitory modification effects on cdk [ ] . akt also has multistep inhibitory modifying effects on cyclin d via gsk- β [ ] . importantly, akt is a major mediator of cell survival through direct inhibition of pro-apoptotic proteins such as bad or inhibition of pro-apoptotic signals generated by transcription factors such as foxo [ ] . based on these associations, we found that akt transcription was significantly suppressed, whereas p , p and foxo transcription was strongly promoted, in hcesp-treated t cells. given that foxo induces apoptosis by upregulating the pro-apoptotic molecule fasl and cdk directly phosphorylates foxo [ ] , the associations among fasl, foxo , cdks, cyclin proteins and akt represent complex regulation by hcesp stimuli that can act via the alteration of mrna transcription of key genes in the akt/pkb pathway to mediate cell cycle arrest and apoptosis. collectively, this could be the mechanism by which hcesps regulate cell apoptosis, the cell cycle and the proliferation of goat t cells. however, due to the lack of available goat immune reagents, we only checked the expression of several key molecules at the transcription level in this study. details regarding the regulation of these molecules at the protein level, along with the associated pathways, merit further investigation. as there is currently no literature revealing the physiological dose range of hcesps, we employed serial dilutions of hcesps ( , , , and µg/ml) for functional studies and demonstrated that µg/ml was the best reference concentration that had a significant impact on cell viability, apoptosis, proliferation and the cell cycle. however, under the circumstance of natural infection, whether the accumulation of hcesps in h. contortus-infected goats could reach this relevant dose ( µg/ml) to induce notable suppression effects on t cells is still unclear, and further efforts are needed to validate this hypothesis. generally, immunomodulation by es proteins of parasitic helminths has several predominant features: mediating th responses as exemplified by the secretion of il- ; inducing the generation of anti-inflammatory cytokines, including il- and tgf-β; inhibiting lymphocyte activation; blocking pro-inflammatory and th cytokines such as il- and ifn-γ; and regulating treg and th responses [ , ] . consistent with these findings, hcesps significantly suppressed the secretion of il- , il- and ifn-γ, indicating that hcesp stimuli exerted critical control effects on th and th responses. although the reduced il- , il- and ifn-γ secretion might be associated with the inhibition of cell viability and proliferation, we could not precisely determine il- /il- /ifn-γ production on a per cell basis here, and we could not determine the exact proportion of il- + /il- + /ifn-γ + t cells among total t cells. thus, whether the reduction in il- /il- /ifn-γ production resulted from hcesp stimuli causing reduced numbers of live il- + /il- + /ifn-γ + t cells, reduced numbers of differentiated il- + /il- + / ifn-γ + t cells, or increased numbers of hyporesponsive il- + /il- + /ifn-γ + t cells remains unclear, and further efforts are needed to address this issue. in a previous study, a galectin- homologue derived from adult toxascaris leonina restrained inflammatory reactions by inhibiting th and th cytokine production by enhancing tgf-β and il- production [ ] . similarly, a high dose of hcesps also upregulated il- and tgf-β secretion to inhibit the host inflammatory response. additionally, tgf-β induces apoptosis via death-associated protein (daxx), another binding receptor of fas [ ] . therefore, increased tgf-β secretion may exacerbate fas-mediated apoptosis via a separate apoptotic pathway. recent studies demonstrated the inhibition of th differentiation and il- secretion by echinococcus granulosus and acanthocheilonema viteae es proteins [ , ] . instead, the secretion of the pro-inflammatory cytokine il- a was notably promoted by hcesp stimuli in this study, indicating that hcesps may not induce apoptosis of the th subset. in addition, tgf-β contributes to th formation and il- secretion [ , ] , and it is likely that increased tgf-β production may function as the key factor to facilitate il- a production. as one of the most intensive research areas, a pleiotropic range of immunomodulatory activities of es proteins has been determined in numerous species of gihelminths, including h. polygyrus [ ] , teladorsagia circumcincta [ ] , a. caninum [ ] , and n. americanus [ ] . however, the role of individual es components is still being elaborated or has yet to be determined in most instances. in our previous work, two novel es proteins, namely, y b a. and 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(hc ) protein of haemonchus contortus: a functional inhibitor of host il- hc ttr: a novel antagonist against goat interleukin derived from the excretory and secretory products of haemonchus contortus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank dr xing-quan zhu for providing valuable and constructive suggestions. ml performed the laboratory tests, analysed the data and drafted the manuscript. xrl directed, coordinated and managed the project and edited the manuscript. xt, zy, ww and alt contributed to the in vitro studies and assisted with cell cultures. cl helped in the implementation of the study. ry, xs and lx provided new analytical reagents and tools. all authors read and approved the final manuscript. supplementary information accompanies this paper at https ://doi. org/ . /s - - - .additional file : primer sequences for the transcription analysis of apoptosis and cell cycle. abbreviations es: excretory-secretory; hcesps: haemonchus contortus es proteins; lc-ms/ ms: liquid chromatography-tandem mass spectrometry; fas: tnf superfamily receptor ; akt: akt virus oncogene cellular homologue; il: interleukin; tgf: transforming growth factor; ifn: interferon; gi: gastrointestinal; pbmcs: peripheral blood mononuclear cells; fcs: foetal calf serum; co-ip: coimmunoprecipitation; go: gene ontology; fadd: fas-associated protein with death domain; bid: a bh domain-only death agonist protein; caspases: cysteine proteases with aspartate specificity; cdk: cyclin-dependent kinase; foxo : forkhead box protein o . key: cord- -jbuuph w authors: lund, morten; røsæg, magnus vikan; krasnov, aleksei; timmerhaus, gerrit; nyman, ingvild berg; aspehaug, vidar; rimstad, espen; dahle, maria krudtaa title: experimental piscine orthoreovirus infection mediates protection against pancreas disease in atlantic salmon (salmo salar) date: - - journal: vet res doi: . /s - - -y sha: doc_id: cord_uid: jbuuph w viral diseases are among the main challenges in farming of atlantic salmon (salmo salar). the most prevalent viral diseases in norwegian salmon aquaculture are heart and skeletal muscle inflammation (hsmi) caused by piscine orthoreovirus (prv), and pancreas disease (pd) caused by salmonid alphavirus (sav). both prv and sav target heart and skeletal muscles, but sav additionally targets exocrine pancreas. prv and sav are often present in the same locations and co-infections occur, but the effect of this crosstalk on disease development has not been investigated. in the present experiment, the effect of a primary prv infection on subsequent sav infection was studied. atlantic salmon were infected with prv by cohabitation, followed by addition of sav shedder fish or weeks after the initial prv infection. histopathological evaluation, monitoring of viral rna levels and host gene expression analysis were used to assess disease development. significant reduction of sav rna levels and of pd specific histopathological changes were observed in the co-infected groups compared to fish infected by sav only. a strong correlation was found between histopathological development and expression of disease related genes in heart. in conclusion, experimentally prv infected salmon are less susceptible to secondary sav infection and development of pd. electronic supplementary material: the online version of this article (doi: . /s - - -y) contains supplementary material, which is available to authorized users. virus infections are a continuous challenge in large-scale aquaculture of atlantic salmon (salmo salar). environmental factors, high intensity production and infectious agents affect both welfare and production [ ] [ ] [ ] . the two most prevalent viral diseases in norwegian atlantic salmon aquaculture are heart and skeletal muscle inflammation (hsmi) and pancreas disease (pd) [ ] . piscine orthoreovirus (prv) is associated with hsmi, is ubiquitous in sea reared atlantic salmon in norway and often detected without any signs of disease [ , ] . pancreas disease is caused by salmon pancreas disease virus, more commonly known as salmonid alphavirus (sav). the two viral diseases have overlapping geographic distributions [ , ] , both target heart and skeletal muscle and may coinfect atlantic salmon [ ] [ ] [ ] . prv is a non-enveloped virus with a segmented, double stranded rna genome, belonging to the genus orthoreovirus in the family reoviridae [ , ] . salmonid erythrocytes are major target cells for prv and more than % of these cells may be infected in the peak phase of the infection [ ] . in later stages of the infection, prv infects myocytes of the heart and skeletal muscles [ ] . the histopathological changes in heart and skeletal muscle gave the condition its name in the late s, and later the association with prv was established [ , ] . sav is an enveloped virus with a single-stranded positive sense rna genome of the family togaviridae [ ] . pancreas, heart and skeletal muscle are the main target tissues. the disease is recognized by growth retardation, reduced slaughter quality and increased mortality [ , , ] . histopathological changes are characterized by acute necrosis of exocrine pancreas, myocardial and skeletal muscle necrosis with subsequent inflammation [ ] . the pancreatic lesions are a hallmark of pd and hence used for diagnostic differentiation from hsmi and cardiomyopathy syndrome (cms) [ ] . however, prv and sav have common target tissues in heart and skeletal muscles, making interactions between the two viral infections possible. sav is divided into six different phylogenetic subtypes [ ] and subtypes and are present in norwegian aquaculture [ , ] . the two subtypes show approximately % differences in nucleotide sequence [ ] , are endemically present in separate geographic areas and differ in virulence [ ] [ ] [ ] . the mechanisms behind the difference in virulence are unknown. no stereotypical difference is described between subtype and [ ] . pd outbreaks vary in duration, severity and accumulated mortality [ ] , indicating that factors other than sav influences disease development. interaction with other infectious agents may be such a factor. protection to a secondary virus infection induced by an unrelated primary virus infection has been recognized since the s [ ] , and has also been described for several viruses infecting salmonid fish [ ] [ ] [ ] [ ] [ ] . however, the duration of the protection of rainbow trout to infectious hematopoietic necrosis after primary infection with the non-virulent cutthroat trout virus was found to be no more than weeks [ ] . in addition, some viral infections in terrestrial animals are shown to aggravate disease development of a secondary viral infection [ , ] . the purpose of this study was to determine if a primary prv infection alters the outcome of a subsequent sav infection. experimental infection trials were performed to compare disease development, viral kinetics and expression of disease associated genes between prv-sav co-infected and sav infected fish. sea water adapted atlantic salmon (n = ) of a sal-mobreed strain (bergen, norway) were used in the study (veso vikan, namsos, norway). the post smolts were transferred to sea water two weeks before prv challenge. prior to challenge, the fish were screened and found to be negative for prv, infectious pancreatic necrosis virus (ipnv) and sav by reverse transcriptase (rt) qpcr. prv shedders (n = ) sampled four weeks after prv challenge were confirmed negative for atlantic salmon calicivirus [ ] . during the challenge trial the fish were kept in filtered and uv-radiated seawater ( ‰ salinity), °c (± °c) and on h light. the fish were fed % of total biomass per day and starved for h prior to handling and sampling. before sampling, the fish were euthanized by bath immersion containing benzocaine chloride ( g/ l water) (apotekproduksjon as, oslo, norway) for min. the challenge trial was approved by the norwegian animal research authority and performed in accordance with the recommendations of the current animal welfare regulations: for- - - - (norway). the inoculum (consisting of pelleted blood cells) was collected in a previous cohabitation trial (veso vikan), i.e. the second passage in experimental fish, and originated from a norwegian field outbreak of hsmi in . rtqpcr was performed as earlier described [ ] and a high level of prv rna was indicated (ct . , using a total rna input of ng in the rtqpcr). the blood pellet was dissolved : in pbs and stored at − °c. on day of the trial, the blood pellet and pbs solution was thawed on ice, diluted : in pbs and . ml of the inoculum was i.p. injected into the anesthetized shedders. the inoculum was confirmed negative for ipnv, infectious salmon anemia virus (isav), sav, piscine myocarditis virus (pmcv) by rtqpcr. after i.p. inoculation, the shedders (n = ) were marked by adipose fin removal and placed in a tank containing naïve fish (n = ). four weeks post prv shedder introduction (wpc-prv), the prv shedders were removed and the cohabitants were distributed into four tanks. as displayed in figure , at wpc-prv, two tanks containing prv cohabitants (n = ) were supplied with either sav (n = ) or sav (n = ) shedders ( : ratio, shedder:cohabitant) starting the early co-infection. while the two other tanks contained prv cohabitants until wpc-prv (indicated by "prv" in figure ) and were sampled as prv controls at and wpc-prv. at wpc-prv, the prv cohabitants (n = ) in the tanks, were supplied with sav (n = ) or sav (n = ) shedders, initiating the late co-infection. hence, cohabitants in the early and late co-infection were challenged with sav shedders and weeks post sea water transfer, respectively. naïve fish (n = ) were kept in a separate tank ("naïve fish ", figure ). sav or sav shedders were i.p. injected with . ml of cell culture medium containing sav or sav at a concentration of tcid /ml. the sav inocula were prepared as described earlier [ ] . sav shedders were marked by maxilla cutting. after injection, the sav shedders were kept in separate tanks for four days before being introduced to the cohabitants. sav or sav shedders (n = ) were placed in tanks with respective sav subtype control tanks (n = in each tank) ( figure ). naïve fish to be i.p. injected with sav at wpc-prv were kept in a separate tank from to wpc-prv ("naïve fish " in figure ). time after introduction of sav shedders will be referred to as weeks post sav shedder introduction (wpc-sav). organ samples from heart on rnalater ™ (ambion inc., usa) and heparinized blood were collected from naïve fish (n = ) before sav challenge. these were confirmed negative for sav and prv by rtqpcr. due to differences in virulence and geographic distribution [ , ] , both sav and sav were included in the study. eight cohabitants were sampled from the prv only and prv-sav co-infected groups, whereas six cohabitants were sampled from the sav control groups at each sampling. eight fish sampled prior to prv challenge served as negative controls. weight and fork length was registered for all sampled cohabitants and fulton's condition factor (k-factor) was calculated (k-factor = weight in grams/ length in cm × ). tissue samples for histopathological evaluation (heart, pyloric caeca including exocrine pancreas and red and white skeletal muscle including the lateral line) were collected and fixed in % phosphate buffered formalin. after h, the formalin was replaced with % ethanol and stored at °c until further handling. two pieces of mm from heart were collected on prefilled . ml tubes (fluidx ® ltd, uk) with . ml rnalater ™ for rtqpcr analysis. heparinized blood was collected from the caudal vein. samples for histopathology were processed and stained with hematoxylin and eosin following standard procedures. the sections from heart, pyloric caeca and skeletal muscle were examined blind and scored for pathological changes in an ordinal system ( , , , and ), based on taksdal et al. and mcloughlin et al. [ , ] . the scoring performed in this study was modified and extended to include a separate score for acute myocardial necrosis and epicarditis as the former is a hallmark in pd development and the latter is observed in both pd and hsmi [ ] . the scoring criteria for exocrine pancreas, myocardial degeneration and inflammation, acute myocardial necrosis, epicarditis and inflammation in skeletal muscle are displayed in additional file . immunohistochemistry for detection of prv and sav in heart tissue were performed as described earlier for detection of prv [ ] ; polyclonal rabbit anti-σ serum ( : ) [ ] for prv and monoclonal murine anti-e ( h ) ( : ) [ ] for sav were used as primary antibodies. both were incubated overnight in a humidity chamber, sav at room temperature and prv at °c. biotinylated goat anti-rabbit ( : ) and biotinylated rabbit anti-mouse ( : ) were used as secondary antibodies (dako, agilent technologies, glostrop, denmark). vectastain abc-ap kit (vector, laboratories, burlingame, ca, usa) was used for visualization. heart tissues from double infected fish tissues were investigated. as negative and positive controls, slides with sav or prv single infected tissues were included. red down arrow indicates introduction of sav shedders. "naïve fish " indicate the experimental fish before virus challenge, "naïve fish " indicate fish dedicated to be sav shedders and sav controls at wpc-prv and "naïve fish " indicate fish dedicated to be sav shedders at wpc-prv. blue box named "prv cohabitation" indicates cohabitant fish exposed to % prv shedders from to wpc-prv. blue box named "prv" indicates prv cohabitants from to wpc-prv without prv shedders. boxes indicating a week period of exposure to % sav shedders are colored red (sav only) and green (prv-sav). co-infection induced at wpc-prv and wpc-prv is denoted prv-sav-early and prv-sav-late, respectively. all samples, including heparinized blood, were shipped cool ( - °c) and arrived within h to the norwegian veterinary institute laboratory after sampling. tissue samples on rnalater ™ were placed at − °c until further analysis. a sub-sample of µl from each heparinized blood sample was subsequently shipped cold, together with heart samples on rnalater ™ , to patogen as for virus analysis. patogen as performed rna extraction and rtqpcr analysis for prv and sav transcripts in heart and heparinized blood. the rtqpcr assay targeting prv is validated to iso standards and was described by glover et al. [ ] . the sav assay is validated and accredited to iso standards and was performed as described earlier [ ] . samples were defined as positive when having a prv or sav ct lower than . . elongation factor α (ef α) served as an internal reference gene [ ] for all rtqpcr assays performed. prv and sav ct values were normalized to ef α ct values (Δct = ct target − ct ef α ). after finalizing the virus analyses, rna (in rnase free h o) extracted from heart and blood by patogen as was shipped frozen on dry-ice, overnight to the nvi. rna quantification and purity was determined using nanodrop uv-vis spectrophotometer (thermo scientific, wilmington, de, usa). finally, µl rnase out ( . u/µl, life technologies) was added and the rna was stored at − °c until gene expression analysis. for gene expression analysis, cdna was produced from ng total rna using quantitect reverse transcription kit (qiagen) according to the manufacturer's instructions. quantitative pcr was performed using ng ( µl of ng/µl) cdna input per reaction using maxima sybr green (thermo scientific) with µm of both primers. primers are listed in table . the cycling conditions used were °c for min, then cycles of °c/ s, °c/ s and °c/ s in a mx p (stratagene, la jolla, ca, usa). a seven-point concentration grade standard curve ( - . ng) was run during testing of the primer pairs. the analyses were carried out using nofima's atlantic salmon oligonucleotide microarray siq- and bioinformatic package stars [ ] . the platform includes k unique probes to protein encoding transcripts; the genes were annotated by functions (go), pathways (kegg) and custom vocabulary. microarrays were manufactured by agilent technologies (santa clara, ca, usa) and unless indicated otherwise, the reagents and equipment were purchased from the same source. the microarray analyses were performed on rna from heart tissue that was shipped overnight from nvi to nofima on dry ice. rna from uninfected hearts, sampled at day , was used as a common reference in all hybridizations. rna amplification and labelling were performed with a two-color quick amp labelling kit and a gene expression hybridization kit was used for fragmentation of labelled rna. total rna input for each reaction was ng. after overnight hybridization in an oven ( h, °c, rotation speed . g), arrays were washed with gene expression wash buffers and and scanned with a genepix a (molecular devices, sunnyvale, ca, usa). genepix pro . was used for spot to grid alignment, assessment of spot quality, feature extraction and quantification. subsequent data analyses were performed with stars. after filtration of low quality spots flagged by genepix, lowess normalization of log -expression ratios (er) was performed. genes that passed the quality control in more than half of the samples were included in the subsequent analyses. differential expression was assessed by criteria: er > . -fold and p < . . the statistical analysis was performed using graphpad prism . (graphpad software inc., usa). differences in viral rna levels were calculated based on Δct values using the non-parametric mann-whitney unpaired rank test. differences in gene transcript levels in heart tissue and histopathological scores between the groups were examined using the non-parametric mann-whitney unpaired rank test. an unpaired student t test was used to examine differences in k-factor and weight. spearman's rank correlation was calculated using stata . (statacorp, usa), for associations between viral rna levels in sav cohabitants and histopathology score of acute myocardial necrosis. in addition, association between gene expression (Δct) and histopathology score of both myocardial degeneration and inflammation and acute myocardial necrosis was calculated. in all calculations of differences, a p < . was considered statistically significant. the cohabitant challenge experiment is displayed in mortality was low during the experiment. three ( . %) prv infected fish died during the first weeks, while one fish died in the prv-sav -early group ( . %) between and wpc-prv. the accumulated mortality at wpc-sav was . and . % in the sav and sav controls, respectively. in the late co-infection there were no mortalities. on day , the mean weight, length and condition (k)factor was . g (range . - . g), . cm (range . - . cm) and . (range . - . ), respectively. at the time of sav shedder introduction, the mean weight at wpc-prv was . g (n = , prv early group) and . g (n = , naïve fish) and at wpc-prv, the mean weight was . g (n = , prv late group). at wpc-prv, all groups had increased average weight and length. however, the mean k-factor was reduced in the prv-sav -early ( . ), sav ( . ) and sav ( . ) groups and increased in prv controls ( . ) and the prv-sav -early group ( . ) . the difference between the sav control groups compared to the prv controls and the prv-sav -early group were significant (p < . ) at wpc-prv. at the end of the trial, i.e. wpc-prv, the prv-sav -late and prv-sav -late groups had a mean k-factor of . and . , respectively. additional file shows detailed range of weight, length and k-factor. at day , the fish were confirmed negative for both prv and sav by rtqpcr. successful transmission and infection of the cohabitants with prv were confirmed by detection of viral rna in blood and heart (figures a-d) . prv was first detected in cohabitant fish at wpc-prv and the level of prv rna peaked at wpc-prv in blood (mean ct . ) and wpc-prv in heart (mean ct . ). high prv rna levels were present in the fish until the end of the experiment at wpc-prv ( figures c and d) . histopathological changes including epicarditis and myocardial degeneration and inflammation in red skeletal muscle were in accordance to experimentally induced hsmi (additional file ), as previously described [ ] . there were no differences in Δct values of prv rna or histopathological changes between the prv controls and prv-sav groups. likewise, there were no differences between the co-infected groups when comparing Δct values of prv transcripts except a significantly lower Δct value in prv-sav -late group compared to prv-sav late group at wpc-prv, p < . ( figures c and d) . sav and sav shedders were confirmed sav positive by rtqpcr in heart and blood. the histopathological changes were characteristic for pd, i.e. loss of exocrine pancreatic tissue, acute myocardial necrosis and myocarditis. in the prv-sav -early group, the levels of sav rna were significantly lower in blood at , and wpc-sav compared to the sav controls (p < . ) and in heart at and wpc-sav (p < . ). at both time points, / fish were sav negative in heart in the co-infected group. the sav rna levels in the sav control group peaked wpc-sav, however the prv-sav -early group did not reach the same level ( figures c and d) . at and wpc-sav, the sav rna levels in blood was undetectable in more than % of the fish in the co-infected group and significantly lower than in the sav control group (p < . ). the sav rna level in heart was significantly lower in the co-infected fish at and wpc-sav compared to the sav controls (p < . ). in the late co-infection, i.e. addition of sav shedders at wpc-prv, sav rna was first detected in the prv-sav -late group in blood and heart at wpc-sav and peaked at week post sav introduction, which was one week later than the control group ( figures a and b) . the sav rna level in blood was significantly lower in the co-infected group at wpc-sav compared to the sav control groups (p < . ). furthermore, the co-infected group had a significantly lower sav rna level in heart compared to the controls at wpc-sav (p < . ). in the prv-sav -late group the sav rna levels did not show the same delay as observed for the prv-sav -late group (figures c and d) . sav rna was first detected wpc-sav and reached peak levels wpc-sav in both heart and blood. however, at and wpc-sav the sav rna level was significantly lower in heart in the coinfected group (p < . ). in blood, the sav rna level was significantly lower at wpc-sav in the prv-sav late group compared to the sav controls (p < . ). during the co-infection, the sav shedders also got infected with prv ( figure ). although not significant, the prv rna levels in the sav shedders after wpc-sav, were higher in heart when compared to sav shedders. histopathological scoring of changes in pancreas and acute myocardial necrosis showed a reduction in the coinfected groups compared with sav control groups (figures and ) . using a non-parametric rank test of the ordinal histopathological score, changes in pancreas were found to be significantly lower at and wpc-sav in both early and late co-infection compared to the sav control groups (p < . ), except at wpc-sav in the prv-sav -late group (figures and ) . the prv-sav -late group had also a significantly lower score in pancreas compared to the sav control group at wpc-sav (p < . ). the prevalence of acute myocardial necrosis was significantly reduced at wpc-sav in the co-infected groups compared to the sav controls (p < . ). at wpc-sav, the prevalence of acute myocardial necrosis was significantly lower in the prv-sav -late and prv-sav -early groups compared to the sav controls (p < . ). the sav rna levels (Δct) and histopathology score of acute myocardial necrosis showed a strong positive spearman's rank correlation (r s = . ) in the sav control group, p < . (n = ), whereas a weaker correlation (r s = . ) was seen in the sav controls, p < . (n = ). immunohistochemistry (ihc) was performed on sections of heart tissue from single infected and co-infected fish. the fish were selected based on high viral levels (indicated by low ct levels) of both viruses. the fish presented in figure were sampled wpc-prv, which for the sav controls and co-infected groups correspond to three weeks post sav introduction ( wpc-sav) in the early co-infection. virus ct values in the respective heart tissues are noted in figure . in the sav infected fish, sparse but distinct staining restricted to single cells was observed using sav antibodies. the prv antibodies yield a weak pink background color in both prv infected and sav infected heart tissues. however, distinct staining was only observed in prv infected heart. staining of both sav and prv antibodies were achieved in two separate sections of a co-infected heart. in the co-infected heart tissue more diffuse staining was observed in epicard, interpreted as unspecific binding for both sav and prv antibodies. when staining the control tissue with prv antibodies, a weak pink background color was observed. additional files , and include more detailed pictures. the ihc demonstrates the presence of prv and sav in the same areas of a co-infected heart section. however, no individual cells could be defined as co-infected. microarray analysis was performed on hearts sampled at and wpc-sav from the late co-infection and differences between the sav control and prv-sav -late group were analyzed (table ) . genes were selected based on their correlation with severity of pathological changes in heart induced by sav infection, as previously reported [ ] . to confirm the array results, rt-qpcr assays were run for seven selected genes, of which six showed significant differences (figure ). the gene regulation relative to wpc-prv (set to zero) is shown in figure . at this time point the heart appeared healthy by histopathological evaluation. gene expression at and wpc-sav in both the early and the late co-infected group were compared to and wpc-sav in the sav control group. significant differences were found between prv-sav late and sav control for calsequestrin, neuropeptide y- and interleukin -receptor accessory protein-like (il r- ) at both and wpc-sav, at wpc-sav for mitochondrial arginase- and at wpc-sav for arginase- and serum amyloid a -protein (saa ) (p < . ). asterisk indicates significant (p < . ) differences between co-challenged groups and sav control fish. the early co-infected groups differed significantly from the sav controls for il r- , mitochondrial arginase- and saa at wpc-sav, and for calsequestrin, il r- , arginase- and saa at wpc-sav (p < . ). no significant differences between the groups were found for matrix metalloproteinase (mmp ) expression. histopathology scores (ordinal variable , , and ) of myocardial degeneration and inflammation and acute acute myocardial necrosis * * * * * * * * figure histopathological evaluation of prv-sav co-infected fish. histopathological scoring of pd associated changes in pancreas and acute necrosis in myocardium detected during the week co-infection. asterisk indicates significant (p < . ) differences between co-infected groups and sav control fish. week represent uninfected control fish. myocardial necrosis were correlated with gene expression levels from the qpcr, using spearman's rank correlation ( table ). the groups investigated (n = /group) were prv controls ( and wpc-prv), prv-sav -early and -late ( and wpc-sav) and sav controls ( and wpc-sav), making a total of n = fish evaluated. significant correlation was found for all gene expression levels toward the score of acute necrosis and inflammation in myocardial tissue, except for mmp against inflammation (table ) . when corrected for multiple comparisons by bonferroni-adjusted significant level, il r , neuropeptide y- , saa , mitochondrial arginase- against necrosis and calsequestrin, il r- , neuropeptide y- , arginase- against inflammation remained significant. this study demonstrates that a primary prv infection reduces disease development of a subsequent sav infection with either sav or sav , as evidenced by lower levels of sav rna, less severe pd pathological lesions and higher condition factors in the co-infected groups. the lack of parallel groups must be accounted for when evaluating the presented results. nevertheless, the observation of a similar reduction in disease development for both sav subtypes in co-infected fish strengthens the validity of the results. the most pronounced evidence of protection was a reduction in pd specific pathological lesions in exocrine pancreas. lesions in the pancreas are a hallmark of sav infection and used diagnostically for separation of pd and hsmi [ ] . the heart is a target organ for both prv and sav, with myocarditis and epicarditis observed in both diseases [ , , ] . this may possibly mask the protective effect on sav induced myocarditis and epicarditis in this study. cardiomyocytic necrosis is a typical pathological finding of early stages of sav infection [ ] , and is not considered a specific feature of hsmi. in our study we found that the early co-infected groups had a significantly lower degree of acute myocardial necrosis compared to sav controls. a recent study indicate a possible difference in susceptibility to sav infection depending on time following sea water transfer [ ] . therefore, we cannot exclude that the size of the fish and time after sea transfer may have an impact on the difference in protection observed between the sav only control groups and the late co-infected groups in our study. co-detection of prv and sav in hearts have been shown by rtqpcr in farmed atlantic salmon escapees in norway [ ] . here, we demonstrate the presence of both viruses in figure histopathological evaluation of prv-sav coinfected fish. histopathological scoring of pd associated changes in pancreas and acute necrosis in myocardium during the week co-infection. asterisk indicates significant (p < . ) differences between co-infected groups and sav control fish. week represent uninfected control fish. neighboring cells in heart tissue by immunohistochemistry along with co-detection by rtqpcr. however, since the protective effect was observed in pancreas which is not a target organ for prv, this indicates that the prv mediated protection is due to systemic responses and not to interaction through co-infection in the same tissue. prv utilizes erythrocytes for replication and dissemination in the fish [ ] , and the salmon erythrocytes can mount innate antiviral responses after prv infection [ , ] . the prv control group had a significantly higher k-factor wpc-prv compared to the sav control groups at the same time point. the k-factors in the sav and sav controls were lower compared to the corresponding double infected groups (prv-sav -early and prv-sav -early), although only significant for the prv-sav -early group. the higher k-factor supports the observed less severe histopathological changes in the co-infected groups, confirming the reduced impact of sav after a preceding prv infection. our results are in accordance with previous co-infections in fish where reduced mortality was observed. preexposure of rainbow trout with either the non-virulent cutthroat trout virus (ctv) (hepeviridae), chum salmon reovirus (csv), or ipnv, gave a four week protection to a subsequent ihnv infection [ - , , ] . a similar effect against isav, lasting eight weeks, was observed for ipnv infected fish. however in that study the isav challenge was given intraperitoneally [ ] . this indicates that long-lasting cross-protection between non-related viruses in fish is a general phenomenon, although the duration of protection may vary. our study suggest an inhibitory effect of prv on a secondary sav infection which may last for at least weeks post prv challenge, and thus a longer duration of protection compared to other interfering virus infections reported in salmonids [ , ] . activation of the antiviral innate immune response, where the type interferon (ifn) system is central [ , ] , is a possible explanation for protection against unrelated viruses after a primary virus infection. studies on sav infection in cell cultures and in vivo have shown upregulation of ifnα and a number of interferon induced genes [ , ] . however, these studies revealed a complex antiviral innate response after sav infection leading to reduced sav propagation. prv infection of atlantic salmon rbc also cause strong up-regulation of antiviral genes of the innate immune response [ ] . two recent studies show that mx expression is upregulated in heart tissue for weeks [ ] , and in erythrocytes for at least eight weeks after prv infection [ ] . interferon type i production was induced at the transcriptional level in erythrocytes for up to weeks [ ] . this suggests that circulating prv infected erythrocytes could play an important role in the observed suppressive effect on sav propagation and pd development by inducing interferon-regulated antiviral responses in most organs prior to sav infection [ ] . therefore, at the time of early sav shedder introduction in our trial, i.e. at wpc-prv, an upregulation of innate antiviral genes is expected. a possible long lasting innate immune response may contribute to the protection seen during the late co-infection. sav rna levels in heart were significantly lower wpc-sav in the early co-infected groups compared to the sav controls. this difference was not present at the same time point in the late co-infected hearts, which could indicate a decreased protection caused by a reduction of innate immunity. however, since fish igm have lower specificity and antigen affinity compared to mammalian serum antibodies, at least up to weeks post infection, a mechanism of low affinity polyreactive natural antibodies cannot be ruled out [ , ] . a possible difference was observed between the two sav subtypes in the ability to handle the consequences of the preceding prv infection. the sav and sav rna levels differed in the co-infected groups. sav replicated more efficiently than sav during the early co-infection, whereas the peak phase of both viruses was lost in the late co-infection with prv, either by a delay (sav ) or by a reduction (sav ). there were large individual variations in levels of sav rna and in prevalence of sav expression of potential pancreas disease marker genes in heart. fold induction or repression of genes identified as potential pd associated genes assessed in sav infected (blue bars) and co-infected (red bars: prv-sav -early, orange bars: prv-sav -late) or weeks after sav shedder introduction. significant differences between sav controls and co-infected groups are indicated with *p < . . boxes indicate mean fold change relative to mean levels at wpc-prv. positive fish in the co-infected groups, which makes it difficult to conclude if the apparent differences in sav and sav kinetics show true different properties of the virus subtypes. sav rna kinetics in the heart tissue of both sav and sav controls were similar when assessed by rtqpcr, which is in accordance with a previous sav challenge trial where several isolates were tested [ ] . an interesting finding, that should be addressed in a suitable study, was the strong correlation between acute myocardial necrosis and sav rna level (r s = . ) compared to sav (r s = . ) in the individual infected fish. if sav is a stronger inducer of acute myocardial necrosis, this may partly explain the observed higher mortality associated with sav compared to sav [ , , ] . a previous study using salmon microarray and rtqpcr analysis have reported that changes in the expression levels of certain genes are specifically associated with sav mediated pathological changes in heart [ ] . we found a similar regulation of these genes in the sav controls in our study and that the expression levels of these genes were less affected in the co-challenge groups. this is in tune with the protective effects of a primary prv infection, supporting histopathological observations and virus kinetics. rtqpcr run on a selected number of genes confirmed the microarray results. in general, the gene expression pattern was more affected in the sav controls compared to the co-infected group. a previous study indicated a difference in gene expression patterns between the two diseases [ ] . our study found that the expression differences between the co-infected and sav control groups changed in line with score of histopathological lesions, with a strong correlation of neuropeptide y- and arginase- expression to the score of myocardial degeneration and inflammation. in mammals, neuropeptide y has been shown to have several effects on inflammatory responses and cardiomyopathy [ , ] . furthermore, expression of il r- , saa and mitochondrial arginase- show a strong and significant correlation towards acute myocardial necrosis. thus, genetic analysis may prove to be an additional tool for evaluation of the severity of salmon heart disease and tissue damage. a peculiar finding was the secondary prv infection detected in the sav shedders who resided with the prv cohabitants. this was observed in both the early and late co-infection. during the early co-infection, sav shedders had a higher prv rna level, although not significant, in heart when compared to sav shedders six weeks after introduction to prv cohabitants. this may suggest that sav yields a stronger protection against prv than sav . a possible explanation is that sav , reported by others to be more virulent [ , , ] , yield a stronger immune response than sav . however, the lack of parallel tanks and number of fish per group (n = ) must be accounted for when interpreting these results. the strong correlation between sav rna levels and myocardial necrosis may be a novel step towards understanding the observed virulence differences between the subtypes. an interesting observation in this perspective is the higher prevalence of hsmi in the geographically separated endemic areas of sav in mid-norway compared to those of sav further south [ , ] . these field observations could potentially be linked to a higher possibility of cross-infection between sav and prv and more prevalent development of both diseases in prv-sav dual infected fish. interactions between viral diseases may be part of the explanation for the large variation in severity described for sav infections in the field [ ] . prv is found to infect fish in fresh water facilities and is ubiquitous in sea farms [ ] . the protective effect in this study could affect the outcome of a prv-sav co-infection after sea transfer. in this study, the experimental fish had high levels of prv rna and developed hsmi. a more subtle prv infection, where there is no hsmi development may cause a difference in strength and duration of the protection. further research should address various field conditions when assessing the implications of prv-sav co-infection. in conclusion, we found that a primary prv infection partially protects against the outcomes of sav infection and pd pathological lesions. host density thresholds and disease control for fisheries and aquaculture site management factors influencing mortality rates in atlantic salmon (salmo salar l.) during marine production methods for investigating patterns of mortality and quantifying cause-specific mortality in sea-farmed atlantic salmon salmo salar the health situation in norwegian aquaculture heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus quantification of piscine reovirus (prv) at different stages of atlantic salmon salmo salar production genetic characterization of salmonid alphavirus in norway pathology of heart and skeletal muscle inflammation (hsmi) in farmed atlantic salmon salmo salar alphavirus infections in salmonids-a review potential disease interaction reinforced: double-virus-infected escaped farmed atlantic salmon, salmo salar l., recaptured in a nearby river sequence analysis of the genome of piscine orthoreovirus (prv) associated with heart and skeletal muscle inflammation (hsmi) in atlantic salmon (salmo salar) piscine orthoreovirus (prv) replicates in atlantic salmon (salmo salar l.) erythrocytes ex vivo immunohistochemical detection of piscine reovirus (prv) in hearts of atlantic salmon coincide with the course of heart and skeletal muscle inflammation (hsmi) heart and skeletal muscle inflammation in atlantic salmon, salmo salar l.: a new infectious disease salmon pancreas disease virus, an alphavirus infecting farmed atlantic salmon, salmo salar l stochastic modelling of direct costs of pancreas disease (pd) in norwegian farmed atlantic salmon (salmo salar l.) the economic benefits of disease triggered early harvest: a case study of pancreas disease in farmed atlantic salmon from norway phylogenetic analyses and molecular epidemiology of european salmonid alphaviruses (sav) based on partial e and nsp gene nucleotide sequences new subtype of salmonid alphavirus (sav), togaviridae, from atlantic salmon salmo salar and rainbow trout oncorhynchus mykiss in norway the first detections of subtype -related salmonid alphavirus (sav ) in atlantic salmon, salmo salar l., in norway mortality and weight loss of atlantic salmon, salmo salar l., experimentally infected with salmonid alphavirus subtype and subtype isolates from norway clinical manifestations of pancreas disease outbreaks in norwegian marine salmon farming-variations due to salmonid alphavirus subtype cross-neutralization studies with salmonid alphavirus subtype - strains: results with sera from experimental studies and natural infections mortality related to pancreas disease in norwegian farmed salmonid fish, salmo salar l. and oncorhynchus mykiss (walbaum) viral interference. some considerations of basic mechanisms and their potential relationship to host resistance induction of protection from ihnv in rainbow trout oncorhynchus mykiss by pre-exposure to the avirulent cutthroat trout virus (ctv) aquareovirus interference mediated resistance to infectious hematopoietic necrosis virus immunological consequences of the coinfection of brown trout (salmo trutta) with infectious hematopoietic necrosis virus (ihnv) and infectious pancreatic necrosis virus (ipnv) study of the viral interference between infectious pancreatic necrosis virus (ipnv) and infectious haematopoietic necrosis virus (ihnv) in rainbow trout (oncorhynchus mykiss) studies on pathogenesis following single and double infection with viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus in rainbow trout (oncorhynchus mykiss) porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral coinfections porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs characterization of a novel calicivirus causing systemic infection in atlantic salmon (salmo salar l.): proposal for a new genus of caliciviridae piscine orthoreovirus (prv) infects atlantic salmon erythrocytes virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus characterization and mapping of monoclonal antibodies against the sleeping disease virus, an aquatic alphavirus molecular genetic analysis of stomach contents reveals wild atlantic cod feeding on piscine reovirus (prv) infected atlantic salmon originating from a commercial fish farm sensitive and specific detection of salmonid alphavirus using real-time pcr (taqman) transcription of reference genes used for quantitative rt-pcr in atlantic salmon is affected by viral infection development and assessment of oligonucleotide microarrays for atlantic salmon (salmo salar l.) comparison of transcriptomic responses to pancreas disease (pd) and heart and skeletal muscle inflammation (hsmi) in heart of atlantic salmon (salmo salar l) atlantic salmon (salmo salar l.) post-smolts challenged two or nine weeks after seawater-transfer show differences in their susceptibility to salmonid alphavirus transcriptome analyses of atlantic salmon (salmo salar l.) erythrocytes infected with piscine orthoreovirus (prv) viral coinfection in salmonids: infectious pancreatic necrosis virus interferes with infectious hematopoietic necrosis virus effects of salmonid fish viruses on mx gene expression and resistance to single or dual viral infections infectious pancreatic necrosis virus infection in atlantic salmon salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or vibrio salmonicida virus interference. i. the interferon the fight between the teleost fish immune response and aquatic viruses immune parameters correlating with reduced susceptibility to pancreas disease in experimentally challenged atlantic salmon (salmo salar) alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype replication in vitro the antiviral innate immune response in fish: evolution and conservation of the ifn system the differential dynamics of antibody subpopulation expression during affinity maturation in a teleost how specific is too specific? b-cell responses to viral infections reveal the importance of breadth over depth a comparative study of marine salmonid alphavirus subtypes - using an experimental cohabitation challenge model the intriguing mission of neuropeptide y in the immune system role of neuropeptides in cardiomyopathies the research was supported by grants # /e and # /o of the research council of norway and # of the norwegian seafood research fund. the sav inoculum was kindly provided by hilde sindre. thanks to sven martin jørgensen (nofima), Øystein wessel (nmbu) and torunn taksdal (norwegian veterinary institute) for expert advises on the challenge design and turhan markussen (nmbu) for valuable comments on the manuscript. the authors declare that they have no competing interests.authors' contributions mkd, va and er launched the project idea. mkd and er participated in the overall design and coordination of the study, interpretation of data and drafting the manuscript. ak and gt performed and interpreted the data from the micro array and revised the manuscript. ibn performed gene expression analysis and revised the manuscript. va participated in the coordination of the study and revised the manuscript. ml coordinated the challenge design and study, all practicalities regarding sample logistics, gathering and interpretation of data and drafted the manuscript. mvr coordinated the challenge design and study, carried out the histopathological scoring, performed immunohistochemical staining, interpretation of data and drafted the manuscript. all authors read and approved the final manuscript. additional file . immunohistochemistry of prv infected heart tissue stained using polyclonal rabbit antiserum targeting prv σ (right panel) and monoclonal murine anti-sav e (left panel). the heart tissue had a prv ct-value of . . key: cord- - gy epzo authors: kumar, pankaj; van den hurk, jan; ayalew, lisanework e.; gaba, amit; tikoo, suresh k. title: proteomic analysis of purified turkey adenovirus virions date: - - journal: vet res doi: . /s - - -z sha: doc_id: cord_uid: gy epzo turkey adenovirus (tadv- ) causes high mortality and significant economic losses to the turkey industry. however, little is known about the molecular determinants required for viral replication and pathogenesis. moreover, tadv- does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. to develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase k treated purified tadv- virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. our analysis resulted in the identification of viral proteins associated with tadv- virions including a novel uncharacterized tav gp protein. further, we detected host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. notably, seven of these host proteins have not yet been reported to be present in any other purified virus. in addition, five of these proteins are known antiviral host restriction factors. the availability of reagents allowed us to identify two cellular proteins (collagen alpha- (vi) chain and haemoglobin) in the purified tadv- preparations. these results represent the first comprehensive proteomic profile of tadv- and may provide information for illustrating tadv- replication and pathogenesis. electronic supplementary material: the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. hemorrhagic enteritis (he) is an economically important disease of turkeys characterized by depression, splenic enlargement, intestinal haemorrhages and sudden death [ ] . the disease is caused by turkey adenovirus (tadv- ), also known as hemorrhagic enteritis virus (hev), a member of genus siadenovirus a [ ] . oral infection of susceptible turkeys with pathogenic tadv- strains results in well-characterized splenomegaly and intestinal bleeding in to days causing subclinical infections and mortality [ ] . although tadv- remains one of the most important causes of economic loss to turkey industry, critical molecular determinants of virulence and factors affecting virus replication are not well understood. this may be in part because of unavailability of an efficient "in vitro" tissue culture system for propagation of tadv- [ ] [ ] [ ] . the genome of tadv- is , bp [ ] . although, tadv- genomic organization of central block of genus-common genes [ ] appears similar to that of other adenovirus genomes [ ] , the left (e ) and right (e ) terminal regions appear absent. interestingly, tadv- encodes a genus specific protein, which shows similarity to bacterial sialidase protein [ ] . although western blot analysis of purified tadv- particles isolated from crude spleen extract revealed presence of eleven structural polypeptides with apparent molecular weight ranging from . to kda [ ] , no systematic study has been performed to identify the precise protein composition of purified tadv- particles. in recent years, mass spectrometry (ms) based proteomic characterization has revealed important insights into viral replication, tropism and virulence for a number of different enveloped viruses [ ] [ ] [ ] [ ] [ ] . in contrast, a few proteomic studies have been reported for nonenveloped viruses [ ] [ ] [ ] [ ] . additionally, there is now compelling evidence suggesting that host cellular proteins incorporated in the virions play an important role in viral replication and pathogenesis [ , , , ] . using ms based approaches, a number of host proteins have been reported to be incorporated into rna viruses ("human immunodeficiency virus- [ , ] "; "simian immunodeficiency virus [ ] "; "respiratory syncytial virus [ ] ; hepatitis c virus [ ] "; "swine hepatitis e virus [ ] "; "coronavirus [ ] " and "influenza [ , ] ") or dna viruses ("herpes simplex virus [ ] "; "african swine fever virus [ ] "; "kshv [ ] "; "marek's disease virus (mdv) [ ] ", and "mimivirus [ ] "). however, to the best of our knowledge, characterization of the host cellular factors integrated into virions for any member of adenoviridae family including tadv- has not been reported so far. here, we report the protein composition of the purified tadv- particles by performing a comprehensive proteomic analysis utilizing liquid chromatography-mass spectrometry (lc-ms/ ms). our analysis resulted in successful identification of viral structural proteins and host-incorporated proteins. moreover, incorporation of two host proteins in purified virions was verified by western blot analysis using available immunological reagents. all turkey procedures were approved by university committee of animal care and supply (protocol # ) at the university of saskatchewan, saskatoon, canada according to guidelines set by the canadian council of animal care. day-old hybrid poults obtained from chinook belt hatcheries, calgary, canada were housed in isolation rooms throughout the experiments. the avirulent tadv- isolate (pheasant origin) was passaged in sero negative turkeys by oral inoculation and purified from crude spleen extracts, as described earlier [ ] . the tadv- virions were purified as previously described [ ] . the proteinase k (pk) treatment of purified tadv- virions was performed as described previously [ ] . briefly, double cscl-purified virions were incubated in ml of mnt buffer ( mm morpholineethanesulfonic acid [mes], mm nacl, and mm tris-hcl [ph . ]) containing proteinase k [ to μg] (roche, mannheim, germany) for min at room temperature and subsequently treated with " mm phenylmethylsulfonyl fluoride" (roche) prior to purification by cscl density gradient centrifugation. purified virions were resuspended in % glycerol and stored at − °c until further use. the experiments were performed in triplicate employing three independent virus preparations. electron microscopy was performed on cscl gradient purified tadv- virions (proteinase k treated or untreated) at em facility at biology department, university of victoria, bc, canada, as described [ ] . briefly, for negatively stained preparation, cscl gradient purified virus was first applied onto carbon and formvar coated grids, washed with h and stained with % aqueous phosphotungstic acid. the specimens were photographed using a charge-coupled device camera (advanced microscopy techniques, amt ccd camera equipped hitachi h tem operating at kv). production and characterization of anti-tadv- serum and monoclonal antibodies (mabs) recognizing tadv- hexon ( g ) and fiber ( - ) proteins has been described earlier [ , ] . chicken polyclonal anti-human hemoglobin serum (ab ) was purchased from abcam (cambridge, ma, usa). rabbit polyclonal antihuman collagen type vi alpha- serum (col a ) was purchased from antibodies-online inc. (atlanta, ga, usa). alkaline phosphatase conjugated goat anti-rabbit (sigma aldrich) and peroxidase-conjugated goat "antiturkey" igg (kpl, maryland, usa) were used as described [ , ] . proteins from purified tadv- were separated by sodium dodecyl sulphate (sds) polyacrylamide gel electrophoresis (page) on - % or - % precast gradient gels (bio-rad),transferred to nitrocellulose membrane and probed with protein specific antibodies as described previously [ ] . proteins from cscl gradient purified virion-enriched (proteinase k treated or untreated) samples were diluted with mm ammonium bicarbonate prior to reduction with mm dithiothreitol and incubated min at °c. cysteine sulfhydryl groups were alkylated with μl of mm iodoacetamide ( min at °c in darkness). each sample was digested with μg of trypsin (promega) at °c for h [ , ] . finally, the samples were de-salted on a waters hlb oasis column, speed vac concentrated and stored at − °c prior to lc-ms analysis. the peptide mixtures were separated by on-line reverse phase chromatography using a easy-nlc ii system (thermo scientific) with a reversed-phase magic c- aq pre-column ( μm i.d., cm length, μm, Å, michrom bio resources inc, auburn, ca, usa) and reversed phase nano-analytical column magic c- aq ( μm i.d., cm length, μm, Å, michrom bio resources inc, auburn, ca, usa) at a flow rate of /min. the resulting peptides were analyzed by the chromatography system, which was coupled on-line with a ltq orbitrapvelos mass spectrometer (thermo fisher scientific, bremen, germany) equipped with a nano-sprayflex source (thermo fisher scientific) as described previously [ , ] . the data was acquired with keratin and trypsin peptide mass exclusion lists. raw files were analysed with proteome discoverer . software suite (thermo scientific). parameters for the spectrum selection to generate peak lists of the collisioninduced "dissociation (cid) spectra were activation type: cid"; (s/n cut-off: . ; total intensity threshold: ; minimum peak count: ; precursor mass: - da). the peak lists were submitted to an in-house mascot . server against "the following databases": uniprot_trembl ( sequences; , , , residues) and uniprot-swissprot ( sequences; residues) all species taxonomy. database search parameters were as follows: precursor tolerance ppm; ms/ms tolerance . da; trypsin enzyme missed cleavages; fourier transform ion cyclotron resonance (ft-icr) instrument type; fixed modification: carbamidomethylation (c); variable modifications: deamidation (n,q); oxidation (m). the decoy database percolator settings: max delta cn . ; target fdr strict . , target fdr relaxed . with validation based on q-value. additional virus only species searches were also performed with tolerances previously mentioned. all data were also searched against ncbi (gallus gallus (chicken)) database to detect viral and host proteins. only sequences identified with a mascot score value greater than were considered as significant. protein identifications were accepted when the peptide probability was greater than . % [ , ] , the protein probability greater than . %, and contained at least identified peptides. peptide identifications were systemically evaluated manually. due to difficulty in propagating turkey adenovirus in cell culture system, tadv- was propagated in six to week old turkeys. tadv- virions were purified from spleens of turkeys inoculated orally with an avirulent vaccine strain of tadv- [ , ] ( figure a ). following cscl density gradient purification, two distinct bands were observed, the upper band (present at lower density) containing capsid and the lower band (at higher density, between . and . ) containing complete infectious viruses ( figure b , left panel). the lower band was subjected to second round of cscl density gradient purification resulting in single band containing purified virions ( figure b, right panel) . virion-enriched preparations were checked for quality by negative stain transmission electron microscopy (tem) (figures c and d) . as seen, virions demonstrated uniform, intact tadv- virus particles of nm diameter. these tem results were consistent with the quality and apparent purity reported earlier [ , ] . the purity of the virion preparation was also determined by western blot analysis using turkey anti-tadv- sera. as seen in figure e , polypeptides of k (hexon), k (iiia), k (penton base), k (fiber) and k (pvi) were detected in cscl purified tadv- virions. these findings suggest that our enrichment procedure yielded a highly purified preparation of tadv- virions. the protein composition of tadv- virions was analyzed by the method of in-solution trypsin "digestion a gel-free approach" to ms that subject the entire sample to sequential one-dimensional reversed-phase chromatography coupled on-line to ms/ms analysis ( d-nanospray-lc-ms/ms). this method eliminates the problems reported with proteins that either enter gel poorly or extracted inefficiently from the gel slices. our lc-ms/ms analysis revealed a total of virus-encoded proteins packaged in the purified tadv- virions. this included proteins, which have been detected in human adenovirus (hadv- ) virions [ ] (table ) , a novel uncharacterized hypothetical viral protein designated as tav gp (table , additional file ) and a non-structural viral protein ( k) to be associated with tadv- virions. in addition to tadv- encoded viral proteins, interestingly cellular proteins appeared to be associated with purified tadv- virions (table ) . to determine if the host proteins are actually incorporated into the virions, the purified tadv- virions were treated with proteinase k ( μg/ml) and subjected to another round (third round) of cscl purification. the proteinase k treated and untreated, purified virions were then analysed by western blotting. proteinase k treatment degrades fiber protein protruding from the capsid but does not degrade hexon protein not protruding from the capsid. as seen in figure a , hexon protein could be detected in proteinase k treated or untreated tadv- virions. in contrast, fiber protein could only be detected in untreated virions, but not in proteinase k treated virions. moreover, tem analysis suggested that the virions were intact and maintained virion integrity after proteinase k treatment and cscl density gradient purification ( figure b ). the lc-ms/ms analysis of proteinase k treated cscl density gradient purified tadv- virions identified eleven virus-encoded proteins (hexon, pvi, pvii, penton base, pviii, sialidase, iiia, adenain, px, iva and dbp) previously reported to be in other adenoviruses (table and figure a ) [ ] . in addition, a novel viral protein tav gp remains an integrated part of proteinase k treated tadv- virions (table , additional file ). as expected, peptides representing fiber protein were not detected in proteinase k treated tadv- virions. in addition, ptp and k virion proteins were not detected in proteinase k treated tadv- ( table ). the high mascot scores and number of peptides observed for hexon, pvi and pvii presumably reflect the fact that they are perhaps the most abundant proteins in the tadv- particles. interestingly only host proteins were exclusively detected in proteinase k treated tadv- virions (table and figure b ). notably, thirteen of these host proteins were the same as detected in the untreated tadv- virions (table , figure b ) indicating that these proteins are part of the tadv- virions. among these proteins, promyelocytic leukemia protein (pml) isoform x (additional file ), collagen alpha- (vi) chain (additional file ), haemoglobin subunit alpha (additional file ) and haemoglobin subunit beta (additional file ) appeared abundant. the pml protein appears as abundant as viral structural protein pviii or penton base peptide. in addition, five host proteins namely, vitronectin, collagen alpha- (vi) chain, collagen alpha- (vi) chain, tyrosine protein phosphatase and turkey heterophil peptide (thp- ) were only detected in proteinase k treated tadv- virions. functional classification of the identified proteins revealed that many of these proteins participate in a common molecular pathway (table and figure c ) and are involved in innate immunity, cell adhesion, cytoskeleton organization and virus replication. non availability of turkey host protein specific antisera made it difficult to verify the packaging of host proteins in tadv- virions. however, human collagen alpha- (vi) peptides showed % identity to turkey collagen alpha- (vi) and chicken collagen alpha- (vi) (additional file ). in addition, human haemoglobin peptides demonstrated % identity to turkey haemoglobin alpha and chicken haemoglobin alpha, % identity to turkey haemoglobin beta and % identity to chicken haemoglobin beta proteins (additional file ). therefore, we attempted to determine the incorporation of collagen alpha- (vi) and haemoglobin in purified tadv- using western blot assays. as shown in figure , anti-collagen alpha- (vi) serum detected collagen alpha- (vi) chain specific band in proteinase k untreated tadv- (panel a, lane ). similar protein could be detected in proteinase k treated purified tadv- (panel a, lane ). antihaemoglobin serum detected haemoglobin specific band in proteinase k untreated tadv- (panel b, lane ). similar protein band could be detected in proteinase k treated purified tadv- (panel b, lane ). viruses exploit multiple host proteins for successful entry, establishment of infection, replication, and immune evasion. for a better understanding of the tadv- -host interactions, we performed a comprehensive analysis of the protein content of tadv- virions, using a lc-ms/ms based proteomic approach. to the best of our knowledge, incorporation of host proteins in adenovirus has not been reported so far. the proteomic analysis of cscl purified tadv- identified a total of virion proteins and host proteins. earlier, proteomic analysis has not reported the detection of host proteins in purified hadv- virions [ , ] . it is possible that the observed host proteins identified by proteomic analysis of cscl purified tadv- virions may not be actually incorporated in the purified virions but are loosely associated on the outside of the tadv- virion capsids. since proteinase k treatment has been traditionally used to remove any contaminating protein from the surface of enveloped viruses [ , ] , we used protease treatment of non-enveloped tadv- to remove the potential contaminating proteins. several lines of evidence validate the approach and suggest that proteinase k treatment of tadv- appears successful in removing contamination proteins. ) intact virions could be detected by tem after proteinase k treatment of tadv- . ) western blot analysis of protease k treated tadv- detected hexon protein but not fiber protein (protruding from the capsid). ) the fiber and k (non structural protein) could not be detected by ms analysis of proteinase k treated tadv- . ) only of the host proteins could be identified in proteinase k treated tadv- . interestingly, all major viral proteins were identified in proteinase k treated virions (table ) except viral ptp, possibly due to its low abundance and least mascot score values observed (table ) . overall sequence coverage observed for different viral peptides ranged from to %, with the majority between and %. earlier, sequence analysis of turkey adenovirus- identified a hypothetical protein orf (named tavgp ) [ ] , which appears to be conserved in raptor adenovirus- [ ] and south polar skua adenovirus [ ] . in contrast, a hypothetical hydrophobic protein was identified in frog adenovirus [ ] , which shows no similarity to similar proteins identified in turkey adenovirus [ ] and raptor adenovirus [ ] . our results suggest that an orf of tadv- encodes a structural protein tavgp , which is incorporated into virion capsid (additional file ). in addition, this is the first report to suggest the existence of tavgp as a structural protein in siadenoviruses particularly of avian origin. the proteomic analysis of proteinase k treated purified virions identified eleven cellular proteins incorporated in tadv- , which have been identified in other viruses (table ). in addition, proteomic analysis identified seven host proteins incorporated in tadv- virions (table ) , which have not been identified so far in any other virus. interestingly, of the detected host proteins, five of the proteins were only detected in proteinase k treated tadv- . it is possible that high abundance non-specific proteins might have masked the detection of these proteins in virions not treated with proteinase k that are truly virion associated, but present in low copy numbers. though earlier reports have demonstrated the packaging of viral [ ] or non viral rnas [ ] into purified adenovirus, recent reports have not described the detection of any cellular protein in purified lizard adenovirus- [ ] , a member of atadenovirus genus and purified hadv- , a prototype of mastadenovirus genus [ ] . the absence of a cellular protein packaged in purified adenovirus virions could be due to variety of reasons. as stated, the difference could be due to the technique used for analysis [ ] . alternatively, it is possible that packaging of the cellular proteins may be dependent on the type of adenovirus (tadv- , a prototype of siadenovirus genus) and origin of cells used for virus cultivation [ ] . the host proteins packaged intadv- are known to play important roles in enhancing the cell-to-cell spread of virus, transcription and virus replication (table , figure ). for example, extracellular matrix (collagen) has been shown to increase infectious sindbis virus titers from bhk cells by enhancing post-infection cell survival [ ] . in another study, rotavirus-induced pi k activation resulted in prolonged adherence of infected cells to collagen and increased virus production [ ] . similarly, extracellular matrix vitronectin has been reported to enhance the growth of human adenovirus (hadv- ) [ ] . however, the incorporation of antiviral host defense factors including, protein pml, haemoglobin and antimicrobial peptide (thp- ) into tadv- virions is particularly intriguing. all of these host defence factors have been implicated in establishing antiviral environments. recent studies have implicated pml in maintaining host antiviral defence and revealed different strategies developed by viruses to disrupt pml nuclear bodies [ ] [ ] [ ] . in addition, protein pml has been shown to be important for the inhibition of adenovirus replication [ ] . similarly, avian antimicrobial peptide thp- , a member of beta-defensin family is effector of the innate defence system and play key functions during host defence by generating vigorous cytokine response [ , ] . on the other hand, a novel role of haemoglobin in innate immunity has been recently reported for classical swine fever virus (csfv) [ ] as silencing of haemoglobin expression using sirna promoted csfv growth and replication, whereas overexpression of haemoglobin antagonized csfv replication and growth by triggering ifn signalling [ ] . although tadv- grows efficiently in spleen of infected turkey, virus grows poorly in primary or established cell lines. it is tempting to speculate that integration of certain established antiviral host restriction factors into viral particles may play a role in determining tadv- replication "in vitro". additional studies need to be performed in order to investigate whether these proteins are functionally required for virus entry, replication and pathogenesis. future availability of reagents and a reliable cell culture system to grow tadv- should make it possible to determine the role of individual host restriction factor in tadv- replication. comparison of turkey hemotthagic enteritis virus isolates allows prediction of genetic factors affecting virulence ictv at paris icv: results of the plenary session and the binomial ballot avian adenoviruses characterization of group ii avian adenoviruses with a panel of monoclonal antibodies evaluation of cell culture propagated and in vivo propagated hemorrhagic enteritis vaccines in turkeys development and application of quantitative real-time pcr for the rapid detection of hemorrhagic enteritis virus in tissue samples the complete dna sequence and genome organization of the avian adenovirus, hemorrhagic enteritis virus genetic content and evolution of adenoviruses characterization of the structural proteins of hemorrhagic enteritis virus proteomic and biochemical analysis of purified human immunodeficiency virus type produced from infected monocyte-derived macrophages alteration of protein levels during influenza virus h n infection in host cells: a proteomic survey of host and virus reveals differential dynamics proteomic changes in hek- cells induced by hepatitis delta virus replication proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins proteomics of herpes simplex virus replication compartments: association of cellular dna replication, repair, recombination, and chromatin remodeling proteins with icp analysis of purified wild type and mutant adenovirus particles by silac based quantitative proteomics analysis of the adenovirus type proteome by liquid chromatography and tandem mass spectrometry methods proteomics strategies to analyze hpv-transformed cells: relevance to cervical cancer proteome analysis of adenovirus using mass spectrometry plunder and stowaways: incorporation of cellular proteins by enveloped viruses cellular proteins in influenza virus particles plasma proteomic analysis of simian immunodeficiency virus infection of rhesus macaques quantitative proteomic analysis of a cells infected with human respiratory syncytial virus ferritin heavy chain is the host factor responsible for hcv-induced inhibition of apob- production and is required for efficient viral infection proteomic analysis of swine hepatitis e virus (shev)-infected livers reveals upregulation of apolipoprotein and down-regulation of ferritin heavy chain proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein quantitative proteomic analyses of influenza virus-infected cultured human lung cells hsv- cgal + infection promotes quaking rna binding protein production andinduces nuclear-cytoplasmic shuttling of quaking i- isoform in human hepatoma cells two-dimensional analysis of african swine fever virus proteins and proteins induced in infected cells proteomic analysis of the kaposi's sarcoma-associated herpesvirus terminal repeat element binding proteins a mass spectrometry-based proteomic approach to study marek's disease virus gene expression mimivirus giant particles incorporate a large fraction of anonymous and unique gene products propagation of group ii avian adenoviruses in turkey and chicken leukocytes proteomic characterization of pseudorabies virus extracellular virions embedding in epoxy resins for ultrathin sectioning in electron microscopy proteomic characterization of bovine herpesvirus extracellular virions complete sequence of raptor adenovirus confirms the characteristic genome organization of siadenovirus full genome analysis of a novel adenovirus from the south ploar skua (catharacta maccormicki) in antarctica viral rnas detected in virions of porcine adenovirus type presence of prepackaged mrna in virions of dna adenovirus molecular characterization of a lizard adenoivirus reveals the first atadenoivirus with two fiber genes and the first adenovirus with either one short or three long fibers per penton effects of collagen matrix on sindbis virus infection of bhk cells rotavirus replication in intestinal cells differentially regulates integrin expression by a phosphatidylinositol -kinase-dependent pathway, resulting in increased cell adhesion and virus yield vitronectin: a possible determinant of adenovirus type tropism for human corneal epithelium effects of promyelocytic leukemia protein on virus-host balance rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies serum-dependent expression of promyelocytic leukemia protein suppresses propagation of influenza virus adenovirus e orf protein inhibits the interferon-mediated antiviral response human beta-defensin induces a vigorous cytokine response in peripheral blood mononuclear cells antimicrobial activity of chicken and turkey heterophil peptides chp , chp , thp , and thp hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www authors thank other members of tikoo's laboratory for helpful suggestions. published as vido-intervac article # . the work was supported by grants from saskatchewan agriculture development fund, alberta livestock and meat agency, saskatchewan chicken industry development fund, canadian poultry research council and agriculture and agri-food canada. the authors declare that they have no competing interests. conceived and designed the experiments: pk, skt. performed the experiments; pk, jv, ag, lea. analyzed the data; pk, jv, skt; ag. wrote the manuscript: pk, lea, skt. all authors read and approved the final manuscript. key: cord- -h slug authors: goossens, evy; verherstraeten, stefanie; valgaeren, bonnie r.; pardon, bart; timbermont, leen; schauvliege, stijn; rodrigo-mocholí, diego; haesebrouck, freddy; ducatelle, richard; deprez, piet r.; van immerseel, filip title: the c-terminal domain of clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis date: - - journal: vet res doi: . /s - - -y sha: doc_id: cord_uid: h slug bovine necrohemorrhagic enteritis is caused by clostridium perfringens and leads to sudden death. alpha toxin, together with perfringolysin o, has been identified as the principal toxin involved in the pathogenesis. we assessed the potential of alpha toxin as a vaccine antigen. using an intestinal loop model in calves, we investigated the protection afforded by antisera raised against native alpha toxin or its non-toxic c-terminal fragment against c. perfringens-induced intestinal necrosis. immunization of calves with either of the vaccine preparations induced a strong antibody response. the resulting antisera were able to neutralize the alpha toxin activity and the c. perfringens-induced endothelial cytotoxicity in vitro. the antisera raised against the native toxin had a stronger neutralizing activity than those against the c-terminal fragment. however, antibodies against alpha toxin alone were not sufficient to completely neutralize the c. perfringens-induced necrosis in the intestinal loop model. the development of a multivalent vaccine combining the c-terminal fragment of alpha toxin with other c. perfringens virulence factors might be necessary for complete protection against bovine necrohemorrhagic enteritis. clostridium perfringens is a gram-positive, spore-forming, anaerobic bacterium. it is a normal component of the intestinal microbiota of animals, including humans. it secretes several toxins and enzymes that cause different forms of tissue damage [ ] [ ] [ ] . consequently, it can cause a variety of diseases in various vertebrates [ ] . the differences in virulence properties between c. perfringens isolates are largely due to differences in toxin production. alpha toxin and perfringolysin o have been identified as the principal toxins involved in gas gangrene caused by c. perfringens as well as in bovine necrohemorrhagic enteritis [ ] . gas gangrene is a frequently lethal histotoxic infection of humans and animals characterized by rapid tissue destruction and impaired immune response [ , ] . bovine necrohemorrhagic enteritis (bovine enterotoxaemia) is an enteric disease of veal calves and beef type suckling calves and is characterized by hemorrhagic to necrotizing enteritis. calves often die without premonitory signs [ , [ ] [ ] [ ] . we recently showed that vaccination of calves with a mixture of native toxins from c. perfringens induces antibodies that protect against c. perfringens challenge in an intestinal loop model of bovine necrohemorrhagic enteritis (goossens et al., provisionally accepted). although both alpha toxin and perfringolysin o are involved in the pathogenesis of gas gangrene, immunization against alpha toxin alone provides good protection against experimental gas gangrene [ , , ] . moreover, evans showed that antiserum raised against alpha toxin was highly effective in protecting guinea pigs against experimental gas gangrene, whereas antiserum to perfringolysin o was not protective against c. perfringens type a infection, and it did not enhance the protective action of alpha toxin antiserum [ ] . studies on gas gangrene cannot be directly extrapolated to bovine necrohemorrhagic enteritis, but these findings indicate that alpha toxin vaccines could provide protection against diseases in which alpha toxin is critically important. here, we tested vaccine preparations based on alpha toxin, the major toxin produced by c. perfringens type a. since native toxins are not safe, we used the enzymatically inactive c-terminal domain of alpha toxin (cpa ). this component is non-toxic and has been shown to provide protection against c. perfringens type a gas gangrene in a mouse model, and it is known to elicit protective immunity against a broad range of clostridial phospholipase c toxins [ , , ] . in addition, mice vaccinated with cpa - were protected against challenge with alpha toxin derived from a calf necrohemorrhagic enteritis isolate [ ] . the aim of this study was to evaluate whether the nontoxic c-terminal fragment of alpha toxin could be a candidate for effective vaccination of calves against bovine necrohemorrhagic enteritis. all experimental protocols were approved by the ethics committee of the faculty of veterinary medicine, ghent university (ec / , ec / , ec / , ec / and ec / ). all animal experiments were carried out in accordance with the approved guidelines. the c. perfringens strains were wild-type strain jir , the plc mutant jir (∆plc), and the c. perfringens jir derivatives carrying either the plc + plasmid (complemented strain jir ) or the empty shuttle vector (complementation control jir ) (table ) [ , ] . the strains were cultured anaerobically at °c in brain heart infusion broth (bhi, oxoid, basingstoke, uk) containing . % glucose. to culture jir [∆plc; (shuttle vector)] and jir (complemented strain), the medium was supplemented with chloramphenicol ( µg/ml). the logarithmic phase cultures used in intestinal loop experiments did not contain antibiotics. to determine the alpha toxin concentration in the culture supernatant, cell-free supernatants were obtained by centrifugation followed by filtration of the supernatants through a . -µm filter. the alpha toxin concentration in the bacterial supernatants was measured using the bio-x α-toxin elisa kit (bio-x diagnostics, jemelle, belgium) and twofold serial dilutions of the alpha toxin standard ( × − - . × − u/ml of phospholipase c type i; sigma-aldrich, st louis, mo, usa) as previously described [ ] . to confirm the role of alpha toxin in the induction of necrotic lesions in an intestinal loop model, seven intestinal loop experiments were conducted using the wild-type c. perfringens strain jir and the alpha toxin-deficient strain c. perfringens jir . in two of the experiments, the c. perfringens jir derivatives carrying the empty shuttle vector (jir ) or the plc + plasmid (jir ) were also included. the number of loops injected with each strain is shown in table . in each calf, an equal number of control loops were injected with sterile bacterial growth medium supplemented with milk replacer. the experiments were performed according to a published protocol using seven healthy male holstein-friesian veal calves aged - months [ ] . briefly, the calves were anesthetized and the small intestine was exteriorized. the loops were ligated and injected with logarithmic phase cultures combined with % commercial milk replacer (vitaspray, nuscience drongen, belgium) in sterile . % nacl solution, as described [ ] . the animals were kept under anesthesia for hours after inoculation, after which they were euthanized and samples were taken. intestinal loop tissue samples were submerged in % (w/v) phosphate buffered formaldehyde. after fixation for h, the samples were processed routinely, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin. sections were evaluated in a blinded ∆plc jir alpha toxin-deficient jir Δplc < . [ ] ∆plc (shuttle vector) jir alpha toxin-deficient with shuttle vector jir (pjir ) < . [ ] complemented jir alpha toxin-complemented jir (pjir ) plc . ± . [ ] manner by a board certified pathologist for the presence of tissue necrosis ( = absence of necrosis, = necrotic lesions present). alpha toxin was expressed in escherichia coli using the pbad topo ® ta expression kit (invitrogen, paisley, uk). a fragment encoding the c. perfringens alpha toxin (plc gene; genbank accession number bab ) was amplified from the dna of c. perfringens jir by pcr using a dna polymerase with proofreading activity (accuzyme, bioline, randolph, ma, usa). the forward primer ( ′-g tga gag gag gat ata aaa atg aaa aga aag att tgt aag gcg - ′) contained an in-frame stop codon and translation re-initiation sequence to remove the n-terminal leader and allow native protein expression. the reverse primer ( ′-g ttt ctt ttt tat att ata agt tga att tcc tga aat cca ctc - ′) excluded the native plc gene stop codon and included the c-terminal v epitope and polyhistidine region for affinity purification. the resulting pcr product was incubated with taq polymerase for min at °c ( u; promega, madison, wi, usa) to add ′ a-overhangs, cloned into the pbad-topo expression vector, and transformed into one shot top ® e. coli. the correct orientation of the alpha toxin insert was verified by sanger sequencing. escherichia coli carrying the pbad-alpha toxin vector was grown at °c to an od of . - . in terrific broth supplemented with µg/ml ampicillin. expression of recombinant c. perfringens alpha toxin was induced for h by adding l-arabinose to a final concentration of . % (w/v). bacteria were harvested by centrifugation and lysed enzymatically using bugbuster (invitrogen). alpha toxin was purified on a ni-sepharose column (his gravitrap, ge healthcare bio-sciences ab, uppsala, sweden) according to the manufacturer's instructions. subsequently, the protein was dialyzed against pbs, purity was analyzed using sds-page, and protein concentration was measured using bca protein assay (thermo fisher scientific, waltham, ma, usa). the recombinant carboxy-terminal domain of alpha toxin fused to glutathione-s-transferase (gst) was kindly provided by prof. titball, university of exeter, uk. this cpa - was produced in e. coli and was therefore devoid of any other c. perfringens proteins [ ] . recombinant native alpha toxin (rcpa) and cpa - were formulated with the adjuvant quila (brenntag biosector, frederikssund, denmark) in pbs. each animal was injected with . ml of the filter-sterilized ( . µm) formulation containing µg antigen and µg quila. control animals received µg quila in . ml pbs. six male holstein-friesian calves aged months were used. they were housed on straw and received water and hay at libitum, and concentrates adjusted to the body weight. for each antigen (rcpa, cpa - or quila control), two calves were immunized subcutaneously in the neck. the calves received a primer vaccination at the age of months, and booster immunizations and days later. the immune response following vaccination was measured using serum samples obtained weeks after the final booster immunization. alpha toxin-specific antibody levels were determined by the end-point dilution method using a blocking elisa (c. perfringens alpha toxin serological elisa kit, bio-x diagnostics). sera were used at a dilution : and assays were performed in duplicate. the specific antibody level was expressed as percent inhibition according to the following formula: % inhibition = [(od negative − od sample)/od negative] × . incubation of cell-free supernatants of the wild-type strain jir (concentrated tenfold using vivaspin, sartorius stedim biotech gmbh, göttingen, germany) on sheep blood agar at °c overnight results in an inner, complete zone of hemolysis caused by perfringolysin o and a less complete outer zone caused by alpha toxin. the sera's ability to neutralize alpha toxin activity was assessed by incubating the jir supernatant with an equal volume of the pooled sera from the two animals that were vaccinated with either a given vaccine or the adjuvant quila for min at °c. ten-microliter drops of these mixtures were spotted on sheep blood agar and hemolysis was assessed after overnight incubation. the test was performed in triplicate using supernatants of c. perfringens jir from three independent biological replicates. alpha toxin activity was determined in duplicate in a -well microtiter plate by evaluating its effect on egg yolk lipoproteins as previously described [ ] . the neutralizing ability of sera was assessed by pre-incubating a twofold dilution series of the sera (two wells per dilution) with a constant amount of alpha toxin ( µg/ml in pbs) for min at °c before adding % egg yolk emulsion. to prepare the yolk emulsion, fresh egg yolk was centrifuged ( × g for min at °c) and diluted : in pbs. after incubation of the -well plates at °c for h, absorbance was measured at nm. alpha toxin activity was indicated by the development of turbidity, which increases absorbance. the inhibitory capacity of the antiserum was expressed as the serum dilution that inhibited % of the alpha toxin activity. this was determined by applying a hill function to the concentration-response data (graphpad prism , graphpad software, san diego, ca, usa). the test was performed in duplicate. primary bovine umbilical vein endothelial cells (buvec) were isolated from umbilical cord veins by an adapted procedure [ ] based on the method of jaffe et al. [ ] . the toxicity of c. perfringens supernatant to cultured bovine endothelial cells has been reported [ ] . the ability of the antisera to neutralize the c. perfringens cytotoxicity to buvecs was determined using a neutral red uptake assay (nru) [ ] . briefly, buvec cells were seeded in -well tissue culture plates at a density of cells per well and cultured for h to obtain cells in the exponential growth phase. the neutralizing ability of the sera was assessed by pre-incubating a twofold dilution series of the sera ( - . %) prepared in serum-free cell culture medium with an equal volume of undiluted c. perfringens supernatant. after min at °c, the cells were treated for h with µl of the supernatant-serum mixture, followed by a standard nru assay. the inhibitory capacity of the antiserum was expressed as the last dilution associated with % cell viability. as a positive control, cells were treated with c. perfringens supernatant pre-incubated with serum-free medium. untreated cells incubated with serum-free medium served as a negative control. the test was performed in duplicate. to study the protection against c. perfringens-induced necrosis provided by the antisera obtained from calves vaccinated with the respective vaccines, three intestinal loop experiments were performed using three male holstein-friesian calves aged months. in each of the three intestinal loop experiments, the sera for each vaccine were pooled. intestinal loops were inoculated with a wild-type strain (jir ) combined with % commercial milk replacer suspended in sterile nacl solution. before inoculation, serum from calves immunized with the different vaccine preparations was added to the nacl solution containing milk replacer to a final concentration of % serum (v/v). in each calf, five intestinal loops were injected with anti-quil a, five with anti-native alpha toxin, and five with anti-c-terminal fragment of alpha toxin. moreover, five control loops per calf were injected with c. perfringens without addition of serum (positive control) and five with sterile bacterial growth medium (negative control). this totaled injected loops per calf. samples were collected and scored as described for the intestinal loop experiments using the alpha toxin-deficient strain. differences in the development of necrotic loops between the wild-type and the mutant c. perfringens strains were analyzed using multivariable logistic regression. the protective effect of the different antisera against development of intestinal necrosis in the loop model was determined by multivariable logistic regression. to account for clustering of loops within a calf, a fixed factor was included describing in which calves the experiments were performed. significance was set at p < . and analyses were performed in spss v. . (ibm corporation, new york, usa). results were reported as means and standard errors of the means (sem). a wild-type strain and an alpha toxin-deficient strain (∆plc) were tested in an intestinal loop model. the wildtype strain caused necrotic lesions in . % ( / ) of the injected loops, whereas the alpha toxin-deficient strain induced necrosis in significantly fewer loops ( . %; / ) (p < . ). to confirm the role of alpha toxin in lesion development by complementing the deficiency, the ∆plc derivatives carrying the empty shuttle vector (jir ) or the plc + plasmid (jir ) were used. necrotic lesions were observed in only one of the ten ( %) loops injected with the alpha toxin-deficient strain carrying the empty shuttle vector. this is significantly fewer than in the loops inoculated with the wild-type strain ( . %; p = . ). the plc-complemented strain induced necrotic lesions in % ( / ) of the loops, which is comparable to the effect of the wild-type strain. no lesions were detected in the control loops treated with sterile bacterial culture medium (figures and ) . after vaccination with native alpha toxin, the non-toxic c-terminal domain of alpha toxin or the adjuvant quila, serum antibodies produced against native alpha toxin were analyzed by elisa. in all calves vaccinated with the native toxin or with the c-terminal domain, a strong antibody response against alpha toxin was detected weeks after the first immunization. the calves vaccinated with the native toxin had antibody titers of . ± . . calves vaccinated with the non-toxic c-terminal domain of alpha toxin had antibody titers of . ± . . no antialpha toxin response was measured in the calves vaccinated with the adjuvant quila. sheep blood agar was used to examine in vitro neutralization of alpha toxin activity of a wild-type c. perfringens strain by sera from calves immunized with the native alpha toxin (rcpa) or the non-toxic c-terminal fragment of the alpha toxin (cpa - ). plates treated with c. perfringens supernatant exhibited both the inner (perfringolysin o) and outer (alpha toxin) zones of hemolysis. incubation of the supernatant with sera against either rcpa or cpa - did not result in an outer zone of hemolysis, indicating neutralization of alpha toxin activity. these sera had no effect on perfringolysin o activity. incubation with sera from the control calves (quila) had no effect on c. perfringens toxin activities ( figure ). to determine whether the antisera against the vaccines can neutralize the lecithinase activity of alpha toxin, serial dilutions of the antisera were incubated with alpha toxin and its activity was measured on egg yolk lipoproteins. the sera of calves immunized with either the native alpha toxin (rcpa) or the c-terminal fragment of alpha toxin (cpa - ) decreased the activity of alpha toxin, with an inhibitory capacity of respectively . ± . for anti-rcpa or . ± . for the sera raised against cpa - . no effect on alpha toxin activity was observed after incubation with sera from calves immunized only with quila. to determine whether the antisera against the vaccines can inhibit the cytotoxicity of c. perfringens, serial dilutions of the antisera were incubated with c. perfringens supernatant. exposure of the endothelial cells to untreated supernatant resulted in % cell death. antisera raised against either the native alpha toxin (rcpa) or the c-terminal fragment of alpha toxin (cpa - ) protected the endothelial cells from c. perfringens cytotoxicity. sera from the control calves did not neutralize the c. perfringens-induced cytotoxicity. pre-incubating the c. perfringens supernatant with a -fold dilution (± ) of the native alpha toxin antiserum resulted in % neutralization of cytotoxicity, whereas a -fold dilution (± . ) of the antiserum against the c-terminal fragment (cpa - ) was needed to neutralize the cytotoxicity. neutralization of the lesion-inducing potential of c. perfringens by sera raised against the respective vaccines was evaluated in the intestinal loop model. thirteen of the fifteen ( . %) positive control loops inoculated with c. perfringens developed necrosis. injection of loops with c. perfringens combined with sera from control calves (immunized with the adjuvant quila) also resulted in a high percentage of necrotic loops ( . % of the loops, / ). injection of loops with c. perfringens combined with antisera raised against native alpha toxin (rcpa) resulted in significantly fewer necrotic loops as compared to the loops containing c. perfringens and the quila antisera (p = . ) and borderline significantly fewer necrotic loops as compared to the untreated loops (p = . ) ( . % of the loops or / ). antisera raised against the non-toxic c-terminal fragment of alpha toxin (cpa - ) did not significantly neutralize the lesioninducing ability of c. perfringens ( / or . % necrotic loops) (figure ). alpha toxin is involved in the induction of necrotic lesions in a calf intestinal loop model and is thus an important toxin in the pathogenesis of enterotoxaemia. we previously showed that alpha toxin production by c. perfringens is required for intestinal virulence by using a double-mutant c. perfringens strain devoid of alpha toxin and perfringolysin o, which was complemented for perfringolysin o to generate an alpha toxin-deficient phenotype [ ] . in the present study, we supported our earlier conclusions by using an alpha toxin-mutant strain. mutant strains are frequently used to evaluate the intestinal loops inoculated with sterile cell culture medium (n = ), the wild-type strain (n = ), the alpha toxin-deficient strain (Δplc) (n = ), the alpha toxin deficient strain carrying the empty shuttle vector [Δplc (shuttle vector)] (n = ) and the alpha toxin-complemented strain (n = ) were histologically examined for the presence of tissue necrosis. the graph represents the percentage of loops in which necrotic lesions were present after h of incubation with logarithmic stage cultures. ** . ≤ p < . and ***p < . indicate a significant difference relative to the loops inoculated with the wild-type strain. virulence effect of c. perfringens genes. a mutant strain was used to show that netb is crucial for the induction of avian necrotic enteritis [ ] . this approach also identified beta toxin as an essential virulence factor of c. perfringens type c in infected rabbits [ ] . moreover, awad et al. used mutant strains to demonstrate that both alpha toxin and perfringolysin o are involved in the pathogenesis of gas gangrene [ , ] . in our study, we confirmed that alpha toxin is required for intestinal virulence in a calf intestinal loop model. this conclusion was based on genetic evidence showing that an alpha toxin-deficient strain has a decreased ability to cause necrotic lesions in this model. the alpha toxin-complemented strain regained the ability to cause the disease, unambiguously fulfilling falkow's molecular koch's postulates [ ] . in the present study, alpha toxin appeared to be a promising vaccine component against bovine necrohemorrhagic enteritis. antisera raised against native alpha toxin reduced the lesion-inducing potential of c. perfringens in the intestinal loop model. however, alpha toxin is a potent dermonecrotic toxin that is not safe for use in calves. alpha toxin can be rendered safe by formaldehyde treatment, but a well-known problem of this treatment is that it might reduce immunogenicity [ , [ ] [ ] [ ] . therefore, a recombinant c. perfringens alpha toxoid may be preferable to a formaldehyde toxoid. the immunogenicity of the c-terminal fragment of alpha toxin in calves was recently reported for the first time [ ] . however, the ability of the antiserum derived after vaccination of calves with cpa - to neutralize the toxicity of c. perfringens to bovine cells or bovine intestine has not been evaluated [ ] . here, we report that the non-toxic c-terminal domain of alpha toxin (cpa - ) may be an effective alternative to the use of native alpha toxin. indeed, calves immunized with the native alpha toxin or with the c-terminal domain of alpha toxin developed a strong immune response against alpha toxin. nevertheless, compared to antisera against the native alpha toxin, sera from calves immunized with the c-terminal fragment of alpha toxin showed weaker inhibition of the alpha toxin activity and weaker neutralization of the c. perfringens-induced endothelial cytotoxicity in vitro. additionally, the lesion-inducing potential of c. perfringens in the intestinal loop model was significantly reduced only by co-administration of antisera from animals vaccinated with the native alpha toxin. the diminished protection afforded by antisera against the c-terminal domain may be attributed to the gst tag fused to the c-terminal domain of alpha toxin for protein purification purposes. distortion of the conformation of the alpha toxin fragment by the gst tag has already been suggested in a previous study reporting that the untagged fragment was more protective against experimental gas gangrene than the c-terminal fragment fused to the gst tag [ ] . in contrast to the c-terminal fragment of alpha toxin, the recombinant native alpha toxin used in this study was fused to a his tag for purification. this his tag is substantially smaller than the gst tag and is less likely to influence the conformation of the alpha toxin. this construct might generate more antibodies against the conformational epitopes that are important for protection. alternatively, it may be that, in addition to antibodies directed to the c-terminal fragment of alpha toxin, also antibodies against the n-terminal fragment are needed to provide protection. however, a previous study showed that immunization with the n-terminal domain of alpha toxin was not protective against experimental gas gangrene [ ] . it is believed that membrane binding induces a conformational change in the n-terminal domain from the closed to open configuration, which could reduce the affinity of antibodies raised against the n-terminal domain and complicates the development of protective antibodies against this n-terminal region [ , ] . moreover, the combination of both toxin domains as vaccine antigen is not straightforward because combination of both non-toxic fragments restores the biological activity of alpha toxin [ ] . total protection was not obtained even after vaccination with native alpha toxin. it is possible that not all alpha toxin was neutralized by the antisera, leaving residual active alpha toxin to exert cytotoxicity. we also do not know whether in the field serum antibodies leaking into the intestinal lumen after intestinal damage will be sufficient to inhibit alpha toxin and the induction of necrotic lesions. this should be tested in a subsequent study by performing intestinal loop experiments in immunized animals without adding antiserum to the ligated intestinal loops. it is possible that total protection against development of intestinal lesions also requires other neutralizing antibodies, for example, against perfringolysin o and/or other c. perfringens proteins. therefore, other c. perfringens proteins in addition to alpha toxin and perfringolysin o might have to be incorporated in a vaccine to obtain complete protection. this is also the case for avian necrotic enteritis, where netb is essential to cause disease, but vaccination with netb provides only partial protection against c. perfringens challenge [ ] [ ] [ ] . endothelial damage is probably a key event in the pathogenesis of bovine necrohemorrhagic enteritis [ , ] . initial epithelial damage could enable alpha toxin to penetrate the epithelial barrier and to act on endothelial cells. in addition to other infectious agents, such as coccidia, enteropathogenic bacteria, coronaviruses and rotaviruses, several c. perfringens factors can contribute to initial epithelial damage, such as collagenase (kappa toxin), hyaluronidase (mu toxin) and mucinase [ ] [ ] [ ] [ ] . more research is needed to investigate the role of these factors in the pathogenesis of necrohemorrhagic enteritis and the protective effect of neutralizing antibodies against these proteins. in this study, we used the calf intestinal loop model to evaluate the vaccine potential of c. perfringens alpha toxin. ideally, vaccinated animals should be challenged with crude toxins or bacterial cultures to obtain conclusive evidence that vaccination against c. perfringens alpha toxin protects against bovine necrohemorrhagic enteritis. however, no challenge model for testing vaccine candidates in calves is yet available [ , , ] . the intestinal loop model remains currently the best available model. in conclusion, this study shows that the non-toxic c-terminal domain of alpha toxin is a promising antigen for vaccine development. although antibodies against c. perfringens alpha toxin neutralize alpha toxin activity and c. perfringens-induced endothelial cytotoxicity in vitro, antibodies against alpha toxin alone are inadequate for complete neutralization of c. perfringens-induced necrosis in the intestinal loop model of bovine necrohemorrhagic enteritis. the development of a multivalent vaccine combining the c-terminal fragment of alpha toxin with other still unidentified c. perfringens virulence factors might be necessary for complete protection against bovine necrohemorrhagic enteritis. clostridium perfringens: toxinotype and genotype virulence genes of clostridium perfringens clostridial enteric diseases of domestic animals the synergistic necrohemorrhagic action of clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells the histotoxic clostridial infections of man clostridial gas gangrene: evidence that alpha and theta toxins differentially modulate the immune response and induce acute tissue necrosis bacterial intestinal flora associated with enterotoxaemia in belgian blue calves cattle enterotoxaemia and clostridium perfringens: description, diagnosis and prophylaxis enterotoxaemia-like syndrome and clostridium perfringens in veal calves immunization with the c-domain of alpha -toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with clostridium perfringens naturally occurring clostridium perfringens nontoxic alpha-toxin variant as a potential vaccine candidate against alpha-toxin-associated diseases the in vitro production of α toxin, θ haemolysin and hyaluronidase by strains of cl. welchii type a, and the relationship of in vitro properties to virulence for guinea-pigs a genetically engineered vaccine against the alpha-toxin of clostridium perfringens protects mice against experimental gas gangrene analysis of protection afforded by a clostridium perfringens alpha-toxoid against heterologous clostridial phospholipases c molecular variation between the alpha-toxins from the type strain (nctc ) and clinical isolates of clostridium perfringens associated with disease in man and animals identification and molecular analysis of a locus that regulates extracellular toxin production in clostridium perfringens virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alphatoxin in clostridium perfringens-mediated gas gangrene in vitro inhibitory effect of hen egg white lysozyme on clostridium perfringens type a associated with broiler necrotic enteritis and its alpha-toxin production lesion development in a new intestinal loop model indicates the involvement of a shared clostridium perfringens virulence factor in haemorrhagic enteritis in calves hemolytic and sphingomyelinase activities of clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway epitope mapping of the alpha-toxin of clostridium perfringens culture of human endothelial cells derived from umbilical veins. identification by morphologic and immunologic criteria neutral red uptake assay for the estimation of cell viability/cytotoxicity netb, a new toxin that is associated with avian necrotic enteritis caused by clostridium perfringens beta toxin is essential for the intestinal virulence of clostridium perfringens type c disease isolate cn in a rabbit ileal loop model synergistic effects of alpha-toxin and perfringolysin o in clostridium perfringensmediated gas gangrene molecular koch's postulates applied to bacterial pathogenicity-a personal recollection years later alpha toxoid of clostridium perfringens. i. purification and toxoiding of alpha toxin of c. perfringens gas gangrene: an open and closed case immunization of broiler chickens against clostridium perfringens-induced necrotic enteritis induction of potential protective immunity against enterotoxemia in calves by single or multiple recombinant clostridium perfringens toxoids crystal structure of the c. perfringens alpha-toxin with the active site closed by a flexible loop region role of the c-domain in the biological activities of clostridium perfringens alphatoxin protection against avian necrotic enteritis after immunisation with netb genetic or formaldehyde toxoids vaccination with recombinant netb toxin partially protects broiler chickens from necrotic enteritis maternal immunization with vaccines containing recombinant netb toxin partially protects progeny chickens from necrotic enteritis increase of clostridium perfringens in association with eimeria in haemorrhagic enteritis in japanese beef cattle extraintestinal stages of eimeria bovis in calves and attempts to induce relapse of clinical disease clostridium perfringens strains from bovine enterotoxemia cases are not superior in in vitro production of alpha toxin, perfringolysin o and proteolytic enzymes genotyping of calves rotavirus in china by reverse transcription polymerase chain reaction bovine "enterotoxemia this research was supported by the agency for innovation by science and technology, flanders, under contract number . the authors thank dr. amin bredan for critical reading and editing of the manuscript. the authors thank christian puttevils, delphine ameye and astra dhanijns for their technical assistance. the authors acknowledge the support of veterinary surgeons from the department of surgery for the surgical interventions and from the department of obstetrics, reproduction and herd health for providing bovine umbilical cords. the authors thank prof. richard titball for the purified nontoxic c-terminal domain of alpha toxin (cpa - ) and prof. julian rood for providing the bacterial strains used in this study. the authors declare that they have no competing interests. key: cord- -vl i b authors: samuel, arthur s; subbiah, madhuri; shive, heather; collins, peter l; samal, siba k title: experimental infection of hamsters with avian paramyxovirus serotypes to date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: vl i b avian paramyxoviruses (apmvs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (apmv- to - ). only in the case of apmv- , the infection of non-avian species has been investigated. the apmvs presently are being considered as human vaccine vectors. in this study, we evaluated the replication and pathogenicity of all nine apmv serotypes in hamsters. the hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. on the basis of one or more of these criteria, each of the apmv serotypes was found to replicate in hamsters. the apmvs produced mild or inapparent clinical signs in hamsters except for apmv- , which produced moderate disease. gross lesions were observed over the pulmonary surface of hamsters infected with apmv- & - , which showed petechial and ecchymotic hemorrhages, respectively. replication of all of the apmvs except apmv- was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. the majority of apmv-infected hamsters exhibited transient histological lesions that self resolved by days post infection (dpi). all of the hamsters infected with the apmvs produced serotype-specific hi or neutralizing antibodies, confirming virus replication. taken together, these results demonstrate that all nine known apmv serotypes are capable of replicating in hamsters with minimal disease and pathology. paramyxoviridae is a large and diverse family whose members have been isolated from many species of avian, terrestrial, and aquatic animal species around the world [ ] . paramyxoviruses are pleomorphic, enveloped, cytoplasmic viruses that have a non-segmented, negative-sense rna genome. the family is divided into two subfamilies, paramyxovirinae and pneumovirinae, based on their structure, genome organization, and sequence relatedness [ ] . the subfamily paramyxovirinae contains five genera: respirovirus, rubulavirus, morbillivirus, henipavirus, and avulavirus, while the subfamily pneumovirinae contains two genera, pneumovirus and metapneumovirus [ ] . all paramyxoviruses that have been isolated to date from avian species can be segregated into two genera based on the taxonomic criteria mentioned above: genus avulavirus, whose members are called the avian paramyxoviruses (apmv), and genus metapneumovirus, whose members are called avian metapneumoviruses. the apmv of genus avulavirus are separated into nine serotypes (apmv- through - ) based on hemagglutination inhibition (hi) and neuraminidase inhibition (ni) assays [ ] . various strains of apmv- , which is also called newcastle disease virus (ndv), have been analyzed in detail by biochemical analysis, genome sequencing, and pathogenesis studies, and important molecular determinants of virulence have been identified [ ] [ ] [ ] [ ] [ ] . as a first step in characterizing the other apmv serotypes, complete genome sequences of one or more representative strains of apmv serotypes to were recently determined, expanding our knowledge about these viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . apmv- comprises all strains of ndv and is the best characterized serotype because of the severity of disease caused by virulent ndv strains in chickens. ndv strains vary greatly in their pathogenicity to chickens and are grouped into three pathotypes: highly virulent (velogenic) strains, which cause severe respiratory and neurological disease in chickens; moderately virulent (mesogenic) strains, which cause mild disease; and nonpathogenic (lentogenic) strains, which cause inapparent infections. in contrast, very little is known about the comparative disease potential of apmv- to apmv- in domestic and wild birds. apmv- strains have been isolated from chickens, turkeys and wild birds across the globe [ , [ ] [ ] [ ] [ ] . apmv- infections in turkeys have been found to cause mild respiratory disease, decreases in egg production, and infertility [ , ] . apmv- strains have been isolated from wild and domestic birds [ ] . apmv- infections have been associated with encephalitis and high mortality in caged birds [ ] [ ] [ ] . apmv- strains have been isolated from chickens, ducks and geese [ ] . experimental infection of chickens with apmv- resulted in mild interstitial pneumonia and catarrhal tracheitis [ ] . apmv- strains have only been isolated from budgerigars (melopsittacus undulatus) and cause depression, dyspnoea, diarrhea, torticollis, and acute fatal enteritis in immature budgerigars, leading to very high mortality [ ] . apmv- was first isolated from a domestic duck and was found to cause mild respiratory disease and drop in egg production in turkeys, but was avirulent in chickens [ , , ] . apmv- was first isolated from a hunter-killed dove and has also been isolated from a natural outbreak of respiratory disease in turkeys. apmv- infection in turkeys caused respiratory disease, mild multifocal nodular lymphocytic airsacculitis, and decreased egg production [ ] . apmv- was isolated from a goose and a feral pintail duck [ , ] . apmv- strains have been isolated from ducks around the world [ , ] . apmv types - , - , and - have been associated with mild respiratory disease and egg production problems in domestic chickens [ ] . there are no reports of isolation of apmv- , - and - from poultry [ ] . but recent serosurveillance of commercial poultry farms in usa indicated the possible prevalence of all apmv serotypes excluding apmv- in chickens [ ] . apmv- (ndv) is known to replicate in non-avian species including humans [ ] [ ] [ ] [ ] [ ] [ ] , although its only natural hosts are birds. apmv- infections in non-avian species are usually asymptomatic or mild. clinical signs in human infections commonly involve conjunctivitis, which usually is transient and self-limiting. presently, apmv- is being evaluated as a vaccine vector against human pathogens [ ] . when administered to the respiratory tract of non-human primates, ndv is highly restricted in replication, but foreign antigens expressed by recombinant ndv vectors are moderately to highly immunogenic. one of the major advantages of this approach is that most humans do not have pre-existing immunity to apmv- . pre-existing immunity is a potential drawback to using vectors derived from common human pathogens, and also can be a concern for any vector if two or more doses are necessary to elicit protective immunity. therefore, we are investigating apmv types to , which are antigenically distinct from apmv- , as alternative human vaccine vectors. also, some of these additional apmv types likely will have differences in replication, attenuation, and immunogenicity compared to apmv- that may be advantageous. however, the replication and pathogenicity of apmv- to - in non-avian species has not been studied. as a first step, we have evaluated the replication and pathogenicity of apmv- to - in hamsters. in this study, groups of hamsters were infected with a prototype strain of each apmv serotype by the intranasal route and monitored for virus replication, clinical symptoms, histopathology, and seroconversion. our results showed that each of the apmv serotypes replicated in hamsters without causing adverse clinical signs of illness, although histopathologic evidence of disease was observed in some cases, and also induced high neutralizing antibody titers. the following nine prototype strains of apmv serotypes to were used, apmv- , ndv lentogenic strain lasota/ ; apmv- , apmv- /chicken/california/ yucaipa/ ; apmv- , apmv- /pkt/netherland/ / ; apmv- , apmv- /duck/hongkong/d / ; apmv- , apmv- /budgerigar/kunitachi/ ; apmv- , apmv- /duck/hongkong/ / / ; apmv- , apmv- /dove/ tennessee/ / ; apmv- , apmv- /goose/delaware/ / ; apmv- , apmv- /duck/new york/ / . all of the viruses were grown in -day-old embryonated specific-pathogen-free (spf) chicken eggs inoculated by the allantoic route, except for apmv- , which was grown in african green monkey kidney (vero) cells (atcc, manassas, va, usa) [ ] . the allantoic fluids from infected eggs were collected h post-inoculation and virus titers were determined by hemagglutination (ha) assay with . % chicken rbc except in the case of apmv- , which was titrated by plaque assay on vero cells. the apmv- samples were inoculated in triplicate onto -well plates of vero cells at % confluency, incubated for h, washed with pbs, overlaid with . % methylcellulose, and observed for plaque production till days post infection (dpi). the cells were fixed with methanol and stained with % crystal violet. values for each tissue sample were based on average plaque count from three wells. vero cells were grown in earle's minimum essential medium (emem) with % fetal bovine serum (fbs). chicken embryo fibroblast (df- ) cells were grown in dulbecco's minimal essential medium (dmem) containing % fbs at °c with % co . for each of the prototype strains, virions were purified on discontinuous sucrose gradients [ ] . the viral proteins were denatured and reduced and were separated on % sds-polyacrylamide gels and negatively stained using e-zinc ™reversible stain kit (pierce, rockford, il, usa). each serotype was analyzed on a separate gel to avoid cross contamination. the n protein bands were excised from the gels and destained with tris-glycine buffer, ph . the excised gel bands were minced in a clean pestle and mixed with elution buffer ( mm tris-hcl buffer ph , mm nacl, . mm edta, mm dtt and . % sds) and transferred to the upper chambers of nanosep centrifugal device (pall life sciences, ann arbor, mi, usa). the proteins were eluted by centrifugation at × g for minutes, and the eluted proteins were quantified and μg of each protein was mixed in complete freund's adjuvant and injected subcutaneously into a rabbit. after two weeks, a booster immunization was given with μg of protein in incomplete freund's adjuvant and two weeks later the hyperimmune sera were collected. the sera were tested by western blot and were found to recognize specifically the homologous n protein (data not shown). the sera were stored at - °c until further use. to study viral replication and pathogenicity, fifty seven -week-old syrian golden hamsters (charles river laboratories inc, wilmington, ma, usa) were housed in negative-pressure isolators under bio safety level (bsl)- conditions and provided feed and water ad libitum. the animals were cared for in accordance with the animal welfare act and the guide for care and use of laboratory animals and the protocols were approved by the institution's iacuc. hamsters in groups of six were inoculated intranasally with μl of infectious allantoic fluid containing hau of each individual apmv, except for apmv- , which contained × pfu/ml, under isoflurane anesthesia. a group of three hamsters served as uninfected controls and were mock infected with normal allantoic fluid. the hamsters were observed three times daily for physical activity and for any clinical signs of illness, and were weighed on day , and dpi. three hamsters from each group were euthanized at dpi and the other three (as well as the three animals in the control group) at dpi by rapid asphyxiation in a co chamber. necropsies were performed immediately postmortem and the following tissue samples were collected for immunohistochemistry (ihc), histopathology, and virus isolation: brain, nasal turbinates, lung, spleen, kidney and small intestine. in addition serum samples were collected on dpi immediately prior to euthanasia, and seroconversion was evaluated by hi assay [ ] . half of each tissue sample was used for virus titration. these samples were collected aseptically in ml of dmem in x antibiotic solution containing units/ml penicillin, ug/ml gentamicin sulfate, and ug/ml amphotericin b (sigma chemical co., st. louis, mo, usa). they were processed immediately to avoid any reduction in virus titers. briefly, a % homogenate of the tissue samples were prepared by using a homogenizer and clarified by centrifugation at × g for min. for all of the serotypes except apmv- , the virus titers in the clarified supernatants were determined by end-point dilution on df- cells. tenfold dilutions of tissue supernatant were inoculated onto df- cells in -well plates and incubated for three days. the plates were fixed in % formalin for min and the cells were permeabilized using % triton x- for min. the plates were washed five times with pbs to remove any residual formalin in the wells. the cells were blocked using % normal goat serum for min and the plates were washed twice with pbs-tween- (pbs-t). the cells were incubated with primary rabbit antiserum raised against the n protein of the homologous apmv serotype at room temperature for h. the plates were washed with pbs twice, min each. the cells were incubated with anti rabbit fitc antibody as secondary antibody for min and washed finally with pbs twice, min each. the slides were visualized and the virus titers were determined by end point titration method using the reed and muench formula, and were photomicrographed using a fluorescent microscope (zeiss axioshop , zeiss, göttingen, germany) [ ] . the virus titers in the supernatants of tissue homogenates from apmv- infected hamsters were determined by plaque assay in vero cells as described above using -fold dilution series. values for each tissue sample were based on average plaque count from three wells. the other half of each tissue sample was used for ihc and histopathology. the tissues were fixed in % neutral buffered formalin, held for approximately seven days, and processed for ihc and histopathology. paraffin embedded -micron sections of all the tissue samples were prepared at histoserve, inc. (maryland, md, usa). the sections were stained with hematoxylin and eosin for histopathology. sections were also immunostained to detect viral n protein using the following protocol. briefly, the tissue sections were deparaffinized in two changes of xylene for min each, hydrated in two changes of % ethanol for min each, changes of % and % ethanol for min each, and finally washed in distilled water. the sections were processed for antigen retrieval in a water bath containing sodium citrate buffer ( mm citric acid, . % tween , ph . ), at - °c for min and then allowed to cool to room temperature for another min. the sections were rinsed in pbs-tween , twice for min each. the sections were blocked with % bsa in pbs for h at room temperature. the sections were then incubated with a : dilution of the homologous primary n-specific rabbit antiserum in pbs for h in a humidified chamber. after three washes in pbs, the sections were incubated with the secondary antibody (fitc conjugated goat anti-rabbit antibody) for min. after a further wash cycle, the sections were mounted with glycerol and viewed under an immunofluorescence microscope. sera were collected from all the hamsters on dpi and evaluated for seroconversion by hi assay [ ] , except in the case of apmv- . in addition, cross-hi tests were performed to investigate cross reactivity with other apmv serotypes. since apmv- does not cause hemagglutination of chicken rbc, the antibody titer was determined by plaque reduction neutralization assay. briefly, the sera were heat inactivated at °c for min. ten-fold dilutions of sera were made and mixed with a constant amount of apmv- ( × pfu), and incubated at room temperature for h in a shaker. the antigen antibody mixtures were analyzed by plaque assay as described above. the serum titer that reduced plaque numbers by % was the end point titer. four-week-old hamsters in groups of six were inoculated by the intranasal route with ha units of each of the apmv serotypes except apmv- , which was administered at a dose of × pfu/ml. three animals were sacrificed at day and the remaining three animals were sacrificed on day ; following sacrifice, the animals were processed for gross pathology, histopathology, ihc, quantitative virology, and seroconversion. three uninfected animals served as controls and were sacrificed and processed on day . all of the animals were observed three times daily and weighed daily. none of the hamsters infected with any of the apmv serotypes showed any visible clinical signs of disease except those infected with apmv- . all the six hamsters infected with apmv- serotype were dull and had rough skin coat at dpi. they appeared weak and lost weight ( table ) till dpi, but later gained body weight and appeared normal by dpi. none of the hamsters died of disease. the uninfected control hamsters appeared healthy and normal. following sacrifice on days and , the followings organs were removed and examined for gross pathology, histopathology, immunohistochemical analysis, and quantitative virology: brain, lungs, nasal turbinates, small intestine, kidney and spleen. gross pathologic findings were limited to the lungs of hamsters inoculated with apmv- ( figure a ) and apmv- (figure b) , and were observed only at dpi. in apmv- -infected hamsters, the entire pulmonary parenchyma was wet, glistening and edematous, with some areas of reddening due to congestion and hemorrhages. there were several diffuse small round shaped red foci on the lungs. similar gross pathology was also seen in the lungs of apmv- infected hamsters, but with foci that were five times larger than those seen in apmv- infected hamsters. no such gross lesions were observed in the hamsters of the other infected groups at dpi. there were no gross visceral pathologic lesions in any of the infected hamsters at dpi. no lesions were detected in the uninfected control hamsters. mean values with standard error are shown. the values of the difference in body weight at dpi are calculated relative to day for the same group and are expressed in percentage gain (+) or loss (-). histopathological lesions at dpi were observed in the nasal turbinates and lungs in all of the infected groups ( figure , and data not shown). figure e and f show representative examples of the nasal turbinates for apmv- and - , respectively. the most predominant histopathological lesions observed in the nasal turbinates were multifocal necrotic/apoptotic epithelial cells and nuclear pyknosis. along the nasal septum were areas of epithelial necrosis, accumulation of nuclear debris in the mucosal epithelium, vacuolation of epithelial cells, and blunting or loss of cilia. small numbers of mononuclear cells and neutrophils multifocally infiltrated the nasal mucosa, with transcytosis and exocytosis into the nasal cavity. there were small accumulations of inflammatory cells and necrotic cellular debris in the nasal cavity. examples of lung tissue are shown for apmv- (figure a and b) , - (figure c ), and - ( figure d ). all the lung samples from the infected groups exhibited interstitial bronchopneumonia of varying severity at dpi. the inflammatory cell infiltrates were mixed populations of lymphocytes, macrophages, and neutrophils. additionally, bronchiolar and type ii pneumocyte hyperplasia in areas of inflammation with variable degrees of cellular atypia were noticed and the degree of cellular atypia was quite prominent. there were no histopathologic findings for any other organs (brain, small intestine, kidney, and spleen) from any infected hamsters on day , or for any of the hamsters at dpi. deparaffinized sections of the virus-infected and uninfected control tissue (brain, lungs, nasal turbinates, small intestine, kidney, and spleen) were immunostained using polyclonal antisera against the n protein of the homologous apmv serotypes. remarkably, animals infected with apmv- did not show any positive immunofluorescence in any of the tissues examined at or dpi. in animals infected with any of the other apmv serotypes, virus-specific antigens were detected on dpi in the lungs and nasal turbinates (figure ). in the nasal turbinates, virus-positive immunofluorescence was noticed throughout the nasal epithelium lining the turbinate bone, and the harderian gland located near the eye showed complete diffuse fluorescence throughout the organ. in the lungs, the viral antigens were mostly localized in the epithelium surrounding the medium and small bronchi. viral n antigens were not detected in any additional organs of infected hamsters at dpi, and were not detected in any of the organs of infected hamsters at dpi. the organs of uninfected control hamsters were also negative by immunohistochemistry assay. turbinates (a, b) , harderian glands (c, d) and lungs (e, f) from hamsters dpi with apmv- (a), apmv- (b), apmv- (c), apmv- (d), apmv- (e), and apmv- (f). nasal turbinates (g) and lung (h) from a representative uninfected control hamster (magnification, × ). (a) section of nasal turbinate infected with apmv- . immunofluorescence was evident in the ciliated epithelium lining the turbinate bone (white arrows). (b) section of nasal turbinate infected with apmv- . immunofluorescence was evident primarily at the apical surface of the ciliated epithelial cells and in the cytoplasm (white arrows). (c) section of harderian gland infected with apmv- . immunofluorescence was evident primarily in the collecting ducts of the gland and also in the cytoplasm of the infected cells (white arrows). (d) section of harderian gland and nasal turbinate infected with apmv- . immunofluorescence was evident primarily the cytoplasm of the infected cells in the harderian gland (white arrows) and in the epithelial cells lining the turbinates (yellow arrows). (e) section of the lung infected with apmv- . immunofluorescence was evident around the bronchial epithelium (white arrows). (f) section of lung infected with apmv- . immunofluorescence was mainly evident around the bronchiolar epithelium (white arrows). (g) section of nasal turbinate from a mock infected hamster (control). (h) section of lung from a mock infected hamster (control). to analyze sites of virus replication, several organs (brain, lungs, nasal turbinate, small intestine, kidney and spleen) were collected on and dpi, for virus isolation and titration. for all of the apmv serotypes except apmv- , titration was performed in df- cells and the titers were expressed in tcid /g tissue; for apmv- , titration was by plaque assay in vero cells (table ) . virus was detected for a number of apmv serotypes on dpi; no virus was detected in any sample for any serotype on dpi. apmv- was isolated from lungs and nasal turbinates of all three animals, with mean titers of . × and × , respectively. apmv- was isolated from lungs ( / animals) and nasal turbinates ( / animals), with mean titers of . × and . × , respectively. apmv- was isolated from lungs and nasal turbinates of all three animals, with mean titers of . × and . × , respectively. apmv- was isolated from lungs and nasal turbinates of all three animals, with mean titers of . × and . × , respectively, and from the kidneys of one animal ( × ). apmv- was isolated from the lungs and nasal turbinates of all three animals, with mean titers of . × and × , respectively, and from the spleen ( animal, × ) and small intestine ( animal, × ). apmv- was isolated from the nasal turbinates only from all three animals, with a mean titer of . × . no virus was isolated from any tissues of the hamsters infected with apmv- , - and - . also, no virus was isolated from the brain of any hamsters infected with apmv- to - . the virus replication in the hamsters infected with apmvs was further investigated by measuring seroconversion dpi. sera were analyzed by hi assay using chicken erythrocytes (table ) in the case of all of the serotypes except apmv- , which was analyzed by plaque reduction assay. the hi titers of the pre-infection hamsters were or less. all hi titers greater than were considered positive. our results showed that all of the infected hamsters seroconverted at dpi, indicating table virus titers in the indicated tissues from hamsters dpi with apmv serotypes to hamster no. brain spleen kidney small intestine replication by all apmv serotypes. the mean hi titers in hamsters for apmv- , - , - , - , - , - , - , - were : , : , : , : , : , : , : , : , respectively. the mean serum antibody titer of apmv- as determined by virus plaque neutralization test was found to be : . the antigenic relationship among apmv serotypes was evaluated by reciprocal hi tests using these sera, which represent convalescent sera obtained by a single infection of hamsters via the intranasal route. each of the antisera exhibited very high hi titer against the homologous serotype and either no or very low hi titer against heterologous serotypes, with the exception of apmv - and apmv- ( table ). the antiserum specific for apmv- and apmv- exhibited substantial cross-reactivity, although the apmv- -specific antiserum had a four-fold higher titer against apmv- than against apmv- , and the apmv- specific antiserum had a two-fold higher titer against apmv- than against apmv- . these reactions indicated the existence of antigenic relatedness between apmv- and apmv- . apmvs are frequently isolated from a wide variety of avian species and are grouped into nine serotypes based on antigenic reactions [ ] . apmv- (ndv) is the most extensively characterized member of the apmv serotypes. apmv- to - have been isolated from both wild and domestic birds, but their disease potential in wild or domestic birds is largely unknown. apmv- has also been shown to infect a number of non avian species [ ] and presently is being evaluated as a potential human vaccine vector for human pathogens [ ] . therefore, there is a possibility that apmv - to - could also be used as human vaccine vectors for human pathogens. however, the ability of apmv- to - to replicate in mammalian hosts was unknown. hence, we have investigated the replication and pathogenicity of apmv- to - in hamsters inoculated intranasally. the intranasal route was intended to resemble the natural route of apmv infection as well as a likely route for vaccine vector administration. in this study, hamsters were chosen as the mammalian animal model because they are widely used to study replication and pathogenesis of a variety of viruses such as adenovirus [ ] , herpes virus [ ] , nipah virus [ ] , human rsv [ ] , human parainfluenza viruses, sars virus [ ] , and eastern equine encephalitis [ ] . clinical disease was not evident with most of the apmv serotypes, and was observed only in the case of apmv- . all of the animals infected with apmv- exhibited clinical disease including weakness and substantial weight loss. only apmv- and - produced gross pathological lesions in the lungs, whereas in hamsters infected with the other apmv serotypes the appearance of the lungs was unremarkable. no gross pathology was evident for any other organs with any of the serotypes. virus was recovered from animals infected with apmv- , - , - , - , - , and - . in all cases, virus was isolated dpi: no virus was detected in any samples for any serotype dpi. the viral titers were moderate and were mostly restricted to the lungs and nasal turbinates; there were isolated incidences of isolation from the spleen (apmv- ), small intestine (apmv- ), or kidneys (apmv- ). no virus was recovered from any tissue from animals infected with apmv- , - and - . however, the lungs and nasal turbinate tissues from animals infected with apmv- and - were positive by ihc, indicating that these viruses infected and replicated at a level that was below detection by our assays for quantitative virology. although apmv- was not detected by virus isolation or by ihc in any of the tissues, all of the animals that were inoculated with apmv- developed table cross-reactivity of sera from hamsters infected with the indicated apmv serotype (top) against the indicated apmv serotype (left column)* *sera were harvested dpi. cross-reactivity was measured by hi assay except when tested against apmv- , in which case a plaque neutralization assay was used. hi titers represent the highest dilution that displayed hi activity, and are expressed in mean hi units. for apmv- , the % plaque reduction titer is shown as the mean. titers of apmv- specific antibodies detected by virus plaque reduction neutralization assay, indicating a low level of replication. by dpi, no virus could be detected in any of the tissues in any the infected hamsters for any serotype, whether by histopathology, histochemistry, or virus isolation. this suggests that the virus was cleared from all tissues and disease was resolved, indicating the self-limited nature of the infections. similar results have been obtained from chickens infected with apmv- , - and - , where no virus could be isolated at dpi [ ] . using ihc, viral n antigen was detected in the same tissues that were positive by virus isolation. an interesting finding was the presence of large amounts of viral antigens at the epithelial cell linings, suggesting that these cells are highly permissive to apmv replication. in addition, the detection of viral antigens, and in most cases infectious virus, in nasal turbinates and lungs of the hamsters indicate that apmv replication is mostly restricted to the respiratory tract. these results show that all apmv serotypes are capable of infecting hamsters using a nasal route of infection and the extensive amount of virus replication in the respiratory tract was not accompanied by severe disease in hamsters. serologic assays demonstrated a humoral response in all the hamsters inoculated with apmv serotypes - to - , a further indication of successful virus replication. our results show that all apmvs (except apmv- , which does not hemagglutinate) produced hi antibody titers that varied between : to : . based on the hi titers, apmv- ( : ) produced maximum antibody titer and apmv- ( : ) produced the least. paradoxically, while apmv- induced the highest titer of hi antibodies, the virus could not be isolated from any of the infected hamsters. conversely, apmv- replicated efficiently in hamsters as observed by virus isolation in nasal turbinates, lungs and kidneys, but produced low levels of virus-specific hi antibodies. whether these examples of incongruity between levels of replication and serum antibody responses are indicative of differences in the antigenicity of the respective hn proteins or to some other factor remains to be determined. warke et al. [ ] have also provided evidence of low hi titers in the case of apmv- , in chickens, which was taken as evidence of a low level of replication of this virus in chickens. the hi test is the most commonly used method to diagnose apmv infections and also is used to measure the antibody response. but chicken antiserum against ndv cross-reacts in hi tests with several of the other apmv serotypes, thus questioning the specificity of hi test in field cases [ ] . warke et al. [ ] indicated that the hi test lacks sensitivity for detecting infection with these apmv serotypes. they also observed that even in the case of a high infectious dose and observed microscopic changes in the infected organs, high hi titers were not observed in the infected birds. on the contrary in our study, most of the apmv serotypes replicated well in hamsters, producing mild respiratory pathology and high neutralizing antibody titers. our results indicated that the antibody developed in hamsters against these serotypes were very specific and no cross reaction was observed between serotypes except between apmv- and - . cross-reactivity between these two serotypes is not completely unanticipated, since they share the highest level of genome nucleotide sequence identity ( %) among the apmv serotypes [ ] . hence, we conclude that the hi test with hamster serum is highly specific and can be used to diagnose different apmv serotypes in field cases. the replication of apmvs in hamsters produced mild rhinitis and mild pathology that was mainly restricted to the respiratory tract. there was a concern that apmvs may also replicate in the intestine and shed in feces, which might act as a source of infection for the other animals. but our results indicated that none of the apmvs replicate in the intestinal epithelial cells of hamsters. importantly, our results also suggest that these viruses do not cross the blood-brain barrier and do not induce neurological symptoms. also, our previous experimental studies of all the apmvs in chickens showed that they were avirulent. taken together, these results show that the apmvs replicate moderately in hamsters and produce either mild or no clinical signs, and elicit substantial antibody responses. therefore, it is possible that apmvs might replicate in other mammalian species including humans. in conclusion, this study is the first comparative report on the replication and pathogenicity of prototype strains of all apmv serotypes in 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golden hamster model for sars-cov infection the hamster as an animal model for eastern equine encephalitis-and its use in studies of virus entrance into the brain antigenic relationships between avian paramyxoviruses. i. quantitative characteristics based on hemagglutination and neuraminidase inhibition tests submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank daniel rockemann, flavia dias and all our laboratory members for their excellent technical assistance and help. "this research was supported by niaid contract no. n a ( % support) and niaid, nih intramural research program ( % support). the views expressed herein do not necessarily reflect the official policies of the department of health and human services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the u.s. government." authors' contributions as carried out the pathogenesis studies, carried out the immunoassays, performed the analysis, and drafted the manuscript. ms carried out the pathogenesis studies. hs participated in the analysis, interpretation of the authors declare that they have no competing interests. key: cord- -v q spmm authors: resende, talita pilar; medida, ramya lekha; guo, yue; vannucci, fabio a.; saqui-salces, milena; gebhart, connie title: evaluation of mouse enteroids as a model for lawsonia intracellularis infection date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: v q spmm lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. the hallmark feature of l. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. a major limitation to the study of l. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. here we investigated the suitability of mouse enteroids as a model to study l. intracellularis infection. mouse enteroids were microinjected with l. intracellularis, filter-sterilized l. intracellularis culture supernatant, or sterile cell culture media (dmem). l. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. there was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in l. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. l. intracellularis infection did not induce changes in gene expression of ki- (proliferation marker), sox (marker for transit amplifying cells) and muc (marker for goblet cells). these results indicate that although l. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine. pig herds and horse farms worldwide are regularly challenged by proliferative enteropathy (pe). this disease is caused by lawsonia intracellularis and is characterized by the thickening of the small intestinal mucosa due to proliferation of intestinal epithelial cells. pe causes diarrhea and compromised weight gain in pigs [ , ] . in weaned foals, pe causes diarrhea and hypoproteinemia, occasionally resulting in death [ ] . other species are also affected by pe, including ratite birds, rabbits, non-human primates, rats and mice [ ] . lawsonia intracellularis is a gram-negative bacterium that requires an intracellular culture system and a specific atmosphere to be propagated in vitro [ ] . traditional single cell cultures (cell lines) are permissive to l. intracellularis propagation, but they have failed to reproduce the increased cellular proliferation observed in pe-affected animals [ ] . the lack of an in vitro model that represents the in vivo progression of pe has limited advancement in knowledge of the pathogenesis mechanisms utilized by l. intracellularis. hence, identification of new alternatives for control and prevention of pe has been difficult, and pig producers and horse owners have been relying on available commercial vaccines (whole cell vaccines) and antimicrobials to prevent and control pe [ ] [ ] [ ] [ ] . the costs associated with producing whole cell vaccines is reflected on the commercial price of the vaccines, hampering the access to some producers to the commercial vaccines, and, therefore preventing pe. in addition, the use of antimicrobials as growth promoters in swine production has raised concerns about the selection of antibiotic-resistant organisms in treated herds [ ] . therefore, a deeper understanding open access *correspondence: resen @umn.edu department of veterinary and biomedical sciences, college of veterinary medicine, university of minnesota, st. paul, mn , usa full list of author information is available at the end of the article of pe pathogenesis is necessary for the development of novel non-antimicrobial based prevention and treatment methods such as recombinant vaccines or antimicrobial alternative strategies [ ] [ ] [ ] [ ] . the currently held hypothesis is that l. intracellularis infects intestinal epithelial cells, especially in the crypt compartment, leading to cellular proliferation and decreased differentiation in goblet cells along with increased apoptotic events [ , , ] . the mechanisms involved in epithelial changes during the course of pe are still unclear. thus, knowledge about interactions between l. intracellularis and intestinal epithelial cells, determined using a controlled environment, could generate a better understanding of how l. intracellularis induces cellular proliferation. a promising alternative to single cell cultures are tridimensional multi-cell type cultures, also known as organoids. one of the most relevant advantages of organoids in relation to the traditional in vitro single cell culture is the similarity of the organoids to their respective tissue of origin [ , ] . enteroids, small intestinal organoids, for instance, possess not only enterocytes, but also enteroendocrine cells, transit amplifying cells, goblet cells and stem cells. all these cells form structures with cell distributions that are similar to crypts and villi found in the small intestine, and possess a lumen where cell debris and secretion are shed continuously [ ] . enteroids have been recently used to study host-pathogen interactions of important enteric bacteria in human medicine [ ] [ ] [ ] [ ] [ ] , but they have not been investigated as a model for bacterial infections in the field of veterinary medicine [ , ] . the objective of this study was to evaluate mouse enteroids as an in vitro model for l. intracellularis infection. infected enteroids were monitored over time by size along with changes in expression of genes that have been reported to change during pe. we found evidence of infection with l. intracellularis in enteroids followed for up to days post-infection (dpi). the genetic profile of infected mouse enteroids, however, diverged from the gene expression changes observed in pe. mouse enteroid preparation and maintenance were performed as described elsewhere [ ] (iacuc approval number - a). the formulation of the enteroid culture medium was as described previously [ ] . a l. intracellularis isolate (phe/mn - , atcc pta- , manassas, va, usa) at low (≤ ) passage [ ] was propagated in mccoy mouse fibroblast-derived monolayers (atcc ® crl- ™ ). mccoy cells were cultured as described elsewhere [ ] . cells were cultured in dulbeco's modified eagle medium (dmem, gibco thermofisher, waltahm, ma, usa) with % fetal bovine serum (fbs; heat inactivated, corning ™ cv, corning, ny, usa). mccoy cells at % confluency were infected with about l. intracellularis and incubated at °c under a controlled microaerophilic atmosphere created with a mixture of gas containing % hydrogen, % carbon dioxide, and % nitrogen [ ] . seven days after infection, cell monolayers were mechanically lysed by passage through a -gauge needle coupled to a syringe after immersion in sterile . % potassium chloride and then centrifuged at × g to separate cell debris from l. intracellularis organisms. the pellets were discarded and the remaining supernatant was then filtered through . µm sterile filters (millipore sigma, burlington, ma, usa) to remove any remaining mccoy cells and nuclei. after a final centrifugation at × g at °c the bacterial pellet was either used to re-infect mccoy cells, or to prepare the inoculum suspended in approximately µl of its own supernatant and used as inoculum. as l. intracellularis growth requires special atmospheric conditions [ ] , we first tested whether the atmosphere conditions required for l. intracellularis growth would impact growth and development of mouse enteroids. one plate with enteroids was maintained under atmospheric conditions specific for l. intracellularis i.e., % hydrogen, % carbon dioxide, and % nitrogen [ ] while another plate was maintained at % co as a control. both plates were incubated at °c. since l. intracellularis manipulation in vitro requires an antibiotic-free sterile system, antibiotics (penicillin/streptomycin) were excluded from both plates to assess sterility of the cultures. the plates were observed every h for weeks, with media replacement and enteroid passage as described previously. to standardize conditions for infection of mouse enteroids with l. intracellularis, we adopted the "incubation and seed" method [ ] . briefly, immediately after enteroid passage and before plating, enteroids were directly mixed with l. intracellularis suspended in µl of enteroid culture medium (resulting in an inoculum with ~ l. intracellularis organisms/ml), with occasional gentle agitation, and incubated at °c under special atmospheric conditions generated with a gas tank of % hydrogen, % carbon dioxide, and % nitrogen for min. the infected enteroids were briefly centrifuged at × g for min, mixed with - µl ice-chilled matrigel (corning, usa), and then seeded into culture plates. enteroids were harvested days post-infection (dpi) by removing culture media and suspending the matrigel in phosphate buffered saline (pbs). after centrifugation for min at × g, the pellet was suspended in % formaldehyde and incubated for h at room temperature. enteroids were then washed in pbs, centrifuged at × g for min, suspended in histogel (thermofischer, usa) and placed into a biopsy mold. histogel blocks were inserted into a cassette and maintained in % alcohol until they were processed for histological evaluation. four-micrometer sections were placed onto glass slides and used for hematoxylin and eosin (h&e) staining and immunohistochemistry [ ] . although l. intracellularis antigen was detected by ihc in the infected enteroids by this method (figure ), we opted to employ the microinjection approach described below to facilitate contact of l. intracellularis with the cellular apical membrane in an effort to improve the level of infection. mouse enteroids were cultured and passaged until a sufficient number of enteroids (average of enteroids per treatment group, per time point) with µm diameter were obtained for the trial. wnt a protein, which regulates the signaling pathways related to the cellular proliferation in the intestinal crypt compartment [ ] , was removed from the enteroid culture media at least days before infection to enable cells to better differentiate. twenty-four hours prior to infection, enteroids were divided into three treatment groups: dmem: sterile antigen is associated mostly with cell debris (arrow heads), × . b l. intracellularis antigen is associated with cell debris (arrow heads) and in close proximity to enteroid cells (black arrows), × . c l. intracellularis antigen is observed in close association with mouse enteroid cells (black arrows), × . d a few l. intracellularis antigens are observed in this image, some close to mouse enteroid cells (black arrow), and some associated with cell debris (arrow head), × . enteroid lumen is indicated by an asterisk (*). culture media; sn: sterile-filtered l. intracellularis-culture supernatant; l. intracellularis: suspension with about l. intracellularis organisms/ml. each enteroid received approximately nl of each respective treatment. treatments were placed directly into the lumen of the enteroids by microinjection using a microinjector (nanoject ii, drumond scientific, broomall, pa, usa) operated by a trained technician. experiments were performed in four independent replicates. microinjected enteroids were monitored for week, from the day prior to infection until dpi. images were captured at × magnification using an inverted microscope coupled with a digital camera immediately after injection ( dpi), and at , and dpi. the enteroid area was measured using an image software (nih imagej software . r, national institute of health, bthesda, md, usa). the relative enteroid area (enteroid growth) was measured by subtracting the enteroid area from the area recorded at its previous measurement. enteroids from each treatment group were harvested at , and dpi and prepared for immunofluorescence. matrigel was disrupted with a sterile pipet tip and the contents in each well were transferred to a . ml tube and centrifuged at × g for min. the supernatant was discarded, and the pellet suspended in ml of pbs. this step was repeated two times to remove matrigel. the resulting enteroid pellet was suspended and fixed in % paraformaldehyde for h at room temperature. after fixation, enteroids were washed three times in pbs and centrifuged at × g for min. fixed enteroids were embedded in histogel (richard-allan scientific histogel, thermofischer, usa) and then either placed in optimum cutting temperature (oct, tissue-tek; sakura finetek, beaver creek, co, usa) for preparing cryosections or dehydrated in % ethanol and embedded in paraffin. paraffin-embedded sections ( μm) on charged slides were used for ihc for detection of l. intracellularis using specific rabbit polyclonal antibodies and a previously described method [ ] . stained sections were evaluated by bright field microscopy. oct-embedded blocks were sectioned ( µm), placed on charged glass slides and stored at − °c until immunofluorescence staining. anti-ki- immunofluorescence was performed as described elsewhere [ ] . briefly, slides were hydrated with tris buffered saline (tbs) containing . % triton-x (tbs-t) for min and then blocked with % normal goat serum (zc , vector laboratories, burlingame, ca, usa) for min to prevent non-specific binding. sections were then incubated with anti-ki- antibody ( : in tbs-t, crm b, biocare medical, pacheco, ca, usa) for h, followed by a min wash in tbs-t and incubation with cy -labeled goat antirabbit antibody ( : , ab , abcam, burlingame, ca, usa) for min. all incubations were carried out at room temperature. slides were washed and mounted with prolongold (thermofisher, usa) and covered with glass coverslips. the number of ki- -positive cells and the total number of cells were counted in at least random fields at × using an olympus bx fluorescence microscope. enteroids were harvested at , , and dpi, released from matrigel as described above, lysed with . ml of trizol (thermofisher, usa) and stored at − °c until rna extraction. rna extraction was performed using chloroform and isopropanol precipitation following trizol protocols and then cleaning with the rneasy mini kit (qiagen, germantown, md, usa) according to the manufacturer's instructions. total rna was quantified using a nanodrop (thermo scientific, usa) and ng of rna were transcribed to cdna using the high capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa) with random hexamer primers. quantitative pcr reactions were performed using sybr green pcr master mix (applied biosystems) according to the manufacturer's instructions. amplification was performed with the following conditions: initial activation at °c for min, followed by cycles of denaturation at °c for s and annealing at °c for s. expression of the housekeeping gene gapdh was monitored as reference. relative gene expressions were normalized to gapdh using the primer efficiencies. the expression levels of ki- (marker for cellular proliferation), sox (marker for transit amplifying cells) and muc (marker for goblet cells) in enteroids of all treatment groups were measured at , and dpi. fold change in each case was calculated in relation to expression in enteroids from day (control). all primers used are listed in table . results are presented as a mean ± standard deviation of the mean (sem) for each group. two-way anova followed by geisser-greenhouse correction was used to verify statistical differences with p < . considered statistically significant. graphpad prism . software for windows (graphpad software, la jolla, ca, usa) was used to perform the analysis. there was no difference in growth or morphology between enteroids maintained for week in the microaerophilic atmosphere relative to those maintained under regular conditions, i.e., % co incubator (data not shown), indicating that growth conditions required for l. intracellularis do not impact enteroid viability or growth. mouse enteroids were infected with l. intracellularis by microinjection. enteroids were harvested and analyzed by ihc for l. intracellularis antigen at , , and dpi. l. intracellularis antigen presence was observed at all time points, indicating successful infection with l. intracellularis. unexpectedly, although l. intracellularis antigen was observed in the cytoplasm of enteroid cells, most of the antigen, especially at dpi, was found in the basal region of epithelial cells (figure ). this is in contrast to the location of l. intracellularis in vivo, which is in the apical region of the epithelial cells [ , ] . to determine whether l. intracellularis infection would lead to increased size or secretory activity of infected enteroids, the area of enteroids in each treatment group was measured at , , and dpi. l. intracellularisinfected enteroids did not have higher area compared with sn or dmem treatment groups ( figure a ). representative images of enteroids from each treatment group are shown in figure b . to determine whether l. intracellularis infection induces changes in the proliferation and differentiation of enteroid epithelial cells, as observed in the swine intestine, we evaluated expression of ki- , sox and muc in enteroids harvested at , and dpi relative to expression in enteroids at dpi. there were no significant differences in expressions of any of the genes analyzed in the treatment groups independent of the time point (figure ) . immunofluorescence staining confirmed that none of the treatment groups showed increased ki- expression overtime, although in the l. intracellularis infected group considerable variability in the in ki- expression throughout was noted ( figure ). classical single cell culture systems have been extensively used in studies to understand host-pathogen interactions. although they provide valuable information, these models are limited by their inability to represent the tissue organization observed in vivo since most of the single cell systems are either cancer-derived or transformed immortalized cell lines [ , ] . these limitations make it difficult to assess the proliferative effects of l. intracellularis infection [ ] . the mechanism by which l. intracellularis causes proliferation of intestinal epithelial cells remains unclear. recently, the effects of l. intracellularis infection on the proliferation of monocultures of non-intestinal, non-epithelial cells, as well as intestinal epithelial cells were investigated under various culture conditions [ ] and the findings supported previous observations that cell monocultures infected with l. intracellularis do not accurately represent proliferation observed in lesions in the intestine of affected animals. in vivo, l. intracellularis accesses the cell cytoplasm via the apical membrane and the organisms are observed in the cytoplasm throughout the course of pe. l. intracellularis is more frequently observed in crypt cells than cells of the villus [ , , ] . therefore, we hypothesized that enteroids would be a suitable model to further investigate l. intracellularis pathogenesis in vitro by providing polarized epithelial cell culture with all the crypt and villus epithelial cell types found in the mammalian intestine and similar cell exchange ratio to the in vivo intestine [ ] . enteroids were firstly developed in [ ] and have been used to study intestinal morphophysiology as well as pathogenesis of some enteric diseases [ , , , ] . human enteroids can be obtained by culture of intestinal crypts isolated from biopsies. mouse enteroids are more widely used in research as the ethical and biosafety regulations for human enteroids are more restrictive. additionally, mouse intestinal physiology is well-characterized and molecular tools to study intestinal cell proliferation and differentiation are available. the presence of l. intracellularis dna has been detected in feces of mice trapped in pig farms and in the surroundings of horse farms [ , ] , suggesting that mice are susceptible to infection. furthermore, mice experimentally infected with l. intracellularis develop intestinal lesions that resemble lesions in affected pigs and horses, although the lesions in mice are less extensive, less severe and mostly localized in the large intestine when compared to the lesions in pigs and horses [ ] [ ] [ ] . in addition, l. intracellularis has been successfully propagated in vitro using mccoy mouse fibroblast cells [ , , ] . based on this knowledge, we hypothesized that mouse enteroids may serve as a feasible model to investigate the progression of l. intracellularis infection and the mechanisms involved during cell proliferation. mouse enteroids in the tridimensional culture conditions, as spheres composed of polarized epithelium, have their cellular apical side oriented to the center of the enteroid. hence, l. intracellularis infection in this study was performed by microinjecting the bacterial suspension directly into the lumen of the enteroid. microinjection is a technique that has inherent limitations in controlling the bacterial inoculum, i.e., the exact number of bacteria each enteroid was exposed to. although we plated enteroids to obtain an average of enteroids per well, the actual number of enteroids per well and their size at the time of injection was found to be variable. these technical limitations may have contributed to the high variability observed in our results and constitute one of the limitations of using tridimensional enteroids as models for the study of luminal pathogens. others have used mouse enteroids [ , ] and human enteroids [ , ] to study bacterial pathogenesis using microinjection. however, none of these studies evaluated infection times longer than h [ , ] . because of the nature of l. intracellularis infection, longer durations, i.e., several days, are needed to observe the expected changes. to our knowledge, this is the first enteroid infection experiment reporting successful bacterial infection of enteroids for up to dpi. whether the length of the infection times had a significant effect on the number of bacteria present and variability on the evaluated parameters remains to be defined. one of the most intriguing findings in the present experiment was the unexpected location of l. intracellularis in the infected enteroids. while l. intracellularis antigen was observed at all time points tested ( , and dpi), the antigen was mostly observed localized in the extracellular space proximal to the basal region of the epithelium of the mouse enteroids. in vivo, l. intracellularis organisms are consistently observed on the apical side of the cytoplasm at similar infection time points to those used in this study [ , ] . a possible explanation for this difference in localization is that, in contrast to conditions in vivo, nutrients necessary for enteroid growth and maintenance are placed over the matrigel, in a region that corresponds to the basal lamina in vivo and not directly in the enteroid lumen. we speculate that the gradient of nutrients on the exterior of the enteroid could be causing the l. intracellularis to move towards the nutrients to support their growth. it is also possible that conditions of the enteroid culture environment could result in less permissible conditions for l. intracellularis closer to the lumen. another possibility is that the fibroblasts and immune cells in the basal region of the intestinal epithelium in vivo may have a regulatory effect on l. intracellularis traffic in the intestine, and the lack of those cells in the mouse enteroid culture would modify l. intracellularis traffic in vitro. immunohistochemistry for l. intracellularis is considered the gold standard method for diagnosis of proliferative enteropathy [ , ] . in the present study, we used immunohistochemistry to verify whether the mouse enteroids, microinjected with l. intracellularis suspension (~ l. intracellularis organisms/ml), were infected and to visually demonstrate the level of infection in these microinjected enteroids. our results were not only surprising in regards to the localization of l. intracellularis antigen, as discussed before, but also in regards to the amount and morphology of the stained antigen. generally, l. intracellularis antigen stained by immunohistochemistry is observed as small bacilli in the cytoplasm of cell cultures [ , , ] and in the apical cytoplasm of intestinal epithelial cells of naturally and experimentally-infected pigs. in the present study, instead of observing similar morphology of l. intracellularis antigen, we observed mostly the antigen as a "bundle" accumulated on the basal region of the enteroid cells. nevertheless, occasional well-shaped l. intracellularis antigens were observed in the cytoplasm of some enteroid cells ( figure d ), which is an indication that after the microinjection in the lumen of the enteroid, l. intracellularis organisms gained entrance into the cell cytoplasm. the events involving the changes in the morphology of the antigen and its final location in the basal side of enteroid cells remains unclear and may deserve more investigation. the homeostasis of the intestinal epithelium involves various signaling pathways of which canonical wnt signaling is responsible for maintaining a balance between the pool of undifferentiated cells with proliferation capacity, named transit amplifying cells, and differentiated cells [ , ] . in vitro, when enteroids are cultured in the presence of wnt a in the media, the transit amplifying cells stay undifferentiated for a longer period of time in relation to in vivo conditions [ ] , resulting in enteroids in a pro-proliferative state. in the present study, one of the main objectives was to verify the effects of l. intracellularis on cell proliferation, therefore we removed wnt a from the culture media days before microinjection and during the experiment to allow the epithelial cells to differentiate, and better reproduce the proportion of proliferative cells of the normal intestinal epithelium. however, we failed to detect increased proliferation in l. intracellularisinfected enteroids by rt-qpcr or immunofluorescence. it is possible that the removal of wnt a days before microinjection was not sufficient to reduce proliferation to a level where we could detect changes induced by l. intracellularis. another possible explanation for this lack of difference in proliferation between treatment groups was the small amount of l. intracellularis delivered to the enteroid lumen, thus requiring a longer time for the bacteria to replicate in numbers to produce a detectable change in proliferation markers. the low amount of antigen detected in the microinjected enteroids indicate that l. intracellularis did not encounter an ideal environment for propagation as it does in mccoy cells and in the intestines of mice and other rodents [ , , , , ] . the infection efficacy of enteroids derived from the intestines of more l. intracellularis susceptible species, such as pigs and horses, would help to confirm this hypothesis. changes in the area of enteroids can indicate an expansion on the number of cells or an increase in secretory activity. secretory activity of enteroids has been demonstrated by swollen enteroids as a result of accumulation of luminal secretions after stimulation with forskolin [ ] . in the present study, we expected to observe increased enteroid areas of infected enteroids that could be due to increased numbers of cells (proliferation) or secretory activity. in contrary to our expectations, we observed an increased volume from dpi to dpi on the sn group without changes in proliferation, suggesting the possibility of a secreted product with capacity to induce intestinal secretion. further confirmation of this phenomenon is required. l. intracellularis is a unique bacterium, requiring intracellular conditions and special atmospheric conditions for propagation in vitro. little is known about l. intracelllaris survival in the cellular cytosol, or the host-cellular pathways that are disrupted during l. intracellularis infection. by using mouse enteroids as an in vitro model for l. intracellularis infectioin, our aim was to develop a tool to further define several aspects of l. intracellularis pathogenesis. the localization of l. intracellularis more abundantly in outside the critical threshold of lawsonia intracellularis in pig faeces that causes reduced average daily weight gains in experimentally challenged pigs proliferative enteropathy lawsonia intracellularis infection and proliferative enteropathy in foals recent advances in understanding the pathogenesis of lawsonia intracellularis infections intracellular bacteria of porcine proliferative enteropathy: cultivation and maintenance in vitro effects of lawsonia intracellularis infection in the proliferation of different mammalian cell lines a novel inactivated vaccine against lawsonia intracellularis induces rapid induction of humoral immunity, reduction of bacterial shedding and provides robust gut barrier function field evaluation of an oral attenuated lawsonia intracellularis vaccine for porcine proliferative enteropathy (ileitis) identifying obstacles to reducing the use of antibiotics to control porcine proliferative enteropathy lawsonia intracellularis: revisiting the disease ecology and control of this fastidious pathogen in pigs antimicrobial stewardship in veterinary medicine novel small molecules affecting cell membrane as potential therapeutics for avian pathogenic escherichia coli small molecules that sabotage bacterial virulence modeling rotavirus infection and antiviral therapy using primary intestinal organoids lawsonia intracellularis exploits β-catenin/ wnt and notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation lawsonia intracellularis in pigs: progression of lesions and involvement of apoptosis growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications modeling development and disease with organoids evaluating shigella flexneri pathogenesis in the human enteroid model a small intestinal organoid model of non-invasive enteric pathogen-epithelial cell interactions convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions interaction of salmonella enterica serovar typhimurium with intestinal organoids derived from human induced pluripotent stem cells intestinal organoids model human responses to infection by commensal and shiga toxin producing escherichia coli characterization of porcine intestinal enteroid cultures under a lipopolysaccharide challenge porcine intestinal enteroids: a new model forstudying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response establishment of human epithelial enteroids and colonoids from whole tissue and biopsy dietary fiber sources and non-starch polysaccharidedegrading enzymes modify mucin expression and the immune profile of the swine ileum attenuation of virulence of lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy comparison of intestinal mucosa homogenate and pure culture of the homologous lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine an alternative method for cultivation of lawsonia intracellularis adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids preparation and characterization of polyclonal and monoclonal antibodies against lawsonia intracellularis extrinsic control of wnt signaling in the intestine the third dimension bridges the gap between cell culture and live tissue evaluation of porcine ileum models of enterocyte infection by lawsonia intracellularis transmissible lleal hyperplasia of hamsters. ii. ultrastructure adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration single lgr stem cells build crypt-villus structures in vitro without a mesenchymal niche human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology adenovirus infection of human enteroids reveals interferon sensitivity and preferential infection of goblet cells lawsonia intracellularis in the feces of wild rodents and stray cats captured around equine farms colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms infection of sparrows (passer domesticus) and different mice strains with lawsonia intracellularis evaluation of the involvement of mice (mus musculus) in the epidemiology of porcine proliferative enteropathy validation of an immunoperoxidase monolayer assay as a serologic test for porcine proliferative enteropathy comparison of different methods for diagnosis of porcine proliferative enteropathy entry of the bacterium ileal symbiont intracellularis into cultured enterocytes and its subsequent release characterization of lawsonia intracellularis gen. nov., sp. nov., the obligately intracellular bacterium of porcine proliferative enteropathy the intestinal crypt, a prototype stem cell compartment infection of different strains of mice with lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy a functional cftr assay using primary cystic fibrosis intestinal organoids publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors acknowledge the technical support provided by marta ferrandis-vila, caitlin kupiec, ashley manicor, amanda g. de souza daniel, erika vasquez, and lacey marshall-lund. of the basal side of the enteroid epithelial cells may indicate nutrients or a microenvironment preferred by l. intracellularis and merits further investigation. likewise, little is known about mouse enteroids maintained with a pathogenic enteric bacterium for a period longer than h [ , ] . more information is needed to determine if long-term infection of enteroids mirrors the dynamics of epithelium in vivo in terms of secretion, differentiation and apoptosis observed in chronic enteric diseases. finally, yet importantly, although we were able to demonstrate infection of mouse enteroids, we did not observe the changes associated with l. intracellularis infection that have been noted in vivo in more susceptible species. testing l. intracellularis infection in enteroids from other more susceptible species will provide further insight to species susceptibility to pathogenesis. authors' contributions tpr contributed to the acquisition, analysis and interpretation of data and preparation of the manuscript. rlm contributed to execution of the experiments, data acquisition and interpretation. yg contributed in the execution of the experiments. cg, fav and mss contributed to the conception, design, data analysis and interpretation of the study. all authors read and approved the final manuscript. the authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: tpr was supported by the brazilian government sponsoring agency coordenadoria de aperfeiçoamento de pessoal de nível superior (capes). the project was partially supported by nifa min- - (mss). the data collected in this project is shown in our results section. further information can be available upon request. mouse manipulation was approved by the university of minnesota institutional animal care and use committee (iacuc approval number - a). the authors declare that they have no competing interests. key: cord- - ib yws authors: xie, xing; lin, yan; pang, maoda; zhao, yanbing; kalhoro, dildar hussain; lu, chengping; liu, yongjie title: monoclonal antibody specific to ha glycopeptide protects mice from h n influenza virus infection date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ib yws canine influenza virus (civ) subtype h n is a newly identified, highly contagious respiratory pathogen that causes cough, pneumonia and other respiratory symptoms in dogs. data indicate that the virus is responsible for recent clinical cases of dog disease in china. however, therapeutic options for this disease are very limited. in this study, seven monoclonal antibodies (mabs) against civ js/ (an h n subtype virus) were produced and characterized. among them, mab d , which is specific for the ha glycopeptide (gp), induced the highest neutralization titers. the protection provided by mab d was evaluated in balb/c mice challenged with homologous or heterologous strains of h n influenza virus, including two strains of civ and one strain of swine influenza virus (siv). the data show that mab d protected the mice from infection with the three viral strains, especially the homologous strain, which was indicated by the recovery of body weight, reduction of viral load, and reduction of tissue damage. moreover, the levels of ifn-γ and tnf-α in the lungs, as detected by elisa, were reduced in the infected mice treated with the mab d compared with those without mab d treatment. thus, our findings demonstrate, for the first time, that a mab could reduce the release of ifn-γ and tnf-α associated with tissue damage by civ infection and that the mab might be of great therapeutic value for civ infection. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. influenza a virus, a highly contagious pathogen, can infect both birds and mammals. it has undergone significant genetic variation to adapt to different hosts [ ] . its interspecific transmission is achieved by the recombination or direct transfer of genetic material [ ] . the first case of dog infection with h n canine influenza virus (civ) was reported in the usa in [ , ] , followed by a report of civ in south korea, which subsequently demonstrated that civ was able to transmit directly from dog to dog [ , ] . recently, the first case of h n civ infection was reported in guangdong province in [ ] . over recent years, infection with h n civ in dogs has developed from scattered cases to wide distribution across the country [ ] [ ] [ ] . dogs have no natural immunity to this virus, thus a number of preventive and therapeutic measures against civ have been attempted to control the prevalence of this virus. among them, vaccination is an important method to prevent and control influenza virus infection [ ] [ ] [ ] . current vaccine research against civ has made some progress. in , the u.s. department of agriculture (usda) approved a list of vaccines against h n civ, which could effectively reduce viral shedding [ ] . in , the patent for an h n civ vaccine in south korea was also approved [ ] . preventive vaccination is historically the primary measure to control influenza virus infection, but it has some limitations [ ] . for example, influenza vaccines may not be effective enough to prevent against divergent viral strains, or may be less immunogenic and effective in certain groups, such as the very young, the old, and the immunocompromised [ ] . therefore, it is crucial to develop other measures to protect animals from infection/disease [ ] . for example, passive immunity by transferring a specific antibody to a recipient could protect animals from infection [ ] . monoclonal antibodies (mabs) can neutralize viruses, thus preventing virus attachment to, or fusion with, the host cell [ ] . many studies have demonstrated that mabs are an effective and preventive treatment against human-origin [ ] [ ] [ ] or avian-origin influenza virus infection [ , , ] . however, to date, there are no neutralizing mabs available to prevent and control h n civ infection. in this study, we identified seven mabs against h n civ, and tested one of them, the d mab, against three different h n subtype virus strains in animal experiments. this is the first description of a neutralizing mab against h n civ. three viral strains of the h n subtype, including a/ canine/jiangsu/ / (js/ ), a/canine/guangdong/ / (gd/ ) and a/swine/shandong/ / (sd/ ) were used in this study. the genbank accession numbers of js/ , gd/ and sd/ are jn to jn , kf to kf and eu to eu , respectively. the three viral strains were adapted to mice by passaging times. they were propagated in -day-old specific-pathogen free (spf) embryonated chicken eggs and stored at − °c before use. madin-darby canine kidney (mdck) cells were cultured in dulbecco's modified essential medium (dmem) containing % (v/v) fetal bovine serum (hyclone, tah, usa) and maintained at °c and in a % (v/v) co atmosphere. balb/c mice ( weeks old, female) were purchased from the animal experiment center, yangzhou university. all animal experiments complied with the guidelines of the animal welfare council of china, and the animal ethics committee of nanjing agricultural university approved the study. fifty-percent tissue culture infective dose (tcid ) assays one day before infection, a -well dish containing a monolayer of mdck cells was prepared. the next day, serial dilutions of the three influenza virus strains were made, and the cell monolayers were laterally inoculated; each dilution had three replicates. the cytopathic effect (cpe) was observed daily and the numbers of wells for a virus dilution that showed more than and less than % pathological changes were recorded. tcid titers were calculated in accordance with the reed-muench method [ ] . canine influenza virus js/ was grown in -day-old spf embryonated chicken eggs at °c for h. allantoic fluids were harvested and the hemagglutination (ha) activity of the allantoic fluids was tested at room temperature using % chicken red blood cells (rbc). ha titers more than or equal to : were selected, and the virus was purified using differential centrifugation and sucrose density gradient centrifugation. preparation of anti-h mabs followed standard hybridoma technology, as previously described [ ] . six-week-old female balb/c mice were injected intracutaneously with μg of purified virus js/ using complete freund's adjuvant (sigma, beijing, china) as the primary adjuvant, followed by incomplete freund's adjuvant. three days before harvesting the splenocytes, μg of js/ were inoculated intravenously. isolation and screening of the hybridomas was performed as described previously [ ] . mabs were prepared by injecting hybridoma cells into the peritoneal cavities of pristane-primed balb/c mice. the ascetic fluid was collected after - days and inactivated at °c for min. the hi test was performed to assess antibody reactivity against three h n strains, js/ , gd/ and sd/ , as previously described [ ] . briefly, μl of serial twofold dilutions of the purified -fold diluted ascetic fluid of the mab were mixed with ha units of virus in disposable hemagglutination plates and incubated at °c for min. then, μl of % chicken rbc were added to each well and incubated at room temperature for min. to rule out non-specific inhibition, in the hi assay, we used the ascetic fluid produced with the injection of sp / myeloma cells as a negative control. the hi titer was expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination of ha units of the virus [ ] . cell-based neutralization assays were performed as previously described [ ] . a dose of tcid of viruses was used in the assays. supernatants of ascites were tested at a starting dilution of : . briefly, two-fold dilutions of hybridoma supernatants were mixed with virus suspension containing tcid of purified h n virus and incubated at °c in a % co incubator for h before their addition to a monolayer of mdck cells in -well plates. one hundred microliters of serum-free dmem was added to each well and incubation at °c continued for h. the cytopathic effect was observed every h for to h. to determine the recognized ha domain of the mabs, we recombinantly expressed the ha, ha and ha proteins of virus js/ . the recombinant proteins were subjected to sds-page under reducing conditions. the proteins were then electro-transferred and immobilized on a nitrocellulose membrane. the membrane was blocked with % nonfat milk in phosphate buffered saline (pbs) containing . % tween (pbst) at °c for h. the membrane was subsequently incubated with the mab prepared in this study, rinsed in pbst, and incubated with horseradish peroxidase (hrp)-conjugated rabbit anti-mouse immunoglobulin (bio-rad, shanghai, china), followed by incubation with chromogenic reagents (tiangen, beijing, china) [ ] . cross-protection by h -specific mab balb/c mice were used to determine the protective efficacy of mab d . intranasal inoculations with tcid of virus strains js/ , gd/ and sd/ were given to experimental groups i (n = ), ii (n = ) and iii (n = ), respectively; the control group received pbs (n = ). each experimental group was divided into three subgroups (n = for each subgroup), which were the virus-infected, mab d and irrelevant mab igg subgroups, respectively. mice injected with pbs or irrelevant mab igg were considered as blank and negative controls, respectively. for the mab d and irrelevant mab igg subgroups, mice were pretreated intraperitoneally with mab d ( μg/ml) or irrelevant mab igg ( μg/ml) against igm from chinese breams developed in our laboratory, at a dose of mg per kg of body weight in μl of pbs before the viral challenge [ , , ] . after h, mice were challenged with three different h n strains. mice were observed daily to monitor body weight and clinical symptoms for up to days. three mice from each subgroup were euthanized humanely according to a pre-designated schedule. at , , , and days post-infection (dpi), blood samples and tissues including heart, spleen, lung, brain and intestine, as well as feces were collected. virus shedding was detected by screening fecal samples. detection of viral rna was used to determine tissue distribution and virus shedding. tissues and feces were homogenized in lysates at a ratio of : (g/ml), respectively, centrifuged at × g for min, and the supernatants were collected for the extraction of viral rna using the virus nucleic acid extraction kit ii (geneaid, taiwan). all tissues collected above, including blood, were used for virus titration; the lung, brain and heart were also used for histological and immunohistochemical analysis at dpi. quantitative assays were carried out to measure viral loads in the blood and main organs [ ] . total rna from tissues and blood samples was reverse transcribed using the primescript™ rt reagent kit with gdna eraser (perfect real time) (takara, dalian, china) and then run on an abi real time pcr system using the sybr premix ex taqtm (perfect real time) kit (takara). reverse transcription and cdna amplification were carried out as described previously [ ] . the primers used were designed against a region of the matrix gene: ′-tctatcgtcccatcaggc/ggtcttgtctttag ccattc- ′. a reference standard was prepared using pmd -t simple vector ( ng/μl; takara) that contained the corresponding target virus sequences. a series of eight -fold dilutions equivalent to × - × copies per reaction were prepared to generate calibration curves and were run in parallel with the test samples [ ] . rna of the amount of the two avian-origin civ and human-origin influenza viruses was calculated from the standard curve by real-time rt-pcr. the detection limit of this assay was copies of rna per ml. after euthanasia at dpi, the heart, brain and lung from the mice inoculated with js/ , gd/ , sd/ or pbs were collected and placed into % neutral buffered formalin. after fixation the tissues were embedded in paraffin, sectioned at μm and stained with hematoxylin and eosin for histological evaluation. sequential slides were stained using an immunoperoxidase method [ ] . expression of hemagglutinin in tissues was examined by immunohistochemical staining of histological sections. in brief, sections were blocked with % bovine serum albumin/pbs, stained with mab d at a dilution of : for one hour at °c, followed by biotin conjugated goat anti-mouse immunoglobulin (bio-rad) at a dilution of : for min at °c. the sections were subsequently incubated with hrp conjugated streptavidin (bio-rad) at °c for min. sections were then developed with hrp-dab chromogenic substrate kit (tiangen) for min and counterstained with hematoxylin. the lungs were assigned a grade of to based on the histological character of the lesions. score criteria of different grades were in accordance with a previous study [ ] . we further investigated if there was a correlation between severe disease and inflammatory cytokine production in virus-challenged mice, and ascertained whether passive immunization with antibodies affected the levels of cytokines involved in defense against three different influenza virus infections. sections of the lungs (alternating right and left lungs) from all the mice were homogenized in ml of pbs per g of lung tissue. the homogenates were centrifuged, and the supernatants were frozen at − °c until tested. the supernatants were assayed for gamma interferon (ifn-γ) and tumor necrosis factor (tnf-α) using elisa kits (sigma-aldrich, beijing). the minimum detection limits of such assays were as follows: pg/ml for tnf-α and pg/ml for ifn-γ, as previously determined by the manufacturer. data were collected and analyzed using ms excel and spss statics v . software. weight loss, viral titers, cytokine levels and histological score were analyzed by analysis of variance (anova), followed by turkey's multiple comparison test with p < . considered to be a significant difference, while p < . was considered to be statistically extremely significant. civ was propagated in mdck cells and the titers of three viral strains, js/ , gd/ and sd/ were determined to be . tcid /ml, . tcid /ml and tcid /ml, respectively. after fusion between spleen cells from h n virusimmunized mice and sp / myeloma cells, we obtained seven mabs against the js/ virus. isotyping tests showed that all of these mabs were igg b isotypes, except for one that was igg a. of the seven mabs identified, four mabs reacted with ha. among these four mabs, mab d reacted with ha and three other mabs reacted with ha ( figure ), as demonstrated by western blotting. hi and neutralization titers of the seven mabs showed that mab d had the highest neutralization activity, but had no hi activity (table ). further analysis indicated that mab d could react with virus strains js/ , gd/ and sd/ , and produce high neutralization activities against the three viral strains, especially against the homologous strain js/ ( table ) . to access the protective efficacy of mab d , we inoculated mice with three different h n strains one day after treatment with mab d . three days after the inoculation, all mice challenged with the three virus strains exhibited clinical signs of infection, including depression, decreased activity and huddling. similar clinical signs were observed in irrelevant mab igg pretreated groups. however, similar to the pbs control group, mice in mab d pretreated groups seemed to be energetic and had a good appetite during the infection. in terms of body weight, mice challenged with js/ after treatment with d showed a similar increase in body weight compared with the pbs control group and there was no significant difference between the two groups ( figures a, b and c). at dpi, mice treated with mab d showed a body weight increase of nearly %. although the body weights in the virus-infected group and irrelevant mab igg group both demonstrated an upward trend, the growth rate was slower than that in the mab d group. the extent of the increase in body weight was significantly slower compared with that of the mab d group at , and dpi (p < . ) (figure a ). in the group of mice infected with gd/ , the body weights of the mice in the three experimental groups all showed an upward trend, but the growth rate of the mice treated with mab d was much higher than in the other two groups. in addition, the body weight changes of mice in the mab d group at , and figure antigen identification of mab d by western blotting using recombinant proteins ha, ha and ha . lane m, protein pre-stained mass markers; lane , mab d reacted with expressed viral protein ha; lane , mab d reacted with recombinant protein ha (not visualized here); lane , mab d reacted with expressed protein ha . viral proteins were identified by dab staining with hrp-labeled goat anti-mouse secondary antibody. dpi were significantly different from those of the other two experimental groups (p < . ). the mice in the mab d group showed weight gains of nearly %, which was not significantly different from the pbs control group ( figure b ). however, after infection with sd/ , mice in the virus-infected group showed a slight , the results are expressed in terms of percent body weight. *p < . , or **p < . , indicates a significant difference in weight data for the mab d treated groups compared with the virus-infected groups and irrelevant mab igg groups. # p < . , indicates a significant difference in weight data for the pbs group compared with the mab d treated group. for viral loads in the lungs (d, e, f), the results are expressed in terms of mean log number of copies/g of rna standard deviation. *p < . , or **p < . , indicates a significantly different virus titer for the mab d group compared with the other two groups. / , and / , indicate the proportion of the lungs in which virus could be detected. decrease in body weight at dpi and mice in the mab igg group displayed a slight decline at dpi. by contrast, mice in the mab d group continued to grow at (p < . ), (p < . ) and dpi (p < . ). the growth rate in the mab d group was significantly higher than that in the virus-infected group and mab igg group, but slightly lower than in the pbs control group at dpi; mice body weight gain in the mab group reached approximately % ( figure c ). real-time pcr was used to evaluate the kinetics of viral rna loads in the lung, heart, brain, spleen, intestine, feces and blood of the infected mice. the viral titers were expressed as the number of copies of viral rna. the dynamic changes of viral titers in the lungs of mice in the virus-infected group, mab d group and irrelevant mab igg group were similar: peak viral titers were observed at , and days after infection, after which the viral titer declined at and dpi (figures d, e and f). however, viral titers in the lungs of mice treated with mab d were significantly lower than in the mice in the other two groups at specific time points. after challenge with js/ , viral loads in lungs of mice treated with mab d were significantly lower than those in the other two groups at , and dpi (p < . ) ( figure d ). for strain gd/ , viral titers of the lungs in the mab d group were significantly lower compared with the other two experimental groups at (p < . ) and dpi (p < . ). in addition, at and dpi, viral rna could not be detected in some lung samples ( / and / , respectively) in the mab d group ( figure e ). for virus sd/ , the viral titer of mice in the mab d group was significantly lower than in the other two groups at (p < . ), (p < . ) and dpi (p < . ) ( figure f ). for mice in the virus-infected group, peak viral titers of three virus strains js/ , gd/ and sd/ were . , . , . copies/g, respectively, while for mice in the mab d group, peak values were . , . and . copies/g, respectively. considering that mab d resulted in a significant reduction in viral titers in the lungs of mice infected with three different virus strains at dpi, we chose that time point to determine the viral rna loads in different tissues and fecal samples. we found that viral loads in collected feces, blood and other tissues at dpi in the mab d group were also lower than those in the other two groups (figure ). after mice were challenged with virus js/ , viral titers of the lung, heart, intestine, feces and blood in the mab d group decreased by (p < . ), (p < . ), (p < . ), (p < . ) and (p < . ) fold, respectively, compared to the other two groups ( figure a ). for mice challenged with virus gd/ , viral titers of the lung, heart, spleen, feces and blood of mice treated with mab d were found to be reduced by (p < . ), (p < . ), (p < . ), (p < . ) and (p < . ) fold in comparison with the other two groups ( figure b ). for virus sd/ , mab d resulted in a reduction in viral titers of the lung, spleen, intestine, feces and blood by (p < . ), (p < . ), (p < . ), (p < . ) and (p < . ) fold, respectively ( figure c) . generally, mab d could reduce viral loads of the three virus strains in the infected mice. notably, in the virus-infected group and irrelevant mab igg group, after infection with virus js/ , even till dpi, virus rna was detected in most tissues, while for the mice treated with mab d , virus rna could not be detected in the brain and almost all the other tissues, except for the lung and blood, at dpi (additional file ). for virus gd/ , at dpi, viral titers in all the detected tissues were much lower compared with the other two viruses, in all three groups. viral titers in some tissues were undetectable. mice in the mab d group showed a much faster virus clearance rate than the other two groups. at dpi, no virus was detected in the intestines and feces of three mice in the mab d group and at dpi, virus was undetectable in all the other tissues in mice treated with mab d , except for the lungs in one mouse (additional file ). however, in mice infected with sd/ , the virus showed the longest retention time. at dpi, nearly all tissues from mice in virus-infected and irrelevant mab igg groups showed detectable virus rna. even in the mab d group, all mice showed positive virus rna in the detected tissues, except for the intestines and feces (additional file ). to compare the above results with pathological findings in mice infected with three different virus strains, and treated with mab d and irrelevant mab igg, we chose the heart, brain and lung from different treatment groups at dpi to perform histopathological and immunohistochemical analysis. all the sampled tissues from mice in the virus-infected group showed significant lesions and viral antigen staining, while those from the control group did not show any lesions. mice treated with mab d had markedly fewer lesions compared with the virus-infected group (figure ) . histological lesions in the mab igg group showed similar results with those in the virus-infected group (data not shown). regardless of virus strain, the lung interstitial space was obviously widened, and the bronchial lumen became narrow, with the alveolar septum thickened by the infiltration of a number of inflammatory cells (figures a and c) . large areas of the lung appeared consolidated, with symptoms of pulmonary congestion ( figure e ). interstitial pneumonia was also obvious, with the alveolar figure viral loads in collected tissues and fecal samples of mice at dpi. mice were pretreated with mg/kg of mab d , mab igg or pbs day before viral challenge with virus js/ (a), gd/ (b) or sd/ (c), respectively. in each virus group, the lung, heart, brain, spleen, intestine, feces and blood of mice were collected for determination of viral loads using real-time pcr at days post-challenge. *p < . , or **p < . , indicates significantly different virus titers compared with the other two groups. septum and proliferation of connective tissue infiltrated with numerous macrophages around the bronchioli and blood vessels (figures c and e) . in brief, histological lesions were characterized by multifocal to coalescing reddish consolidation in mice infected with the virus js/ , gd/ or sd/ in both the virus-infected and irrelevant mab igg groups. however, the degree of histological lesions observed for sd/ was the most severe, and gd/ showed the least severe lesions among these three viruses. mice treated with mab d showed only mild necrotizing bronchiolitis and ciliated tracheal epithelium with mild hyperplasia ( figure f ). in addition, very small gaps were observed between the alveoli and there were no excessive amounts of alveolar macrophages in the lung (figures b and d) . for virus strain gd/ , mab d demonstrated the best protective efficacy compared with the other two viruses. mice in the pbs group showed bronchia with a simple ciliated columnar epithelium and the alveolar cavity as a vacuolated thin-walled structure ( figure g ). viral antigen staining was present in almost all bronchiolar epithelial cells and some alveolar cells at dpi after virus challenge ( figures a, c and e) , while mice in mab d showed only a little virus antigen staining surrounding vessels near the alveoli (figures b, d and f) . the pbs control group had no viral staining ( figure g ). lung grades for degree of injury at dpi are shown in figure . there was significantly less injury to the lungs in the mab d group than in the virus-infected group and mab igg group for all virus strains. similar to the lung, all mice in the virus-infected groups showed histological lesions in the brain. the extent of histological lesions infected was the most severe with sd/ and was the least severe for gd/ . the severity of the infection may depend on the differences in virus titer. in the cerebrum, congestion and hemorrhage were evident. nerve fibers were dissolved and neurons had necrolysis-like vacuoles; glial nodules and neuronophagia were also observed ( figures o and q) . dilation and hyperemia were found in the capillaries ( figure s ). moreover, microglial cells and nerve cells showed a satellite phenomenon. the cytoplasm of the neurons was basophilic because of contraction ( figures o and q) . viral antigens could be detected in glial nodules and microglial-gathered areas (figures o, q, and s) . the brains of mice treated with mab d had only mild lesions surrounding microglial cells and nerve cells ( figures p and t) , and almost no antigen staining was found (figures r and t) . the pbs control group showed no histological findings ( figure u ) and no viral staining ( figure u ). in the heart, for all mice in virus-infected groups, the cardiac striated muscle was disordered and full of vacuoles, characterized by myocarditis, and lymphocyte infiltration was observed ( figure h ). lymphoproliferation was also found among the muscle fibers ( figure j ). the nuclei showed pyknosis, with some myocardial cells showing coagulative necrosis ( figures j and l) to gain a better understanding of the effect of civ on the innate immune response and to ascertain whether passive immunization with monoclonal antibody affected the levels of cytokines, we examined the levels of ifn-γ and tnf-α in the lungs of mice in the virus-infected and mab d groups. as shown in figure , the level of ifn-γ in response to all three virus strains showed an identical trend, higher at , and dpi than at and dpi. the ifn-γ levels in mab d group in all three virus strains were significantly lower than the virus-infected group or mab igg group, especially at , and dpi. however, the cytokine levels differed with the various virus strains. for virus strain js/ , the ifn-γ level of all groups reached its peak at dpi, and then exhibited an overall downward trend. the cytokine levels in the mab d group at (p < . ) and dpi (p < . ) were significantly lower than that in the virus-infected or mab igg group; however, the ifn-γ level did not show any significant difference among the three groups at other time points ( figure a ). the ifn-γ level in the mab d group returned to normal after dpi, while the other two groups returned to the normal level at dpi. for virus strain gd/ , the ifn-γ levels of the three experimental groups were significantly higher than in the mice in the pbs group at (p < . ), (p < . ) and dpi (p < . ), sustaining a relatively high level until dpi, after which it decreased. the peak level of ifn-γ in the mab igg group was lower compared with the other two virus strains at dpi ( figure b ). although the ifn-γ levels in the three experimental groups challenged with gd/ did not show much difference compared with each other, the ifn-γ level in the mab d group at dpi returned to a normal level, which was significantly lower (p < . ) compared with the other two groups; all the three groups showed normal levels at dpi. for virus strain sd/ , this virus induced the largest rise in ifn-γ levels. the level in the sd/ -infected the degree of lung injury after infection with virus js/ , gd/ and sd/ at dpi. the lungs were assigned a grade to based on the histological character of the lesions. *p < . , or **p < . , indicates a significantly different score for the mab d group compared with the other two groups. group was significantly higher than that of the other two viruses at dpi. in addition, mice in virus-infected and mab igg groups both demonstrated the same trend in the period of to dpi (p < . ), i.e., significant elevation followed by a downward trend ( figure c ). although the ifn-γ level in the mab d group at to dpi was relatively high compared with the pbs group, the ifn-γ levels were significantly lower than those of the other two groups at (p < . ) and dpi (p < . ), and then declined to the normal level at dpi. figure characterization of ifn-γ and tnf-α secretion from lung tissues of mice challenged with virus. cytokine concentrations were measured by elisa in supernatants of homogenates from the lungs infected with three virus strains. mice were pretreated with mg/kg of mab d , mab igg or pbs day before challenged with virus js/ (a, d), gd/ (b, e) or sd/ (c, f), respectively. the cytokine levels were measured in the infected mice on days , , , and post challenged. the results are expressed in terms of pg/ml. *p < . , or **p < . indicates significantly different changes for mab d group compared with the virus-infected group or the mab igg group. # p < . , or ## p < . , indicates a significant difference between this group and the pbs control group. changes in tnf-α level were not the same as those for ifn-γ. after infection with the three virus strains, tnf-α levels of mice in all groups were only slightly higher than those of the pbs group at dpi and increased at and dpi. levels reached their maximum at dpi and decreased at dpi, however, the cytokine level was still higher than that of the control group at dpi which was quite different compared with the ifn-γ level. the increase in tnf-α level was lower compared with the ifn-γ level. in addition, the tnf-α level in the mab d group was apparently lower than the virusinfected group or mab igg group, especially at and dpi. for virus strain js/ , tnf-α levels in the virusinfected group and mab igg group showed a small rise at dpi, reaching its peak at dpi, and then declined; however, the cytokine level was still significantly higher (p < . ) than that of the pbs group ( figure d ). the tnf-α level in the mab d group at dpi (p < . ) was remarkably lower than that in the virus-infected group and mab igg group, and decreased to a similar level as the pbs group. for virus strain gd/ , the tnf-α level of all three experimental groups increased from to dpi, and then returned to normal at dpi ( figure e ). the tnf-α level of the mab d group at dpi was significantly lower (p < . ) than in the other two groups, while the cytokine level did not show much difference in these three groups at the other time points. for virus strain sd/ , the tnf-α levels in the virusinfected group and mab d group were markedly higher than those of the pbs group from to dpi ( figure f ). although the tnf-α level in the mab d group was also significantly higher (p < . ) than the pbs group, except at and dpi, the level in the mab d group was significantly lower (p < . ) compared with the virus-infected and irrelevant mab igg groups at and dpi. h n civ is a newly identified avian influenza virus (aiv) subtype that can infect dogs and transmit directly from dog to dog [ , ] . civ infection has been reported in several countries, including south korea and china [ , , ] . it is important to develop a set of measures to prevent and control civ infection in dogs. antibody-mediated passive immunity can provide protection against invading pathogens [ , ] . in this study, we developed seven mabs against js/ , whose pathogenicity has been characterized both in mice [ ] and dogs [ ] . among them, four mabs reacted with ha. the ha glycoprotein is the primary target of antibodies that confer protective immunity to influenza viruses [ ] . therefore, the generation of neutralizing antibodies against antigenic sites on the ha glycoprotein is regarded as a criterion for evaluating immunity to influenza viruses and is believed to constitute the main correlate of protection [ , ] . anti-ha globular head mabs have potent neutralizing activity against homologous strains, but have very limited breadth of reactivity because of the high variability of amino-acid changes in the ha globular head [ ] . ha , which is the ha stalk, however, is a conserved region of ha among all influenza a virus subtypes [ , ] and is responsible for the fusion of the virus and the endosomal membrane during the entry of the virus into the cell [ ] . here, western blotting showed that mab d recognized the ha domain of h , and had highest neutralization activities. although mab d lacked hi activity, some previous studies reported that a lack of in vitro hi activity of anti-ha mabs does not rule out protective activity in vivo [ , ] . therefore, we selected the anti-ha mab d for further evaluation in regards to protection against different influenza virus strains. to investigate the protection of mab d against homologous and heterologous strains of h n influenza viruses, we selected three virus strains to perform the challenge experiment in mice, including two strains of civ (js/ and gd/ ) and one strain of swine influenza virus (siv) (sd/ ). considering that almost all h n civ isolates reported were not lethal to mice or dogs in challenge experiment [ , , , , ] , we evaluated the protection efficacy by body weights, viral loads and histological lesions. body weight loss is the parameter most commonly used to evaluate influenza viral pathogenicity in mice [ ] . our study shows that all three virus strains could remarkably reduce the growth rate of the mice after infection, while pretreatment with mab d helped to control the declination to some extent. from the pathological point of view, the lung, heart and brain in mab d treated groups showed markedly fewer lesions compared with the virus-infected group and mab igg group, with all virus strains. the pathological scores of the lungs in mab d group were lower than those in the virus-infected group and mab igg group, suggesting that mab d could mitigate the damage caused by influenza virus. these results suggest that mab d could offer a protective effect against the three virus strains. to further evaluate the effects of the anti-influenza virus mab, we monitored viral loads by real-time pcr. the mab d decreased the viral loads in the lungs to significantly lower levels, relative to those in the virusinfected group and mab igg group. similar results were also found in other tissues. for js/ , the virus in the brain and other organs of all three mice treated with mab d had been cleared by dpi, except for the lungs. for virus gd/ , the application of mab d caused virus clearance from the intestine and feces days earlier than that in the other two groups; moreover, there was no detectable virus rna at dpi. for virus sd/ , a slower rate of clearance of viral load was observed. this virus could persist and be detected in most tissues in the mab d group until dpi, but virus in the intestine and feces had been cleared by dpi. these results indicate that protection against the virus strains provided by mab d might be caused by earlier clearance of the virus from the tissues or shortening the time of virus shedding. a previous study has reported that mabs could reduce the period of virus clearance [ ] . our study indicates that mab d could provide good protection against challenge with homologous, as well as heterologous, virus strains of h n influenza virus. this finding was in accordance with a previous report [ ] that ha mabs are highly cross-reactive among strains of the same subtype, and even within different subtypes. in spite of this, we found that this mab was relatively less effective against the swine-lineage than caninelineage h virus strains. sequence analysis of amino acids showed that js/ had higher sequence identity to canine-lineage gd/ ( . %) than to swine-lineage sd/ ( . %), with and different amino acids, respectively, in ha (data not shown). the difference in amino acid sequence may affect antigen-antibody recognition. this may explain why the mab induced by canine-lineage influenza virus strain js/ is not strong enough to react against more distantly related strains within the same subtype. cytokines are important in establishing an innate immune response, as well as in determining the magnitude of the inflammatory response to influenza virus infection. the most important feature of the mechanism of immune suppression with influenza virus h n is the cytokine storm [ , ] . here, to gain a better understanding of how virus infection and mab treatment affected host immune response, we analyzed the levels of ifn-γ and tnf-α in the lung. an important finding in this study is that the tnf-α levels peaked two days later than ifn-γ levels in all groups. a previous study showed similar results: ifn-γ and tnf-α in the lungs of pigs infected with human h n influenza virus peaked at dpi, and dpi respectively [ ] . ifn-γ has important immune-regulatory functions and antiviral activity, and is primarily produced by natural killer (nk) and t cells. nk cells are a major player in innate immune responses. we speculated that early production of ifn-γ during infection probably arises from nk cells, whereas tnf-α functions relatively late in the inflammatory cycle induced by infection, at a time when virus is already being contained and the response is centered on resolution of the inflammation [ , ] . tnf-α in mice challenged by js/ and sd/ maintained higher levels until dpi. a previous study demonstrated that the depletion of tnf-α in influenza or respiratory syncytial virus-infected animals significantly reduced pulmonary inflammation and cytokine production, without compromising viral clearance [ ] . we speculated that the elevated level of tnf-α at dpi might be correlated with the uncleared virus loads in the lungs. in the present study, three strains of h n civ were shown to induce elevated levels of cytokines in the lungs. this was in agreement with previous reports on h n civ [ , ] . during h n influenza virus infection, the elevated pro-inflammatory cytokine response has been proposed as the main cause of the increased severity of the disease [ ] . the study of lee et al. [ ] reported that the levels of ifn-γ and tnf-α in the lungs of dogs infected with h n influenza virus increased quickly, while the infected dogs developed severe bronchointerstitial pneumonia accompanied with massive infiltration of immune cells. this result suggests that the dysregulation of chemokines during h n civ infection might contribute to viral pneumonia characterized by extensive immune cell infiltration. in support of this hypothesis, we observed that elevated levels of cytokines accompanied the clinical manifestations in the civ infected dogs. we found that ifn-γ and tnf-α levels in the mab d group were significantly lower than in the virus-infected group or mab igg group, while the histopathological findings showed more significant lesions in lungs of mice from the latter two groups than in the mab d group. these observations indicate that mab d treatment may reduce the virus-induced cytokine production and pathological lesions caused by virus infection. the effect is likely to be mediated by inhibition of civ replication by the mab. fritz et al. [ ] reported that active influenza virus replication is required for the induction of potent proinflammatory, regulatory and chemotactic factors. our study and a previous report [ ] demonstrate that active replication of civ in the canine respiratory system results in intense inflammatory responses. considering that it is important for the host to maintain a balance of the cytokine levels, we speculated that inhibition of the inflammatory cytokine response might offer a therapy for civ infection. however, salomon et al. [ ] demonstrated that inhibition of the cytokine response during h n influenza virus infection is not sufficient to protect against death, and proposed that therapies targeting the virus would be preferable. in conclusion, our results suggest that the ha -specific mab d could contribute to early recovery from influenza infection with different h n virus strains. this mab will further our understanding of the antigenic properties of h n virus and might contribute to the prevention and control of h n virus epidemic in dogs. pandemic and seasonal human influenza virus infections in domestic cats: prevalence, association with respiratory disease, and seasonality patterns host range restriction and pathogenicity in the context of influenza pandemic transmission of equine influenza virus to dogs influenza a virus (h n ) in dogs with respiratory disease transmission of avian influenza virus (h n ) to dogs experimental infection of dogs with avian-origin canine influenza a virus (h n ) 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pathology in dogs with experimental canine h n influenza virus infection role of host cytokine responses in the pathogenesis of avian h n influenza viruses in mice severe canine influenza in dogs correlates with hyperchemokinemia and high viral load inhibition of the cytokine response does not protect against lethal h n influenza infection additional file : viral rna detection in collected samples of mice inoculated with virus strain js/ . numbers , and represent the three mice euthanized from each group at different dpi. + represents viral loads in terms of mean log number of copies/g of rna. numbers of +'s represent the magnitude of rna copies.additional file : viral rna detection in collected samples of mice inoculated with virus strain gd/ . numbers , and represent the three mice euthanized from each group at different dpi. + represents viral loads in terms of mean log number of copies/g of rna. numbers of +'s represent the magnitude of rna copies.additional file : viral rna detection in collected samples of mice inoculated with virus strain sd/ . numbers , and represent the three mice euthanized from each group at different dpi. + represents viral loads in terms of mean log number of copies/g of rna. numbers of +'s represent the magnitude of rna copies. the authors declare that they have no competing interests. key: cord- - ntdb yf authors: mair, kerstin h; müllebner, andrea; essler, sabine e; duvigneau, j catharina; storset, anne k; saalmüller, armin; gerner, wilhelm title: porcine cd α(dim/-)nkp (high) nk cells are in a highly activated state date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: ntdb yf natural killer (nk) cells play a crucial role in the early phase of immune responses against various pathogens. in swine so far only little information about this lymphocyte population exists. phenotypical analyses with newly developed monoclonal antibodies (mabs) against porcine nkp recently revealed that in blood nkp (-) and nkp (+) cells with nk phenotype exist with comparable cytotoxic properties. in spleen a third nkp -defined population with nk phenotype was observed that was characterised by a low to negative cd α and increased nkp expression. in the current study it is shown that this nkp (high) phenotype was correlated with an increased expression of cd and cd compared to the cd α(+)nkp (-) and nkp (+) nk-cell subsets in spleen and blood. additionally nkp (high) nk cells expressed elevated levels of the chemokine receptor cxcr on mrna level. functional analyses revealed that splenic nkp (high) nk cells produced much higher levels of interferon-γ and tumor necrosis factor-α upon stimulation with cytokines or phorbol- -myristate- -acetate/ionomycin compared to the other two subsets. furthermore, cross-linking of nkp by nkp -specific mabs led to a superior cd a expression in the nkp (high) nk cells, thus indicating a higher cytolytic capacity of this subset. therefore porcine splenic nkp (high) nk cells represent a highly activated subset of nk cells and may play a profound role in the immune surveillance of this organ. natural killer (nk) cells were initially characterised by their spontaneous lytic activity against certain tumor and virus-infected cells [ , ] . besides their role as cytotoxic cells through the production of perforin and granzymes, nk cells are potent producers of cytokines like interferon (ifn)-γ and tumor necrosis factor (tnf)-α [ ] and thus play important roles in immunomodulation and the defence against viral, parasitic and bacterial pathogens [ ] . a considerable number of phenotypically and functionally different nk-cell subsets have been identified up to date [ ] . for example, human nk cells can be divided into functionally and also developmentally distinct subsets according to their differing expression of cd in combination with cd [ , ] and more recently cd b and cd [ ] . in the mouse likewise cd and cd b (mac- ) are used to dissect nk cells into functionally and developmentally different subsets [ ] . additionally, the chemokine receptor cxcr is used in combination with cd to distinguish nk-cell subsets in the mouse [ ] . for porcine nk cells a perforin + cd + cd -cd -cd -cd -cd α + cd ß -cd b + cd + phenotype has been described and it was shown that these lymphocytes can perform immediate cytotoxicity against nk-susceptible targets [ ] [ ] [ ] . moreover, in parasitic as well as in viral infections increases in nk cell number and activity have been reported [ , ] , but also inhibitory effects on nkcell mediated cytotoxicity and cytokine production by viral infections are described [ ] [ ] [ ] [ ] . despite these hints on important functions of porcine nk cells in vivo, so far no investigations on the existence of functionally differing nk-cell subsets have been reported. nevertheless, a recent study from our group with newly developed monoclonal antibodies (mabs) against the activating receptor nkp enabled a more comprehensive insight into the phenotype of porcine nk cells and putative subsets [ ] . nkp (cd , ncr ) is a member of the natural cytotoxicity receptor (ncr) family, which is involved in the control of tumors and viral infections [ ] [ ] [ ] [ ] [ ] [ ] . moreover, it has been used as a marker for nk cell identification in different species like humans [ , ] , monkeys [ , ] , rodents [ ] [ ] [ ] , cattle [ ] and more recently in sheep [ ] and horses [ ] . in contrast, nkp in the pig was shown to divide porcine cd -cd α + nk cells into nkp and nkp + subsets in blood and all organs tested [ ] . cd -cd α + nkp -nk cells show phenotypic and functional properties of nk cells although they produce reduced levels of ifn-γ compared to the nkp + subset after in vitro stimulation. additionally, a third nk cell population with elevated nkp expression levels was identified in high frequencies in spleen and liver, pointing towards a special role of nk cells with this phenotype. therefore, in this study we focused on functional and phenotypical properties of cd α dim/-nkp high nkcells in the spleen. we observed that these cells differ in their expression of various nk-cell associated markers including cd , the tnf-receptor family member cd and the chemokine receptor cxcr compared to the cd α + nkp and nkp + nk-cell subsets. additionally, this nk-cell subset showed an increased cytokine production and cytolytic activity. thus, our data indicates that cd α dim/-nkp high nk cells in the pig are in a highly activated state. blood and spleens were obtained from - month-old healthy pigs from an abattoir. animals were subjected to electric high voltage anaesthesia followed by exsanguination. this procedure is in accordance to the austrian animal welfare slaughter regulation. peripheral blood mononuclear cells (pbmc) were isolated using density gradient centrifugation (lymphocyte separation medium, density: . g/ml, paa, pasching, austria) as described previously [ ] . dissected spleen was cut into small pieces and mechanically dissociated by forcing through a sieve. after a washing step in phosphate buffered saline (pbs, paa) cells were applied to density gradient centrifugation to isolate mononuclear cells. isolated lymphocytes were finally resuspended in culture medium or pbs containing % (v/v) porcine plasma for analysis by flow cytometry (fcm). isolated porcine pbmc and splenocytes were cultivated in rpmi (paa) with stable glutamine supplemented with % (v/v) heat inactivated foetal calf serum (fcs, paa), iu/ml penicillin and . mg/ml streptomycin (paa). medium for sorted nk cells was additionally supplemented with mm sodium pyruvate (paa), nonessential amino acids (paa) and μm -mercaptoethanol (sigma-aldrich, vienna, austria). where indicated, cells were additionally cultured in the presence of various cytokines as outlined below. freshly isolated pbmc or splenocytes were resuspendend in pbs containing % (v/v) porcine plasma and labelled for flow cytometric analysis. cultured cells were resuspended in pbs containing % (v/v) fcs for fcm staining. all incubation steps were performed for min on ice. the following primary antibodies were used for cell surface staining: unconjugated or alexa -conjugated anti-nkp (igg , clone viv-km , [ ] ), anti-cd (igg b, clone bb - e , southern biotech, birmingham, al, usa), percp-cy . -conjugated anti-cd (igg a, clone bb - e - c , bd biosciences, san jose, ca, usa), efluor -conjugated anti-cd (igg , clone ppt , custom-conjugation by ebioscience, san jose, ca, usa), unconjugated or fitc-conjugated anti-cd α (igg a, clone / / ), pe-conjugated anti-cd α (igg a, clone - - , bd biosciences), anti-cd (igg , clone g , serotec, raleigh, nc, usa), biotin-conjugated anti-cd (igg , clone b c , [ ] ). all non-commercial monoclonal antibodies were produced in-house [ ] . where indicated, these antibodies had been purified and covalently conjugated to fluorochromes or biotin. alexa fluor- protein labelling kit (life technologies, carlsbad, ca, usa) was used for conjugation of anti-nkp mabs according to manufacturer's instructions. fitc conjugation for anti-cd α mabs was performed as described elsewhere [ ] . anti-cd mabs were biotinylated using sulfo-nhs-lc-biotin (thermo scientific, pierce, vienna, austria) following manufacturer's instructions. unspecific binding was assessed by appropriate isotype-matched control antibodies. for indirect labelling, anti-mouse anti-igg -pe (southern biotech) and streptavidin-brilliant violet conjugate (biolegend, san jose, ca, usa) were used as second-step reagents. to discriminate between live and dead cells, fixable near-ir dead cell stain kit (life technologies) was used according manufacturer's protocol with . μl reactive dye per reaction. if unconjugated and conjugated antibodies with the same isotype were used in combination, a sequential staining was performed. unconjugated primary mab was used in a first step, followed by isotype-specific dye-conjugated antibodies. after secondary incubation, free binding sites of mouseisotype specific antibodies were blocked by whole mouse igg molecules ( μg per sample, jackson immunoresearch, suffolk, uk) followed by a further incubation step with fluorochrome-conjugated primary mabs. fcm analyses were performed on a facscanto ii or facsaria (bd biosciences). data of at least × lymphocytes per sample were recorded. data were analysed with facsdiva software (version . . , bd biosciences) and flowjo software (version . . ., tree star, ashland, or, usa). box plots were created by sigmaplot software (version . , systat software inc., erkrath, germany). for intracellular staining of ifn-γ, pbmc and splenocytes were stimulated in -well round-bottom plates at × cells per well in a final volume of μl. cells were either stimulated with iu/ml recombinant human interleukin (rhil)- (roche, vienna, austria) in combination with ng/ml recombinant porcine interleukin (rpil)- and ng/ml rpil- (both r&d systems, minneapolis, mn, usa) overnight, or left in medium alone as negative control. for ifn-γ labelling, brefeldin a (golgiplug, bd biosciences) was added to microcultures at a final concentration of μg/ml, h prior to harvest. cells were labelled with antibodies against cd , cd α and nkp as stated above. afterwards cells were fixed and permeabilized as described elsewhere [ ] and labelled with anti-ifn-γ-pe (igg , clone p g , bd biosciences) as well as corresponding isotype control mab (mouse-igg -pe, clone mopc- , bd biosciences). for sorting of cd -cd α + nkp and cd -cd α + nkp + nk cells of blood as well as cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen, isolated mononuclear cells were labelled with primary antibodies against cd , cd α and nkp as described above. as secondary antibodies anti-igg -pe (southern biotech), anti-igg a-alexa and anti-igg b-alexa (both life technologies) were used. pbs containing % (v/v) fcs and mm edta was used for all washing steps. sorting was performed on a facsaria (bd biosciences). purity of sorted cell populations was at least . % or higher. sorted cells were either transferred directly into cell culture or resuspended in tri reagent (sigma-aldrich) and stored at − °c for subsequent mrna analysis. cd a degranulation assay nk cell receptor mediated degranulation was assessed by measuring the expression of cd a on the cell surface in combination with four-color flow cytometry to discriminate between the different nk-cell subsets. degranulation assays were performed according to a protocol for human nk cells [ ] and modified as follows. triggering of nkreceptors was performed by using monoclonal antibodies against nkp (igg , clone viv-km , [ ] ), cd (igg , clone g , serotec) or a combination of both. monoclonal antibodies were coated on -well round-bottom wells by incubation overnight at °c at a concentration of μg/ml each in pbs in a total volume of μl per well. isotypematched irrelevant antibodies served as control ( μg/ml in μl per well). plates were washed with pbs for three times before cells were added. freshly isolated pbmc and splenocytes were stimulated with rhil- ( iu/ml) and rpil- ( ng/ml, biosource, nivelles, belgium) overnight with × cells in a total volume of μl per well, using -well round-bottom plates. since nkp was rapidly internalised after receptor triggering, cells were labelled with alexa -conjugated anti-nkp mab prior to transfer into antibody-coated plates. the simultaneous use of viv-km for fluorescence-staining and viv-km for coating of plates was possible because the two mabs bind to different sites on nkp [ ] . after two washing steps to eliminate unbound anti-nkp -alexa antibodies, cells were used at a concentration of × cells in a total volume of μl per well (mab-coated -well round-bottom plate) in the degranulation assay. additionally, microcultures were supplemented with fitc-conjugated anti-cd a mab (igg , clone e / , serotec) at a final concentration of μg/ml and the two protein transport inhibitors brefeldin a (golgiplug, final concentration μg/ml) and monensin (golgistop, final concentration μg/ml) (both bd biosciences). after an incubation of one hour at °c, cells were re-labelled with alexa -conjugated anti-nkp in combination with pe-conjugated anti-cd α and efluor -conjugated anti-cd monoclonal antibodies for fcm as described above. analysis of ifn-γ and tnf-α production by elisa facs-sorted nk-cell subsets from blood and spleen were stimulated in -well round-bottom plates at × cells in a final volume of μl per well for cytokine production. for ifn-γ and tnf-α production cells were stimulated with a combination of rhil- ( iu/ml), rpil- ( ng/ml) and rpil- ( ng/ml). tnf-α production was also analysed after stimulation with ng/ml phorbol- -myristate- -acetate (pma) and ng/ml ionomycin (both sigma-aldrich). after h, supernatants were collected and tested for cytokine production with commercially available elisa kits for ifn-γ (mabtech, nacka strand, sweden) and tnf-α (r&d systems) according to manufacturers' protocols. optical densities (ods) were measured at / nm with an elisa reader (tecan, sunrise, crailsheim, germany). total rna from facs-sorted nk-cell subsets from blood and spleen was isolated using tri reagent (sigma-aldrich) according to manufacturer's protocol. rna quality control and cdna synthesis were performed as described elsewhere [ ] . expression of target genes was determined by real-time pcr, using an internal standard as calibrator. the internal standard (is) was generated by pooling equal aliquots of the cdna samples investigated in this study. primers for target genes were designed using either the public domain programmes primer [ ] or primer-blast [ ] for cxcr . all primers were synthesised commercially (eurofins mwg operon, ebersberg, germany). sequence information of used primers is listed in table . whenever possible, primers were forced to span over exon junctions in order to increase specificity. for amplification of target genes sybr w green i ( . ×, sigma-aldrich) was used as reporter dye. the qpcr reaction-mixes contained itaq w dna polymerase ( . u/reaction, bio-rad, hercules, ca, usa), gene specific primers ( nmol/l each), a final concentration of μmol/l dntp each and mmol/l mgcl (for cxcr . mmol/l were used) within provided reaction buffer ( ×, bio-rad). qpcr was performed on a cfx ™ (bio-rad), pcr conditions are listed in additional file . optimisation and validation of the qpcr assays with target gene-specific primers are likewise described in more detail in additional file . specificity of the generated pcr products using a cdna pool of samples was further verified by automated sequencing using the pgem-t easy vector system (promega, madison, wi, usa) and m standard sequencing primer (eurofins mwg operon). the multiplex qpcr assay for the reference genes (β-actin, cyclophilin a and gapdh) that were used to normalise each target-gene expression was performed as previously described [ ] . each plate contained corresponding randomly assigned rt-minus controls ( % of all samples investigated), the no-template controls (ntc), as well as the is. all samples were measured in duplicates. data were analysed using the cfx manager software (bio-rad) in the linear regression mode. for the quantification we applied the method described elsewhere [ ] . target gene expression was displayed as ^-ΔΔcq values representing the fold changes relative to is. data was analysed for statistical significance by spss w (spss statistics version . , ibm corp., armonk, ny, usa). datasets with two groups (pbmc) were analysed using paired two-tailed student's t-test. for datasets containing more than two groups (spleen) one-way variance analysis with bonferroni correction for paired sample means was applied. if sample size per group was < , no statistical evaluation was performed. three different levels of significance were defined: p < . (indicated by *), p < . (indicated by **) and p < . (indicated by ***). to expand the knowledge about the phenotype of previously described nkp -defined nk-cell populations in swine [ ] , we performed flow cytometric analyses of lymphocytes isolated from spleen and blood. a gating hierarchy was used throughout the experiments to exclude doublets, dead cells as well as cd + t cells. remaining cd lymphocytes were further analysed for cd α and nkp expression (see additional file ). as previously described [ ] , among cd lymphocytes two nk populations could be found in blood, namely nkp and nkp + cells that were both cd α + ( figure a , upper graph). the third nkp -defined subset that was found in spleen ( figure a , lower graph) was characterised by a low to negative cd α expression and increased expression of the activating receptor nkp . this applied to all animals analysed, resulting in a mean fluorescence intensity (mfi) for nkp that was - times higher than in the splenic nkp + subset ( ± to ± respectively, figure b , upper graph). cd α + nkp + cells in blood and spleen showed comparable expression levels of the activating receptor ( ± and ± respectively). we then investigated differences in the expression level of cd α in the respective nk-cell subsets. splenic nkp high nk cells showed a significantly reduced level of cd α expression compared to the other splenic nk-cell subsets in all animals analysed sequences of primers ( ´- ´) for target genes as well as primer positions on (+) strand, length of specific product in base pairs (bp) and product melting temperature in°c are indicated. (nkp high : ± , nkp + : ± , nkp -: ± , figure b , lower graph). of note, nkp and nkp + nk cells in blood as well as spleen also showed a differential expression of cd α, although it was not as obvious as for the nkp high subset. within each location nkp -nk cells showed the highest expression of cd α, thus indicating a correlation between an increase of nkp and a decrease of cd α expression on porcine nk-cell subsets. to get more insight into the phenotype of the nkp defined nk cells we further analysed the expression of cd and the tnf-receptor family member cd . the latter is an important marker to distinguish between nk-cell subsets in mouse and human [ ] [ ] [ ] ] and the porcine orthologue of cd was recently identified [ ] . no marked difference in cd or cd expression between blood nkp and nkp + nk-cell subsets could be observed, although nkp + cells showed a slightly increased cd expression ( figure c) . a clear difference between the three splenic nkp -defined nk-cell subsets could be observed for cd (nkp high : ± , nkp + : ± , nkp -: ± , figure c ) and was even more obvious for cd expression (nkp high : ± , nkp + : ± , nkp -: ± , figure c ). nkp -nk cells showed the lowest and nkp high nk cells the highest expression of both markers, thus indicating a positive correlation between nkp , cd and cd expression levels. in human and mice higher cd expression on nk cells is associated with an increased cytokine production [ , , , ] . if the same holds true for porcine nk cells, splenic nkp high cd high nk cells should be the most prominent cytokine producers compared to the other subsets. we therefore compared ifn-γ and tnf-α production between the different nk-cell populations in blood and spleen of several individuals (n = ). intracellular staining for ifn-γ in total pbmc and splenocytes was performed after in vitro stimulation with a combination of rhil- , rpil- and rpil- overnight. cells cultured in medium alone served as negative control and did not show any ifn-γ production. the percentage of ifn-γ + nk cells was significantly higher in the blood nkp + nk-cell subset compared to blood nkp cells after cytokine stimulation ( . % ± . versus . % ± . , respectively, figure a and b). nevertheless, the produced amount of ifn-γ per cell, investigated by the mfi, was similar for both blood nk-cell subsets ( ± for nkp + and ± for nkp -, figure b , right graph). splenic nkp high nk cells clearly showed the highest frequency of ifn-γ + cells compared to the other two splenic subsets whereas nkp cells had the lowest frequency (nkp high : . % ± . %, nkp + : . % ± , nkp -: . % ± . , figure a and b) . additionally, the amount of ifn-γ produced per cell was considerably higher in the nkp high subset (nkp high : ± , nkp + : ± , nkp -: ± , figure b , right graph). yet, stimulated splenic nk-cell subsets showed an overall lower frequency of ifn-γ producing cells compared to blood. results of intracellular cytokine staining were confirmed by elisa ( figure c ). facs-sorted cd -cd α + nkp and cd -cd α + nkp + nk cells from blood and cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen were stimulated with rhil- , rpil- and rpil- overnight and supernatants were tested for ifn-γ production. blood nkp + nk cells produced higher levels of ifn-γ ( to -fold) compared to the blood nkp subset ( ± ng/ml versus ± ng/ml, respectively, figure c ), which is consistent with the data obtained by flow cytometry. in spleen differences between the nk-cell subsets were much more pronounced as nkp high nk cells showed a to -fold higher ifn-γ production compared to the nkp + ( ± ng/ml to ± ng/ml, figure c ), and to -fold higher production compared to the nkp -nk cells ( ± ng/ ml to ± ng/ml). in addition to ifn-γ, tnf-α production of the different facs-sorted nk-cell subsets was measured by elisa after cytokine or pma/ionomycin stimulation overnight (figure ). after stimulation with rhil- , rpil- and rpil- , splenic nkp high nk cells likewise showed the highest levels of tnf-α. thus tnf-α production was to -fold higher in the nkp high nk-cell subset compared to splenic nkp + nk cells ( ± pg/ml to ± pg/ml, figure a ) and to -fold compared to nkp -nk cells ( ± pg/ml to ± pg/ml, figure a ). blood nkp + nk cells showed a . to -fold higher tnf-α production compared to the blood nkp -nkcell subset ( ± pg/ml to ± pg/ml, figure a ). no obvious differences could be observed for tnf-α production between blood or spleen nkp and nkp + nk-cell subsets after pma/ionomycin stimulation ( figure b ) whereas splenic nkp high nk cells again showed an increased tnf-α production compared to splenic nkp + ( to -fold, ± pg/ml to ± pg/ml, figure b ) and splenic nkp -nk cells ( to -fold, ± pg/ml to ± pg/ml). data from both, ifn-γ as well as tnf-α production indicated that splenic nkp high nk cells, that also displayed an increased cd expression, are the most potent cytokine producing nk cell subset. it was already shown that blood nkp and nkp + nkcell subsets show comparable cytolytic activity against xenogeneic and allogeneic target cells in a nkp independent manner [ ] . to investigate whether the nkp high phenotype was also correlated with an increased cytolytic activity we performed cd a degranulation assays in combination with multi-colour flow cytometry. we used monoclonal antibodies against nkp or cd to mimic receptor-specific ligands to look at a possible correlation between receptor density and cytolytic activity of the different nk-cell subsets in blood ( figure ) and spleen ( figure ). background degranulation was determined by using irrelevant-isotype matched control antibodies. blood nkp + nk cells showed a clear cytolytic activity after triggering with anti-nkp mabs indicated by the induction of cd a ( figure ). as expected, nkp -nk cells showed no obvious increase in cd a expression after anti-nkp stimulation. although blood nkp as well as nkp + nk cells got activated by triggering of the fc receptor cd , this stimulation led to a higher cytolytic activation in the nkp + nk-cell subset ( . % ± . compared to . % ± . in the nkp -nk cells, figure b ). co-crosslinking of both receptors also led to a higher cd a expression in the blood nkp + nk-cell subset. interestingly, a comparison of data for the three different stimulations of the blood nkp + nk-cell subset revealed no co-stimulatory effect after co-crosslinking of nkp and cd ( figure a+b ). similar results were obtained for spleen nkp and nkp + nk-cell subsets ( figure ). splenic nkp + nk cells showed a higher cytolytic activity after triggering with anti-nkp and/or anti-cd mabs compared to nkp -nk cells ( % ± . to . % ± . for nkp and . % ± . to . % ± . for cd , figure a+b ). figure a+b) . a slight increase of both read-outs after co-receptor triggering with cd and nkp could be observed for this nk-cell subset compared to the nkp + cells where no obvious additive effect on degranulation could be found. the comparison of cytotoxic capability by degranulation assays demonstrated that blood nkp + nk cells showed an overall higher proportion of cd a + cells than splenic nkp + nk cells, regardless which receptor had been activated (mean of all stimulated fractions, blood nkp + cd a + : . % versus spleen nkp + cd a + : . %). however, cd a expression levels per cell, analysed by mfi, did not differ strongly (mean of all stimulated fractions, blood mfi nkp + cd a + : versus spleen mfi nkp + cd a + : ). instead, again regardless which receptor had been activated, the blood nkp + nk-cell subset showed more similar frequencies of cd a + cells to the spleen nkp high nk-cell subset (mean of all stimulated fractions, blood nkp + cd a + : . % versus spleen nkp high cd a + : . %). so far, our functional data indicated that splenic nkp high nk cells are in an elevated stage of activation. therefore we further analysed the expression of other nk-associated markers like the nk-receptors nkp and nkg d in this nk-cell subset. moreover, expression of the chemokine receptor cxcr was investigated since the expression of cxcr in combination with cd can be used for the identification of nk-cell subsets with different functional properties in the mouse [ ] . therefore, facs-sorted nkp -defined nk-cell subsets of blood and spleen were analysed for expression of these markers by quantitative rt-pcr ( figure ). additionally, nkp mrna levels in the different nk-cell subsets derived from blood and spleen were analysed. results confirmed data obtained from protein figure splenic nkp high nk cells produced the highest levels of tnf-α. analysis of tnf-α production of nkp -defined nk-cell subsets (cd α + nkp -: blue, cd α + nkp + : green, cd α dim/-nkp high : red) isolated from blood and spleen. facs-sorted cd -cd α + nkp and cd -cd α + nkp + nk cells from blood and cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen were stimulated with rhil- , rpil- and rpil- (a) or pma and ionomycin (b) for h. supernatants were tested for tnf-α production in elisa. bar graphs on the left show data from one representative animal and are displayed as the mean of duplicates ± sd. tnf-α production of four animals analysed are shown on the right. mean values are represented by a black bar. significant differences between the subsets in blood or spleen are indicated (* = p < . , ** = p < . ). expression, showing the same differential expression of nkp mrna in the respective subsets ( figure ). interestingly, despite high nkp expression, for the ncrfamily member nkp a reduced expression was found for splenic nkp high nk cells compared to the other two splenic subsets. blood nkp -nk cells seemed to express slightly higher levels of nkp as blood nkp + nk cells. nkg d mrna levels were very homogeneous among the different nkp -defined subsets. also, this receptor showed the lowest variation in expression levels between different individuals. the most prominent difference in rna expression was observed for the chemokine receptor cxcr . splenic nkp high nk cells showed an overall higher expression of this receptor compared to nkp and nkp + nk-cell subsets from both spleen and blood. for nkp and nkp + nk cells from blood and spleen similar expression levels of cxcr were observed. recently, the phenotype of porcine nk cells was revisited in a study from our group using newly developed mabs against porcine nkp . interestingly, cd -cd α + nk cells in the blood were shown to be either nkp + or nkp - [ ] . additionally, a third nk-cell subset with elevated nkp expression was found in high frequencies in spleen and liver that was associated with a cd α dim/phenotype. in the current study we aimed to investigate this splenic nkp high nk-cell subset in more detail and elucidate possible functional as well as phenotypical differences to the nkp and nkp + nk-cell subsets in the pig. figure nkp high nk cells in spleen showed the highest cytolytic capacity. the cytolytic capacity of nkp -defined nk-cell subsets (cd α + nkp -: blue, cd α + nkp + : green, cd α dim/-nkp high : red) isolated from spleen was analysed after receptor-mediated degranulation. cells were stimulated and gated for flow cytometric analysis as outlined in figure . splenic nkp high nk cells showed highly elevated expression of this activating receptor compared to the other two nkp -defined subsets in spleen as well as in blood in all animals analysed, which could be demonstrated on protein as well as on mrna level. additionally, splenic nkp high nk cells displayed a strongly decreased expression of cd α, a receptor that was so far described to be expressed by all nk cells in the pig [ , ] . of note, also the nkp and nkp + nk cells in blood and spleen showed minor differences in their cd α expression. we observed a negative correlation, thus an increase of nkp expression was accompanied by decreased expression level of cd α. furthermore, expression levels of cd and the tnf-receptor family member cd differed between the three nkp defined nk-cell subsets. cd expression was clearly enhanced in splenic cd α dim/-nkp high nk cells and this subset also displayed slightly higher levels of cd compared to the other two splenic subsets. in contrast, nkp -nk cells showed the lowest expression of both receptors, thus indicating a positive correlation in the expression levels of nkp , cd and cd . only few reports highlight on differential expression levels of nkp on nk cells in other species, although the existence of nkp dull and nkp bright nk cells was already described in the late s on human nk cells [ ] . additionally, it was reported that cells within distinct human nk-cell subsets show different levels of nkp expression. thus, higher cd expression [ , , ] as well as higher cd expression [ , ] was associated with higher surface density of nkp on human nk cells. obviously, the latter is akin to the correlation of nkp and cd we observed in the pig. more recently a phenotype of human nk cells with elevated expression of nkp was reported, which show an activated phenotype and seem to play a profound role in hepatitis c virus infection [ ] . likewise to our findings, in that study nkp high nk cells showed a higher expression of cd . furthermore, the authors of this report used the different expression levels of nkp to separate nk cells into distinct subsets, similar to the approach of our study. cd is used to distinguish between different nk-cell subsets in mouse and human. in the mouse, cd in combination with cd b is used to differentiate functionally as well as developmentally different nk-cell subsets [ , ] . cd divides the mature cd b high murine nk cells into two distinct subsets. cd high nk cells show a higher proliferative capacity and additionally an increased cytokine production compared to the cd low nk-cell subset [ , ] . in regard to cytotoxicity, differing reports exist. it was shown that murine cd high nk cells show elevated cytolytic activity compared to the cd low nkcell subset [ ] . however, another report describes the cd low/-cxcr -nk cells to be the more cytolytic subset in the mouse [ ] . in human nk cells, high cd expression is also linked to higher cytokine production and the cd low/phenotype correlates with an overall higher cytolytic activity [ , , ] . in contrast, the recent study on human nk cells that used nkp to distinguish between different nk-cell subsets showed that nkp high cd high nk cells have an enhanced cytokine production and cytolytic activity [ ] . we likewise observed this bifunctionality in the nkp high cd high nk-cell subset in spleen, with an elevated ifn-γ and tnf-α production figure varying mrna expression of nk-associated markers on nkp -defined nk-cell subsets in blood and spleen. facs-sorted cd -cd α + nkp and cd -cd α + nkp + nk cells derived from blood and cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen were analysed for their mrna expression of various nk-markers by quantitative rt-pcr. messenger-rna expression of the nk receptors nkp , nkp and nkg d as well as the chemokine receptor cxcr were analysed in the different nk-cell subsets (cd α + nkp -: blue symbols, cd α + nkp + : green symbols, cd α dim/-nkp high : red symbols). the ^-ΔΔct values for each individual animal (n = ) are shown as the fold differences relative to an internal standard (is = , black dotted line). geometric mean values of the three animals are represented by a black bar. after in vitro stimulation but also higher cytolytic capacity after triggering of the activating receptors cd or nkp compared to the other two splenic nk-cell subsets. consistent with this data, it was already suggested that higher density of the activating receptor nkp on the nk-cell surface is associated with higher cytolytic function [ , , ] . interestingly, we could not observe an obvious synergistic effect after co-crosslinking of both receptors in our study. nevertheless, similar results were shown for cd and nkp co-triggering in resting human nk cells [ ] . we observed an overall lower cytotoxic activity in the nkp subset in blood and spleen when stimulated with anti-cd antibody, although the expression level of cd was only slightly lower as in the nkp + nk cells. in our previous study we showed that blood nkp porcine nk cells had a cytotoxic capacity comparable to blood nkp + nk cells in killing assays using xenogeneic and allogeneic cell lines as targets [ ] . the lower killing capacity we observed in this study may be caused by the triggering of only a single activation pathway, whereas the killing of target cells is likely to result from the triggering of several activating receptors and/or lack of inhibitory signals. although splenic nkp high nk cells produced higher levels of ifn-γ compared to the other two subsets as shown by elisa, the overall proportion of ifn-γ producing cells was lower compared to blood nk cells. therefore, the higher cytokine production seems to result from a superior cytokine production on a single cell level as indicated by the higher mfi for ifn-γ in the splenic nkp high nk-cell subset. similar results could be observed for the cytolytic activity determined by cd a degranulation assays after receptor triggering. although the proportion of cd a + cells was not higher in the splenic nkp high subset compared to blood nkp + nk cells, nkp high nk cells showed the highest cd a expression levels on a per cell basis. thus, these data may suggest also a higher killing capacity on a single cell level within the splenic nkp high nk cells. taken the functional findings together, our data indicate that nkp high nk cells are in a highly activated state and can readily release high amounts of cytokines or cytolytic granules upon stimulation. to get further insight into the phenotype of the nkp defined nk-cells subsets in the pig we finally performed rt-qpcr analyses of distinct nk-cell associated markers. expression analyses of other activating receptors, namely nkp and nkg d, showed no marked differences between the nkp -defined nk cell subsets in blood as well as in spleen. splenic nkp high nk cells showed slightly lower levels of the ncr-family member nkp . nkg d that is described as important nk-cell receptor involved in target recognition in other species [ ] was very uniformly expressed between the different nk-cell subsets in the pig, which is consistent with data in mouse and human where nkg d shows an overall similar expression pattern between different nk-cell subsets [ , ] . the most prominent difference in expression between the nkp defined nk-cell subsets was observed for cxcr . splenic nkp high nk cells showed elevated levels of this chemokine receptor compared to the other two subsets. likewise, mouse cd high nk cells showed the highest levels of cxcr and recently a further sub-division of murine nk-cells by these two markers has been proposed [ ] . the chemokine receptor cxcr is associated with the recruitment of nk cells into the lymph node [ ] and accumulation in tumors [ ] . in the murine spleen, cxcr is important for intrasplenic trafficking of nk cells upon inflammatory signals [ ] as well as the mobilisation and migration of nk cells from the spleen into the periphery [ ] . thus, one can speculate that the elevated expression of cxcr on porcine splenic nkp high nk cells may indicate that this nk-cell subset is in a "ready-to-go" state for recruitment of nk cells into the periphery or within the spleen. in conclusion, our data shows that the splenic nkp high nk cells are in a highly activated state and can be readily activated upon in vitro stimulation. furthermore nkp high nk cells are characterised by a distinct receptor expression pattern compared to the nkp and nkp + porcine nk-cell subsets in blood as well as in spleen, including the tnf-receptor family member cd that can be used to separate functionally and developmentally different nk-cell subsets in other species. additional file : optimisation and validation of qpcr assays for nk-associated gene-specific primers. the suitability of the newly designed primers was verified in separate experiments by performing dilution series of pcr products in : or cdna pools in : steps in quadruplicates. the dilution series, in conjunction with the melt characteristics of the pcr product, were used to optimise the assays regarding the primer concentration, annealing and extension times and the efficiency for the pcr. the optimised pcr conditions including annealing and extension conditions as well as the reaction parameters (slope of the regression analysis corresponding to the efficiency of the qpcr) and the dynamic range for detecting % positive of the lowest dilution are indicated in the table. a product was detected in the rtminus control of some samples, nevertheless these showed at least . cqs or more difference to the respective rt-plus sample (Δct values are indicated in the table). calibration curve, melt curve and amplification blot for each target is illustrated. natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. ii. characterization of effector cells natural" killer cells in the mouse. ii. cytotoxic cells with specificity for mouse moloney leukemia cells. characteristics of the killer cell regulation of human nkcell cytokine and chemokine production by target cell recognition natural killer cells as an initial defense against pathogens natural killer cell subsets in man and rodents human natural killer cells: a unique innate immunoregulatory role for the cd (bright) subset cd bright cells differ in their kir repertoire and cytotoxic features from cd dim nk cells cd b and cd reflect distinct population and functional specialization in human natural killer cells cd dissects mature nk cells into two subsets with distinct responsiveness and migratory capacity murine cxcr +cd bright nk cells resemble the human cd bright nk-cell population expression of t-cell associated antigens by porcine natural killer cells discrimination between two subsets of porcine cd + cytolytic t lymphocytes by the expression of cd antigen perforin expression can define cd positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic t, natural killer t and mhc un-restricted cytotoxic t-cells host environment as a modulating factor of swine natural killer cell activity changes in lymphocyte populations in suckling piglets during primary infections with isospora suis infection with classical swine fever virus: effects on phenotype and immune responsiveness of porcine t lymphocytes natural killer cell dysfunction during acute infection with foot-and-mouth disease virus porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr infected pigs nkp expression discriminates porcine nk cells with different functional properties enhanced in vivo growth of lymphoma tumors in the absence of the nk-activating receptor nkp /ncr recognition and prevention of tumor metastasis by the nk receptor nkp /ncr natural killer p (high) expression defines a natural killer cell subset that is potentially involved in control of hepatitis c virus replication and modulation of liver fibrosis lethal influenza infection in the absence of the natural killer cell receptor gene ncr killing of avian and swine influenza virus by natural killer cells expansion of b + natural killer (nk) cells and decrease in nkp + nk cells in response to influenza p , a novel natural killer cell-specific surface molecule that mediates cell activation molecular cloning of nkp : a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity moretta l: identification, molecular cloning and functional characterization of nkp and nkp natural cytotoxicity receptors in macaca fascicularis nk cells identification, activation, and selective in vivo ablation of mouse nk cells via nkp the murine homologue of the human nkp , a triggering receptor involved in the induction of natural cytotoxicity rat nkp activates natural killer cell cytotoxicity and is associated with fcεriγ and cd ζ nkp defines a subset of bovine leukocytes with natural killer cell characteristics nkp defines ovine cells that have characteristics corresponding to nk cells generation and characterization of monoclonal antibodies to equine nkp monoclonal antibodies reactive with swine lymphocytes. ii. detection of an antigen on resting t cells down-regulated after activation porcine cd : identification, expression and functional aspects in lymphocyte subsets in swine characterization of swine leukocyte differentiation antigens preparation of cells and reagents for flow cytometry detection of intracellular antigens in porcine pbmc by flow cytometry: a comparison of fixation and permeabilisation reagents a rapid method for assessment of natural killer cell function after multiple receptor crosslinking porcine t-helper and regulatory t cells exhibit versatile mrna expression capabilities for cytokines and co-stimulatory molecules primer on the www for general users and for biologist programmers primer-blast: a tool to design target-specific primers for polymerase chain reaction quantitative simultaneous multiplex real-time pcr for the detection of porcine cytokines ra: cd defines phenotypically and functionally different human nk cell subsets application of cd as a marker for distinguishing human nk cell subsets nkp is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human nk cells. correlation between surface density of nkp and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells cd surface density identifies a functional intermediary between the cd bright and cd dim human nk-cell subsets analysis of natural killer cells in tap -deficient patients: expression of functional triggering receptors and evidence for the existence of inhibitory receptor(s) that prevent lysis of normal autologous cells maturation of mouse nk cells is a -stage developmental program synergy among receptors on resting nk cells for the activation of natural cytotoxicity and cytokine secretion roles of the nkg d immunoreceptor and its ligands induced recruitment of nk cells to lymph nodes provides ifnγ for t(h) priming natural killer cell accumulation in tumors is dependent on ifn-γ and cxcr ligands intrasplenic trafficking of natural killer cells is redirected by chemokines upon inflammation ifn-γ acts on t cells to induce nk cell mobilization and accumulation in target organs porcine cd α dim/-nkp high nk cells are in a highly activated state the authors thank maria stadler, katharina reutner, sandra groiß and sarah rosenthaler for their technical support. kerstin h. mair and part of this work were funded by the phd program "host pathogen interaction in the pig", university of veterinary medicine vienna, austria. the authors declare that they have no competing interests.authors' contributions khm carried out the laboratory work except qpcr analyses and participated in the design of the study. am performed practical work and data analyses for the qpcr assays. see and jcd performed qpcr assay design, validation and carried out qpcr data analyses. aks contributed to the design of experiments and drafting of the manuscript. as and wg were responsible for conception and design of the study. khm and wg wrote the manuscript and analysed the data. all authors read and approved the final manuscript. key: cord- -f jtufja authors: benedictus, lindert; otten, henny g; van schaik, gerdien; van ginkel, walter gj; heuven, henri cm; nielen, mirjam; rutten, victor pmg; koets, ad p title: bovine neonatal pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the mhc haplotype date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: f jtufja bovine neonatal pancytopenia (bnp), a bleeding syndrome of neonatal calves, is caused by alloantibodies absorbed from the colostrum of particular cows. a commercial bvd vaccine is the likely source of alloantigens eliciting bnp associated alloantibodies. we hypothesized that the rare occurrence of bnp in calves born to vaccinated dams could be associated with genetic differences within dams and calves. we found that the development of bnp within calves was a heritable trait for dams, not for calves and had a high heritability of %. to elucidate which genes play a role in the development of bnp we sequenced candidate genes and characterized bnp alloantibodies. alloantigens present in the vaccine have to be presented to the dam’s immune system via mhc class ii, however sequencing of drb showed no differences in mhc class ii haplotype between bnp and non-bnp dams. mhc class i, a highly polymorphic alloantigen, is an important target of bnp alloantibodies. using a novel sequence based mhc class i typing method, we found no association of bnp with mhc class i haplotype distribution in dams or calves. alloantibodies were detected in both vaccinated bnp and non-bnp dams and we found no differences in alloantibody characteristics between these groups, but alloantibody levels were significantly higher in bnp dams. we concluded that the development of bnp in calves is a heritable trait of the dam rather than the calf and genetic differences between bnp and non-bnp dams are likely due to genes controlling the quantitative alloantibody response following vaccination. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. since an increase in newborn calves with the bleeding syndrome bovine neonatal pancytopenia (bnp) was observed all over europe [ ] [ ] [ ] . epidemiological studies showed a strong association between the occurrence of bnp in calves and vaccination of their dams with the pregsure© bvd vaccine (pfizer animal health) [ ] . symptoms of bnp are severe internal and external bleeding, first seen around - days of age. hematological signs are severe leukopenia and thrombocytopenia. in addition, trilineage hypoplasia of the bone marrow can be observed upon post-mortem examination [ ] [ ] [ ] . colostrum of dams that had previously given birth to a calf which developed bnp contained alloantibodies recognizing bovine leukocytes [ ] [ ] [ ] [ ] . feeding this colostrum to healthy neonatal calves induced the symptoms of bnp [ , , ] . proteins from the bovine kidney cell line mdbk [ ] , used to grow the bvd type virus present in pregsure© bvd, are the likely source of alloantigens that induce alloantibody production in vaccinated dams. the alloantibodies bind mdbk cells and it was shown that an important target of these antibodies were mhc class i proteins [ , , ] . moreover, mdbk derived mhc class i proteins were detected in the pregsure© bvd vaccine [ , ] and immunization of calves with pregsure© bvd induced alloantibodies recognizing mdbk cells [ , ] . since the incidence of bnp calves born to pregsure© bvd vaccinated dams was estimated to be lower than . % [ , , ] , it was hypothesized that factors other than vaccination per sé play a role in the etiology of bnp. the prevailing hypothesis is that the pathogenesis of bnp resembles a histocompatibility (mis)match between dam and calf and is based on immunization of the dam with mdbk derived mhc class i [ , ] . first, in the dam mdbk cell derived proteins, present in the preg-sure© bvd vaccine, are presented in the context of mhc class ii. the resulting t cell help to b cells recognizing allogeneic differences between mdbk cells and the dam will result in the generation of alloantibodies which are also present in the colostrum. due to tolerance to self-antigens, dams do not exhibit adverse effects after vaccination, i.e. the vaccine induced alloantibodies do not recognize alloantigens expressed in the dam. the maternal alloantibodies transferred to the calf via the colostrum will recognize alloantigens in case of a partial alloantigen match between mdbk cells and the calf. we hypothesized that the rare occurrence of bnp after pregsure© bvd vaccination may depend both on the capability of the dam's immune system to present the mdbk alloantigens via mhc class ii, as well as the degree of alloantigen (mis)match between the dam and the mdbk cell line (and the calf and the mdbk cell line, respectively) and the ensuing immune response of the dam. since alloantigens (including mhc i and mhc class i associated b m) and mhc class ii are genetically determined and therefore heritable, we studied whether differences in these genes between dams and/or calves may explain why bnp only occurs in part of the calves born to pregsure© bvd vaccinated dams. first we studied the heritability of the development of bnp in the calf as a potential dam or calf trait. next, to elucidate if these genes genes play a role in the development of bnp we sequenced and compared the mhc and b m candidate genes and characterized bnp associated alloantibodies. the data used for the heritability study were a subset of data from a large multi country epidemiological study on bnp [ ] and concerned dutch farms that participated in this study. data on herd matched bnp and non-bnp calves were collected by on farm questionnaires. we looked at the heritability of the development of bnp within the calf as a trait of pregsure© bvd vaccinated dams as well as of calves born to these dams. the definitions for bnp and non-bnp calves used, were according to jones et al. [ ] . a bnp calf was defined as a calf that showed one or more bnp clinical signs on or before days of age; bone marrow depletion as assessed by histopathology and/or thrombocytopenia (< × /litre) and leucopenia (< × /litre). a non-bnp calf was defined as a calf on the same farm as a case, aged - days at the time of case reporting, no clinical signs of bnp up to days of age, and normal blood parameters (thrombocytes ≥ × /litre, leucocytes ≥ × /litre). to ensure that the correct phenotype, bnp or non-bnp, was assigned to the dam, only calves that were fed colostrum from their own dam were included. furthermore, dam-calf combinations without pedigree information were excluded. pedigrees of calves and dams were provided by the dutch cattle improvement organization (crv, arnhem, the netherlands). the pedigree of dam-calf combinations meeting the inclusion criteria were traced back up to generations and the final pedigree included records. the first generation of the pedigree was a % complete for calves and % complete for dams. the data were analyzed using the software package asreml [ ] , a statistical package that fits generalized linear mixed models using residual maximum likelihood. the heritability of the development of bnp within the calf as a dam and calf trait was estimated from the dam and sire variance components of a sire-dam model. only alloantigens inherited from the sire can be recognized by maternal alloantibodies and therefore the heritability of the development of bnp as a calf trait was estimated by calculating bnp as a sire trait. variables included in the data set were: bnp was fitted as a binomial variable using the logistic link function to relate binomial outcome of bnp to the linear predictor used for the generalized linear mixed model. the following general model was used: where bnp is the outcome of bnp, μ is the general mean, (x i ) n is one or more of the aforementioned variables, sire j is the random effect of the jth sire; dam k is the random effect of the kth dam and e (i)n jk is the vector of residuals. heritability was calculated using the variance components of the model, as follows: bnp as dam trait h = σ dam /σ p ; bnp as a sire trait h = σ sire /σ p ; σ p = σ dam + σ sire + (π )/ , where σ p is the phenotypic variance, σ dam is the dam variance, σ sire is the sire variance and the residual variance was fixed at (π )/ . first we looked at the effect of each individual variable on bnp in a sire-dam model. next all variables with a p-value < . were included in the final sire-dam model. immune responses normally decline with time and to test if the incidence of bnp after the last preg-sure© bvd vaccination also declines with time, the variable time since last pregsure© bvd vaccination was forced into the final model despite having a p-value higher than . in the univariate model. because the estimates for time since last pregsure© bvd vaccination appear to have a linear effect on bnp, the variable was added as a linear covariable in the final model. there were only eight dams with one pregsure© bvd vaccination and because the vaccination scheme consists of an initial prime and subsequent boost vaccination which may have been interpreted as one vaccination by the farmer, in the final model animals with one or with two pregsure© bvd vaccinations were grouped. blood of calves was drawn as part of the multi country epidemiological study on bnp [ ] . farms with more than one living bnp dam were revisited in to collect blood-and colostrum-samples from dams. throughout our study we used the following definitions for dams and calves: non-bnp dam -dam that had been vaccinated with pregsure© bvd and had not given birth to a calf that developed bnp following colostrum feeding. -bnp dam -dam that had been vaccinated with pregsure© bvd and had given birth to a calf which developed bnp following colostrum feeding. -non-bnp calf -calf born to a pregsure© bvd vaccinated dam, that upon receiving colostrum from its dam did not show signs of bnp, confirmed via hematology and/or pathology. -bnp calf -calf born to a pregsure© bvd vaccinated dam, that upon receiving colostrum from its dam showed clear signs of bnp, confirmed via hematology and/or pathology. this study was approved by the animal ethical committee of utrecht university and conducted according to their regulations. sequence based typing of mhc class i was done using gene specific primers aligning with intron and intron of mhc class i genes , , and [ ] (additional file ). these primers amplify exon and , which encode the most polymorphic regions of the mhc class i gene. for genes , and pcr was carried out in μl containing . u expand high fidelity taq (roche diagnostics, indianapolis, usa), . mm mgcl , . mm each dntp and . μm, or . um in the case of primers with ambiguous nucleotide, of each primer. the thermal cycling profile was °c for min, cycles of °c for s, °c for s, °c for s followed by °c for min. for gene pcr conditions were similar, except . u of amplitaq® (applied biosystems, life technologies) was added, the mgcl concentration was mm and the annealing temperature was °c. sequencing of pcr's resulting in a product were performed on the dna analyzer (applied biosystems) using the same primers used for the pcr and the bigdye® terminator v . cycle sequencing kit (applied biosystems). sequence products were analyzed using seqscape© (v . , applied biosystems). forward and reverse sequences were aligned to a reference sequence to produce a consensus sequence. using the ipd mhc database [ ] a library of the exon and sequences of known mhc class i alleles was constructed using seqscape©. seqscape© is able to cope with ambiguous nucleotides and, in the case of heterozygous pcr products, matches the consensus read to the best combinations of alleles from the library. consensus read basecalling of the amplified genomic dna and library matches to known full length mhc class i cdna sequences were checked. using the assigned mhc class i alleles, mhc class i haplotypes were determined using haplotypes defined in codner et al. [ ] and this study (additional file ). mhc class i haplotypes define a set of mhc class i alleles that are inherited together and haplotype differences between animals do not give information on differences in mhc class i as an alloantigen. to better estimate allogeneic differences between mdbk cells and dams/calves we looked at mhc class i protein differences between mdbk cells and dams/calves. alloantibodies recognize the extracellular part of expressed proteins and we therefore looked at protein differences within the extracellular part of mhc class i (exon - ). dna sequences of exon - were translated into protein sequences and the difference in protein sequence between two mhc class i alleles was calculated, expressed as percentage of the protein sequence that was different. dams can recognize mdbk alleles (listed in additional file ) as non-self if there are differences between the dam and mdbk mhc class i and for dams we calculated the difference between the mdbk allele that was most different to the dam mhc class i alleles. for alloantibodies to recognize mhc class i in the calf, there has to be a (partial) match between the mdbk and paternally inherited calf mhc class i and for calves we calculated the difference between the most similar mdbk and paternally inherited calf mhc class i allele. beta- -microglobulin (b m) primers (additional file ) flanking exon were designed using the bovine whole genome assembly umd . . pcr was carried out in μl containing . u pfuturbo cx hotstart dna polymeras (agilent, santa clara, usa), mm mgcl , . mm each dntp and . μm each primer. the thermal cycling profile was °c for min, cycles of °c for s, °c for s, °c for s followed by °c for min. sequencing was performed as described for mhc class i. forward and reverse sequences were aligned to the umd . reference sequence using seqscape©. drb sequence based typing was based on the method described by miltiadou et al. [ ] . primers aligning with intron and of the drb locus (additional file ) amplify exon , the most polymorphic region of the drb gene. pcr was carried out in μl containing . u amplitaq gold (applied biosystems), . mm mgcl , . mm each dntp and . μm each primer. the thermal cycling profile was °c for min, cycles of °c for s, °c for s, °c for s followed by °c for min. sequencing was performed as described for mhc class i. sequence reads were analysed using seqscape© as described for mhc class i. total alloantibody levels were assessed as serum antibody levels specific for mdbk cells. the latter were suspended in serum diluted : in pbs supplemented with % fcs and . % sodium azide. bovine igg binding was detected using polyclonal biotinylated sheep anti-bovine igg antibodies (abd serotec, bio-rad laboratories inc, hercules, usa) and streptavidin-phycoerythrin (bd biosciences, franklin lakes, usa). isotype specific alloantibodies were measured in a similar way. mdbk cells were suspended in serum or colostrum diluted : and alloantibody binding was detected by bovine isotype specific mouse monoclonal antibodies [ ] and fitc conjugated polyclonal goat anti-mouse antibodies (bd biosciences). total leukocytes were isolated from blood collected from ten healthy randomly selected dams at the slaughterhouse by hypotonic lysis of erythrocytes. whole blood was suspended in parts of distilled water, after lysis of erythrocytes isotonicity was restored using volume of x pbs. total leukocytes, used to detect alloantibody binding to peripheral blood mononuclear cells (pbmc), were suspended in serum or colostrum diluted : . alloantibody binding was detected by anti-bovine igg mouse monoclonal antibodies and fitc conjugated polyclonal goat anti-mouse antibodies. in all alloantibody binding experiments serum from non pregsure© bvd vaccinated dams were used as (isotype) controls. flow cytometry (bd facscanto™, bd biosciences) was used to measure alloantibody binding and data was analyzed using flowjo software (tree star inc., ashland, usa). pbmc were selected based on forward and sideward scatter. data are depicted as geometric mean fluorescent intensity (gmfi). in the case of alloantibody binding to pbmc depicted gmfi values are gmfi values subtracted by the gmfi of the isotype controls. in order to be able to compare alloantibody binding of pbmc irrespective of total alloantibody levels in serum or colostrum, relative alloantibody binding was calculated by dividing the gmfi of each sample by the gmfi of alloantibody staining of mdbk cells, representing total alloantibody binding. a positive pbmc sample was defined as a sample that had a higher geometric mean fluorescent intensity (gmfi) than the average of all measured samples or in the case of alloantibody level compensated values defined as having a higher relative signal than the average of the relative signal of all samples. the wald test was used to test whether a variable improved the fit of the sire-dam model. haplotype/allele frequencies were analyzed using fisher's exact test. alloantibody binding levels were compared by two tailed simple t-tests for unequal variance. to adjust for multiple comparisons the false discovery rate (fdr) was controlled using the method by benjamini and hochberg [ ] . this method controls the chance of falsely declaring the result of a statistical test as significant. the largest p-value lower than its fdr-derived significance threshold and all p-values smaller were considered to be significant. the number of significant p-values that are false positive was controlled at %. correlation was tested with pearsons correlation. normality was tested with d' agostino and pearsons omnibus normality test. effects were considered significant at p < . . when applicable, values were given as mean ± the standard error of the mean, with the latter between brackets. heritability of the development of bnp within the calf as a trait for pregsure© bvd vaccinated dams and for calves based on the inclusion criteria dam-calf combinations were selected for the heritability analysis. the calves were born from dams, fathered by sires and comprised bnp cases. the effect of each individual variable on bnp is summarized in additional file . the parameter estimates and odds ratios for the final model are shown in table . for pregsure© bvd vaccinated dams the heritability estimate for the development of bnp within the calf was . ( . ) and for sires it was . ( . ). the odds of bnp increased with an increased number of pregsure© bvd vaccinations. the odds of bnp increased up to the third lactation and was lower for the fourth and fifth lactation. the effect of time since last pregsure© bvd vaccination on bnp was not significant. sequence based typing was used to determine mhc class i haplotypes in vaccinated non-bnp and bnp dams ( table ). the largest frequency differences between dams were seen for variants of the a mhc class i haplotype, but with a p-value of . , which was much higher than the fdr-threshold of . , this was not significant. assuming an incidence of bnp of . % for pregsure© bvd vaccinated dams [ ] , the positive predictive value of the a haplotypes was . . implying that bnp only occurred in . % of calves born to preg-sure© bvd vaccinated dams with the a mhc class i haplotype. the difference in protein sequence between the extracellular parts of the mdbk and dam mhc class i alleles was . % ( . %) for vaccinated non-bnp dams and . % ( . %) for bnp dams, with a p-value of . this was not significantly different between both groups (additional file ). the paternal mhc class i haplotype frequencies of non-bnp and bnp calves are shown in table . based on the fisher's exact test the frequency of the a haplotype was significantly higher in bnp calves, however with a p-value of . this value was higher than the fdr threshold of . . assuming an incidence of bnp of . % for pregsure© bvd vaccinated dams [ ] , the positive predictive value of the a haplotype was . . which implies that only . % of calves with a paternally inherited a mhc class i haplotype born to pregsure© bvd vaccinated dams get bnp. in five non-bnp and three bnp calves fathered by the same sire, the mhc class i haplotypes were also typed (additional file ). since all eight calves had the a mhc class i haplotype, it is likely that the sire was a homozygous. in that case both non-bnp and bnp calves inherited the a haplotype from their father and for these calves there was no association between the paternally inherited a haplotype and the development of bnp. the protein difference between the extracellular part of the mdbk mhc class i alleles and paternally inherited to adjust for multiple comparisons the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant. calf mhc class i alleles was . % ( . %) for non-bnp calves and . % ( . %) for bnp calves, with a p-value of . this was not significantly different between both groups (additional file ). exon of the beta- -microglobulin (b m) gene, encoding % of the mature protein, was sequenced in mdbk cells and in five vaccinated non-bnp dams and five bnp dams that were farm matched. the b m sequences of all vaccinated non-bnp dams, bnp dams and mdbk cells were identical. results of the drb typing of vaccinated non-bnp dams and bnp dams are shown in table . the largest frequency differences were seen for the drb alleles and both with a higher frequency in vaccinated non-bnp dams. however, the p-values were well above the fdr threshold and not significant. total alloantibody levels in dams not vaccinated with pregsure© bvd and in pregsure© bvd vaccinated non-bnp and bnp dams were assessed as serum antibody levels specific for mdbk cells using flow cytometry ( figure ). alloantibody levels in bnp dams were significantly higher than in both non-bnp dams and dams not vaccinated with pregsure© bvd, levels in vaccinated non-bnp dams are significantly higher than in dams not vaccinated with pregsure© bvd. isotype specific alloantibody binding of mdbk cells is shown in figure . igg alloantibodies were most abundant and the levels were significantly higher in serum of non-bnp dams and in serum and colostrum of bnp dams compared to dams not vaccinated with pregsure© bvd. igg alloantibody levels were significantly higher in serum and colostrum of bnp dams compared to dams not vaccinated with pregsure© bvd. igg alloantibody levels tended to be higher in non-bnp dams as well, but due to higher variation among dams, did not differ significantly from that in dams not vaccinated with pregsure© bvd. for igm and iga there were no significant differences between groups. antibodies present in serum and colostrum from non-bnp and bnp dams bind pbmc ( figure a ). alloantibody binding of pbmc was significantly higher for both serum and colostrum of bnp dams compared to non-bnp dams. the number of pbmc samples that were positive were also higher for both serum and colostrum of bnp dams. however, average alloantibody binding of pbmc and alloantibody binding of mdbk cells had a high correlation ( figure b ) and when alloantibody binding of pbmc was compensated for mdbk specific alloantibody levels to enable comparison of the binding of pbmc irrespective of total alloantibody levels, the relative signal was to adjust for multiple comparisons the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant. c denotes a local name and is not included in the ipd bovine mhc class ii database. the same for bnp dams and non-bnp dams for serum as well as colostrum ( figure c) . also, the number of pbmc samples that were positive were similar in both groups for serum as well as colostrum. results of individual serum and colostrum samples are shown in additional file . we hypothesized that the rare occurrence of bnp after pregsure© bvd vaccination depends both on the capability of the dam's immune system to present the mdbk alloantigens via mhc class ii, as well as the degree of alloantigen (mis)match between the dam and the mdbk cell line (and the calf and the mdbk cell line, respectively) and the ensuing immune response of the dam. as a corollary we hypothesized that genetic differences in mhc class ii in dams and alloantigens in dams and calves (e.g. mhc i and mhc class i associated b m) would then explain why bnp only occurs in part of the calves born to pregsure© bvd vaccinated dams. the present study demonstrates that the development of bnp in calves is a heritable trait for pregsure© bvd vaccinated dams with the high heritability estimate of %, which shows that genetic differences between dams explain in part why only the colostrum of some pregsure© bvd vaccinated dams cause bnp in the calf. genetic variation in the paternal haplotype of the calves is not related to the development of bnp in the calf, since the heritability of the development of bnp in calves born to pregsure© bvd vaccinated dams is %. demasius et al. [ ] found that in an experimental german holstein x charolois crossbred herd with a limited number of sire lines, all bnp cases were restricted to a single maternal grandsire, also indicating the importance of the genetic background of the dam. in addition from a limited number of bnp dams was shown to induce bnp in randomly selected healthy calves [ ] [ ] [ ] which supports the notion that the genetic background of the calf is not critical. the phenotype of the calf was based on very strict objective criteria, whereas the phenotype of the dam was based on the phenotype of the calf. bnp is caused by alloantibodies present in the colostrum and the phenotype of the calf therefore depends on the quality, quantity and source (own dam or other dam) of the ingested colostrum. this means that the phenotype of the calf, may not always be the proper phenotype of the dam. much of this information was farmer reported and although we have tried to control for these aspects, the possibility exists that non-differential misclassification of the phenotype of the dam occurred in this study and implies that the heritability for the development of bnp within calves of % for dams is potentially underestimated. results were compared by two tailed simple t-tests for unequal variance. to adjust for multiple comparisons, the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant and are depicted by an asterisk (*). in our more in depth analyses of the genetic differences between pregsure© bvd vaccinated non-bnp and bnp dams we sequenced a number of specific candidate genes. an important target of bnp alloantibodies is mhc class i [ , ] , a highly polymorphic alloantigen [ ] . hence mhc class i was genotyped to see if differences in mhc class i alloantigen repertoire of dams and/or calves were associated with the development of bnp in the calf. we did not find an association between the mhc class i of the pregsure© bvd vaccinated dams and the occurrence of bnp. although the number of bnp calves in the mhc class i haplotyping analysis was limited, it showed that bnp calves do not have a single paternal mhc class i haplotype and that most of the paternal haplotypes are shared between bnp and non-bnp calves ( table , additional file ), together indicating that the paternally inherited mhc class i of calves is not associated with the occurrence of bnp. this result supports our finding that the heritability of the development of bnp in calves is zero and shows that bnp and non-bnp calves do not have a different allogeneic background. ballingall et al. [ ] found no differences in drb allele frequencies between bnp and non-bnp calves. since drb and mhc class i are in linkage disequilibrium, this corroborates our mhc class i typing result in calves. the binding of certain monoclonal antibodies to the b m-mhc class i heavy chain heterodimer can depend on the associated b m allele [ ] or mhc class i allele [ ] . although polymorphisms within the bovine b m gene are known, none lead to changes in the amino acid sequence [ ] . nevertheless we wanted to exclude the possibility that an unknown rare allelic variant of b m influences the recognition and immune response to mdbk mhc class i proteins present in the vaccine. since sequences of b m were identical in the mdbk cell line and all typed pregsure© bvd vaccinated non-bnp and bnp dams, it is highly unlikely that allelic variations of b m play a role in the etiology of bnp. another aspect of immune recognition of mdbk alloantigens present in the pregsure© bvd vaccine is their presentation to the dam's immune system via mhc class ii. mhc class ii haplotypes have been associated with disease resistance and susceptibility [ , ] and influence antibody responses after vaccination [ , ] . we found no association between mhc class ii haplotypes, as assessed by sequencing the highly polymorphic drb locus, and the occurrence of bnp in pregsure© bvd vaccinated dams. pregsure© bvd vaccinated bnp dams had significantly higher serum alloantibody levels compared to pregsure© bvd vaccinated non-bnp dams. nonetheless figure isotype characterization of alloantibodies from pregsure© bvd vaccinated dams. flow cytrometry was used to measure the isotype of alloantibodies binding to mdbk cells in serum (ser) or colostrum (col) from i) dams not vaccinated with pregsure© bvd (bnp-vacc-) ii) pregsure© bvd vaccinated non-bnp dams (bnp-vacc+) and iii) pregsure© bvd vaccinated bnp dams (bnp + vacc+). all results were compared by two tailed simple t-tests for unequal variance. within each isotype, all groups are compared to the non pregsure© bvd vaccinated dams (ser bnp-vacc-). to adjust for multiple comparison, the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant and are depicted by an asterisk (*). gmfi = geometric mean fluorescent intensity. alloantibodies were produced both in pregsure© bvd vaccinated non-bnp and bnp dams, confirming results from a previous study [ ] . alloantibody production by all pregsure© bvd vaccinated dams indicated there were allogeneic differences between the bovine mdbk proteins and both pregsure© bvd vaccinated non-bnp and bnp dams. this corroborated the sequencing results, where we did not find a difference between mhc class i or b m between pregsure© bvd vaccinated non-bnp and bnp dams. it also indicated that all dams were able to present alloantigens from the pregsure© bvd vaccine in the context of mhc class ii and fitted with the lack of an association between drb and the occurrence of bnp within pregsure© vaccinated dams. the antibody isotype produced by b-cells depends on the cytokines that are produced during an (vaccine induced) immune response [ , ] . the type of vaccine induced immune response may therefore influence the quality of the ensuing antibody response. as different antibody isotypes induce different biological effector functions, such as complement activation and neutralization, we studied the quality of the antibody response in pregsure© bvd vaccinated non-bnp and bnp dams to determine if bnp dams only differ in alloantibody levels or also in the isotype and specificity of alloantibodies produced. bnp is caused by alloantibodies from colostrum and for bnp dams serum and colostrum alloantibodies were compared to see if results for serum alloantibodies can be extrapolated to colostrum derived alloantibodies. alloantibody isotypes were similar in serum of pregsure© bvd vaccinated non-bnp dams and serum and colostrum of bnp dams, indicating a similar response to vaccination in both groups. likewise, studying cattle responding with high or low antibody levels after vaccination with hen-egg white lysozyme or candida albicans extract heriazon et al. [ ] also did not find any differences in igg and igg levels between animals, whereas antibody levels following vaccination varied significantly. when stained with serum or colostrum from pregsure© bvd vaccinated bnp dams higher numbers of (random) pbmc samples were positive for alloantibody binding and on average staining intensity was higher than when stained with serum or colostrum from pregsure© bvd vaccinated non-bnp dams. however, when compensated for alloantibody binding of mdbk cells to enable comparison of binding to pbmc irrespective of total alloantibody levels, numbers of positive pbmc samples and the relative staining intensity with alloantibodies were comparable between pregsure© bvd vaccinated bnp and non-bnp dams, indicating that the specificity for allogeneic cells was also comparable. based on the similar antibody isotypes and relative staining of pbmc we argue that alloantibodies from pregsure© bvd vaccinated non-bnp and bnp dams are qualitatively similar and that only the level of alloantibodies is higher in pregsure© vaccinated bnp dams. the high correlation between binding of alloantibodies to mdbk cells and pbmc corroborates the notion that the most important alloantigens in the pregsure© bvd vaccine are derived from the producer cell line. bastian et al. [ ] found that bnp dams also had higher bvd neutralizing antibody levels than pregsure© bvd vaccinated non-bnp dams, showing that bnp dams generally respond with higher antibody levels to components in the pregsure© bvd vaccine. in combination with the high heritability estimate for the development of bnp in calves for preg-sure© bvd vaccinated dams, it is likely that genetic differences between vaccinated non-bnp and bnp dams are due to genes that determine the level of antibody production after pregsure© bvd vaccination. in cattle high heritability estimates have been found for antibody production after vaccination, these ranged from % to % [ , ] . high antibody production after brsv vaccination was associated with single nucleotide variants of tlr and tlr [ ] . likewise, differences in responsiveness of the innate immune system of bnp dams to the adjuvant of the pregsure© bvd vaccine may have led to higher antibody production to antigens in the vaccine. the occurrence of bnp shows that in an outbred population some individuals may respond very differently to vaccination than the general population. this emphasizes the importance of monitoring adverse effects of both existing and new vaccines, but may on the other (see figure on previous page.) figure binding of peripheral blood mononuclear cells by alloantibodies from pregsure© bvd vaccinated dams. a: peripheral blood mononuclear cells (pbmc) from ten random dams were stained with serum (n = ) and colostrum (n = ) of different pregsure© bvd vaccinated non-bnp dams (bnp-vacc+, n = ) and with serum (n = ) and colostrum (n = ) of pregsure© bvd vaccinated bnp dams (bnp + vacc+, n = ). igg alloantibody binding was measured by flow cytometry. gmfi subtracted by isotype control is plotted on the y-axis. the horizontal dotted line depicts the overall average geometric mean fluorescent intensity (gmfi) and the number above the plots describes the number of samples with a signal above the horizontal line. b: correlation between the average igg alloantibody binding of pbmc's from ten dams to igg alloantibody binding of mdbk cells by serum or colostrum samples as in figure a . c: the data from figure a were divided by the gmfi signal of the alloantibody staining of mdbk cells by the respective serum or colostrum. the horizontal dotted line depicts the overall average relative signal and the number above the plots describes the number of samples with a signal above the horizontal line. mean ± standard error of the mean is depicted in all graphs. two tailed simple t-tests for unequal variance was used to compare serum or colostrum alloantibody binding of pbmc's between pregsure© bvd vaccinated non-bnp and bnp dams. correlation was tested with pearsons correlation. normality was tested with d'agostino and pearsons omnibus normality test. hand also provide opportunities for selective breeding for an increased humoral immune response. bovine mhc class i has an unusual organization, with six putative genes of which a variable number of genes are functionally present per haplotype [ ] , making mhc class i typing in cattle difficult. several techniques with different (dis)advantages have been used to type mhc class i in cattle, including serology [ ] , cloning and sequencing of full length cdna [ ] and next generation sequencing of polymorphic regions [ ] . in this study we use gene specific primers for four of the six mhc class i genes to amplify exon and [ ] , encoding the most polymorphic region of the mhc class i. alleles are distinguished based on exon and sequence and full length sequences are imputed from the ipd bovine mhc class i database [ ] , a method commonly used for hla typing (e.g. [ ] ). advantages of this typing method are that the amplified gene specific sequence normally only contains two alleles, allowing the use of traditional sanger sequencing, and that a relatively large number of samples can be typed, as was necessary for the present study. however, there are also some limitations to this method. the gene specific primers are only validated for holsteins, which was not a problem in this study as all dams were of holstein origin, and alleles from mhc class i gene and are not directly typed. however, genes and are the least polymorphic of the bovine mhc class i genes with only seven documented alleles of which only two have been reported in holsteins [ ] . one of these alleles ( * ) can be imputed based on haplotype and the other ( * ) is amplified by gene specific primers. mhc class i haplotypes define a set of mhc class i alleles that are inherited together and because different haplotypes can define very similar mhc class i alleles, haplotype differences between animals do not accurately represent allogeneic or immunological differences between animals. it has been hypothesized that the occurrence of bnp depends on allogeneic (mis)matches between the dam, the mdbk cell line and the calf [ , ] . the likelihood of an alloimmune response is directly related to the number of epitope mismatches between the foreign alloantigen and the host [ ] and in order to better estimate allogeneic (mis)matches between animals and mdbk cells, we analyzed protein differences in the extracellular domain of the mhc class i protein between mdbk cells and dams/calves. although this method is potentially a better estimate of allogeneic differences between animals than only relying on mhc class i haplotypes, accurate prediction of antibody epitopes is much more complicated and depends on many other factors, such as conformation, non-linear epitopes and flanking residues [ ] . since genes and are not directly typed in the mhc class i method used in this paper, in some animals the presence of certain alleles was imputed from the defined haplotype for that animal and for newly defined haplotypes the presence of additional alleles cannot be excluded, giving an extra level of uncertainty to the analysis of the mhc class i protein differences. however, previously published haplotypes comprise the majority of the haplotypes typed in this study and the results from the mhc class i halpotyping corroborates the results from other experiments in this study. together, the results indicate that the occurrence of bnp is not associate with a specific allogeneic background of bnp dams or calves. the only difference we found between pregsure© bvd vaccinated non-bnp and bnp dams were in the alloantibody levels and this would imply that the development of bnp in the calf primarily depends on the alloantibody dose the calf absorbs. the finding by jones et al. [ ] that the odds of bnp increases with increased colostrum intake, and thus alloantibody intake, strengthens this hypothesis. the risk of bnp increases with increased number of pregsure© bvd vaccinations and this can be explained by boosting of antibody production, increasing the alloantibody levels in the colostrum and thus increasing the alloantibody dose of the calf after colostrum ingestion. furthermore, our findings that the heritability for the development of bnp in the calf was % for calves, whereas as a dam trait the heritability was % and the observation that bnp can be induced in unrelated healthy calves by alloantibodies/ colostrum from bnp dams [ ] [ ] [ ] show that the dam and not the calf plays a pivotal role in determining whether a calf gets bnp or not. we conclude that the development of bnp in calves is a heritable trait of the dam rather than the calf and that genetic differences between bnp and non-bnp dams are likely due to genes controlling the quantitative alloantibody response following vaccination. additional file : primers used for the amplification of mhc class i genes, bèta- -microglobulin and drb . the table lists the sequences and the location of the forward and reverse primers used to amplify the mhc class i genes, bèta- -microglobulin and drb genes. additional file : list of mhc class i haplotypes and mhc class i typing results of the mdbk cell line. list of mhc class i haplotype definitions used in this article and the mhc class i typing results of the mdbk cell line. the newly defined haplotypes, containing an uu prefix or suffix, are provisional haplotypes. these haplotypes have not been confirmed using different mhc class i typing methods and because the gene specific primers used in this study do not amplify gene and and have not been validated for all known mhc class i alleles, the presence of additional alleles cannot be excluded. in some cases previously defined haplotypes [ ] have been renamed to accommodate for additional haplotype variants within a group. allele nomenclature refers to the ipd bovine mhc class i database [ ] . additional file : summarizing results of the univariable analysis of the effect of independent variables on bnp, including sire and dam heritability estimates (n = ). the table provides results from the univariable analysis of the effect of independent variables on bnp. the number of records per category, the estimate (β), the odds ratio and the p-value from the wald test are given. the heritability estimates for bnp as a sire trait and bnp as a dam trait are also depicted. additional file : comparison of the difference in protein sequence of the extracellular part of the mhc class i protein (exon - ) between the the mdbk mhc class i allele that is most different to the mhc class i alleles of pregsure© bvd vaccinated non-bnp and bnp dams. dna sequences of the extracellular part of mhc class i, exon - , were translated into protein sequences and the percentage of sequence difference between the mdbk mhc class i allele that was most different to the dam mhc class i alleles was calculated. results for pregsure© bvd vaccinated non-bnp and bnp dams were compared using an unpaired t-test for unequal variance. additional file : mhc class i haplotypes of calves (n = ) fathered by the same sire. the table lists sequence based mhc class i haplotyping results for three bnp and five non-bnp calves fathered by the same sire. additional file : comparison of the difference in protein sequence of the extracellular part of the mhc class i protein (exon - ) between the most similar mdbk and paternally inherited mhc class i allele from non bnp and bnp calves. dna sequences of the extracellular part of mhc class i, exon - , were translated into protein sequences and the percentage of sequence difference between the most similar mdbk and paternally inherited calf mhc class i allele was calculated. results for non-bnp and bnp calves were compared using an unpaired t-test for unequal variance. additional file : binding of peripheral blood mononuclear cells by alloantibodies from pregsure© bvd vaccinated dams. a: peripheral blood mononuclear cells (pbmc) from ten random dams were stained with serum (ser, n = ) and colostrum (col, n = ) of different pregsure© bvd vaccinated non-bnp dams (bnp-vacc+, n = ) and with serum (n = ) and colostrum (n = ) of pregsure© bvd vaccinated bnp dams (bnp + vacc+, n = ). igg alloantibody binding was measured by flow cytometry. gmfi subtracted by isotype control is plotted on the y-axis. the horizontal dotted line depicts the overall average geometric mean fluorescent intensity (gmfi) and the number above the plots describes the number of samples with a signal above the horizontal line. b: the data from additional file a were divided by the gmfi signal of the alloantibody staining of mdbk cells by the respective serum or colostrum. the horizontal dotted line depicts the overall average relative signal and the number above the plots describes the number of samples with a signal above the horizontal line. mean ± standard error of the mean is depicted in all graphs. two tailed simple t-tests for unequal variance was used to compare alloantibody binding of pbmc's between pregsure© bvd vaccinated non-bnp and bnp dams. this study was funded by zoetis. authors' contributions lb participated in the design of the study, carried out the experiments, the data collection and analysis and prepared the manuscript. hgo and wgjvg participated in the design, the experiments, the data collection and analysis of the sequencing experiments and revised the manuscript. hcmh, mn and gvs participated in the design, the experiments, the data collection and analysis of the heritability study and revised the manuscript. vpmgr and apk participated in the design of the study, the experiments, the data collection and analysis and prepared the manuscript. all authors read and approved the final manuscript. gehäuftes auftreten von hämorrhagischer diathese infolge knochenmarkschädigung bei jungen kälbern calf-level factors associated with bovine neonatal pancytopenia -a multi-country case-control study haemorrhagic diathesis in neonatal calves: an emerging syndrome in europe reproduction of bovine neonatal pancytopenia (bnp) by feeding pooled colostrum reveals variable alloantibody damage to different haematopoietic lineages bone marrow depletion with haemorrhagic diathesis in calves in germany: characterization of the disease and preliminary investigations on its aetiology immunophenotyping and characterization of bnp colostra revealed pathogenic alloantibodies of igg subclass with specifity to platelets, granulocytes and monocytes of all maturation stages bovine neonatal pancytopenia: is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies? vaccine detection of colostrum-derived alloantibodies in calves with bovine neonatal pancytopenia alloantibodies against mhc class i: a novel mechanism of neonatal pancytopenia linked to vaccination ingestion of colostrum from specific cows induces bovine neonatal pancytopenia (bnp) in some calves established kidney cell lines of normal adult bovine and ovine origin vaccine-induced antibodies linked to bovine neonatal pancytopenia (bnp) recognize cattle major histocompatibility complex class i (mhc i) effect of the vaccination scheme on pregsure (r) bvd induced alloreactivity and the incidence of bovine neonatal pancytopenia asreml user guide. release . . hemel hempstead, uk: vsn international ltd generation and maintenance of diversity in the cattle mhc class i region constraints on haplotype structure and variable gene frequencies suggest a functional hierarchy within cattle mhc class i establishment of a sequence-based typing system for bola-drb exon monoclonal-antibodies against bovine immunoglobulins and their use in isotype-specific elisas for rotavirus antibody controlling the false discovery rate -a practical and powerful approach to multiple testing bovine neonatal pancytopenia (bnp): novel insights into the incidence, vaccination-associated epidemiological factors and a potential genetic predisposition for clinical and subclinical cases lack of evidence for an association between mhc diversity and the development of bovine neonatal pancytopenia in holstein dairy cattle acquisition of hla class i w / defined antigenic determinant by heavy-chains from different species following association with bovine beta- -microglobulin biochemical characterization of activation-associated bovine class i major histocompatibility complex antigens beta- -microglobulin haplotypes in us beef cattle and association with failure of passive transfer in newborn calves association of blv infection profiles with alleles of the bola-drb . gene characterization of lymphocyte subpopulations and major histocompatibility complex haplotypes of mastitis-resistant and susceptible cows genes controlling vaccine responses and disease resistance to respiratory viral pathogens in cattle a single amino acid deletion in the antigen binding site of bola-drb is predicted to affect peptide binding type and type responses in regulation of ig isotype expression in cattle isotype-specific antibody responses of cattle to salmonella dublin lipopolysaccharide and porin following salmonella dublin vaccination and acute and chronic infection immunoglobulin isotypes of lactating holstein cows classified as high, average, and low type- or- immune responders quantitative evaluation of genetic and environmental parameters determining antibody response induced by vaccination against bovine respiratory syncytial virus a quantitative approach to classifying holstein cows based on antibody responsiveness and its relationship to peripartum mastitis occurrence polymorphism of bovine mhc class i genes cdna sequence of cattle mhc class i genes transcribed in serologically defined haplotypes a and a two-way calf to dam major histocompatibility class i compatibility increases risk for retained placenta in cattle hla class i sequence-based typing using dna recovered from frozen plasma the number of amino acid triplet differences between patient and donor is predictive for the antibody reactivity against mismatched human leukocyte antigens hlamatchmaker: a molecularly based algorithm for histocompatibility determination bovine neonatal pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the mhc haplotype we thank the dutch dairy farmers and veterinary practitioners who enabled the sample collection. we also thank matthijs schouten for taking the on farm questionnaires, crv (arnhem, the netherlands) for supplying the pedigrees and gd animal health service (deventer, the netherlands), especially ingrid den uijl and iwan kristens, for help with processing the serum samples and data of the calves. submit your next manuscript to biomed central and take full advantage of: key: cord- - w hivv authors: vidaña, beatriz; martínez, jorge; martorell, jaime; montoya, maría; córdoba, lorena; pérez, mónica; majó, natàlia title: involvement of the different lung compartments in the pathogenesis of ph n influenza virus infection in ferrets date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: w hivv severe cases after ph n infection are consequence of interstitial pneumonia triggered by alveolar viral replication and an exacerbated host immune response, characterized by the up-regulation of pro-inflammatory cytokines and the influx of inflammatory leukocytes to the lungs. different lung cell populations have been suggested as culprits in the unregulated innate immune responses observed in these cases. this study aims to clarify this question by studying the different induction of innate immune molecules by the distinct lung anatomic compartments (vascular, alveolar and bronchiolar) of ferrets intratracheally infected with a human ph n viral isolate, by means of laser microdissection techniques. the obtained results were then analysed in relation to viral quantification in the different anatomic areas and the histopathological lesions observed. more severe lung lesions were observed at h post infection (hpi) correlating with viral antigen detection in bronchiolar and alveolar epithelial cells. however, high levels of viral rna were detected in all anatomic compartments throughout infection. bronchiolar areas were the first source of ifn-α and most pro-inflammatory cytokines, through the activation of rig-i. in contrast, vascular areas contributed with the highest induction of ccl and other pro-inflammatory cytokines, through the activation of tlr . electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. in , the swine-origin h n influenza a virus (iav) emerged and caused outbreaks of respiratory illness in humans around the world. after the pandemic, outbreaks of that strain have continued to cause serious illness and increased mortality, particularly in young adults and children [ ] . the physiopathology of pandemic h n (ph n ) infection differs between individuals. whilst most patients develop mild upper respiratory-tract infection [ ], some patients progress to develop severe lower respiratory tract complications [ ] . in addition, high rates of clinically unapparent infections have been reported by seroepidemiological studies [ , ] . this evidence suggests that the severity of influenza is, at least, partially determined by host factors. severe cases after ph n infection are consequence of diffuse alveolar damage (dad) [ ] , also termed interstitial pneumonia, triggered by the spread of ph n infection from the upper to the lower respiratory tract [ , ] and an exacerbated inflammatory host immune response [ ] [ ] [ ] [ ] [ ] . several hypotheses have been made about the mechanisms involved in the dysregulation of the host inflammatory response after ph n and iav infection. however, all of them involve the up-regulation of proinflammatory cytokines [ , ] and the influx of inflammatory leukocytes to the lungs [ , ] . while leukocytes have traditionally been considered the major source of pro-inflammatory cytokines, there is also a consensus calling attention to the role of epithelial and endothelial cells as important sources of pro-inflammatory cytokines during various infectious processes [ ] [ ] [ ] . in a recent study, brandes et al. [ ] showed that lethality in a murine model of acute influenza arose directly from the damage caused by constrained innate inflammation primarily involving neutrophils co-acting with monocytes. however, the first influx of neutrophiles and monocytes needs to be induced by the release of innate immune mediators from lung resident cell populations (respiratory epithelium, endothelium and alveolar macrophages), which first come in contact with the virus. during ph n infection, leukocyte influx into the lungs is regulated by the signalling effects of cytokines and chemokines released by different lung cell populations in response to infection [ , , ] . although cytokines have specific functions and are released in a cell-type-dependent manner, all of them are produced/ activated via common mechanisms involving the activation of pattern recognition receptors (prrs) [ , ] . by these means, several studies have implicated either lung endothelial or epithelial cells as key regulators in the initial release of pro-inflammatory cytokines after ph n infection [ , , ] . however, their relative contribution to cytokine release regulation mechanisms and the dynamics of their release have not entirely been elucidated. understanding which cellular types and inflammatory dynamics are involved in the development of the detrimental inflammatory response after iav infection, is important for the future development of therapeutic strategies, designed to the control of the host immune response. in this way, the modulation of the host immune response will have the potential advantage of exerting less selective pressure on viral populations, an important factor in the development of iav infection therapies. with the aim to unravel the mechanisms involved in the regulation of the innate immune response against iav infection, and the relative contribution of the different lung compartments, we investigated the early induction of innate immune molecules, observed in different lung compartments. we tried to discern the cytokine production of the main cell type present in each lung compartment (endothelial cells, epithelial bronchiolar cells and alveolar epithelial cells) by means of a laser capture microdissection (lcm) technique. as the limitations of the technique did not allow us to completely individualize the target cells, we assessed the cytokine expression associated to the viral replication in the different lung compartments (vascular, alveolar and bronchiolar). the human ph n isolate a/castillalamancha/ rr / was used in the present study. the virus was isolated at the national influenza centre, centro nacional de microbiología, instituto de salud carlos iii (cnm, isciii) from a respiratory sample sent by the spanish influenza surveillance system for virological characterization. it was isolated from a -years old woman, without co-morbidities, who developed a severe disease and died. the viral isolate was passaged in madin-darby canine kidney (mdck) cell cultures four times and had a final titre of . tcid /ml. titre was determined using the reed and muench method [ ] . eleven neutered male ferrets between and months of age, and seronegative for iav (influenza a antibody competition multi-species elisa, id screen ® , france), were randomly selected from a stable, purposely bred colony (euroferret, denmark). upon arrival at the centre de recerca en sanitat animal (cresa), the animals were placed in biosafety level (bsl- ) facilities. ferrets were randomly assigned to different experimental groups, separated into experimental isolation rooms and then acclimated during week. the animals were inhabited in standard housing cages for laboratory ferrets (f-suite ferret housing, tecniplast, italy) and they were provided with commercial food pellets and tap water ad libitum throughout the experiment. all experiments were performed under a protocol (no. ) that was reviewed and approved by the animal and human experimentation ethics commission of the universitat autònoma de barcelona. animals were divided into two groups. the control group included two ferrets and the infected group included nine ferrets which were intratracheally inoculated with tcid / ml of the a/castillalamancha/rr / virus diluted in . ml of phosphate buffer saline (pbs). during inoculation, animals were deep sedated with butorphanol ( . mg/kg) (torbugesic ® vet, s.a., spain) and medetomidine ( . mg/kg) (domtor ® pfizer, s.a., spain) administered subcutaneously. after inoculation, animals were reverted from sedation with atipamezole ( . mg/kg) subcutaneously administered and monitored by a specialized veterinarian until complete recovering from sedation. control and infected animals were euthanized at and , and h post infection (hpi) respectively. early times post infection were selected in order to evaluate the innate immune response and assess early immune viral recognition and induction of pro-inflammatory cytokines and chemokines throughout infection. animals were euthanized by an intravenous injection of sodium pentobarbital ( mg/kg), under anaesthesia with ketamine ( - mg/kg) (imalgene ® merial, s.a., spain) and medetomidine ( . mg/kg) (domtor ® pfizer, s.a., spain), administered subcutaneously. necropsies were performed in control and infected animals after euthanasia according to a standard protocol. right lung lobe sections (cranial and caudal lobes), were taken for histological examination. the tissues were fixed for - h in neutral-buffered % formalin, and then embedded in paraffin wax in two different blocs containing one portion of the cranial and the caudal right lung lobes, consecutively taken. one of the paraffin blocks was sectioned at µm, and stained with haematoxylin and eosin (he) for examination under light microscopy; the second paraffin block was used for microdissection studies. cross sections of the cranial and caudal pulmonary lobes for each animal were histopathologically and separately evaluated. semiquantitative assessment of iav-associated microscopic lesions in the lungs was performed. the lesional scoring was graded on the basis of lesion severity as follows: grade (no histopathological lesions observed), grade (mild to moderate necrotizing bronchiolitis), grade (bronchointerstitial pneumonia characterised by necrotizing bronchiolitis and alveolar damage in adjacent alveoli), and grade (necrotizing bronchiolitis and diffuse alveolar damage in the majority of the pulmonary parenchyma). microscopic lesional scores were assigned for each lobe, and the means of the two lobes were used for the final histopathological score for each animal. lung samples from infected and control animals were used for the microdissection study. for each animal, μm sections were cut from formalin-fixed paraffinembedded (ffpe) lung tissue and mounted in penmembrane slides (two sections per slide). prior to deparaffinization, slides were placed into an oven at °c for min. for each lung sample, sections were cut, deparaffinised, and rehydrated using standard protocols with rnase-free reagents, and stained with % cresyl violet acetate (sigma, c ), and alcoholic % eosin (alvarez, - ). stained slides were then dehydrated through a series of graded ethanol steps prepared with depc treated water (ambion, p/n am g) to % ethanol. the slides were then air-dried for min, and individually frozen at − °c in ml parafilm sealed falcons, before being transferred to the lcm microscope (lmd ; leica microsystems) for simple microdissection. bronchiolar, vascular, and alveolar areas were separately selected for analysis using the leica lmd (leica) system ( × magnification, laser microdissection software version . . . ). selected areas were chosen by a pathologist as observed in figure . in infected animals, areas which exhibited pathological lesions were preferably selected. the total dissected area per selected lung compartment and animal rose approximately to . mm . one cap was used per anatomic dissected compartment and animal. the different dissected areas were then collected separately into rnase-free . ml pcr tubes, per lung compartment. viral titration from lungs samples and nasal swabs was performed on control and infected animals, and determined by plaque forming units (pfu) as described previously [ ] . a portion of around . g of the right caudal lung lobe and nasal swabs were placed in . ml of dulbecco's modified eagle's medium (dmem) (bio-whittaker ® , lonza, verviers, belgium) with µg/ml penicillin and streptomycin, and frozen at − °c until further processing. for the detection of iav antigen by immunohistochemistry (ihc), paraffin sections were stained with a primary antibody against the influenza a nucleoprotein (np), as previously described [ , ] . briefly, an antigen retrieval step was performed using protease xiv (sigma-aldrich, usa) for min at °c, and then blocked for h with % bovine serum albumin (bsa) ( c, sigma-aldrich química, s.a., spain) at room temperature (rt). samples were then incubated with a commercially available mouse-derived monoclonal antibody (atcc, hb- , h l- - r ) concentration ( mg/ml), as a primary antibody, at a dilution of : at °c overnight, followed by h incubation with biotinylated goat antimouse igg secondary antibody (dako, immunoglobulins as, denmark). finally, an abc system (thermo fisher scientific, rockjord, il, usa) was used and the antigen-antibody reaction was visualized with , ′-diaminobenzidine dab as chromogen. sections were counterstained with mayer's haematoxylin. the positive control consisted of a ffpe lung from a ferret and of a ffpe heart from a chicken, experimentally infected with influenza. the same sections in which the specific primary antibody was substituted with pbs or an irrelevant antibody [anti-porcine circovirus type (pcv ), diluted : ] were used as negative controls. semiquantitative assessments of iav antigen expression in the lungs were performed. the positive cells in six arbitrarily chosen × objective fields in alveolar areas, and five arbitrarily chosen × objective fields in bronchial or bronchiolar areas, were quantified separately in each lung lobe (cranial and caudal) for every animal. the mean of the total cell counts per field across two lobes was calculated for each animal. viral rna from total lung samples (proximal mediastinic right caudal lobe section of mm ) was extracted with nucleospin ® rna virus kit (macherey-nagel, düren, germany), following the manufacturer's instructions. iav matrix (m) gene was then quantified from the rna extracted by the taq-man one-step quantitative real time pcr (rrt-pcr) using the primers and probe described in additional file [ ] . one-step rrt-pcr master mix reagents (applied biosystems, foster city, ca, usa) were used following the manufacturer's instructions using μl of eluted rna in a total volume of μl. the amplification conditions were as follows: reverse transcription at °c for min; initial denaturation reaction at °c for min and pcr-cycles of °c s, and °c min. reaction was performed using fast equipment (applied biosystems). samples with a ct value ≤ were considered positive for influenza viral rna detection. viral rna detection from different lung anatomic compartments was extracted using the mirneasy ffpe kit (no. , qiagen, valencia, ca, usa) and the rna stabilisation and on-column dnase digestion protocols (qiagen), following the manufacturer's instructions. briefly, the transfer film with the attached dissected material, per animal and lung compartment, was placed in deparaffinization melting buffer at °c for min, and then treated with proteinase k at °c for min. isolated rna was concentrated using ethanol precipitation method and membrane column attachment. rna was eluted twice in μl total depc water, yielding between approximately and ng/μl of rna, per sample. rna extracts were stored at − °c until required. reverse transcription was performed using an improm-ii reverse transcription system, with random primers (promega, madison, wi, usa), using µl of the eluted rna. iav m gene was then quantified on the rna, extracted by two-step rrt-pcr using the forward and reverse primers for detection of the m gene, described in additional file . briefly, rrt-pcr was performed using a power sybr green kit, (applied biosystems) and fast equipment (applied biosystems). pcr reactions were performed in μl reaction volumes, in quadruplicates; amplification cycles were used, and the annealing temperature was °c. results are expressed as inverted ct values. samples with a ct value ≤ were considered positive for influenza viral rna detection. total rna was isolated from lcm tissues, using the mirneasy ffpe kit (no. , qiagen), as described in the above section. relative mrna expression levels of ifnα, il- , tlr- , il- , rig-i, ifnγ, tnfα, ccl , ccl and the housekeeping gene β-actin, at each different lung compartment, were assessed by two-step rrt-pcr. primer sequences and source are illustrated in the additional file . amplicon sizes of the target genes range between and base pairs. briefly, rrt-pcr was performed using a power sybr green kit (applied biosystems) and fast equipment (applied biosystems). rna extraction was performed on the ferret lung tissue samples with an rneasy mini kit (qiagen), using the rna stabilization and on-column dnase digestion protocols (qiagen). reverse transcription was performed using an improm-ii reverse transcription system (promega, madison, wi, usa), at . µg rna. pcr reactions were performed in µl reaction volumes in quadruplicates; amplification cycles were used, and the annealing temperature was °c. the expression levels were normalized using the house-keeping gene β-actin using the relative standard curve method and taking into account primer efficiency. the results are expressed as arbitrary units. relative mrna expression levels of selectin p-ligand (selplg), and the housekeeping gene β-actin in the vascular compartment, were assessed by two-step rrt-pcr, as described in the above section. briefly, rrt-pcr was performed using a power sybr green kit (applied biosystems) and fast equipment (applied biosystems). pcr reactions were performed as described in the above section and primer sequences and the genbank accession number are illustrated in additional file . the expression levels were normalized using the house-keeping gene β-actin using the relative standard curve method and taking into account primer efficiency. the results are expressed as arbitrary units. primer sequences for the selplg gene was designed as described previously [ ] . the ferret-specific selplg gene is available in the ncbi nucleotide database [ ] . the amplification product was detected by electrophoresis to validate the size of the product, in accordance with the primer design, and the products were purified using a qiaquick pcr purification kit (qiagen). sequencing reactions were performed with abi prism bigdye terminator cycle sequencing v. . ready reaction (applied biosystems), and analysed using an abi prism model automated sequencer (applied biosystems). the amplified sequence correlated with the ferret specific target sequences. data visualization was performed with graphpad prism (graphpad software, la jolla, ca, usa). all statistical analysis was performed using spss . software (spss inc., chicago, il, usa). for all analysis, ferret was used as the experimental unit. the significance level (α) was set at . . the shapiro-wilk's and the levene test were used to evaluate the normality of the distribution of the examined quantitative variables, and the homogeneity of variances, respectively. no continuous variable that had a normal distribution was detected. thus, a non-parametric test (wilcoxon test) using the u mann-whitney test was used to compare the different values obtained for all the parameters (histopathology and viral load), between groups (control versus infected), and between different compartments of the infected group (alveolar, bronchiolar and vascular), for viral load and gene expression profiles, at all sampling times. histopathological evaluation identified lesions mainly localized in bronchial/bronchiolar and alveolar areas ( figure ) . at hpi, histopathological lesions were characterised by mild bronchiolitis, consisting of bronchial/bronchiolar epithelium necrosis, and the presence of a mild macrophagic infiltrate in the bronchial/bronchiolar lumen. lesions were similar in both cranial and caudal pulmonary lobes at this stage. at hpi, lesions were consistent with bronchointerstitial pneumonia, or, interstitial pneumonia in the caudal lobes. bronchointerstitial pneumonia was characterised by bronchial/ bronchiolar necrosis and alveolar epithelial necrosis, lymphoplasmacytic infiltration, with mucus and cell debris filling the bronchioli and the adjacent alveoli. in addition, two of the three animals presented acute interstitial pneumonia (dad) in caudal lobes, at this time point. as expected, after infection, inflammation affected firstly the bronchioli (bronchiolitis) at hpi followed by extension to surrounding alveoli (bronchointerstitial pneumonia) at and hpi. however, some animals developed a more extended inflammation of alveolar septa (dad) at hpi. dad was characterised by a moderate to severe bronchiolar necrosis with lymphoplasmacytic infiltration in the lamina propria, and a variable number of macrophages and neutrophils in the lumen. vascular and alveolar oedema was also observed accompanied by inflammatory infiltrates in alveolar and perivascular areas. the alveolar septa were also congested, and presented necrosis of type- pneumocytes cells accompanied by interstitial inflammatory infiltrates in the septa and lumen, mainly characterised by macrophages and neutrophils. at hpi, lesions were milder than at hpi and consisted of mild bronchointerstitial pneumonia. higher histopathological scores were observed in the infected group in comparison with the control group, which did not present any histopathological lesion. of infected animals, lower histopathological scores were observed at hpi, while higher histopathological scores were observed at hpi. at hpi, histopathological scores slightly decreased in comparison to hpi (figure ). viral detection and quantification by ihc was performed in lung tissues. in general, viral antigen was observed in the nucleus of bronchial and bronchiolar epithelial and glandular cells of infected animals at hpi (figure ) . at hpi, iav antigen was observed in the nucleus of the respiratory epithelium (bronchial, bronchiolar and alveolar), and in the nuclei and cytoplasm of macrophages. in the alveolar epithelium, positive reaction was mainly observed in type ii pneumocytes, but also in type i pneumocytes (figure ). quantification of iav positive cells in the lungs of infected animals revealed that higher numbers of positive cells were observed in bronchiolar areas at hpi. statistically, significant higher numbers of positive cells in bronchial areas were observed in comparison with alveolar areas at and hpi (figure ) (p < . ). viral rna detection by rrt-pcr in the total lung, and in the different lung compartments, and viral titration by pfu in the total lung was performed. control animals were negative to viral titration and to viral rna detection in the total lung, and in the different lung compartments (figure ). in the total lung, higher viral rna detection was observed at and hpi, and then levels decreased at hpi. viral rna detection in the different anatomic compartments of infected animals, showed higher viral rna levels in bronchiolar and alveolar areas at hpi, followed by hpi. vascular areas showed higher levels of viral rna in comparison to control animals, at alltime points. viral titration in the total lung showed higher viral titers at hpi, which decreased to similar levels at and hpi. viral secretion on nasal swabs was only observed in animals at hpi (figure ). innate immune gene expression levels observed in the different anatomic lung compartments are represented in (figure ). innate immune gene expression levels were higher in all anatomical compartments, in comparison with control animals. higher levels of il- and tnfα gene expression were observed in bronchiolar areas, in comparison with alveolar and vascular areas along the experimental period. il- gene expression levels were higher at hpi while the highest tnfα levels were detected at hpi. higher il- , ccl and ccl gene expression levels were observed at hpi. il- and ccl were more expressed in bronchiolar areas, while ccl was more expressed in vascular areas. regarding the ifn response, ifnα gene expression levels were higher in bronchiolar areas at early stages post-infection ( dpi), while ifnγ was highly expressed also at bronchiolar areas, but in later stages of infection ( and hpi). interestingly after bronchiolar expression, high levels of il- and ifnγ were also observed in alveolar and vascular areas at and hpi. besides, a higher expression of il- in alveolar areas was observed at hpi. with regard to prrs expression, rig-i gene was very highly expressed r in bronchiolar areas at hpi, while no major differences in expression were observed in alveolar or vascular areas throughout infection. in contrast, tlr was highly expressed in all the compartments, but significantly in alveolar and vascular areas at hpi. vascular adhesion was evaluated by the expression of selplg gene in vascular areas. infected animals showed higher levels of selplg gene expression when compared to control animals. selplg expression was upregulated throughout the infection (see additional file ). three main mechanisms are suggested to be involved in ph n pathogenicity: (i) alveolar epithelial necrosis, caused by viral cytopathogenicity, and the release of cytokines by infected cells; (ii) activation of lung vasculature, which increases endothelial permeability, releases cytokines, and triggers leukocyte migration; and (iii) damage triggered by the inflammatory infiltrates, mainly formed by neutrophils and macrophages, plus the release of cytokines from these cells [ ] . these mechanisms work in a feed forward manner, eventually increasing the inflammatory damage produced. in this study, inflammation was firstly observed in bronchiolar compartments, in association with viral replication in bronchiolar epithelial cells and the higher expression of ifnα and il- , early after infection. these findings were expected, since the bronchiolar epithelium is one of the first targets after iav infection [ , ] . later, more severe histopathological lesions were observed in correspondence with higher presence of leukocytes and the infection of alveolar epithelial cells. concurrently, bronchiolar areas presented the higher expression of tnfα, ccl , il- and ifnγ. alveolar areas were also shown to contribute to the influx of inflammatory cells by the up-regulation of ifnα, il- and il- , at hpi, and ccl and ifnγ, at hpi. the up-regulation of il- , ifnγ and ccl has been related to lung recruitment of neutrophils, nk and t cells and the development of severe influenza infection [ , , , ] . in addition, tnfα and il- up-regulation mediate phagocytic inflammatory infiltration and the apoptosis of infected cells [ , ] , which has also been associated with severe lung lesions after iav [ , , ] . our results indicate that early infection of bronchiolar epithelial cells followed by alveolar epithelial cells are the primary source of inflammatory mediators and contribute to the severity of the lung lesion after iav infection. several studies have pointed to the protective role of ifnα release after iav and other viral infections [ ] [ ] [ ] [ ] . here, ifnα was highly upregulated in bronchiolar areas in association with the presence of iav antigen detected by ihc in bronchiolar epithelial cells. in contrast, the expression of this cytokine in alveolar areas was not as relevant as in the bronchiolar compartment, even though viral replication was extensive in the alveolar compartment at hpi. this differential ifnα expression observed in the lung compartments might reflect diverse intracellular signalling pathways in different cell types, with distinct physiological functions (bronchiolar epithelial cell versus pneumocyte). in that sense, the prrs tlr and rig-i were also up-regulated differently in the lung compartments studied, which may indicate that the different cell types recognise viral incursions and activate the release of cytokines through different signalling pathways. in bronchiolar areas, the up-regulation of different immune mediators was associated to a strong up-regulation of rig-i. rig-i is activated by the detection of viral rna in replication and its activation has been associated with protective and enhanced t cell responses against infection and the induction of ifn responses [ , ] . in contrast to bronchiolar areas, vascular and alveolar areas presented a higher induction of tlr but did not show up-regulation of rig-i at any time point. after iav infection, tlr recognizes infected cells and induces an antiviral state that recruits damage-inducing inflammatory cells contributing to lung pathology [ ] . different studies reported that tlr up-regulation was associated with a higher induction of different pro-inflammatory molecules, such as ccl , particularly in vascular areas [ ] . the detrimental role of ccl in iav pathogenesis was proven in a study where ccr deficient mice infected with iav inhibited macrophage migration to the lungs, increasing survival rates [ , ] . other studies have also associated ccl upregulation with poorer outcomes of infection [ , , [ ] [ ] [ ] . in our study, vascular areas showed most of ccl expression, particularly at hpi, when leukocyte migration was most observed. in addition to ccl , a higher up-regulation of il- and ifnγ was also observed at hpi in vascular areas, which expression has also been related with severe pathology after iav infection [ ] . these data establish a link between tlr signalling and the activation of endothelial and alveolar epithelial cells, leading to the release of different pro-inflammatory mediators, after iav infection. in the present study, viral active replication was not observed in vascular areas and only low viral antigen was detected by ihc in alveolar areas at early stages of infection, when tlr was highly upregulated. tlr is expressed in the endosomal compartments of various cellular types, whose activation is mediated by the recognition of double stranded rna (dsrna), entered into the cell by endocytosis [ , , , ] . since dsrna is a universal pathogen-associated molecular pattern (pamp), it has been assumed that tlr would play a key role in antiviral immunity. however, it has been demonstrated that tlr also contributes to pulmonary damage through the induction of pro-inflammatory molecules and the recruitment of leukocytes, during iav virus infection [ , [ ] [ ] [ ] [ ] [ ] . furthermore, tlr polymorphisms have been shown to be related to severe cases of influenza infection in humans and mice [ , , ] and its upregulation has been associated with severe pulmonary lesions in the ferret model after iav infection [ , [ ] [ ] [ ] [ ] . moreover, it is known that tlr signalling is not required for the initial cell-autonomous recognition of viral infection or the induction of ifnα, which is induced via viral recognition by rig-i [ ] . these data support the results observed in this study and suggests that viruses actively replicating in the cytoplasm are recognized by rig-i, but not tlr , irrespective of their route of entry [ ] . besides, it has been proved that influenza virus-infected cells do not generate dsrna [ ] , due to the activity of the cellular rna helicase uap [ ] . therefore, we hypothesize that tlr may recognize unidentified rna structures, from dying influenza virus-infected cells (cell-derived extracellular rna or erna), or viral components from non-replicative viruses. supporting this hypothesis, replication-deficient iav, arisen from internal deletions of iavs, significantly activated endothelial lung cells inducing a strong host cell response [ ] . besides, it has been recently proved, in vitro and in vivo, that various types of non-self erna can be sensed by recipient cells during infection, inducing the release of pro-inflammatory cytokines from recipient cells [ , [ ] [ ] [ ] . in summary, our data points to the role of the respiratory bronchiolar epithelium as the first source of proinflammatory cytokines and the influx of phagocytic cells into the lungs, early after iav infection, through the activation of rig-i. in addition, alveolar and vascular areas contributed to the induction of the pro-inflammatory innate immune response, through the activation of tlr . in vascular areas, this activation was observed to be independent of active viral replication. we hypothesize that the innate immune response induced by vascular areas, may be consequence of signals triggered by the detection of viral rna, or, through a complex crosstalk mechanism with the infected lung epithelium. innate immunity to influenza virus infection increased lung neutrophil apoptosis and inflammation resolution in nonresponding pneumonia endothelial cells are central orchestrators of cytokine amplification during influenza virus infection endothelial activation and dysfunction in the pathogenesis of influenza a virus infection into the eye of the cytokine storm switch from protective to adverse inflammation during influenza: viral determinants and hemostasis are caught as culprits evasion of influenza a viruses from innate and adaptive immune responses innate immune recognition in infectious and noninfectious diseases of the lung a simple method of estimating fifty per cent endpoints clinical response to pandemic h n influenza virus from a 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the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia matrix metalloprotease mediates neutrophil migration into the airways in response to influenza virus-induced toll-like receptor signaling sensing of viral infection and activation of innate immunity by toll-like receptor role of human toll-like receptors in naturally occurring influenza a infections involvement of toll-like receptor in the immune response of lung epithelial cells to double-stranded rna and influenza a virus toll-like receptor gene polymorphisms and severity of pandemic a/h n / influenza in otherwise healthy children attenuation of the influenza virus sickness behavior in mice deficient in toll-like receptor does toll-like receptor play a biological role in virus infections pathogen recognition and innate immunity rig-i-mediated antiviral responses to single-stranded rna bearing ′-phosphates the cellular rna helicase uap is required for prevention of doublestranded rna formation during influenza a virus infection impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production the lung microvascular endothelium as a therapeutic target in severe influenza extracellular rna promotes leukocyte recruitment in the vascular system by mobilising proinflammatory cytokines the authors declare that they have no competing interests.authors' contributions bv, mm, jom, jam and nm conceived and designed the study. bv, jom, nm, jam, mm, lc and mp carried out sample collection and laboratorial analysis. bv, jom and nm wrote the manuscript. all authors read and approved the final manuscript. additional file . primer sequences. in this table, primer sequences used for amplification of mrna by rrt-pcr are shown. comparisons of the gene expression levels of selplg in vascular areas of infected and control animals at , , and hpi. the data expresses the mean with the sems. no statistically significant differences were observed between groups.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -vhmi authors: lannes, nils; python, sylvie; summerfield, artur title: interplay of foot-and-mouth disease virus, antibodies and plasmacytoid dendritic cells: virus opsonization under non-neutralizing conditions results in enhanced interferon-alpha responses date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: vhmi foot-and-mouth disease virus (fmdv) is a highly infectious member of the picornaviridae inducing an acute disease of cloven-hoofed species. vaccine-induced immune protection correlates with the presence of high levels of neutralizing antibodies but also opsonising antibodies have been proposed as an important mechanism of the immune response contributing to virus clearance by macrophages and leading to the production of type-i interferon (ifn) by plasmacytoid dendritic cells (pdc). the present study demonstrates that the opsonising antibody titres mediating enhanced ifn-α responses in pdc were similar to neutralizing titres, when antigenically related viruses from the same serotype were employed. however, sera cross-reacted also with non-neutralized isolates of multiple serotypes, when tested in this assay. both uncomplexed virus and immune complexed virus stimulated pdc via toll-like receptor . an additional finding of potential importance for strain-specific differences in virulence and/or immunogenicity was that pdc activation by fmdv strongly differed between viral isolates. altogether, our results indicate that opsonising antibodies can have a broader reactivity than neutralizing antibodies and may contribute to antiviral responses induced against antigenically distant viruses. foot-and-mouth disease virus (fmdv) is a highly contagious infectious agent inducing disease of cloven-hoofed animals including cattle, swine, goats and sheep. due to the significant economic impact on livestock, a tight disease control is required. however, its high mutation rate contributes to immune escape and the presence of seven serotypes (o, a, c, asia- , south african territories , and ) each containing a large variety of isolates with high antigenic variability. current conventional vaccines, consisting of inactivated virus, provide a short-term serotype specific protection. however, vaccination does not induce protection against all isolates within one serotype [ ] . protection is related to the presence of high level of neutralizing antibody in serum. however, animals with low levels of neutralizing antibodies can also be protected [ , ] . furthermore, non-neutralizing concentrations of monoclonal antibodies (mab) can induce protection in mice [ ] . thus, other mechanisms than neutralization could be involved in protection. it has been shown that opsonisation of fmdv enhances phagocytosis by monocytes and macrophages in vitro [ ] . more recent in vivo data emphasize the potential role of opsonising antibodies in a mouse model, in which protection was mediated in a macrophage-dependent manner [ ] . while these studies indicate that immune complexed virus could be eliminated after phagocytosis by macrophages bearing fc receptors (fcr), other studies also indicate a participation of dendritic cells (dc), at least in vitro. immune complexes induce the activation of porcine plasmacytoid dc (pdc) resulting in the production of interferon (ifn)-α [ ] . the possible involvement of opsonising antibodies in fmdv immunity was also confirmed with bovine pdc [ ] . pdc represent . - . % of porcine peripheral blood mononuclear cells (pbmc), with similar functional characteristics to their human and murine counterparts [ ] . they are characterized by producing high amount of antiviral type-i ifn in response to a wide range of pathogens. pdc activation is mediated by sensing pathogen-associated molecular patterns (pamp) through pattern recognition receptors (prr). by expressing prr, such as toll-like receptors (tlr) and c-type lectin receptors, pdc contribute to the innate and adaptive antiviral immunity [ ] . antibody-dependent fmdv entry in pdc occurs via the fcγrii receptor (cd ) [ ] , linking pdc to the adaptive immunity [ ] . considering the possible importance of opsonising antibodies and pdc in the protection against fmdv, the main aim of this study was to characterize the relationship between neutralizing and opsonising activities of polyclonal sera from immunized pigs. although neutralization and opsonisation occurred at similar serum dilutions when antigenically related viruses were employed, opsonisation also occurred in the absence of neutralization and across different serotypes. we also discovered differences in the ability of various fmdv isolates to activate pdc. for pdc enrichment, monoclonal antibodies against following cell surface markers were used: cd a (mab - - a), cd (mab cam a), cd (mab e ) and cd (mab pt a). for phenotyping, mab against cd a and cd were used. hybridoma for mab - - a was kindly provided by dr a. saalmüller (veterinary university, vienna, austria). mabs cam a, e and pt a were purchased from vmrd (pullman, wa, usa). unsorted and sorted (see below) pbmc were cultured in dulbecco's modified eagle's minimal essential medium (dmem) plus glutamax ™ -i (gibco, life technologies, basel, switzerland) supplemented with μm of βmercaptoethanol (life technologies) at °c at % co . baby hamster kidney (bhk) cells were grown in glasgow's minimum essential medium (gmem, life technologies) supplemented with % v/v fetal bovine serum (fbs, south america origin, biowest, nuaillé, france). for virus preparation and serum neutralization test, cells were cultured in fbs-free gmem at °c, % co . blood was collected alternatively from a total of specific pathogen-free (spf) pigs of - months old kept at our institute. pbmc were isolated from citrated blood using ficoll paque ( . g/l, amersham pharmacia biotech ag, dubendorf, switzerland) density gradient [ ] . for pdc enrichment, pbmc were separated using magnetic sorting system (macs) with depletion (ld) and selection (ls) columns (miltenyi biotech gmbh, bergisch-gladbach, germany). pdc were enriched either using cd a positive selection with ld columns or by a first depletion of cd + cells with a subsequent positive selection for cd a + cells. alternatively, pbmc were isolated using ficoll paque and optiprep ( % w/v solution of oidixanol in water, sigma-aldrich, saint louis, mo, usa) density gradients followed by a depletion of cd + cells and a final enrichment of cd + cells [ ] . purity of the sorted population was verified by flow cytometry detection, after staining with anti-cd a and anti-cd mabs and isotype-specific r-phycoerythrin (r-pe) and fluorescein isothiocyanate (fitc) conjugates (southern biotechnology associates, birmingham, al, usa) as described [ ] . the pdc population was identified as cd high cd a low cells by flow cytometry [ ] . isolates of fmdv were propagated in bhk- cells as previously described [ ] and viral titres were determined by end-point titration on bhk- cells [ ] . o ukg , c noville, o bulgaria / , o vietnam / , a brazil / , a turkey/ and asia- turkey/ were kindly provided by drs. nigel ferris and satya parida (institute for animal health, pirbright, uk). in order to avoid heparin sulphate adaptation of the virus, the isolates were not passaged more than three times in bhk- cells [ ] . mock antigen was prepared from uninfected bhk- cells in the same manner as fmdv. two spf pigs were immunized intra-muscularly with the full-dose of a monovalent fmdv vaccine consisting of inactivated type-o manisa antigen (merial, lyon, france). animals received prime injection at day and a booster at day . at day , , , , and , blood samples were taken and serum prepared for storage at - °c. serum samples from a naïve animal of the same litter were used as controls. in certain experiments, serum was complement inactivated by heat-treatment at °c for min. serial dilutions of heat-treated serum were incubated at room temperature (rt) for min with tcid of fmdv isolate to form immune complexes in final volume of μl/well and then added to confluent bhk- cells, grown in -well plate for days. the neutralization titre was calculated according to kaerber [ ] based on fmdv-induced cytopathogenic effect (cpe). isolated pbmc and sorted cells ( × cells/ml) were stimulated in μl of serum-free medium with fmdv, fmdv/immune serum mixture or fmdv/naïve serum mixture. sera were used either untreated or heat-treated. fmdv/serum mixtures were previously formed for min at °c. as control, cells were stimulated with cpg d ( μg/ml, invitrogen, basel, switzerland). influenza virus strain pr was employed to test the specificity of the tlr inhibitor irs ( '-tgcttgcaagcttgcaagca- '). as control, the oligonucleotide (odn) sequence ( '-tcctgcaggttaagt- ') was used [ ] . irs and ctrl-odn were purchased from eurofins mwg operon (ebersberg, germany). influenza virus was grown on days embryonated chicken eggs and titrated as previously described [ ] . pdc activation by influenza virus employed an multiplicity of infection (moi) of tcid / cell [ ] . supernatants from enriched pdc were harvested after h and ifn-α was detected by specific elisa as described [ ] . data were collected using a versamax photometer with softmax pro software. significant differences were determined with sigmaplot v using the sum rank test (p < . ). we previously documented that pdc activation by fmdv occurs only in presence of specific antibodies [ ] . however, these experiments were performed with pdc, which were only enriched about~ - fold from pbmc using a cd a positive selection by magnetic cell sorting ( figure a ). therefore, more efficient purification methods were evaluated in this study. pdc are non-t cells (cd ) [ ] and are found within the cd alow cd high cd pbmc [ ] . based on this, pbmc were first depleted from cd + monocytes and subsequently enriched using cd a. this resulted in a frequency of - % of pdc (~ fold enrichment), depending on the experiment. a third protocol employed a cd depletion and subsequent cd enrichment of pbmc resulting in a pdc population of - % ( figure a ). type a cpg oligonucleotides, like cpg odn d , are potent activators of ifn-α secretion from porcine pdc [ ] and were employed as positive controls. while cpg induced only a few hundred units of ifn-α from unpurified pbmc, after magnetic cell sorting the production was increased by~ fold but with little differences between the different purification protocols ( figure b ). this contrasted with pdc stimulation by fmdv o ukg . as reported previously, no stimulation of pbmc or cd a-enriched pbmc in terms of ifn-α production was observed. only the protocols employing either the cd -depleted and subsequent cd a-enriched pbmc or the cd -depleted and subsequently cd -enriched pbmc permitted the induction of ifn-α after stimulation with fmdv o ukg ( figure c ). it is important to note that these responses were in the range of times lower than those seen after cpg stimulation. as the ifn-α level was not significantly higher when cd -depleted/cd -enriched pbmc were compared with the cd -depleted/cd a-enriched pbmc while the number of cells obtained upon enrichment was higher with the latter protocol, the cd depletion/ cd a enrichment method was employed for the following experiments. while testing for the capacity of different fmdv isolates to activate pdc, we found remarkable differences, exemplified for three different fmdv isolates employed at mois of , . and tcid /cell (figure a ). at the lowest moi, ifn-α was only detected after stimulation with o bulgaria / . with the moi of . tcid /cell, asia- turkey induced a weak response and o bulgaria / a relatively strong response. these responses were further increased when the virus dose was doubled, but o ukg remained a poor ifn-α inducer ( figure a ). considering these results, we tested more fmdv isolates for their ability to activate pdc at an moi of tcid /cell ( figure b ). the results confirmed that the ability of fmdv to activate pdc varies considerably, even within one serotype. relatively strong inducers were o bulgaria / and asia- turkey ; weak inducers were o vietnam / and o ukg , and the most inefficient inducers were c noville, a brazil / and a turkey . as previous studies used purified subtype-specific antibodies [ ] or high concentration of serotype-specific immune serum [ ] to form fmdv immune complexes, we were interested to determine the relationship between serum concentration and ifn-α enhancement. opsonisation of fmdv o ukg by a serum with a neutralization titre (nt) of . log at dilutions below log did not induce pdc activation ( figure a ). in fact, such high serum concentrations showed suppressive effects on pdc stimulation by fmdv, which were independent on the immune status of the donor animal (data not shown). they were therefore omitted in the remaining experiments. by increasing serum dilutions, immune complexes enhanced pdc-produced ifn-α. even with an moi as low as . tcid /cell, immune complexes activated pdc ( figure a) . the same approach was applied for o bulgaria / , a high ifn-α inducing fmdv isolate ( figure b) serum employed showed a nt of . log against this virus. with o bulgaria / at an moi of tcid /ml, immune serum enhanced pdc-derived ifn-α response in the expected concentration-dependent manner with maximum ifn-α level at a serum dilution of . log and a gradual loss of the enhancing activity with further serum dilutions ( figure b ). in contrast, when o bulgaria / was used at mois of . and tcid /cell, this serum concentration-dependent relationship was not observed ( figure b ). in particular, with the highest virus dose a clear enhancement of ifn-α responses was not observed. we concluded that low mois have to be employed to measure antibody-dependent enhancement of ifn-α responses by pdc when high ifn-α-inducing fmdv isolates are used. results obtained with various serum dilutions and various fmdv isolates indicated that other serum factors than specific immunoglobulin (ig) influenced pdc activation. for this reason a naive serum was titrated in parallel to the immune serum, and the possible impact of complement tested by heat-treatment of the sera before forming immune complexes. figure shows a representative experiment in which immune serum enhanced the ifn-α response induced by fmdv o ukg relative to naive serum. nevertheless, this enhancement was only clear at a serum dilution up to log ( figure a ). with heattreated sera, immune complexes enhanced pdc-derived ifn-α were more clearly visible with statistical differences at serum dilutions up to log . we concluded that complement is not required for the enhancement of ifn-α by immune-serum, and heat-labile serum factors could even have a suppressive effect. considering this, all further experiments were performed with heat-treated serum. our previous work demonstrated that fmdv immune complex-mediated pdc activation required live virus and was associated with expression of non-structural fmdv proteins. this indicated that a virus replication cycle is initiated with formation of double stranded rna, a potential trigger of ifn-α responses [ ] . although tlr is known to be the main sensor for rna viruses in pdc, it represents a receptor for single stranded rna. we were therefore interested to determine the role played by tlr in sensing fmdv and fmdv immune complexes. to this end we used the immuno-regulatory sequence (irs ) representing an odn inhibitor of tlr which had been previously established for human and murine immune systems [ ] . we employed influenza virusstimulated pdc, a known ligand for tlr to test the efficiency of irs to inhibit the tlr pathway of porcine pdc. as shown in figure complexes were employed irs inhibited pdc responses by - % ( figure b ). similar levels of inhibition were also observed with other fmdv isolates (data not shown). the control odn did not influence ifn-α production of fmdv-stimulated pdc (data not shown). pdc cultures permit to detect efficient opsonisation of fmdv in the absence of neutralization we were next interested to determine whether opsonising activity can also be detected in the absence of neutralizing activity. to this end, an anti-o manisa serum was employed and tested against fmdv o ukg (nt of . log ) and against a brazil / (nt below detection limit). interestingly, despite the inability to neutralize the a serotype virus, opsonising activity against both viruses was observed at a similar serum dilution between to log . also the maximum ifn-α production was reached at the same serum dilution of log with both fmdv isolates ( figure a ). these experiments were repeated with five different batches of pdc preparations, and the enhanced ifn-α responses by the immune serum compared to the effect of naive serum were consistently observed despite some variability in the height of the ifn-α responses (figures b and d) . similar results were also obtained with another serum sample obtained from a second vaccinated pig (data not shown). furthermore, both sera were also able to opsonize a/turkey/ confirming the existence of opsonizing antibodies cross-reacting with other serotypes. considering these surprising results, neutralizing and opsonising activities of an anti-o manisa serum were measured against a collection of fmdv isolates of serotypes o, a, c and asia- and the results are summarized in table as nt and opsonisation titres (ot) representing the highest serum dilution able to opsonise fmdv for enhanced ifn-α responses. as expected the immune serum was only able to neutralize the o serotype viruses o ukg and o vietnam / . with these viruses we found a similar ot of and log , respectively. as shown in figure c , d, non-neutralized a brazil / could be opsonised to a titre of log . also fmdv a turkey and asia- turkey/ , albeit to a lesser extent, were opsonised although not being neutralized. only, c -noville was neither neutralized nor opsonised by an anti-o manisa serum. these results demonstrate that opsonisation of fmdv can occur in the absence of neutralization. pdc are professional sensors of viruses producing large amount of type-i ifns. however, in particular nonenveloped viruses such as fmdv have been shown to be unable to trigger pdc activation in vitro, unless the cells are stimulated with immune complexed virus [ , ] . however, in the present study we demonstrate that fmdv can activate pdc also in absence of specific antibodies if improved purification methods are employed. type-c c -noville < . * opsonizing titres representing minimum serum dilution able to enhance ifnα responses by pdc (calculated from - independent experiments as described in figure ). we also demonstrate that both immune-complexed fmdv and uncomplexed fmdv activate pdc via tlr . as expected, the responsiveness of pdc to fmdv is possible with more pure populations of pdc but our results also indicate that other factors could be important such as the percentage of other cell types present in the cell culture. the observation that in vivo ifn-α responses occur in the early innate phase of the immune response of cattle and pigs [ , ] support the idea of an early activation of pdc by fmdv in absence of specific antibodies and support the in vivo relevance of our data. in fact, in cattle it has been demonstrated that pdc are the source of early ifn-α responses [ ] . our study also demonstrates remarkable differences of different fmdv isolates to activate pdc. the reason for the differences in activation of pdc could be either related to the uptake of the virus by pdc or to the function of viral genes such as the ifn antagonist l pro , the viral proteinase. fmdv interacts with immune and nonimmune cells [ ] via arg-gly-asp (rgd)-dependent and rgd-independent mechanisms, after cell culture adaptation to heparin sulphate binding [ ] . certainly, the differences we observed with pdc are not related to a cell culture adaptation of certain fmdv isolates to heparin sulphate binding since all viruses employed in this study had a maximum of three passages. furthermore, we have compared cell culture adapted heparinsulphate binding virus to its non-adapted original wild-type fmdv (viruses as described in [ ] ), and found no differences in the ability to activate pdc (data not shown). on the other hand, the leader proteinase l pro inhibiting the type-i ifn pathway [ ] , is variable among serotypes [ ] and could contribute to a isolate-specific fmdv counteraction on the ifn-α induction in pdc. the potential of some fmdv isolates to consistently induce ifn-α also at low mois would support this hypothesis. overall, it is important to note that in comparison to the ifn-α levels in response to influenza viruses [ ] and cpg [ ] , fmdv is a poor stimulator of pdc. nevertheless, considering that ifn-α is known to efficiently inhibit fmdv replication in vitro and in vivo [ ] , it is now important to investigate the relationship of the ability of an fmdv isolate to induce ifn-α in vitro and to promote in vivo innate and adaptive immune responses. previous studies have demonstrated that antibodydependent internalization of fmdv via the fcγrii represents an important pathway to enter macrophages [ ] and pdc [ , ] . one of the main objectives of this study was to determine the relationship between opsonisation and neutralization. non-neutralized serotype-a and asia- isolates could be efficiently opsonised using an anti-o serum while others were not, such as a serotype c isolate. from phylogenetic studies based on vp amino acid sequence, which contains the most antigenic site [ ] , serotypes o and asia- belong to the same branch [ ] . serotypes a and c are more distant, partially explaining our findings [ ] . interestingly, this opsonisation efficiently occurred with serum dilutions similar to those required for neutralization, when antigenically related viruses from the same serotypes were employed. this would indicate that the type of antibodies and their specificities is similar for both functions. however, we also found opsonisation in nonneutralizing conditions contradicting this idea. it could be speculated that opsonising activity is possible at lower avidity compared to neutralization, in which the antibody binding to the virus is in competition to virus binding to the cellular receptor. this competition can only occur indirectly for opsonisation, since the cellular fcr will bind to the fc portion of the antibody. the observation that opsonisation can occur at similar serum dilutions with a neutralized and non-neutralized virus would favour the presence of non-neutralizing epitopes, which can be conserved between different serotypes. the answer to these questions needs to be addressed using monoclonal antibodies rather than a polyclonal serum such as in the current study. the broad activity of opsonising antibodies is interesting in the frame of vaccine-induced immune responses. non-neutralizing vaccine-induced antibodies could enhance innate immune responses against antigenically less related fmdv challenge strains and restrict their ability to replicate. furthermore, other fcr-mediated functions such as virus phagocytosis and more efficient antigen presentation of viral antigens to t cells could be promoted. although this is not sufficient to mediate complete protection against disease, it could limit virus replication, the severity of clinical diseases and transmission within the heard. future studies are required to determine how such responses correlate to protection (partial or complete), and if this is the case how vaccines can be improved to promote broadly reactive antibody responses able to link innate and adaptive immunity 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act as endogenous ligands for toll-like receptors and may promote systemic lupus erythematosus hemagglutinindependent tropism of h n avian influenza virus for human endothelial cells efficient sensing of avian influenza viruses by porcine plasmacytoid dendritic cells characterization of blood mononuclear cells producing ifn alpha following induction by coronavirus-infected cells (porcine transmissible gastroenteritis virus) natural interferon-alpha producing cells: the plasmacytoid dendritic cells novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease aspects of the persistence of foot-and-mouth disease virus in animals-the carrier problem innate immune responses against foot-and-mouth disease virus: current understanding and future directions foot-and-mouth disease virus: a long known virus, but a current threat the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase the non-structural leader protein gene of foot-and-mouth disease virus is highly variable between serotypes foot-and-mouth disease submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this present work was supported by eu fp project fmd-disconvac (grant agreement ) and r research foundation switzerland (project - ). we thank drs. nigel ferris and satya parida for providing some of the fmdv isolates. we also thank heidi gerber for mab preparation and help with virus preparation as well as andreas michel and hans-peter lüthi for regular bleeding and care of the animals. http://www.veterinaryresearch.org/content/ / / the authors declare that they have no competing interests. key: cord- -smlq y authors: dhakal, santosh; renukaradhya, gourapura j. title: nanoparticle-based vaccine development and evaluation against viral infections in pigs date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: smlq y virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. the economic burden caused by virus infections such as porcine reproductive and respiratory syndrome virus, swine influenza virus, porcine epidemic diarrhea virus, porcine circovirus , foot and mouth disease virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. pigs can also have a role in zoonotic transmission of some viral infections to humans. inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. thus, improvements over existing vaccines are necessary to: ( ) increase the breadth of protection against evolving viral strains and subtypes; ( ) control of emerging and re-emerging viruses; ( ) eradicate viruses localized in different geographic areas; and ( ) differentiate infected from vaccinated animals to improve disease control programs. nanoparticles (nps) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. nps help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. some of the nps-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (m) cells and dendritic cells (dcs). in conclusion, nps-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. this review highlights the recent developments in nps-based vaccines against porcine viral pathogens and how the nps-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy. viruses are the obligate intracellular nano-sized particles, which depend on host cell machinery for propagation and survival. they carry deoxyribonucleic acid (dna) or ribonucleic acid (rna) as their genomic material. there are several viruses from both dna and rna virus families that infect and produce disease in pigs [ ] . there are many economically important swine viral infections which cause considerable morbidity and mortality, and responsible for significant economic losses to the pork industry (table ). depending on their cellular and tissue tropisms, viruses cause pathological changes and clinical signs associated with respiratory system, reproductive and gastrointestinal tracts, nervous system, skin and extremities, alone or in combinations [ , ] . porcine reproductive and respiratory syndrome virus (prrsv), an enveloped and positive-stranded rna virus of arteriviridae family, causes porcine reproductive and respiratory syndrome (prrs) [ ] . prrs is responsible for over one billion dollar loss per year through direct and indirect costs in the us swine industry [ ] . two entirely distinct genotypes of prrsv circulate in european (genotype /prrsv ) and north american countries (genotype /prrsv ) and cause tremendous economic loss. prrsv is transmitted through oral-nasal secretions and semen. the clinical signs include fever, anorexia, mild to severe respiratory problems, abortion and reproductive failures. it is the most common pathogen associated with porcine respiratory disease complex (prdc) [ ] . swine influenza (flu) constitutes another persistent health challenge to the global pig industry. flu infection is caused by influenza a virus of orthomyxoviridae family which has negative-sense, single-stranded, segmented rna genome. influenza virus is transmitted through direct contact with infected animals or contaminated fomites, aerosols and large droplets [ ] . the clinical signs of influenza infection include fever, anorexia, loss of weight gain and respiratory problems. influenza associated economic losses are due to morbidity, loss of body weight gain, increased time to market, secondary infections, medication and veterinary expenses [ ] . influenza of swine origin occasionally infect humans and can even lead to pandemics as of [ ] . porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) are enteric pathogens of young pigs [ ] . these viruses belong to coronaviridae family and have positive-sense, single-stranded rna genome. tgev did serious economic damage to the swine industry in s but with the advent of vaccines it has been largely controlled [ ] . pedv still results in high morbidity and mortality in neonatal piglets with clinical signs like severe diarrhea, vomiting, dehydration and death. in / , pedv outbreak in the us led to over a billion-dollar loss [ ] . rotaviruses are double-stranded rna viruses of reoviridae family, cause enteric infections in pigs. rotavirus of groups a, b, c, e and h are involved in porcine enteric infections. some of these porcine rotaviruses also have zoonotic potential [ ] . foot and mouth disease (fmd) is another highly contagious, acute viral disease in pigs. the etiologic agent, fmd virus (fmdv), is a positive-sense, single-stranded rna virus of picornaviridae family [ ] . fmdv is transmitted through direct contact with infected animals or contaminated sources. clinical signs include high fever, appearance of vesicular lesions on the extremities, salivation, lameness and death. fmdv causes frequent epizootics in many parts of the world resulting in severe economic loss, food insecurity and trade restrictions [ ] . classical swine fever (csf) or hog cholera can result in high morbidity and mortality in pigs. it is caused by csf virus (csfv), an enveloped, positive-sense, singlestranded virus of flaviviridae family. transmission of csfv occurs through oral-nasal routes after contact with infected pigs or contaminated resources and even vertically from infected sows to piglets [ ] . clinical signs include fever, anorexia, respiratory problems, neurological disorders, reproductive failures and death. csf is a notifiable disease to world organization for animal health (oie). the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . for example, the / outbreak of csfv in the netherland resulted in death of million pigs and economic losses of . billion dollars [ ] . united states is free of csfv; however, this virus is endemic in many parts of the world including central and south america, africa and asia. porcine circovirus (pcv ), a single-stranded dna virus of circoviridae family, causes multi-systemic disease referred as porcine circovirus-associated disease (pcvad). pcv is transmitted horizontally as well as vertically. direct contact is the most efficient way of horizontal transmission of this virus. the clinical signs of pcv infection include poor weight gain, respiratory problems, dermatitis, enteritis, nephropathy and reproductive failures [ ] . five genotypes of pcv (pcv a to pcv e) are identified and circulate with high prevalence in swine herds causing significant economic losses worldwide [ ] . porcine parvovirus (ppv) is the common cause of reproductive failure in swine herds. this single-stranded dna virus of parvoviridae family is transmitted through oral-nasal routes. stillbirths, mummification, embryonic death, and infertility (smedi syndrome) are linked to ppv infection. conventionally, ppv was considered genetically conserved but recent evidences suggest that several virulent strains have emerged due to its high mutation rate [ ] . aujeszky's disease or pseudorabies in pigs is caused by suid herpesvirus , a double stranded dna virus belonging to herpesviridae family. the causative agent is spread primarily through direct animal-to-animal (nose-to-nose or sexual) contact. pseudorabies is characterized by nervous disorders, respiratory problems, weight loss, deaths in younger piglets and reproductive failures; and is one of the most devastating infectious diseases in pig industry [ , ] . african swine fever (asf) causes hemorrhagic infection with high morbidity and mortality. the etiologic agent, asf virus (asfv), is a double stranded dna virus of asfarviridae family [ ] . virus transmission occurs through direct contact with infected animals, indirect contacts with fomites or through soft tick species of the genus ornithodoros. clinical disease may range from asymptomatic infection to death with no signs. acute infections are characterized by high fever, anorexia, erythema, respiratory distress, reproductive failure in pregnant females and death [ ] . asf is oie notifiable disease. united states is free of asfv, however, this virus is endemic in domestic and wild pig population in many parts of the world with possibility of transmission to the us and other nonendemic regions through animal trades [ ] . the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . besides the rna and dna viruses described above, many other emerging and re-emerging viruses such as porcine hepatitis e virus, porcine endogenous retrovirus, porcine sapovirus, japanese encephalitis virus, encephalomyocarditis virus and others cause variable degree of impact in swine health and economic losses in pig industry globally [ , , ]. different types of vaccines that are available against economically important swine viruses are listed in table . vaccines against prrsv are being used in the us since s [ ] . both inactivated and modified-live virus vaccines are available and used globally. these vaccines are effective in reducing clinical disease and viremia mainly against homologous but not against heterologous infections [ ] . therefore, different strategies are ongoing to develop live, inactivated, subunit and mucosal prrsv vaccines to induce better immunity and broader protection [ , [ ] [ ] [ ] . swine influenza vaccines are also most effective when the vaccine strains closely match with the circulating strains [ , ] . to increase the immunity and protection, vaccines containing multiple strains of influenza a virus (iav) and autogenous vaccines are widely used [ , ] . cocirculation of multiple lineages of iav and frequent antigenic drift are responsible for reduced field efficacy of current swine influenza vaccines. moreover, the most commonly used whole inactivated iav vaccines administered via intramuscular route do not induce adequate mucosal antibody and cellular immune responses, suffer maternal antibody interference in young piglets and even can cause enhanced respiratory disease [ , ] . the emergence of highly virulent strains of pedv in recent years has highlighted the need of safe and effective vaccines against porcine enteric coronaviruses that prevents clinical disease, mortality and virus shedding in neonates [ ] . modified live vaccines against rotavirus are available for use in pigs against rotavirus a but their efficacy under field conditions is questionable indicating the need of alternatives for porcine rotavirus management [ ] . the available inactivated vaccines provided great help in prevention and control of fmd outbreaks in many countries. however, the development of these vaccines needs high level biocontainment facilities. further, the fmdv serotypes undergo continuous antigenic drift and escape the vaccine-induced immunity [ ] . thus, fmd vaccines with less stringent regulatory procedures and multi-serotype protective efficacy are needed in the future. safe and highly efficacious live-attenuated vaccines are available against csfv but differentiation of infected from vaccinated animals (diva) is not possible with these vaccines, which limit their use during outbreak control or disease eradication programs [ ] . inactivated whole virus or subunit vaccines based on pcv a are highly adopted in pig farms and are efficacious in reducing clinical signs and improving the production parameters. however, infections are still widespread in vaccinated farms [ , ] . further, the replacement of pcv a to pcv b and recently to pcv d is in part contributed by the selection pressure exhibited by pcv a-based vaccines [ ] which highlights the need of vaccines that protect against multiple genotypes. the currently used inactivated vaccines of porcine parvo virus protect against old ppv strains but not against the newly emerging strains demanding for more efficacious vaccines [ , ] . fortunately, pseudorabies has been eradicated in many countries including the us by using inactivated and attenuated vaccines together with stringent biosecurity measures. however, it is still a problematic disease in many countries including china and is also maintained in feral swine populations in other countries [ , ] . the frequent emergence of virulent strains even in the vaccinated herds demands updated vaccine technology to achieve efficient control and ultimate global eradication of pseudorabies [ , ] . vaccine is not available so far against asfv, and the control measures depend entirely on early identification and culling of infected herds and adoption of strict sanitary measures [ ] . vaccine development is hindered by the antigenic diversity and multitude of immune-evasion strategies used by the virus. an effective vaccine will definitely help in control and eradication of asfv from endemic countries and prevent its geographical expansion [ ] . [ , ] porcine epidemic diarrhea (ped) rna particle, inactivated and live-attenuated virus (in asia) protective immune response in sows better mucosal immunity [ , ] foot and mouth disease (fmd) inactivated virus less stringent requirements in vaccine production protection against multiple serotypes [ ] classical swine fever (csf) live-attenuated virus diva potential [ ] porcine circovirus associated disease (pcvad) inactivated, recombinant subunit multi-genotype protection [ , ] porcine parvovirus infection inactivated virus protection against novel strains [ , ] pseudorabies inactivated, live-attenuated virus protection against novel emerging strains [ , ] african swine fever (asf) none novel cross-protective vaccine [ ] importance of nanoparticle-based vaccine delivery platforms development of vaccines has made significant impact on reducing the viral infectious disease burden in both humans and animals. however, there are still many diseases for which either we do not have vaccines or need substantial improvements over existing ones [ , ] . in the past few decades, nanoparticles (nps)-based technologies have elicited significant interests in the development of novel vaccine candidates as they offer multiple benefits over inactivated virus or subunit soluble antigens. nps-based vaccines (nanovaccines) are prepared either by encapsulating vaccine components within the nps or by decorating the particle surface with viral antigens. nps protect antigens from proteolytic degradation, prolong their bioavailability and maintain slow and sustained antigen release. all of these properties help in induction of better immune responses compared to soluble antigen vaccines [ ] . the different mechanisms used by various nps to facilitate immune modulation of antigen presenting cells (apcs) are depicted graphically in figure . briefly, nps can enhance antigen adsorption and uptake by apcs; they can also facilitate antigen processing mechanisms; nps can induce maturation of dcs and promote antigen cross-presentation through major histocompatibility complex (mhc) class i to cd + t cells; and induce production of different innate cytokines that regulate humoral and cellular immune responses. nps-loaded antigens are readily phagocytosed by apcs; soluble antigens are not [ ] . moreover, dendritic cells (dcs), the key player involved in bridging innate and adaptive immunity, preferentially internalize nps compared to microparticles (> nm). for example, when poly(lactic-co-glycolic acid) (plga) particles of size nm to µm encapsulating ovalbumin were tested on mouse bone-marrow derived dendritic cells, nm sized particles were taken up efficiently compared to larger ones [ ] . the nm sized plga nps resulted in greater activation of dcs and stronger antigen-specific t cells responses in immunized mice compared to soluble antigens and larger particles [ ] . besides controlled delivery of antigens, nps also provide adjuvant-like functions. vaccine adjuvants either work as antigen delivery systems facilitating antigen uptake and presentation by apcs or they activate innate immune receptors for cytokine production and maturation/migration of dcs [ ] . adjuvant-induced innate immune responses determine the type of adaptive immune responses generated such as t helper (th ) versus t helper (th )-biased immunity [ ] . alum, the most widely used adjuvant in humans, is safe and inexpensive. its compatibility has been proved favorable with different vaccine antigens. however, despite inducing potent antibody responses, alum is a weak-inducer of cell-mediated immunity. adverse reactions are observed at injection site with alum-based adjuvants [ , ] . in veterinary vaccines, oil-in-water emulsions or saponins are the most common adjuvants. these can also cause adverse reactions at the injection sites [ , ] . while number of adjuvants are available for parenteral vaccinations, very limited options are available for intranasal (in) or other alternative routes of immunization [ , , ] . nps can serve as an alternative adjuvant for human and animal use as they act both as antigen delivery system and activate the innate immune responses [ ] [ ] [ ] . further, the modern vaccination approach has shifted from traditional whole pathogen-based antigens to small fraction (subunit) of the pathogen. however, purified whole inactivated pathogen and subunit or recombinant antigens by themselves are poorly immunogenic and require a potent immunostimulatory platform to augment the immune response. this can be achieved through nps-based technologies [ , ] . nps-based platforms can be used to deliver multiple antigens or antigen/adjuvant combinations, which improves antigen uptake and concurrent activation of apcs leading to innate immune programming [ , ] . co-delivery of cpg oligodeoxynucleotide and tetanus toxoid in nanospheres induced significantly greater t cell proliferative response and to times greater igg antibody isotypes in mice after subcutaneous immunization compared with the group that received tetanus toxoid and cpg oligodeoxynucleotide in soluble form [ ] . likewise, co-delivery of melanoma antigen and toll-like receptor (tlr) agonist in plga nps induced therapeutic anti-tumor effects that are mediated through potent cd + t cell activation [ ] . nps can be surface modified to target microfold (m) cells, macrophages or dcs, and could be used for mucosal vaccination through oral, nasal or other mucosal routes of immunization. in mice, surface coating of plga nps encapsulating hepatitis b virus vaccine antigens with lectin resulted in efficient targeting of oral delivered nps to mucosal m cells and induced secretary iga antibody response in mucosal surfaces [ ] . likewise, dcs targeted chitosan nps loading plasmid dna encoding nucleocapsid protein of severe acute respiratory syndrome coronavirus (sars-cov) induced better nucleocapsid protein-specific mucosal iga antibody response compared to soluble unentrapped antigens after nasal immunization in mice [ ] . a targeted t-cell mediated immune response is critical in protection against intracellular pathogens such as viruses. beneficially, nps-delivered antigens are useful in antigen cross-presentation to cytotoxic t lymphocytes (ctls) and development of robust cell-mediated immune response [ , ] . plga-based particulate vaccines are shown to induce efficient t-cell immunity in mice and pigs [ ] [ ] [ ] [ ] . similarly, rodent and pig studies have shown that polyanhydride nps-based vaccines also enhance cellular immunity [ , ] . thus, immunogenic properties of different polymer-based nps could be exploited to improve the efficacy of vaccines for use against porcine viral infections. in this review, only studies conducted in pigs related to the development and evaluation of nps-based vaccine candidates by using virus-like particles (vlps), biodegradable polymers, polysaccharides and liposomes against porcine viral infections are included (table ) . vlps are constructed using viral structural proteins, which can self-assemble but are non-infectious as they lack the viral genomic material. vlps mimic the virion and can effectively induce innate and adaptive immune responses [ ] . vlps are produced using different bacterial, insect, yeast or mammalian expression systems [ ] . due to their smaller size and particulate nature, vlps-based vaccines are processed and presented not only through mhc class ii but also through mhc class i pathway leading to the generation of antibodies as well as ctl responses [ , ] . the potential use of vlps in porcine viral vaccine development is evident through the success in commercialization of human papilloma virus (hpv), hepatitis b virus and malaria vaccines by adapting this technology [ ] . in one study, prrsv vlps containing five (gp , gp , gp , gp a and m) and two (gp and m) viral surface proteins were generated using the baculovirus expression system. prrsv vlps vaccine was mixed at : ratio with mycobacterium tuberculosis whole cell lysate (m. tuberculosis wcl) adjuvant and administered into pigs. vlps-vaccinated pigs were partially protected with -log reduction of virus titers in lungs. vlps-vaccinated pigs also had enhanced ifn-γ response compared to mock challenge pigs [ ] . however, in another study, when pigs were vaccinated in with prrsv vlps expressing n, m, gp and e proteins, enhanced viremia accompanied with higher level of ifn-α cytokine response was observed [ ] . the contrasting results in prrsv vlps study suggest the need for further research to fully evaluate the potential of vlps-based prrsv vaccines for swine. influenza-associated vlps expressing ha, na and m proteins of pandemic (h n ) virus were inoculated twice intramuscularly with or without emulsigen (mvp lab, usa) adjuvant to pigs. this vaccine induced robust serum igg, mucosal iga and virus neutralizing antibody responses in pigs. after homologous virus challenge, vlps-vaccinated pigs had significantly reduced pneumonic lesions and virus titers were substantially lowered in upper and lower respiratory tracts compared to mock vaccinated animals [ ] . many studies have been conducted with the goal to develop vlps-based fmdv vaccine using various expression systems encoding different viral antigens. rabbit hemorrhagic disease virus (rhdv) vlps expressing t-cell epitope of a protein of fmdv (rhdv- a-vlps) was generated. this vlps vaccine induced maturation of bone marrow derived dendritic cells in vitro [ ] . pigs immunized im with rhdv- a-vlps together with montanide isa adjuvant (seppic, france) induced higher serum igg and iga antibody responses. this vaccine also increased number of ifn-γ secreting cells and lymphoproliferative responses in pbmcs compared to vaccine delivered without adjuvant and in rhdv- a-vlps inoculated pigs; however, challenge experiments were not performed [ ] . guo et al. constructed fmdv vlps expressing capsid proteins vp , vp and vp and immunized pigs by im route [ ] . vlps-vaccinated pigs produced virus-specific neutralizing antibodies and ifn-γ response in peripheral blood mononuclear cells (pbmcs) as good as the inactivated fmdv vaccine control. after challenge with homologous virus, vlps-vaccinated pigs did not show specific clinical signs [ ] . in another study, vp epitope peptides (ep - ) of fmdv were inserted into the coat protein genes of male-specific coliphage (ms ) (cp-ep - vlps) and injected im to pigs. this formulation resulted in induction of virus neutralizing antibodies and protected % of the immunized pigs compared to only % protection in peptide alone vaccinated animals. however, the protection was lower than inactivated vaccine ( %) indicating the need : of further improvement in this vlps either by using longer sequence of epitope or addition of other adjuvants [ ] . vlps generated by insertion of vp epitopes of fmdv into porcine parvovirus vp were administered im to pigs. this vlps-vaccine induced higher virus neutralizing antibodies compared to synthetic peptide vaccine and resulted in better protection to challenge fmdv infection [ ] . vlps have also been developed and tested against porcine neurotropic viruses [ , ] . porcine encephalomyocarditis virus (emcv) vlps containing structural protein p , nonstructural protein a and protease c were generated. after im administration together with montanide ims n vg adjuvant (seppic), vlps-vaccine induced sustained production of virus neutralizing antibodies comparable to commercial vaccine control. there was absence of any severe injection site reactions in vlps-vaccinated pigs [ ] . this suggests the potential of developing vlps-based vaccine against emcv disease in pigs. likewise, in a recent study, japanese encephalitis virus genotype i (gi) vlps encoding premembrane (prm) and envelope (e) proteins were constructed. after subcutaneous immunization, this vaccine formulation induced robust neutralizing antibody response and protection against both homologous gi and heterologous giii jevs viruses. this finding indicates the cross-protection potential of vlps-based jev vaccine in pigs [ ] . early study on pcv vlps used full length cap protein in escherichia coli expression system [ ] . pigs vaccinated against pcv using cap vlps and isa adjuvant (seppic) by im route induced cap-specific igg antibodies. vaccinated animals were apparently healthy with normal body weight gain and absence of any clinical signs of disease [ ] . li et al. [ ] showed induction of cap-specific igg antibodies in pigs vaccinated by subcutaneous (sc) injection of cap vlps. vaccinated pigs demonstrated reduced fever, viremia and mild pathological changes in lungs and lymph nodes compared to unvaccinated challenge animals. in another study, vlps co-expressed with cap protein and porcine gm-csf were administered im to pigs. this vaccine formulation induced significantly higher virus neutralizing antibodies in pigs. after virus challenge, vlps-vaccinated pigs had normal body weight gain compared to cap protein alone and commercial pcv vaccine groups. virus clearance, however, was observed in equally in vlps as well as other control vaccine groups [ ] . only a single vlps-based vaccine study for porcine parvovirus was found [ ] . ppv-vlps expressing major structural protein vp were administered im with double oil emulsion (doe) mineral oil adjuvant to weaned pigs. there was an induction of significantly higher neutralizing antibodies in vlps-vaccinated animals compared to inactivated vaccine group. further, when gilts immunized with this formulation were challenged with virulent ppv, virus was not detected in any of the fetuses. thus, ppv-vlps can be a potential vaccine candidate to prevent ppv-induced reproductive failure [ ] . in summary, vlps of various origin can be used to develop more efficient vaccines against porcine viral infections. further studies are needed to evaluate their immunogenicity and protective efficacy under field conditions. plga is a co-polymer of lactic acid and glycolic acid. it is the most widely explored synthetic polymer in vaccine studies. it is a safe and non-toxic compound, and its hydrolysis products are readily assimilated into existing metabolic pathways [ ] . plga nanoparticles are prepared either by oil in water emulsification or nanoprecipitation methods [ , ] . plga nps bear a net negative charge. they enter apcs through pinocytosis and endocytosis, undergo reversal of charge and endolysosomal escape of entrapped vaccine cargo leading to antigen processing in cytoplasm, resulting in cross-presentation of antigen to cd + t cells through mhc class i pathway [ , ] . plga nps are involved in maturation of dcs of mice and human origin, and controlled release of entrapped antigens leading to efficient expansion and differentiation of memory t-cells [ , ] . in rodent studies, induction of robust t-cell immunity is observed with plga nps-based vaccines containing various vaccine antigens [ , ] . further, plga is approved for drug deliveries in humans by the us food and drug administration (fda) and european medicine agency (ema) [ ] . plga nps enhance antigen uptake and induce maturation of porcine apcs [ , , ] . single dose of in immunization with plga nps-encapsulated inactivated/ killed prrsv antigen (nps-kag) induced activation of innate natural killer (nk) cells, γδ t-cells and secretion of innate cytokine ifnα [ ] . nps-kag vaccine also induced greater frequency of cd + t cells; increased secretion of ifn-γ; lowered frequency of t-regulatory cells; and reduced secretion of inflammatory cytokines compared to control kag-vaccinated animals [ , ] . in a subsequent study, when nps-kag was co-administered in with m. tuberculosis wcl adjuvant, a balanced th / th immune response and augmentation of mucosal iga antibody response was observed. after heterologous prrsv challenge, pigs that received nps-based vaccine showed no clinical signs and also had significant reduction in lung virus load [ , ] . plga nps were also used to encapsulate highly conserved influenza peptides and evaluated for efficacy in pigs after in administration. plga nps-based subunit vaccine resulted in induction of epitope-specific t-cell response but not the antibody response [ ] . the t-cell biased immune response was also observed in pigs after in immunization with plga nps-encapsulated inactivated/killed influenza virus (plga-kag) vaccine in pigs [ ] . in plga-kag vaccine administered animals observed reduced fever; lowered pneumonic lesions; and increased virus clearance from lungs after heterologous virus challenge compared to kag vaccine controls [ ] . in another study, pedv kag was encapsulated in plga nps and used to immunize pregnant sows by in route. this nanovaccine induced higher virus-specific igg and neutralizing antibodies in serum and greater igg, iga and neutralizing antibody responses in colostrum. it also induced greater cell proliferation and ifn-γ responses in restimulated pbmcs compared to kag vaccine controls. importantly, piglets born to nps-vaccinated sows had higher virus neutralizing antibodies and were better protected against homologous virus challenge than kag controls [ ] . these studies suggest that plga nps can be used as an efficient means of enhancing virus-specific cell-mediated immune responses in pigs. polyanhydrides are another type of synthetic polymer widely studied for vaccine deliveries [ ] . polyanhydride nps are synthesized by polycondensation or emulsification processes and are biodegradable, biocompatible and safe for vaccine delivery [ , ] . they activate innate immune responses in a manner similar to lipopolysaccharides (lps) [ ] . the surface-eroding nature of polyanhydride nps provides safe microenvironment for the encapsulated antigens and facilitates slow and sustained antigen release [ , ] . induction of better antibody and cell-mediated immune responses by polyanhydride npsbased vaccines has been reported against viral, bacterial and parasitic infections [ , ] . inoculation of polyanhydride nps-based siv kag vaccine (kag-nanovaccine) by in route enhanced cell-mediated but not the antibody responses in pigs [ ] . after heterologous virus challenge, kag-nanovaccine group had six to eightfold reduction of nasal virus shedding compared to kag vaccine controls [ ] . in a subsequent study, when kag-nanovaccine formulation was supplemented with cpg-odn adjuvant, both cell-mediated as well as mucosal iga antibody responses were improved [ ] . after heterologous virus challenge, cpg-odn-adjuvanted kag-nanovaccine provided better protection through a significant reduction in influenza-induced fever, -fold reduction of nasal virus shedding and -fold reduction in lung virus titers compared to pigs immunized with five-times greater quantity of soluble killed antigen (kag) vaccine [ ] . this study also indicates the dose-sparing ability of polyanhydride nps. thus, polyanhydride nps can also be used to induce better cellular as well as humoral immune responses in pigs. chitosan, alginate and other polysaccharides have also attracted attention as materials for nps formulation and drug delivery studies. chitosan is a natural polymer derived from deacetylation of chitin and is composed of glucosamine and n-acetylglucosamine residues [ ] . due to the availability of amino and carboxyl groups in an acidic microenvironment, chitosan nps have net positive surface charge which makes them highly mucoadhesive and increases their half-time of antigen retention on mucosal surfaces [ , ] . further, chitosan nps can reversibly open the epithelial cell tight junctions thereby improving paracellular and intracellular antigen transport across mucosal epithelial surfaces [ , ] . chitosan nps also enhance antigen uptake by apcs, induce apc maturation and active secretion of innate cytokines [ , ] . thus, chitosan nps form an attractive mucosal vaccine delivery vehicle. chitosan-based nps are used in pigs to deliver adjuvants such as bee venom and plasmid encoding porcine il- and il- /il- genes, which improved induction of better virus-specific immune responses of respective vaccines against prrsv and pcv [ , ] . chitosan nps enhance antigen uptake by porcine apcs and activate them to produce innate cytokines including ifn-alpha, tnf-alpha and il- β [ ] . chitosan nps encapsulated siv kag (cnps-kag) vaccine administered twice through in route without any additional adjuvant in pigs induced the cross-reactive mucosal iga antibodies. chitosan nps-based vaccine also induced ifn-γ response in pbmcs and tracheobronchial lymph nodes (tbln) better than kag vaccine controls. this vaccine formulation substantially reduced the challenge heterologous virus titers by up to -fold in both the upper and lower respiratory tracts compared to soluble kag vaccine. this finding emphasizes the potential benefits of using chitosan nps in future development of mucosal swine influenza vaccine for pigs [ ] . recently, dendrimer-like-alpha-d-glucan (nano- ) nps derived from sweet corn variety sugary- was examined as an alternative, safe, cost-effective and potent adjuvant [ , ] . nano- are positively charged nps which efficiently adsorb negatively charged antigens through electrostatic interactions. rodent studies have shown that nano- nps enhance antigen uptake by dcs, induce their maturation, activate them to produce pro-inflammatory cytokines and help in induction of antigen-specific antibodies [ , ] . in a recent study, we observed that nano- nps with or without addition of siv killed antigen (kag) can stimulate porcine apcs and produce cytokines such as ifn-α, tnf-α and il- β [ ] . pigs immunized via in route with nano- nps adsorbed siv kag at two-to-one ratio (nano- + kag) resulted in cross-reactive mucosal iga responses better than kag controls. moreover, pigs immunized im with nano- adsorbed ovalbumin (nano- + ova) had significantly greater igg and igg antibodies in serum compared with pigs vaccinated with ova alone [ ] . these findings highlight the possibility of using cornderived nano- nps as a potential adjuvant in porcine viral vaccine development. liposomes can encapsulate both hydrophilic and hydrophobic molecules in aqueous and non-aqueous phases of their vesicles [ ] . liposome vesicles protect antigens from enzymatic degradation, enhance antigen internalization by apcs and maintain controlled release of antigens [ ] . liposome-encapsulated antigens can enhance both cellular and humoral immune responses [ , ] . in a pig study, liposome nps were used as an im adjuvant for a pcv dna vaccine [ ] . liposome nps-adjuvant induced higher neutralizing antibodies and ifnγ response in pigs and reduced viremia of a challenge virus compared to alum-adjuvanted vaccine, providing the evidence that liposome nps can be a potent adjuvant in pigs [ ] . in our recent study, we used liposome nps to encapsulate ten highly conserved peptides of different influenza viruses of human and pig origin and immunized pigs through in route co-administered with monosodium urate (msu) crystal adjuvant [ ] . the liposome-adjuvant based vaccine enhanced the mucosal iga antibody response and induced peptide and virusspecific t-helper/memory cells and ifnγ responses resulting in reduced fever and modest reduction in virus titers in the respiratory tract of pigs [ ] . these studies highlight the fact that liposome-based nps can be used as an attractive vaccine delivery platform against porcine viral infections. virus infections have significant impact on pig industry worldwide. use of available vaccines have definitely helped in achieving strong control over some of the porcine viral infections such as food and mouth disease, transmissible gastroenteritis, classical swine fever and pseudorabies. vaccination also helped in reducing the clinical signs and increasing the production parameters in pcv -associated disease. however, for many other porcine viruses, further improvements in existing vaccine platforms and development of novel vaccine delivery systems are necessary to: ( ) induce better mucosal and cell-mediated immunity; ( ) protect against emerging and re-emerging strains; ( ) enhance the breadth (heterologous, cross-genotype and heterosubtypic) of immunity; and ( ) differentiate between infected and vaccinated animals. nps-based vaccine delivery platforms such as vlps, biodegradable polymers and liposomes have great potential as they-( ) protect vaccine antigens from degradation; ( ) facilitate antigen uptake and processing by apcs; ( ) impart adjuvant potential; ( ) can be used in mucosal and other alternate routes of immunizations; and ( ) induce effective mucosal and cellular cross-protective (broader) immunity. research efforts are ongoing to develop porcine viral vaccines using nps-based technologies. however, more collaboration(s) and in-depth studies are warranted to make this innovative vaccine antigen delivery technology successful and practical for application in food animal industry. to date, almost all of the immunomodulatory mechanisms of nps-based vaccine delivery platforms have been studied in rodent disease models, which may or may not reflect the situation in pigs or other domestic animal species [ ] . likewise, proper understanding of effect of size, charge and other physicochemical properties of nps after delivery through different routes of immunization in pigs is 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different adjuvants and comparison with commercial pcv subunit vaccine in an experimental challenge liposomal nanoparticle-based conserved peptide influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs intranasal delivery of influenza antigen by nanoparticles, but not nkt-cell adjuvant differentially induces the expression of b-cell activation factors in mice and swine porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the research reviewed in this article was supported by agriculture and food research initiative competitive grant no. - - from the usda-nifa and nanovaccine institute ( - ), iowa state university to rgj. salaries and research supports were provided by the state and federal funds appropriated to oardc. we thank dr. steven krakowka for scientific editing of the manuscript. authors' contributions sd wrote the article; gjr edited and revised the article. both authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - uw xcxw authors: sugiarto, sarah; spiri, andrea m.; riond, barbara; novacco, marilisa; oestmann, angelina; de miranda, luisa h. monteiro; meli, marina l.; boretti, felicitas s.; hofmann-lehmann, regina; willi, barbara title: passive immunization does not provide protection against experimental infection with mycoplasma haemofelis date: - - journal: vet res doi: . /s - - -x sha: doc_id: cord_uid: uw xcxw mycoplasma haemofelis (mhf) is the most pathogenic feline hemotropic mycoplasma. cats infected with mhf that clear bacteremia are protected from mhf reinfection, but the mechanisms of protective immunity are unresolved. in the present study we investigated whether the passive transfer of antibodies from mhf-recovered cats to naïve recipient cats provided protection against bacteremia and clinical disease following homologous challenge with mhf; moreover, we characterized the immune response in the recipient cats. ten specified pathogen-free (spf) cats were transfused with pooled plasma from cats that had cleared mhf bacteremia; five control cats received plasma from naïve spf cats. after homologous challenge with mhf, cats were monitored for days using quantitative pcr, hematology, blood biochemistry, coombs testing, flow cytometry, dnak elisa, and red blood cell (rbc) osmotic fragility (of) measurement. passively immunized cats were not protected against mhf infection but, compared to control cats, showed significantly higher rbc of and b lymphocyte (cd r/b (+)) counts and occasionally higher lymphocyte, monocyte and activated cd (+) t lymphocyte (cd (+)cd (+)) counts; they also showed higher bilirubin, total protein and globulin levels compared to those of control cats. at times of peak bacteremia, a decrease in eosinophils and lymphocytes, as well as subsets thereof (b lymphocytes and cd (+), cd (+) and cd (+) t lymphocytes), and an increase in monocytes were particularly significant in the passively immunized cats. in conclusion, passive immunization does not prevent bacteremia and clinical disease following homologous challenge with mhf, but enhances rbc osmotic fragility and induces a pronounced immune response. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. hemotropic mycoplasmas (hemoplasmas) are noncultivable epierythrocytic bacteria that infect a variety of mammalian species worldwide [ ] . in recent years, hemoplasmas have attracted scientific attention due to their host diversity and pathogenic potential [ ] . the main pathogenic feature of hemoplasmas is hemolysis, and clinical signs such as lethargy, anorexia, pale mucosal membranes, pyrexia, jaundice and pigmenturia may be present in severely affected animals [ ] . reports of hemoplasma infections in humans emphasize the need to characterize these agents in more detail [ ] [ ] [ ] [ ] [ ] [ ] . feline hemoplasmas can thereby serve as a model because of their extensive molecular and clinical characterization within this group of organisms. feline hemoplasmas comprise at least three different species: mycoplasma haemofelis (mhf), "candidatus mycoplasma haemominutum" (cmhm) and "candidatus mycoplasma turicensis" (cmt) [ ] [ ] [ ] . concurrent infections with two or three hemoplasma species have been documented [ ] [ ] [ ] [ ] , suggesting that no immunological cross-protection exists between the feline hemoplasmas, which has recently been experimentally confirmed [ ] . mhf is the most pathogenic of the three feline hemoplasma species and can induce severe hemolytic anemia, which is potentially fatal if left untreated. in contrast, the other two feline hemoplasmas may induce mild anemia, and the infection often remains subclinical [ ] . the natural route of hemoplasma transmission between cats is still unresolved, but aggressive interactions and blood-sucking arthropods have mainly been implicated [ ] [ ] [ ] . for experimental transmission, the intraperitoneal, intravenous or subcutaneous inoculation of hemoplasma-containing blood has been successful [ , [ ] [ ] [ ] . recently, a low-dose infection model for mhf that aimed to more accurately mirror the natural transmission of hemoplasmas was developed [ ] . different antibiotic regimens reduce hemoplasma blood loads and alleviate clinical signs but, so far, no treatment protocol has successfully and consistently cleared feline hemoplasma infections [ , [ ] [ ] [ ] [ ] . this limitation emphasizes the need to further investigate protective immune mechanisms against these agents. recently, cats that were experimentally infected with mhf or cmt and overcame bacteremia were shown to be protected from reinfection with the same hemoplasma species [ , ] . a study by novacco et al. [ ] suggested a significant role for the humoral immune response in protecting against cmt reinfection: nine out of the ten cats that were protected from reinfection showed intermediate to high antibody levels against cmt before challenge. furthermore, a transient decrease in antibody levels was observed in the protected cats immediately after attempted reinfection, which could be due to the binding of antibodies to the inoculated antigens. in the early phase after re-challenge, compared with the control group, the protected cats exhibited significantly higher il- /il- ratios and cd + t lymphocyte counts and a pronounced eosinophilia. therefore, the authors concluded that an early th immune response, prior to the onset of bacteremia, is beneficial for protection against cmt reinfection [ ] . this result was not found in the study by hicks et al. [ ] , where an early increase in the pro-inflammatory cytokines tumor necrosis factor-α (tnf-α) and interleukin- (il- ) was observed in cats protected from mhf reinfection. furthermore, the immune response seemed to be skewed towards a th response after primary mhf infection, whereas a switch from an initial th to a delayed th response was observed after primary cmt infection [ , ] . these results suggest that cats respond to infection by different feline hemoplasma species with different immune mechanisms. important data on the immune response elicited by hemoplasma infection have been provided by previous studies [ , ] , but the mechanisms that confer protection against re-infection have yet to be clarified. passive immunization transfers humoral immunity to a nonimmune individual in the form of antibodies and allows the protective role of antibodies in the absence of cellular immune mechanisms to be assessed. the present study aimed to investigate whether the passive transfer of antibodies from mhf-recovered to naïve recipient cats induced partial or complete protection against bacteremia and clinical disease following homologous challenge with mhf. different parameters addressing the humoral and cellular immune response were monitored in passively immunized and control cats. in the main experiment, -month-old male specified pathogen-free (spf) cats from liberty research, inc. (waverly, new york, usa) were used as recipient cats. five adult spf cats served as blood donors: three of these cats had overcome a previous mhf infection (hbu , hbz and hcd ) [ ] , while two cats were naïve adult spf cats (gcn and jcr ). moreover, in a pre-experiment, a -year-old male castrated spf cat (hcc ) was used. all cats were housed in groups in a confined university facility under ethologically and hygienically ideal conditions [ ] . all experiments were approved by the veterinary office of the canton of zurich (tvb / ) and were conducted in accordance with swiss laws. fifty days prior to plasma transfusion and experimental infection, the spf status of all the cats was confirmed by testing for the absence of infection with feline hemoplasmas, feline calicivirus, feline herpesvirus- , feline coronavirus, feline leukemia and feline immunodeficiency virus, as well as bartonella henselae and chlamydia felis, as recently described [ , ] ; the absence of mhf infection was again confirmed by real-time taqman ® quantitative (q)pcr [ ] on day prior to plasma transfusion and mhf inoculation. all donor cats and the cat from the preexperiment were blood-typed using a commercial immunochromatography technique (feline lab test a + b, alvedia, france). the recipient cats had been bloodtyped by liberty research, inc. (waverly). a standard saline-agglutination cross-matching procedure was performed to ascertain the compatibility of transfusion [ ] . for passive immunization, a total of ml of plasma was collected from the three cats that had overcome previous mhf infection (hbu , hbz and hcd ) [ ] . the plasma was collected from these cats on day post mhf inoculation. hbu had cleared the infection without treatment, whereas hbz and hcd had received antibiotic treatment to clear the infection (hbz : mg/kg/day doxycycline for days; hcd : mg/ kg/day doxycycline for days, followed by mg/kg/ day marbofloxacin for days); in the latter two cats, antibiotic treatment was stopped - days prior to plasma collection. all cats tested negative for mhf for at least eight consecutive weeks following antibiotic treatment and prior to the plasma collection, as determined using weekly collected blood samples that were analyzed in triplicate with a mhf-specific qpcr assay [ ] . for plasma transfusion of the control cats, a total of ml of plasma from two adult naïve spf cats (gcn and jcr ) was collected. prior to plasma collection, the two cats were confirmed to be negative for all three feline hemoplasmas using qpcr [ ] and to be free from all commonly known feline pathogens, as described [ ] . for plasma preparation, whole blood was collected from the donor cats from the jugular vein under shortduration general anesthesia ( mg/kg ketamine, narketan ® , vétoquinol ag, belp, switzerland; . mg/kg midazolam, dormicum ® , roche pharma ag, reinach, switzerland); the blood was anti-coagulated with acidcitrate-dextrose (acd) to a final concentration of . %. plasma was separated from rbcs by centrifugation at g for min and stored at − °c until use. prior to transfusion, the plasma was thawed, pooled (pool a: plasma from the mhf-recovered cats hbu , hbz and hcd ; pool b: plasma from the naïve spf cats gcn and jcr ), and filtered through a . µm pore-size filter (jet biofil, madrid, spain), and ml aliquots were prepared. before freezing and after thawing and filtration, the plasma was tested in triplicate with a mhf-specific qpcr assay for the absence of mhf organisms [ ] . for the plasma transfusion, all recipient cats were put under short-duration general anesthesia ( mg/kg ketamine, narketan ® , vétoquinol ag; . mg/kg midazolam, dormicum ® , roche pharma ag) and ml of whole blood was collected for baseline analysis and to prevent circulatory volume overload. the plasma was prewarmed to °c and intravenously administered over a duration of min using standard blood transfusion sets (hemo-nate ® , micron, utah medical products, inc., utah, usa). during transfusion, the cats were closely monitored for transfusion reactions. in the pre-experiment, an adult naïve spf cat (hcc ) was intravenously transfused with a ml aliquot of plasma pool a that was later used for the passive immunization of the cats in group a. after the transfusion, weekly blood samples were collected from the cat for weeks and analyzed using mhf-specific qpcr [ ] . for the main experiment, the recipient cats were assigned to two groups: group a (n = , passive immunization, cats dhr , dhr , jhw , jhw , jhw , jhw , jhx , jhx , jhz and jic ) and group b (n = , control group, cats dhp , dht , jhv , jhw and jic ). the cats of each group were housed together during and after the end of the study period. on day , plasma transfusion was performed, and the cats were experimentally inoculated with mhf. for the passive immunization, each cat in group a was transfused with a ml aliquot of plasma pool a. each cat in group b was transfused with a ml aliquot of plasma pool b. ten minutes after the completion of the transfusion, all cats in groups a and b were inoculated with mhf by a subcutaneous injection of µl of % dimethyl sulfoxide (dmso)-preserved blood containing copies of mhf diluted with µl of phosphatebuffered saline (pbs), as previously described [ ] . an aliquot of the same mhf inoculum was used to infect the recipient cats in this study that had been used to infect the plasma donor cats (hbu , hbz and hcd ) in a previous study [ ] . clinical condition, body temperature and body weight were recorded, and blood samples collected prior to plasma transfusion and mhf inoculation (day ), twice weekly post inoculation (pi) until week and weekly thereafter up to day pi.; an additional blood collection to measure red blood cell (rbc) osmotic fragility (of) was performed on day pi because rbc of was still increased compared with baseline values in several cats of group a and b at day pi. all samples were collected without anesthesia, with the exception of the samples collected at day . samples for pcr analysis and serology were stored at − °c until analysis. flow cytometry, hematology, blood biochemistry, coombs tests and rbc of assays were carried out within h of blood collection. hematological parameters were determined on day , twice weekly pi until week and weekly thereafter up to day pi on a sysmex xt- iv instrument (sysmex corporation, kobe, japan), as previously described [ ] . white blood cell differential analysis was confirmed using manual evaluation of wright-giemsa stained blood smears. severity of anemia was defined as mild ( - %), moderate ( - %) and severe (< %). bilirubin, total protein and albumin concentrations were measured on day , on days and pi and weekly thereafter until day pi using a cobas integra system (roche diagnostics, rotkreuz, switzerland). globulin values were calculated by subtracting the albumin value from the total protein concentration. reference intervals (mean ± standard deviation, sd) for pcv and total protein concentrations for kittens of and weeks of age were obtained from the literature [ ] . because bilirubin concentrations reach adult values after week of age [ ] , the laboratory's device-specific reference interval was used for this parameter. rbc of was determined on day and on days , and pi using a published protocol [ ] . the reference interval was determined in nine healthy cats during a previous study ( % hemolysis in nacl . - . % w/v) [ ] . direct coombs tests were performed on day and on days and pi. rbcs from edta-anticoagulated blood samples were washed, diluted in nacl . % w/v solution and incubated for h at °c with feline antiglobulin reagent (mp biomedicals, llc., solon, ohio, usa) in dilutions ranging from : to : , as described [ ] . agglutination in dilutions of ≥ : was defined as positive. quantification of mhf blood loads by qpcr was performed on day , twice weekly pi until week and weekly thereafter up to day pi. total nucleic acid (tna) was extracted from μl of edta anti-coagulated blood using the magna pure lc (roche diagnostics ag, rotkreuz, switzerland) and the magna pure lc tna isolation kit (roche diagnostics) following the manufacturer's instructions. tna was eluted in µl elution buffer and stored at − °c until use. with each batch of extraction, a negative control consisting of µl of phosphate-buffered saline (pbs) was used to monitor for cross-contamination. all tna samples were tested with a mhf-specific qpcr assay to detect and quantify mhf as previously described [ ] . for absolute quantification, tenfold serial dilutions of a standard plasmid were used as described [ ] . positive and negative controls were included in each pcr run. antibodies against the recombinant dnak protein of mhf were measured on day , on days and pi and weekly thereafter until day pi using a previously described elisa [ ] . the serum was diluted : , and ng of recombinant protein per well was used as described [ ] . all samples collected from one cat were measured on the same plate, and all plates were antigen coated within the same batch. absorbance was measured at a wavelength of nm (od ) using a spectramax plus microplate spectrophotometer (molecular devices, sunnyvale, ca, usa). an od ≤ . was defined as negative based on dnak elisa results obtained from spf cats, as described [ ] . flow cytometric analysis was performed on day and on days , , , , , , , and pi. three different staining combinations of primary antibodies were used: ( ) fluorescein isothiocyanate (fitc)-conjugated mouse anti-feline cd antibody (f , southern biotech, allschwil, switzerland); ( ) an r-phycoerythrin (rpe)conjugated mouse anti-feline cd antibody (vpg , abd serotec, puchheim, germany) and a fitc-conjugated mouse anti-feline cd antibody; and ( ) a fitc-conjugated mouse anti-feline cd antibody (fcd , southern biotech) and a rpe-conjugated rat anti-mouse cd r/ b antibody (ra - b , abd serotec). cd is a marker for feline t lymphocytes [ ] , and cd + -and cd +positive t lymphocytes represent helper and cytotoxic t lymphocytes, respectively [ ] . cd + cd + t lymphocytes represent activated cd + t lymphocytes, and cd r/b antibody was used to identify b lymphocytes [ , ] . prior to the start of the experiment, the antibodies were titrated for optimal dilution as follows: cd and cd , : dilution; cd and cd , : dilution; and cd r/b , undiluted. a µl aliquot of edta anticoagulated blood was incubated with µl of the aforementioned antibodies. to rule out non-specific binding, isotype control antibodies (rpe-conjugated mouse igg and fitc-conjugated mouse igg , abd serotec) and unstained blood samples from each cat were used as negative controls. blood samples were stained according to published protocols [ , ] . flow cytometry was performed using a guava easy-cyte ™ ht flow cytometer (millipore, darmstadt, germany) using the guavasoft . software. gates representing the lymphocyte population were set based on forward and side scatter, and events were acquired for each sample. the absolute number of each lymphocyte subset was calculated by multiplying the absolute lymphocyte number from hematology by the subset percentage as previously published [ ] . up to different parameters per cat were statistically analyzed using analyse-it ® for microsoft excel version . . . (analyse-it software, ltd., leeds, uk) and graph-pad prism . (graphpad software, inc., ca, usa). the mann-whitney u test (p mwu ) was used to compare the two groups a and b at each time point. one cat (jhw ) in group b stayed pcr-negative and seronegative throughout the -day study period. to investigate whether significant differences observed between groups a and b were related to the inclusion of this cat, the mann-whitney u tests were repeated without the data of this cat for all significant parameters. the results from these analyses were reported if they differed from the original results including cat jhw . friedman's test (p f ) followed by dunn's post test (p d ) was used to analyze the parameters over time when more than two time points were considered. multiplicity adjusted p values for each comparison in a family of comparisons were computed. fisher's exact test was used to determine significant differences of proportions. p values < . were considered significant. all donor cats, the cat from the pre-experiment and all recipient cats were confirmed to be blood type a. crossmatching revealed no incompatibilities between the donor cats, the cat of the pre-experiment and the recipient cats. all plasma samples used for transfusion tested mhf pcr-negative. the adult cat (hcc ) transfused with an aliquot of plasma pool a during the pre-experiment tested mhf pcr-negative in each of the samples collected during the weeks after the transfusion (data not shown). the cat stayed clinically healthy throughout the entire experiment. prior to plasma transfusion and experimental infection (day ), all cats in both groups tested mhf pcr-negative. all cats in groups a and b became mhf pcr-positive within - days and - days pi, respectively ( figures a and b) , except for one cat (jhw ) in group b. cat jhw remained pcr-negative throughout the -day follow-up period ( figure b ) but became mhf pcr-positive after the end of the experiment (day pi, data not shown). peak hemoplasma loads (group a: . × - . × copies/ml blood; group b . × - . × copies/ml blood) were reached between days and pi in group a and days and pi in group b. there was no significant difference in mhf blood loads between the two groups during the -day follow-up period, except for day pi, when group a showed significantly higher mhf blood loads (p mwu = . ; figure c ); this difference was not significant when the pcr-negative cat jhw in group b was excluded from the analysis (p mwu = . ). the cats in both groups tested negative in the dnak elisa (defined as an od ≤ . ) prior to the plasma transfusion (figures a and b) . after passive immunization and subsequent infection, group a cats showed slightly but significantly higher dnak elisa od values at days , and pi compared with the od values of cats in group b (p mwu < . ; figure c ). from day until day pi, no significant difference in the dnak elisa od values was evident between the two groups. when cat jhw was excluded from the analyses, the anti-dnak antibody level in cats in group a was still significantly higher early after passive immunization (days and pi, p mwu < . ), but not at day pi and thereafter until day pi when compared to cats in group b. there was no significant difference in the time of seroconversion (od > . ) between the two groups; cats in both groups, a and b, seroconverted between days and pi, with the exception of cat jhw (group b), which did not develop anti-mhf dnak antibodies throughout the study ( figure b ). two other cats (jhw , group a, and jhv , group b) were seronegative (od ≤ . ) by the end of the experiment ( figure b ); one of these cats (jhw ) was treated with doxycycline between days to pi because of a pronounced decrease in packed cell volume (pcv) to % (day pi, see below). seven out of ten cats in group a became moderately anemic (pcv between and %) at days to pi ( figure a ). in group b, one cat showed moderate anemia (pcv %) and one cat severe anemia (pcv %) at days and pi, respectively ( figure b ). the lowest pcv observed during the study was found for cat dht (pcv %, days pi) in group b. there was no significant difference in the mean pcv values between the two groups ( figure c ). there was a significant change in pcv over time for both groups (group a, p f < . ; group b, p f = . ); pcv values of the cats were higher towards the end of the study period when compared to the first weeks pi (figures a and b) . this finding may be attributed to the increasing age of the cats; reference intervals of pcv (±sd, %) were reported to increase from the age of weeks ( . ± . ) to weeks ( . ± . ) [ ] . three cats in group a (cats jhw , jhw and jhz ) and two cats in group b (cats dht and jhv ) developed elevated body temperature > . °c during the course of the experiment: jhv at day pi, jhw at day pi, dht at day pi, jhw at day pi and jhz at day pi. in addition, apathy, anorexia and weakness were observed in three cats in group a (jhw and jic at day pi; and jhx at day pi) and in one cat in group b (dht at day pi). three cats necessitated oral antibiotic treatment with doxycycline ( mg/kg/day, supracycline, grünenthal gmbh, mitlödi, switzerland) for - days. the decision to treat the cats was based on the presence of severe anemia (group a: dht , pcv of %, day pi) or a pronounced decrease in pcv within h (jhw , pcv of %, day pi; jhx , pcv of %, day pi). all three cats showed clinical signs (apathy, anorexia, weakness and fever) when the treatment was initiated. at day , rbc of was significantly lower in cats in group a when compared to cats in group b (p mwu = . ), but mean rbc of of both groups remained within the reference interval (group a and b, % hemolysis in nacl . and . % w/v, respectively; reference interval, % hemolysis in nacl . - . % w/v, figure c ). thereafter, at days and pi, rbc of was significantly higher in cats in group a compared to cats in group b (p mwu < . ; figure c ). when cat jhw was excluded from the analyses, there was no longer a significant difference between the groups on day pi. remarkably, compared with baseline values, rbc of was still increased in several cats in groups a and b at day pi (figures a and b ) ; therefore, one further measurement was obtained at day pi. rbc of significantly changed during the course of infection only in cats in group a (p f = . , figure a ). the rbc of reached maximal values at day pi in eight out of ten animals in group a (range, % hemolysis in nacl . - . % w/v) and at day pi in cat jhz ( % hemolysis in nacl . % w/v) and at day pi in cat jhw ( % hemolysis in nacl . % w/v; figure a ). cat jhw in group b, which turned mhf pcr-positive only after the end of the experiment (day pi), showed a pronounced increase in relative rbc of at the time of mhf bacteremia ( % hemolysis in nacl . % w/v, day pi; reference interval, % hemolysis in nacl . - . % w/v; figure b ). all cats in both groups tested negative in the direct coombs test at all time points (data not shown). cats in both groups showed an increase in leukocyte counts immediately after infection, followed by a significant decrease, with the nadir being reached three to four weeks pi (data not shown). there was no significant difference in leukocyte counts between the two groups during the -day follow-up period. the lymphocyte cell counts showed an initial increase followed by a significant decrease, with the nadir reached at day pi, coinciding with the onset of peak bacteremia in cats in group a (p f < . , figure a ); this pattern was less pronounced in cats in group b (p f = . , figure a ). furthermore, compared with cats in group b, cats in group a had significantly higher blood lymphocyte counts at days and pi (p mwu < . ), as well as a tendency for higher counts at day pi (p mwu = . , figure a ). when cat jhw was excluded from the analyses, the lymphocyte cell counts tended to be mhf at day . significant differences between groups are indicated with asterisks (p mwu < . ). the reference interval for rbc of based on measurements in nine healthy cats is shaded grey ( % hemolysis in nacl . - . % w/v) [ ] . rbc of significantly changed during the course of infection in cats in group a (p f = . , significantly higher levels at days and pi compared with day pre-inoculation, p d < . ), but not in cats in group b. higher in group a compared to group b during the study period, but significance was not achieved. the eosinophils showed a similar pattern to the lymphocyte counts in cats in group a, but the decrease in cell counts was delayed, and the nadir was reached at weeks pi (p f < . , figure b ); again, this pattern was less pronounced in cats in group b (p f = . , figure b ). in contrast, the monocyte counts increased after infection in cats in group a, with maximal values reached at day pi (p f < . , figure c ); again, this pattern was less pronounced in cats in group b (p f = . , figure c ). when compared to the counts in group b, the monocyte counts in group a were higher at most of the time points pi, with significance reached at days , , , and pi (p mwu < . , figure c ). the monocyte cell counts were higher in group a compared to group b at most time points pi when cat jhw was excluded, with significance reached at days and pi (p mwu < . ). the neutrophils significantly changed over time only in cats in group a (p f = . , figure d ). there were no significant differences in the neutrophil counts between the two groups. when the course of the different white blood cell subsets was compared with the course of pcv and mhf bacteremia, most cats showed a decrease in the leukocyte, lymphocyte, and neutrophil cell counts just prior to the decrease in pcv and the onset of maximal mhf blood loads (data not shown). a similar pattern was also observed for the eosinophils, whereas the monocyte counts peaked at the onset of maximal anemia in most of the cats (data not shown). in the passively immunized cats, all analyzed lymphocyte subsets [cd + , cd + and cd + t lymphocytes, activated cd + t lymphocytes (cd + cd + ) and b lymphocytes (cd r/b + )], showed a significant change over time (p f < . ). in the control cats, a significant change during the course of infection was only observed for the activated cd + t lymphocyte subset (p f < . ). specifically, cats in group a showed a significant decrease in the cd + , cd + and cd + t lymphocyte counts, with the nadir reached at days , and pi, respectively, coinciding with the onset of peak bacteremia (cd + , p f = . ; cd + , p f = . ; cd + , p f < . , figures a-c) . the cd + /cd + ratio was significantly increased at - weeks pi in the passively immunized cats (p f = . , figure d ). cats in group a showed a significant decrease in their b lymphocyte counts during maximal bacteremia, with the nadir reached at day pi (p f = . , figure e ), and the b lymphocyte counts were significantly higher at days , , and pi compared with cats in group b (p mwu < . ; figure e ). when cat jhw was excluded from the analyses, the b lymphocyte counts were also higher in cats in group a compared to cats in group b, with significance reached at days and pi (p mwu < . ). the activated cd + t lymphocyte subsets showed a somewhat different pattern than the other lymphocyte subsets in group a. the activated cd + t lymphocyte counts increased early during the course of infection into the onset of pcrpositivity and peak bacteremia, and then, they markedly decreased and reached a nadir at day pi (p f < . ); this pattern was also present but less pronounced in group b (p f = . , figure f ). furthermore, compared with cats in group b, cats in group a showed significantly higher activated cd + t lymphocyte counts at day pi (p mwu = . ) and significantly lower levels at day pi (p mwu = . ; figure f ). when cat jhw was excluded from the analyses, a tendency for higher activated cd + t lymphocyte counts in cats in group a compared to group b was found at day pi (p mwu = . ); the difference at day pi was still significant (lower counts in cats in group a when compared to cats in group b; p mwu = . ). bilirubin concentrations significantly increased in both groups during the course of the experiment and peaked at day pi (group a, p f < . ; group b, p f = . ; additional file a). bilirubin concentrations were lower in group a than in group b at days and pi (p mwu = . ) but tended to be higher from day until the end of the experiment, with significance reached at day pi (p mwu = . ; additional file a). the mean bilirubin concentrations in both groups remained within the reference interval (bilirubin concentration < . μmol/l) throughout the study period; bilirubin concentrations exceeding the reference interval were observed in three cats in group a (dhr , day pi, jhw , day pi; jhw , day pi, bilirubin concentrations, range . - . μmol/l). the total protein concentrations significantly changed over time in groups a (p f < . ) and b (p f = . ; additional file b) and peaked at days and pi, respectively. higher total protein concentrations were observed in cats in group a compared to cats in group b, with significance reached at days , and pi (p mwu = . ; additional file b). when cat jhw was excluded from the analyses, total protein concentrations of group a were also higher compared to cats in group b with significance reached at days , and pi (p mwu < . ). the mean total protein concentrations of both groups remained within the reference interval (total protein, reference interval for - weeks of age, - g/l) throughout the study period; total protein concentrations above the reference interval were observed in two cats in group a and one cat in group b (total protein concentrations between . - . g/l). the globulin concentrations significantly increased over time only in group a (p f < . ; additional file c). compared with cats in group b, cats in group a showed significantly higher globulin concentrations at days and pi (p mwu < . ; additional file c). this is the first study to investigate the protective role of the humoral immune response against hemoplasma infection using a passive immunization experiment. our study demonstrated that the passive transfer of antibodies from mhf-recovered to naïve spf cats does not prevent infection, high bacterial loads and the development of clinical signs following homologous challenge with mhf. the passively immunized and control cats showed no differences in the onset and extent of bacteremia and anemia during the course of mhf infection. hicks et al. [ ] recently documented that mhf-recovered cats were protected from reinfection following re-challenge with a homologous mhf isolate. our study indicates that the presence of antibodies to mhf cannot mediate protection against homologous challenge, but that cellular or innate immune mechanisms may be necessary to provide protection against mhf infection. this is also in line with the study by hicks et al. [ ] , which reported protection from mhf reinfection in the absence of a pronounced th -type response in the cats after re-challenge. in the latter study, the protected cats showed no increase in anti-dnak antibody and il- mrna levels following mhf re-challenge. the anti-dnak antibody levels in the passively immunized cats were only higher in the first weeks after transfusion than in the control cats, and they remained below the threshold for seropositivity up to day pi. it is possible that the level of antibodies in the passively immunized cats following transfusion was not sufficiently high to prevent bacteremia. however, hicks et al. [ ] documented protection from mhf although the antibody levels to dnak did not rise after mhf challenge infection and remained at rather low levels (mean log-transformed relative antibody level (ral) < . ) when compared to the control cats with de novo mhf infection (peak mean ral > . ). they concluded that if mhf-specific antibodies had been responsible for immune protection in these cats, then low levels of anti-dnak antibodies could be sufficient to provide protection against mhf infection. because the assay and the calculations used to quantify anti-dnak antibodies in that study were different from those used in the present study, a direct comparison of antibody titers was not possible. furthermore, it needs to be considered that antibodies with different epitope specificities that are not detected with current dnak elisa could mediate immune protection. however, assays to measure such antibodies are not available. infection enhancement, i.e. earlier onset of bacteremia and anemia, was recently documented in cats that had recovered from cmt infection and were re-challenged with mhf [ ] . all cmt-recovered cats were serologically positive before mhf inoculation. the study suggested that the pre-existing antibodies against cmt might have had enhanced infection kinetics in the cmt-recovered cats [ ] . although infection kinetics were not different between the passively immunized and control cats in this study, our results still suggest that the transfer of antibodies from mhf-recovered cats could potentially be harmful. the passively immunized cats showed a significant increase in rbc of during the course of infection. this effect was not observed in the control cats, indicating that the increase was not simply due to a non-specific effect of plasma transfusion on rbc fragility. it can be hypothesized that the antibodies bound to the mhf organisms attached to rbcs. rbc-bound antibodies have been found after primary mhf infection [ , , ] and can induce receptor-mediated rbc phagocytosis or the activation of the complement system [ ] . in the present study, rbcbound antibodies were not detected in the cats by coombs test, but the sensitivity of this assay may be limited [ ] . the bilirubin concentrations were also higher in the passively immunized versus control cats at some time points during infection. rbc destruction leads to the formation of free hemoglobin, which is processed by the mononuclear phagocytic system. during this process, bilirubin is formed, released in the blood stream and further metabolized and excreted by the liver. during pronounced rbc destruction, the capacity of the liver to metabolize bilirubin is overwhelmed, and hyperbilirubinemia occurs. hyperbilirubinemia was observed in three cats of group a, although mean bilirubin concentrations in both groups remained within the reference interval. nevertheless, the higher rbc of values and occasionally higher bilirubin concentrations could point towards a more pronounced rbc destruction in the passively immunized cats following subsequent mhf challenge exposure. the passively immunized cats showed signs of a more pronounced immune response when compared to control cats. cats of group a showed higher lymphocyte, monocyte and b lymphocyte counts and higher total protein and globulin concentrations than the control cats at several time points during the experiment. furthermore, peak globulin concentrations coincided with peak anti-dnak antibody levels in both groups. this result was also found in our recent study, where we documented polyclonal hypergammaglobulinemia during mhf infection coinciding with high anti-dnak antibodies [ ] . we speculated that a large portion of the immune globulin pool was not hemoplasma-specific and was potentially due to a polyclonal b lymphocyte activation. the induction of autoreactive antibodies has recently been reported for the porcine hemoplasma mycoplasma suis and is thought to be caused by the upregulation of b lymphocytes in response to changes to the rbc surface of the infected host [ ] . it could be argued that the use of the homologous mhf isolate for experimental challenge of the recipient cats that had been used to infect the plasma donor cats contributed to the more pronounced immune response in the passively immunized cats. this has been reported for other feline pathogens, i.e. feline coronavirus (fcov) which can cause feline infectious peritonitis (fip) [ ] . during the pathogenesis of fip, the virus targets macrophages and infection of these cells can be enhanced in the presence of antibodies (antibody-dependent enhancement, ade) [ ] . takano et al. [ ] showed that cats passively immunized with antibodies to serotype i fcov showed an enhanced onset of disease following inoculation with the homologous serotype; this was not found when cats passively immunized with antibodies to serotype ii fcov were challenged with serotype i. the authors concluded that fcov re-infection with the same serotype might induce ade and could advance the development of fip. although ade has also been suspected in bacterial infection [ ] , it is unknown whether it might play a role in the pathogenesis of feline hemoplasma infections [ ] . signs of infection enhancement have recently been documented in "candidatus m. turicensis"-recovered, seropositive cats following a challenge with mhf [ ] . the study suggested that the presence of antibodies directed against cmt could enhance mhf infection. however, to the best of our knowledge, disease enhancement after rechallenge with the same feline hemoplasma species has not been documented. in contrast, two studies showed that cats that had recovered from mhf or cmt infection were protected from re-infection following re-challenge with the same hemoplasma species, respectively [ , ] . both studies had used an aliquot of the same mhf or cmt isolate for re-infection that had been used for primary infection of the cats (personal communication, rhl). the passively immunized cats showed a significant decrease in lymphocyte counts near the onset of peak bacteremia (around weeks pi). this comprised decreases in absolute counts of cd + t lymphocytes, cd + t lymphocytes and b lymphocytes around three to weeks pi, and was followed by an increase of the lymphocyte subsets until the end of the study. in two recent studies we reported a very similar pattern in the lymphocyte subsets following mhf infection of naïve and cmt-recovered cats. the decrease was explained by the migration of the cells from the peripheral blood to the draining lymph nodes, where they become activated and proliferate [ , ] . in the individual cats, the decrease in leukocyte, lymphocyte and neutrophil counts occurred just prior to the development of anemia and the onset of peak bacteremia. a similar pattern was also observed for the eosinophils, whereas the monocyte counts peaked at the onset of maximal anemia. a decrease in leukocyte and neutrophil counts could be caused by the increased consumption of these cells, which are involved in bacterial killing or, less likely, by reduced production due to undefined inhibitory factors [ ] . monocytosis could be found due to hemolysis [ ] and was shown to be associated with mhf infection in naturally infected cats [ ] . we documented leukopenia and neutropenia, as well as decreased eosinophils, lymphocytes and increased monocytes, at times of peak mhf bacteremia also in a recent study [ ] . it is difficult to judge whether the decrease in the white blood cell subsets was due to bacterial replication and cell consumption or, conversely, whether the decrease in white blood cells facilitated bacterial replication. interestingly, one cat in group b (jhw ) stayed pcrnegative and seronegative during the -day follow-up period, but it developed bacteremia and seroconverted days after mhf inoculation. although low-level bacteremia, below the detection limit of the pcr assay, cannot be completely excluded in this cat, we would expect a productive infection to result in seroconversion [ , ] . because all cats in this study were kept under strictly controlled hygienic conditions, vector-borne transmission between the cats can be excluded. however, all cats of group b were housed together and four out of five cats in group b tested mhf pcr-positive at the time that bacteremia and seroconversion occurred in cat jhw . therefore, direct transmission between the cats, i.e., by aggressive interactions, seems most likely, although no obvious clinical signs of aggressive interactions were observed in the cats in this study at any time during the experiment. a direct transmission of mhf through saliva seems unlikely because only low hemoplasma loads can be detected in saliva of mhf-infected cats [ ] and transmission by oral or subcutaneous inoculation of saliva has not been successful for another feline hemoplasma species, cmt [ ] . the present study had some limitations. no masking was used during data collection and analysis. furthermore, the relatively small groups size used in this study for animal welfare reasons could have masked significant differences between the passively immunized and control cats. however, as no protection but rather signs of infection enhancement were found in the passively immunized cats, the limited group size should not have affected the principal hypothesis addressed in this study. the present study used an experimental set-up to address the protective role of passively transferred antibodies in mhf infection. experimental infection studies do have some inherent limitations when results are generalized to natural infections. however, to mirror the natural transmission of hemoplasmas most accurately, a published low-dose infection model was applied in this study [ ] . the inoculated dose contained only copies of mhf, which corresponds to approximately . μl of infectious blood from a naturally infected cat [ ] . this small blood volume can easily be transmitted by bloodsucking arthropods or via aggressive interaction between cats-both of the latter transmission routes are assumed to be natural ways of transmission for feline hemoplasmas [ ] [ ] [ ] . therefore, the infectious dose applied to the cats should not be the reason for the lack of protection in the passively immunized cats. several measures were undertaken to ensure that the plasma used for passive immunization did not contain viable mhf organisms that could transmit infection. the donor cats were tested weekly for at least eight consecutive weeks before the plasma was collected. all these samples and the plasma pool itself were tested in triplicate with a highly sensitive mhf-specific qpcr assay [ ] and revealed pcr-negative results. furthermore, an aliquot of the plasma pool used for passive immunization was transfused into an adult spf, the cat was followed for weeks after transfusion and stayed pcr-negative. in conclusion, passive immunization did not provide protection against experimental infection with mhf but instead enhanced rbc fragility and was associated with a more pronounced immune response after infection. this result suggests that a humoral immune response in the absence of cellular immune mechanisms is insufficient to provide protection from mhf infection. potential vaccine candidates should include the induction of a cellular immune response against mhf. and the plasma transfusions. fsb was responsible for the clinical aspects of the study and supported the plasma transfusions of the cats. rhl supported the statistical analyses. ss and bw drafted the manuscript. bw and rhl edited the 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different markers of disease progression cold agglutinins in cats with haemobartonellosis western immunoblot analysis of the antigens of haemobartonella felis with sera from experimentally infected cats immune-mediated anemias in the cat the coombs' test in veterinary medicine: past, present, future pathobiology of mycoplasma suis antibodydependent enhancement occurs upon re-infection with the identical serotype virus in feline infectious peritonitis virus infection an update on feline infectious peritonitis: virology and immunopathogenesis antibody-dependent enhancement of infection: bacteria do it too molecular biology and pathogenicity of mycoplasmas interpreting absolute wbc counts diagnosis of feline haemoplasma infection using a real-time pcr assay detection of humoral response using a recombinant heat shock protein , dnak, of mycoplasma haemofelis in experimentally and naturally hemoplasma-infected cats the mouse anti-feline cd antibody was kindly provided by m. b. tompkins, north carolina state university, nc, and g. dean, colorado state university, co, usa. the authors thank e. gönczi, t. meili, b. weibel and the technicians of the clinical laboratory, vetsuisse faculty, for excellent laboratory assistance, as well as m. stirn, v. cattori, c. asquith and k. helfer-hungerbühler for helpful assistance in laboratory work and analyses. the authors also thank s. hartnack for helpful discussion and the animal caretakers m. zihlmann and f. dreier for the dedicated animal care. the laboratory work was performed with logistical support from the center for clinical studies at the vetsuisse faculty, university of zurich. ams was supported by a research grant (forschungskredit, fk- - - ) from the university of zurich. the study was supported by the kids and career program of the vetsuisse faculty, university of zurich. this study was the doctoral thesis of s. sugiarto. the authors declare that they have no competing interests.authors' contributions bw, rhl and fsb conceived and coordinated the study. ss was responsible for the training and clinical care of the cats and the sample and data collection. ams and ao were involved in sample collection and cat training. mn and lhm supported the flow cytometric analyses. mlm was responsible for the molecular analyses. br was responsible for the management of the spf cat facility and animal welfare, the clinical pathology data, the plasma collection • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -b yikvj authors: giotis, efstathios s.; robey, rebecca c.; skinner, natalie g.; tomlinson, christopher d.; goodbourn, stephen; skinner, michael a. title: chicken interferome: avian interferon-stimulated genes identified by microarray and rna-seq of primary chick embryo fibroblasts treated with a chicken type i interferon (ifn-α) date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: b yikvj viruses that infect birds pose major threats—to the global supply of chicken, the major, universally-acceptable meat, and as zoonotic agents (e.g. avian influenza viruses h n and h n ). controlling these viruses in birds as well as understanding their emergence into, and transmission amongst, humans will require considerable ingenuity and understanding of how different species defend themselves. the type i interferon-coordinated response constitutes the major antiviral innate defence. although interferon was discovered in chicken cells, details of the response, particularly the identity of hundreds of stimulated genes, are far better described in mammals. viruses induce interferon-stimulated genes but they also regulate the expression of many hundreds of cellular metabolic and structural genes to facilitate their replication. this study focusses on the potentially anti-viral genes by identifying those induced just by interferon in primary chick embryo fibroblasts. three transcriptomic technologies were exploited: rna-seq, a classical ′-biased chicken microarray and a high density, “sense target”, whole transcriptome chicken microarray, with each recognising – regulated genes (curated for duplication and incorrect assignment of some microarray probesets). overall, the results are considered robust because of the compiled, curated list of regulated genes were detected by two, or more, of the technologies. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. the interferon (ifn) response is one of the most important arms of host innate immunity against virus infection [ , ] . infected cells are able to recognise foreign nucleic acids and induce the synthesis and secretion of type i ifn (ifn-α and ifn-β) and type iii ifn (ifn-λ), which bind to receptors on the surface of neighbouring cells and trigger the transcriptional regulation of genes involved in the antiviral state. studies in mammals have demonstrated that there are several hundred such ifn-regulated genes (irgs). because the vast majority are up-regulated they are overwhelmingly referred to as ifn-stimulated genes (isgs) so, hereafter, they will be referred to generically as isgs (or specifically as chicken isgs, chisgs), except where the more generic term avoids confusion. induction of isgs involves the jak/stat signalling pathway: stat is either recruited directly to target promoters for a relatively weak activation or, more commonly, is recruited in a complex called isgf in association with stat and irf [ , ] . isgs are the focus of considerable current attention with regard to: (i) their antiviral activity, (ii) an increasing appreciation of the complexity of their regulation and (iii) their targeting by virus-encoded modulators of ifn-induced responses [ , , ] . these studies require comprehensive catalogues of the isgs, especially where system-wide approaches are undertaken. even though many key mammalian isgs have been known for some time, it is with the relatively recent advent of transcriptomic technologies that the full complement has been catalogued (mainly using microarrays [ ] ; see also schoggins et al. [ ] ). in contrast to the mammalian ifn system our equivalent knowledge of the avian system has lagged behind. although ifn was discovered in chickens in [ ] the first chicken ifn gene was characterised in [ ] and the key chicken isg, pkr, was identified in [ ] . the derivation of the chicken genome sequence, first drafted in [ ] , did not greatly advance our understanding of chicken isgs because of the incomplete nature of the gallus gallus genome assembly, even at v (galgal ), which might be partly due to the fact that the chicken karyotype has six pairs of macrochromosomes (but pairs of microchromosomes), and the difficulties in annotating immunity genes, which are some of the most divergent between mammals and birds [ ] . however, it has become apparent that key genes of the innate immune system, such as the transcription factors irf and one member of the irf /irf dyad [ , ; unpublished] , are absent from avian species, indicative of significant functional differences between them and mammals. moreover, for reasons that are not understood, the cytosolic pattern recognition receptor, rig-i, appears to have been lost from chicken as well as other galliformes [ , ] . to generate a chicken isg database we have compared data from three transcriptomic technology platforms: (i) the classical ′-biased genechip chicken genome array ( k; affymetrix, high wycombe, uk), (ii) the chicken gene . sense target (st) whole transcriptome array (affymetrix) and (iii) illumina (little chesterford, uk) rna-seq. this three-way comparison allowed a high level of cross-validation of data from each technology, beyond what would normally be achieved by qrt-pcr. it also allows subsequent studies, constrained to use any particular technology, to be more broadly compared. we monitored irg expression in chicken embryo fibroblast (cef) induced for h with units recombinant chicken ifn-α (rchifn ; hereafter routinely referred to as ifn), a time chosen to reflect predominantly primary signalling targets. the expression data for selected genes were also validated by pcr and qrt-pcr. overlapping data show generally high degrees of concordance in the identity of the irgs and their relative levels of regulation by ifn, with disparity mainly where multiple microarray probes exist for single genes. the study was presented in a preliminary form as a poster at the international cytokine and interferon society (icis) meeting ("cytokines "; october - , ) in bamberg, germany [ ] . freshly isolated cef were provided by the former institute for animal health (compton, uk, now the pirbright institute, pirbright, uk). cells were seeded in t flasks (greiner bio one, kremsmünster, austria; . × cells/flask) and cultured overnight in . ml media (gibco thermo fisher scientific, paisley, uk) supplemented with % heat-inactivated newborn bovine serum (nbcs; gibco), % tryptose phosphate broth (tpb; sigma-aldrich, gillingham, uk), % nystatin (sigma-aldrich) and . % penicillin streptomycin (gibco). recombinant chicken ifn-α (rchifn ) was prepared as previously reported [ ] and was added in culture media to a final concentration of units/ml. confluent cells were treated with ifn or mock-treated and incubated for six hours before harvesting. cells were stored at − °c in rnalater (sigma-aldrich) until rna extraction. the experiment was repeated in triplicate with three different batches of cef. total rna was extracted from cells using an rneasy kit (qiagen, crawley, uk) according to the manufacturer's instructions. on-column dna digestion was performed using rnase-free dnase (qiagen) to remove contaminating genomic dna. rna samples were quantified using a nanodrop spectrophotometer (thermo fisher scientific, paisley, uk) and checked for quality using a bioanalyzer (agilent technologies, wokingham, uk). all rna samples had an rna integrity number (rin) ≥ . . rna samples were processed for microarray with the genechip ® chicken genome array (affymetrix) using the genechip ® ′ ivt express kit (affymetrix) or for microarray with the chicken gene . st array (affymetrix) using the ambion (paisley, uk) wt expression kit for affymetrix genechip ® whole transcript (wt) expression arrays (ambion) and the genechip wt terminal labelling and controls kit (ambion), following the manufacturers' instructions, as described previously [ ] . total rna ( ng) was used as input and quality checks were performed using the bioanalyzer at all stages suggested by the manufacturer. rna samples were processed in two batches of but batch mixing was used at every stage to avoid creating experimental bias. hybridisation of rna to chips and scanning of arrays was performed by the medical research council's clinical sciences centre (csc) genomics laboratory (hammersmith hospital, london, uk). rna was hybridised to genechip chicken genome array chips (affymetrix) in a genechip hybridization oven (affymetrix), the chips were stained and washed on a genechip fluidics station (affymetrix), and the arrays were scanned in a gene-chip scanner g with autoloader (affymetrix). cdna was synthesised from rna samples from untreated and ifn-treated cef using the quantitect ® reverse transcriptase system (qiagen) according to the manufacturer's instructions. the glyceraldehyde -phosphate dehydrogenase (gapdh) was used as a reference gene. all target gene expression levels were calculated relative to gapdh expression levels and the target gene expression level in − h uninfected cef using the comparative c t method (also referred to as the −ΔΔct method). triplicate untreated (control) and ifn-treated cef were processed for transcriptome analysis by rna-seq. the cell samples used were identical to those used for the microarray analyses. total rna was extracted as for microarrays (above) and rna libraries were prepared for deep sequencing using the truseq rna sample preparation kit (illumina) according to the manufacturer's instructions. total rna ( . μg) was used as an input for each library. a total of six rna adapter indices were randomly assigned to the samples to allow multiplexing of libraries. at the end of the protocol, libraries were quantified using a nanodrop spectrophotometer and checked for quality using a bioanalyzer high sensitivity dna chip (agilent technologies). rna library qpcr quantification, multiplexing and sequencing was performed by the medical research council's clinical sciences centre (csc) genomics laboratory, hammersmith hospital, london, uk. libraries were quantified using the kapa biosystems (london, uk) library quantification kit (kk ) on an abi fast qpcr machine (applied biosystems). libraries were then diluted to a nm stock solution, pooled for multiplexing, denatured and diluted to a final molarity of pm. libraries were loaded on to the flow cell ( - pm per lane) for clustering and cluster generation was performed by the illumina cbot using version kits. sequencing of the flow cell was then carried out on the illumina hiseq using the version kits. data were processed using microarray data were processed using workflows in genespring ™ (agilent) and partek ™ (partek inc., st louis, mo, usa) commercial software suites. data (.cel files) were analysed and statistically filtered using either partek genomic suite . (partek gs) or genespring version . (agilent technologies) software. input files were normalized with either gcrma or genespring algorithms for gene array on core metaprobesets. a one-way anova was performed using either software across all samples. statistically significant genes were identified using mixed model analysis of variance with a false discovery rate (benjamini-hochberg test) of p < . . fold-change values <± . were removed. rna-seq data were imported into clc bio's genomics workbench (clc bio, aarhus, denmark; now qiagen), quality-controlled and thereafter processed using that package (versions and ). after quality control, the reads were subjected to quality trimming then mapped against ensembl galgal annotated genes (release [ ] ) for quantitative analysis of expression. fold change and false discovery rates (fdr) were calculated using kal's z test [ ] , with pooled data, or baggerly's test [ ] , using separate triplicates. initially, we used the k genechip ® chicken genome array (affymetrix) because, as well as displaying probes for chicken transcripts, it displays probes for transcripts from different viral pathogens of chickens, which offers advantages to those studying virus infections in a chicken background. subsequently, we used the more refined chicken gene . st array (affymetrix) because it offers a higher probe density against chicken genes and should allow detection of transcript isoforms, including non-polyadenylated and alternatively polyadenylated, though it does not include probes for viral genes. separate weekly batches of cef, produced from pools of eggs from the same flock (rhode island red) held in spf-like conditions at the former compton laboratory of the institute for animal health (now the pirbright laboratory) served as biological replicates. principal component analysis of the microarray data (data not shown) indicated limited variation between batches so, thereafter, biological triplicates were used routinely. irgs were identified from expression analysis data determined using the k genechip following ifn treatment ( units, h) of cef. after quantile normalization, significant hits were identified with genespring using an unpaired t test with asymptotic p-value computation and benjamini-hochberg multiple testing correction to generate false discovery rates (fdr). a matrix of fdr (from < . to ) plotted against fold change (fc; from . to > ) is shown in table . a relatively conservative fdr of < . returned differentially expressed probesets. overlaying this with a value for fc for which changes in expression might reasonably be expected to be readily and reliably assayed using other technologies, namely > , reduced the number of selected, significant probesets to a manageable ( up, down). these settings were therefore chosen for further analysis. for of these probe sets, no currently recognised genes were automatically assigned. of the remaining probe sets, were assigned to genes recognised in duplicate by other probe sets. consequently recognised genes were identified as differentially expressed (the down-regulated transcript was not, at that time, assigned to a recognised gene). with the chicken gene . st array, probe sets demonstrated differential expression ( up, down) at the same settings (fc > , fdr < . ). amongst these, there were five duplicated probe sets and that were not automatically assigned to recognised genes therefore recognised genes were uniquely identified as differentially regulated. illumina rna-seq yielded a total of million reads ( bases; paired) for the mock-treated cef triplicate samples and million for the ifn-treated samples. upon quality trimming and mapping to ensembl galgal annotated genes (release ), using clc bio's genomic workbench, recognised genes were identified as differentially regulated ( up, down) using kal's proportion-based z test [ ; as implemented in the clc bio package] at the same settings (fc > , fdr < . ). kal's is performed on the pooled reads from ifn-treated and untreated samples. it is perhaps, therefore, more widely applicable; it also returned a number of irgs comparable to those returned by the microarrays. triplicate-based analysis using baggerly's proportionbased beta-binomial test [ ; as implemented in the clc bio package] at the same settings (fc > , fdr < . ) returned an additional up-regulated genes. comparison of the complete raw gene lists from the three technologies using the most compatible identifier (essentially the gene symbol) with an online venn diagram tool (venn diagram generator; [ ] ) demonstrated that recognised genes were identified as differentially regulated. of these, were identified in common by all three technologies and a further were identified by two out of three technologies, meaning that were identified by at least two technologies. a total of were therefore each identified only by individual technologies ( figure a) . as well as comparing the identities of the differentially regulated genes, the correlation of expression of the genes identified by the different platforms was examined in terms of both level and rank of fc (figures a and b) . for instance, comparing rna-seq data with the k genechip data, spearman correlation values were . for fc level and rank. considering the current state of assembly and annotation of the chicken genome, the correlation of isgs in terms of gene identity as well as the level and rank of induction as indicated by all three technology platforms is reassuring. nevertheless the platform transcriptomic data were validated for selected genes by rt-pcr (data not shown) and by qrt-pcr ( figure a) . a h time point was chosen for microarray and rnaseq analysis of ifn treatment as it has been widely used and is known to result in significant levels of a broad range of isgs in mammals, making it suitable for defining the chicken interferome. use of this single time point does not, however, provide unequivocal insight into mechanistic interpretation of isg induction; for instance, it does not discriminate between strictly isre-dependent induction of isgs and isre-independent induction of isgs by mechanisms that might include immediate high-level induction of irf , which has been observed in mammalian systems [ ] [ ] [ ] . kinetic analysis of the induction of expression of a subset of isgs was therefore conducted at , , and min post application of ifn (see figure b ). even among highly-induced isgs, different temporal profiles were observed, from the rapid accumulation of ifit ( -fold by min) and rsad (which remain at steady levels to min) to the steadier, sustained accumulation of mx and the more modestly induced stat ; with lgp and trim peaking at min. although differences in mrna stability and turnover will influence the profiles, this identification of the isgs will allow detailed analysis of their promoters to investigate elements (and the factors that bind them) that contribute to the complexity of the observed induction patterns. of the irgs initially identified by all three technologies, ). this suggests either that the mammalian equivalents are isgs but that they are not included as such in interferome or that they are not isgs in mammals. the raw lists were refined by manual "curation", allowing for synonyms of recognised genes (for instance isg - versus isg ( )) and, after bioinformatic analysis using blast, etc., assigning recognised gene identifiers to probe sets that previously lacked them. at the end of this process ( figure b ; additional files , ), it was apparent that some (n = ) differentially regulated genes identified by the microarrays were also identified as differentially regulated by rna-seq but that they fell outside of the strict fc > and fdr < . parameters, reflecting unsurprising disparity in the sensitivity of the three technologies. those genes that were expressed down to fc > . or with an fdr up to < . were, therefore, also incorporated to produce a final list ( figure c ; additional files , ). it is obvious that this manual curation of the data, to allow for alternative gene id nomenclature used by the three technologies and for differences in sensitivity, introduced minor changes to the figures from the automatic comparisons cited above (figure ; additional files , ). curation, therefore, reduced the number of irgs from to . it also increased the number of differentially expressed genes detected by two out of three technologies from to (compare figures a and b) . relaxing the criteria for detection of differentially regulated genes by rna-seq (to fc > . and/or fdr < . ) further increased the number of genes detected by all three technologies from to (representing %) or by at least two of the technologies from to ( %), leaving genes detected by single technologies (compare figures b and c) , with of those detected by rna-seq alone (using the kal's test, at fc > . and fdr < . ; additional files , ). of the additional isgs identified by rna-seq as significant (fc > fdr < . ) by the more sensitive baggerly's test but not by kal's (table ) , two were also identified as significant by kal's using the relaxed criteria (fdr < . ). baggerly's, therefore, identified isgs additional to those described in the above analyses using rna-seq (kal's analysis) and the microarrays (table ) analysis of rna-seq data depends directly on the extant annotated genome sequence. perhaps not surprisingly therefore, rna-seq identified the largest proportion of genes amongst the set of unique irgs that we compiled ( ; %). nevertheless, the microarrays each identified % of the genes ( and ) . congruence was highest, and almost identical, between rna-seq and each microarray ( and ; ± %; all percentages referring to the total of unique irgs). between microarrays it fell to % ( ). for two-way-only comparisons, the distribution of unique genes between the microarrays was symmetrical ( and ; %). between rna-seq and each microarray, unique genes were biased > -fold towards rna-seq: ( %) versus ( %) against the genechip and ( %) versus ( %) against the st array. clearly in simple terms of numbers of irgs identified, rna-seq outperforms the microarrays. this is probably attributable to the historic nature of the array design based on earlier genome assemblies and annotations, with consequent effects on overall coverage (which might disproportionately affect conditionally expressed genes such as those of the innate immune responses). nevertheless, the ability of microarrays to quantify expression of % (about ) of such a large pool of important genes will often prove sufficient for the experimental objectives where other considerations might affect the choice of technology (see below). moving away from actual numbers of genes, it is worth noting that deeper analysis (in the form of validation by alternative approaches) will, by definition, be required to determine which of the genes identified uniquely as irgs by individual technologies are actually irgs. genomic loci for each of the predicted isgs were visually inspected using genomic workbench's genome browser, displaying tracks showing: gene, transcript, exon and orf annotations for the current chicken genome build as well as read-mapping for control and ifn-treated reads [ ] . on occasions, such inspection revealed the presence of non-annotated, inducibly-transcribed regions, representing exons, whole genes or even gene families. examples include those previously described at the chicken ifitm locus [ ; data not shown], at the herc locus (described below) or downstream of ccl (loc ; "c-c motif chemokine -like"; figure ). systematic analysis of these isgs is outside the scope of this manuscript but the data deposited from this study (european nucleotide archive (ena) study number prjeb [ ] ) will facilitate ongoing study and improved annotation. in some cases, although not currently annotated on the ensembl chicken genome, the genes have ids in ncbi and were identified as isgs by one of the microarrays. examples of these include loc , loc ("guanylate-binding protein -like") and loc ("hect domain and rld -like", a member of the herc family, discussed below). about % of the reads from cefs did not map to the current chicken genome. the unmapped reads combined from the control and ifn-treated samples were assembled into contigs using the de novo assembly function of genomic workbench. the rna-seq function of genomic workbench was then used to quantitate expression of the contigs in control and ifn-treated samples. one of the most highly-expressed contigs was one which, when analysed by blast, proved to represent a homologue of stat , which is missing from the current ensembl annotated reference chicken genome in (b) pyurf shows -fold suppression by ifn but the sequence surrounding pyurf shows -fold induction from the right-hand end of the unannotated, antisense loc and considerably higher upregulation from the left-hand end (due to its lower uninduced levels), consistent with these representing homologues of ifn-inducible human genes herc and herc . assembly (galgal ; release ), though at ncbi it has recently been placed as a refseq gene on chromosome in the new assembly galgal (an annotated form of which has not yet been released and is currently not scheduled for release). the de novo assembled contig sequence was used to derive primers for rt-pcr; characterisation of chicken stat will be reported elsewhere. the data on differential expression showed an overwhelming over-representation of genes up-regulated by ifn. for each of the technologies, only one gene was detected as down-regulated. corresponding geneids were pyurf (pigy upstream reading frame; ensgalg ) for rna-seq and pigy (phosphatidylinositol glycan anchor biosynthesis, class y; ncbi geneid: ) for the st array. the down-regulated k genechip probe (gga. . .s _at), though not mapped to a known gene at the time of initial processing, according to the affymetrix netaffx ™ analysis center [ ] is now also assigned as pyurf. in humans, pigy and pyurf represent different open reading frames on the same spliced transcript of a gene on hs chromosome located downstream of herc then herc . the pyurf/pigy gene is overlapped on the opposite strand by herc , which extends downstream to be followed by fam a. similarly, the chicken pigy (ncbi) and pyurf (ensembl) genes map to a locus lying upstream of herc then fam a on gg chromosome (see figure ) , with herc-like loc ("hect domain and rld -like") starting upstream but spanning and extending downstream of the chicken pyurf. our rna-seq data ( figure ) indicate that this locus is poorly annotated and demonstrates complex regulation of the component genes by ifn. thus, although the pigy/ pyurf transcript is down-regulated by ifn, as recorded by all three technologies, it appears to be closely flanked upstream and downstream by still unannotated multiple exons that are clearly strongly induced by ifn (figure ). sequences within these upstream and downstream regions (which are represented by the single ncbi refseq (gal-gal ) gene, loc , but appear as though they may represent two separate genes, figure ) bear homology with genes of the herc family, consistent with the fact that herc neighbours the human pigy/pyurf gene and that herc neighbours the chicken pigy/pyurf gene. the chicken herc gene shows no evidence of induction by ifn. description of the interferon-inducibility of the chisgs serves as the first step in understanding the regulation of their expression and their role in anti-viral (and potentially broader anti-microbial) activities. there is considerable current interest in the antiviral responses of particular cell types, particularly those of the lymphoid, myeloid and dendritic lineages. however, the definition of a wide variety of these cell types is not so advanced in avian species so we felt it best to produce baseline data for readily available, primary cells, namely chick embryo fibroblasts (cef) as they are highly responsive to ifn. they also remain important for commercial production of vaccine viruses (including human vaccines) as well as for the routine isolation and diagnosis of avian pathogens. given the currently incomplete nature of the chicken genome assembly (even at galgal ) and of its annotation (as currently available for galgal and even as awaited for galgal ) it is inevitable that updates will continue to be released but the primary data reported here, and publicly-available, for microarrays and rna-seq, can always be applied to updated microarray assignments as well as to subsequent genome assemblies and annotations. all things being equal, rna-seq would seem to be the method of choice for transcriptomic analysis of chicken ifn responses, particularly given its ability to produce high-resolution quantitative and qualitative data. moreover the data are readily portable and can be easily mined by others with different research focus. they can also be applied immediately to newly released genome assemblies and annotations (whether global or local), whereas microarray analysis must await the generation of annotation updates for each technology. however, although the cost of sequencing has fallen, and will probably continue to do so, there remain considerable overheads to handling large data sets from extensive, complicated experiments, especially in terms of computing and data storage capacity, as well as speed of processing and archiving. for such experiments, microarrays continue to offer a tractable approach, capable of quickly quantifying and comparing the expression of the central core of irgs producing relatively compact data for rapid analysis and easy archiving. induction of innate responses with pamps will trigger different or broader ranges of responses by virtue of the fact that they will trigger other or more pathways than just the ifn-pathway. for instance we (giotis et al. unpublished) and others [ ] have begun to analyse the responses induced by the dsrna analogue poly[i:c]. regulation of isg expression might affect the innate responses observed in different cell lines or tissues so it will be important to understand the mechanisms involved. additionally, we have observed suppression of isg induction in the spontaneously immortalized chicken fibroblast cell line, df- [ ] , due to their enhanced basal expression of the regulatory isg, socs (giotis et al., unpublished) . identification of the isgs means that their promoters, enhancers and other regulatory elements can be systematically analysed to help understand the complex kinetics of expression of their expression (figure ). several studies have investigated changes in host gene expression in response to infection in vivo or in culture with particular avian viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although many of these genes will represent innate (and potentially antiviral) host responses, the majority will be involved in the metabolic, cell cycle and ultrastructural changes that the virus has to induce to facilitate replication. furthermore, it is not unusual for viruses to modulate the expression of signalling molecules key to the antiviral responses or of antiviral effectors themselves. for instance, we have shown that even an attenuated strain of fowlpox virus blocks induction of ifn-β (chifn ) and is highly resistant to the antiviral activity induced by ifn [ , ] . the results of existing and future studies of infection in vivo or in culture with particular avian viruses can now be compared with data presented here for isg induction by ifn to look for evidence of modulation of isg expression by viruses, whether that be modulation of individual isgs, subsets [ ] or the complete set. for instance, fowlpox virus blocks essentially all isg expression but a mutant defective in the fpv ankyrin repeat/f-box protein identified by laidlaw et al. [ ] induces modest levels of a subset of the isgs (giotis et al., unpublished) . such analyses can be extended to important avian zoonotic viruses and pathogens with huge impact on the global poultry industry. although this study relates to type i ifn, extensive comparison with the effects of type iii ifn could now be conducted, extending on the qrt-pcr comparison made by masuda et al., who looked at induction of mx and oas by ifn-β, ifn-γ and ifn-λ [ ] . interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures inborn errors of anti-viral interferon immunity in humans interferon-stimulated genes: a complex web of host defenses pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses functional classification of interferon-stimulated genes identified using microarrays a diverse range of gene products are effectors of the type i interferon antiviral response virus interference. i. the interferon chicken interferon gene: cloning, expression, and analysis characterization of the chicken pkr: polymorphism of the gene and antiviral activity against vesicular stomatitis virus international chicken genome sequencing c ( ) sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution evidence of the adaptive evolution of immune genes in chicken functional analysis of chicken irf in response to dsrna analog poly(i:c) by integrating overexpression and knockdown defense genes missing from the flight division innate sensing of viruses by pattern recognition receptors in birds id: : transcriptomic analysis of the chicken interferome genetic screen of a library of chimeric poxviruses identifies an ankyrin repeat protein involved in resistance to the avian type i interferon response species difference in anp a underlies influenza a virus polymerase host restriction dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources differential expression in sage: accounting for normal between-library variation involvement of the irf- transcription factor in antiviral responses to interferons constitutive expression of an isgf /irf transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection ifn regulatory factor- bypasses ifn-mediated antiviral effects through viperin gene induction interferome v . : an updated database of annotated interferon-regulated genes chicken interferon-inducible transmembrane protein restricts influenza viruses and lyssaviruses in vitro the df- chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses transcriptomic profiling of virus-host cell interactions following chicken anaemia virus (cav) infection in an in vivo model molecular responses to the influenza a virus in chicken trachea-derived cells early host responses to avian influenza a virus are prolonged and enhanced at transcriptional level depending on maturation of the immune system transcriptional analysis of host responses to marek's disease viral infection analysis of the early immune response to infection by infectious bursal disease virus in chickens differing in their resistance to the disease a comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferon-induced transmembrane proteins in viral resistance analysis of the crow lung transcriptome in response to infection with highly pathogenic h n avian influenza virus integrated analysis of microrna expression and mrna transcriptome in lungs of avian influenza virus infected broilers differential expression of micrornas in marek's disease virus-transformed t-lymphoma cell lines genetic screen of a mutant poxvirus library identifies an ankyrin repeat protein involved in blocking induction of avian type i interferon biological effects of chicken type iii interferon on expression of interferon-stimulated genes in chickens: comparison with type i and type ii interferons we are grateful for the skilled support of laurence game, nathalie lambie and adam giess of the medical research council's (mrc) clinical sciences centre's (csc) genomics facility in conducting microarray analysis and illumina sequencing. we gratefully acknowledge sarah butcher and geraint barton of the bioinformatics support service at imperial college london for their advice. the datasets supporting the conclusions of this article are available from the following repositories: european bioinformatics institute (ebi) arrayexpress accession numbers e-mtab- (for the k genechip; [ ] ) and e-mtab- (for the st array; [ ] ). european nucleotide archive (ena) study number prjeb (for illumina rna-seq; [ ] ). additional file . table of additional file . detailed information on chisgs identified by rna-seq, and microarray technologies ( ). technologies identifying significant irgs are listed as " " rna-seq (using kal's z test); " " affymetrix k genechip chicken genome array and " " chicken gene . st array' . chisgs significant by one or both microarrays and rna-seq using kal's z test under relaxed criteria (fc > . or fdr < . ) are indicated by "( )". "+" after the technology identifier indicates that ifn-induced rna-seq read density was observed at the location of the unannotated gene. ( ) interferome status [ ] . ( ) human homologue data (hugo) [ ] . ( ) mouse orthologue data (mgi) [ ] . ifn: interferon; irgs: ifn-regulated genes; isgs: ifn-stimulated genes; cef: chicken embryo fibroblasts; rchifn : recombinant chicken ifn-α; rin: rna integrity number; qrt-pcr: quantitative real-time pcr; gapdh: glyceraldehyde -phosphate dehydrogenase; fc: fold change; fdr: false discovery rate. the authors declare that they have no competing interests. esg and rcc design of the study, data acquisition and analysis, drafting the manuscript. ngs data compilation and analysis, drafting the manuscript. cdt design, production, curation and maintenance of chisg browser website. sg design of the study, critically reviewing the manuscript. mas design of the study, data analysis, finalizing manuscript. all authors read and approved the final manuscript. key: cord- -xg foch authors: tanaka, yoshikazu; sato, yuka; osawa, shuichi; inoue, mai; tanaka, satoka; sasaki, takashi title: suppression of feline coronavirus replication in vitro by cyclosporin a date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: xg foch the feline infectious peritonitis virus (fipv) is a member of the feline coronavirus family that causes fip, which is incurable and fatal in cats. cyclosporin a (csa), an immunosuppressive agent that targets the nuclear factor pathway of activated t-cells (nf-at) to bind cellular cyclophilins (cyp), dose-dependently inhibited fipv replication in vitro. fk (an immunosuppressor of the pathway that binds cellular fk -binding protein (fkbp) but not cyp) did not affect fipv replication. neither cell growth nor viability changed in the presence of either csa or fk , and these factors did not affect the nf-at pathway in fcwf- cells. therefore, csa does not seem to exert inhibitory effects via the nf-at pathway. in conclusion, csa inhibited fipv replication in vitro and further studies are needed to verify the practical value of csa as an anti-fipv treatment in vivo. coronaviruses are single-stranded rna viruses that generally cause respiratory or intestinal infections such as severe acute respiratory syndrome (sars) in humans and transmissible gastroenteritis (tge) in pigs. feline coronaviruses (fcov) have been classified into two biotypes, comprising the ubiquitous feline enteric coronavirus (fecv) and infectious peritonitis virus (fipv). the widely accepted theory in vitro is that fipv arises by mutation of parental fecv in the gastrointestinal tract of infected cats, then systemically spreads and causes fip that is fatal in cats [ , ] . although the mutation sites are not fully understood, some accessory genes are candidates for the site of the critical mutations responsible for fip [ , ] . approximately % of all purebred cats are infected with feline coronavirus and among these, - % develop the classical symptoms of effusive/ wet fip, the non-effusive/dry form of fip, or a combination of the two [ ] . many strategies have been developed to cure fip. interferon ω inhibits fipv in vitro but is ineffective in vivo [ ] . various other immunosuppressants, such as glucocorticoids and cyclophosphamide, have been studied, but although these drugs prolong life, the outcome of fipv infection remains fatal [ ] . thus, an effective vaccine and therapeutic medicine against fipv are still needed. investigators have reported that the immunosuppressant cyclosporin a (csa) can suppress the genomic replication and transcription of several viruses [ ] [ ] [ ] [ ] . cyclophilins (cyp) are cellular factors with high affinity for csa [ ] and comprise a family of cellular peptidyl-prolyl cis-trans isomerases (ppiases) that catalyze the cis-trans interconversion of the amino-terminal of peptide bonds to proline residues, thus facilitating changes in protein conformation as a chaperone protein [ ] . cyclosporin a blocks both the enzymatic activities of cyp that lead to the calcineurin (cn)-nf-at and p-glycoprotein pathways. the capsid protein of human immunodeficiency virus type (hiv- ) possesses a cyclophilin a (cypa) binding site for incorporation into the virion [ , ] . a csa-induced reduction in cypa inhibits transport of the reverse-transcribed viral genome to the nucleus [ ] . cyclophilin b (cypb) is another target of csa that promotes hepatitis c virus (hcv) replication by regulating the rna-binding ability of the hcv ns b protein. in addition, cypa facilitates mouse cytomegalovirus (mcmv) replication and cypb is required for the infectious entry of human papillomavirus type . here, we show that csa inhibits intracellular replication of the fipv genome and viral protein expression in vitro independently of the nf-at pathway. felis catus whole fetus- (fcwf- ; american type culture collection, va, usa) cells were maintained in dulbecco's modified eagle's medium (d-mem, sigma-aldrich, tokyo, japan) supplemented with % fetal bovine serum (jrh, nissui, tokyo, japan). we propagated fipv ( - strain; a gift from dr tsutomu hodatsu, kitasato university, japan) in fcwf- cells and then purified them by linear sucrose gradient ultracentrifugation. we inoculated fcwf- cells with fipv - at a multiplicity of infection (moi) of plaque-forming unit (pfu) per cell to study their effects on fipv infection. after adsorption for h at °c, the medium containing the virus was removed, and the cells were rinsed three times with phosphate-buffered saline [pbs (−)] and incubated with or without various concentrations of csa (sigma-aldrich), cyclosporin h (csh; cosmobio, tokyo, japan) and fk (sigma-aldrich) for h. the cells were then processed for photography. the cells were adsorbed with fipv, rinsed three times with pbs (−), overlaid with dmem containing % fetal bovine serum and % agarose s (nippon gene, toyama, japan). after a -h incubation, the cells were stained with giemsa solution and the plaques were counted. we isolated the nucleocapsid (n) gene of fipv from the plasmid pbrdi (provided by dr peter j. m. rottier, institute of biomembranes, utrecht university, the netherlands) containing the fipv - genome, using the polymerase chain reaction (pcr) with the primers '-acaaggac gacgacgacaaggccacacagggacaacgcg- ' and '-ccggaattcttagttcgtaacctcatcaa- ' for the first amplification. the products of the first amplification were purified by electrophoresis and gel extraction, and then a second pcr proceeded with the purified products and primers containing a flag-tagged sequence: ( '-gccaccatggactacaaggacgacgacgacaag- ' and '-ccggaattcttagttcgtaacctcatcaa- '). the second pcr products were cut with nco i and eco ri and subcloned into the sites between nco i and eco ri of the ptri-ex . vector (novagen, takara bio, kyoto, japan) to express flag-tagged n protein. the fcwf- cells were infected at an moi of pfu per cell and then incubated with or without csa, csh or fk . the medium was removed at h post-infection, and rnaiso-plus (takara bio) was added to the cells for rna preparation according to the manufacturer's protocol. total rna ( ng) was reverse-transcribed using the pri-mescript rt-pcr kit (perfect real time; takara bio). viral cdna were quantified by real-time pcr using the forward and reverse primers to the fipv-n gene ( '-tggccacacagggacaac- ') and ( '-agaac gaccacgtcttttggaa- '), and the taqman probe (fam-gttgca gcacagccagcataaacaa-bhq- ). reaction mixtures were prepared according to the manufacturer's protocol using premix extaq (takara bio), and sequences were amplified using a sequence detection system (applied biosystems, tokyo, japan) under the following cycling conditions: initial denaturation at °c for s and cycles at °c for s and °c for s each. complementary dna to the fipv-n gene was cloned into the ptri-ex . vector, which was serially diluted to provide standards for fipv gene quantification. the viral rna copy number was normalized using the feline β- -microglobulin (β m) gene (genbank; nm_ ). the β m gene derived from fcwf- cells was cloned by pcr amplification using the following primers: fβ m-f '-ggcgcgttttgtggtcttggtc- ' and fβ m-r '-cacttaacgaccttgggctc- '. the amplified pcr products were subcloned into ptac- plasmids (biodynamics laboratory inc. tokyo, japan) to provide standards for the β m gene. we then quantified the feline β m gene by real-time pcr using the forward ( '-cgcgttttgtggtcttggtcttggt- ') and reverse ( '-aaacctgaacctttggagaatgc- ') primers for the β m gene and detected the gene using the taqman probe, tamra-cggactgctctatctgtc ccacctgga-bhq- . luciferase activities were quantified using pgl . [luc p/nfat-re/hygro] (promega, tokyo, japan), prl-sv vectors for the nf-at response assay and the interferon stimulation response was determined using the plasmid pisre-tk hrluc (f) provided by riken bioresource center (tsukuba, japan) and the pgl promoter (promega). both reporter assays proceeded using the dual-luciferase reporter assay system (promega). briefly, the two reporter plasmids were co-transfected into fcwf- cells with or without csa or fk for each assay. recombinant feline ifnα (pbl biomedical laboratories, nj, usa) was added at a concentration of units/ml to the culture medium to evaluate the response to interferon (ifn). total cell lysates were prepared with reporter lysis buffer provided with the dual-luciferase reporter assay system at h before the assay. luciferase activities were quantified in triplicate assays using a lumat lb (berthold technologies, tokyo, japan). the cell membranes were disrupted with cell lysis buffer [ mm tris-hcl, ph . , mm ethylenediamine tetraacetic acid (edta), % np- , . m nacl] including complete mini (roche diagnostics, tokyo, japan) at h after infection. the cell lysates were resolved by electrophoresis on - % bis-tris gels (invitrogen, ca, usa) and western blotted onto immobilon-p membranes (millipore, tokyo, japan). non-specific protein binding was blocked with % non-fat dry milk for h and then the membranes were incubated with anti-feline coronavirus nucleocapsid (n) antibody (fipv - ; mybiosource, ca, usa) or anti-β-actin (sigma-aldrich) for h. antigen signals were visualized by reacting proteins on the membranes with horseradish peroxidaseconjugated anti-mouse igg antibody (promega) followed by an enhanced chemiluminescence substrate (supersignal west femto maximum sensitivity substrate; thermo scientific, tokyo, japan) according to the manufacturer's protocol. we assayed wst- to evaluate cytotoxicity using the cell counting kit- (dojin chemical inc., wako, japan) according to the manufacturer's directions. statistical significance was determined using the student's t test. for all data analyzed, a significance threshold of p < . was assumed. the values are expressed in some figures as means ± standard deviation (sd). we initially assessed the effects of csa on fipv rna replication using cytotoxicity assays. cyclosporin a at concentrations of - . μm (cytotoxic dose , . ± . μm) did not affect fcwf- cell viability ( figure a ) and dose-dependently reduced the numbers of fipv plaques ( figure b) , whereas μm csa was slightly cytotoxic. cyclosporin a blocks both the enzymatic activities of cyp that lead to the calcineurin (cn)-nf-at and the p-glycoprotein pathway. we therefore assessed the effect of various concentrations of fk , which also blocks the nf-at pathway, on cell viability to confirm that csa inhibited fipv through this pathway. the cytopathic inhibitory effects did not significantly differ in the presence or absence of . - μm fk ( figure b ) and cell viability was not affected by μm fk (figure a ). quantitative rt-pcr showed that . - μm csa dose-dependently suppressed fipv rna replication, whereas fk did not exert significant inhibitory effects, except at μm fk (approximately % reduction compared to μm fk , p < . ; figure a ). western blotting showed that csa, but not fk dosedependently decreased fipv-n protein ( figure b) . we then examined whether the suppressive effects of csa on fipv replication depend on the inhibitory nf-at pathway or p-glycoprotein pathway by incubating fipv-infected cells with csh, which specifically blocks the p-glycoprotein pathway. the results show that no inhibition occurred (data not shown). to determine whether the action of csa and fk involves activation of interferon-stimulated gene responses in fcwf- cells, the isre-luciferase reporter plasmid, pisre-tk hrluc (f) and pgl promoter plasmid as a normalization-control plasmid were transfected into fcwf- cells and cultured with feline interferon α, csa or fk . the results of the dual-luciferase assay showed that none of these factors significantly affected luciferase activities at h after transfection. these results indicate that fcwf- cells are unresponsive, even to interferon α ( figure a) . consequently, the action of csa on intracellular fipv replication does not involve the activation of interferonstimulated genes on fcwf- cells. moreover, to evaluate the effects of csa and fk on the calcineurin-nf-at pathway in fcwf- cells, the nf-at luciferase reporter plasmid, pgl . [luc p/nfat-re/hygro] and prl-sv as a normalization-control plasmid were transfected into fcwf- cells that had been incubated with csa or fk . neither csa nor fk affected nf-at luciferase activities in fcwf- cells ( figure b ). these findings show that csa does not influence the nf-at pathway in fcwf- cells and that the inhibition of fipv rna replication by csa is independent of the calcineurin nf-at pathway. we discovered that csa inhibits intracellular fipv replication in vitro. the results of qrt-pcr and western blotting showed that viral proliferation was strikingly inhibited at csa concentrations between . and μm. the results of cell viability assays showed that μm csa was slightly cytotoxic. the inhibitory effects at this concentration should therefore be considered in the light of cytotoxicity. in contrast, immunosuppressive fk did not inhibit fipv replication except at μm according to the qrt-pcr findings. however, the inhibitory effects of . - μm fk did not significantly differ on western blots and the number of plaques did not significantly differ within the same concentration range of fk . these findings might be related to the difference of the analytical detection sensitivity between qrt-pcr and western blot assays. cyclosporin a binds cyclophilins, whereas fk binds cellular fkbp. each complex independently inhibits the phosphatase activity of calcineurin that mediates the nf-at pathway, which is critical to the expression of cytokines and their receptors [ , ] . the results of nf-at reporter assays indicate that neither csa nor fk influenced nf-at activities on fcwf- cells under our experimental conditions. thus, the antiviral activity against fipv is not involved in the suppression of gene responses regulated by nf-at but instead is exerted through distinct mechanisms that are independent of fk . pfefferle et al. recently described that sarscoronavirus nsp overexpression increases signaling through the nf-at pathway and enhances the induction of interleukin [ ] . our data were somewhat inconsistent with these findings. the discrepancy might be explained in part by the fact that their experimental system included the addition of -o-acetylphorbol -myristate (pma) and ionomycin to the culture medium. further knockdown studies of the nf-at gene would clarify its role in fipv proliferation. furthermore, ifnα did not stimulate isre-promoter activities in fcwf- cells under these conditions. these data suggest that the action of csa on the intracellular replication of fipv is independent of the ifn pathway. this cell line might not be responsive to ifnα and would thus be useful for isolating feline coronavirus. cyclophilins have ppiase activity [ , [ ] [ ] [ ] , contribute to the maturation of several proteins, and are involved in cell signaling, mitochondrial function (atp synthesis), molecular chaperone activity, rna splicing, stress response, gene expression, cholesterol transport, and the regulation of kinase activity [ ] [ ] [ ] [ ] . surface plasmon resonance technology and bioinformatics tools have found that the sars coronavirus n protein binds to cypa [ ] [ ] [ ] . therefore, the n protein of fipv probably binds to cyp proteins to regulate viral replication. the roles of cyp in virus replication and the inhibitory effect by csa on hcv and several other viruses have been studied [ ] [ ] [ ] [ ] . the proposed mechanisms of the inhibitory effect of csa mainly involve cypa and cypb in virus replication [ , , ] . based on these earlier findings, we believe that interaction between the fipv genome is likely or that viral and cyp proteins play critical roles in viral replication and transcription. further studies are required to resolve which cyp is critical for viral replication and which viral protein is required to form replication complexes with cyp and/or other cellular proteins. cats with clinically diagnosed fip have very rarely been cured, although several therapeutic strategies have been attempted. some cats treated with prednisolone and phenylalanine mustard or cyclophosphamide have gone into remission [ ] . various immunosuppressants, such as glucocorticoids and cyclophosphamide, may prolong life but do not alter the fatal outcome [ ] . further investigations using ppiase dominant-negative assays and rna interference methods are warranted to clarify the role of csa against ppiase in fipv replication in vitro. in addition, whether csa could be useful as fip treatment in vivo remains to be determined acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis a review of feline infectious peritonitis virus infection: - cellular composition and interferongamma expression of the local inflammatory response in feline infectious peritonitis (fip) effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis treatment of cats with feline infectious peritonitis sdz pri , an orally bioavailable human immunodeficiency virus type proteinase inhibitor containing the -aminobenzylstatine moiety mode of action of sdz nim , a nonimmunosuppressive cyclosporin a analog with activity against human immunodeficiency virus (hiv) type : interference with hiv protein-cyclophilin a interactions requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype cyclophilin b is a functional regulator of hepatitis c virus rna polymerase cyclophilin: a specific cytosolic binding protein for cyclosporin a active site mutants of human cyclophilin a separate peptidyl-prolyl isomerase activity from cyclosporin a binding and calcineurin inhibition sdz - , a new picornavirus capsid-binding agent with potent antiviral activity in vivo efficacy of sdz - , a new picornavirus capsid-binding agent inhibition of human immunodeficiency virus type replication in human cells by debio- , a novel cyclophilin binding agent handschumacher re: calmodulin, cyclophilin, and cyclosporin a the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors cyclosporin a inhibits an initial step in folding of transferrin within the endoplasmic reticulum cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins a and b overexpression, purification, and characterization of yeast cyclophilins a and b cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins multiple cyclophilins involved in different cellular pathways mediate hcv replication immunophilins: switched on protein binding domains? peptidyl-prolyl cis-trans isomerase is the cyclosporin a-binding protein cyclophilin cyclophilin a-induced alterations of human immunodeficiency virus type ca protein in vitro cyclophilin a is required for an early step in the life cycle of human immunodeficiency virus type before the initiation of reverse transcription cyclophilin a modulates processing of human immunodeficiency virus type p gag: mechanism for antiviral effects of cyclosporin a purified matrix protein of vesicular stomatitis virus blocks viral transcription in vitro nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a suppression of feline coronavirus replication in vitro by cyclosporin a this study was supported by a grant-in-aid for scientific research (c) from the japan society for the promotion of science and the strategic research base the authors declare that they have no competing interests. key: cord- -h p kmss authors: follet, jérôme; guyot, karine; leruste, hélène; follet-dumoulin, anne; hammouma-ghelboun, ourida; certad, gabriela; dei-cas, eduardo; halama, patrice title: cryptosporidium infection in a veal calf cohort in france: molecular characterization of species in a longitudinal study date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: h p kmss feces from animals were collected on farms in the region of brittany, france. each sample was directly collected from the rectum of the animal and identified with the ear tag number. animals were sampled three times, at , and weeks of age. after dna extraction from stool samples, nested pcr was performed to amplify partial s-rdna and kda glycoprotein genes of cryptosporidium. the parasite was detected on all farms. one hundred out of calves ( . %) were found to be parasitized by cryptosporidium. amplified fragments were sequenced for cryptosporidium species identification and revealed the presence of c. parvum ( . %), c. ryanae ( . %), and c. bovis ( %). one animal was infected with cryptosporidium ubiquitum. the prevalence of these species was related to the age of the animal. c. parvum caused . % of cryptosporidium infections in -week-old calves but only . % in -week-old animals. the analysis of the results showed that animals could be infected successively by c. parvum, c. ryanae, and c. bovis for the study period. c. parvum gp genotyping identifies iia subtypes of which . % were represented by iiaa g r . this work confirms previous studies in other countries showing that zoonotic c. parvum is the dominant species seen in young calves. cryptosporidium is a genus of protozoan parasites infecting a wide range of hosts [ ] . all groups of vertebrates are susceptible to cryptosporidium infection worldwide. this parasite is the etiological agent of cryptosporidiosis, which is mainly characterized by diarrhea in humans and livestock. massive outbreaks of enteritis in people such as in milwaukee, wisconsin (usa) have increased public awareness of this parasite [ ] . in humans, the prevalence and severity of infection increase in immunodeficient individuals such as aids patients. in immunocompetent patients, the disease is self-limited [ ] . no drug therapy is yet available and the high resistance of oocysts to environmental conditions and chemical treatment make cryptosporidiosis difficult to control [ ] . cattle have been considered to be a primary reservoir for cryptosporidium oocysts for zoonotic c. parvum [ ] . these animals could be a risk factor via environmental contamination from their manure being spread on farmland or their grazing on watersheds [ ] . on farms, transmission of cryptosporidium spp. can result from ingestion of contaminated food or water, by direct transmission from host to host, or through insect vectors [ ] . in cattle, infection by cryptosporidium spp. was first reported in [ ] . since vaccines have become commercially available against escherichia coli k , rotavirus, and coronavirus, cryptosporidium has emerged as the main neonatal diarrheic agent in calves [ ] . in farm animals, the economic impact is related to morbidity, mortality and growth retardation [ ] . among the species previously described (if the three fish species are accepted without complete genetic characterization) [ , [ ] [ ] [ ] , c. parvum, c. bovis, c. ryanae and c. andersoni usually infect cattle. c. parvum has zoonotic potential and is a frequent cause of human cryptosporidiosis [ ] . c. bovis and c. ryanae have not been found in humans and there is only one description of c. andersoni in a patient [ ] . recent reports have described an age-related distribution of these aforementioned species in dairy cattle on the east coast of the united states [ ] [ ] [ ] , india, china, georgia [ ] , malaysia [ ] , and denmark [ ] . the most prevalent species were c. parvum in preweaned calves, c. ryanae and c. bovis in postweaned calves and c. andersoni in adult cows [ , ] . in france, previous studies on the prevalence of cryptosporidium in cattle were based on microscopic determination [ ] or coproantigen detection using elisa [ ] . these studies on dairy calves reported a within herd prevalence of cryptosporidium without identifying species or the relation to the host's age. the present study was conducted in farms in brittany, france to determine the prevalence of cryptosporidium in veal calves. we used genotyping and subtyping for the molecular study of cryptosporidium isolates. follow-up of the same animal allowed us to determine the outcome of the infection and the age distribution of cryptosporidium species. fifteen fattening units in brittany (france) were included in this work. they belonged to three industrial veal producers representative of integrators in france and did not present any known history of cryptosporidium infection. these farms were located in four administrative regions ( figure ): côtes d'armor (ca -ca ), morbihan (mo ), ile-et-vilaine (iv -iv ), and mayenne (ma -ma ). during the summer and autumn of , all farms were visited three times and fecal samples were taken from animals exhibiting diarrhea at the age of weeks old. calves arrived in fattening units at the age of weeks old and were confined in small groups from four to six animals per pen. because of a concomitant welfare study [ ] , calves had to stay to weeks without any external stress despite the farmer's presence. at the age of weeks old, calves were finally sent to the slaughterhouse. consequently, sampling was done at the ages of weeks, weeks, and weeks (table ). these points of sampling corresponded to the beginning, the middle and the end of the fattening period. fecal samples were collected and shipped by a veterinarian. collectors respected the following criteria: use of a single pair of latex gloves per animal, a single plastic sterile cup per animal, and collection of at least g of feces per sample. feces were collected directly from the rectum of each animal and stored at °c in potassium dichromate ( . % wt/vol) until processed. cups were capped, labeled with the animal's ear tag number, and accompanied by a form recording the date of sampling, the animal's sex, breed, identification number, and the mean age of the herd. after washing steps in water to eliminate potassium dichromate from the samples, dna was extracted according to the method previously described [ ] without the cetyl trimethylammonium bromide (ctab) and polyvi-nylpyrrolidone (pvp) treatment steps. an s rna gene fragment was amplified by nested pcr according to xiao et al. [ ] . the partial gp gene was amplified according to gatei et al., [ ] . pcr products were analyzed on % agarose gel and visualized by ethidium bromide staining. to ensure purity and limit the presence of pcr inhibitors, all pcr-negative samples were reprocessed. samples were treated for oocyst purification by immunomagnetic separation (dynabeads ® anti-cryptosporidium, invitrogen ™, norway) according to the manufacturer's instructions. these samples were finally processed as previously for dna extraction and pcr amplification. cryptosporidium species identification pcr products were purified on an ultracel ym membrane (microcon, millipore, bedford, ma, usa) according to the manufacturer's instructions. dna sequencing reactions were performed using internal primers of the nested pcr with the abi prism big dye terminator cycle sequencing kit (applied biosystem, foster city, ca, usa). capillary electrophoresis was performed by genoscreen (lille, france). sequences were analyzed using blast at ncbi [ ] . the prevalence of cryptosporidium infection on farms from four administrative regions in brittany (france) was studied ( figure ). all cryptosporidium-positive specimens generated the expected ssu-rna products in nested pcr and revealed that no farm was free of cryptosporidium. the molecular analysis of fecal samples revealed that ( . %) were positive for cryptosporidium. as shown in table , the overall prevalence of infected animals was . % ( / ) and ranged from % on a farm in morbihan (mo ) to % on farms in ile-et-vilaine (iv , iv ) and in mayenne (ma ). amongst the specimens sampled from -week-old and -week-old animals, cryptosporidium prevalence was . % and . %, respectively (range, %- . %). in -week-old calves, the prevalence decreased to . % (range, %- . %). the prevalence of infection decreased as the age of the calves increased. for species identification, the positive nested pcr products were sequenced. sequence analysis from readable electrophoregrams revealed the presence of c. parvum, c. bovis, and c. ryanae. one additional cryptosporidium genotype showing % identity with cryptosporidium ubiquitum (eu ) (previously identified as * a calf is considered to be positive if at least one out of the three samples is positive. **the number of animals is because one calf died between the age of and weeks. cryptosporidium cervine genotype [ ] ) was detected in one calf. this sequence was deposited in genbank under the accession number gu . sixty ( . %) samples were identified as c. parvum as follows: forty-six sequences had % identity with the genbank af nucleotide sequence, had % identity with the af nucleotide sequence and three had % identity compared to both references. these sequences were deposited in genbank under the accession numbers gu to gu . for the other positive specimens, ( . %) were identified as c. ryanae (previously described as cryptosporidium deer-like genotype). thirtyone of these had % identity with the ay sequence [ ] and eight were % identical to this reference. these nucleotide sequences were deposited in gen-bank under the accession numbers gu to gu . for the last positive samples, ( %) had an identical nucleotide sequence with c. bovis (genbank accession number, ay ) formerly known as the cryptosporidium bovine b genotype. within these sequences, had % identity to the reference deposited in genbank, three sequences had % identity. these last sequences were deposited in genbank under the accession numbers gu to gu . prevalence of c. parvum, c. ryanae, and c. bovis in relation to calf age the distribution of cryptosporidium species identified in animals at the age of , , and weeks is shown in figure . the prevalence of each species changed with the age of the calves. c. parvum prevalence was . % in the -week-old calves and decreased to . % in -week-old animals. this species was not identified in -week-old calves. c. ryanae and c. bovis were identified in -weekold calves in . % and . % of the specimens, respectively. the prevalence of these species in -week-old animals increased to . % and . %, respectively. this prevalence evolved to % and % in -week-old animals. the presence of one, two, or three species of cryptosporidium was determined in each animal (n = ) for which the sequences were readable in all positive samples. three calves positive for c. parvum at the age of weeks were excluded because cryptosporidium species could not be identified in all of the following samples collected in these animals. as shown in in the time lapse of this study, % of the animals ( / ) were found to excrete two different species of cryptosporidium successively. indeed, . % ( / ) produced c. parvum and c. ryanae, . % ( / ) excreted c. parvum and c. bovis, and . % ( / ) excreted c. ryanae and c. bovis. finally, . % ( / ) of the animals studied were detected to produce c. parvum, c. ryanae, and c. bovis. the subtyping analysis was performed on c. parvum positive specimens. from targeted samples, could be used for sequence analysis. as shown in table , all alleles identified belong to the iia family. the most common subtype iiaa g r ( % identity with reference strain ab ) was found in out of samples ( . %). six samples ( . %) were typed as subtype iiaa g r ( % identity with reference strain gq ), three samples ( . %) as subtype iiaa g r ( % identity with reference strain dq ) and two samples ( . %) as subtype iiaa g r ( % identity with reference strain dq ). finally one sample ( . %) was subtyped as iiaa g r ( % identity with reference strain dq ) and another one ( . %) as subtype iiaa g r ( % identity with reference strain dq ). discussion calves under month of age are frequently infected with cryptosporidium sp [ ] which results in economic loss [ ] . in france, up to date, the prevalence of cryptosporidium in diarrheic calves has been studied only by elisa and microscopic strategies [ , , ] . no data are available on a molecular basis to study cryptosporidium species in calf herds in that country. the present study based on s rdna and gp gene analysis is the first in france to include molecular characterization to describe the prevalence and the host age related susceptibility to different cryptosporidium species after a follow up of the same animal. our results showed that all fifteen farms were contaminated with cryptosporidium. the parasite prevalence on farms ranged from % to % of the sampled animals. this observation was in accordance with results in michigan (usa) where this parameter ranged from % to % [ ] . the prevalence of . % obtained in this work tended toward the upper end of the scale compared to other investigations done in france which ranged from . % in beef herds [ ] to % in suckling calves [ ] and in other european countries where prevalence ranged from . % to % [ , ] . however, the sampling program did not allow the study of animals under weeks of age. indeed, the animals arrived in these structures at the age of to weeks and farmers did not allow sampling before two complete resting weeks for each animal. therefore, our results could underestimate the real prevalence as huetink et al. showed that the percentage of parasite excreting animal declines after the third week of age [ ] and that the first peak of prevalence is at the age of days [ ] . in our study, the higher prevalence of cryptosporidiosis was observed in calves weeks old ( . %) and the lowest ( . %) in the -week-old animals. this observation shows that prevalence of cryptosporidium infection decreases with increasing age of the cattle in france as in many other countries [ , , [ ] [ ] [ ] [ ] [ ] [ ] . additionally, our data confirmed the presence in france of a host age-related susceptibility to the infection with different cryptosporidium species. c. parvum was predominantly detected in -week-old calves ( . %) compared to c. ryanae or c. bovis detected in . % and . % of the positive samples respectively. it is noteworthy that these results are very similar to data obtained in ireland on calves under days of age with %, . %, and . % of prevalence of the same species, respectively [ ] and in the uk on animals over weeks old with % c. parvum, % c. bovis, and % c. ryanae [ ] . in contrast to previous studies [ , ] , c. ryanae and c. bovis were found with similar prevalence predominantly in week and week old calves. this association between the age of the cattle and the cryptosporidium species identification has been supported by several studies [ , , , , ] but different reports suggest that cryptosporidium species repartition regarding the age of the host could be due to a technical artifact. despite the fact that the methodological strategy based on pcr using genus specific primers and partial direct sequencing of the s rdna is commonly used to identify cryptosporidium species [ ] , this molecular tool is limited in the case of mixed infections. feng et al., [ ] suggested that the important shedding of c. parvum in preweaned calves had probably masked the concurrent infection of these animals by c. bovis or c. ryanae. furthermore, previous reports suggested that a dominant cryptosporidium species in a sample can be preferentially amplified by pcr [ , ] . it is noteworthy that this situation of mixed cryptosporidium species infection in farm animals would be more prevalent than originally believed [ ] [ ] [ ] . mixed cryptosporidium species could also explain sequencing difficulties encountered in this work. the simultaneous presence of several species in the same sample could lead to amplification and sequencing of different genetic fragments leading to unreadable superimposition of electrophoregrams. consequently, in our work based on the utilization of cryptosporidium generic primers, the amplification of a single fragment with a single sequence is not conclusive evidence that the sample contains only a single species. however, based on our results, it is possible to confirm the predominance of different species of cryptosporidium by group of age among the calves. particularly, our data showed that animals can be sequentially infected with c. parvum, c. ryanae and c. bovis as well as c. parvum, c. bovis and c. ryanae. this observation provides evidence that a previous infection with c. parvum did not protect calves against an infection with other cryptosporidium species. fayer et al. suggested that the peak of cryptosporidiosis prevalence in young calves could reflect the immaturity of the immune status [ ] . it was also suggested that the low excretion of c. parvum oocysts in older calves might be related to the development of immunity that also protected the animal against a secondary challenge [ ] . it has been reported that immunity arises in the first two weeks after infection [ ] . interestingly, fayer et al. [ ] described that calves previously challenged with c. parvum were able to excrete oocysts after a second challenge with c. bovis but not with c. parvum. the authors concluded that immunity to c. parvum was not extended to c. bovis. consistently, in our study, the presence in the same animal during sequential sampling of c. parvum, c. bovis and c. ryanae suggests that immunity against c. parvum and against c. bovis did not extend to c. ryanae. furthermore, the observation that one animal excreted sequentially c. parvum, c. ryanae and c. bovis suggests that immunity against c. ryanae did not extend to c. bovis as well. finally, the risk to human health posed by cryptosporidium infected cattle in france was assessed. the detection of c. ubiquitum (a rare infectious agent detected in humans [ ] ), c. ryanae and c. bovis (which are mainly specific for cattle) led to consider that the -week-old calves are not likely a public health concern. however, the major detection of c. parvum, a prevalent zoonotic species, in -week-old calves was in agreement with the report of atwill et al., who considered that the contribution of cattle to human cryptosporidiosis is limited to calves under months of age [ ] . to determine c. parvum subtypes, the sequence analysis of a fragment of the gp gene was done. our results show that in the region of brittany, all identified c. parvum gp subtypes belonged to the iia family which was previously found in both animals and humans [ ] . particularly, human infections with the iia subtype are commonly seen in areas with intensive animal production [ ] . among the gp subtypes formerly described in cattle [ ] , only six were identified in this work, being iiaa g r the most commonly found. this subtype has been widely reported in calves and humans in different countries such as in portugal [ ] , slovenia [ ] and the netherlands [ ] . this observation confirms previous works and suggests a zoonotic transmission of the parasite also in this region. it is noteworthy that the three predominant subtypes (iiaa g r , iiaa g r , and iiaa g r ) found in this work were also described in cattle with an equivalent distribution in the netherlands [ ] and england [ ] . thus, the subtype iiaa g r was found in . % of the samples in this work, . % in the netherlands and . % in england. the iiaa g r was identified in . % of the samples in this report, . % in the netherlands and . % in england. the iiaa g r determined in . % of our samples, was characterized in . % in the netherlands and . % in england. it is remarkable that subtypes, iiaa g r , iiaa g r and iiaa g r were equivalently underrepresented in these three countries. this observation could suggest that the proportion of a gp subtype would not be randomly represented in a population. finally, the zoonotic transmission assessment of c. parvum in france would require a comparative investigation of variable genetic loci both in human and animal samples. this is the first report on the molecular identification of cryptosporidium species or genotypes in veal calves in france. according to data reported previously in many countries, a sequential distribution of species is observed in cattle according to age. c. parvum was mainly observed in the youngest calves, while c. ryanae and c. bovis became predominant in stool specimens collected in older animals. in some cases, several cryptosporidium species were successively detected in the same calf, suggesting that the immune defense against c. parvum is not efficient against c. ryanae or c. bovis. finally, the major identification of the iiaa g r subtype in france suggests that -week old calves could be a reservoir for zoonotic parasites transmissible to humans. fayer r: taxonomy and species delimitation in cryptosporidium a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply the cell biology of cryptosporidium infection advances in the epidemiology, diagnosis and 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classification of cryptosporidium isolates from humans and calves in slovenia molecular epidemiology of cryptosporidium in humans and cattle in the netherlands submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was supported by the catholic university of lille through the "projet grande campagne ensemble innovons" genotyping program. we would like to thank the veal unit managers who participated in this study. authors' contributions jf and kg participated in the conception and design of the study, carried out the experiments and drafted the manuscript. hl designed the sampling strategy and collected samples on farms. jf, kg and afd designed the protocol for molecular assay and participated in the analysis result. ohg carried out molecular assays. edc, gc and ph participated in the coordination of the study and helped draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -jiauk fq authors: risalde, maría a; molina, verónica; sánchez-cordón, pedro j; romero-palomo, fernando; pedrera, miriam; garfia, bartolomé; gómez-villamandos, josé c title: pathogenic mechanisms implicated in the intravascular coagulation in the lungs of bvdv-infected calves challenged with bhv- date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: jiauk fq resistance to respiratory disease in cattle requires host defense mechanisms that protect against pathogens which have evolved sophisticated strategies to evade them, including an altered function of pulmonary macrophages (mΦs) or the induction of inflammatory responses that cause lung injury and sepsis. the aim of this study was to clarify the mechanisms responsible for vascular changes occurring in the lungs of calves infected with bovine viral diarrhea virus (bvdv) and challenged later with bovine herpesvirus type (bhv- ), evaluating the role of mΦs in the development of pathological lesions in this organ. for this purpose, pulmonary lesions were compared between co-infected calves and healthy animals inoculated only with bhv- through immunohistochemical (mac , tnfα, il- α, inos, cox- and factor-viii) and ultrastructural studies. both groups of calves presented important vascular alterations produced by fibrin microthrombi and platelet aggregations within the blood vessels. these findings were earlier and more severe in the co-infected group, indicating that the concomitance of bvdv and bhv- in the lungs disrupts the pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment modulated by inflammatory mediators released by pulmonary mΦs. in this regard, the co-infected calves, in spite of presenting a greater number of imΦs than single-infected group, show a significant decrease in inos expression coinciding with the presence of more coagulation lesions. moreover, animals pre-inoculated with bvdv displayed an alteration in the response of pro-inflammatory cytokines (tnfα and il- ), which play a key role in activating the immune response, as well as in the local cell-mediated response. the bovine respiratory disease complex (brdc) is an important problem for the cattle industry, often resulting in severe economic losses [ , ] . this fatal bovine respiratory infection is a multi-factorial disease associated with a primary viral infection followed by a secondary bacterial infection. it is frequently characterized by concurrent infections of several pathogens. the etiologic agents related to feedlot pneumonias include bovine viral diarrhea virus and (bvdv- and bvdv- ), bovine herpesvirus type (bhv- ), bovine parainfluenza- virus, bovine respiratory syncytial virus, bovine adenovirus, bovine coronavirus, mannheimia haemolytica, pasteurella multocida, histophilus somni and mycoplasma spp. [ ] [ ] [ ] [ ] . resistance to respiratory disease in cattle requires host defense mechanisms that protect against viral and bacterial pathogens that have also evolved sophisticated strategies to evade the host immune responses, including among others an altered pulmonary macrophages (mΦs) function or the induction of profound inflammatory responses that cause lung injury and sepsis [ ] . the inflammatory process is a protective mechanism that occurs in response to trauma, infection or tissue injury [ , ] . increased blood supply, enhanced vascular permeability and migration of immune cells occur at damaged sites. in this process, mΦs play a central role in managing different immunopathological phenomena through the secretion of inflammatory mediators such as nitric oxide (no), prostaglandins, and the pro-inflammatory cytokines tumor necrosis factor-α (tnfα) and interleukin (il)- [ ] [ ] [ ] . no is a potent vasodilator, acting to maintain vascular tone and function within the vessel wall, generated from l-arginine by nitric oxide synthase (nos) enzymes. there are three main isoforms of nos with distinct functions and patterns of expression: endothelial nos, neuronal nos and inducible nos (inos) highly expressed in mΦs [ , ] . prostaglandins are other important inflammatory mediators implicated in the vascular homeostasis since its low levels can prevent or reverse aggregation of platelets and induce vasodilation [ , ] . these molecules are produced from arachidonic acid metabolites by the catalysis of cyclooxygenase- (cox- ) [ ] . pro-inflammatory cytokines induce integrin expression and redistribution of leukocytes, which contribute to their recruitment and activation at the site of inflammation [ , ] , as well as increasing vascular permeability and tissue injury [ , ] . there is evidence that inflammatory cytokines are the main orchestrators of the inflammatory cascade in brdc, detecting high levels of tnfα and il- in the airways of cattle infected with respiratory pathogens [ ] [ ] [ ] [ ] . bvdv is a pestivirus that although it is not a primary agent in the pathogenesis of brdc, it can suppress the host immune system and increase the risk of secondary infections, thus enhancing pulmonary colonization by other pathogens such as bhv- [ ] [ ] [ ] . the mechanisms of the immunosuppressive action of bvdv are object of debate, including changes related to decreased lymphocyte proliferation [ , ] , severe lymphoid depletion in lymphoid tissues [ ] [ ] [ ] , decreased chemotaxis and phagocytic activity [ ] , increased production of prostaglandin e [ , ] , increased no synthesis in response to lipopolysaccharide [ , ] and impaired production of pro-inflammatory cytokines [ , [ ] [ ] [ ] [ ] . therefore, the aim of this study was to clarify the mechanisms responsible for ultrastructural and histopathological changes occurring in the lungs of calves pre-infected with bvdv and challenged later with bhv- , as well as to analyze the role of mΦs in the appearance of the lesions. thus, this work will contribute to gaining a better understanding about how this virus predisposes to secondary airborne infections. the experimental design and collection of samples have been described by risalde et al. [ ] . briefly, twenty-four friesian calves ( - months old), bvdv and bhv- antigen and antibody (ab) free, were housed in the animal experimental center of cordoba university (spain). the animals had an adjustment period of one week before the study started and were separated into three groups based on the inoculation protocol: twelve calves were inoculated intranasally with ml of a suspension of non-cytopathic bvdv- strain with a titration of tissue culture infective dose % (tcid )/ml (courtesy of the institute für virologie, tiho, hannover, germany). twelve days later, when the calves showed neither clinical signs nor bvdv viraemia, they were challenged with ml of bhv- . strain iowa containing tcid /ml (courtesy of the hipra laboratories, girona, spain) (bvdv/bhv- group). two calves of this group, bhv- . -free, were used as bvdv infection controls. at the same time, ten calves were inoculated only with bhv- . (bhv- group). two un-infected (ui) animals were used as negative controls and received intranasally ml of tissue culture fluid free of viruses. the time point of bhv- . inoculation was defined as day . after virus inoculation, clinical examination was performed daily. in order to determine the number of platelets, edta blood obtained from coccygeal vein and nasal swab samples were collected at , , , , , , and hours post-inoculation, , , , , , , and days post-inoculation (dpi) with bhv- . . the infected calves were sedated with xylazine (rompun w % solution; bayer healthcare, kiel, germany) and euthanized by overdosing with thiopental-sodium (thiovet w ; vet limited, leyland, lancashire, uk) in batches of two at , , , and dpi. two animals of the bvdv/bhv- group were killed at dpi just before bhv- . inoculation (bhv- . -ui), being used as bvdv infection controls for this group. on the contrary, the two negative control animals were euthanized at the end of the study, being used as ui controls for the bhv- group. the experimental procedure was carried out in accordance with the code of practice for housing and care of animals used in scientific procedures, approved by the european economic community in ( / /eec amended by the directive / /ec). all euthanized calves were subjected to necropsy examination. the samples were collected from the lungs and immediately frozen at − °c for virological study; likewise, they were fixed in % buffered formalin solution for histopathological and immunohistochemical studies as well as in . % glutaraldehyde in . m pbs for ultrastructural analysis. lung tissue samples were analyzed to discard any infection with secondary bacterial pathogens, being subjected to microbiological routine cultures (xylose lysine deoxycholate agar, macconkey agar and blood agar) by using standard procedures. no bacteria were isolated from these samples. rneasy lipid tissue kit (qiagen) was used to extract bvdv ribonucleic acid (rna) from lung samples, according to the manufacturer's instructions. this rna was subjected to a one step real-time reverse transcription plus polymerase chain reaction (rt-pcr) using primers and taqman probes (at the same concentration) from conserved regions of the '-utr of bvdv- a [ ] . the reaction was performed using the real time ready rna virus master (roche, mannheim, germany) in a lightcycler . , according to the manufacturer's instructions. the samples with a cycle threshold value less than or equal to were considered as positive. the positive control was the ncp bvdv- strain at tcid /ml. a real-time pcr analysis of the bhv- deoxyribonucleic acid (dna), extracted from lung samples using genomic dna from a tissue kit (macherey-nagel, germany), was performed according to the oie terrestrial manual [ ] . after analyzing the results on an applied detector (applied biosystems, usa), any sample with a cycle threshold value less than or equal to was considered as positive. the positive control was the bhv- . strain iowa at . tcid /ml. glutaraldehyde-fixed samples were post-fixed in % osmium tetroxide, dehydrated in acetone and embedded in epon w (fluka chemie ag, buchs, switzerland). sections ( nm) were counterstained with uranyl acetate and lead citrate, and examined with a philips cm- transmission electron microscope. formalin-fixed samples were dehydrated through a graded series of alcohol to xylol and embedded in paraffin wax by routine techniques for light microscopy. samples were sectioned ( μm) and stained by different methods as haematoxylin-eosin and the fraser lendrum technique, or processed for their immunohistochemical study using the avidin-biotin-peroxidase complex (abc) method. the abc method for immunohistochemistry (ihc) was performed on serial sections of formalin-fixed samples, which were dewaxed and rehydrated as previously described by pedrera et al. [ , ] . briefly, endogenous peroxidase activity was exhausted by incubation with . % hydrogen peroxide in methanol for min at room temperature. the samples were subjected to different methods for antigen retrieval (table ) . after pretreatment, the sections were rinsed three times in pbs (ph . ) for min and then covered with % normal horse serum (pierce-endogen, woburn, usa) in . m tris buffered saline (tbs) (ph . ) or % normal goat serum in pbs for min at room temperature, for primary monoclonal ab(mab) and polyclonal ab(pab), respectively. after this blocking stage, sections were incubated with primary ab at °c overnight. after primary incubation, the slides were washed in pbs (three times for min each) and then incubated with the secondary antibodies for min at room temperature. biotinylated horse anti-mouse igg secondary ab (pierce-endogen) diluted : in tris buffer containing normal horse serum % was used for the primary mab. biotinylated goat anti-rabbit igg secondary ab (vector laboratories, burlingame, ca, usa) diluted : in pbs containing normal goat serum . % was used for the primary pab. after three further min washes in tbs, samples were incubated with the abc complex (vectastain w abc elite kit, vector laboratories, ca, usa) for h at room temperature. all tissue sections were finally rinsed in pbs and incubated with chromogen solution (novared w substrate kit, vector laboratories). slides were counterstained with mayer's haematoxylin. details of the primary mab and pab are summarized in table . tissue samples from cattle, in which reactivity for primary ab against cytokines and cellular markers used in this study had been demonstrated, were used as positive controls in ihc [ , ] . tissue sections for which the specific primary ab were replaced by rabbit or mouse non-immune sera (dakocytomation, glostrup, denmark) were used as negative controls. in order to evaluate the number of immunolabeled cells and to correlate the results obtained using different ab, two paraffin-wax blocks from the lungs of each animal were selected. cell counts were carried out on randomly chosen fields of . mm from tissue sections from these blocks. the results are given as the number of positive cells per . mm . identification of different types of immunolabeled cells was based on morphological features, location and size of the cells. the different pulmonary mΦs exhibited rounded or elongated morphology, an indented nucleus and abundant cytoplasm. interstitial mΦs (imΦs), pulmonary intravascular mΦs (pim) and pulmonary alveolar mΦs (pam) are localized in distinct anatomical compartments of the lung, including connective tissue, being adhered to the endothelium in the pulmonary capillaries and air spaces, respectively. pim and imΦs were grouped together and are described as "septal mΦs". a semiquantitative estimation of platelet aggregation factor-viii-positive was performed on randomly chosen . mm areas. the results are given as the presence of positive clusters of platelets per area as follows: absent (−), mild (+), moderate (++) and abundant (+++). for graphical representation and statistical analysis, they were scored from absent to severe ( to ). quantifications were performed by two experienced observers but with no previous knowledge of which group was being analyzed. data were assessed to calculate mean ± standard error values and were analyzed with the sas system for windows, version . (sas institute, cary, north carolina, usa). chi-square analysis was employed for the estimation of platelet aggregation factor-viii-positive for which a p < . was accepted as statistically significant, using a fisher's exact test in the instance of an expected value below . the duncan's multiple range test (p < . ) was used to analyze significant differences of the values in the same group at various time points (*) and non-paired student's t-test (p < . ) was used between both inoculated groups at the same time point (**). the main respiratory signs are given in risalde et al. [ ] . briefly, the calves pre-infected with bvdv show more intense respiratory signs such as cough, mucopurulent nasal discharge, dyspnoea and open-mouth breathing mainly between and dpi, while the calves of the bhv- group only presented a moderate serous nasal discharge. platelet numbers were within the clinically normal range in all calves throughout the study, except for animals from both inoculated groups, where an important decrease of platelet numbers to approximately % was observed at hpi. the presence of bvdv in the lungs of co-infected calves was detected by rt-pcr between and dpi ( - dpi bvdv), presenting the samples cycle threshold values between and ; whereas bhv- was detected by pcr from to dpi in these calves with cycle threshold values from to . on the contrary, bhv- was only detected between and dpi in the bhv- group, showing the samples higher cycle threshold values (between and ), while bvdv was not detected in these calves throughout the study. there were no remarkable lesions in the negative control animals. the pulmonary parenchyma of inoculated calves was affected by interstitial pneumonia with alveolar septal thickening produced by interstitial aggregates of mononuclear cells. the appearance of this alteration was earlier in the co-infected calves and was associated with occasional haemorrhages. the fraser lendrum technique revealed fibrin microthrombi in some pulmonary venules and capillaries of inoculated calves, being more severe in co-infected calves, mainly from dpi onwards ( figure a) . these changes were confirmed by the ultrastructural study together with the presence of fibrin in alveoli associated with pam in both inoculated groups at early stages ( figure b) . moreover, these animals displayed an intense hyperaemia as well as interstitial and alveolar oedema during this period. these findings were associated with a great quantity of platelets, whose detection was performed by ihc using anti-factor-viii ab. thus, in the negative control calves, this ab prompted positive granular immunostaining among sheathed capillary cells, free in the interstitium and occasionally in mΦs cytoplasm. however, in the inoculated groups, clusters of immunostained granular material were observed in blood vessels. moreover, numerous imΦs and periarterial mΦs, together with some pam, were swollen and displayed an intense positive granular and cytoplasmic reaction in both inoculated groups between and dpi (figure a, b) . in the bvdv/bhv- group, clusters of platelets were observed from dpi, being more evident throughout the study associated with mΦs engulfing platelets that peaked at dpi (score value of in all evaluated fields) ( figure ). subsequently, the positive reaction in mΦs and vascular lumina diminished considerably without recovering thereafter ( figure c ). there was a significant difference in the presence of platelet clusters in this group throughout the study (p = . ). in the bhv- group, these findings were observed from dpi with similar values to the co-infected group, decreasing the number of platelet clusters until the end of the study (figures d and ) . however, there was no significant difference in the incidence of these clusters in the lungs of animals infected only with bhv- . ultrastructural studies confirmed that there was an increase in the number of activated platelets forming multiple aggregations within the blood vessels, whose vascular lumina usually appeared completely occluded. the changes indicative of this activation were an enlarged and deformed shape of the cells, a partial or total decrease in granule numbers and a dilation of the open canalicular system. the most common form of platelet aggregation was the appearance of mosaic-like clusters comprising fully degranulated platelets with fusion of cytoplasmic membranes. occasionally, these membranes completely disappeared, giving rise to a finely granular structure with low electron density, containing vestiges of platelet organelles and surrounded by a membrane layer. in the bvdv/bhv- group, platelet aggregation occurred sooner and was more intense, increasing between and dpi ( figure a ) and not showing a total recovery at the end of the study ( figure c ). in the bhv- group this lesion was moderate, presenting retrieval signs from dpi ( figure b, d) . subcellular changes indicative of a slight secretory activation in imΦs such as enlargement, proliferation and dilation of rough endoplasmic reticulum and golgi complex cisternae were mainly observed in the bhv- group at dpi. moreover, from dpi, some pim and pam of both inoculated groups appeared enlarged and rounded, with loss of filopodia, increased number of lysosomes and varying amounts of cell debris in their cytoplasm, characteristic signs of phagocytic activation ( figure c) . immunolabeling of septal mΦs displayed similar kinetics in single and dual infections after bhv- . inoculation, although presenting differences in the magnitude of (see figure on previous page.) figure fibrin microthrombi in pulmonary venules of animals pre-infected with bvdv and challenged later with bhv- . . co-infected animals presented vascular alterations that facilitate the occlusion of the pulmonary venules as fibrin microthrombi observed with fraser lendrum technique (a) and tem (b) at dpi, as well as pim enlarged and rounded engulfing cell debris (arrowheads) observed with tem at dpi (c). their response. thus, the bvdv/bhv- group showed a higher number of these cells during the study, peaking at dpi (p < . ). the number of pam was also significantly higher in the co-infected calves at the start of the study, showing a decrease from dpi ( figure ). secretory activity of mΦs observed ultrastructurally was confirmed by ihc, which enabled the detection of mΦs-secreted inflammatory mediators. tnfα and il- α-producing cells, identified as septal mΦs and pam, were detected immunohistochemically in the lungs of control and infected animals. these proinflammatory cytokines presented differences in magnitude and kinetics between single and dual infections. tnfα-positive septal mΦs were associated with sites of inflammation in the bvdv/bhv- group (figure a ), showing only a slight peak at dpi (p < . ), whereas the bhv- group presented a longer response of this chemical mediator in peribronchial areas (from dpi; p < . ) ( figure b ). on the contrary, il- α-reactive septal mΦs were significantly different between both infected groups before bhv- inoculation ( dpi bhv- ). bvdv/bhv- group calves maintained lower numbers of il- α-positive septal mΦs throughout the study (figure c) , showing a delayed response to bhv- inoculation (from dpi onwards). by contrast, bhv- group calves displayed an early increase of this cytokine associated with peribronchial areas (at dpi; p < . ) ( figure d ). the number of pam positive for studied cytokines was low in both inoculated groups, presenting only a slight response at the end of the study, with the exception of an il- α peak in the single infected group between and dpi (figure ) . numerous groups of immunolabeled septal mΦs presenting inos-positive cytoplasmic granules, mainly imΦs, were observed in the pulmonary parenchyma of animals infected only with bvdv ( figure a dpi ( figure b ). in healthy calves, septal mΦs presented a similar response after bhv- inoculation; although in these animals the decrease of this mediator was observed later than in the bvdv/bhv- group. on the contrary, the number of inos-positive pam was higher in the bvdv/bhv- group and did not suffer changes throughout the study, while bhv- group calves showed a significant decrease from dpi (figure ). intracellular localization of cox- was perinuclear and cytoplasmic in the mΦs, being its expression scarce in the lungs during the study ( figure c ), except for septal mΦs of the bvdv/bhv- group at dpi (p < . ) (figure ). this intense expression occurred at sites of inflammation and injury, mainly oedemas, and was correlated with the degree of pulmonary inflammation ( figure d ). the objective of this study was to evaluate and characterize the vascular changes observed in the lungs of healthy calves and calves with subclinical bvd both experimentally inoculated with bhv- . , and to clarify the role of the pulmonary mΦs in the local response to the secondary pathogen. the results show that following bhv- inoculation, both groups of calves displayed a mononuclear cell infiltrate and prompted major vascular alterations in the lungs, marked by intense intravascular coagulation in small and medium-sized blood vessels from an early stage of the disease. three mechanisms, jointly known as virchow's triad, can induce thrombosis [ ] : ) endothelial wall injury; ) abnormalities of blood constituents (platelets, coagulation and fibrinolytic pathways); and ) abnormalities of blood flow. with regard to endothelial damage, infection by human herpes simplex virusbelonging to the same subfamily that bhv- [ ] has been shown to cause endothelial injury, favoring the exposure of subendothelial tissue and the release of procoagulant mediators [ , ] . here, however, neither histopathological nor ultrastructural examination disclosed any morphological evidence of endothelial damage in either of the inoculated groups. analysis of the cell components involved in coagulation pathways revealed fibrin deposits and intense platelet aggregation in the pulmonary microvasculature of both groups; these findings were particularly marked in the bvdv/bhv- group at dpi, coinciding with a significant increase of the rectal temperature and severe clinical respiratory symptoms [ ] . according to this, in the course of certain acute viral infections, platelets may be activated in vivo, leading to their degranulation, figure pro-inflammatory cytokines in the lungs of calves with and without pre-existing bvdv challenged with bhv- . . an immunohistochemical study revealed the presence of septal mΦs and pam positive for tnfα (a) and il- α (c) associated with sites of inflammation in the pulmonary parenchyma of the bvdv/bhv- group at dpi. however, the calves of the bhv- group presented a higher number of imΦs reactive to tnfα (b) and il- α (d) in the peribronchial areas of the lung at dpi. aggregation and withdrawal from circulation [ ] [ ] [ ] . the procoagulant activity of bvdv and bhv- has been reported in vitro [ ] . in our experimental study, it was increased in the co-infected calves due to the concomitance of both agents in the lungs between and dpi. despite the absence of bvdv in the blood of these calves at the moment of bhv- inoculation, it does not completely disappear, being detected by pcr in the lungs and by ihc in lymphoid tissues throughout the experience [ ] . however, there was no evidence in any stage of direct interaction between these viruses and platelets, which would suggest that platelet activation may be enhanced by indirect mechanisms including the expression of inflammatory mediators released by mΦs, which are known to play a major role in the maintenance of tissue homeostasis [ , ] . the study of pro-inflammatory cytokines revealed alterations in the kinetics and magnitude of tnfα and il- expression; both mediators can prompt changes in coagulation by increasing the number of endothelial adhesion molecules or increasing vascular permeability [ , ] . thus, the bvh- group calves display an increase in il- synthesis by septal mΦs coinciding with the onset of platelet aggregation in the lungs ( dpi). this, together with the subsequent action of tnfα, would favor the maintenance of the procoagulant setting. by contrast, bvdv/bhv- group calves, whilst exhibiting a higher number of imΦsthe main producers of these cytokinesdisplayed inhibited il- expression until dpi, along with a minimal tnfα response. this impaired cytokine production, also observed at the systemic level [ ] , indicates that the synergic action of both chemical mediators can be ruled out as a potential mechanism for inducing platelet aggregation in the co-infected group. calves inoculated only with ncp bvdv ( dpi for the bvdv/bhv- group) displayed greater expression of inos by septal mΦs than healthy calves [ , , ] . however, following bhv- inoculation, bvdv/bhv- group calves exhibited an early decline in inos ( dpi), an inflammatory mediator that limits the extent and duration of pathogen-induced platelet activation [ ] . this finding, together with the moderate response of tnfα, may have favored the appearance of platelet aggregates in the early stages of the disease, and intense aggregation coinciding with the greatest decrease in inos levels ( dpi). the intense platelet aggregation observed in the lung microvasculature of the bvdv/bhv- group at dpi, together with the increase in number and size of pim as a result of phagocytic and secretory activation would indirectly prompt a slowdown in blood flow and a subsequent response by the cox- enzyme aimed at reversing that process [ ] . however, in view of the damage observed at later stages, this action was presumably unable to counter the procoagulant events associated with the drop in inos expression, these being additionally enhanced by the delayed action of il- in co-infected animals. slowed blood flow, together with cytokine release, may lead to increased vascular permeability and extravasation of leukocytes into the pulmonary parenchyma [ , , , ] . therefore, the results of this study indicate that the concomitance of bvdv and bhv- in the lungs enhances a synergic action of their pathogenic mechanisms, disrupting the maintenance of pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment, characteristic of the brdc, which appears to be modulated by inflammatory mediators released by pulmonary mΦs. in this respect, further research is required into the possible involvement of this concomitance, through the use of live bvdv and bhv- vaccines, in the triggering of an impaired pulmonary immune response. on the contrary, animals pre-inoculated with bvdvdespite suffering a transient infection -exhibit an alteration in the response of pro-inflammatory cytokines which play a key role in activating the immune response. ab: antibody; abc: avidin-biotin-peroxidase complex method; bhv- : bovine herpesvirus- bhv- group: calves inoculated only with bhv- ; brdc: bovine respiratory disease complex; bvdv: bovine viral diarrhea virus bvdv/bhv- group: calves inoculated with bvdv and bhv- dna: deoxyribonucleic acid; dpi: days post-inoculation ihc: immunohistochemistry; il: interleukin; imΦs: interstitial macrophages; inos: inducible nitric oxide synthase; mab: monoclonal ab mΦs: macrophages; no: nitric oxide; nos: nitric oxide synthase pcr: polymerase chain reaction; pim: pulmonary intravascular mΦs; rna: ribonucleic acid; rt-pcr: reverse transcription-polymerase chain reaction; tbs: tris buffered saline; tcid : tissue culture 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immunology of bovine viral diarrhea virus distinct role and location of the endothelial isoform of nitric oxide synthase in regulating platelet aggregation in males and females in vivo filovirus-induced endothelial leakage triggered by infected monocytes/ macrophages increased release of interleukin- beta, interleukin- , and tumor necrosis factoralpha by bronchoalveolar cells lavaged from involved sites in pulmonary tuberculosis submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by grants from the junta de andalucía-feder (p -agr- ). ma risalde and v molina were supported by a pre-doctoral grant from the spanish ministry of education and science. the authors thank the institute für virologie, tiho (hannover, germany) and laboratorios hipra sa (girona, spain) for providing the bvdv and bhv- . , respectively, and the farm "las rozuelas del valle" (torrecampo, spain) for providing the animals. we specially thank to a. muñoz for his technical assistance with the statistical analysis. the authors declare that they have no competing interests.authors' contributions mar participated in the design and coordination of the experiment, performed post-mortem and ihc examinations, analyzed the data and drafted the manuscript. vm participated in the design of the experiment, carried out tissue processing and participated in the ihc examinations and data analysis. pjsc participated in the design and coordination of the experiment, coordinated tissue sample collection at post-mortem and tissue sampling. fr carried out clinical monitoring and post-mortem examinations and helped with tissue sampling. mp carried out the clinical monitoring and tissue sampling at post-mortem. bg performed the molecular techniques. jcg conceived and coordinated the study, carried out the ultrastructural study and coordinated the drafting of the manuscript. all authors read, commented on manuscript drafts and approved its final version. key: cord- -mkm ygh authors: kirchhoff, jana; uhlenbruck, sabine; goris, katherina; keil, günther m; herrler, georg title: three viruses of the bovine respiratory disease complex apply different strategies to initiate infection date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: mkm ygh bovine respiratory disease complex (brdc) is the major cause of serious respiratory tract infections in calves. the disease is multifactorial, with either stress or reduced immunity allowing several pathogens to emerge. we investigated the susceptibility of bovine airway epithelial cells (baec) to infection by the three major viruses associated with the brdc: bovine respiratory syncytial virus (brsv), bovine herpesvirus type (bhv- ) and bovine parainfluenza virus type (bpiv ). for this purpose, two culture systems for well-differentiated baec were used: the air-liquid interface (ali) system, where filter-grown baec differentiate into a pseudostratified respiratory epithelium and precision-cut lung slices (pcls) where baec are maintained in the original tissue organisation. comparative infection studies demonstrated that entry and release of bpiv occurred specifically via the apical membrane with ciliated cells being the major target cells. by contrast, airway epithelial cells were largely resistant to infection by bhv- . when the epithelial barrier was abolished by opening tight junctions or by injuring the cell monolayer, bhv- infected mainly basal cells. respiratory epithelial cells were also refractory to infection by brsv. however, this virus infected neither differentiated epithelial cells nor basal cells when the integrity of the epithelial barrier was destroyed. in contrast to cells of the airway epithelium, subepithelial cells were susceptible to infection by brsv. altogether, these results indicate that the three viruses of the same disease complex follow different strategies to interact with the airway epithelium. possible entry mechanisms are discussed. virus infections of the respiratory tract are the most common cause of viral diseases worldwide ranging from common colds to life-threatening pneumonia [ ] . a large number of both rna and dna viruses uses the respiratory tract to initiate host infection. infection may be restricted to or most evident in certain sections of the airway system such as trachea, bronchi or alveoli. for some viruses, the respiratory tract may just serve as a primary entry site from where infection spreads to other organs or tissues. all these viruses encounter the respiratory epithelium as a primary barrier against invading pathogens. this barrier is composed of differentiated epithelial cells that have different functions. an important defence strategy is the mucociliary clearance system. while some epithelial cells are specialized to produce and release mucins, other cells are equipped with cilia that enable them to contribute to the transport of the mucus out of the respiratory tract. viruses differ in their ability to infect the differentiated respiratory epithelial cells. productive infection by human influenza viruses has been reported to preferentially occur in mucus-producing cells [ ] . other viruses, e.g. parainfluenza virus (piv ), may have a preference for ciliated cells [ ] . characteristic features of differentiated epithelial cells such as ciliary activity are not maintained in any of the available immortalized cell lines. analysis of such cells requires primary cell cultures. when tracheal or bronchial epithelial cells are grown on filter supports under air-liquid interface (ali) conditions, they differentiate and acquire properties of specialized cells, i.e. mucus production or ciliary activity [ , ] . another culture system for differentiated respiratory epithelial cells are precision-cut lung slices (pcls), where the epithelial cells are maintained in their original setting. in addition to mucus production and ciliary activity, this culture system provides another characteristic feature of the airway, bronchoconstriction. as submucosal cells are also present in pcls, they can be included in the investigation [ ] [ ] [ ] . bovine respiratory disease complex (brdc) is a leading cause of morbidity and mortality in feedlot cattle. the disease is considered as a multifactorial disorder caused by a combination of viral and bacterial pathogens together with environmental risk factors. the most important viral pathogens associated with brd are bovine respiratory syncytial virus (brsv), bovine parainfluenza virus (bpiv ) and bovine herpesvirus (bhv- ), a member of the subfamily alphaherpesvirinae [ ] [ ] [ ] [ ] . brsv and bpiv are paramyxoviruses that use the fusion protein f on the cell surface to mediate fusion of the viral envelope with the cellular membrane and thus to get the viral genome into the cell. prior to fusion, the virions attach to the cell surface. attachment of piv is accomplished by another surface protein, the hn protein which interacts with sialic acid-containing cellular glycoconjugates and thus binds to cells in a sialic acid-dependent manner [ , ] . attachment of human rsv (hrsv) can be mediated by the surface protein g that binds to glycosaminoglycan structures [ ] as well as by the fusion protein f that may bind to both glycosaminoglycans as well as to a specific protein receptor [ , ] . nucleolin has recently been reported to promote infection though the authors did not exclude the possibility that another so far unknown protein may serve as a receptor for entry of hrsv [ ] . no data on receptors for brsv have been reported yet. alphaherpesviruses like bhv- , herpes simplex virus (hsv- ) or pseudorabies virus use the surface glycoprotein gc for initial attachment to heparan sulphate structures. for entry, virions have to bind to a specific receptor (nectin- or herpesvirus entry mediator (hvem)) which is accomplished by the surface protein gd. the subsequent fusion reaction is mediated by the combined action of the surface proteins gb/gd/gh/gl [ , ] . we have reported recently that well-differentiated respiratory epithelial cells are susceptible to infection by bpiv whereas they are rather resistant to infection by brsv [ ] . here we have included another important virus of the brdc, bhv- and analysed virus entry into and release from differentiated respiratory epithelial cells in more detail. our results show that the three viruses have developed different strategies to infect the cells of the airway epithelium. the generation of recombinant brsv expressing green fluorescent protein (brsv-gfp) and preparation of viral stocks has been described in detail elsewhere ( ; brsv atue was kindly provided by karl-klaus conzelmann, max-von-pettenkofer-institut, munich). bpiv was provided by friedrich-loeffler-institut (insel riems, germany). bpiv stocks were prepared in kop-r cells [ ] . bhv- -gfp was generated from the strain bhv- /aus as described previously [ ] and propagated in kop-r cells. for this purpose, cells were grown in cm -flasks till % confluence and then infected with an moi of . for h. after - days a cytopathic effect was observed and supernatants were collected and centrifuged by low speed centrifugation ( × g). all virus stocks were frozen in liquid nitrogen and stored at − °c. mdbk (madin-darby bovine kidney cells; kindly provided by wolfgang garten, philipps-universität marburg, marburg, germany), vero cells (african green monkey cells; atcc, ccl- ) as well as kop-r cells (bovine oropharynx tissue, rie ; friedrich-loeffler-institut, insel riems, germany) were cultured in dulbecco's modified eagle medium (dmem) supplemented with or % fetal calf serum (biochrom ag, berlin). all cell lines were maintained in -cm culture flasks at °c and % co and passaged twice a week. primary baec (bovine airway epithelial cells) were derived from bovine bronchi of - months old calves and epithelial cells were prepared as described previously [ , ] . briefly, cells were seeded on collagen coated, semipermeable membrane supports (greiner, well, . -μm pore size) at a density of . × cells per filter support. after baec had reached confluence, the apical medium was removed and cells were cultured under ali conditions for at least weeks to establish a polarized and differentiated epithelium. prior to infection, the transepithelial electrical resistance (teer) was determined to verify an intactness of the epithelial barrier. for this purpose, a millicell ers- volt-ohm meter (millipore corporation, billerica, ma, usa) was used. to obtain the actual resistance of the epithelial cell layer, the resistance value of a cell-free filter support was subtracted from the teer values determined across the filter-grown cultures. teer values were above Ω • cm prior to infection. pcls were prepared from the lobus accessorius of - months old calves as described previously [ , ] . lobes were filled with low-melting agarose (agarose lm gqt, gerbu, gaiberg, germany). after the agarose had solidified, cylindrical portions containing a section of an airway were stamped out. using a krumdieck tissue slicer (tse systems, model md - ) slices, μm thick, were generated and incubated in ml of rpmi medium (invitrogen/gibco, germany) in -well plates at °c and % co . the viability of the slices was determined by screening for ciliary activity using a light microscope (zeiss axiovert ). baec were washed extensively with phosphate-buffered saline (pbs) to remove secreted mucus. subsequently, the virus suspension, diluted in dmem, was added at μl per filter to the cells for h (bpiv , bhv- -gfp) or h (brsv-gfp) at °c and % co . for infection of the cells from the basolateral surface, filters were inverted for the duration of infection. after unbound virus had been removed by washing, the cells were further incubated at °c for to days. to open tight junctions, the cells were washed thrice with ca + /mg + -free pbs and then apically treated with ca + /mg + -free pbs containing . m egta (applichem, darmstadt) at °c. after min, egta was removed and cells were infected with either bhv- -gfp or brsv-gfp. teer was determined before and after egta treatment. pcls were washed three times with pbs to remove mucus and then infected with virus ( ffu/ml) diluted in μl of rpmi medium. after h (bhv- -gfp, bpiv ) or h (brsv-gfp), the inoculum was removed and the slices were washed twice with pbs followed by the addition of ml of rpmi medium. slices were incubated for - days in % co at °c. to analyze the importance of sialic acids for bpiv infection, the slices were washed with pbs followed by incubation with mu/pcls of neuraminidase (na) type v from clostridium perfringens (sigma/aldrich, munich) for h at °c. after removal of na, the slices were rinsed in pbs prior to virus infection. to assess viral shedding, supernatants of apically infected ali cultures were taken post-infection over a period of week. for this purpose, μl aliquots of prewarmed emem were applied to the apical surface of the filter inserts and harvested after a min incubation period at °c. for basolateral virus release, μl of the basolateral medium was collected and replaced by fresh medium. supernatants were combined with × viral stabilizing solution ( m mgso , . m hepes, ph . ), quick-frozen on dry ice and stored at − °c. teer was determined during the course of the experiment. in the case of pcls, μl of the supernatant was collected at each time point and replaced by the same amount of rpmi to avoid differences in virus concentration. for each virus, cultures from at least animals were used and experiments were performed in duplicate. cells grown on membrane filters and pcls were fixed for min at room-temperature with % paraformaldehyde in pbs. fixed cells were washed twice with . m glycine in pbs and then permeabilized with . % triton x- diluted in pbs for min. all antibodies were diluted in pbs containing % bovine serum albumin at room temperature. bpiv -infected cells were stained with a polyclonal antiviral antiserum (vmrd, caprine origin) followed by a fitc-labeled secondary antibody (sigma-aldrich). cilia were stained by using a cy -labeled monoclonal antibody against β-tubulin (sigma-aldrich). basal cells were visualized by a monoclonal mouse anti-human cd antibody (serotec) and a primary antibody specific for ttf- (clone bgx- a mouse monoclonal, bio-genex) was used to stain pneumocytes type ii. nuclei were visualized with dapi ( ′, ′-diamidino- -phenylindole) which was added to the apical surface of the cells and removed after incubation for min ( °c) followed by three washing steps with pbs. after washing, cells were embedded in mowiol resin and photomicrographs were generated using a leica tcs sp aobs confocal laser scanning microscope. for image processing, adobe photoshop (version . , adobe systems, usa) was used. one day prior to infection, mdbk (bpiv , bhv- -gfp) or vero cells (brsv-gfp) were grown to confluence in well plates and then exposed to serial tenfold dilutions of virus suspensions in duplicate. for virus adsorption, cells were incubated at °c for h (bpiv , bhv- -gfp) or h (brsv-gfp). after inoculation, cells were overlaid with % methylcellulose and plaques were counted (bhv- -gfp, bpiv ) or (brsv-gfp) days post-infection. in case of bpiv , the cells were stained for virus antigen. the viral titer is indicated in foci-forming units per ml (ffu/ml). the three viruses associated with the bovine respiratory disease complex were compared for their efficiency to infect differentiated respiratory epithelial cells. for this purpose, baec were grown for about four weeks under ali conditions to build up a polarized and pseudostratified epithelium. cells were infected by bpiv , brsv-gfp or bhv- -gfp at an moi of . and analyzed two (bpiv and bhv- -gfp) or three (brsv-gfp) days post infection (dpi) for the presence of infected cells. as described in earlier studies [ ] , bpiv efficiently infected the respiratory epithelium. co-staining of β-tubulin indicated that ciliated cells were the target cells ( figure a ). on the other hand, infection by brsv-gfp was very inefficient ( figure b) ; only a few infected cells were detectable and those cells were identified as ciliated cells using an antibody directed against β-tubulin. only occasionally non-ciliated cells were infected by brsv-gfp. when bhv- -gfp was applied to the apical surface, the epithelium was found to be refractory to infection ( figure c ). not a single infected cell was detectable at two dpi; if the incubation time was increased up to five days, only occasionally infected cells were found. the same result was obtained when the titer was enhanced tenfold and infection was monitored up to five days. only when an moi of was applied, a few cells were infected and appearance of infected foci suggested that infection spread to adjacent cells (see additional file ). infection of baec by the three bovine viruses was also evaluated by determining the kinetics of virus production ( figure ). apical release of infectious bpiv was detected at hours post infection (hpi) and increased exponentially by hpi; the maximum infectivity titer of~ × ffu/ml was determined at hpi. brsv-gfp was detectable in the supernatant only at hpi and the maximum titer of × ffu/ml was reached after seven days. the apical release of infectious bhv- -gfp was as slow as that of brsv-gfp but proceeded only to a maximum titer of . × ffu/ml. basolateral release of all three viruses was below detection level. for any of the three viruses, we did not observe a general decrease in the transepithelial resistance values during the course of the experiment. despite some fluctuation of individual teer values post-infection and variability between individual trans-wells, apical infection of differentiated cultures did not affect the integrity of the epithelium (see additional file a). since brsv-gfp and bhv- -gfp were not able to establish an efficient infection of baec when applied from the apical side, we analyzed whether they could initiate infection from the basolateral side of the cells. for this purpose, the device containing the membrane was turned upside down. under these conditions, bhv- -gfp was able to infect baec as indicated by gfp expression after a h incubation time. the efficiency of infection could be enhanced by increasing the moi tenfold ( figure a ). in contrast, brsv-gfp was not able to establish an infection after basolateral inoculation ( figure b ). the results presented above indicate that bhv- -but not brsv-gfp is able to infect baec from the basal side. to rule out that the pore-size of the filter membrane and the size and morphology of the virions prevented access to the basal side, we used an epithelium injury model to investigate infection. well-differentiated baec were mechanically injured with a sterile needle and subsequently infected by either bhv- -gfp or brsv-gfp from the apical surface for two or three h. as shown in figure a , infection occurred exclusively within the region of injury and the underlying cells are the site of infection ( figure c ). further staining with anti-cd which is specifically expressed on bronchial epithelial basal cells [ ] showed that bhv- -gfp infected this cell type ( figure b ). in contrast, in case of brsv-gfp, hardly any cells at the injury site expressed gfp (data not shown). as shown above, a few infected cells were detected in the undamaged region; these cells were identified as ciliated cells by staining for β-tubulin. to further analyze bhv- entry into baec, egta was used to transiently open the tight junctions. cells were treated with . m egta for min prior to infection with either bhv- -gfp or brsv-gfp. in contrast to untreated cultures, teer values dropped to the baseline after egta treatment (see additional file b). as shown in figure a , pretreatment with egta enabled bhv- -gfp entry from the apical side as indicated by gfp-positive foci distributed all over the filter-grown cells. by contrast, the disruption of the epithelial barrier had no effect on the efficiency of brsv-gfp infection or on the cell type infected by this virus. virus titration revealed that the yield of bhv- -gfp released into the supernatant was pcls provide an alternative culture system for differentiated respiratory epithelial cells and have the advantage that they contain baec in the original setting. moreover, connective tissues and alveolar regions are present within these slices as well as immunomodulatory cells. to analyze the infection strategies of bpiv , brsv-gfp as well as bhv- -gfp, pcls were prepared from the lungs of - months old calves. for infection studies, only those slices were used, thatupon microscopic inspection -did not show any loss of the ciliary activity. pcls were infected with the same infectious dose ( ffu/ml) of any of the three viruses. the patterns of infected cells were quite different. bpiv exclusively infected ciliated cells of the respiratory epithelium, whereas bhv- -gfp-infected cells were located underneath the uppermost epithelial cell layer (figures and a) . it is known that sialic acids serve as receptor determinants for entry of bpiv . to determine the importance of sialic acids in the infection of differentiated respiratory epithelial cells by bpiv , slices were treated with neuraminidase from c. perfringens prior to infection. as shown in figure , desialylation almost completely abolished infection confirming that infection of differentiated respiratory epithelial cellslike that of immortalized cells (data not shown) -is sialic aciddependent. infection of the slices by bhv- -gfp or brsv-gfp was not affected by neuraminidase treatment. staining for cd revealed that basal cells, the progenitor cells of the differentiated respiratory epithelial cells, are primarily infected by bhv- -gfp ( figure b ). when the incubation time was increased to one week, infection spread to the periphery of the slice ( figure c ). in contrast, basal cells were not targeted by brsv-gfp. the latter virus predominantly infected subepithelial cells within the peribronchiolar connective tissue at three dpi and only a low number of ciliated cells were gfp-positive ( figure d ). the morphologies of at least some infected cells in the submucosal figure bpiv , bhv- -gfp and brsv-gfp infection of pcls before or after neuraminidase treatment. pcls were first incubated for h with or without mu/pcls neuraminidase type v and then infected with bpiv or bhv- -gfp with ffu/ml for h or with brsv-gfp for h. at dpi, the slices were fixed and in case of bpiv stained for virus antigen (green). cilia were visualized by staining against β-tubulin (red). scale bar = μm. area resembled those of dendritic cells ( figure e ). after one week of incubation, brsv-infected cells were also detected in the peripheral part of the slice comprising the alveoli ( figure f ). incubation with anti-ttf- (thyroid transcription factor − ), which stains nuclei of type ii pneumocytes revealed that both, ttf- positive and negative cells, were infected. stained cells were cuboidal in shape and found at the intersections of two or more alveoli whereas infected cells that were not stained by anti-ttf- had a flattened morphology (see additional file ). the efficiency of infection was also determined with respect to infectious progeny virus released into the culture medium. for this purpose, supernatants were collected at different time points after infection. as shown in figure , bpiv reached a viral titer of . × ffu/ml after h, which increased to × ffu/ml by day seven post infection (pi). infection of pcls by brsv-gfp resulted in titers in the supernatant that were hardly above the detection level. virus release of bhv- -gfp was intermediate with titers that were -to -fold lower than those determined for bpiv . consistent with the differences in the amount of virus released into the supernatant, there was also a difference in the kinetics of virus release: bpiv was detectable in the supernatant at hpi followed by bhv- -gfp at hpi; brsv-gfp was released into the supernatant not until hpi. culture systems for differentiated respiratory epithelial cells are models of choice to investigate how respiratory viruses interact with the epithelial barrier when they enter the airways. they comprise cell types like ciliated cells that are important for airway function and cannot be cultured as immortalized cells. using these culture systems, it is possible to determine which of the different cell types is susceptible to infection. these results may be different from those obtained with immortalized cells. a striking example is brsv which infects almost all immortalized cells [ , ] , whereas airway epithelial cells are rather refractory to infection ( [ ] , this paper). membrane-grown respiratory epithelial cells cultured under air-liquid interface conditions undergo mucociliary differentiation to generate a pseudostratified epithelium including ciliated cells, secretory cells and progenitor cells. they allow targeted access of reagents or microorganisms to the apical and basal membrane. though we succeeded in establishing ali cultures from bovine airway epithelial cells, most studies were performed so far with human cells. establishing ali cultures for cells of different species is still challenging [ , ] . the alternative culture system for differentiated respiratory epithelial cells, pcls, can be applied to lungs from different species [ , , , ] . they allow analyzing infection of not only the epithelial cells but also of submucosal cells. brsv, bpiv as well as bhv- are important viral pathogens within the bovine respiratory disease complex [ , ] . therefore, it was of interest to compare the entry strategies of these viruses. bpiv efficiently infected the airway epithelial cells using sialic acids on the apical surface as a receptor determinant for virus entry from the apical side. as virus egress also proceeds via the apical plasma membrane, bpiv -like human piv (hpiv )has the characteristics of several other viruses that cause a localized infection of the respiratory tract, e.g. influenza viruses [ , , ] . it remains to be further elucidated why bpiv preferentially targets the ciliated cells. this is a question that can be addressed in the future with culture systems for differentiated airway epithelial cells. bhv- showed a completely different infection pattern. when bhv- -gfp was applied to the apical side of the well-differentiated airway epithelial cells, infected cells were detected only occasionally. recently, an explant culture system derived from the upper trachea of cows was used to analyze bhv- infection. single sites of infection were detected in the epithelium when the virus was applied at tcid /ml [ ] . this infectious dose is about -fold higher than that required for efficient infection by bpiv . thus, differentiated epithelial cells from both the proximal trachea and the bronchi are rather resistant to infection by bhv- . successful infection required the access to the basal side of the epithelium either after opening of the tight junctions by calcium depletion or after mechanical injuring of the cell monolayer. infection studies with ali cultures indicated that the cells that are sensitive to bhv- -gfp infection are not the differentiated epithelial cells but cells with the phenotype of basal cells, i.e. the progenitor cells of the differentiated epithelial cells. also in pcls, basal cells were found to be the primary site of infection. when the inoculation time was increased up to one week, virus was also found in the periphery of the slice, and the distribution pattern suggests direct cell-to-cell spread. this infection pattern could not be predicted from studies with polarized immortalized cells. our results most closely resemble those reported for polarized mdck cells where infection was not possible via the apical surface, but via the basolateral side after opening of the tight junctions or after injuring the cell monolayer [ , ] . however, this cell system could not reveal the preference for the basal cell phenotype. other studies with polarized cells reported virus entry from both sides [ , ] or preferentially via the apical membrane [ ] . these conflicting reports underline the importance of infection studies with differentiated airway epithelial cells. from our results, the question arises how bhv- gets across the epithelial barrier. a model that has been proposed for hsv- suggests entry through small lesions within the epithelium [ , , , ] . further possible explanations will be discussed below. a third infection pattern was observed when differentiated epithelial cells were infected with brsv-gfp. though brsv-gfp -like bpiv -preferentially infected ciliated cells, the overall efficiency of infection was much lower. whereas bpiv readily infected airway epithelial cells, only single brsv-infected cells were observed even when the virus was applied at an increased moi. the difference in infection efficiency was also evident from the amount of infectious virus released by the infected cultures. the titers of infectious progeny brsv-gfp were up to -fold lower when compared to bpiv . in contrast to bhv- -gfp infection, the relative resistance of the airway epithelium against brsv-gfp infection could not be overcome by opening the tight junctions or by injuring the cell monolayer. the infection pattern observed with brsv-gfp resembles that reported for hrsv. a previous study reporting the release of a substantial amount of progeny virus had applied an unusual high multiplicity of infection [ ] . later on, it was reported also for hrsv that airway epithelial cells are infected with low efficiency [ , ] . a striking feature of the brsv infection is the susceptibility of submucosal cells to infection. some of these cells have morphologies that resemble those of dendritic cells. others were located within the epithelium lining the alveolar lumen. replication of brsv within the alveolar epithelium in both type i and type ii pneumocytes has been reported in an ultrastructural analysis of brsv-infected calves [ ] . staining of pcls for the presence of anti-ttf- , which was identified as a marker for type ii pneumocytes in sheep and swine [ , ] , demonstrated that type ii pneumocytes are susceptible to infection by brsv-gfp. alveolar cells that were negative for ttf- were also infected by brsv-gfp. these latter cells had a flattened morphology that is characteristic for type i pneumocytes. therefore, both type i and type ii pneumocytes appear to be susceptible to infection by brsv-gfp. unfortunately, the identity of the infected cells in the submucosal area could not be determined. immunological reagents available for bovine cells are limited and those few that were suitable for staining of pcls did not provide an unambiguous result. as differentiated airway epithelial cells are refractory to infection by brsv-gfp and bhv- -gfp, it remains to be elucidated how these viruses get across the epithelial barrier. as mentioned above, passage through leaky sites within the epithelial monolayer is a possibility proposed for hsv- . an alternative explanation has been suggested for measles virus. here, the major receptor, slam, is present on lymphoid cells but absent from epithelial cells. the recently identified measles receptor on epithelial cells, nectin , has a polarized surface distribution enabling infection via the basolateral side but not via the apical plasma membrane. therefore, it has been suggested that measles virus infection is initiated in alveolar macrophages or dendritic cells which are used as a ferry through the epithelial barrier [ , ] . we cannot exclude that dendritic cells that uptake antigen from the airway lumen via their dendrites capture brsv and carry infection to the submucosal infection sites as well. invitro studies have demonstrated rsv infection and replication within dendritic cells [ ] . also it has been suggested that the virus may persist in lymphoid or other tissues [ , , ] . a further way to get across the epithelium without infecting the epithelial cells is transcytosis. this entry option has been shown for several viruses, e.g. for epstein-barr virus to get across the oral epithelium ( [ ] , and references therein). there are no data available supporting transcytosis as an entry strategy for bhv- and brsv. an alternative explanation of the infection pattern of brsv and bhv- may be that the viruses enter differentiated bronchial epithelial cells without immediate initiation of replication. we do not have any evidence for this possibility, because brsv and bhv- infection of differentiated bronchial epithelial cells was not enhanced even when the infection time was extended to seven days. we would like to suggest another entry strategy that may apply to brsv and bhv- . both viruses have been associated with the bovine respiratory disease complex, a multifactorial disease that involves the interplay of different factors such as stress and microbial co-infections. bacterial pathogens associated with brd are mannheimia haemolytica, pasteurella multocida or mycoplasma bovis [ , , ] . infection of the epithelium by either of these microorganisms may induce a response reaction in the epithelial cells that make them susceptible to virus infection. for example, if the cells are responding by expressing suitable virus receptors that are otherwise absent from the cell surface, brsv or bhv- may initiate infection. thus, in the future it will be interesting to analyze the interaction of bacterial pathogens with differentiated airway epithelial cells and to determine whether bacterial infection predisposes the epithelium to virus infection. the analysis of the entry mechanism of the three bovine viruses is hampered by the fact that suitable antibodies against bovine nectin- , the receptor for bhv- , are not available commercially and the receptor for brsv has not been identified. however, when bacterial co-infection increases the susceptibility to infection by brsv, this approach may help to identify the receptor for brsv. also, our finding that pneumocytes are susceptible to brsvinfection, may be helpful in this respect. taken together, our data provide new insights in the airway infection by bovine respiratory viruses that may help not only to understand the pathogenicity of viruses involved in brdc but also to develop new intervention strategies. additional file : infection of well-differentiated baec by bhv- -gfp at different mois. bhv- -gfp was applied to the apical surface of ali cultures at an moi of . , . or for h. cultures were fixed at - dpi. virus-infected cells are shown in green. additional file : teer time course of infected ali cultures. cultured cells were apically infected with either of the three viruses or mock-infected and the transepithelial resistance was measured before infection, post infection and further at daily intervals (a). teer was also measured in egta-treated cultures before infection, directly after egta treatment, and at the indicated time points post-infection (b). values were corrected for the blank resistance. mean values and standard deviation of three independent cultures are shown. additional file : ttf- labeling of pcls. brsv-infected pcls were stained for ttf- followed by a mouse-cy second antibody (red). upper panels show a ttf- positive, infected cell typically located at the intersection of two or three alveoli. the middle panel represents an example of an infected cell with ttf- negative staining and a characteristic flattened shape. lower panels show ttf- positive 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tieraerztliche hochschule hannover. we are grateful to prof. klaus conzelmann for providing recombinant brsv and dr wiebke köhl for generating brsv-gfp. we thank gyula balka for information about ttf- as a marker for pneumocytes. financial support was provided by a grant of deutsche forschungsgemeinschaft (sfb , tpa ). authors' contributions jk and gh conceived and designed the experiments; jk, su and kg performed the experiments; jk, su, gk, kg and gh analyzed the data; gk contributed reagents/materials/analysis tools; jk and gh wrote the paper. all authors read and approved the final manuscript.submit your next manuscript to biomed central and take full advantage of: key: cord- -ux shvji authors: saade, georges; deblanc, céline; bougon, juliette; marois-créhan, corinne; fablet, christelle; auray, gaël; belloc, catherine; leblanc-maridor, mily; gagnon, carl a.; zhu, jianzhong; gottschalk, marcelo; summerfield, artur; simon, gaëlle; bertho, nicolas; meurens, françois title: coinfections and their molecular consequences in the porcine respiratory tract date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ux shvji understudied, coinfections are more frequent in pig farms than single infections. in pigs, the term “porcine respiratory disease complex” (prdc) is often used to describe coinfections involving viruses such as swine influenza a virus (swiav), porcine reproductive and respiratory syndrome virus (prrsv), and porcine circovirus type (pcv ) as well as bacteria like actinobacillus pleuropneumoniae, mycoplasma hyopneumoniae and bordetella bronchiseptica. the clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. in this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. we discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health. bacterial and viral respiratory diseases are a major health issue in species reared under confined conditions in large groups. most often multiple infectious agents are involved in the development of these clinical conditions making unsuited the common reductionist approach of host-pathogen interactions by the study of single infection [ ] . infection by more than one type of pathogen (viruses, bacteria and parasites amongst others) is described as a mixed infection. however, the term coinfection is frequently used to describe concomitant infection of a cell or a host by separate pathogens [ ] . since in the literature the definitions of coinfection and mixed infection have been both used to describe the same events, we will use the term "coinfection" in the current review. additionally, in virology, the term superinfection is used if one virus infects the cell or the host before infection by the second superinfecting virus. we will also use the term "superinfection" in the review. finally, an opportunistic pathogen is usually considered as a pathogen that would not have infected animals in absence of the primary infection, or alternatively, "pathogen" that would have been asymptomatic in the absence of the primary infection. in some studies, however, the use of the terms "coinfection" is not suitable and "superinfection" should be used instead, as we will see later. this semantic point is responsible for a lot of confusion and makes comparisons between studies sometimes tricky. the outcome of any coinfection or superinfection can be affected by the interactions taking place between the infectious agents, the nature of the cell/host, adverse environmental and management conditions, intestinal and respiratory microbiomes, and the triggered immune response-innate and adaptive-developed afterwards [ , ] . when occurring at the same time or with a delay, infections can impact the virulence of causative pathogens with subsequent consequences on the host immune response and its ability to clear the infections [ ] . the first contact with a pathogen can change the cell/host response against any other second pathogen, possibly causing a more virulent infection, reducing its severity or suppressing it completely [ ] . thus, different scenarios concerning the pathogen interactions can be observed, the first infectious agent can promote the second one, attenuate its effects or simply prevent its establishment. conversely, the second pathogen may also influence the first one directly or indirectly. coinfections have been described in both humans and animals [ , ] . moreover, bacterial and viral infections might be followed by secondary bacterial or viral infections, which in some cases are responsible for the pathology development and the observed clinical signs. in this review, the current knowledge regarding frequent coinfections that occur in the porcine respiratory tract and particularly in the lungs are reviewed. when possible, we focused on the interactions between the mentioned pathogens and the various mechanisms justifying these interactions and their consequences on the host's response. we especially discussed coinfections involving main bacteria and viruses associated with the so-called porcine respiratory diseases, excluding coinfections involving parasites and fungi (including their metabolites, such as mycotoxins). moreover, we do not discuss the impact of adverse environmental and management conditions which have been shown to be of major importance in the modulation of respiratory infections' severity [ ]. respiratory diseases have been formally described in pigs as early as the ′s [ ] and several studies have been carried out to identify associated agents. the role of the infectious pathogens has been assessed by using two main approaches: direct research of the pathogens (by culture or polymerase chain reaction-pcr for instance) from tissue samples of diseased (acute or chronic stage) and non-diseased pigs or indirect detection by serological tests to look for antibodies produced after exposure to specific pathogens. these studies indicated that frequently under field conditions, several infectious pathogens are simultaneously detected from lung lesions (see [ ] [ ] [ ] causative respiratory infectious agents can be divided into primary and secondary or opportunistic pathogens. primary pathogen being defined here as pathogen that can infect the animal as first unique pathogen and then facilitate secondary or opportunistic coinfection. these primary pathogens include common bacteria such as highly virulent actinobacillus (a.) pleuropneumoniae, m. hyopneumoniae, bordetella (b.) bronchiseptica in young piglets and common viruses such as swiav [ ] . prrsv and pcv are not strictly respiratory pathogens as swiav, however, since they also frequently affect the respiratory system and since they can act as facilitators of secondary respiratory infections, they must be considered too. other primary pathogens such as aujeszky's disease virus (adv) and prcov are reported but they are far less frequently encountered today or they have less impact on porcine health [ ]. then, some viruses like the porcine cytomegalovirus can also inhibit host immune functions-particularly the action of t lymphocytes-and promote respiratory diseases such as the porcine reproductive and respiratory syndrome [ ] . among the secondary pathogens common bacteria such as lower virulence strains of a. pleuropneumoniae, a. suis, glaesserella parasuis, pasteurella multocida, and streptococcus (s.) suis are reported. together primary and secondary pathogens are involved in the "porcine respiratory disease complex" (prdc) [ ] . several studies have assessed the nature of the infectious agents directly or indirectly associated with respiratory diseases in pigs [ , , , ] . in one of these studies involving breeding sows in five french farrow-to-finish herds [ ] , results indicated that s. suis, a secondary pathogen, was quite widespread among sows- . % of the animals being positive using a pcr assay-and pcv and swiav infections were highly prevalent ( % of the sows with antibodies against pcv and between . % and % of the sows with antibodies against swiav). other infectious agents such as a. pleuropneumoniae, g. parasuis and p. multocida were detected in %, %, and % of the sows, respectively [ ] . in another study evaluating infectious agents associated with respiratory diseases in farrow-to-finish pig herds in france, it has been shown that m. hyopneumoniae, prrsv, and swiav subtype h n were the major pathogens involved in pneumonia-like gross lesions [ ] . for extensive pleuritis, prrsv was frequently associated with a. pleuropneumoniae [ , ] . regarding bacteria associated with lung lesions in french slaughter pigs [ ] , a report mentioned lesions of pneumonia and pleuritis as the most frequent lesions. in these lesions, bacteria such as m. hyopneumoniae, p. multocida, a. pleuropneumoniae, s. suis, and g. parasuis were detected in . %, . %, . %, . %, and . % of the lungs, respectively [ ] . in a retrospective analysis of the etiologic agents associated with respiratory diseases in pigs in usa, two or more infectious agents were identified in . % of the analyzed cases [ ] . prrsv ( . % of the samples), p. multocida ( . %), m. hyopneumoniae ( %), swiav ( . %), g. parasuis ( . %) and pcv ( . %) were the infectious agents most frequently encountered [ ] . in korean pigs, prrsv and pcv were frequently identified associated or not to various bacteria such as s. suis ( . %), m. hyopneumoniae ( . %), p. multocida ( . %), and a. pleuropneumoniae ( %) [ ] . below we review the main primary pathogens as defined above, common viruses such as prrsv, pcv , swiav, prcov and adv as well as bacteria like a. pleuropneumoniae, m. hyopneumoniae and b. bronchiseptica. conversely, other pathogens involved in the prdc are not presented in the following sections while considered in additional file presenting the different coinfections' situations. prrsv is an enveloped single stranded positive rna virus belonging to the arteriviridae family. two different species, prrsv- (also known as betaarterivirus suid ), from european origin, and prrsv- from american origin, are now distinguished [ ] . this enveloped virus replicates mainly or exclusively in macrophages such as alveolar macrophages (ams), but also macrophages from the nasal mucosa and pulmonary intravascular macrophages (pims) [ , ] . in vitro, prrsv can also replicate in cultured monocytes and monocyte-derived cells including macrophages [ ] and in vitro-derived dendritic cells (dcs) generated either from bone marrow hematopoietic cells (bmdcs) or blood monocytes (modcs), depending on the in vitro culture conditions [ , ] . however, such in vitro generated dcs are not representative of in vivo primary dcs which do not seem to be permissive to viral replication [ ] . in fact, modc and bmdc (at least when generated using granulocyte macrophage colony-stimulating factor, gm-csf) although possessing functional overlaps with the dc family, do not represent bona fide dcs, which represent an own lineage of hematopoietic cells distinct from the monocytic lineage [ ] . different cell surface molecules are involved in prrsv entry and infection of cells: heparan sulfate, porcine sialoadhesin-also known as sialic acid-binding immunoglobulin-type lectin (siglec- ), siglec- , cd and cd [ , ] . heparan sulfate is a glycosaminoglycan (gag) that seems to play a modest or secondary role in prrsv infection since the blocking of this receptor on ams induced only a mild decrease in prrsv infectivity. moreover, this effect was not observed with all the prrsv isolates tested, suggesting that the involvement of heparan sulfate depends on the antigenic diversity of prrsv [ ] . siglec- /cd is a member of the sialic acid-binding lectins (siglecs) family and is expressed on macrophages [ ] and siglec- has been identified as an alternative receptor to siglec- [ ] . binding of prrsv to siglecs induces its internalisation by clathrin-mediated endocytosis. expression of recombinant porcine sialoadhesin is sufficient to induce the internalisation of prrsv by non-permissive cells, but not replication [ ] . cd is a scavenger receptor involved in prrsv infection [ ] . its expression on nonpermissive cells makes them susceptible to infection with prrsv and allows productive replication of the virus [ ] . moreover, cd -ko animals are still susceptible to prrsv- infection [ ] , whereas cd -ko animals are resistant to prrsv- and prrsv- [ , ] . finally, myh has been recently identified as an indispensable partner of cd for prrsv cell entry for both prrsv- and prrsv- [ ] . prrs clinical signs can be nearly absent to severe depending on the considered prrsv species and strains. when observed, there are, amongst the most frequent, lethargy, dyspnea, tachypnea, as well as a reproductive disease [ ] . prrsv can persist in infected pigs for several months after the initial infection particularly in lymphoid tissues and has the ability to alter the host's immune system to escape it (for review see [ ] ). prrsv interferes with the porcine innate immune response through downregulation of type i interferons (ifns-ifnα and ifnβ mostly), which are cytokines known for their antiviral properties [ ] . prrsv-infected macrophages also had a reduced capacity to produce the pro-inflammatory cytokines tnfα and il β [ ] and the production of the anti-inflammatory cytokine il was found enhanced during infection [ ] . nevertheless, the role of cytokine modulation during prrs is unclear considering that the effects appeared to depend on the prrsv species, as well as on the prrsv isolates, since opposite results can be found with different prrsv strains [ , ] . in fact, some prrsv- isolates were shown to enhance ifnα production while other prrsv- isolates suppressed it. results seemed also very variable for the immunoregulatory il along different isolates of prrsv- [ , ] , making general conclusions about how prrsv alters innate immune responses difficult. prrsv impact on adaptive cellular immunity seems also to be highly variable according to the species and the strain [ ] . conversely, whereas non-protective antibody response against the viral nucleocapsid is found within a week post-infection, neutralizing antibodies appearance is highly delayed for all prrsv species and strains, appearing only after or weeks of infection and peaking even later [ ] . pcv is a naked circular single stranded dna virus belonging to the circoviridae family and responsible for porcine circovirus disease (pcvd). the attachment of pcv to target cells occurs through chondroitin sulfate b and probably other receptors [ ] . internalisation is not fully known but it does not seem to involve a specific receptor and the gags could mediate internalisation and binding to the target cells [ ] . most of the time the infection is subclinical but in some circumstances such as coinfections with other respiratory pathogens it can cause the post-weaning multisystemic wasting syndrome (pmws), clinically characterized by wasting respiratory disease, and enteritis [ ] . infection with pcv can occur in utero, resulting in stillborn piglets and mummified fetuses, or death at different ages after birth [ ] . in young and older animals, pcv was found in cells expressing monocytes (cd + ), and t and b cells (cd + , cd + , igm + ) markers [ ] . further results showed that active replication of the virus was supported by t and b cells, with enhanced replication in proliferative cells [ ] . in vitro, pcv can also infect many other cell types including endothelial cells, gut epithelial cells, fibrocytes, and dcs [ ] . in dcs the virus seems to persist and remain infective for a prolonged period without replication indicating that these cells might serve as a vehicle for virus spread in the host [ ] . pmws is characterized by the depletion of lymphoid cells affecting t cells, b cells, and nk cells [ ] . this lymphopenia was also associated with impaired responses of peripheral blood mononuclear cells (pbmcs) to mitogen stimulation with lower levels of il , ifnγ, and il production compared to pbmcs from non-infected pigs [ ] . another feature of pmws is an elevated level of il found in lymphoid organs, especially in the t cells rich areas [ ] . il -mediated immunosuppression could play an important role in the pcv infection and the development of pmws. pcv has also the ability to alter the innate immune response [ ] . even though the virus does not productively infect dcs, evidence shows that it can interfere with the normal plasmacytoid dcs (pdcs) response. upon stimulation with cpg-odn, pdcs' ability to produce ifnα and tnfα was impaired in cells previously infected with pcv [ ] . pcv dna isolated from infected cells induced the suppression of pdc ifnα production [ ] . influenza a viruses are enveloped single stranded negative rna viruses belonging to the orthomyxoviridae family. these enveloped viruses can infect a broad range of hosts, with pigs being one of their natural hosts (for a review see [ ] ). the three main iav subtypes encountered in pigs are h n , h n , and h n [ ] , but many genetic lineages and antigenic variants within these subtypes are co-circulating in the pig population worldwide. subclinical infections with swiavs are common in pigs, but they can also induce a disease similar to what is observed in humans, with upper respiratory tract distress associated with fever, cough, rhinitis, high morbidity, and low mortality [ ] . the main targets of swiavs are epithelial cells of the respiratory tract but iavs can also non-productively infect alveolar macrophages [ ] . two major glycoproteins are present at the surface of the virus: hemagglutinin (ha) and neuraminidase (na). binding of ha with the sialic acid molecules at the surface of the host cells will induce the endocytosis of the viral particle [ ] . the na molecule plays the main role in the budding of the virus by removing the sialic acid, allowing the release of neoformed virus particles from the infected cell [ ] . the innate response against the virus includes production of high levels of pro-inflammatory cytokines such as ifnα, tnfα, and il . dcs, in particular pdcs play an important role in this response [ ] . an important observation was that the production of these cytokines correlated to the viral loads and the severity of the disease. infection with swiav induces cellular and humoral specific immune responses in pigs recovering from the disease and the serum igg and the mucosal iga can protect the animal from re-infection [ ] . ns and pa-x are the main viral proteins that alter the innate immune response, mainly by blocking the type i ifn response [ ] as well as the nlrp inflammasome activation [ ] in infected-epithelial cells and alveolar macrophages. finally, the main mechanisms through which the swiav escapes the adaptive host immune system are the antigenic drift and the antigenic shift concerning mainly ha and na which are also the two major antigenic proteins expressed on the surface of the virus and against which the neutralizing humoral response is directed [ ] . prcov is an enveloped single stranded positive rna virus belonging to the coronaviridae family. in pigs, four alphacoronavirus, one betacoronavirus and one deltacoronavirus have been described [ , ] . thus, most of the porcine coronaviruses are from the genus alphacoronavirus. the only respiratory porcine coronavirus, prcov, is a variant of transmissible gastroenteritis virus (tgev) where a large ′ region deletion (nucleotides - ) in the spike gene of the virus altered the tropism and the virulence. even if pigs have been shown to be susceptible to the first sars-cov (serological evidence and isolation of the virus in a pig farm in the xiqing county of tianjin, china) [ ] they have not been successfully experimentally infected, at this stage, by sars-cov- [ ] . prcov uses aminopeptidase-n (cd ) domain iv to enter cells [ ] and replicates to high titers in the lungs ( × - tissue culture infectious dose -tcid ) specifically in type and pneumocytes. moreover, it can infect epithelial cells of the nares, trachea, bronchi, bronchioles, alveoli, and, occasionally, alveolar macrophages [ ] . infections with the prcov are usually subclinical, but there is variation between strains and some can induce a more severe disease. prcov can infect pigs of all ages by direct contact transmission or aerosol [ ] . the clinical signs are associated to the respiratory system and are mild to severebronchointerstitial pneumonia-depending the strain and the context (environmental and management factors as well as the presence of other pathogens). suid herpesvirus , usually known as prv or adv is the responsible agent of aujeszky's disease in pigs. it is a double stranded enveloped dna virus from the herpesviridae family and alphaherpesvirinae subfamily targeting respiratory and/or genital mucosae for its replication [ ] . adv has a very broad host range varying from domestic animals like pigs, cattle, goats, sheep, cats and dogs to wild animals such as ferrets, foxes, hares, raccoons, and wild deer, and where it induces different diseases [ ] . infected animals usually show fever, sneezing, coughing and vomiting accompanied occasionally with typical nervous manifestations like convulsions, aggressiveness and lack of coordination. mortality rate can reach % in suckling piglets while in mature pigs the infection is inapparent or mild [ ] . adv possesses eleven types of envelope glycoproteins playing major roles in the interaction with host cells and the induction of immune response [ ] . viral binding and fusion with the plasma membrane of the target cell-epithelial cells, neurons and alveolar macrophages-are controlled by a cascade of events orchestred by glycoproteins c (gc), gb, gd, gh and gl. the binding process starts with an interaction of gc with heparin sulfate proteoglycans [ , ] . stabilization of this interaction is then assured by the binding of gd to specific cellular receptors known as herpesvirus entry mediators such as hvea (tnfrsf ), hveb (prr , nectin ), hvec (prr , nectin ), hved (pvr, cd ), and -o-sulfated heparin sulfate [ , ] . at this stage, tyrosine-based or dileucine-based endocytosis in parallel with clathrin-mediated endocytosis occur by the mediation of gb, gh and gl, leading to the penetration of the capsid and the tegument into the cellular cytoplasm. finally, the interaction of the capsid with dynein leads to the release of viral dna into the cellular nucleus after a transport along microtubules from the periphery to the nuclear pores [ ] . porcine humoral immune response is induced by adv and neutralizing antibodies are mainly directed against gc [ ] . specific cell mediated immune responses are also triggered and mhc class i restricted, gc-specific, cytotoxic cells are induced. adv also alters the ifn signaling pathway by suppressing stat tyrosine phosphorylation leading to an inhibition of ifn-stimulated genes (isgs) expression [ , ] . adv may be involved in the prdc and can be isolated alone or with other pathogens. accordingly, a study conducted in taiwan reported the association of adv with pcv in . % of the evaluated pigs using a multiplex pcr [ ] . animals affected with this gram negative bacterium develop a pleuropneumonia characterized by fibrinohemorrhagic necrotizing bronchopneumonia and fibrinous pleuritis which can reach a high mortality rate [ , ] . although the disease is best known in its acute/peracute forms, subacute and/or chronic presentations with low or no mortality are highly prevalent, especially in the presence of antibiotic treatments. many herds are subclinically infected without previous or present episodes of clinical disease and in the absence of suggestive lesions at the slaughter house. animals are, nevertheless, carriers of the pathogen. this happens in several conventional herds which may be simultaneously infected not only with several low/intermediate virulent strains, but also, in some cases, with strains highly likely to cause disease. in the latter case, outbreaks may suddenly appear in the presence of concomitant diseases or as a consequence of changes in management and/or environment [ , ] . eighteen serotypes of the bacterium have been described, which can all induce disease, although clear differences in virulence have been described [ , ] . these bacteria can be found mainly in tonsils of carrier animals; virulent strains have a tropism for the lower respiratory tract where they preferentially bind to ciliated cells of the terminal bronchioli and pneumocytes [ , ] . different virulence factors expressed by a. pleuropneumoniae are involved in the colonization and the development of the disease. adhesion to cells could be mediated by type iv fimbriae that are expressed upon contact with respiratory epithelial cells in vitro and during lung infection [ , ] . adhesion of a. pleuropneumoniae to respiratory epithelial cells also involves the binding of bacterial lipopolysaccharides to glycosphingolipids on the surface of the cells [ , ] . the formation of biofilm by the bacteria is likely to play an important role in the colonization of the host [ ] . after attachment to the target cells, the bacteria can produce four different pore-forming exotoxins (apx i, ii, iii and iv) inducing the lysis of alveolar epithelial cells, thus allowing the acquisition of nutrients by the bacteria, but also participating in the development of the lesions [ , ] . some of the virulence factors expressed by a. pleuropneumoniae interfere with the host's immune response. the toxins apx i, ii and iii induce the lysis of not only respiratory epithelial cells, but also of cells involved in the innate immune response such as macrophages and neutrophils [ , ] . at lower concentrations, these toxins lose their lytic properties but can still impair macrophages chemotactic activity and their phagocytic abilities [ ] . the capsular polysaccharides of a. pleuropneumoniae interfere with macrophage phagocytosis and enable resistance to complement-mediated killing [ ] . a. pleuropneumoniae may also interfere with the antibody response by producing proteases that can degrade porcine iga and igg [ , ] . this cell wall-free bacterium is considered to play a primary role in prdc and is the causative agent of porcine enzootic pneumonia (ep), a disease with high morbidity but low mortality rates [ ] . the main pathological mechanisms involved in m. hyopneumoniae infections are: (i) adhesion to the ciliated cells of the tracheal epithelium inducing ciliostasis, loss of cilia and exfoliation, dysregulation of cellular homeostasis (with increased intracellular calcium concentration) and secretion of cytotoxic factors, (ii) alteration of the mucociliary tract, (iii) inflammatory reactions sometimes exacerbated and prolonged, and (iv) manipulation of the innate and adaptive immune responses [ , ] . among the adhesins described in m. hyopneumoniae, p is reported to be a major determinant of cell adhesion [ ] [ ] [ ] . several other adhesins were reported: p linked to p , lpps, lppt, mgpa, p , p , p , p , p , and p [ , ] . most adhesins are transcribed and translated during m. hyopneumoniae infection and then undergo posttranslational cleavage to result in diverse products on the membrane surface [ , , ] . the diversity of surface proteins can also derive from the variation in the number of repeats in genes encoding adhesins [ ] . these mechanisms of antigenic variation enable the bacterium to escape from immune system recognition and to invade the host [ ] . adhesins can also recruit extracellular matrix components (plasminogen, fibronectin and actin amongst others), and therefore can promote invasion and inflammatory response [ , ] . the immune response induced against m. hyopneumoniae, may have a double action: over-activation of the local immune response resulting in a pathologic inflammatory reaction or local immunosuppression explaining the chronic nature of the associated pathologies [ , ] . acute m. hyopneumoniae infection leads to the recruitment and activation of various innate immune cells, essentially through the involvement of a large range of cytokines: il , il and tnfα in lungs; cxcl , il , il , il , il , tnfα and il in bronchus-associated lymphoid tissue (balt) or tracheobronchial lavage fluid (tblf) [ , , ] . some of these inflammatory cytokines (tnfα, cxcl , il β, il ) are produced chronically in the lungs and play powerful roles in apoptosis (tnfα), differentiation and chemotaxis of neutrophils (respectively, il and il ), and macrophage activation (tnfα, il β). chronic infections are typically associated with intense lymphoid hyperplasia [ ] and are characterized by an accumulation of igg-and iga-expressing plasma cells, cd + t cells, macrophages and dcs in the balt of inflamed lung tissue [ ] . involvement of t cell activation in chronic inflammation is also supported by the presence of t-cell cytokines such as il- and il- in bronchoalveolar exudates [ ] . in vitro studies conducted with macrophages cocultured with m. hyopneumoniae highlighted a strong activation of inflammatory pathways inducing the production of cytokines and chemokines, and expression of receptors or pathways inducing cell apoptosis [ , , , ] . moreover, m. hyopneumoniae is described as an inhibitor of macrophages phagocytic activity, which may explain the chronicity of m. hyopneumoniae infections and the greater host susceptibility to other pathogens [ , , ] . mycoplasma hyopneumoniae was found to activate costimulatory molecule expression on bona fide dcs with poor tnfα production, contrasting with monocytes. interestingly, a strong mitogenic activity for b cells was observed [ ] . altogether, these data indicate that m. hyopneumoniae is well sensed by the innate immune system, but the presence of immune evasion mechanisms targeting antigen presenting cells remains a possibility that needs further investigations. antibody responses after infection develop slowly and do not appear to correlate with protection [ , ] . the literature on m. hyopneumoniae infections coupled with information from mouse models indicates that adaptive immune responses represent a fragile balance between pathogenic and protective th-cell responses, probably belonging to the th or th types [ , ] . this aerobic gram-negative bacterium can be found in the respiratory tract of several animal species and it presents a worldwide distribution in the porcine rearing [ ] . b. bronchiseptica has a strong tropism for ciliated cells from the respiratory tissue and is mostly detected in the apical portion of the ciliated cells of turbinates, trachea and lungs [ , ] . it can also be found in the cytoplasm of neutrophils and macrophages and rarely in the alveolar lumen associated with small tufts of cilia [ , ] . hence, infected pigs show cilia loss in the bronchial and bronchiolar epithelium associated with multifocal erosion, fibrosis, and hyperplasia. neutrophil infiltrates are noted in the peri-conchal meatus and the submucosa of the bronchioles and alveoli, while lymphocyte and plasma cell infiltrations occur at the level of the lamina propria [ , ] . cell adhesion of b. bronchiseptica is a multifactorial process involving two main virulence factors; filamentous hemagglutinin (fha) and pertactin (prn) [ , ] . the expression of both adhesins is controlled by the bordetella virulence genes (bvg)as signal transduction system. fha is an adhesin with several binding domains including a carbohydrate-recognition domain responsible of the adhesion to macrophages and ciliated epithelial cells, a heparin-binding domain that mediates the binding to sulfated polysaccharides, and an arg-gly-asp domain (rgd) regulating the intercellular adhesion molecule (icam ) by epithelial cells after interaction with the nf-κb signalling pathways [ ] . this rgd domain is also present in the structure of prn and contributes to the binding process [ , ] . on the other hand, non-opsonic adhesion mechanisms play a role in binding to the host cells such as carbohydrate-specific mechanisms and those involving sialic acid-containing compounds [ ] . virulence of the bacteria depends on the strains; therefore, clinical signs can be different going from sneezing and transient nasal discharge for moderate and non-toxic strains to bronchopneumonia and atrophy of the nasal turbinate bones for virulent strains, especially if they are associated to other bacteria such as p. multocida [ ] . thus, b. bronchiseptica is usually described as primary lung pathogen in young pigs where it causes necrohemorrhagic bronchopneumonia whereas in older pigs this bacterium is mostly known as an opportunistic pathogen contributing to the prdc [ ] . the immune response against b. bronchiseptica is mainly triggered by the different toxins expressed such as adenylate cyclase, tracheal cytotoxin and dermonecrotic toxin (dnt). in the following section and additional file , we have used the published studies evaluating multiple infections including viral-viral, bacterial-viral, viral-bacterial and bacterial-bacterial respiratory coinfections and superinfections in swine. both in vivo and in vitro studies comparing single to multiple infections were included. studies evaluating vaccinations and the development of diagnostic techniques such as elisa or qpcr were excluded as well as trials testing antiviral or antibacterial molecules when there was no clear comparison between single and multiple infections. an attempt to present a synthetic view of coinfections is depicted in the heat maps (see figures , , ) . however, we recommend readers to refer to additional file for each coinfection couple to get a more detailed view. in these heat maps, we were interested in the effect of the first pathogen on the multiplication of the second one (named "assessed pathogen" in the figures) and on the host immune response and/or clinical signs. these effects were evaluated and a grade from − to + was given to every pathogen depending on the intensity of its impact on the multiplication of the second agent and on the immune response or on the clinical signs. negative grades were given to pathogens decreasing the multiplication of other pathogens, while positive grades were given to those inducing an increase. similarly, negative grades were attributed to pathogens with a tendency to decrease clinical signs or immune response related to the other pathogen. positive grades were given in case of an increase. the sum of the grades was calculated if the same pathogen combination was evaluated in several studies except in the case of discordant results. this grading was represented in the following heat maps and the number of the identified studies for the same pathogen combination is shown on the maps. in the heat maps, other pathogens, that are less associated to prdc such as g. parasuis, m. hyorhinis, m. floculare, p. multocida, and s. suis or even not considered as respiratory pathogens like staphylococcus aureus, classical swine fever virus, hepatitis e virus, porcine rubulavirus, ppv, and torque teno sus virus have also been included. indeed, these pathogens can also impact the outcome of respiratory infections and deserve, at least, to be mentioned. viral/viral respiratory coinfections have always had an important role in the porcine respiratory disease complex [ ] . several studies assessed the presence of two or more viral pathogens in pigs showing respiratory clinical signs in farms located in endemic regions [ , , ] . the main viruses contributing to the porcine respiratory disease are swiav, prrsv, pcv , and to a lower extent the prcov and the adv. due to their fast-spreading and their economic consequences, some viruses were more studied than others in the last years, especially pcv , prrsv, and swiav as shown in additional file a and b. we will thus put more emphasis on these three viruses as causes of primary infections. many in vivo studies were carried out to assess the severity of the clinical signs and the development of the microscopic/macroscopic lesions. this approach enabled a comparison between coinfection/superinfection and single-infection conditions. then, viral interference was progressively more frequently measured as a way to better understand the consequences of coinfections. in the last decades, the strong development of molecular biology and various tools enabled the evaluation of the immune response developed following polyviral infections. in additional file , the selected studies that were carried out on viral coinfections are presented from the oldest in vivo experiments to the latest in vitro and ex vivo experiments (additional file a and b). this data synthesis highlights the major impact of prrsv primary infection, which can both increase the titre of the following virus (pcv , hepatitis e virus-hev) in vitro [ ] and in vivo [ ] [ ] [ ] (figure a ), but can also worsen the clinical score associated to the disease ( figure a ). interestingly, even when the prrsv does not increase the viral production of the other virus, as observed in coinfections involving swiav [ ] or prcov [ ] (figure a) , it can also worsen the associated clinical signs. swiav and pcv as primary infectious agents have been less studied. however, it can be observed that swiav can interfere with other virus productions (prrsv and prcov) [ , ] whereas pcv has some detrimental impact on the clinical outcome of secondary viral infections (prrsv, swiav, and ppv) [ ] [ ] [ ] (see additional file and figure a) . then, regarding the inflammation induced in coinfection conditions, various outcomes were observed depending which viruses were considered (figure a) . many in vitro and in vivo experiments, with different bacterium-virus and virus-bacterium combinations, have been performed to identify the underlying mechanisms of the prdc (see figures b, c, b , c, and b). the main studies are presented in additional file c and d. bacterium-viral coinfections can also involve various primary respiratory pathogens. among them, the most frequently studied bacterium is m. hyopneumoniae, a pathogen that induces a chronic respiratory disease and can influence the outcome of a subsequent viral infection. however, mycoplasma infection needs to be already well established in the respiratory tract at the time of the viral infection to potentiate it. indeed, m. hyopneumoniae inoculated to pigs simultaneously or shortly before the virus did not strongly impact the severity of the viral infections (pcv , swiav, prrsv) [ ] [ ] [ ] , while its impact was clearly evidenced when inoculated weeks before viral infections [ ] . it is well-known that viral infections can induce an ideal environment for a bacterial superinfection through different mechanisms such as the destruction of the epithelial barrier, the over-expression of the receptors involved in the bacterial adhesion to the cells, and the alteration of the host immune response [ , , , ] . the swiav infection has been shown, for instance, as a way to facilitate the colonization of epithelial cells by s. suis, but only for the serotypes containing sialic-acid in their capsule [ ] . the swiav infection induces a loss of ciliated cells leading to the impairment of the mucociliary clearance function, but induces also the presence of the viral ha on the surface of infected cells that interacts with the sialic acid of the bacterial capsule, leading to increased adherence of s. suis [ , ] . although these swiav effects on s. suis have been clearly shown in vitro, no clear in vivo impact of swiav infection on s. suis pulmonary load has been described [ ] . it was clearly shown that the presence of both pathogens significantly induces more inflammation than single infections [ , ] . overall, studies carried out in pigs showed that a bacterium-virus or a virus-bacterium coinfection frequently induces an aggravation of pulmonary lesions ( figure b ) and a higher inflammation ( figure b ) and immune response, with increased production of pro-inflammatory cytokines. in many bacterium-virus and virus-bacterium associations, this worsened outcome seems to be the result of additive effects from both pathogens rather than a real synergy [ , ] . however, a potentiation of the viral infection by bacteria can also be observed in other cases, such as in the m. hyopneumoniae-prrsv coinfection [ ] . in that case, higher amounts of prrsv genomes were detected in lymphoid tissue and blood [ ] and a slower viral clearance was observed [ ] (figure b) , suggesting that the recruitment of immune cells in the lung parenchyma upon established m. hyopneumoniae infection may provide a steady supply of susceptible cells for prrsv [ ] . then, in porcine ams and in the "african green monkey" (originally described as porcine origin) st-jude porcine lung (sjpl) cell line, prrsv infection has been shown to be blocked by a pre-infection with a. pleuropneupmoniae, this antiviral activity being due to the a. pleuropneumoniae metabolites [ ] ( figure b) . given the fact that in vivo studies involving prrsv and a. pleuropneumoniae did not always investigate the impact of an a. pleuropneumoniae pre-infection on the subsequent prrsv infection [ , ] , as done in experiments performed in vitro [ ] , it cannot be easily concluded if this interference would be observed in the target species. however, in vivo, prrsv-a. pleuropneumoniae interactions were reported as absent or mild [ , ] (figure b ). in virus-bacterium coinfections, the dogma usually encountered is that viruses play an immunomodulatory role, which favors bacterial superinfections. nevertheless, a pre-disposing effect is also described for m. hyopneumoniae, which promotes viral but also bacterial superinfections [ ] (figure d ). few studies of experimental coinfections or superinfections with m. hyopneumoniae and/or other bacteria involved in prdc were performed compared to coinfections involving viruses. these studies are reported in additional file e. overall, these coinfections or superinfections induce more clinical signs and lung lesions and poorer technical performances when compared to single infections with the same infectious pathogens ( figure c ). the bacterialbacterial coinfections are also responsible for immune response alterations (for reviews see [ , ] ). for example, macrophages from pigs infected by m. hyopneumoniae decrease their phagocytosis capacity against a. pleuropneumoniae [ , ] . m. hyorhinis and m. flocculare, two mycoplasmas commonly co-isolated with m. hyopneumoniae in gross pneumonia-like lesions, may also impact the immune response by inducing the cytotoxicity of immune cells and/or the secretion of cytokines affecting its outcome [ ] . co-stimulation of porcine bmdcs with m. hyopneumoniae and m. hyorhinis induces a strong il production. in this last in vitro model, m. hyopneumoniae associated with m. flocculare reduces tnfα production compared to bmdcs stimulation by m. flocculare alone producing a tnfα concentration greater than that observed after stimulation with m. hyopneumoniae alone and m. hyorhinis alone [ ] . therefore, m. flocculare might play an initial role in pulmonary inflammation by inducing the production of tnfα by resident myeloid cells. supplementary investigations will be needed to elucidate the role of this cytokine in the pathogenesis of the disease [ ] . other examples of bacterium-bacterium in vivo coinfections are presented in additional file e. regarding coinfections and superinfections, most studies assessed the clinical outcome of the process but less is known about the mechanisms of interactions between pathogens and the consequences for the pathogens themselves, the infected cells and more generally for the host. the outcome of dual infection is variable depending on the antagonism, neutrality or synergy between the infectious agents. on the host side, coinfection can make the host response ineffective, and vice versa. if we look now at the possible interactions that can occur between pathogens we have to consider the nature of the infectious agents (summary provided in figure ) . different situations can be observed and coinfections can involve virus with virus, bacterium with virus and vice versa, and bacterium with bacterium. regarding virus-virus interactions, consequences are diversified and many studies looking at virus replication in coinfection situations have been carried out [ ]. the first consequence of coinfection could be the so-called viral interference, a situation whereby one virus interferes with the replication of the other one making the cells resistant to the superinfecting virus [ ] . the most common way for viral interference is indirect and based on the production of type and ifns which induce the expression of isgs after interacting with their cognate receptors [ , ] . these proteins then activate numerous mediators of the cellular antiviral system that may non-specifically block the replication of viruses. they may also interfere, to a certain extent, with bacterial multiplication since ifn can also be induced by intracellular bacterial pathogens (ibps) or some extracellular bacteria [ , ] . nevertheless, in some situations type ifns can also increase the host susceptibility to subsequent bacterial infection [ ] through impaired macrophage recruitment with a reduced cxcl and cxcl transcription [ ] and a reduced il [ ] production. then, there is also the non-interferon-mediated viral interference (or intrinsic interference) which is a cellular state of resistance induced by the virus to new viral infection by closely related or unrelated viruses [ ] . various mechanisms are described to explain this cellular state of direct or indirect resistance (for examples see [ ]). in this type of interference, which can occur between viruses but also between viruses and bacteria [ , ] , there is a competition between pathogens for the metabolites and all the host factors that allow their multiplication. besides the mechanisms involving a competition for common cellular factors, there are also several other mechanisms of interference described. these relies on viral defective interfering (di) particles [ ] , rna interference (rnai) [ ] , non-specific double stranded rna (dsrna) [ ] as well as trans-acting proteins [ ] . interference can occur at specific steps or multiple steps of the viral replication cycle (attachment, penetration, genome replication and/or budding) and can be direct or indirect. inhibition of superinfection (superinfection exclusion and superinfection suppression) is one of several consequences that can be observed in the interference between related and unrelated microorganisms. in superinfection exclusion, an established infection interferes with a subsequent, closely related infection [ , ] . an example of this phenomenon in pigs is the exclusion of highly virulent classical swine fever virus strain margarita in wild boars persistently infected with this virus upon a challenge infection with the same margarita strain [ ] . superinfection suppression is a quite close concept where this time persistently infected cells resist to a challenge with a heterologous virus [ ] . furthermore, when the host immune response-innate or adaptive-is considered in the study of the complex interactions taking place in viral coinfections, additional mechanisms of indirect interference linked to cellular and humoral cross-protection-resulting from a first viral contact with a wild-type or a vaccine strain-can be described. conversely, in some situations, viral coinfections can directly or indirectly result in enhanced replication and virulence for one or both pathogens as observed in several studies involving porcine viruses [ , , [ ] [ ] [ ] . in other cases, coinfection/superinfection has no effects on virus replications and the viruses can coexist in a relation called accommodation [ ] . besides consequences in terms of viral replication, there are also consequences for the genetic of the viruses and their evolution through events of recombination between closely related viral genomes. recombination, the parameters influencing it and its consequences were reviewed in rna and dna viruses [ , ] . then, as a result of all these possible interactions between viruses, the severity of the resulting disease and its epidemiology can be altered as exemplified in additional file . in the pig studies, most often, however, the exact mechanisms controlling interactions between viruses were not elucidated. several mechanisms explaining bacterium-virus and virus-bacterium interactions have been identified (for reviews see [ , , ] ). the interactions can have either a positive or a negative impact on both pathogens depending on the bacterial and viral species involved. usually, when the interactions are direct they promote viral infection without affecting the bacterial species [ , , ] . examples of these direct interactions are (i) direct binding of the virus to a bacterium or (ii) the utilization of a bacterial product by the virus. an example of direct interactions in the respiratory tract is the cleavage of the iav ha into ha and ha by a staphylococcus aureus protease helping the viral particle to become infectious [ ] . on the contrary, when interactions are indirect they often provide an advantage to bacterial infections. four mechanisms dealing with indirect interactions have been described: (i) viral alteration of the epithelial barrier, (ii) reduction or suppression of the host immune response, (iii) viral alteration/displacement of the microbiota, and (iv) virus-induced alteration of bacterial cellular receptor expression [ ] . all these mechanisms can operate together for the benefit of the superinfecting bacteria. a typical example of these indirect interactions is provided by pcv and swiav and porcine pathogenic bacteria such as a. pleuropneumoniae [ ] and s. suis [ , , ] where the bacteria benefit from the prior viral infections. however, bacteria can also directly benefit from a previous viral infection as observed in a study demonstrating that staphylococcus aureus was able to bind viral ha [ ] . the consequence of that binding was an enhanced bacterial internalisation by two mechanisms: (i) binding to ha exposed at the surface of infected cells, and (ii) binding to free extracellular virions. in some other situations, non-pathogenic bacteria can also directly or indirectly protect the host from viral infection as typically observed with probiotic bacteria which can show antiviral activity through the binding/ capture of the viruses and/or the competition for cell adhesion (for a review see [ ] ). this type of interaction has been frequently observed with enteric bacteria [ ] and an example in pigs is the reduced infection of ipec-j cells by vesicular stomatitis virus after pre-incubation of the cells with multiple probiotic bacteria [ ] . an intriguing relationship is occurring between ibp and viruses where metabolic reprogramming in host cells triggered by viruses might support or conversely limit coinfection by an intracellular bacterial partner (for a review see [ ] ). different possibilities can be identified in that type of interaction [ ] : (i) the first pathogen can reprogram cellular metabolism related to cellular immunity and decrease the defense against the other pathogen, (ii) the metabolic changes triggered by the first pathogen can facilitate the adhesion, the penetration, and the replication of the other, and (iii) the coinfection transform the active replicative state of the first pathogen into a stable persistent state. the first possibility associated to a decrease of the cellular defense is a commonly accepted mechanism [ ] while the second and the third possibilities are less experimentally demonstrated [ ] . looking at bacterium-bacterium interactions, they are extremely complex to assess because of the large diversity of the bacterial world and because little is known about the mechanisms underpinning these interactions during infections. moreover, it is now also clear that intestinal and respiratory microbiomes affect the interactions between pathogenic bacteria and the porcine host [ ] . some examples of the complex interactions occurring in bacteria-bacteria coinfections are presented in additional file e, but little is known about the mechanisms controlling these interactions. however, some mechanisms were provided above and interesting reviews dealing with that subject were published recently [ , ] discussing the possible direct interactions between bacteria-mainly chemical and physical. indirect interactions between bacteria were not reviewed in these articles but were discussed to some extent in other review papers focusing on polymicrobial infections [ , ]. the first observation coming from this review must be, even if several studies have been carried out on the subject, a lack of data about some specific coinfections and many discrepancies between studies. for instance, there are only a few in vivo studies about pcv in virus/virus coinfections and about pcv and prrsv in virus/bacterium coinfections (additional file and heat maps in figures , , ) . discrepancies are not surprising because of the definition of coinfection is not always the same between studies in addition to huge variations in the coinfection parameters amongst studies. in this review we focused on experimental (in vivo and in vitro) coinfections, it is worth to underline that these studies are inspired by field veterinarians and epidemiologists observations. however, the definition of epidemiologist coinfection can also vary between studies. indeed, in some cases there is concomitant direct identification of two microorganisms in the same animals, sometimes in the same farms, while in other cases it is just an indirect identification of the microorganisms' presence at some points through indirect serological assays. moreover, as stated before, the term coinfection is sometimes used to describe some situations of superinfections where the delay can be significant. regarding the experimental parameters, the multiplicities of infection (moi), the strains, the potential delays between successive infections, the routes of inoculations, the types of cellular hosts considered (more or less susceptible to one of the microorganism), the genetic background (breeds) and the sanitary status (specific pathogen free or conventional breeding) of the pigs, and the readout to assess coinfection outcome varied a lot between studies. to fully compare studies, a standardization of the assays would be needed. interestingly also, whereas in vitro studies' usefulness is not in question, it is important to underline here that in vitro observed interplay between pathogens cannot be automatically applied in vivo. for instance, whereas m. hyopneumoniae decreases the prrsv titre in vitro [ ] , it increases prrsv shedding in vivo and indeed worsens the clinical signs upon coinfection [ ] . consequently, the use of intermediary settings, such as co-culture of different cell types (see [ ] for examples), precision cut lung slices (pcls) [ ] or organoids [ ] , could help to understand the complexity of coinfections in the respiratory tissue. in vitro approaches usually consider one or a few cell types with some limitations during the evaluation process of the coinfection consequences. some viruses can contribute to the elimination of other viruses just because of their ability to replicate faster on a particular cell type [ ] . thus, results obtained in vitro cannot mimic the field situation when both agents coinfect the same pig, providing inaccurate conclusions about the coinfection dynamics. under such circumstances, pathogens may simply have different host cells and no longer be under direct competition for resources [ ] . besides the different interactions that infecting agents can have between them through a competition to resources, studies showed clearly that the immune system and the immunological responses can highly affect these interactions by inducing the competitive power of a pathogen or abolishing it and making it less competitive on the resource [ , ] . the effects of the immune system (especially humoral parameters) are often not taken into consideration in selected in vitro models [ ] . on the other hand, in vivo coinfection experiments have to deal with numerous constraints (health status of the animals used, cost, husbandry, and ethics amongst others) and therefore are not always easy to perform. hence, although in vivo experiments are required in this very complex field, they surely need to be combined to in silico/in vitro/ex vivo analyses of potential interactions between pathogens. moreover, multiple parameters of the coinfection protocols appeared difficult to set without any a priori such as the choice of the pathogen that will be inoculated first and the delay between infections. one possibility to deal with multiple parameters is to use intra-host infection mathematical modelling [ ] allowing to play, at limited cost, with the different parameters of the coinfections. however, these models need to be fed with data coming from conventional in vitro experiments as well as more complex in vivo studies. the other possibility is to rely on field prevalence studies monitoring the very presence of the pathogens (isolation, pcr) instead of the sero-conversion, in order to have a clear epidemiological picture of when and where coinfections occur. consequently, ex vivo models such as pcls generated from freshly sacrificed pigs [ ] or organoids [ ] are developing. these models are closer to mimic the in vivo situation than usual in vitro approaches, combining different types of cells and providing the pathogens with a wider range of cell hosts. however, the contribution of the immune response to the interaction between different pathogens is rarely considered [ ] . furthermore, the moi cannot be controlled because the number of infected cells in the slice or the organoid cannot be monitored easily either. another limiting factor in coinfection studies is the cell regeneration, which can vary between in vivo and in vitro models. cell regeneration can highly affect the dynamics of a coinfection, giving some pathogens extra target cells guarantying their longer existence while contributing to the clearance of others [ ] . finally, other potential technical limitations could always be discussed such as the lack of precision or sensitivity in the different diagnostic techniques especially in the presence of multiple agents. hence, the detection of coinfecting pathogens could be compromised or reduced as compared to their detection level in the context of single infections. as shown in this review many works have been dedicated to the study of coinfections and superinfections in pigs. usually, when the experiments were carried out in vivo, the researchers were more interested in the clinical outcome than in the interactions occurring between pathogens. indeed, in most of the cases the fine interactions between pathogens and especially the mechanisms behind these interactions and its potential consequences, at the molecular level, on the immune response were not studied for several reasons including technical limitations. also, in the studies assessing the occurrence of coinfections/superinfections in the fields, coinfection identification based on molecular tools such as pcr would be more accurate than sero-prevalence approaches which are less prone to identify currently present pathogens and thus coinfective pathogens. then, a better knowledge of each pathogen involved is crucial. we thus would like to make recommendations for future studies dealing with respiratory coinfections in pigs: (i) authors should clearly summarize their coinfection or superinfection experimental setup-doses of pathogens, delays between infections-in their materials and methods section; (ii) in this summary they would need to clearly present the pathogens they use and they should, as often as possible, select well-characterized strains; (iii) environmental and management conditions would need a strict control and monitoring; (iv) animal genetic and sanitary status would need to be carefully described and monitored during the study; and (v) the multiplications of all the pathogens shall be followed during the experiment using highly sensitive and specific assays. a clear description of all these parameters would help the scientific community to compare studies and progress in the understanding of the complex interactions between microorganisms. in the last years, the concept of innate immune memory or trained immunity has gained a lot of interest. this concept is coming from old observation, in [ ] , recognizing that the bacterial vaccine strain "bacille de calmette et guérin" (bcg) was protecting not only against mycobacterium tuberculosis but also against antigenically different microorganism causing childhood mortality, suggesting an "adaptation" of the cellular innate immune system. since then, many interesting studies about innate immune memory or trained immunity have been published (for a review see [ ] ) and it is recognized that cells such as myeloid cells, nk cells, and even epithelial cells [ ] can have a higher and quicker response upon re-exposure to a pathogen. trained immunity is accompanied by epigenetic changes and most often associated with modifications in cellular metabolism. a close look at potential epigenetic changes and cellular metabolism modifications would be of high interest in respiratory coinfection studies in the porcine species. recently an alternative to the mechanism of trained immunity in resident lung innate immune cells named "epigenetic legacy" has been described [ ] . in that study, the authors demonstrated that following iav clearance and clinical recovery 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be considered at the within-host scale? a modelling contribution to prrsv infection results of bcg immunization in new york city trained immunity: a program of innate immune memory in health and disease trained immunity carried by non-immune cells influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the colleagues who shared their experience regarding coinfections in pigs. fm is supported by an establishment grant from the région pays de la loire (rfi food for tomorrow-cap aliment). gs phd is funded by foundation philippe jabre (liban). all the authors were involved in the writing of the review. gs, nb, and fm generated the figures. gs, cd, jb, cf, cm-c, gsi, nb and fm prepared the additional file . all the authors read and approved the final manuscript. key: cord- -dw qsl s authors: porter, emily; tasker, séverine; day, michael j; harley, ross; kipar, anja; siddell, stuart g; helps, christopher r title: amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: dw qsl s recent evidence suggests that a mutation in the spike protein gene of feline coronavirus (fcov), which results in an amino acid change from methionine to leucine at position , may be associated with feline infectious peritonitis (fip). tissue and faecal samples collected post mortem from cats diagnosed with or without fip were subjected to rna extraction and quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) to detect fcov rna. in cats with fip, % of tissue, and % of faecal samples were pcr-positive, as opposed to % of tissue, and % of faecal samples in cats without fip. relative fcov copy numbers were significantly higher in the cats with fip, both in tissues (p < . ) and faeces (p = . ). pcr-positive samples underwent pyrosequencing encompassing position of the fcov spike protein. this identified a methionine codon at position , consistent with the shedding of an enteric form of fcov, in % of the faecal samples from cats with fip, and in % of the samples from cats without fip. in contrast, % of the tissue samples from cats with fip and % from cats without fip had a leucine codon at position , consistent with a systemic form of fcov. these results suggest that the methionine to leucine substitution at position in the fcov spike protein is indicative of systemic spread of fcov from the intestine, rather than a virus with the potential to cause fip. feline coronavirus (fcov) infection is ubiquitous in domestic cats, particularly in multi-cat households where up to % of animals may be infected [ ] [ ] [ ] . the majority of fcov infections are asymptomatic or are associated with mild enteric disease [ ] . however, approximately - % of infected cats develop the invariably fatal disease, feline infectious peritonitis (fip) [ ] [ ] [ ] . one of the most important questions in fcov research is why some fcov-infected cats develop fip, whereas others remain healthy. one current model of fip pathogenesis proposes that cats are infected with fcov by the faecal-oral route. subsequently, the virus mutates into the virulent form. this form has an enhanced tropism for monocytes/macrophages, and in vitro studies suggest that this is reflected as sustainable replication in, and subsequent activation of, monocytes [ , ] . these activated monocytes carry the virus in the blood and, as a result of complex interactions with endothelial cells, induce the granulomatous phlebitis that is the pathogenic hallmark of fip [ , ] . the age, breed, gender, reproductive status and immune response of individual cats also influence the development of fip [ ] . currently, there is intense interest in determining which mutations alter the virulence of fcovs. a recent paper published by chang et al. [ ] derived full genome sequence data from a collection of fcovs obtained from the faeces of healthy cats and from the tissues of cats diagnosed with fip. they provided evidence of an association between fcov virulence and an amino acid substitution (methionine to leucine at position , m l) within the putative fusion peptide of the fcov spike (s) protein. specifically, the authors concluded that the m l substitution distinguished fip from non-fip associated fcovs in % of cases. a second substitution, two amino acids downstream of m l (serine to alanine at position , s a) distinguished a further % of fip from non-fip associated fcovs. the s protein fusion peptide is a critical element in the fusion of viral and cellular membranes during virus entry [ ] and it is reasonable to think that amino acid substitutions within this peptide may alter the tropism of the virus. in addition, a study by licitra et al. has shown that it is possible to distinguish between fcovs from animals with and without fip on the basis of one or more substitutions in the amino acid sequence that comprises the furin cleavage motif within the s protein [ ] . this furin cleavage site (consensus motif r-x-k/r-r, where r is the basic arginine residue, x is any residue and k is the basic lysine residue) delineates the border of the receptor-binding (s ) and fusion (s ) domains of the s protein and is distinct to the m l substitution site described above. mutation at this site is proposed to alter proteolytic cleavage of the s protein and modify s protein fusogenic properties, which again may relate to the tropism of the virus [ ] . finally, pedersen et al. [ ] concluded that truncating and non-truncating mutations in the c gene occur in a significant proportion of fcovs associated with fip. chang et al. [ ] suggest that functional c protein expression is crucial for fcov replication in the gut but is dispensable for systemic replication. however, they also do not exclude the possibility that the loss or alteration of the c protein may enhance the fitness of the virus in the monocyte/macrophage environment. over the past years, the university of bristol has collected a large number of post-mortem tissue and faecal samples from a cohort of thoroughly examined cats. these include cats with a definite diagnosis of fip, confirmed by the presence of the typical histological fip lesions, in which immunohistochemistry (ihc) demonstrated fcov antigen within macrophages [ ] , and cats with diseases other than fip that completely lacked any histological changes consistent with fip. importantly, this long-term study has enabled both faecal and tissue samples to be collected from fcov-infected cats with and without fip, allowing comparable samples from naturally infected cats to be examined. samples were screened for fcov rna by quantitative reverse transcriptase-polymerase chain reaction (qrt-pcr) [ ] and, if positive, were assessed for the m l substitution by pyrosequencing. post-mortem tissue samples, and faeces whenever possible, were collected from cats that were euthanized with suspected fip, or due to other diseases. fip was then definitively diagnosed or excluded by histopathology and, in the case of fip, the demonstration of fcov antigen in fip lesions by ihc [ ] . tissues of cats without fip were also tested by ihc for the presence of viral antigen. tissue samples were collected into rnalater (life technologies) within h of death for subsequent molecular analysis. the tissue samples were left in rnalater for - h at room temperature or °c before the rnalater was discarded, and the tissue samples stored at − °c. the faecal samples were stored at − °c until use. further samples were collected into % neutral-buffered formalin for histology and ihc. the tissues collected comprised primarily mesenteric lymph node, liver, kidney, spleen and omentum, while other tissues (e.g. intestine, brain, lung, pericardium, pancreas or other lymph nodes) were included based on gross pathological findings or reported clinical signs. the formalin-fixed tissue samples were subjected to standard processing for histopathology. they were embedded in paraffin wax and sections prepared and stained by haematoxylin-eosin. sections were examined by a boardcertified veterinary pathologist (mjd) at the university of bristol for histopathological changes. selected wax blocks were then sent to veterinary laboratory services, university of liverpool for ihc analysis as previously described [ ] . for a cat to be assigned to the "fip group", it needed to have histopathological changes consistent with fip in which fcov antigen was demonstrated within macrophages in lesions [ ] . for a cat to be assigned to the "non-fip group", histopathological changes consistent with fip needed to be completely absent, and fcov antigen within macrophages needed to be absent for all tissues. only individual tissue samples with a positive qrt-pcr result, and lesions consistent with fip on histopathology were used for pyrosequencing. faecal samples were classified on the basis of the diagnosis attributed to the cat from which the sample originated. total rna was extracted from mg of tissue or mg of faeces with a nucleospin rna ii kit (macherey-nagel) using methods based on previous work by dye and siddell [ , ] . reverse transcription was done using a mj mini gradient thermal cycler and improm ii reverse transcriptase (promega). nine microlitres of total rna solution were combined with μl improm ii × reaction buffer, . μl mm mgcl , μl dntps ( mm each), μl random hexamers ( . μg/μl) and μl improm ii reverse transcriptase. the reaction was made up to a total volume of μl with rnase-free water. the following thermal profile was used; °c for min, °c for min, °c for min and °c hold. the resulting μl of cdna was added to μl of rnase-free water and stored at − °c. randomly selected samples were checked for inhibition of the rt reaction using an rna internal amplification control. no inhibition was detected (results not shown). the qpcr was done on an agilent mx p qpcr system (agilent technologies). a qpcr master mix was made for each reaction with . μl × gotaq master mix (promega), . μl of μm forward and reverse primer (p /p ) ( table ) , . μl of μm taqman probe (table ), . μl mm mgcl and made up to μl with rnase-free water. the primers and probe were produced by metabion (metabion international) and were described previously by dye et al. [ ] . one-tenth of the randomly primed cdna ( μl) was added to the pcr master mix. the reaction plate was heat sealed and the following thermal profile was used: °c for min, cycles of °c for s, °c for s and °c for s. fluorescence was detected at nm during the extension phase. feline cov cdna was used as a positive control and rnase-free water as a negative control. reactions that failed to reach the threshold cycle (ct) value by cycle were deemed to be negative. a ct value of was assigned a relative copy number of [ , ] , and the following equation, which takes into account the % efficiency of the qrt-pcr assay [ ] , was used to calculate the relative copy number of each qrt-pcr positive sample: . ( -ct value) . all samples that were positive by fcov qrt-pcr underwent conventional pcr to amplify a base-pair dna fragment encompassing position in the s protein gene. pcr was done using a mj mini gradient thermal cycler. briefly, for each reaction, a pcr mix was made that included . μl × gotaq master mix, . μl of μm forward and reverse primer (f /r ) (table ) , μl of randomly primed cdna reaction products and water to a volume of μl. the following thermal profile was used; °c for min, cycles of °c for s, °c for s and °c for s, before being held at °c. the pcr products were used for the pyrosequencing reaction or stored at - °c until required. samples that failed to produce definitive sequence data were pyrosequenced, following repeat amplification using the same pcr protocol with cycles of amplification. single strand sequencing templates were produced by binding the biotinylated pcr product to streptavidincoated sepharose beads (fisher), followed by chemical denaturation and neutralisation. for each sample, the following mix was prepared; μl streptavidin beads, μl pyromark binding buffer (qiagen) (ph . containing mm tris-hcl, m nacl, mm edta, . % tween ) made up to μl with water. the bead mixture was added to the pcr product and shaken at rpm for min. the sequencing primer mix contained . μl μm sequencing primer (table ) and . μl pyromark annealing buffer (qiagen) ( mm tris-oac, mm mg-oac ph . ) for each sample. the control oligonucleotide (a self-sequencing oligonucleotide) mix contained μl μm oligonucleotide c and μl annealing buffer. twenty five microlitres of the sequencing primer mix was added to the appropriate wells of a pyrosequencing plate, μl of control oligonucleotide was added to one well, and the plate was placed on the pyrosequencing workstation. the pyrosequencing workstation was prepared with trays containing wash buffer ( mm tris-oac ph . ), denaturing buffer ( . m naoh), % ethanol and distilled water. the streptavidin bead bound pcr product was taken up using a vacuum pump and the pyrosequencing bead collector. the bead collector was then placed in the % ethanol for s, in the denaturing buffer for s and in the wash buffer for s. after turning off the vacuum, the bead collector was placed in the pyrosequencing plate and agitated for s to dislodge the beads. the pyrosequencing plate was heated on a plate holder at °c for min, before being placed into the pyromark q (qiagen) and left to cool for min. while the plate was cooling, the pyrosequencing cartridge was prepared. pyromark gold q enzyme, substrate and dntps (qiagen) were added into the appropriate wells of the cartridge. volumes were as outlined by the pyromark q software. during the experimental set up, the dispensation order of the nucleotides was defined as; cgctcatg. the cartridge was placed into the pyromark q instrument and the protocol run. all primers used in the pyrosequencing assay were designed using a combination of pyromark assay design software (qiagen), primer ' software [ ] and mfold [ ] , and were made by eurofins (mwg operon) ( table ). the primer positions were based on those used by chang et al. [ ] , and numbered according to the fcov c je genome [genbank:dq ]. degeneracies were added to the primers, and the location of the primers optimised, based upon a sequence alignment comprised of all available type i fcov genomes (data not shown). conventional pcr to amplify a base-pair dna fragment encompassing position in the s protein gene was carried out as described above, (see pyrosequencing methods) on samples that did not show a m l substitution in the pyrosequencing assay. the pcr primers (f /r ) were then used as sequencing primers in a standard sanger sequencing protocol (eurofins, mwg operon). the fcov relative copy numbers were entered into a database (excel , microsoft) and exported into ibm spss statistics software (version . ). the data sets were evaluated for normal distribution using the kolmogorov-smirnov (k-s) test. non-normally distributed data were described as median and range (minimum and maximum values). data evaluating fcov relative copy numbers in tissue and faecal samples from cats with and without fip were analysed using a multilevel modelling approach (mlwin v . ) [ ] , to account for the repeated measures within cats, and a non-parametric mann-whitney u test. the conclusions drawn from both analyses were in full agreement, so the simpler mann-whitney u test analysis is presented here. relative copy numbers were compared between the fip and non-fip samples for tissue and faecal samples combined, for faecal samples only, and for tissue samples only. significance was assigned at a level of p < . . historical samples were collected with full informed consent from owners that samples could be used for research purposes. the project has been approved under ethical review by the university of bristol animal welfare and ethical review board (vin/ / ). a total of samples were analysed and full details of the samples and results are shown in table . in cats with fip, the diagnosis was confirmed by histopathology and the demonstration of fcov antigen within macrophages in fip lesions by ihc. in cats without fip, the diagnosis was made by histology; neoplasia (e.g. lymphoma, astrocytoma, chemodectoma, or biliary cystadenoma), and inflammatory processes (e.g. chronic lymphoplasmacytic infiltrates of unknown aetiology in liver and kidney, and bronchopneumonia) predominated. a total of faecal samples were analysed by fcov qrt-pcr, and ( %) were positive. these comprised of ( %) faecal samples from cats with fip, and of ( %) faecal samples from cats without fip (table ) . a total of tissue samples were analysed by fcov qrt-pcr, and ( %) were positive. these comprised of ( %) tissue samples from cats with fip, and of ( %) tissue samples from cats without fip (table ) . relative fcov rna copy numbers were not normally distributed (p < . ). the relative copy numbers in pooled faecal and tissue samples in the fip group (median; range: ; - ) were significantly higher (u = . , p < . ) than in the non-fip group ( , - ). when only tissue samples were considered, the relative copy numbers in the fip group ( ; - ) were also significantly higher than those in the non-fip group ( ; - ) (u = , p < . ). finally, analysis of faecal samples alone showed that the relative copy numbers in the fip group ( ; - ) were significantly higher than those in the non-fip group ( ; - ) (u = . , p = . ). the faecal and tissue samples with positive qrt-pcr results were subjected to the pyrosequencing assay. of the faecal samples successfully sequenced, were obtained using the cycle pyrosequencing assay, whereas required the cycle assay. of the tissue samples successfully sequenced, were obtained using the cycle pyrosequencing assay, whereas required the cycle pcr. in of the ( %) faecal samples positive by qrt-pcr, a methionine (aug) codon alone was found at position (table and figure a ). in of the ( %) samples, a leucine codon (uug) alone was found at position (table and figure b) . additionally, ( %) sample (cat , faeces) showed a mixed population of rna coding for either methionine (aug) or leucine (cug) at this position (table and figure c ). importantly, methionine and leucine codons were identified in faecal rna samples from both cats with and without fip (table ) . specifically, a methionine codon was identified in samples from fip cats, and a leucine codon was identified in samples from fip cats. overall, a methionine codon was found in the majority ( / ; %) of faecal samples from cats with fip, but a significant number had a leucine codon ( / ; %). similarly, a methionine codon was identified in samples from cats without fip (including one mixed infection), and a leucine codon was identified in sample from cat without fip (which had the mixed infection). overall, a methionine codon was found in all faecal samples from cats without fip, and sample in cat had a leucine codon ( / ; %, which also had a methionine as a mixed infection). in of the ( %) tissue samples positive by qrt-pcr, a leucine codon ( uug, cug) alone was found at position (table and figures a and b ). in the remaining of the ( %) tissue samples, a methionine codon (aug) was found at this position ( table and tissue types are defined by superscripts: a omentum, b liver, c kidney, d lung, e stomach, f lymph node, g cerebrum, h abdominal lymph node, i heart, j spleen, k colon, l mesenteric lymph node, m brain, n mesentery, o pleura, p pericardium, q medulla, r pons, s small intestine, t intestine. the amino acid coded at position in the s protein of fcov rna from tissue samples without the m l substitution is defined by the superscripts: ucu (ser) and gcu (ala). figure c ). no mixed infections were found in tissue samples. importantly, leucine and methionine codons were identified in rna from tissue samples from both cats with and without fip (table ) . specifically, a leucine codon was identified in samples from fip cats, and a methionine codon was identified in samples from fip cats. overall, a leucine codon was found in the majority ( / ; %) of fip tissue samples, but a significant number had a methionine codon ( / ; %). similarly, a leucine codon was identified in samples from cats without fip, and a methionine codon was identified in sample from cat without fip. overall, a leucine codon was found in the majority ( / ; %) of tissue samples from cats without fip, with a minority ( / ; %) having a methionine codon. in one specific case (cat ), we noted that the sample from one tissue (spleen) contained a leucine codon, whereas samples from three other tissues (liver, lung and mesenteric lymph node) all contained a methionine codon. conventional rt-pcr amplification products of rna from tissue samples that did not contain the m l substitution (from cats , and ) were analysed by sanger sequencing for the s a substitution. one of the samples (cat , pleura) showed an alanine codon at position . the remaining four samples ( from cat and three from cat ) all showed a serine codon at this position (table ). the most important finding in this study is that the m l substitution in the fcov s protein does not correlate with fip disease phenotype, as was proposed by chang et al. [ ] . we reach this conclusion because of two observations. first, although a leucine codon was found in the majority ( %) of tissue samples with fip lesions, a leucine codon was also found in the majority ( %) of non-fip tissue samples. second, a significant number ( %) of fip tissue samples had a methionine codon at this position. tissue samples from naturally fcov infected cats without fip have not been previously evaluated and provide an important insight into fcov infection in the absence of fip. we believe that the m l substitution is more likely to be a marker of systemic fcov infection, as opposed to a marker of fip or the development of disease. as our tissue samples were collected post-mortem, we cannot exclude the possibility that the cats without fip ( samples, from cats , , , , and ) and the leucine codon at position , would have gone on to develop fip if they had not been euthanized due to other reasons. however, histopathological changes consistent with fip were absent in all cats, and ihc did not identify fcov antigen. the finding that the majority of tissue samples from both cats with and without fip have a leucine codon at position does not challenge the idea that systemic spread of fcov is an important step in the development of fip. indeed, the latter is supported by the findings of our study, since fcov rna was found in a far greater proportion of tissue samples with fip lesions ( %) than tissue samples from cats without fip ( %), and, in those samples that were qrt-pcr positive, significantly higher fcov relative copy numbers were found in the fip samples, as has been found in a previous study in naturally infected cats [ ] . however, as sampling in our study took place post-mortem, it could also be argued that the elevated fcov levels in fip tissues were a consequence of the massive immunological dysregulation that results from the disease, rather than a contributing factor towards the development of disease. another viewpoint is that the low levels of fcov rna from the tissues of cats without fip are a result of fcov infecting only fully differentiated macrophages and monocytes, among which are tissue-specific macrophages. this view is supported by our results as ihc, a detection method of low sensitivity, did not detect fcov antigen anywhere in the pcr-positive tissues from cats without fip, providing further evidence of low level viral infection either of tissue macrophages (in persistently infected animals) or in monocytes that were in vessels in the respective organ at the time of sampling [ ] . these findings are also in accordance with recent in vivo and in vitro studies that showed only the virulent form of fcov can effectively and sustainably replicate in monocytes [ , , ] . the m l substitution was not the only s protein substitution that was proposed by chang et al. [ ] to correlate with the fip disease phenotype. they also showed that a second substitution, s a, distinguished a further % of fip from non-fip associated fcovs. we confirmed this result in so far as rna obtained from one of five tissue samples that did not show the m l substitution showed the s a substitution. we believe that, as was proposed by chang et al. [ ] , changes such as the m l and s a substitutions, and potentially others, could be representative of a class of mutations that influence the fusogenic activity of the fcov s protein and, as such, deserve particular attention with regard to the pathogenesis of fip [ ] . it is noteworthy that the substitutions identified by licitra et al. [ ] that are proposed to distinguish between fcovs from animals with and without fip are also suggested to have an effect upon the fusogenic activity of the s protein. with regard to faecal samples, our study found that an unexpectedly high percentage ( %) of faecal samples from cats with fip were fcov qrt-pcr positive and their relative copy numbers were significantly higher than those of faecal samples from cats without fip. moreover, the majority ( %) of fcov rna sequences in faecal samples from cats with fip had a methionine codon at position in the fcov s protein gene, suggesting that these animals were shedding an enteric form of the virus. it seems reasonable to suggest that these cats were infected with an enteric, and a systemic, virulent form of fcov. whether one form was derived from the other following a single infection or whether these cats were infected twice with different fcovs cannot be determined. it has been proposed that the severe immune dysregulation in cats with end-stage fip might create an opportunity for super-infection by enteric fcov circulating in surrounding carriers [ ] . more interestingly, a smaller but significant proportion of faecal samples from cats with fip ( %) provided fcov rna samples that encoded leucine at position . the current model of fip pathogenesis proposes that once the enteric form of the virus has mutated to a virulent form, it is generally no longer horizontally transmitted via the faeces [ , ] . this view has been challenged [ ] , and the current results also suggest that a systemic form of the virus can be found in the faeces of fip cats. however, we accept that this does not mean that excreted virus is necessarily able to infect further cats by the enteric route, as has recently been shown in some experimental studies [ ] . further research is necessary to resolve these issues. in contrast to the pattern shown by the analysis of faecal samples from cats with fip, the analysis of the faecal samples from cats without fip seemed more straight-forward. all "non-fip" faecal samples that were fcov qrt-pcr positive encoded methionine at position , indicative of infection with the enteric form of the virus. also, as found in our study, a shedding proportion of % by cats without fip is not unexpected [ , ] . an interesting faecal sample from a cat without fip (cat ) showed a mixed population of rnas encoding for either methionine or leucine at position . there was no evidence of fcov rna in the single tissue sample taken from cat , and, therefore, one interpretation could be that the m l substitution in the faecal sample was a relatively recent event and the virus had not yet spread systemically. however, this interpretation has to be considered as tentative because we have observed examples of negative qrt-pcr results in tissue samples from cats that were clearly fcov infected due to their fip grouping; cat , liver; cat , liver. as histopathology results and ihc for these two liver samples showed changes consistent with fip, these negative qrt-pcr results are likely to have arisen due to an absence of fcov in the particular samples taken for molecular analysis, as a variable distribution of fcov in affected tissues has been reported [ ] . our study importantly also demonstrated that a pcrbased pyrosequencing [ ] approach is a rapid and accurate method to identify single nucleotide differences at a specific position within a dna fragment, or in our case a viral genome. however, it has some limitations. some samples required , rather than , cycles of pcr amplification to generate adequate amounts of dna for sequencing. this was especially true for pcr products generated from samples that contained low amounts of viral rna, e.g. tissue samples from cats without fip. there were also several samples which, despite containing quantities of viral rna measurable by qrt-pcr, did not produce sufficient pcr products for pyrosequencing, even after amplification cycles. these samples were excluded from the results as they did not contribute any additional sequence data to the study. however, one explanation may be that, despite the degeneracy of the primers used (f /r ), differences in the viral primer binding sites may have precluded efficient amplification in these samples. in summary, we have used a pyrosequencing assay to determine the distribution of a specific m l substitution in the s protein of fcov rna obtained from a large number of post-mortem tissue and faecal samples from cats with and without fip. additionally, sanger sequencing was used to determine whether the s a substitution was present in tissue samples that did not contain the m l substitution. this represents the first study that compares similar samples from cats with and without fip with regard to the viral phenotype. our results contribute to a better understanding of fcov genomic mutations and how they may, or may not, be used as markers of the virus phenotype. the results also make clear that 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a taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type ii feline coronavirus use of a reverse-transcriptase polymerase chain reaction for monitoring the shedding of feline coronavirus by healthy cats pyrosequencing: an accurate detection platform for single nucleotide polymorphisms amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis the authors thank the veterinary practices, cat breeders and rescue centres that helped in the acquisition of samples used in this study. we also thank our colleagues, dr emi barker, dr chris palgrave, louise dawson and debra fews at the feline centre and the veterinary pathology unit, langford veterinary services, university of bristol, who have assisted in obtaining post mortem samples. we would also like to thank members of the histology laboratory, veterinary laboratory services, school of veterinary science, and university of liverpool for technical assistance. professor toby knowles of the school of veterinary sciences, university of bristol, is also thanked for his help with statistical analyses. this research was supported by a project grant from the petplan charitable trust. the authors declare that they have no competing interests. key: cord- - ti mru authors: wei, xiaona; she, gaoli; wu, tingting; xue, chunyi; cao, yongchang title: pedv enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ti mru with the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (pedv) has led to significant economic loss in the global swine industry. many studies have described how coronaviruses enter cells, but information on pedv invasion strategies remains insufficient. given that the differences in gene sequences and pathogenicity between classical and mutant strains of pedv may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the pedv gi and gii subtype strains in vero cells and ipec-j cells. we first characterized the kinetics of pedv entry into cells and found that the highest invasion rate of pedv was approximately % in the ipec-j cells and approximately % in the vero cells. to clarify the specific endocytic pathways, systematic research methods were used and showed that pedv enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin ii, clathrin heavy chain, eps , cholesterol, and caveolin- were indispensably involved. in addition, lipid raft extraction assay showed that pedv can also enter cells through lipid raft-mediated endocytosis. to investigate the trafficking of internalized pedv, we found that pedv entry into cells relied on low ph and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. the results concretely revealed the entry mechanisms of pedv and provided an insightful theoretical basis for the further understanding of pedv pathogenesis and guidance for new targets of antiviral drugs. as a type of alphacoronavirus, porcine epidemic diarrhea virus (pedv) has caused enormous economic loss to the global pork industry, especially after the emergence of highly pathogenic pedv variant strains in . pedv was first reported in in the uk [ ] , and afterward was also discovered in europe and asia [ ] [ ] [ ] [ ] . although pedv has persisted in asian swine-producing countries, it does not attract enough global attention. after october , severe ped outbreaks occurred even in chinese pig farms that were already vaccinated with cv inactivated or live-attenuated vaccines [ , [ ] [ ] [ ] . in , the first pedv outbreak occurred in the us and rapidly spread across the entire country [ ] . molecular epidemiological results show the genetic differences between classical (gi subtype) and new pedv variant strains (gii subtype) [ , , , , , ] . pedv is presently recognized worldwide due to dramatic changes observed in its epidemic character, pathogenic properties, and gene drift [ ] . studies focused on its pathogenesis [ , ] , immune evasion [ ] and developing effective vaccines [ , ] are progressing. a virus is a non-cellular life form that must rely on cells to complete its life cycle. the first step in virus infection is successful entry into cells. most enveloped viruses enter cells through cellular endocytosis [ , ] . the endocytic pathways utilized by viruses vary, including clathrin-mediated endocytosis (cme), caveolaemediated endocytosis, lipid raft-mediated endocytosis, and macropinocytosis, among others. cme is the most classical and well-known endocytic pathway utilized by viruses. after binding to cell surface receptors, the virus is packaged by clathrin-coated pits (ccps) and transported to clathrin-coated vesicles (ccvs), in which virus particles as the "cargo" will be transported to the early endosomes [ , ] . caveolae is a plasma-specific invagination structure with a diameter of - nm. when viral particles interact with receptors, caveolae coated with caveolin- invaginates and pinches off plasma membrane, then the caveolae vesicles mature into caveosomes and deliver "cargoes" to early endosomes [ , ] . lipid raft are plasma membrane microdomains enriched in sphingolipids and cholesterol that participate in the lateral organization of the cell surface. raft-mediated endocytosis is the process of internalization of ligands and receptors by these domains [ ] . the mechanisms of some coronaviruses entry into cells have already been studied, such as severe acute respiratory syndrome coronavirus (sars-cov), murine hepatitis virus (mhv), and human coronavirus (hcovs). entry of sars-cov into hepg and cos cells is clathrindependent while entry into vero e cells is clathrin-and caveolae-independent [ , ] , but the lipid raft plays an important role in the process [ ] . mhv entry into cells needs clathrin [ ] [ ] [ ] , the same as hcov-nl [ ] . for hcov- e, caveolae-mediated endocytosis is utilized to enter human fibroblast cells [ ] . sars-cov and mhv-cov can induce continuous micropinocytosis, but this occurs in the later phase during infection and is not associated with virus entry [ ] . coronaviruses enter host cells via various endocytic pathways after viral spike glycoprotein (s) interacts with receptors and then initiates the endocytic process. internalized viruses are trafficked like cargoes to membrane fusion sites through specific transport routes. different covs have varying fusion sites [ ] . the fusion site of the middle east respiratory syndrome coronavirus (mers-cov) takes place in the early endosome, while mhv and the feline infectious peritonitis virus (fipv) are transported to the lysosome to fuse. although there have been many studies on the invasion mechanism of cov, the invasion strategy of pedv has not yet been fully elucidated. in , park et al. [ ] revealed that pedv entry followed clathrin-mediated endocytosis and was dependent on a low ph for successful entry into cells. in their research, one pedv strain was studied in vero cells in the presence of trypsin and only the chemical inhibitors and confocal method were used to reveal the pedv entry. however, there are still important questions to address. considering that covs take advantage of different pathways to enter cells, whether different subtypes of pedv invade cells by different ways and whether pedv enter different types of cells through different ways remains to be determined. to concretely clarify the entry and transportation routes of pedv, we used vero and ipec-j cells as models for pedv entry and chose cv -like strain gds and highly pathogenic variant strain gds to compare the invasion strategies of different pedv subtypes. our results will advance the understanding of the pathogenesis and immune evasion of pedv. vero cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs, gibco) and antibiotics ( u/ml penicillin and μg/ml streptomycin). ipec-j cells were grown in dmem/nutrient mixture f- (dmem/f ) supplemented with % fbs and antibiotics. the pedv strains used in this study were cv -like strain gds (gi subtype, genbank id: mh . ) and highly pathogenic variant strain gds (gii subtype, genbank id: km . ). exogenous trypsin ( μg/ml) was added to proliferate pedv strains in vero cells and μg/ ml trypsin was added in ipec-j cells. sbti (soy bean trypsin inhibitor type i; sigma no. t ). the endocytic inhibitors used included dynasore (sigma-aldrich, no. ), chlorpromazine (cpz, sigma-aldrich, no. c ), methyl-β-cyclodextrin (mβcd, sigma-aldrich, no. c ), nystatin (sigma-aldrich, no. ), ammonium chloride (nh cl, sigma-aldrich, no. a ), and bafilomycin a (baf a , sigma-aldrich, no. ). antibodies against clathrin heavy chain, caveolin , eea , rab , and lamp coupled with secondary goat anti-rabbit alexa fluor and goat anti-mouse alexa fluor were purchased from abcam. the mouse anti-pedv-s monoclonal antibody [ ] and anti-pedv-n polyclonal antibody (prepared in our laboratory) were used in the immunofluorescence analysis and western blotting analysis, respectively. the overexpression plasmids of wild-type and mutant dynamin ii (gfp-dyn-wt and gfp-dyn-m), eps (gfp-eps -wt and gfp-eps -m), and caveolin- (gfp-cav-wt and gfp-cav-m) were provided by prof. mark mcniven, mayo center for biomedical discovery (rochester, mn, usa). to investigate the trypsin dependency of pedv strains, vero cells were seeded in -well plates until confluence. after washed with pbs, cells were infected with pedv strains at a multiplicity of infection (moi) of . with or without trypsin ( μg/ml) or with trypsin and μg/ml sbti for h before the quantification of the viruses by qrt-pcr. to test the dynamics of pedv internalization, vero or ipec-j cells were seeded in -well plates until confluence. the cells were pre-chilled for min and inoculated with pedv at a moi of . at °c for h for virus binding. the cells were washed three times with ice-cold pbs to remove unbounded viruses and immediately warmed to °c to initiate internalization. after incubation for the indicated time intervals, the cells were treated with proteinase k ( mg/ml) at °c for min and then washed with pbs to inactivate and remove the non-internalized pedv particles. the control cells were then washed with pbs. the cells were collected and subjected to qrt-pcr analysis [ ] . to test the effect of inhibitors on pedv internalization, it was necessary to evaluate the cytotoxicity of cell inhibitors. the cells were seeded in -well plates at a density of × cell/well, grown for h, and treated with endocytic inhibitors at the indicated concentration for h. then μl of cck- solution was added to each well and incubated at °c for h. an absorbance of nm was measured. the experiments were repeated three times independently. the concentration of each used inhibitor did not cause significant cytotoxicity to the cell viability. to test the effect of inhibitors on pedv internalization, the cells were pre-treated with different concentrations of drugs for h and then infected with gds or gds strains at moi = in the presence of drugs for h. after washing with citrate buffer (ph . ) [ ] and pbs, the cells were incubated with medium containing trypsin for h or h at °c and collected for qrt-pcr and western blotting analysis, respectively. the expression of pedv n protein was detected by qrt-pcr and western blotting with gapdh as the reference. total rna was extracted using trizol (invitrogen) according to the manufacturer's instruction and cdna was synthesized with a revertra ace qpcr rt master mix with gdna remover kit (toyobo, osaka, japan). qpcr reaction was performed using a sybr premix ex taq ii kit (takara, tokyo, japan) using a light cycler real-time pcr system (roche diagnostics, indianapolis, in, usa). for the western blotting analysis [ ] , the cells were washed with pbs and lysed in ripa lysis buffer on ice for min. after sds-page electrophoresis, proteins were transferred onto polyvinylidene fluoride (pvdf) membrane via the semidry method and immunoblotted with the corresponding antibodies. transfection of vero cells and ipec-j cells with the overexpression plasmids of wild-type and mutant dynamin ii (gfp-dyn-wt and gfp-dyn-m), eps (gfp-eps -wt and gfp-eps -m), and caveolin- (gfp-cav-wt and gfp-cav-m) were performed using lipofectamine (invitrogen) transfection reagents according to the manufacturer's protocol. the cells were seeded in -well plates until % confluence. h after transfection, the cells were infected with pedv at moi = for h. virus was moved with citrate buffer and pbs and replaced with fresh medium containing trypsin, and virus internalization was evaluated by confocal fluorescence microscope. for the rna interference assay, sirnas against dynamin ii (sidyn, sus scrofa: ′-cac ctc atg atc aat aac a- ′, chlorocebus sabaeus ′-cct aca tca aca cga acc a- ′), clathrin heavy chain (sichc, sus scrofa ′-ccc ata cca tga ctg atg a- ′, chlorocebus sabaeus ′-gat gaa cct tat gca tgc a- ′), eps (sieps , sus scrofa ′-cct gtg gat att ctt gga a- ′, chlorocebus sabaeus ′-ccc aga aac agc aag tac a- ′), and caveolin- (sicav, sus scrofa ′-caa cat gca gaa aga aat a- ′, chlorocebus sabaeus ′-cct tca ctg tga cga agt a- ′) were designed and synthesized based on the corresponding full-length mrna sequences of sus scrofa and chlorocebus sabaeus, sir-nas against rab a (sirab , ′-gat ggt gga tga cag act a- ′), and vps (sivps , ′-gct tca aga gag act act a- ′) were designed and synthesized based on the corresponding mrna homologous sequences of sus scrofa and chlorocebus sabaeus. the control sirna (sicontrol) was designed and synthesized irrelevantly to all the known genes of sus scrofa and chlorocebus sabaeus genome, respectively, by ribobio (guangzhou, china). the cells were seeded in -well plates until % confluence. to ensure transfection efficiency, a second transfection was carried out at h after the first transfection. at h post-first transfection, the cells were infected with pedv at moi = for h. virus was moved with citrate buffer and pbs and replaced with fresh medium containing trypsin, and virus internalization was evaluated by qrt-pcr and western blotting at hpi and hpi, respectively. alexa- labeled transferrin (trf ) or alexa- labeled cholera toxin b subunit (ctb) were diluted at : and mixed with pedv at moi = . the cells were washed three times with pbs and added to the mixture of pedv and trf or ctb at °c for h and then incubated at °c for min for internalization. after washing with pbs, the cells were fixed, permeabilized, blocked, incubated with mouse anti-pedv-s monoclonal antibody, incubated with alexa -conjugated goat anti-mouse igg (h + l), stained with dapi, and analyzed using a confocal fluorescence microscope. light exposure was avoided throughout this experiment. cells cultured in glass-bottom dishes for h were washed with ice-cold pbs and incubated with pedv at °c for h. cold viruses were replaced with pre-warmed medium, and the cells were immediately shifted to °c. at specific time points, the cells were fixed in % paraformaldehyde at rt for min after washing three times with pbs. permeabilization was carried with . % triton x- at rt for min. after washing with pbs, the cells were blocked with % bsa in pbst at rt for min to block unspecific binding sites. the specific primary antibodies against chc, eea , caveolin- , rab , lamp , and anti-pedv-s antibody were used to probe the cells at °c overnight. the cells were incubated with secondary antibodies (goat anti-rabbit igg antibody conjugated to alexa fluor and goat anti-mouse igg antibody conjugated to alexa fluor ) at °c for h. fluorescent images were acquired using the light-scanning module of a leica tcs sp sted × confocal microscope. the cells ( × ) were incubated or not incubated with pedv at °c for h, washed three times with ice-cold pbs, and lysed in ml tne buffer ( mm tris, mm nacl, mm edta, and ph . ) containing % triton x- and % phenylmethanesulfonyl fluoride (pmsf) on ice for min. the homogenized cell lysates were centrifuged at °c for min at g and the supernatant was mixed with isometric ml containing % sucrose in tne buffer. the lysates-sucrose mixture was placed at the bottom of ultracentrifugal tubes and overlaid with ml % and ml % sucrose in tne buffer. the cell lysates were ultracentrifuged at °c for h at g in a sw rotor (beckman). after centrifugation, twelve ml fractions were collected from the top to the bottom of the tubes. the fractions were concentrated with % peg at °c overnight, and the pellets were resuspended in μl of tne buffer after centrifuging at °c for min at g. the localizations of lipid raft-associated protein caveolin- and pedv n protein were analyzed by western blotting. all the graphs were created with graphpad prism software. all the data are presented as the means ± standard deviations (sds) from at least three independent experiments. significance was estimated using one-way anova with multiple comparisons to control. p values less than . were defined as the threshold for statistical significance. p values between . and . were marked with one asterisk, p values between . and . were marked with two asterisks, p values between . and . were marked with three asterisks, and p values less than . were marked with four asterisks. coronavirus entry is inextricably linked with proteolytic processing of the s protein. in most cases, pedv is trypsin dependent. thus, we investigated the trypsin dependency of both strains used in our research. as shown in figure a , gds strain needed trypsin while gds strain is trypsin independent. so, we added trypsin in the following assays to explore the invasion mechanism of pedv. the dynamics of viruses invading different kinds of cells vary, and there may be differences among various subtypes of the same virus. thus, it is necessary to know the entry dynamics of pedv before studying the endocytic pathways. the cells were incubated with pedv at moi = . at °c for adsorption and shifted to °c to initiate internalization. the adsorbed but not internalized virions were removed with proteinase k, and the pedv invasion rates at different time points were detected using qrt-pcr. the invasion kinetics ( figure ) showed that most of the pedv particles were detached from the cells by proteinase k at the beginning of invasion. after min in the vero cells ( figure b ) and min in the ipec-j cells ( figure c ), nearly maximum proportions of viral particles completed the internalization. approximately % of the pedv particles entered the vero cells, while only % entered the ipec-j cells. notably, the gds strain demonstrated less efficient invasion than the gds strain, but there was no significant difference. dynamin ii plays an essential role in cellular membrane fusion during vesicle formation due to its gtpase activity, and it is necessary for clathrin-and caveolae-mediated endocytosis [ , ] . thus, we explored the essentiality of dynamin ii in pedv entry using specific chemical inhibitors, overexpression of domain-negative mutants of dynamin ii, and sirna interference. dynasore [ ] , a cell-permeable non-competitive inhibitor of dynamin ii, was used to pre-treat cells at different concentrations to analyze the effect on pedv entry. the cytotoxicity test showed that μm of dynasore had no effect on the viability of the vero and ipec-j cells (additional file ). cells were pre-treated with μm and μm dynasore for h before pedv infection. dmso was used as a negative control. the effect of dynasore on pedv entry was quantified by qrt-pcr at hpi. pedv invasion was significantly inhibited by dynasore. at a concentration of μm, the invasion rates of the gds and gds strains into the vero cells were approximately % and %, and the invasion rates into the ipec-j cells were % and %, respectively ( figure a) . a comparison of the invasion rates of the gds and gds strains indicated that the gds strain was more sensitive to dynasore than the gds strain when invading the ipec-j cells but there was no significant difference between them when invading the vero cells. many studies used the overexpression of dominant negative mutants to explore the role of dynamin ii in virus entry [ , ] . mutation of dynamin ii from k to a can inhibit gtpase activity and reduce endocytosis [ ] . cells were transfected with wild-type and mutant types of dynamin ii respectively and infected with gds and gds strains at h after transfection. the confocal results showed that the vero and ipec-j cells overexpressing wild-type dynamin ii (gfp-dyn-wt) were infected with pedv while the cells overexpressing mutant dynamin ii (gfp-dyn-m) were barely infected ( figure b ). sirna interference was also used to identify the importance of dynamin ii on virus entry [ , , ] . sus scrofa and chlorocebus sabaeus sirnas of dynamin ii (sidyn) were designed and synthesized. the interference efficiency of sirna on the dynamin ii expression in the vero and ipec-j cells was obvious at both the mrna and protein levels (additional file ). cells were infected with pedv after transfection twice and the internalized virions were quantified at hpi and hpi by qrt-pcr and western blotting assay, respectively. the qrt-pcr results ( figure c ) showed that the knockdown of dynamin ii expression reduced the pedv internalization. the internalization rates of the gds and gds strains into vero cells were approximately % and %, the internalization rates into the ipec-j cells were approximately % and %, respectively, but there was no significant difference between the gds and gds strains in the two cells ( figure c ). the same results were confirmed by western blotting ( figure d ). taken together, the results suggested that pedv entry relies on dynamin ii. clathrin-mediated endocytosis is the most commonly used and classical endocytic pathway for virus entry. to identify whether pedv utilized cme to enter cells, we co-inoculated the vero and ipec-j cells with pedv and trf, which is the most typical biomolecule that uses cme to enter cells [ ] . the co-inoculation results figure trypsin-dependency and kinetics of pedv entry into cells. a vero cells were seeded in -well plates until confluence. cells were washed with pbs and infected with pedv strains (moi = . ) without trypsin or in the presence of trypsin ( μg/ml) or trypsin and μg/ml sbti. cells were collected for qrt-pcr at hpi. b, c vero cells (b) and ipec-j cells (c) were incubated with pedv gds and gds strains, respectively, at °c for h and shifted to °c immediately to initiate internalization. at , , , , , , , , and min after incubation, the cells were treated with proteinase k ( mg/ml) at °c for min to inactivate the non-internalized virions. the control cells were washed with pbs. the invasion rates were calculated by qrt-pcr analysis. ****p < . . dynamin ii involved in pedv entry. a cells were pre-treated with μm and μm dynasore at °c for h, respectively, and incubated with gds or gds strain for h. dmso was used as a negative control. the cells were collected at hpi for qrt-pcr assay to test the invasion efficiency of pedv. b vero and ipec-j cells were transfected with gfp-dyn-wt and gfp-dyn-m, respectively, and infected with pedv strains at h after transfection. the cells were fixed at hpi and stained for confocal analysis. c, d vero and ipec-j cells were transfected with sidyn twice and infected with pedv strains at h after the second transfection. the invasion rates of pedv into the cells were detected at hpi and hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. scale bars indicate μm. ** . < p < . ; *** . < p < . ; ****p < . . showed that pedv co-located with trf in the two types of cells ( figure a ), which means that pedv might utilize clathrin-mediated endocytosis to enter cells. to prove this conjecture, we used specific chemical inhibition, the overexpression of domain negative mutants of eps , the knockdown expression of chc and eps by sirna, and the location of pedv in the cells to estimate the role of clathrin-mediated endocytosis in pedv entry. cpz is a specific chemical inhibitor used to block the cme pathway by preventing the assembly of ccps at the plasma membrane [ ] . the cytotoxicity test showed that μm cpz have no effect on the viability of the vero cells and μm cpz have no effect on the viability of the ipec-j cells (additional file ). vero and ipec-j cells were treated with cpz at different concentrations for h and then infected with pedv. the internalization rates of pedv after cpz treatment were quantified by qrt-pcr and western blotting at hpi and hpi, respectively. the qrt-pcr results (figure b ) showed that pedv invasion was significantly inhibited by cpz. the invasion rates of the gds and gds strains at the highest drug concentrations were nearly % and % in the vero cells and % and % in the ipec-j cells, respectively. notably, there were no significant differences between the gds and gds strains in the vero cells but the gds strain was more sensitive than the gds strain in the ipec-j cells, reflecting the gds strain's significantly decreased invasion rates ( figure b ). the same results were also observed by western blotting ( figure c ) and ifa assay (additional files , ) . the role of cme in endocytosis was also identified by the overexpression of gfp-tagged dominant negative mutants of eps [ ] . eps is a critical component of ccps by interacting with adaptor protein (ap- ), a major clathrin adaptor complex [ ] . cells transfected with wild-type (gfp-eps -wt) and mutant eps (gfp-eps -m) were infected with pedv strains at h after transfection. the confocal results of the pedv invasion showed that the cells overexpressing wild-type eps were infected with pedv while few infections were observed in the overexpressed gfp-eps -m cells ( figure d ). sirna was also used to explore the role of cme in pedv entry by interfering with the expression of clathrin heavy chain (chc) and eps . chc and clathrin light chain form a triskelion shape, which is a key component for regulating the formation and disassembly of the clathrin lattice [ ] . cells were infected with pedv strains after transfection twice, and the invasion rates of the viruses were assessed using qrt-pcr and western blotting assay at hpi and hpi, respectively. the quantitative experiments showed that knockdown of the expression of chc significantly reduced the invasion rates of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells, respectively, and there was no significant difference between the gds and gds strains ( figure e ). knockdown of the expression of eps also significantly reduced the invasion rates of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells, respectively, and there was no significant difference between the gds and gds strains ( figure g ). the significant inhibition of sichc and sieps on pedv entry was also observed by western blotting assay (figures f, h) . to estimate whether pedv directly entered the cells through cme, we analyzed the localization of pedv and chc in the vero and ipec-j cells, respectively. pre-cooled cells were incubated with pedv at °c for h for adsorption and shifted to °c for internalization. five min later, the cells were washed and fixed for observation using an ultrahigh-resolution laser confocal microscope. the confocal results showed that pedv particles co-located with chc protein in the vero and ipec-j cells ( figure i ), but some virions were not co-localized with chc. the results indicated that pedv can enter cells through the cme pathway, but cme may not be the only pathway utilized by pedv. cholesterol, an important component of cell membranes, embeds phospholipid bilayers and plays a crucial role in the fluidity of cell membranes [ ] . many studies showed that most enveloped virus relied on cholesterol to invade cells [ , ] . if a virus invades cells, depending on the presence of cholesterol, it will be sensitive to cholesterol extractants. mβcd can eliminate cholesterol on the plasma membrane of cells [ ] . nystatin can bind to the cholesterol-enriched regions of cell membrane and then decompose cholesterol and impair cholesterol synthesis [ ] . the cytotoxicity test showed that the maximum tolerance concentrations of vero and ipec-j cells to mβcd were mm and . mm, respectively, and the maximum tolerance concentrations to nystatin were μm and μm, respectively (additional file ). cells were pre-treated with different concentrations of mβcd and nystatin for h, then infected with pedv. the effects of drugs on pedv entry were estimated by qrt-pcr and western blotting at hpi and hpi, respectively. mβcd showed a significant inhibition of pedv entry. the internalization rates of the gds and gds strains after mβcd treatment were approximately % and % in the vero cells and approximately % and % in the ipec-j cells. there were no significant differences between the gds and gds strains in the mβcd-treated vero figure pedv entry relies on the cme pathway. a vero cells and ipec-j cells were incubated with mixture of alexa- labeled trf (red) and pedv (green) at °c for h, and then shifted to °c for min. the cells were fixed and stained for pedv using monoclonal antibody against pedv s protein. the cellular localizations of trf and pedv were observed with a confocal fluorescence microscope. light exposure was avoided throughout this process. b, c. the vero cells were pre-treated with μm and μm of cpz, and the ipec-j cells were pre-treated with μm and μm of cpz, respectively, at °c for h and incubated with gds or gds strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. d the vero cells (left) and ipec-j cells (right) were transfected with gfp-eps -wt and gfp-eps -m, respectively, and infected with pedv strains at h after transfection. the cells were fixed at hpi and stained for confocal analysis. e-h the vero cells and ipec-j cells were transfected with sichc and sieps and infected with pedv strains at h after the second transfection. the cells were collected at hpi and hpi for qrt-pcr and western blotting analysis, respectively. i the cells were pre-cooled at °c for min, incubated with pedv strains at °c for h, shifted to °c for min to initiate internalization, and washed for three times to remove un-internalized viral particles. the cells were fixed and stained with anti-pedv-s (red) and anti-chc (green) primary antibodies. ctrl means control. scale bars indicate μm in a, μm in d, and μm in i. ** . < p < . ; *** . < p < . ; ****p < . . cells but in the ipec-j cells, the gds strain was more sensitive to mβcd ( figure a ). the results were confirmed by western blotting ( figure b ) and ifa assay (additional files and ). pedv entry was significantly inhibited by nystatin treatment. the internalization rates of the gds and gds strains after nystatin treatment were approximately % and % in the vero cells and approximately % and % in the ipec-j cells. the gds strain was more sensitive to nystatin than the gds strain in both the vero and ipec-j cells ( figure c ). the same results were confirmed by western blotting ( figure d ) and ifa assay (additional files and ). to further evaluate the importance of cholesterol, cells pre-treated with mβcd were supplemented with exogenous cholesterol and then infected with pedv, and the changes in the viral invasion rates were quantified by qrt-pcr at hpi. the results showed that adding exogenous cholesterol could significantly increase the invasion rate of pedv. the average invasion rate of the gds and gds strains increased from % to over % in the vero cells and from % and % to approximately % in the ipec-j cells ( figure e ). the results indicated that pedv invading and entering cells depended on cholesterol but the two pedv subtypes showed different degrees of dependence on cholesterol when entering the vero and ipec-j cells. as both caveolae and lipid raft are rich in cholesterol, they are sensitive to cholesterol inhibitors [ , ] . to identify whether caveolae-mediated endocytosis was involved in pedv entry, cells were co-inoculated with pedv and ctb, which entered the cells after interactions with specific receptors [ ] . the localization results showed that pedv co-localized with ctb in vero and ipec-j cells ( figure a ), which means pedv may utilize caveolae-mediated endocytosis to enter cells. as the caveolae is mainly coated with caveolin- [ ] , knocking down the expression and separating the interaction factors with caveolin- blocked the caveolae-mediated endocytic pathway. overexpression of the domain-negative mutant of caveolin- [ ] blocked its interaction with the interaction factors. cells were transfected with wild-type caveolin- (gfp-cav-wt) and mutant caveolin- (gfp-cav-m), then infected with pedv at h after transfection, and fixed for confocal observation at hpi. the results showed that pedv infected gfp-cav-wt-overexpressing cells but barely infected gfp-cav-m-overexpressing vero or ipec-j cells ( figure b ). sirnas (sicav) were designed and synthetized to knockdown caveolin- expression. cells were transfected with sicav twice and then infected with pedv. after the second transfection, the invasion rates of pedv were measured by qrt-pcr and western blotting at hpi and hpi, respectively. the qrt-pcr results showed that the knockdown of caveolin- expression reduced the internalization of pedv. the inhibition rates in the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells, respectively (figure c) . compared with the gds strain, the gds strain showed a higher degree of reduction in the invasion rate in the vero cells but there was no significant difference in the ipec-j cells ( figure c ). the same results were confirmed by western blotting assay ( figure d ). to identify the role of caveolae in pedv entry, we investigated the cellular localization of pedv with caveolin- . pre-cooled cells were incubated with pedv at °c and then shifted to °c for internalization. the cells were then washed and fixed for min for observation with an ultrahigh-resolution laser confocal microscope. the cellular localization results showed that pedv was colocated with caveolin- in the vero and ipec-j cells ( figure e ). pedv can enter cells through the caveolaemediated pathway. if pedv can enter cells through the lipid raft pathway, the viral components should be contained in lipid raft enrichment layer after isolated by sucrose density gradient centrifugation [ ] . after incubation with pedv, the cells were lysed and subjected to sucrose gradient centrifugation. the products were extracted from the top down and a total of fractions were obtained for western blotting analysis. caveolin- was used as the protein marker representing the lipid raft layer [ ] . the results showed that pedv could be detected in the upper lipid raft enrichment layer. almost all the virions were concentrated in the lipid raft enrichment layer in the vero cells ( figure a ) and virions were detected in both the upper and lower components in the ipec-j cells ( figure b ). the differences between the gds and gds strains were the proportion of virions in the upper and lower components in the ipec-j cells. the gds particles were mainly present in the upper layer while amounts of gds particles were present in the lower layer (figure b) . the results indicated that pedv utilized lipid rafts to enter cells. viruses that enter cells via endocytosis are usually trafficked by endocytic vesicles to early endosomes for sorting and are transported to late endosomes or fused with early endosomes [ ] . if viruses do not fuse in the early endosomes and release genomes into the cytoplasm, they will enter the late endosomes with the further acidification and maturation of the early endosomes. similarly, figure pedv entry relies on cholesterol. a, b vero cells and ipec-j cells were pre-treated with . mm and mm and mm and . mm mβcd, respectively, at °c for h and incubated with gds or gds strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j cells were pre-treated with μm and μm and μm and μm of nystatin, respectively, at °c for h and incubated with gds or gds strains for h. dmso was used as a negative control. the cells were collected at hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. e the vero cells and ipec-j cells were pre-treated with different concentrations of mβcd at °c for h, supplemented with μg/ml of soluble cholesterol at °c for h, and infected with pedv strains for h. the cells were collected at hpi for qrt-pcr assay to test the invasion efficiency of pedv. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . a vero cells and ipec-j cells were incubated with a mixture of alexa- labeled ctb (red) and pedv (green) at °c for h, and then shifted to °c for min. the cells were fixed and stained for pedv using monoclonal antibody against s protein. the cellular localizations of ctb and pedv were observed with a confocal fluorescence microscope. light exposure was avoided throughout this process. b vero cells (up) and ipec-j cells (down) were transfected with wild-type caveolin- (gfp-cav-wt) and domain negative mutant of caveolin- (gfp-cav-m), respectively, and infected them with pedv strains at h after transfection. the cells were fixed at hpi and stained for confocal analysis. c, d the vero cells and ipec-j cells were transfected with sicav twice and infected with pedv strains at h after the second transfection. the cells were collected at hpi and hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. e cells were pre-cooled at °c for min, incubated with pedv strains at °c for h, shifted to °c to initiate internalization for min, and washed for three times to remove viral particles that were not internalized. the cells were fixed and stained with anti-pedv-s (red) and anti-caveolin- (green) primary antibodies. scale bars indicate μm in a, μm in b, and μm in e. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . if the ph environment in the late endosomes does not meet the requirements for conformational changes of viral glycoproteins to cause membrane fusion, viruses will enter the lysosomes with the transportation of the late endosomes and finally achieve membrane fusion. viruses require acidic ph to traffic between endosomes. therefore, it is necessary to clarify whether pedv relies on a low ph environment. nh cl is an inhibitor of endosome acidification [ ] . baf a is a v-atpase inhibitor that can block the traffic of endocytic cargoes from early endosomes to late endosomes and inhibit the stability of low ph environments in the lysosome lumen [ ] . cells were pre-treated with different concentrations of nh cl and baf a (additional file ) and infected with pedv (additional file ). the invasion rates of pedv were measured by qrt-pcr and western blotting at hpi and hpi, respectively. the results showed that cells pretreated with nh cl significantly inhibited pedv entry. the invasion rates in the gds and gds strains were approximately % and % in the vero cells and % and % in the ipec-j cells ( figure a ). this significant inhibition was also confirmed by western blotting (figure b ) and ifa assay (additional files and ). baf a can significantly inhibit pedv entry. the invasion rates in the gds and gds strains were approximately % and % in the vero cells and % and % in the ipec-j cells ( figure c ). western blotting ( figure d ) and ifa assay (additional files , ) also confirmed the inhibition of baf a . the results proved that pedv entry requires low ph. early endosomes mature into late endosomes by increasing intraluminal acidity through proton pump activity. late endosomes can become larger vesicles by fusing with the same type of endosomes, so they mostly exist in the form of a multivesicular body (mvb). late endosomes release rab , incorporate rab , and prepare to fuse with lysosomes [ ] . to explore whether pedv particles are trafficked after internalization, we interfered with the expression of rab [ ] involved in late endosomes and vps [ ] involved in late endosometo-lysosome maturation and identified whether viral particles co-located with early endosomes, late endosomes and lysosomes. knockdown of the expression of rab (sirab ) can significantly inhibit the invasion efficiency of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells ( figure a) . similarly, the knockdown of the expression of vps (sivps ) also significantly inhibited the invasion efficiency of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells ( figure b ). for cellular location by pedv observation, pre-cooled cells were incubated with pedv at °c and then shifted to °c for internalization. the cells were washed and fixed at different time points after shift for observation using an ultrahigh-resolution laser confocal microscope. cellular localization assays showed that internalized pedv could co-locate with eea , the early endosome protein marker, in the vero were incubated or not with pedv at °c for h and then lysed in tne buffer containing % triton x- and % pmsf on ice for min. after mixing with isometric % sucrose, the homogenized cell lysates were subjected to ultracentrifugation after being overlaid with % and % sucrose. after centrifugation, a total of fractions were collected from the top to the bottom of the tubes. the localizations of the lipid raft-associated protein caveolin- and pedv n protein were analyzed by western blotting after being concentrated with % peg . and ipec-j cells min after endocytosis ( figure c ) and could co-locate with rab , the late endosome protein marker, min after endocytosis ( figure d ), while the late endosomes were mostly in the form of mvb. colocalization of pedv with lamp (lysosomal associated membrane protein ), an important lysosome membrane component, was also observed min after endocytosis in the two types of cells ( figure e ). the results demonstrated that pedv was trafficked to the lysosomes after entering the cells through endocytosis, and there were no differences between the two pedv genotypes and cells. since highly pathogenic variant strains emerged in , pedv has attracted global attention. many studies have reported that vaccines based on cv or cv -like strains have low protection efficiency against re-emerging variant strains [ , , [ ] [ ] [ ] . genotyping showed that pedv strains can be sorted into two genotypes, gi subtypes (classical) and gii subtypes (variant). the nucleotide sequence of s subunit of s protein is - % similar between gi and gii, which shows high variability [ , ] . as the main antigen of pedv, the s protein incubated with gds or gds strains for h. double-distilled water was used as a negative control. the cells were collected hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j cells were pre-treated with nm and nm baf a at °c for h and then incubated with gds or gds strains for h. dmso was used as a negative control. the cells were collected hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. ** . < p < . ; *** . < p < . ; ****p < . . vero cells and ipec-j cells were transfected with sirab and sivps twice, respectively, and then infected with pedv strains at h after the second transfection. the cells were collected at hpi for qrt-pcr analysis. ctrl means control. c-e the vero cells and ipec-j cells were pre-cooled at °c for min, incubated with pedv strains at °c for h, shifted to °c to initiate internalization. the non-internalized viral particles were removed by washing. min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-eea (green) primary antibodies (c). min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-rab (green) primary antibodies (d). min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-lamp (green) primary antibodies (e). scale bars indicate μm in c-e. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . plays an important role in inducing immune responses and viral entry. mutation of the s gene may lead to different mechanisms of virus invasion, which may help elucidate the pathogenesis and immune evasion of pedv. as sars-cov utilizes varying endocytic routes to invade different cells [ , ] , we wondered whether pedvs can enter cells through different pathways. burkard et al. clarified that coronavirus entered cells through the endosome/lysosome pathway and was proteolytic dependent. the furin cleavage site just upstream of the fusion peptide (fp) of the s protein was the key to determining the fusion site of the viral membrane [ ] . while the s protein of pedv does not have the furin cleavage site, a conserved arginine just upstream of the putative fp as the potential cleavage site can be cleaved by trypsin [ ] . whether the theory mentioned above is applicable to pedv thus needs further study. here, we explored the gi and gii subtype pathways of pedv entry into vero and ipec-j cells, respectively, and the transportation route after internalization. our results showed that two the subtypes of pedv utilized clathrin-, caveolae-, and lipid raft-mediated endocytosis to enter the vero and ipec-j cells, but the utilization efficiency of each endocytic pathway varied depending on the different genotypes and types of cells. to describe the dynamic curve of pedv entry, the appropriate viral scavenger is extremely important to remove virus particles effectively adsorbed on the cell surface. we compared the effects of citrate buffer (ph . ) with proteinase k ( mg/ml), and the latter exhibited a stronger capacity to remove viruses. the results of dynamic invasion showed that the virus invaded vero cells % within min, while the invasion efficiency of the ipec-j cells was only approximately %. although ipec-j cells are considered the host cells of pedv, the cell lines cultured in vitro lost their polar growth state in vivo [ ] , which possibly affects the viral recognition and reduces the infection efficiency of the virus. gds showed a lower invasion rate than gds , but there was no significant difference. dynamin ii, a gtpase, plays an important role in endocytosis by pinching the endocytic vesicles off the plasma and is necessary in clathrin-and caveolae-mediated endocytosis. we found that pedv entry is dynamin iidependent, although the sensitivity of the gds and gds strains to dynasore varied. considering the low specificity of chemistry inhibitor, dominant negative mutant and sirna-mediated knockdown of dynamin ii were carried out to evaluate dynamin ii for pedv infection. both the gds and gds strains were inhibited by sidynamin ii with no significant difference. clathrin-mediated endocytosis is a classical and commonly used pathway for most enveloped viruses. many coronaviruses use cme to enter cells, such as sars-cov entry into hepg cells and cos cells and phev entry into neuro- a cells [ , ] . to ascertain whether pedv utilized cme to enter cells, the chemistry inhibitor cpz was used to prevent clathrin assembly and further block cme. clathrin is composed of light chain (clc) and heavy chain (chc) which form clathrin lattices under the interaction of ap- and eps . both cpz pre-treatment and sirna-mediated knockdown of chc and eps can significantly reduce the invasion rate of pedv into cells, with no significant difference between the gds and gds strains. dominant negative mutants can provide a more specific method to study endocytic pathways by separating the prototype protein from their interaction regulatory factors. in this study, we also showed that cells overexpressing dominant negative mutant of eps were barely infected with pedv but cells overexpressing wild-type eps were infected as normal. however, the inhibition of endocytosis by overexpressing dominant negative mutants may be compensated through other clathrin-independent endocytic pathways. when viruses enter cells through cme, they are carried by clathrincoated vesicles. co-localization of viral particles and chc indicated that pedv entry relies on cme. although caveolae and lipid rafts have the same components, such as caveolin- , gm , and cholesterol, they are two completely different endocytic pathways. before investigating whether pedv can use these two pathways to enter cells, we first examined whether pedv invasion depends on cholesterol. the cholesterol inhibitors nystatin and mβcd had significant inhibitory effects on pedv entry. nystatin had higher inhibitory effects on gds entry than gds . the inhibitory effects of mβcd on vero cells were similar for gds and gds strains. mβcd effects were lower on gds than gds in ipec-j cells. we hypothesized that gds cell invasion mainly depended on cholesterol on the cell surface, while gds depended on the presence of cholesterol on the cell surface and cholesterol synthesis. exogenous cholesterol supplementation also confirmed the importance of cholesterol in pedv entry. endocytic vesicles formed in the caveolae-mediated pathway were coated with caveolin- , which plays a critical role in the process. dominant negative mutant, rna interference, and the cellular co-localization of caveolin- with viral particles provided further evidence that pedv entry needed caveolin- . collectively, both subtypes of pedv entered the vero and ipec-j cells through caveolae-mediated endocytosis. however, park et al. [ ] showed that pedv entry was independent of caveolae-coated pit assembly by treating vero cells with nystatin. the different results may be explained by different operational details. firstly, nystatin was used before and during the incubation of pedv in this study, while only before incubation in the research of park et al. the concentrations of nystatin used in the two studies were different. the highest concentration of nystatin used in park's research was μm [ ] , while the highest concentration we used was μm in vero cells. when the concentration is μm, nystatin does not inhibit the entry of pedv, which is consistent with park's results. secondly, park et al. added methyl cellulose to block second-cycle infection [ ] , while we added nothing except trypsin in medium. whether these reasons cause two different results needs further study. lipid raft acted as a platform for cell signal transduction and viral invasion, distributed in an island form on the plasma membrane of cells, and was isolated by sucrose density gradient centrifugation. western blotting analysis showed that pedv n protein located in the lipid raft (upper layer) with caveolin- in the cells. as shown in figure b , pedv n protein is also present in the bottom layer when infected with ipec-j cells, which may be due to the different composition of the plasma membranes of the two kinds of cells. we demonstrated that pedv gi subtype gds and gii subtype gds strains could enter vero and ipec-j cells via the clathrin-, caveolae-, and lipid raft-mediated endocytosis pathways. furthermore, we also found that the invasion efficiency of the two strains was different with different endocytosis pathway. these differences between gds and gds strains may be due to the difference of s gene especially the s region of s gene (homology was about %), which is responsible for cell entry and membrane fusion by binding with receptor. the difference of gene may lead to the difference of binding ability or affinity between s protein and receptor, thus leading to the different utilization or initiation efficiency of different endocytosis pathways. however, whether the different gene sequence causes different invasion efficiency between gds and gds strains needs further study. after internalization, viral particles are transported by specific endosomes for membrane fusion. the classical transit route is the endo-/lysosomal pathway, in which endocytic cargoes are transported along endocytic vesicles, early endosomes, and late endosomes-lysosomes. park et al. have confirmed that nh cl and baf-a could inhibit pedv entry [ ] , which is consistent with our results, but needs to be confirmed by different methods. in this study, in addition to chemical inhibitors, we also used sirna interference and cellular localization of virus particles to identify the role of ph and endosomes. the results of this study revealed that pedv entry relied on low ph, which means that internalized pedv particles are transported to endosomes and lysosomes, as demonstrated by the co-localization of viral particles with eea , rab , and lamp . liu et al. [ ] reported that pedv s protein was activated by lysosomal cysteine proteases to activate pedv entry. however, based on the data, we could not conclude that membrane fusion occurred at the lysosomes; more technical methods are necessary to demonstrate the mechanism. in conclusion, studying the internalization and intracellular trafficking mechanism of pedv are important to understand viral pathogenesis and benefit to the development of future therapies strategies. this study demonstrated that both the gi and gii subtypes of pedv enter vero and ipec-j cells via the clathrin-, caveolae-, and lipid raft-mediated endocytosis pathways, but the efficiency of each endocytosis pathway varies depending on the different genotypes and types of cells. the internalized pedv entered the lysosomes through the early and late endosomes. the results of this study provide a theoretical basis for the further understanding of pedv pathogenesis to find new targets of antiviral drugs. virus-like particles 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jurisdictional claims in published maps and institutional affiliations we would like to thank prof. qiang yang, college of veterinary medicine, nanjing agricultural university, china, for providing us with ipec-j cells. we would also like to thank prof. mark mcniven, mayo center for biomedical discovery, usa, for providing us with overexpression vector of wild-type and domain negative mutant of dynamin ii and caveolin- . supplementary information accompanies this paper at https ://doi. org/ . /s - - - . and ipec-j cells were treated with different concentrations of dynasore at °c for h. cck- solution was added to each well at °c for h, and absorptions of nm were detected. dmso was used as a negative control. (b) the vero cells and ipec-j cells were transfected with sidyn, and the second transfection was carried out at h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at h after the first transfection. ctrl means control. ** . < p < . ; *** . < p < . ; ****p < . .additional file . clathrin-mediated endocytosis is involved in pedv entry. (a) vero cells and ipec-j cells were treated with different concentrations of cpz at °c for h. cck- solution was added to each well at °c for h, and absorptions of nm were detected. double-distilled water was used as a negative control. (b, c) the vero cells and ipec-j cells were transfected with sichc and sieps , and the second transfection was carried out at h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at h after the first transfection. ctrl means control. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . cells were seeded in -well plates until confluence. cells were pre-treated with μm cpz, mm mβcd, μm nystatin, mm nh cl and nm baf a respectively, at °c for h and incubated with gds (a) and gds (b) strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate μm. cells were seeded in -well plates until confluence. cells were pre-treated with μm cpz, . mm mβcd, μm nystatin, mm nh cl and nm baf a respectively, at °c for h and incubated with gds (a) and gds (b) strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate μm. the authors declare that they have no competing interests.received: october accepted: january key: cord- - pyuy os authors: miyazaki, ayako; kuga, kazufumi; suzuki, tohru; kohmoto, mariko; katsuda, ken; tsunemitsu, hiroshi title: genetic diversity of group a rotaviruses associated with repeated outbreaks of diarrhea in a farrow-to-finish farm: identification of a porcine rotavirus strain bearing a novel vp genotype, g date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: pyuy os group a rotaviruses (gars) are one of the most common causes of diarrhea in suckling pigs. although a number of g and p genotypes have been identified in porcine gars, few attempts have been made to study the molecular epidemiology of these viruses associated with diarrhea outbreaks within a farm over an extended period of time. here, we investigated the molecular characteristics of gars that caused four outbreaks of diarrhea among suckling pigs in a farrow-to-finish farm over the course of a year. g and p genotyping of gars detected at each outbreak demonstrated genetic diversity in this farm as follows: g p[ ] was detected at the first outbreak, g p[ ]/[ ] and g p[ ] at the second, g p[ ] at the third, and g p[ ], g p[ ]/[ ], and p[ ] combined with an untypeable g genotype at the fourth. sequence analysis of the detected gars revealed that such genetic diversity could have resulted not only from the introduction of new gar strains, but also from gene reassortment between gar strains within the farm. further, the gar strain carrying the untypeable g genotype was shown to be a novel porcine gar bearing a new g genotype, as confirmed by the rotavirus classification working group. group a rotaviruses (gars) are the most common etiological agent of severe diarrhea in infants and young children worldwide, as well as being a common cause of acute enteritis in young farm animals [ ] . they are members of the rotavirus genus, within the reoviridae family, and their genome consists of segments of double-stranded rna (dsrna) encased in a triple-layered capsid. these segments encode six structural (vp -vp , vp , and vp ) and five or six non-structural proteins. amongst these, the outer capsid proteins vp and vp , which independently elicit the production of neutralization antibodies, define the g and p serotypes, and form the basis of a binomial nomenclature [ ] . in recent years, the serotype classification system has been almost completely replaced by a genotyping classification system based on sequence differences of the respective gene segments [ , ] . prior to the present study, at least g genotypes and p genotypes were identified in humans and animals [ , ] . among them, at least g genotypes (g -g , g -g ) and p genotypes (p [ ] , p [ ] -p [ ] , p [ ] , p [ ] , p [ ] , p [ ] / [ ] , p [ ] , p [ ] , p [ ] , and p [ ] ) have been described in pigs [ ] [ ] [ ] [ ] [ ] [ ] . the zoonotic potential of animal gars is a great concern [ ] . gars with unusual g genotypes commonly found in pigs and cattle have been detected in sporadic and epidemic cases of diarrhea in human populations [ ] [ ] [ ] [ ] [ ] [ ] . a full genome-based genotyping system proposed by matthijnssens et al. revealed the existence of reassortant strains and the close relationship between human and animal strains [ , ] . in addition, a number of new g and p genotypes have recently been identified in animals [ , , , , ] . in light of these findings, animal gars have come to be regarded as a potential reservoir for genetic diversity in human gars, and studying them is therefore critical for understanding the evolution and ecology of human gars. besides their zoonotic potential, as the most frequently detected enteropathogen associated with diarrhea among suckling pigs, gars also pose an economic threat to the pig industry due to poor growth performance, and increased morbidity and mortality rates [ , ] . given the present lack of a commercially available vaccine for gar-associated diarrhea in japan, the only means of prevention has been hygiene management. however, although effectively managing gar infections requires an ecological understanding of gars, few attempts have been made to examine the molecular epidemiology of porcine gars associated with diarrhea within a farm over an extended length of time. we therefore investigated the genotypic characteristics of gars causing repeated outbreaks of diarrhea among suckling pigs in a farrow-to-finish farm over the course of a year. outbreaks of epidemic diarrhea occurred among suckling pigs four times between february and march at a large farrow-to-finish farm with sows located in the miyazaki prefecture, japan. a total of fecal samples (one sample per litter, to samples per outbreak) were collected from the rectum of diarrheal pigs that had not been treated with antibiotics. all animal experiments were approved by the animal ethical committee and the animal care and use committee of national institute of animal health. bacteriological examinations were carried out for escherichia coli, salmonella enterica, and clostridium perfringens, and parasitological examinations were carried out for coccidia and cryptosporidium parvum, as described previously [ ] . fecal specimens were diluted with eagle's minimum essential medium to % suspensions and clarified by centrifugation at × g for min. the supernatant was collected and total rna was extracted from μl of the fecal suspensions using trizol ls (invitrogen corp., carlsbad, ca, usa). recovered total rna was suspended in μl of rnase/dnase-free water and stored at - °c until use for reverse transcription-polymerase chain reaction (rt-pcr) and polyacrylamide gel electrophoresis (page). page of the extracted rna was performed with . % precast gels (e-pagel; atto corp., tokyo, japan). the gels were stained using a silver stain plus kit (bio-rad laboratories, hercules, ca, usa). gar electropherotypes were determined by comparing the individual rna migration patterns of genome segments on the gel [ ] . the gar vp gene and the vp * fragment of the vp gene were amplified using a qiagen onestep rt-pcr kit (qiagen, valencia, ca, usa) with primer pairs beg / end and con /con , respectively [ , ] . rt-pcr was conducted to detect group b and c rotaviruses (gbrs and gcrs), transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine sapovirus as described previously [ ] [ ] [ ] [ ] [ ] . the amplicons were analyzed in % agarose gel electrophoresis and visualized by uv after ethidium bromide staining. the amplicons of the gar vp and vp genes were purified using microspin s- hr columns (ge healthcare, uppsala, sweden). the purified pcr products were used as a template for sequencing on an applied biosystems automated dna sequencer using dye terminator cycle sequencing chemistry (applied biosystems, foster city, ca, usa) and sequenced from both directions. to determine the complete coding region of the vp gene of a tj - strain, primers tj - f ( '-gta acc caa tgg aca tta cac tg- ') and tj - r ( '-cta ctg aaa atg atg cga tgt c- ') were also used as sequencing primers. the sequences were assembled, edited, and analyzed using mega software [ ] . the nucleotide sequences of the vp and vp genes from the detected gars were compared with those of reference strains available in the genbank. multiple nucleotide sequence alignments were carried out using the clustal w algorithm. genetic distances were calculated using the kimura- correction parameter, and phylogenic dendrograms were constructed by the neighbor-joining method with bootstrap replications [ ] . the nucleotide sequences determined in this study have been submitted to genbank under the following accession numbers: tj - (vp :ab , vp : ab ); tj - (vp :ab , vp :ab ); tj - (vp :ab , vp :ab ); tj - (vp :ab , vp :ab ); tj - (vp :ab , vp :ab ); tj - (vp : ab , vp :ab ); tj - (vp :ab , vp : ab ). epidemic outbreaks of diarrhea affecting almost all suckling pigs born to % to % of lactating sows occurred in february, march, and may , and march . common clinical signs included profuse watery diarrhea and dehydration lasting about one week. mortality rates were less than %. age and sow parity of the sampled pigs are summarized in table . while no pattern was observed for the first two outbreaks, the third and fourth outbreaks mostly affected pigs less than seven days old that were born to gilts. more than % of the samples collected at each outbreak were positive for gars, as determined by rt-pcr targeting vp , and are listed in table . a small minority of samples contained other enteropathogens: gbr was detected in two samples at the first outbreak, isospora suis and eimeria porci were detected in one sample at the second outbreak, and gcr was detected in one sample at the fourth outbreak. these results indicate that gar was the most common enteropathogen associated with the repeated outbreaks of diarrhea and most likely the cause as well. all segments of gar genomic rna were visualized in samples out of the samples subjected to rna-page. these rna migration patterns were classified into six electropherotypes designated ei to evi ( figure ). all electropherotypes displayed a long migration pattern resembling that of the porcine osu strain (a reference porcine gar strain). a different electropherotype was observed in each outbreak, as summarized in table . rna-page found no samples to be infected with more than one gar strain, although combined infection of gar and gbr was observed in two samples (data not shown). the nucleotide sequences of the vp gene ( bp, corresponding to nucleotides through of the porcine g osu strain's vp gene) were successfully determined in samples. blast search analysis and phylogenic analysis of these vp genes with those of established g genotypes led to classification of the gar strains into four genotypes: g , g , g , and an untypeable g genotype ( figure a ). eleven g strains were identified in this study, with eight strains detected at the first outbreak, two at the second, and one at the fourth (table ) . a phylogenic tree based on a selection of human and animal g strains was generated as described by collins et al. [ ] . these eleven strains constituted a separate branch distantly related to human and porcine g strains comprising g lineages iii and vi (figure b ). nucleotide identities for the eleven strains were . % to % compared to each other, . % to . % to the old japanese porcine g isolates jp - , jp - , jp - , jp - , jp - , jp - , and hokkaido- that belong to g lineage vi, and . % to . % to human and porcine g strains belonging to g lineages iii and vi (figure b and additional file , table s ). the two g strains tj - and tj - , both of which were identified at the third outbreak, constituted a separate branch distantly related to porcine g strains, comprising lineage i (figure c ) [ ] . the nucleotide identities of their vp genes were % compared to each other, . % to . % to the porcine g strains comprising lineage i, . % to . % to human g strains comprising lineage i, and . % to . % to the g strains comprising other lineages (figure c and additional file , table s ). the g isolate tj - , identified at the fourth outbreak, constituted a separate branch distantly related to human and porcine g strains comprising lineage iii (figure d ) [ ] . the nucleotide identities of its vp gene were . % to . % compared to the g strains comprising lineage iii, . % to . % to lineage i, and . % to . % to lineage ii (figure d and additional file , table s ). phylogenic analysis revealed that the untypeable g genotype strain tj - , identified at the fourth outbreak, did not cluster with any reference sequence from the group of g genotypes (figure a) . in a blast search analysis, the nucleotide identities of its vp gene were . % compared to vp w, the uncommon human g strain identified in thailand ( bp, [genbank:dq ]) [ ] , up to . % to other g strains, and up to . % to the strains of other g genotypes. we therefore characterized the tj - strain by analyzing the complete coding sequence of the vp gene, which turned out to be -bp long and included a predicted protein of amino acids. we compared the nucleotide and deduced amino acid sequences with those from the reference strains of the established g genotypes ( table ) and found that the highest identity was to the g rrv strain ( . % nucleotide identity and . % amino acid identity), with lower nucleotide and amino acid identities to other strains ranging from . % to . %, and . % to . %, respectively. the rotavirus classification working group (rcwg) employs a cutoff value of % nucleotide identity to define a novel g genotype [ ] , and on submitting the sequence to the rcwg, it was subsequently assigned as a new g genotype: g . the nucleotide sequences of the vp * fragment of the vp genes ( bp, corresponding to nucleotides through of the porcine p[ ] osu strain's vp gene) were successfully determined in of samples in which the vp gene sequences had been previously determined. blast search analysis and phylogenic analysis of these vp genes with those of established p genotypes led to classification of the gar strains into three genotypes: p [ ] , p [ ] , and p[ ]/ [ ] (table ). eight p [ ] strains were identified, with six strains detected at the first outbreak, one at the second, and one at the fourth ( table ) . these strains had nucleotide identities of . % to % compared to each other, . % to the porcine gub strain identified in japan, and . % to . % to other porcine p [ ] strains (additional file , table s ). two p [ ] strains (tj - and tj - ) were identified at the third outbreak and one (tj - ) at the fourth, having . % to % sequence identities with each other. they were clustered in one branch related to the table s . porcine osu strain and in another to porcine and bovine p [ ] strains (isolated in china and korea respectively) with . % to . % nucleotide identity ( figure and additional file , table s ). in contrast, one p[ ]/ [ ] strain identified at the second outbreak (tj - ) and one at the fourth (tj - ) showed low nucleotide identity ( . %) with each other (additional file , table s ). the tj - strain belonged to a lineage comprised of porcine p [ ] strains of japanese origin (gub , fgp and fgp ) and global origin (cmp and hp ) with . % to . % nucleotide identity ( figure and additional file , table s ). the tj - strain was clustered with the porcine p[ ]/ [ ] strains of ireland origin ( b/ /ire) and japanese origin (jp - and jp - ) with . % nucleotide identity (figure and additional file , table s ). based on the sequence and phylogenic analysis of gar strains detected among the four outbreaks, at least five combinations of g and p genotypes (g/p combinations) were identified: g p [ ] at the first outbreak, g p[ ]/ [ ] and g p [ ] at the second, g p [ ] at the third, and g p [ ] , g p [ ] , and g p [ ] / [ ] at the fourth ( table ) . although the vp genes from the g p [ ] and g p [ ] / [ ] strains were closely related to each other genetically, they combined with different p genotypes. similarly, the vp genes from the g p [ ] and g p [ ] strains were highly homologous, yet they combined with different g genotypes. based on fecal analyses, we demonstrated that the cause behind repeated outbreaks of diarrhea among suckling pigs over a one-year period was primarily gar infection. sequence and phylogenic analysis of the vp and vp genes from detected gars identified a spectrum of g and p genotypes, including g , g , g , p [ ] , p [ ] / [ ] , and p [ ] . in addition, we identified the tj - strain, which contained a new vp genotype g , as confirmed a) the reference strains are shown with host species/strain name/g genotype. abbreviations for host species are shown in the legend of figure . b) the highest identity to the reference strain is shown in bold. table s . and assigned by the rcwg. although the detected g and p genotypes (excluding g ) were common in pig populations worldwide [ , , , ] , the strains identified in this study displayed marked genetic variation when compared to others around the world as well as older japanese strains, suggesting genetic variability in gar strains circulating in the pig population of japan. several limitations to the present study warrant mention. not only was the total number of samples low, but also, roughly half of them could not be successfully analyzed for either the vp or vp genes. however, despite these limitations, we found at least five different g/p combinations and six electropherotypes during the four outbreaks, indicating considerable genetic diversity of gar strains within a farm continuing over one year. co-circulation of different gar strains in the same herd has also been reported in several studies: collins et al. identified at least to gar strains among asymptomatic pigs in each farm at a same time [ ] , and barreiros et al. detected at least three strains during a diarrhea outbreak on a single swine farm [ ] . while such genetic diversity in the same herd may have resulted from the introduction of new gar strains, our results support the additional possibility of genetic reassortment between gar strains within the herd. indeed, the g p [ ] and g p [ ] / [ ] strains contained almost identical g sequences, and the g p [ ] and g p [ ] strains had nearly identical p [ ] sequences. a previous study has shown that herds with gar-associated diarrhea tend to be managed in an all-in all-out system and to have large numbers of sows [ ] . because diarrhea occurs in pigs when the oral challenge level of gar exceeds the protective level of passive immunity acquired through colostrum and milk, the onset age and diarrhea severity are influenced by management practices that affect these factors, such as housing type, sanitation, and crate design [ ] . by maintaining strict sanitation management and housing sows and gilts in separate units (about sows were kept in gestation and farrowing barns), the farm in the present study might reduce the uniformity of exposure levels and immunity to gars. the variation in affected pigs across the outbreaks appears to reflect the variation in the level of lactogenic immunity passed from sows to piglets (table ) . therefore, it cannot be ruled out that differing immunity levels among sows or gilts may be associated with the existence of the genetically diverse gar strains and the repeated diarrhea outbreaks. based on accumulated evidence, it is clear that gars can transfer between pigs and humans, making pigs a potential reservoir for the emergence of unusual or novel strains of human gars [ ] [ ] [ ] [ ] [ ] [ ] . in fact, the novel g strain we found was most similar to the human vp w strain, which can also be considered to be a g strain. the vp w strain was reported as a rare g p [ ] human strain identified in a clinical sample collected in thailand between and [ ] . although the origin of g remains unclear, the vp sequence of vp w [genbank: dq ] is genetically closely related to those of porcine p [ ] strains detected in thailand [ ] . further, genetic analysis of several genome segments of the tj - strain other than vp and vp revealed that, except for the vp gene, this strain shares a similar genetic background with the porcine ym strain [miyazaki a, in preparation]. taken together, these findings suggest that vp w may be a porcine-human reassortant or a porcine strain. considering that the newly identified g has been detected in both pigs and humans, and the scarcity of animal gar strains in the genetic database, further surveillance and epidemiological study on gars in domestic animals will be needed to better understand the origin and circulation of human gars. here, we demonstrated the genetic diversity of gars associated with repeated outbreaks of diarrhea among suckling pigs within a farm over a one-year period. such genetic diversity within a single farm might pose a challenge for developing effective methods of prevention against diarrhea caused by gar infection. additional file : table s : comparison of the vp genes of the gar strains identified in this study to those of reference and selected strains. comparison of the vp genes of the gar strains identified in this study to those of reference strains and a selection of g , g and g strains. additional file : table s : comparison of the vp * fragment of vp genes of the gar strains identified in this study to those of reference and selected strains. comparison of the vp * fragment of vp genes of the gar strains identified in this study to those of reference strains and a selection of p [ ] , p [ ] , and p[ ]/ [ ] strains. rotaviruses. in fields virology full genome-based classification of rotaviruses reveals a common origin between human wa-like and porcine rotavirus strains and human ds- -like and bovine rotavirus strains van ranst m: recommendations for the classification of group a rotaviruses using all genomic rna segments reassortant group a rotavirus from straw-colored fruit bat (eidolon helvum) whole genome characterization of new bovine rotavirus g p[ ] and g p[ ] strains provides evidence for 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g in japanese piglets with diarrhea: close relationship of their vp genes with those of recent human g strains predominance of porcine p[ ] genotype rotaviruses in piglets with diarrhea in northern thailand an outbreak of diarrhoea in one-week-old piglets caused by group a rotavirus genotypes relationship between group a porcine rotavirus and management practices in swine herds in ontario rotavirus and reovirus genetic diversity of group a rotaviruses associated with repeated outbreaks of diarrhea in a farrow-tofinish farm: identification of a porcine rotavirus strain bearing a novel vp genotype, g much appreciation is extended to the members of the rotavirus classification working group for their input regarding strain classification and the identification of the novel vp genotype. we appreciate dr t. murai's efforts in collecting samples and making helpful suggestions. we would like to thank n. hattori, h. kunifuda, and drs n. kawanishi, t. miyamoto, s. ogawa, and k. hirano for their technical support. this work was supported in part by a research grant from the national institute of animal health, japan. authors' contributions am participated in designing the study, carried out the examination and molecular characterizations of gars, and drafted the manuscript. ht conceived this study and participated in its design and coordination. kk and ts participated in the study design and coordination and carried out the virological examinations. mk and kk participated in designing the study and carried out the parasitological and bacteriological examinations. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -zf tcwe authors: ge, shikun; xu, long; li, ben; zhong, fagang; liu, xiang; zhang, xiaoying title: canine parvovirus is diagnosed and neutralized by chicken igy-scfv generated against the virus capsid protein date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: zf tcwe canine parvovirus (cpv) can cause acute and highly contagious bloody enteritis in dog. to obtain antibodies against cpv, hens were immunized with virus-like particles (vlp) of cpv-vp . the igy single chain fragment variables (scfv) were generated by t phage display system and expressed in e. coli system. the titer of the primary scfv library reached to . × ( ) pfu/ml, and % of the phages contained the target fragments. the cpv-vlp and cpv-vp protein showed similar reaction values to the purified scfv in the elisa test, and the results of elisa analysis using igy-scfv toward cpv clinical samples were consistent with commercial immunochromatographic assay (ica) and pcr detection, the scfv did not show cross reactivity with canine distemper virus (cdv) and canine coronavirus (ccv). igy-scfv was successfully expressed in crfk cells, and in the virus suppression assay, % of cpv infections were eliminated within h. docking results demonstrated that the number of amino acids of the binding sides between scfv and vp were aa and aa , respectively. this study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian igy-scfv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine. canine parvovirus (cpv) was first identified in [ ] ; it has a single-stranded dna genome of negative polarity, about bp in length. the cpv capsid is a nm diameter icosahedron containing three structural viral proteins (vp , vp and vp ) and two non-structural proteins (ns and ns ), among them, vp accounts for % of the viral capsid and represents the major determinant of host range and virus-host interactions [ ] . although the conventional attenuated and inactivated cpv vaccines have been successful in reducing the disease outbreaks, the genus of parvovirus still causes severe epizootics worldwide and leads to severe economic losses in dogs [ ] . antibody based approach is promising in cpv disease diagnosis, therapy and prevention, and the relevant hyper immunoglobulin g (igg) and full-length monoclonal antibody (mabs) have been routinely applied in the veterinary practices [ ] . at present, most commercially available mabs are produced in mammalian system using hybridoma technology. functional antibody fragments (i.e.: scfv, fab) generated by phage-display technology have been not yet routinely evaluated and applied in the veterinary medicine despite it has been well confirmed that such engineered antibodies offer a series of advantages over polyclonal and full-length monoclonal antibodies. antibody phage display technology is to display polyclonal antibodies on the surface of the phage shell protein and to screen the specific monoclonal antibodies open access *correspondence: zhang@bio.uminho.pt department of biology, centre of molecular and environmental biology, university of minho, campus de gualtar, - braga, portugal full list of author information is available at the end of the article by bio-panning procedure [ ] . phages are more stable and can be stored for years at °c; it can be re-produced rapidly, successively and inexpensively with a confirmed sequence in the prokaryote or eukaryote systems without hybridoma and immunization procedure. furthermore, higher affinity mutants of scfv can be generated through site directed mutagenesis which is much easier and simpler to be performed [ ] . chicken (gallusb gallus domesticus) igy antibody generated by igy-technology is another conventional approach in generating large amount of high specific antibody and have been used for broad biomedical purposes owning to a series of advantages such as higher productivity, better animal welfare, higher immunogenicity to mammal conserved proteins and lower cross-reactivity, as compared to the generation and application of mammalian serum igg [ ] . our previous work confirmed that the generated polyclonal igy targeted to cpv-vp could apply into the immunotherapy and immunoprophylaxis for the cpv infection [ ] . in recent years, as an interesting tendency in igy technology, generation of igy-scfv in order to better combine the biological superiorities of both igy and recombinant antibody fragment, is gaining increasing attention and application [ , ] . our previous studies demonstrated that igy-scfv can be generated and applied in different immunoassays for the detection of small molecules gentamicin [ ] and large molecules pifn-γ [ ] in the veterinary practice. as an attempt to understand the feasibility of chicken igy-scfv in diagnosis and therapy of veterinary diseases, this study aimed to construct and characterize chicken sourced scfv against cpv-vp virus like particles (vlp) using phage display technology, and to evaluate the specificity, sensitivity and virus inhibition ability of the obtained scfv. twelve-week old white leghorn hens were immunized intramuscularly with cpv-vlp (provided by dr. shiqi sun [ ] ) mixed with freund's adjuvant (sigma-aldrich, st. louis, mo, usa) at four different sites of breast muscles. cpv-vlp protein ( µl, mg/ml) in equal volume of phosphate buffered solution (pbs, . m, ph . ) was emulsified with freund's complete adjuvant (fca; sigma-aldrich, st. louis, mo, usa) in the first immunization, and four booster immunizations were followed up with freund's incomplete adjuvant (fia; sigma-aldrich, st. louis, mo, usa) at -week intervals. all experimental animal protocols were reviewed and approved by the institutional ethics committee for the use of laboratory animals. the hen's spleen was collected to extract the total rna by total rna kit (tiangen biotech, beijing, china), and the first-strand cdna was synthesized by hiscript q select rt supermix for pcr (+gdna wiper, vazyme biotech, nanjing, china). the heavy variable fragment (v h ) and light chain variable fragment (v l ) genes were amplified by pcr with primers ( table ). the v h and v l were assembled with primers hf-ecor i & lr-hind iii by overlap pcr. the products of overlap pcr (scfv) were purified through gel extraction kit (omega, norcross, ga, usa). this protocol was performed as described in [ ] . the scfv gene products were digested with ecor i and hind iii restriction enzyme and ligated to t select - b ( . pmol; novagen, darmstadt, germany) by t dna ligase (takara biotechnology, dalian, china) in a work volume of µl at °c overnight. the ligation products were directly added to t package extract ( µl) and the mixtures were incubated at °c for h in vitro to create the primary library by add luria-bertani medium ( µl) to stop the reaction. the primary scfv library was amplified by liquid lysate amplification refer to the t select system manual. the titers of the primary and amplified library were evaluated by plaque assay. this protocol was performed as described in [ ] . the amplified library was subjected to four rounds of bio panning on microplates for the enrichment of the specific scfv phages refer to the t select system manual. the microplates were coated with cpv-vlp overnight ( µg, µg, µg and µg per well in each round) at °c, and washed with tween-pbs (pbst, tween . %; µl/well) and pbs, respectively. after blocking with skimmed milk ( %) in pbst, the amplified phages from the initial library of each round of panning were added to the microplates and incubated at °c for h, and the bound phage was eluted with sds ( %) in each round. in the first three rounds, eluted phages were amplified by infect bl bacterial culture (novagen, darmstadt, germany). the enrichment of specificity was determined from the input/ output ratio of the phage. a total of random clones of on the plates were selected from the fourth round to identify the positive rate by using a pair of universal primers ( table ). the amplified library was added with % chloroform and stored at °c. this protocol was performed as described in [ ] . the phage-scfv with the highest affinity was selected to ligate with pet- a (+) vector use t dna ligase (takara biotech, dalian, china). the recombinant plasmids were transformed into bl (de ) competent cells, and the scfv expression was induced with isopropylβ-d-thiogalactopyranoside (iptg). the bacterial cells were collected by centrifugation and sonication, and the supernatants and pellets were used for sds-page analysis, respectively. protein was purified by histrap hp histidine-tagged protein cultured columns (ge, pittsburgh, pa, usa). this protocol was performed as described in our previous reports [ ] . the cpv-vp proteins were separated by sds-page ( % spacer gel and % separation gel) and blotted on nitrocellulose membranes ( western blot was performed as described in [ ] . to determine the accuracy of scfv, a total of clinical dog stool samples were collected using sterile swabs in an animal hospital (xinger, xi'an, china). among them, samples were confirmed cpv positive and were negative by the hospital using commercial colloidal gold test strip (ica). the samples were homogenized ( %, w/v) in pbs ( ml, ph . ) and centrifuged, the supernatants were used for indirect elisa and pcr. in order to verify whether scfv has specificity in clinical practice detection, canine distemper virus (cdv) and coronavirus (ccv) were used to detect the cross-reaction by elisa. the crfk cells (lmai bio, shanghai, china) were seeded into -well plates with optimum culture medium and transfections were performed when the cell monolayer density reached %. the transfection mixture (containing the pcmv- -scfv) was added into each well and incubated for h at °c in moist % co . at h post-transfection, the cells were fixed with paraformaldehyde ( %), permeabilized with triton x- ( . %) and incubated with bsa (beyotime, shanghai, china) to block nonspecific binding sites. they were incubated with diluted his-tag antibody ( µl; affinity biosciences, oh, usa) for h then washed with pbs. goat anti-mouse igg (h + l) fluor -conjugated ( : ; affinity biosciences, oh, usa) was added. the stained cells were visualized by use a nikon eclipse i fluorescence microscope (nikon, sendai, japan). total protein of the cells transfected h were extracted for western blot; his-tag antibody and goat anti-mouse igg hrp (biosharp, hefei, china) were used as primary and secondary antibodies, respectively. ica was performed as described in [ ] . normal crfk cells ( docking was performed by discovery studio . software (biovia, san diego, ca, usa) and the antibody model cascade program in discovery studio software was used to build the molecular model of the scfv. then, to obtain the docking conformation, the scfv molecular model was docked with vp using the zdock molecular dock program. finally, the residues contact frequency (rcf) algorithm was used to analyze the predicted results, and the bind surfaces of amino acids in scfv and vp were obtained. the lengths of vh-linker, vl-linker and scfv were approximately bp, bp and bp, respectively ( figures a and b) . the titer of the primary anti-cpv scfv library ( figure c ) and the amplified library were . × pfu/ml, and . × pfu/ml, respectively. there were % of the phages containing the target fragments in the primary scfv library. after rounds of "bind-elute-amplify" bio-panning procedure, the data of input and output phages in each round indicated that the phage library had been enriched times ( figure and table ). a total of scfv genes showed relatively high binding capacity to cpv-vlp in the reactivity detection of phages, and the sequencing confirmed that all the scfv genes have complementary determining region (cdr ) that was the main mutation region in both vh and vl. there were few clones having limited mutations in framework region (fr, figure ). the phage-scfv (no. the solubility analysis showed that scfv mainly existed in the form of inclusion body ( kda; figure a , lane ). the denaturation and purification of scfv inclusion body protein showed a single protein band with high purity ( figure b ). the vp protein could bind to scfv with a single binding strip ( kda; figure c ). the immunoreactivity of scfv against soluble vp was examined by elisa. scfv bound in a dose-dependent manner to the soluble vp ( figure ). the minimum antibody concentration for the detected antigen was ng/µl. the coincidence of elisa ( figure a ) and pcr (figure c) with ica (data not show) was % and . %, respectively. the scfv showed no cross reactivity with cdv and ccv ( figure b ). the sequencing results showed that the scfv and pcmv- vectors were successfully connected (figure a) , ifa confirmed that scfv was significantly expressed in crfk cells ( figure b ). immunoblotting further confirmed that the scfv protein was correctly folded and modified in the cell ( figure c ). the crfk cells expressing scfv showed a small amount of cytopathic effect (cpe) after cpv infection; the cells without scfv expression were significantly broken away from the bottom wall, became round, and some cells even broke up ( figure d ). virus tcid at different time points was determined; the growth rate of virus in cells expressing scfv was significantly lower than that in cells not expressing scfv ( figure e ). the inhibition rates of scfv on virus growth at h, h, and h were %, % and %, respectively ( figure f ). the main structural domains of scfv were vlcdr , vlcdr , vldr , vhcdr , vhdcr , and vhcdr , with antigen-binding sites on scfv ( figure a ). the dimensional scfv model was subsequently constructed ( figure b ). a total of highly similar vp homologous sequences were simulated by software ( figure c ), the vp molecular stereo model was established by combining the characteristics of each sequence ( figure d ). after analysis on scfv and vp binding mode ( figure e ), the interacting amino acids at the binding sides of scfv (aa ) and vp (aa ) were confirmed ( figure f and g). the mabs have been widely applied in the biomedical areas owning to their high specificity and homogeneity. however, mabs produced by mammals may have the side effects of immunogenicity, thrombocytopenia and hypersensitivity reactions, etc., which has greatly limited their application in target detection and treatment [ , ] . with the development of antibody library technology and humanized antibody modification technology, humanized recombinant antibodies have been gradually developed and entered clinical trials [ ] . diversified antibody generation strategies could be a future tendency in antibody engineering in order to better combine the characteristics and advantages of antibodies from different sources. as a notable example, brolucizumab (beovu) is the first fda approved rabbit-derived scfv used as vascular endothelial growth factor (vegf) inhibitor for the treatment of exudative (wet) age-related macular degeneration (amd), diabetic macular oedema and macular oedema secondary to retinal vein occlusion, which could better overcome the possible side effects (discomfort and increased tears in the affected eyes, itchy or watery eyes, dry eyes, swelling of the eyelids, etc.) of murine-derived igg-fab fragment (lucentis) [ ] . in avian igy, similar attempts have also been made. for instance, the snake venom contains neurotoxic proteins, the urgent administration of hyperimmune serum from horse used to be the most efficient treatment, which can recognize many different antigenic determinants. however, generation of equine anti-venom is costly and associated with several potential side effects. chicken igy-scfv has been generated against glutaraldehyde-attenuated daboia russelii formosensis (drf) venom proteins for passive immunization, which can identify and neutralize the toxic activity of the venom components, with only small quantity of antigens required to induce a significant antibody response in hens, and can be also used as a rapid diagnostic tool for wound secretions to determine snake types [ ] . as summarized by previous authors, owning to their unique structure, phylogenetic distance and tmechanisms of molecular diversification, igy antibodies provide a series of important advantages over mammal igg, including the stronger immune responses of chicken system to the proteins conserved among mammals, decreased/no cross-reactivities (i.e.: rheumatoid factor, human anti-mouse igg antibody, complement system, fc receptors) in the mammal systems [ , ] , and more convenient design of primer against igy-scfv (table ) , as igy only has one isotype and lacks hinge region [ ] . furthermore, it is noteworthy to address, recent studies confirm that from the glycobiology point of view, recombinant igy antibody could be a potentially promising immune-therapeutic candidate after proper antibody engineering and expression as igy is more heavily glycosylated [ ] , and has higher sialic acid content [ ] as compared to mammal igg. recombinant functional antibody fragments remove or reduce irrelevant structures, while retain the specificity and main biological activities of natural antibodies, which offers a wider application prospect than natural antibodies [ ] . recombinant igy-scfv could combine the advantages of both igy molecular and functional antibody fragment [ ] . designing on chimeric antibody could be the next step for igy-scfv study in order to provide better compatibility of the antibody in the host system, and to recoup the possibly decreased specificity and affinity of antibody fragments as compared to full length antibody. recent study confirmed that mammalian igg and avian igy shared compatible v-c region interfaces, which may be conducive for the design and utilization of mammalianavian chimeric abs [ ] . in our study, the high consistency of elisa analysis to pcr and ica on clinical samples confirmed the specificity of the obtained igy-scfv (figure ) , which offers the potential using igy-scfv for rapid detection of cpv. scfv neutralized the virus ( figure d ), inhibited cpv replication with significantly reduced growth rates of the cpv observed in the crfk cells ( figure e , f), which provides the value of further therapeutic investigation of obtained igy-scfv. as alternative to viral components, cpv-vp -vlp was used as immunogen in this study. with increasing applications in vaccine design and immunization, vlp has been recognized as safe and effective particle to stimulate adequate immune responses for both viral and non-viral diseases by inducing lymphocyte proliferation and specific antibody with high titer [ ] . the docking of scfv-vp shows that there were antigenbinding sites on scfv, with high binding force to vp . according to residues contact frequency (rcf) algorithm analysis, binding amino acids on scfv and binding amino acids on vp were involved in the binding (figure ), these results provide us confidence that a well-designed cpv-vlp can be used as a potent immunogen to induce qualified specific antibodies. in conclusion, we demonstrated that specific igy-scfv can be generated with high specificity and significant inhibition to cpv growth. as a preliminary evaluation, our work revealed the potential of igy-scfv as a novel approach in veterinary diagnosis and therapy. ccv: coronavirus; cdv: canine distemper virus; cpv: canine parvovirus; ica: immunochromatographic assay; igg: immunoglobulin g; igy-scfv: chicken igy single chain variable fragments; iptg: isopropyl-β-d-thiogalactopyranoside; mabs: monoclonal antibody; scfv: single chain fragment variables; vlp: viruslike particles. canine gastroenteritis associated with a parvovirus-like agent. the can veterin the three-dimensional structure of canine parvovirus and its functional implications a survey of diagnosis and treatment of pet canine parvovirus disease in china generation and validation of canine single chain variable fragment phage display libraries antibody phage display: overview of a powerful technology that has quickly translated to the clinic recombinant antibody fragment production use of igy antibodies in human and veterinary medicine production of egg yolk antibody (igy) against recombinant canine parvovirus vp protein chicken monoclonal igy antibody: a novel antibody development strategy insights into the chicken igy with emphasis on the generation and applications of chicken recombinant monoclonal antibodies preclinical studies and clinical evaluation of compounds from the genus epimedium for osteoporosis and bone health generation and characterization of chicken-sourced single-chain variable fragments (scfvs) against porcine interferon-gamma (pifn-gamma) convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc self-assembly of virus-like particles of canine parvovirus capsid protein expressed from escherichia coli and application as viruslike particle vaccine construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scfv) antibody against staphylococcus aureus developed with the t phage display system preparation of a chicken scfv to analyze gentamicin residue in animal derived food products antibody validation by western blotting immunofluorescent cell assay of infectious pancreatic necrosis virus the safety and side effects of monoclonal antibodies therapeutic antibodies, vaccines and antibodyomes phage display technology for human monoclonal antibodies agents for the prevention and treatment of age-related macular degeneration and macular edema: a literature and patent review single chain antibody fragment against venom from the snake daboia russelii formosensis chicken egg yolk antibodies (igy-technology): a review of progress in production and use in research and human and veterinary medicine egg yolk antibodies (igy) and their applications in human and veterinary health: a review the impact of n-glycosylation on conformation and stability of immunoglobulin y from egg yolk publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations dr. xy zhang acknowledges the adjunct position and supports offered by the university of guelph, canada. authors' contributions xyz, fgz and skg conceived this project. skg, lx and bl performed the experiments. skg, lx, xl and xyz wrote the manuscript. all authors read and approved the final manuscript. this work was finically supported by the grand of china natural science foundation ( , ) and the incubation project on state key laboratory of biological resources and ecological environment of qinba areas (slgpt kf - ). all data supporting our findings are included in the manuscript. all experimental animal protocols were reviewed and approved by the institutional committee of shaanxi university of technology for the use of laboratory animals (approval number - ). not applicable. the authors declare that they have no competing interests. key: cord- -ov fy b authors: nazki, salik; khatun, amina; jeong, chang-gi; mattoo, sameer ul salam; gu, suna; lee, sim-in; kim, seung-chai; park, ji-hyo; yang, myoun-sik; kim, bumseok; park, choi-kyu; lee, sang-myeong; kim, won-il title: evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ov fy b the host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (prrsv) from infected pigs is currently poorly understood. to better understand the dynamics of host–pathogen interactions, seventy-five of pigs infected with prrsv-ja and control pigs were euthanized at , , , and days post-challenge (dpc). blood, lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected to evaluate the cellular immune responses. the humoral responses were evaluated by measuring the levels of anti-prrsv igg and serum virus-neutralizing (svn) antibodies. consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between and dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by dpc. at peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived dc/macrophage and conventional dc frequencies were increased, and these effects coincided with the early induction of local t-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, bal, and bln as early as dpc. conversely, the systemic t-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between and dpc. taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining prrsv through the early induction of t-cell responses at the sites of virus replication. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded positive-sense rna virus with an approximate . -kb genome, belongs to the genus betaarterivirus of the family arteriviridae (ictv ). in pigs, prrsv causes porcine reproductive and respiratory syndrome (prrs), which is characterized by reproductive failure in breeding sows and severe respiratory distress in young and growing pigs [ ] . prrs results in colossal economic losses in the swine industry worldwide, and these losses are still observed three decades after its emergence in the united states and europe. after the exposure of pigs to prrsv, the virus replicates in alveolar macrophages (am) and further spreads rapidly throughout the body via a lymphohematic route. this viral spread results in acute infection characterized by viremia that lasts for approximately month [ ] , and a few studies have reported a nonviremic persistent infection of secondary lymphoid tissues lasting for approximately days or longer [ ] . in general, the viremia peaks at approximately - days post-infection (dpi) and is almost cleared by dpi depending on the viral strain and age of the pigs [ , ] . additionally, the immune response against prrsv depends on the strain, but the virus usually has immunosuppressive properties [ , ] , which leads to the increased susceptibility of pigs to secondary microbial infections [ ] . the interactions between prrsv and host immune responses have been widely studied, but most studies investigated systemic immune responses using pbmc and/or serum [ ] . previous studies have shown that interstitial pneumonia constitutes the major lung lesions in prrsv-infected pigs and that significantly decreased numbers of alveolar macrophages are found in bronchoalveolar lavage (bal) and lung parenchyma samples from prrsv-infected pigs [ ] . however, to the best of our knowledge, the kinetics of local immune responses in the lungs or lymph nodes during the course of infection compared with those of peripheral immune responses have not been previously studied. this information would provide a more in-depth understanding of the sequential activation of both immune compartments and the correlation between local or peripheral immune responses and virus clearance in infected pigs. as a result, achieving a comprehensive understanding of the immune responses against prrsv infection remains an important goal in prrsv research. during prrsv infection, the pig immune system is capable of escalating an immune response to ultimately clear the virus from the body [ ] . for clearance, proper stimulation of the pig innate immune system is required to direct the development of protective adaptive immunity against prrsv. interestingly, the preferential sites for prrsv replication are alveolar macrophages present in the lungs, which form the major component of the respiratory dc/macrophage network. this network is predominantly involved in sensing foreign antigens, controlling inflammation, and initiating the adaptive immune response [ ] . different dc subsets with specific functional specializations exist in the respiratory dc/macrophage network in the lungs and are reportedly resistant to prrsv infection [ , ] . however, upon activation, these dc travel to lymphoid tissues to present antigen to t lymphocytes and thereby serve as the link between innate and adaptive immunity [ , ] . t cells, in turn, play a critical role in the development of anti-prrsv immunity due to their cytotoxic effector functions in clearing infected cells from the body and developing and regulating antigen-specific immune responses [ ] . however, whether the peripheral virusspecific immune response is appropriately correlated with the local immune response during infection, which could result in the precise use of the peripheral response as a surrogate for scrutinizing the local immune response during viral clearance, remains unclear. therefore, discerning the local and peripheral immune responses during prrsv infection is important for understanding the basic mechanism of viral clearance from the host. cytokines secreted by immune cells act on their targets in an autocrine, paracrine, and/or endocrine manner to prompt local and/or systemic immune responses. in porcine respiratory diseases, proinflammatory cytokines play a key role in activating and synchronizing the adaptive immune responses to clear the virus from the body [ ] . however, the tissue damage caused by excessive production of these cytokines is controlled by the secretion of anti-inflammatory cytokines, which results in the maintenance of homeostasis in the body [ ] . moreover, effective instigation of the local inflammatory response in the lungs accompanied by significant changes in the proinflammatory cytokine levels in serum has been observed in pigs with respiratory diseases [ , ] . nevertheless, whether the local or the systemic cytokine/ chemokine response plays the primary role in the clearance of prrsv from pigs during the acute phase of infection remains unclear. in this context, the present study aimed to investigate the trend of host immune responses against prrsv infection during disease progression and to elucidate the innate and adaptive immunological mediators modulated by the prrsv-ja strain both systemically in peripheral blood and locally in the bronchoalveolar lavage, lung parenchyma and bronchial lymph nodes (bln) of infected pigs. marc- cells, an african green monkey kidney cell line that is highly permissive to prrsv [ ] , were used for virus propagation and assays. these cells were maintained in rpmi- medium (gibco ® rpmi- , life technologies, carlsbad, ca, usa) supplemented with % heat-inactivated foetal bovine serum (fbs, life technologies), mm l-glutamine, and an antibiotic-antimycotic cocktail (anti-anti, life technologies) containing iu/ml penicillin, µg/ml streptomycin, and . µg/ml fungizone ® [amphotericin b] in a humidified chamber with % co at °c. in this manuscript, this medium is designated rpmi growth medium. the north american prrsv- strain ja (genbank: ay . ) was used in the present study. one hundred -week-old piglets purchased from a prrsv-seronegative farm were randomly assigned to two groups and housed in separate animal rooms. after days of acclimatization, the pigs in the infected group (n = ) were intramuscularly inoculated with ml of the prrsv-ja strain ( × tcid /ml) diluted in sterile pbs. the control pigs (n = ) remained uninfected. feed and water were provided ad libitum to all the pigs. five pigs from the control group and , , , and pigs from the infected group were humanely euthanized on days , , , and post-challenge (dpc), respectively. euthanasia was performed by electrocution after the intramuscular injection of ml of azaperone ( mg/ml, stressguard ® , dong bang inc., seoul, south korea). three infected pigs died during the course of the experiment due to high fever and reduced body growth. an overview of the animal study is presented in figure . after euthanasia, the lungs, trachea and bronchi were aseptically extracted and lavaged with ml of sterile pbs. the collected lavage fluid was centrifuged at × g and room temperature for min to separate the bronchoalveolar lavage fluid (balf) and cells (bal), and these samples, along with the lung parenchyma and bln, were also used for immune cell analysis. these tissues and balf were collected in tubes, snap-frozen using liquid nitrogen and stored immediately at − °c for rna extraction and cytokine analysis, respectively. for histopathology, the lung tissues were also collected in % neutral-buffered formalin. blood was collected from the euthanized pigs at , , , and dpc, and the serum and pbmc were separated. in addition, blood samples from pigs, including uninfected and infected pigs that were going to be euthanized on and dpc, were also collected at , , , , , and dpc, and the serum and pbmc were separated. the body weights of all the pigs were measured at dpc, and the body weight gains of the euthanized pigs were measured at , , , and dpc. serum viremia was measured at , , , , , , and dpc, and the viral load in the lungs was quantified in the euthanized pigs at , , , and dpc. viral rna was extracted from serum using a magmax viral rna isolation kit (ambion; applied biosystems, life technologies, inc.) according to the manufacturer's instructions. real-time reverse transcription-polymerase chain reaction (rt-qpcr) employing the prime-q pcv prrsv detection kit (genet bio, daejeon, south korea) was performed for the quantification of serum viremia. one-step rt-qpcr was performed in accordance with the manufacturer's instructions. pcr amplification was performed using a model fast real-time pcr system (applied biosystems, foster city, ca, usa). the cycling conditions were as follows: (i) cdna synthesis for min at °c; (ii) -min predenaturation step at °c; and (iii) cycles of denaturation for s at °c figure overview of the animal study. prrsv-negative piglets (n = ) were purchased and acclimatized for days. seventy-five pigs were then infected, and pigs were used as a negative control (nc). pigs belonging to both the nc and infected (i) groups were euthanized at , , , and days post-challenge (dpc), and lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected. blood samples for sera and peripheral blood mononuclear cells (pbmc) were collected weekly post-challenge. the body weight of the pigs was also monitored from the day of purchase to the end of the experiment. and annealing/extension for s at °c. to calculate the amount of prrsv in each sample, the cq values were converted to virus titres (tcid /ml) by generating a standard curve through the titration of prrsv- strain ja . in addition, marc- cells were used to quantify the virus titres in lung tissues using a microtitration infectivity assay [ ] . briefly, tissue homogenates ( % [weight/ volume]) of finely chopped lung pieces were prepared in dulbecco modified eagle medium (dmem) with antibiotics, and these mixtures were vortexed vigorously for - min and then centrifuged at ~ × g and °c for h. the collected supernatant was filtered through a . -μm sterile syringe filter and used as an inoculum for the measurement of virus titres. the virus titres were calculated at to days post-inoculation based on the cytopathic effect (cpe) and are expressed as tcid /ml [ ] . the lungs of the necropsied pigs in both groups were subjected to pathological evaluation on each day of necropsy. the microscopic lung lesions were given a score on a scale from to to reflect no lesion, mild interstitial pneumonia, moderate multifocal interstitial pneumonia, and severe interstitial pneumonia, respectively. the microscopic lesions were examined from five different lobes of the lungs, and the average value was ultimately utilized for scoring purposes. the serum samples from uninfected and challenged pigs were tested for anti-prrsv antibody (igg) using a commercially available elisa kit (prrs ab elisa . ; bionote inc., hwaseong-si, republic of korea) according to the manufacturer's instructions. samples with an s/p ratio (the ratio of the net optical density of the test samples to the net optical density of the positive controls) ≥ . were considered to be positive for the prrsv antibody. the serum virus-neutralizing (svn) antibodies were measured through a fluorescent focus neutralization (ffn)-based svn assay with marc- cells as described previously [ ] , with some modifications. after heat inactivation at °c for h, the serum samples were serially (twofold) diluted using rpmi- growth medium. two-hundred-microlitre mixtures were prepared by mixing each diluted serum sample with fluorescent focus-forming units per ml (ffu/ml) of prrsv-ja at a ratio of : and were then incubated for h at °c in a humidified atmosphere with % co . each mixture was transferred onto a monolayer of marc- cells in -well plates and incubated for another h at °c. the medium was replaced with µl of fresh rpmi growth medium per well and further incubated for h at °c. the cells were later fixed using ice-cold % (v/v) acetone, air-dried, and stained with mouse anti-prrs nc mab a (median diagnostic, gangwondo, korea) and fitc-conjugated goat anti-mouse igg (h + l) (bethyl laboratories, tx, usa). subsequently, the plates were washed at least three times with pbs and observed under a fluorescence microscope to examine the prrsv-specific ffu. the svn titre is expressed as the reciprocal of the highest dilution at which a % or higher reduction in the number of ffu was observed. pbmc were isolated from the blood samples ( ml) by the density gradient method using leucosep ™ centrifuge tubes (greiner bio-one north america inc., nc, usa) and leucoprep ™ lymphocyte separation media (greiner bio-one north america inc.) according to the manufacturers' instructions. the blood samples were briefly stratified on leucoprep ™ solution at a ratio of : (blood:leucoprep) and centrifuged at × g for min. the purified pbmc were collected, washed twice with sterile pbs (ph . ) and resuspended in . ml of sterile pbs supplemented with % heat-inactivated fbs (gibco, carlsbad, ca, usa). contaminating red blood cells (rbc) were removed by treatment with rbc lysis buffer (ebioscience, ca, usa). for pathological evaluation, the right-sided lobes were clamped to collect specimens for rna extraction and histopathology, and the left lobes of the lungs were used for bal collection according to a previous study [ ] . the lungs were lavaged with - ml of pbs containing µg/ml ampicillin (usb corporation cleveland, oh, usa) and an antibiotic-antimycotic cocktail (anti-anti, life technologies), and the harvested fluid was centrifuged for min at × g. the resulting supernatant was collected as bal fluid (balf), whereas the cell pellet (bal cells) was washed three times with pbs after rbc lysis. the cells were resuspended in facs buffer ( % fbs in phosphate-buffered saline and . % sodium azide). the bln were passed through a -μm cell strainer (spl life sciences, pocheon, korea) in pbs and then washed with facs buffer according to a previous study [ ] . the single cell suspension obtained was used for flow cytometric analysis. mononuclear cells from lung parenchyma were prepared based on a previous study [ ] , with few modifications. briefly, lung tissue was collected, washed in sterile ice-cold pbs and suspended in serum-free rpmi media containing dnase i ( u/ml, sigma, st. louis, mo, usa) and collagenase d ( mg/ml, roche diagnostics, mannheim, germany). single-cell suspensions were prepared using the gentlemacs octo dissociator (miltenyi biotec, san diego, ca, usa) and incubated at °c for min. subsequently, the cells were passed through a -μm cell strainer, washed, and resuspended in facs buffer for flow cytometry analysis after rbc lysis. finally, the cells were counted with a countess ™ automated cell counter (invitrogen, carlsbad, ca, usa), and their viability was tested by trypan blue (sigma-aldrich, st. louis, mo, usa) exclusion [ ] . for cell surface staining, single-cell suspensions were incubated on ice for min with specific antibodies as listed in additional file , and the cells were then washed three times with facs buffer. when necessary, secondary antibodies conjugated with fluorochrome were used. natural killer (nk) cells, dc and macrophages required only cell surface staining, whereas the different subsets of t cells required intranuclear and intracellular staining. two subsets of nk cells have been phenotypically defined based on nkp marker expression: nkp + and nkp − nk cells [ , ] . following pbmc staining, a similar gating hierarchy was followed by excluding the unstained cells, doublets and cd + cells, and the cd − lymphocytes were further analysed for cd α and nkp expression. among cd − cells, two populations were found in the pbmc, namely, nkp + and nkp − nk cells, and both of these cells were cd α + (additional file a). bal cells were subjected to cell surface staining for the cd surface marker, and the viability of these cells was analysed using propidium iodide (pi) staining (additional file b). additionally, dc and macrophages were segregated from the bal cell population based on staining and gating strategies outlined previously (additional file c) [ , ] . from the mhc-ii + cell population, five phenotypically and functionally defined subpopulations were distinguished using the cd and cd a (sirpα) surface markers. among the mhc-ii + cells, cd a + / cd high cells were defined as am, whereas cd a + / cd int , cd a + /cd low , cd a + /cd − and cd a − /cd − cells were defined as monocytederived macrophages (momɸs), monocyte-derived dendritic cells (modc), conventional dendritic cells (cdc ) and conventional dendritic cells (cdc ), respectively, based on a previous study [ ] . regulatory t cells (tregs), which require intranuclear staining of foxp after cell surface staining, were fixed with cold fixation/permeabilization buffer (ebioscience, thermo fisher scientific, seoul, korea) at °c for min and were then stained for foxp at °c for min. based on a previously described staining and gating strategy [ ] , tregs (cd + foxp + cells) were apparent among the cd + cd − population (additional file d). the t-cell subsets subjected to intracellular staining were stained according to previous studies [ , ] , with few modifications. briefly, single-cell suspensions were treated with a mixture of × cell stimulation cocktail (ebioscience, thermo fisher scientific, seoul, korea) and × brefeldin a (ebioscience, thermo fisher scientific, seoul, korea) in rpmi growth media and incubated at °c in a humidified chamber with % co for - h. the cells were then stained with antibodies for various cell surface markers in cold facs buffer for min at °c, properly washed twice with cold facs buffer, and fixed with intracellular (ic) fixation buffer (ebioscience, thermo fisher scientific, seoul, korea) at °c for min. for intracellular staining, the cells were washed twice with permeabilization buffer ( μl/well) and stained with cytokine-specific antibodies in cold permeabilization buffer at °c for min. subsequently, the cells were washed twice with permeabilization buffer. the gating strategy employed for obtaining various t-cell phenotypes after the gating of singlet lymphocytes is demonstrated in additional file e. a -μl suspension of the stained cell populations in facs buffer was run on an accuri c flow cytometer (bd accuri ™ c plus, bd biosciences, md, usa). bd accuri ™ c plus software version . . . (bd biosciences, md, usa) was used to analyse the data after setting compensation settings according to monocolour and isotype control stains. the data are presented as percentages of all the cell subsets. the cytokine levels in the sera and balf of the uninfected and infected pigs at , and dpc were measured using a porcine-specific procartaplex ™ multiplex immunoassay (thermofisher scientific, vienna- , austria) according to the manufacturer's instructions. magnetic microsphere technology based on porcine cytokine/chemokine antibody-immobilized magnetic beads was employed in the immunoassay for cytokine quantification [ ] . the concentration of each cytokine was measured by running the samples on the luminex ® ™ system (luminex corporation, austin, tx, usa). appropriate standards provided in the kit were utilized to determine the concentration of each cytokine. the machine was verified and calibrated using a luminex ® / ™ verification kit and a luminex ® / ™ calibration kit (luminex corporation, austin, tx, usa) prior to use. graphical presentations of the data were prepared using graphpad prism . (graphpad, san diego, ca, usa), and the data were statistically analysed using spss advanced statistics . software (spss, inc., chicago, il, usa). a nonparametric t-test (mann-whitney u test) was used to compare the viral loads in the lung tissues, the average daily weight gain (adwg), the phenotypes of various cell subsets and the cytokine responses between two groups. the normalized dead cd + cells were analysed by repeated anova (tukey post hoc test) to determine the overall difference, and pairwise comparisons were also performed at different days post-challenge. spearman rank correlation and linear regression were used to determine the associations between two parameters. differences were considered statistically significant if p < . and are indicated by asterisks and different letters over the bars. the highest viremia and lung viral loads in prrsv-ja challenged pigs were detected between and dpc (figures a, b) . the mean peak virus titre in serum at dpc was recorded as . tcid /ml, whereas the mean peak live viral load in the lungs at dpc, which was measured through the microtitration infectivity assay, was . tcid /ml. the virus was gradually cleared by dpc, at which point, the serum and lung viral loads had decreased to mean values of . and . tcid / ml, respectively. as expected, the control group maintained an uninfected state throughout the experiment. the effect of viremia on body weight gain was observed in the infected pigs by calculating the adwg of the pigs ( figure c ). the adwg in the prrsv-ja -challenged pigs was significantly lower than that in the control pigs at , and dpc. the adwg per control pig was recorded as . kg, whereas the adwg of the infected pigs was reduced to . kg. as expected, the growth rate of the pigs was negatively affected by viremia following infection, and this negative correlation was significant (r = − . ; p ≤ . ) ( figure d ). moderate to severe interstitial pneumonia with alveolar wall thickening due to type pneumocyte proliferation and inflammatory cell infiltration was detected in the infected pigs during the period of peak viremia. thus, the highest microscopic lung lesion score in the infected pigs was recorded at dpc, and the scores of the lung lesions in these pigs decreased at later time points but remained at significantly higher levels compared with those in control pigs (figures a, b) . an elisa based on the nucleocapsid (n) protein was employed to measure the prrsv-specific antibody (igg) response in the infected and noninfected pigs ( figure a ). at dpc, prrsv-ja -specific igg were detected in the serum of the infected pigs. additionally, the sample-to-positive (sp) value gradually increased in the infected group from to dpc, whereas in the control group, the pigs did not produce any prrsv-specific igg at any time point. a low svn titre was observed in the challenged pigs at and dpc, when most of the virus was already cleared from the body ( figure b ). in general, the svn antibody responses predominantly appear at the later stages of infection, which is the phase at which most of the virus is cleared from the body, and might play a minor role in the clearance of virus. nk cells, which are a specialized subpopulation of lymphocytes, are the innate immune cells critically responsible for directly killing virus-infected cells, which ultimately leads to viral clearance in the host [ ] . to observe the effect of prrsv infection on nk cells in pigs, the frequencies of two different nk cell subsets in the pbmc population were analysed. the percentages of nkp − nk cells (cd + nkp − in cd − ) in the pbmc populations of the uninfected and infected pigs were higher than those of nkp + nk cells (cd + nkp + in cd − ) (additional file a). moreover, compared with the control pigs, the infected pigs exhibited a significantly (p ≤ . ) increased frequency of nkp + nk cells at the early time point of dpc, whereas the frequency of nkp − nk cells was slightly increased at dpc and significantly increased at dpc. the frequency of nkp + nk cells returned to normal values at dpc, but the frequency of nkp − nk cells in the infected pigs remained at significantly higher values up to dpc ( figures a, b) . the associations between nk cells in pbmc and serum viremia were evaluated post-infection ( figures c, d) . the nkp + nk cell population revealed a significant (r = . ; p < . ) positive correlation with viremia; however, no statistically significant correlation between the levels of nkp − nk cells and viremia was observed. the receptors of host cells determine the cell tropism of prrsv. among others, cd , a cysteine-rich scavenger receptor (srcr), acts as the determinant receptor for prrsv entry and infection [ , ] . the bal cells were subjected to cd staining, and the results show that although the virus did not affect the cd + cells until dpc, the infected pigs exhibited a significant (p ≤ . ) reduction in the cd + cell percentage at dpc, showing a gradual recovery with the decline in the viral loads at dpc ( figure a ). the reduction in the cd + cell population was attributed to the death of cd + cells, which was confirmed by observing the viability of these cells through pi staining. the mean frequencies of dead cd + cells in the infected pigs, which were normalized to those in the uninfected pigs, was significantly (p ≤ . ) higher at and dpc ( . % and . %, respectively) ( figure b ). the class ii major histocompatibility complex (mhc-ii) is essential for the presentation of antigens to t cells and is constitutively expressed on macrophages and dc [ , ] . the mhc-ii + cells were analysed to observe the changes in the dc/macrophage network in the lungs. the mhc-ii + cells, such as cd + cells, show significant (p ≤ . ) decreases at and dpc ( figure c) . interestingly, the percentage of am (cd a + /cd high /mhc-ii + cells), which constituted the majority of mhc-ii + cells, decreased significantly at dpc after infection (p ≤ . ) (figure d ). in contrast, the cd a + /cd int /mhc-ii + cell (momɸ) and cd a + /cd low /mhc-ii + cell (modc) frequencies were significantly (p ≤ . ) reduced in the infected pigs early during the infection process ( dpc) ( figures e, f) . however, the cd a + / cd int /mhc-ii + cell percentages in the infected pigs were significantly (p ≤ . ) increased at and dpc, and a higher (p ≤ . ) cd a + /cd low /mhc-ii + cell frequency was also detected at , , and dpc in the infected pigs compared with the control pigs. cd a + /cd − /mhc-ii + and cd a − /cd − / mhc-ii + cells were not frequently detected among bal cells and accounted for less than % of the five subsets ( figures g, h) . at dpc, a significantly higher (p ≤ . ) percentage of these cell populations was observed in the infected pigs, and this value decreased with the reduction in the viral load and the recovery of am. the associations between the lung viral loads and different subpopulations of macrophages and dc in bal were evaluated after pooling the results of infected pigs at each time point (figure ) . during the infection, mhc-ii + cells and am displayed a significant negative correlation (p < . ) with lung viral loads; however, other subsets exhibited a significant (p < . ) positive correlation. during infection, prrsv-ja alters the dynamics of the respiratory dc/macrophage network by destroying am while increasing the populations of antigen-presenting cells (apc), which bridge the gap between innate and adaptive immune systems by presenting the foreign antigen to t cells. the peripheral immune response was measured by analysing the t-cell populations in the pbmc of uninfected and infected pigs at , , , and dpc (additional file a). the th (ifn-γ + in cd + cd − ) response in the infected pigs was significantly higher (p ≤ . ) than that in the control pigs at dpc, and these responses continued to increase until dpc. the cytotoxic t lymphocyte (ctl) (ifn-γ + in cd − cd + ) response in the infected pigs started to increase at dpc and was significantly higher (p ≤ . ) compared with that in the control pigs at dpc ( figure a ). at dpc, the th (il + in cd + cd − ) cell response was significantly higher (p ≤ . ) in the infected pigs compared with the control pigs, and the response was further escalated at later time points. the il- -producing cd − cd + cell population was significantly higher (p ≤ . ) in the infected pigs compared with uninfected pigs at dpc. the delay in the induction of effector t cells was mainly perceived peripherally in blood. to observe the activation of the local adaptive immune responses by innate immune cells at the sites of replication and the persistence of prrsv, the t-cell phenotypes in the bln, bal and lung parenchyma of the euthanized control and infected pigs were analysed at , , and dpc. the percentages of various immune cells in the lungs, bal and bln of uninfected and prrsv-ja infected pigs at different stages of infection are summarized in additional files b, c, and d. overall, various t-cell responses were significantly induced in the lungs as early as dpc, and significant responses in bal and bln were first detected at dpc and were maintained until dpc. the th cell (ifn-γ + in cd + cd − ) frequency in all the local tissues was significantly (p ≤ . ) induced in the challenged pigs as early as dpc, and the frequency in bal cells tended to be higher. the ctl (ifn-γ + in cd − cd + ) frequency was significantly (p ≤ . ) higher in the lungs of the infected pigs compared with that of the control pigs at dpc, whereas the frequency in bln and bal cells was higher at the early time point and significantly higher at dpc. the prrsv-ja -infected pigs also displayed a higher induction (p ≤ . ) of th cells (il + in cd + cd − ) in the lymph nodes and lung tissues at dpc, whereas a slight increase in these cells was observed in bal cells. the il- -producing cd − cd + population was significantly (p ≤ . ) induced in the lungs and bal cells of the infected pigs at dpc, whereas in bln, this cell population showed a slight increase at dpc and a significant increase (p ≤ . ) at dpc (figures b-d) . therefore, compared with the weak and delayed peripheral responses, early and effective cellular immune responses were triggered in local tissues by prrsv-ja infection. tregs are well known for their immunosuppressive activities, and previous studies have demonstrated that prrsv infection induces treg responses that might be responsible for ineffective adaptive immune responses [ ] [ ] [ ] . unlike previous studies, no upregulation of tregs (cd + foxp + in cd + cd − ) was detected in pbmc isolated from prrsv-infected pigs. moreover, tregs were significantly (p ≤ . ) reduced in the bal throughout the course of infection, but no such decline was observed in the lungs (figure ). however, the treg frequencies in bln of the infected pigs initially exhibited a significant decline at dpc but then increased significantly at dpc. the levels of seven different innate and adaptive cytokine/chemokine proteins in the sera and balf at , and dpc were compared between the uninfected and infected pigs ( figure ) . the results reveal that interferon-α (ifn-α) was significantly (p ≤ . ) induced in the sera and balf of the infected pigs at dpc. however, at peak viremia ( dpc), the ifn-α level was reduced peripherally in the sera of the infected pigs, whereas the cytokine level was elevated locally in the balf. nevertheless, significant increases in the level of this cytokine (p ≤ . ) were maintained both locally and systemically in the infected pigs compared with the uninfected pigs. furthermore, proinflammatory cytokines/chemokines, such as tumour necrosis factor-α (tnf-α), interleukin- β (il- β), il- , il- and il- , show a similar pattern of early induction at dpc in the sera of the infected pigs followed by decreases as viremia increased. in the sera, significant (p ≤ . ) changes in il- β were only observed in the infected pigs at and dpc. however, significant (p ≤ . ) induction of il- β and il- locally in the balf was observed in the infected pigs at dpc. in addition, elevations in all the proinflammatory cytokine/chemokine levels were observed in the infected pigs as the viral load increased, and significant increases in the levels of il- β, il- , il- and il- were observed in the infected pigs compared with the uninfected pigs at dpc. moreover, il- , an anti-inflammatory cytokine, was significantly induced (p ≤ . ) at and dpc in the balf of the infected pigs. overall, the clearance of the virus from the pigs also coincided with the increased secretion of anti-viral and proinflammatory figure local and systemic levels of cytokines/chemokines in the pigs. the concentrations of cytokines/chemokines in the a sera and b balf of pigs belonging to both groups at , , and dpc were measured using a multiplex luminex-based cytokine immunoassay. the bars in the graphs represent the mean ± sem of the cytokine levels, and the asterisks (*) indicate a statistically significant difference between the averages found for the uninfected (nc) pigs and those obtained for the infected pigs at each time point (* indicates p ≤ . and ** indicates p ≤ . ). cytokines locally in the balf of prrsv-infected pigs, although no considerable shifts were detected in the serum. despite exhaustive research, the understanding of the protective immune responses against prrsv in pigs remains limited. previous studies have mainly focused on certain aspects of immune responses and/or have used a narrow time window to study the immune responses during prrsv infection; therefore, the available data offer a limited picture of the host defence system. to the best of our knowledge, the present study constitutes the first investigation of various aspects of immune responses, such as local vs. systemic and innate vs adaptive, during the course of prrsv infection to obtain a broader picture of the host defence against prrsv. here, we demonstrate the critical role of local immune responses in the clearance of prrsv from pigs due to the early induction of dc/macrophage subsets, the activation of protective t cells in local lymphoid and lung tissues, and the stimulation of proinflammatory cytokines locally in the lungs. in pigs that were intramuscularly inoculated with the biologically characterized prrsv-ja strain [ ] , the viral titre reached its peak value in the serum and lungs between and dpc. in addition, moderate to severe interstitial pneumonia and a significant decrease in the adwg, which are characteristics of prrsv infection [ ] , were detected in the challenged pigs by dpc. mild histopathological lesions (with a lesion score lower than ) characterized by alveolar wall thickening due to type pneumocyte proliferation were also observed in some of the uninfected healthy pigs that were free of other respiratory pathogens. these observations were likely obtained due to the stress induced by environmental factors, such as housing conditions, weaning, individual space, and ambient temperature variations, as previously reported [ , ] . similar to previous reports [ ] , the prrsv-ja strain induced delayed (≥ dpc) and weak (≤ ) svn titres in infected pigs. an svn antibody titre of offers sterilizing immunity, whereas a titre greater than is considered protective against prrsv infection [ ] . the anti-prrsv antibody response induced by prrsv-ja in the infected pigs crossed the threshold of . (s/p ratio) between and dpc, and this response was in accordance with the response produced by other prrsv strains [ ] , but the role of these antibodies in protection against prrsv infection is unknown [ ] . nk cells, an important component of the innate host defence system, play a critical role in the resolution of viral infections [ ] . in general, the potential roles of nk cells in relation to prrsv immunity are poorly understood [ , ] . in the current study, two defined nk cell subpopulations were distinguished in pbmc based on an approach similar to one previously described [ ] . similar to previous observations, the nkp + nk cell frequency in pbmc collected from both control and infected pigs was lower than that of nkp − cells. nkp + and the nkp − nk cells execute analogous cytolytic activities but produce different levels of ifnγ, with nkp + nk cells producing higher amounts of ifn-γ. moreover, nkp can be expressed in nkp − nk cells after stimulation with interleukins (il)- , il- and il- [ ] . in the current study, early increases in the frequencies of nkp + and nkp − nk cells in prrsv-ja -challenged pigs were clearly detected. previous studies have shown similar increases in the cd − cd − cd + cell population after infection with different strains of prrsv [ , ] . induced proliferation of nk cells has also been perceived after influenza infection, suggesting that this subset of lymphocytes is capable of antigen-specific clonal expansion [ ] . prrsv, however, significantly suppresses nk cell-mediated cytotoxicity to evade the host immune response [ ] , but these previous studies did not consider the subsets of nk cells. the specific action of different subsets of nk cells on prrsv has not yet been explored, and further studies are needed to explain the role of nk cells in the containment of the virus during infection. the main targets of prrsv are cells belonging to the monocyte and macrophage lineages, particularly am. however, dc are also reportedly vulnerable to prrsv infection [ ] . the dc/macrophage network, which senses the foreign antigen and initiates the immune response, constitutes one of the main components of the respiratory immune system [ ] . it is plausible to expect that the viral infection of these cells alters this network and thus affects downstream immune responses. therefore, dissecting how these immune cells respond to prrsv infection is important to better understand the nature of prrs. until now, limited studies have investigated the alterations in the respiratory dc/macrophage network in pigs during disease progression [ , ] , and the association of the alteration in this immune network with the t-cell response has not been reported. here, we attempted to explore the dynamics of this important host defence mechanism in relation to prrsv infection. during infection, prrsv replicates in the cd + cells in the lungs and induces apoptosis at the early stages of infection [ ] . in the current study, a flow cytometric analysis of bal cells revealed that prrsv-ja decreased the cd + cell population in the infected pigs at the time when peak viral loads were detected. after the viral load decreased, the cd + cell population recovered to the normal levels. pi staining of the cd + bal cells revealed that the decrease in the cd + cell population was due to cell death. a similar reduction in the macrophage population after prrsv- infection was previously reported [ ] . following the strategy described previously [ ] , different subsets of dc and macrophages were identified in bal cells, and the changes in the populations of these subsets during disease progression were studied. previous reports verify that prrsv infection impairs dc function directly by downregulating mhc-ii expression [ , ] . intriguingly, among the five cell subsets in mhc-ii + cells, only the cd a + /cd high / mhc-ii + (am) cell population decreased significantly at peak viremia, revealing a significant negative correlation between the population of these cells and the viral loads in the lungs, whereas significant positive correlations were found for the other subset populations. the increases in the cd a + /cd int /mhc-ii + and cd a + /cd low /mhc-ii + cell populations can be attributed to the higher influx of monocytes and their differentiation in the lungs during inflammation. moreover, cd a + /cd − /mhc-ii + and cd a − /cd − / mhc-ii + cells exhibit strong migration and antigenpresenting capabilities [ ] , which can be credited to the increased influx of these cells into the lungs during the course of infection. our results were in agreement with those obtained in previous studies, which found that the populations of these cells are increased in the bal after prrsv- infection [ ] . interestingly, cdc s activate allogenic naïve t cells and aid induction of the th response, whereas cdc s induce a th response in pigs [ ] . after sensing antigen, the mature migrating dc recruit nk cells to drain ln, thus providing an early source of ifn-γ and thereby promoting th polarization [ ] . thus, dc work together with nk cells to regulate innate immunity and further dictate the direction and intensity of the adaptive immune response [ ] . the effective immune response to counter viral infections is governed by the proper activation of t lymphocytes by apc [ ] . in the current study, we analysed the dynamics of t lymphocytes in pbmc, lung parenchyma, bal, and bln to detect the central cause of the clearance of prrsv from the body. delayed induction of th , th and ctl responses was observed in the pbmc of the infected pigs after dpc, when most of the virus had been cleared from the blood, whereas in bln, the virus persists for a longer period, which leads to early and sustained (> dpc) induction of t cell responses. previous studies also found a delayed induction of effector t cells in the peripheral blood of pigs infected with prrsv [ , ] . intriguingly, significant increases in the frequencies of these t lymphocyte subpopulations were detected in the lung parenchyma and lymphoid tissues of the infected pigs at early stages of infection ( dpc), when the virus levels were at their peak in the body. in agreement with our findings, a higher frequency of t-helper cells and ctl has been observed in local tissues, such as the bal, lymph nodes and lung parenchyma, of pigs after swine influenza infection, and this induction has been linked to viral clearance [ ] . the local cell-mediated immune responses are considered crucial for the clearance of the influenza virus [ ] . moreover, during human respiratory syncytial virus infection, increased populations of cd + and cd + t cells exhibiting effector functions have been observed in the bal of infected patients [ ] . a recent study investigating t-cell proliferation in prrsv- -infected pbmc at dpi and the expression of lymph node-homing receptors revealed that the t-helper cell response plays a main role in viral clearance and that the ctl response is strongest at the site of infection [ ] . in addition, the induction of th cells in local tissues has also been found to be essential for the resolution of respiratory infections [ ] . in the present study, the cell responses in bln and bal were found to be significantly increased from or dpc and were maintained until dpc, when the virus was completely cleared. therefore, it can be concluded that local t-cell responses in the lungs, bln and bal are induced markedly faster than systemic responses and are maintained at significantly high levels, even after virus clearance, substantiating the critical role of local immune responses in the clearance of prrsv from pigs. the treg lineage is responsible for the maintenance of homeostasis in the immune system by suppressing the activation of various immune cells, including other t cells, nk cells and dc [ ] . treg play a vital role in the pathogenesis of some viral infections that result in severe inflammatory lesions, such as influenza [ ] . however, the treg response in pigs after prrsv infection is controversial. although some studies have revealed increased frequencies of tregs after prrsv infection [ , , ] , other studies revealed that the induction of tregs was not changed and even suppressed after infection [ , ] . these conflicting results could be due to the strainspecific response of the pigs to the virus after challenge or to the method used to evaluate the changes in the treg response. several studies have revealed that tregs are induced due to prrsv infection, but most of these studies were performed using in vitro or ex vivo assays in which pbmc and mononuclear cells isolated from the lungs and lymph nodes of pigs were infected or reinfected with prrsv and subsequently observed to monitor the proliferation of various t-cell subsets [ , , ] . however, some ex vivo assays have also demonstrated the inability of certain strains of prrsv to induce tregs [ ] . in the current study, the treg frequencies were directly evaluated in the peripheral blood, bal, lung and lymphoid tissues collected from prrsv-ja -infected pigs. during the acute phase of infection, the treg frequencies largely remained unchanged, but suppression was observed in the bal throughout the infection period and in bln at dpc. comparable results were obtained in a previous in vivo study, revealing that the treg numbers remained unchanged in the pbmc, lymph nodes and tonsils of the infected pigs up to dpc [ ] . similar results were observed in another study, which showed no significant upregulation of tregs in the lymph nodes and pbmc at the acute phase of prrsv infection [ ] . furthermore, the decrease in treg frequencies in ja infected pigs could be attributed to phenotypic plasticity, which resulted in most of the naïve cd + t cells differentiating into effector cells, such as th and th , after being antigenically stimulated during the acute infection [ ] . a previous study revealed that the influenza virusinfected pigs showed a similar suppression of tregs in the bal and lymph nodes and an increased frequency of t-helper cells during the acute phase of infection [ ] . moreover, in some acute infections of mice with lymphocytic choriomeningitis virus (lcmv) and influenza virus, little or no effect on the immune response was observed after the removal of tregs from mice [ , ] . based on the findings from these studies, it can be deduced that tregs play only a minor role during many acute infections, but further studies are needed to understand the ultimate cause of this decline in the treg populations, specifically in bal cells during prrsv infection. cytokine secretion by immune cells plays a major role in protection against invading pathogens or in the induction of pathology [ ] . the levels of proinflammatory cytokines have often been associated with the severity of the disease. however, lower mrna and protein expression levels of proinflammatory cytokines have been associated with protection against viruses in pigs infected with prrsv strains that typically induce mild to moderate clinical signs [ ] . prrsv has been shown to reduce or suppress ifn-α production during infection [ ] . similarly, in the current study, ifn-α was peripherally suppressed in the infected pigs with increasing viral replication; however, in the balf, the levels of ifn-α were maintained, even at peak viremia, possibly playing a role in local viral clearance. similar to our observations, the induction of ifn-α with increasing viremia has also been observed locally in lymphoid tissues and the balf in prrsv-infected pigs [ ] . moreover, local induction of the proinflammatory cytokines il- β and il- in lymphoid tissues is reportedly linked to the clearance of prrsv from infected pigs [ ] . similar induction of the proinflammatory cytokines il- α, il- , il- , and tnf-α figure chronological order of the immune responses elicited in prrsv-infected pigs. this figure shows an overall representation of the immune responses elicited in prrsv-infected pigs during the acute phase of infection, which is the stage when the virus reached its maximum titre. most of the virus is cleared from blood and tissues by dpc due to the pig immune responses. the delayed induction of serum virus-neutralizing antibodies and the t-cell response in peripheral blood at days post-exposure, when most of the virus was cleared from the body, indicate that, in addition to the other factors, the early induced local t-cell response plays a key role in the clearance of the virus. has been observed in local tissues in previous studies [ ] . in the current study, the induction of proinflammatory cytokines locally in the balf at peak viremia could thus be linked to their contribution to the clearance of the virus from the pigs. in contrast, the induction of il- in local tissues during early infection was observed in the present study. the production of il- , which has been previously observed, reportedly contributes to the persistence of prrsv by suppressing the immune response in pigs [ ] . however, il- production also protects pigs from the tissue damage caused by proinflammatory cytokine overexpression [ ] . tregs are thought to produce il- , but in the current study, the treg frequencies did not change significantly. however, in addition to tregs, monocytes, th cells, mast cells and b cells are also known sources of il- [ ] . consequently, measuring the systemic cytokine response might not provide a full picture of the protective events orchestrated by cytokines locally in the lungs of prrsv-infected pigs. in conclusion, the early stimulation of the dc/macrophage frequencies coincided with the induction of a protective t-cell response in local lymphoid tissues and lung parenchyma, which are known sites of prrsv replication. moreover, the local proinflammatory cytokine responses at peak viremia augmented the clearance of the virus from the infected pigs. a temporal sequence of immunobiological events after prrsv infection in pigs is proposed in figure , and the observed schematic suggests that local t lymphocytes might play an important role in the clearance of the virus from pigs during the acute phase of infection. in addition, early nk cell induction and delayed t-cell responses in peripheral blood were also perceived, and these might play a complementary role in viral clearance. future studies are needed to further elucidate and refine the understanding of the anti-prrsv immune response in pigs, and these future studies should primarily focus on obtaining a better understanding of and deciphering the local immune responses with the aim of developing an effective strategy to restrain the virus. characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination duration of infection and 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in gnotobiotic pigs porcine reproductive and respiratory syndrome: neb- prrsv infection did not potentiate bacterial pathogens comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr ), atcc vr , and two recent field isolates of prrsv convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus porcine 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jurisdictional claims in published maps and institutional affiliations the authors are pleased to acknowledge the undergraduates at the college of veterinary medicine and the laboratory technicians at the veterinary diagnostic center of jeonbuk national university (jbnu) for their constant assistance throughout the study. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . key: cord- -k dqe lk authors: hoelzer, karin; bielke, lisa; blake, damer p.; cox, eric; cutting, simon m.; devriendt, bert; erlacher-vindel, elisabeth; goossens, evy; karaca, kemal; lemiere, stephane; metzner, martin; raicek, margot; collell suriñach, miquel; wong, nora m.; gay, cyril; van immerseel, filip title: vaccines as alternatives to antibiotics for food producing animals. part : new approaches and potential solutions date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: k dqe lk vaccines and other alternative products are central to the future success of animal agriculture because they can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations. to assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the united states department of agriculture, with support from the world organisation for animal health, organized the second international symposium on alternatives to antibiotics. it focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. this article, the second part in a two-part series, highlights new approaches and potential solutions for the development of vaccines as alternatives to antibiotics in food producing animals; opportunities, challenges and needs for the development of such vaccines are discussed in the first part of this series. as discussed in part of this manuscript, many current vaccines fall short of ideal vaccines in one or more respects. promising breakthroughs to overcome these limitations include new biotechnology techniques, new oral vaccine approaches, novel adjuvants, new delivery strategies based on bacterial spores, and live recombinant vectors; they also include new vaccination strategies in-ovo, and strategies that simultaneously protect against multiple pathogens. however, translating this research into commercial vaccines that effectively reduce the need for antibiotics will require close collaboration among stakeholders, for instance through public–private partnerships. targeted research and development investments and concerted efforts by all affected are needed to realize the potential of vaccines to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks. alternatives to antibiotics can help minimize the need for antibiotics by helping to prevent and control infectious diseases in animal populations. as such, safe and effective alternatives are crucially important to the future success of animal health and production. to assess scientific advancements in the research and development of alternatives to antibiotics, highlight promising research results and novel technologies, assess challenges associated with their commercialization and use, and provide actionable strategies to support their development, the united states department of agriculture (usda), with support from the world organisation for animal health (oie), organized the second international symposium on alternatives to antibiotics. the symposium focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the licensure and development of alternatives to antibiotics. this two-part manuscript synthesizes and expands on the scientific presentations and expert panel discussions from the symposium regarding the use of vaccines as alternatives to antibiotics that can reduce the need for antibiotic use in animals. part synthesizes and expands on the expert panel discussions regarding the opportunities, challenges and needs related to vaccines that may reduce the requirement for use of antibiotics in animals, while part two focuses on highlighting new approaches and potential solutions. a general discussion of the importance of antibiotic resistance and the opportunities, challenges and needs related to vaccines as alternatives that may reduce the need for use of antibiotics in animals is provided in part of this review, including a discussion of the properties of ideal vaccines, how current vaccines compare to these ideal vaccines, and how investment decisions around research and development of vaccines are made. this second part of the manuscript will highlight specific research advancements in the area of veterinary vaccines. as mentioned in part one of this manuscript, most pathogens invade the host at the mucosal surfaces, such as the gastro-intestinal (gi) tract. the gi tract constitutes the largest surface area of the body and is exposed daily to vast numbers of foreign antigens derived from feed, the microbiota and pathogens [ ] . within the intestine a complex cellular network has evolved to prevent unwanted immune responses to innocuous antigens, for instance feed or microbiota, while allowing swift protective responses against agents that cause infectious disease. key to keeping enteric pathogens at bay is the presence of protective pathogen-specific secretory iga (siga) at the site of entry, which prevents the adhesion of micro-organisms to the intestinal surfaces and neutralizes their enterotoxins. triggering robust and protective intestinal siga responses usually requires the local administration of vaccines [ ] . although live attenuated oral vaccines have had tremendous success, resulting for instance in the near global eradication of poliovirus [ ] , concerns on the dissemination of vaccine strains into the environment and rare cases of reversion to virulence, leading to vaccine-induced disease, have driven oral vaccine development to nonliving or vectored vaccines [ ] . however, oral vaccination is challenging due to several hurdles imposed by the cellular and molecular architecture of the gut: (i) the harsh environment of the stomach and small intestine, including the low ph, digestive enzymes, and bile salts, required to digest feed also easily destroys vaccines, (ii) a poor uptake of vaccine antigens by the intestinal epithelial barrier and (iii) the tolerogenic mechanisms that pervade the intestinal tissues, leading to peripheral and oral immune tolerance upon oral administration of antigens via the induction of foxp + regulatory t cells. this often results in a low immunogenicity of oral vaccines and requires innovative strategies to deliver the vaccine antigens to the intestinal immune system as well as the inclusion of adjuvants that promote innate and adaptive immunity [ ] . the mucosal immune system in the gut can be divided in inductive sites, where sampled antigens stimulate naive t and b cells, and effector sites, where effector cells perform their functions, e.g. assisting in the production of siga. in the small intestine, the inductive sites comprise the gut-associated lymphoid tissues (galt) and the mesenteric lymph nodes, while the effector sites constitute the lamina propria and the surface epithelium [ ] . the galt itself is composed of peyer's patches (pp), appendix and isolated lymphoid follicles. the presence of other galt-like structures, such as lymphocyte-filled villi (rat, human) and cryptopatches (mouse) is dependent on the species. interestingly, while in birds and most mammals pp or their equivalent are scattered throughout the small intestine, in pigs, ruminants and dogs the pp in the distal small intestine (ileum) are continuous. fish and reptiles on the other hand lack pp and the intestinal immune system in these species is composed of epithelial leukocytes . . next generation anticoccidial vaccines autogenous vaccines to reduce the need for antibiotic use conclusions references and rare, small non-organized lymphoid aggregates. it remains largely unknown how these species-specific differences might affect the efficacy of oral vaccines. from their entry point, which is typically the oral cavity, to their delivery site, most commonly the small intestine, the integrity of delivery systems and the stability of vaccine components are at risk. lysozyme in saliva, the low gastric ph together with pepsin and intestinal proteases can degrade oral vaccines. enteric coating of vaccine components with ph-responsive polymers with a dissolution threshold of ph might protect against gastric degradation and results in the release of their contents in the small intestine [ ] . in this context, ruminants pose an additional problem to vaccine stability as their polygastric gastro-intestinal tract effectively degrades substances including vaccines. site-specific delivery of oral vaccines to the small intestine is favorable as the mucus layer covering the small intestinal epithelium consists of only one layer, which is loosely adherent, less thick and patchy as compared to the colonic mucus layers and might promote their access to the intestinal epithelium. in addition, the small intestine is less densely populated by the microbiota, which might further disrupt the integrity of the delivery systems and the stability of vaccine components. underneath the mucus layer, a single layer of intestinal epithelial cells prevents uncontrolled access of the luminal content to the underlying intestinal tissues, further restricting uptake of oral vaccine antigens. crossing of the epithelial barrier by vaccines could be enhanced by exploiting antigen sampling routes in the small intestine or by adopting strategies used by enteric pathogens to colonize or invade the host [ ] . the best-known sampling route in the gut is associated with microfold (m) cells. these specialized intestinal epithelial cells reside within the follicle-associated epithelium covering the peyer's patches and take up macromolecules, particulate matter and microorganisms [ ] . many enteric pathogens hijack m cells to invade the host by binding to apical receptors. for instance, the invasin protein of yersinia species interacts with β integrin on m cells, leading to infection [ ] . likewise, gp marks m cells in many species and binds to fimh, a subunit of type i pili on escherichia coli and salmonella enterica. this interaction results in uptake of fimh + bacteria and initiates mucosal immunity [ ] . although many groups have focused on improving antigen uptake by targeting oral vaccines to m cell-specific receptors, these cells represent only a small, species-specific percentage of the total intestinal epithelial cell population. although m cell numbers increase from the cranial to caudal small intestine and m cell targeting strategies work quite well in rodent models, they mostly fail in larger animals due to the long passage time needed to reach the distal small intestine, where the gut-associated immune system is most pronounced. besides m cells, sampling of luminal antigens also occurs by intestinal mononuclear phagocytes via transepithelial dendrites. this sampling mainly occurs by cd c + cx cr + macrophages, which transfer the antigens to cd + dendritic cells (dcs). these dcs then drive the differentiation of regulatory t cells (tregs), which subsequently induce tolerance to these proteins [ ] . in the steady state, goblet cells can also transport small soluble proteins (< kda) across the epithelium to tolerogenic dcs via so-called goblet cell-associated antigen passages [ ] . absorptive intestinal epithelial cells or enterocytes, constituting > % of the small intestinal epithelium, may also sample the luminal content through receptor-mediated transcytosis. for instance, the neonatal fc receptor (fcrn), an mhc class i-like fcγ receptor, is expressed on the apical surface of enterocytes and transcytoses igg, immune complexes or fc-coated nanoparticles from the lumen to the basolateral surface of the epithelium [ ] . similar to m cells, it might be worthwhile to target apical receptors exploited by enteropathogens on small intestinal enterocytes to promote uptake of antigens by the epithelial barrier. a potential candidate would be aminopeptidase n (anpep), a zinc-dependent peptidase present in the brush border of small intestinal enterocytes, which serves as an entry receptor for several coronaviruses and also binds f fimbriae, a colonisation factor produced by porcine-specific enterotoxigenic e. coli. anpep also transports f fimbriae as well as microparticles functionalised with anpep-specific monoclonal antibodies across the intestinal epithelial barrier, resulting in robust intestinal siga responses, at least in piglets [ , ] . although the selective targeting of vaccine antigens to apical receptors might promote their uptake by the epithelium via transcytosis, this process is in itself insufficient to trigger protective intestinal immunity upon oral vaccination and explains the need to include adjuvants. these adjuvants should act on antigen presenting cells as well as intestinal epithelial cells to promote the induction of protective siga and cell-mediated immune responses. indeed, enterocytes not only provide a physical barrier separating the intestinal lumen from the host tissues, but also relay information on the luminal content to the underlying immune cells through the secretion of inflammatory or tolerogenic mediators. for instance, during the steady state, enterocytes produce thymic stromal lymphopoëtin (tslp) and transforming growth factor (tgfβ), which imprint a tolerogenic phenotype on intestinal dendritic cells [ ] . in contrast, upon infection enterocytes secrete il- and il- [ ] . this probably facilitates a switch from a tolerogenic to an immune-inductive environment, allowing activation of intestinal antigen presenting cells. as yet the most effective adjuvants for oral application are the enterotoxins from vibrio cholera (ct) and enterotoxigenic e. coli (etec) (lt). due to inherent toxicity, dmlt was developed, a nontoxic lt mutant retaining its adjuvanticity. this dmlt triggered intestinal memory responses upon oral vaccination with a nonliving etec vaccine and seems a promising candidate to be included as adjuvant in oral vaccines [ , ] . similarly promising strategies have been reported for eimeria [ ] . recent studies have shown that eimeria-induced il- production is critical in the initiation of early innate immune response in coccidiosis and blocking of il- production by exogenous il- -neutralizing antibody reduced both the intracellular development of eimeria and the severity of intestinal lesion [ ] [ ] [ ] . in summarizing this part, future design of oral vaccines should be tailored to the needs of the target species, focus on the selective delivery of vaccines to epithelial receptors to promote their transport across the epithelial barrier, induce protective immune response in the target tissues, and should include a mucosal adjuvant able to trigger memory siga responses. endospores, or spores, are produced by many bacteria as a response to nutrient deprivation. the spore is a dormant entity about μm in size that can germinate, allowing a nascent cell to emerge and enter vegetative cell growth [ ] . the spore carries remarkable resistance properties, being typically resistant to high temperatures (typically - °c), desiccation, irradiation, and exposure to noxious chemicals [ ] . the two principal sporeforming bacterial genera are bacillus and clostridia with the latter being exclusively anaerobic. members of the bacillus genus are being used as probiotics, that is, microorganisms that are added to the diet to improve the balance of microbial communities in the gi-tract and are therefore beneficial to human or animal health [ , ] . typical species include bacillus clausii, bacillus coagulans and bacillus subtilis. for a long time, it has been assumed that bacillus spores are soil organisms yet the evidence supporting this is actually rather sparse. instead, spores are found in the soil in abundance but live, vegetative cells, are rarely if ever found other than in association with plants or in the animal gut. mounting evidence shows that spores, although found in the soil, are mostly dormant and are shed in the feces of animals, which are their natural hosts [ ] . the consumption of spores associated with soil-contaminated plant matter enables spores to enter the gi-tract, transit the gastric barrier unscathed and then germinate and proliferate in the intestine before excretion as dormant spores [ ] . evidence suggests that spore forming bacteria comprise as much as % of the gut microbiota, indicating that the ability to form spores enables bacteria to survive in the environment as well as entering and transiting the gastric barrier of animals [ ] . the extraordinary resistance properties of bacillus spores coupled with their ease of genetic manipulation, and their successful use as probiotics, makes them attractive candidates for the delivery of heterologous antigens for vaccination. spores have been used as vaccine vehicles in a number of ways, differing principally in whether spores are genetically modified or not. in all cases b. subtilis has been utilized due to the excellent genetics available. using genetic modification, a chimeric gene consisting of a fusion between a b. subtilis anchor gene and an open reading frame encoding a putative protective antigen is first constructed. the next step is introduction of the chimera into the b. subtilis chromosome using a gene transfer technique, typically dna-mediated transformation, a process in b. subtilis that is straightforward. typically, the anchor is the ′-end of a gene encoding a spore coat protein such that the chimera is displayed on the spore coat. surprisingly, heterologous antigens displayed on b. subtilis spores are mostly stable and do not appear to suffer extensive degradation. using this approach a number of candidate antigens have been displayed and then evaluated in animal models. for example, spores displaying a tetanus antigen ttfc conferred protection to a lethal dose of tetanus toxin when administered orally [ , ] . mice dosed orally with spores expressing part of the alpha toxin of clostridium perfringens were protected to challenge with alpha toxin [ ] . a more recent example is that of clostridium difficile where a c-terminal fragment of the toxin a (tcda) could be stably expressed and when administered orally to hamsters conferred protection to c. difficile infection [ , ] . this particular vaccine has now entered clinical evaluation in humans [ ] . using a non-genetically modified organism (gmo) approach it has been shown that spores can adsorb antigens efficiently onto their surface and surprisingly this is both strong and stable, and reflects the unique biophysical properties of the spore [ ] . for the adsorption approach, it has been shown that the gastric barrier is particularly corrosive and adsorbed antigens are labile, but for intranasal delivery this method appears satisfactory. using this approach inactive (killed) spores can be used and success has included studies showing protection to influenza (h n ) [ ] and significant reduction in lung counts of animals challenged with mycobacterium tuberculosis [ ] . a unique feature of spores is their ability to enhance immune responses and this adjuvant effect has been characterized in depth [ - ]. however, the use of spores as mass-delivery vehicles for vaccines has several limitations. oral delivery clearly is the preferred approach but appears to work effectively only for the gmo approach. oral delivery also raises issues of tolerance and may prove to be a limiting factor. sublingual delivery has also been explored; this approach appears to provide levels of protection that are equivalent to oral delivery, but requires more doses [ , ] . nasal delivery is suitable but raises potential safety issues. for animal vaccines, spores are attractive since they are currently used as feed probiotics but also because they can survive the high temperatures used for feed production and may offer long-term utility. as mentioned already, spores have been manipulated for protection against c. perfringens but there now exists the opportunity to develop spores for protective vaccination to necrotic enteritis, an important poultry disease caused by c. perfringens that has been identified as a high vaccine research priority by the oie ad hoc group (see additional file in http://doi.org/ . /s - - - ). one application that is particularly promising is the use of spore vaccines in aquaculture. with intensive fish farming, bacillus spores are being used as probiotic feed supplements. for shrimp farming, viral diseases have devastated the industry and one of the most important shrimp pathogens is white spot syndrome virus (wssv) that causes seasonal outbreaks of disease [ ] . a number of groups have developed b. subtilis spores that display the vp capsid protein of wssv and when administered in feed appears to protect against white spot disease [ - ]. the mechanism for protection is intriguing; even though shrimp are not thought to produce antibodies, it is clear that presentation of the viral antigens does produce some level of specific immunity. despite the progress being made with spore vaccines one key issue remains: the containment of gmos. because spores are dormant with the potential to survive indefinitely in the environment, the use of recombinant spores in spore vaccines is likely to raise environmental concerns and successful regulatory approvals may be slow or impossible to secure. for human use, it is likely that a case can be made that the recombinant spore vaccines addresses an unmet clinical need, but for animal use devising a method for biological containment will be crucial. technological advancements now make it possible to genetically engineer bacteria and other microorganisms that deliver heterologous antigens in a way that can stimulate mucosal as well as humoral and cellular systemic in veterinary medicine, oral vectored vaccines have been instrumental in the eradication or control of rabies in wildlife reservoirs [ , ] . oral vectored vaccines have also been developed for several other veterinary applications, including some economically important diseases of food-producing animals that are associated with considerable antibiotic use such as porcine circovirus type- (pcv- ); in some cases, the vaccine vector is a chimera containing parts of multiple microorganisms-for instance, an attenuated live vaccine may be used as the vector-and the resulting vaccine simultaneously confers protection against multiple diseases, for instance marek's disease and infectious bursal disease or newcastle disease and avian influenza [ , ] . the development of some vaccine vector systems has been very successful and numerous veterinary vaccines have been developed based on them; the canarypox virus vector system alvac, for instance, has been used for the development of a range of veterinary vaccines including against rabies, influenza, and west nile virus [ ] . similarly, adenovirus vectors have also been widely used in veterinary medicine, both in companion and food-producing animals [ ] . vaccine platforms such as these are particularly valuable as they can allow for the rapid development of vaccine candidates in response to emerging vaccine needs, but the possibility of anti-vector immunity can restrict their usefulness [ ] . research and development of additional vaccine vector platforms is therefore needed. salmonella strains that express foreign antigens, either chromosomally or plasmid-based, have yielded promising results in several species including mice, humans, pigs and chicken [ ] [ ] [ ] [ ] [ ] [ ] . diseases for which these salmonella vectored vaccines were investigated include influenza, brucella abortus, post-weaning diarrhea and heterologous strains of salmonella [ ] [ ] [ ] [ ] . the use of pasteurellaceae as vectors for modified live vaccines against shipping fever in calves is currently under investigation, with promising preliminary findings [ ] . use of this vector system for other diseases including pinkeye has been suggested [ ] . in-ovo vaccination is a mass-vaccination strategy that is mainly used in broiler chickens albeit occasionally also in broiler-breeder and layer chickens [ ] . eggs are injected in the hatchery, typically during the third week of embryonic development around day or . to vaccinate, a small hole is made in the shell at the blunt end of the egg and the vaccine is injected below the chorion-allantoic membrane into the amniotic cavity or directly into the embryo. commercial in-ovo vaccination systems that automatically inject the eggs have been available since the early s. more than % of broiler chickens in the us are vaccinated in ovo, and in brazil that fraction equals % [ ] . the most common use of in-ovo vaccination is for marek's disease, potentially combined with vaccines against other diseases such as gumboro or newcastle disease. the ability to deliver a clearly defined vaccine dosage to every single chick and to invoke early protection in the chicks is among the main benefits of this technology, but it is labor-intensive, causes stress for the chicks, and high sanitary standards need to be followed during vaccine preparation and injection to manage infection risks [ , ] . in addition, the location of the vaccine injection is critical for efficacy. it has been shown, for instance, that if marek's disease vaccine is accidentally deposited into the air cell or allantoic fluid, adequate protection is not achieved [ ] . the stage of embryonic development can have profound effects on vaccine safety and efficacy [ ] . one study, reported that vaccination of - dayold embryos with herpes virus of turkeys (hvt) led to pronounced lesions and embryonic deaths, while vaccination on days did not cause detectable lesions [ ] . embryonic age at vaccination has also been shown to be correlated with antibody titers [ ] . maternal antibody titers actually increase after the typical age for in-ovo vaccinations and peak just after hatch [ ] . this can interfere with proper vaccine responses. however, evidence suggests that some vaccine strains are more affected by maternal antibodies than others [ ] . deliberate vaccine development may therefore limit the often disruptive effects that can be caused by maternal antibodies [ ] . other factors that need to be considered in the development of a successful in-ovo vaccination program include the characteristics of the vaccine or vaccines to be used, the type of incubator in which the eggs are housed in the hatchery, and the breed and age of the parent flock [ ] . in-ovo vaccination strategies are promising means of reducing antibiotic use in poultry production and have been the subject of intense research. importantly, they can provide robust and early protection against immune suppressive diseases such as infectious bursal disease [ , ] and vaccines against multiple diseases have been successfully combined. for instance, studies have shown that in-ovo vaccination strategies can simultaneously confer protective immunity against marek's disease, infectious bursal disease, newcastle disease, fowl poxvirus, coccidiosis, and necrotic enteritis [ , ] . other combination vaccines under investigation include vectored vaccines that simultaneously provide protection against newcastle disease and infectious bursal disease [ ] . in-ovo vaccination strategies have also been explored for other poultry diseases with promising results. this included an avian influenza vaccine based on a non-replicating human adenovirus vector [ ] , a recombinant viral vector vaccine against infectious laryngotracheitis [ ] , recombinant protein eimeria vaccines [ , , ] and a fowl adenovirus vectored vaccine against inclusion body hepatitis [ ] , among many others. a mycoplasma gallisepticum vaccine for in-ovo vaccination of layer chickens has also recently been evaluated, even though high chick losses at hatch were reported for the medium and high doses of the vaccine that were investigated [ ] . therefore, in-ovo vaccination strategies are capable of controlling several economically important poultry diseases. many of these diseases are viral, but can predispose animals to secondary bacterial infections. therefore, in many cases, in-ovo vaccines are promising alternative approaches to the use of antibiotics. clostridium perfringens is widespread in the environment and in the gastrointestinal tract of most mammals and birds. however, this bacterium is also one of the most common pathogens of food-producing animals, causing disease only under circumstances that create an environment which favors growth and toxin production, such as stress, injury, or dietary changes [ ] . the bacterium itself is not invasive, but causes disease through the production of a wide array of toxins and enzymes. however, no single strain produces this entire toxin repertoire, resulting in considerable variation in the toxin profiles and disease syndromes produced by different toxinotypes of this bacterium [ ] . while some of these toxins act only locally, other toxins which are produced in the gut exert their action in other internal organs or can act both locally and systemically [ ] [ ] [ ] . to date, efficacious vaccines are only available for the diseases caused by systemic action of the toxins and vaccination against enteric diseases still remains a challenge. however, some of these enteric diseases caused by c. perfringens are of major economic importance and lead to considerable use of antibiotics. amongst them are necrotic enteritis in broilers and necro-haemorrhagic enteritis in calves. despite the fact that much research is being directed to the development of novel vaccines against these c. perfringens-induced enteric diseases, several key barriers still have to be overcome. in general, clostridial vaccines require multiple doses to achieve full immunity. unfortunately, parenteral booster immunizations are impossible in the broiler industry, where mass parenteral vaccination is only feasible at the hatchery, either in ovo or on day-old chicks. because single parenteral vaccination at day of hatch offers no protection, other delivery methods need to be developed [ ] . oral vaccines can more easily be administered to birds, without the need of individual handling of the chicks and are therefore recommended. however, some questions arise when developing an oral vaccine as compared to the parenteral administration route. in addition to the fact that maternal antibodies can block the immune response in young chicks, also the induction of oral tolerance has to be circumvented and an efficient way to present the antigens to the mucosal immune system has to be developed. oral tolerance is a common problem in mammals and fish when developing oral vaccines. this is in contrast to chickens, where oral tolerance is age-dependent, and only an issue in -to -day-old chicks. after that age, protein antigens have been shown to induce a robust immune response and oral vaccination schemes are thought to be feasible [ ] . one appealing strategy for the delivery of vaccine candidates to the mucosal immune system is the use of attenuated or avirulent bacteria as antigen vehicles [ ] . attenuated recombinant salmonella strains which express c. perfringens antigens have been tested in several studies as oral vaccine vectors, leading to some promising results. however, the amount of protection afforded by these vaccines is not as high as compared to multiple doses of parenteral vaccination, and seems to depend on the colonization level and persistence of the vaccine strain [ ] [ ] [ ] [ ] . this indicates that the use of live vectors to express antigens derived from c. perfringens strains in the gut of broilers is a promising approach, but the vaccine delivery strategy still needs to be optimized to achieve optimal antigen presentation to the mucosal immune system and provide improved protection. alternatives to attenuated salmonella strains can be bacillus subtilis spores or lactobacillus casei, which both have a gras status and have the potential to be used as vaccine carriers for clostridium antigens [ , ] . b. subtilis has the advantage that the heat-stable spores can easily be incorporated in the feed and l. casei has known probiotic effects that facilitate the development of mucosal immunity. however, these types of vectors still have to be tested for their capacity to induce a good immune response, in particular against heterologous antigens, in broilers and whether they are able to provide protection against necrotic enteritis. another issue to be addressed when developing a vaccine against c. perfringens-induced enteric diseases is the choice of the antigens to be included in the vaccine. c. perfringens-induced diseases are the result of the toxins and enzymes that are produced and vaccination of chicks with c. perfringens supernatants provides protection against experimental necrotic enteritis [ , ] . however, the protective capacity of the supernatants depends on the strain used for supernatant preparation, indicating that full protection might be determined by an effective combination of different bacterial immunogens [ ] . in order to elucidate the optimal mixture of antigens to protect against necrotic enteritis, challenge trials are being performed mostly using parenteral vaccination schemes. once the ideal combination of antigens is known, this will have to be adapted to oral delivery strategies. several c. perfringens antigens have been evaluated as potential vaccine candidates. the tested antigens include both c. perfringens toxins (e.g. alpha toxin and the netb toxin) and highly immunodominant proteins identified in postinfection serum from birds immune to necrotic enteritis [ ] . in general, immunization studies of broilers with a single antigen all resulted in some level of protection against experimental necrotic enteritis. remarkably, immunization with netb toxin, which is essential to cause disease in broilers, does not afford higher levels of protection than vaccination with other toxins or proteins. however, when birds were vaccinated either via the parenteral or the oral route, with a combination of both netb toxin and alpha toxin, higher levels of protection were obtained [ , ] . in order to obtain full protection against c. perfringens-induced enteric diseases, not only antibodies that inhibit toxin activity might be needed; a combination of antigens targeting also bacterial proliferation, colonization and/or nutrient acquisition could be more efficient than either one of the individual approaches. indeed, in a recent study disruption of the putative adhesin-encoding gene cnaa resulted in a reduced ability to colonize the chicken intestinal mucosa and to cause necrotic enteritis [ ] . this strengthens the idea that vaccine antigens that target bacterial colonization might be indispensable to obtain a working vaccine against c. perfringens-induced enteric diseases. additional vaccine targets might be enzymes that aid in breakdown of the host tissue and nutrient acquisition, such as, amongst others, mucinases, collagenases and hyaluronidases. in contrast to the extensive efforts to develop a vaccine against necrotic enteritis in chickens, considerably less research has been directed to vaccination against necro-haemorrhagic enteritis in calves. the recent demonstration of the essential role of alpha toxin in necro-haemorrhagic enteritis and the proposition of a pathogenesis model will allow for the more targeted development of a vaccine [ , ] . in calves as in chickens, protection against c. perfringens-induced necrosis can be obtained by antibodies against a mixture of toxins, at least in an experimental model for bovine necro-haemorrhagic enteritis [ ] . furthermore, antibodies against alpha toxin alone, which is essential to cause intestinal disease in calves, are not sufficient to provide the same level of protection as antibodies directed against a mixture of c. perfringens proteins, indicating that a mixture of different antigens will be needed to provide full protection [ ] . in order to fully protect calves against c. perfringens-induced enteric diseases, antigens that target bacterial colonization and proliferation might be of equal importance as antigens targeting the toxin activities. next, it has to be explored whether parenteral vaccination is sufficient to induce a protective immune response or if a combination of systemic and mucosal immunity is needed when not only the bacterial toxins but also bacterial colonization is targeted. as administration of multiple parenteral doses of a vaccine to calves is more feasible than for chicken, it may be assumed that the development of a vaccine against necro-haemorrhagic enteritis is more straightforward and that c. perfringens supernatants can be used as a vaccine preparation. however, native toxins cannot be used as vaccine antigens due to safety issues. inactivation of clostridial toxins is generally achieved by formaldehyde treatment, which risks residual formaldehyde in the vaccine preparation, incomplete inactivation of the toxins, and batch-to-batch variation. moreover, formaldehyde inactivation can induce changes in the tertiary protein structures of relevant antigens and influence the immunogenicity of the vaccines. indeed, vaccination of both chickens and calves with formaldehyde inactivated c. perfringens supernatants or toxins have resulted in a good antibody response, but these are unable to protect against intestinal disease [ , ] . to overcome the need of chemically inactivating the c. perfringens toxins, current research focusses on the use of recombinant toxoids to develop a vaccine against c. perfringens-induced diseases. while this may be a good strategy to obtain a safe and protective vaccine on a laboratory scale, the production process is more laborious and time-consuming than production of conventional toxoids, especially because of the required purification steps [ ] . therefore, recent studies have explored the use of efficient low-cost alternatives, such as non-purified recombinant clostridial toxins and even recombinant bacterins, with success [ ] [ ] [ ] . in summary of this section, considerable progress has recently been made in the development of efficacious vaccines against c. perfringens-induced enteric diseases. the main issue that hampers a breakthrough in this field is the identification of a defined combination of antigens that is able to provide full protection against disease. these antigens will most likely target both the bacterial toxins and the bacterial colonization and proliferation. for the broiler industry, once the ideal vaccine antigens have been identified, development of an oral vaccine is needed. coccidiosis, an enteric disease cause by protozoan parasites of the genus eimeria, remains a major economic and welfare concern for the poultry industry globally. seven species (eimeria acervulina, e. brunetti, e. maxima, e. mitis, e. necatrix, e. praecox and e. tenella) are known to infect chickens, and at least six others infect turkeys [ , ] . the costs associated with coccidial disease are difficult to calculate, but have been estimated to exceed billion us dollars for the chicken industry alone, worldwide [ ] . because coccidiosis is a predisposing factor for the occurrence of necrotic enteritis, the true economic burden is likely even higher. all eimeria species can cause disease but the severity and clinical symptoms vary among species, and there is little or no cross-protection across species or some strains [ , ]. modern poultry production systems require effective control of coccidian parasites, typically through the routine use of anticoccidial drugs in feed or water. in the european union, eleven different anticoccidial drugs are currently licensed and between and tonnes are sold for use in animals for markets such as the uk every year [ ] . anticoccidial drugs can be divided into two groups, synthetic or chemical anticoccidials and ionophores, which are products of fermentation [ ] . in some countries such as the us, ionophores are classified as antibiotics, albeit with low human medical importance. the ionophores currently dominate the anticoccidial drug market, largely because they provide incomplete protection, even against naïve field strains without any drug resistance. low levels of parasites survive and induce protective immunity against the prevailing local parasite strains, without causing clinical disease [ ] . anticoccidial drugs provide an efficient means of controlling coccidial parasites and are highly cost-effective. however, drug resistance is widespread and increasing consumer concerns related to drug use in livestock production and residues in the food chain encourage the use of alternatives such as vaccination. notably, because coccidiosis is a predisposing factor for necrotic enteritis and other secondary bacterial infections, efficient control of this parasite is important to minimize the use of medically important antibiotics, including those deemed critically important for human health, in poultry production. the first anticoccidial vaccine was marketed in [ ] . it is a live parasite vaccine which includes multiple wild-type (i.e., non-attenuated) eimeria species. exposure to limited levels of such non-attenuated parasites permits the induction of a natural immune response in the chicken, resulting in protection against subsequent coccidial challenge. however, because protective immune responses against eimeria are fully species specific, the inclusion of each individual target species is necessary if comprehensive protection is to be achieved, which results in relatively complex vaccine formulations. such vaccines commonly include between three and eight parasite species or strains. the approach has been highly successful, although the lack of attenuation has been associated with reduced flock performance following vaccination and occasional clinical disease (reviewed elsewhere [ ]). in response to this limitation, a second generation of live eimeria vaccines has been developed using attenuated parasite lines. for most of these vaccines, attenuation was achieved by selecting for so-called precocious strains, which typically exhibit reduced pathogenicity with fewer and/or smaller rounds of asexual replication. these attenuated strains retained their ability to immunize. the first live attenuated anticoccidial vaccine was launched in , and several similar vaccines have been developed since using the same approach [ ]. nonattenuated and attenuated anticoccidial vaccines have become popular in the breeder and layer sectors, but are less widely used in the much larger broiler sector due to their relatively high cost compared to anticoccidial drugs and their limited availability. because eimeria cannot replicate effectively in vitro, the production of these live vaccines can only be achieved in eimeria-free chickens and separate chickens have to be used for each species or strain to be included in a vaccine. despite these production concerns billions of anticoccidial vaccine doses are sold every year, but more would be required to fully meet the growing demand. efforts to improve on first and second generation live anticoccidial vaccines have included extensive attempts to identify antigens that are appropriate for use in subunit or recombinant vaccines. in addition, progress has been made on the preparation of novel adjuvants and some promising results have been obtained, although data on their use in poultry has so far remained fairly limited [ ] . as an example, one vaccine is formulated from a crude mix of affinity purified e. maxima gametocyte antigens [ ], although the levels of protection achieved have remained controversial and production of the vaccine still requires parasite amplification in chickens. numerous studies have suggested that defined antigens such as apical membrane antigen , immune mapped protein , lactate dehydrogenase and so are highly promising vaccine candidates (reviewed elsewhere [ ] ). studies of eimeria field populations have reported limited diversity in many of these antigens, indicating that recombinant vaccines for eimeria may succeed even though antigenic diversity has undermined equivalent vaccines for related parasites such as plasmodium [ , ] . however, at present no recombinant anticoccidial vaccine is close to reaching the market. one of the biggest remaining challenges is how to deliver the antigens in an affordable, effective, and, most importantly, scalable manner. a range of vectored expression/delivery systems have been suggested including fowlpox virus (fwpv), hvt, salmonella typhimurium, yeasts such as saccharomyces cerevisiae and the tobacco plant nicotiana tabacum, with several showing promise [ ] . most recently, it has been suggested that eimeria itself might function as an expression/delivery vector for vaccine antigens [ ] [ ] [ ] . the ability to express and deliver anticoccidial vaccine antigens from multiple parasite species in a single transgenic line could provide an opportunity to streamline anticoccidial vaccine production from as many as eight lines to just one or two. using an attenuated vector species such as e. acervulina can improve productive capacity enormously and reduce vaccine cost. the parasite vector may also provide some ability as an adjuvant and methods for on-farm delivery are well established [ ] . in summary of this section on new coccidiosis vaccines, as pressure to reduce antibiotic drug use in livestock production increases it is clear that the demand for coccidial vaccines is stronger than ever. in the us, approximately - % of broiler companies use programs that include vaccination to control coccidiosis [ ] . this trend is primarily driven by demands to produce "no antibiotics ever" poultry products. however, it has also been shown that some coccidial vaccines provide an opportunity to replace drug-resistant field parasites in a poultry house with susceptible vaccine strains. while current european attenuated vaccines are limited by their lower reproductive potential, live vaccines do retain considerable unexplored potential. a better understanding of the underlying immune mechanisms through which these nontraditional approaches operate is needed to allow further progress. ultimately, it is clear that novel vaccines must be cost-effective, compatible with high standards of animal welfare, scalable and easy to deliver. autogenous vaccines (av) are also known as emergency, herd-specific or custom made vaccines. although the legal basis and exact definition differs from country to country, avs are used worldwide (e.g. eu, usa, canada, brazil, china, indonesia, australia, egypt) and have a long history of use. the use of avs for the control of fowl cholera has been well-documented [ , ] . as a common definition, all avs are made from inactivated bacterial or viral strains which were isolated from the same flock in which the vaccine is to be used. the use of avs is only allowed if no licensed vaccine is available, or it is respectively ineffective or does not cover the current pathogen strains in the flock. the definition of a flock varies and may include integrated concepts of production chains in different places; to address the issue, the concept of an epidemiological link has recently been proposed by the co-ordination group for mutual recognition and decentralised procedures [ ] . licensed vaccines have advantages compared to avs, including obligatory good manufacturing practice (gmp) production. licensed vaccines are also produced in bigger batches with defined strains and a high level of quality which makes their efficacy and safety predictable. however, licensed vaccines are not available in all cases. to generate avs, selected bacterial or viral strains are usually combined with a proper adjuvant. several viral or bacterial species can be used in a combination vaccine and different serotypes can also be combined in a polyvalent vaccine. the combination of inactivated viruses and bacteria is also an option. bacterial avs are accepted in all countries of the economic european area, whereas viral avs are not allowed in european countries including france, denmark and spain [ ] . a critical role in the successful production and use of an av falls to the isolation of vaccine strains. therefore diagnostic samples must be carefully obtained, based on appropriate choices regarding which sick and untreated animals to select for sample collection, which necropsy material to select, and which cultivation conditions and strains to use after results from sero-, toxo-or virulencetyping. for that purpose several methods like pcr, maldi-tof ms, slide agglutination or dna sequencing are available. because of the fundamental importance of the strain choice for the production of an adequate av, close collaboration between diagnostic laboratory and vaccine production is critical. each production is custom-made and numerous adjuvants, viral and bacterial isolates, including serotypes, toxins and species, provide countless combinations. this underlines the importance of experience as the basis in the production of high quality avs. the veterinarian also has obligations regarding diagnosis, ordering and responsibility for the administration of the vaccine. a variety of bacterial components are often used in avs. these include for poultry: depending on the animal species and age at vaccination different adjuvants can be used. as a standard adjuvant with good safety and efficacy, aluminium hydroxide is often used for production. polymer and other gel-like adjuvants are also available for production in aqueous mixtures. oily adjuvants, especially for water-in-oil emulsions, require a more sophisticated mixing procedure because of the need of a stable emulsion. furthermore oily vaccines might pose safety concerns. however, these induce a promising long lasting immune response because of a depot effect. in the case of organic animal production use of plant oil might be an option in order to avoid unwanted hydrocarbons. the risk of adverse effects, which depend on the adjuvant-antigen combination, can be decreased by standardization of the protocols. more data regarding the efficacy and safety of avs in field studies should be collected because clinical safety and efficacy is not regulated. . further steps in quality control include the inactivation test, endotoxin content or stability tests. some producers offer gmp production, and gmp production is required in some countries such as finland or sweden [ ] . in most countries gmp is only recommended. this example shows the vast differences in national legislation regarding the definition and interpretation of avs. because of worldwide circulation of animals and their pathogens a harmonization of manufacture, control and use of immunological veterinary medicinal products like av is important, and the aim at the economic european area [ ] . in summary, avs are a valuable option in certain situations where commercial vaccines are either not available or expected to lack efficacy because of a mismatch between circulating and vaccine strains. the selection of adequate clinical isolates and vaccine formulations requires considerable expertise and the effective use of avs depends on adequate manufacturing and appropriate veterinary oversight. regulatory differences among countries create a highly fragmented legal landscape that would benefit from further harmonization. vaccines are proven strategies for the prevention or control of infectious diseases in animal populations. therefore, they are promising alternatives that can reduce the need to use antibiotics in food-producing animals and their direct mitigating impact on antibiotic consumption has been demonstrated in a number of studies, even though the relationship between antibiotic use and vaccination is not in all cases clear-cut. the ideal vaccine is safe, effective against a broad range of pathogens, and easily adapted to mass-application. at the same time, it is cheap to produce and use, easy to register across key jurisdictions, and generates durable protection, ideally after a single administration. existing vaccines still fall short of these ideals. in fact, many current vaccines have a number of shortcomings with regard to safety, efficacy and/or user-friendliness that limit their ability to replace antibiotic use. overcoming these challenges will take close collaboration and innovative new approaches. public-private partnerships represent one promising governing structure for assuring such close collaboration across public and private sectors. investments in basic and applied research are equally needed to overcome these challenges, and research needs will have to be prioritized to ensure scarce resources will be preferentially dedicated to areas of greatest potential impact. research to characterize and quantify the impact of vaccination on antibiotic use is equally needed. yet, some data demonstrating the ability of vaccines to reduce antibiotic consumption are already available. similarly, key research breakthroughs and a number of highly promising vaccination approaches are already in development. these include new oral vaccines based on bacterial spores, live vectors, or new delivery strategies for inactivated oral vaccines; they also include new vaccination strategies in-ovo, combination vaccines that protect against multiple pathogens, the use of recent biotechnological advances, and comprehensive approaches to manage diseases caused by ubiquitous pathogens. therefore, further reductions in the need for antibiotic use through the use of new vaccines are all-but-certain, and investments in research and development of new vaccines will be vital for the sustained success of animal agricultural production around the world. intestinal epithelial cells: regulators of barrier function and immune homeostasis induction of secretory immunity and memory at mucosal surfaces vaccines: the fourth century crossing 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microparticles targeted to epithelial apn as oral antigen delivery system intestinal epithelial cells promote colitis-protective regulatory t-cell differentiation through dendritic cell conditioning enterotoxigenic escherichia coli (k ) induce proinflammatory responses in porcine intestinal epithelial cells evaluating the a-subunit of the heat-labile toxin (lt) as an immunogen and a protective antigen against enterotoxigenic escherichia coli (etec) induction of long term mucosal immunological memory in humans by an oral inactivated multivalent enterotoxigenic escherichia coli vaccine induction of protective immunity against eimeria tenella, eimeria maxima, and eimeria acervulina infections using dendritic cell-derived exosomes il- a regulates eimeria tenella schizont maturation and migration in avian coccidiosis chicken il- f: identification and comparative expression analysis in eimeria-infected chickens downregulation of chicken interleukin- receptor a during eimeria infection roles 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-beta fusion toxin in mice, sows and cows protective potential of recombinant non-purified botulinum neurotoxin serotypes c and d host specificity of turkey and chicken eimeria: controlled cross-transmission studies and a phylogenetic view we would like to thank the organizers, sponsors, and participants of the nd international symposium on alternatives to antibiotics on which this manuscript is based, in particular the united states department of agriculture (usda) and the world organisation for animal health (oie). the authors declare that they have no competing interests.authors' contributions kh, fvi, and cg planned the manuscript. kh led the drafting of the manuscript. lb, dpb, ec, smc, bd, eev, eg, kk, sl, mm, mr, mcs, nmw, cg, and fvi provided additional information and contributed to writing the manuscript including drafting selected sections and reviewing the manuscript. fvi and cg revised the manuscript. all authors read and approved the final manuscript. key: cord- -gonx taq authors: pignatelli, jaime; alonso-padilla, julio; rodríguez, dolores title: lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (ptov-he) protein date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: gonx taq hemagglutinin-esterases (he) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. he proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. the role of the he protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. the phylogenetic analysis of he coding sequences from porcine torovirus (ptov) field strains revealed the existence of two distinct he lineages. in a previous study, ptov virus strains with he proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. in this work, we report antigenic differences between the two he lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of ptov infection in the farms. toroviruses (tov) are enveloped, positive single-stranded rna viruses of cattle, horses, pigs and humans [ ] . they have been associated with enteric infections and diarrhea, especially in young animals and children [ ] , and are considered a potential zoonotic threat [ ] . the torovirinae subfamily of the coronaviridae family (order nidovirales) comprises four species: equine torovirus (etov), bovine torovirus (btov), porcine torovirus (ptov) and human torovirus (htov). the few epidemiological studies performed in different countries indicate high prevalences of these viruses [ ] [ ] [ ] . the torovirus's genome size (~ kb) and organization are very similar to those of other coronaviruses, with two huge overlapping open reading frames (orf) shaping the ′-end of the genome, where the replicase/transcriptase machinery is encoded, and a final third of the rna molecule hosting the coding sequences for the four structural proteins (from ′ to ′): spike (s), hemagglutinin-esterase (he), membrane (m), and nucleocapsid (n) [ ] . by analogy to other nidoviruses, the s protein is considered to be the putative receptor binding molecule. copies of this molecule form the large spikes protruding from the viral particles as shown by electron microscopy [ , ] . the he protein forms homodimers which make up the smaller spikes [ , ] . this he protein is a class i membrane glycoprotein of about kda that belongs to the receptor destroying enzyme (rde) protein family. as an rde protein he provides reversible binding to glycosylated surfaces [ ] due to its ability to bind sialic acids and catalyze the disruption of that binding by means of its acetyl-esterase activity. the d structure and sialic acid preference of ptov-(strain markelo) and btov-(strain breda) he proteins have been solved [ ] . two main domains have been defined in the he monomer of both proteins: the enzymatic acetyl-esterase region (e) and the receptor binding (r) or lectin domain. despite the great amount of knowledge acquired on this protein family, it is not yet clear what is the exact role of he during the torovirus infectious cycle. the etov, the only cell culture adapted torovirus, as well as different btov strains that could be isolated in cell culture, all lack a functional he protein [ ] [ ] [ ] [ ] due to deletions or mutations in the he gene, acquired during their adaptation to in vitro growth conditions. thus, he expression seems to be detrimental for in vitro culture of these viruses, as it occurs with the coronavirus murine hepatitis virus (mhv) [ ] . however, the maintenance of the he protein in a vast majority of new ptov and btov field isolated strains indicates that it has to play an important function during in vivo infections. several hypothetical functions have been postulated for the tov-he: (i) being a viral co-receptor, (ii) digestion of mucus layers to allow the virus to reach the target cells in the respiratory and/or enteric tracts, (iii) release of viral particles bound to decoy receptors, (iv) influencing host/cell tropism, or as recently proposed by de groot's group, (v) acting as a molecular timer in the virus pre-attachment step to the host-cell [ ] . two ptov-he lineages have been identified, with representative strains being markelo and p [ ] . they share an amino acid sequence homology of %. recently, during a longitudinal survey of ptov in a spanish pig herd, several ptov-he isolates representative of both lineages were identified [ ] , and similar findings were described from a survey performed in korea [ ] . in the first case, the two ptov-he lineages were detected even within the same animal at two sequential sampling time points, indicating that both ptov strains carrying different he proteins coexisted on the same farm infecting the same piglets, and suggesting that the immune response generated against one ptov strain did not protect the animals against the infection by the other strain. to further investigate this hypothesis ptov-he proteins corresponding to each he lineage were expressed, characterized, and used to track the anti-he response in the animals from the farm where the ptov strains were obtained. the analysis of serum samples by hemagglutination inhibition assay and elisa revealed antigenic differences between the two he lineages. cells, viruses and antisera bsc (african green monkey kidney cells) cells were grown in dulbecco's modified eagle's medium (dmem), supplemented with % heat-inactivated neonatal calf serum (ncs), non-essential amino acids ( %), gentamicin ( μg/ml), penicillin ( iu/ml), streptomycin ( μg/ml) and fungizone ( . μg/ml). vaccinia virus (vacv), strain western reserve (wr) [ ] , and the recombinant vacv (rvv) derivatives were propagated and titrated in bsc cells. to obtain a polyclonal antiserum against ptov-he (αhe), a new zealand rabbit was inoculated with two different peptides coupled to keyhole limpet hemocyanin (klh). peptides comprising residues - (ctnpstpn sldipqqlc) and - (ltppenipshc) of ptov-he-bres [genbank: fj ] [ ] were chemically synthesized at the proteomics facility of our institution after selection of target ptov-he antigenic regions by bioinformatic analysis with protean software (dnastar inc. madison, wi, usa). the serum sample collection and their treatment were previously described [ ] . the samples corresponded to animals from three different litters (a, b and c; four animals per litter) of the same farm, and were collected at -, -, -, -, and -weeks of age. serum samples from the corresponding sow of each litter (n = ) collected at the first day of sampling were also obtained. . the amino acid residues that are conserved in all sequences from one lineage but are different from those in the strains corresponding to the other lineage are depicted in blue. other residues that are conserved in all sequences or are non-lineage specific are depicted in red. pgemt plasmids with ptov-he coding sequences from isolates . and . [ ] were used to sub-clone the indicated ptov-he sequences into the pjr vacv transfer vector [ ] by asymmetric digestion with restriction endonucleases bamhi and ncoi. the resulting plasmids, pjr-he . and pjr-he . , carried the he genes under the control of the vacv synthetic early/late promoter (pe/l). insertion of the he genes into the vacv hemagglutinin locus (ha) was achieved by homologous recombination between the transfer vectors and the virus genome following standard procedures [ ] . the resulting rvv-he . and rvv-he . viruses were selected and grown following standard procedures to yield viral stocks that were titrated and stored at − °c until use. a control rvv harboring a disrupted ha locus (rvv-ha -) was generated in parallel under the same procedures using the empty pjr vector. rvv expressing soluble forms of he . and he . proteins fused to the c-myc tag were also generated to facilitate protein purification by affinity chromatography. for this purpose, the sequences coding for the fusion proteins were cloned into the pjr vector, and the corresponding rvv were obtained as described above. bsc cells infected with the corresponding rvv at a multiplicity of infection (moi) of plaque-forming units per cell (pfu/cell) were collected at hours postinfection (hpi) in laemmli sample buffer. expression of ptov-he proteins in infected cells was analyzed by polyacrylamide gel electrophoresis (sds-page) and western blot with the rabbit polyclonal αhe serum. after incubation with a horseradish peroxidase-conjugated secondary antibody (sigma-aldrich, saint-louis, mo, usa), the reactive bands were detected by chemoluminescence using the commercial ecl reagent (ge healthcare, uppsala, sweden). subconfluent bsc cell monolayers were grown in mm-diameter coverslips and infected with rvv-he . , rvv-he . , or the control virus rvv-haat an moi of pfu/cell. at hpi, cells were washed, fixed with % paraformaldehyde and permeabilized with . % triton x- in pbs. after a blocking step with % fetal calf serum (fcs) in pbs, cells were incubated for h with αhe polyclonal antiserum diluted ( : ) in pbs containing % fcs, washed three times and stained with alexa fluor goat anti-rabbit igg (molecular probes™, life technologies, carlsbad, ca, usa) at : dilution. dapi reagent ( ′, ′-diamidino- -phenylindole) (molecular probes™) was used to stain cell nuclei. after washing with pbs, coverslips were mounted on microscope slides using the prolong® gold anti-fade reagent from molecular probes™. images were captured with a confocal radiance system (bio-rad, münchen, germany) and processed using imagej [ ] and adobe photoshop cs (adobe system inc., san josé, ca, usa). confluent bsc cell monolayers seeded in mm plates were infected with rvv-he . and rvv-he . at a moi of . at hpi cells were rinsed with pbs and harvested. after a centrifugation step at × rpm for min, cell pellets were resuspended in tne buffer ( mm tris-cl, mm edta and mm nacl) and subjected to three successive freeze-thaw cycles and sonication. the soluble fractions were stored at − °c until needed. to obtain esterase inactivated ptov-he cell extracts, rvv infected bsc monolayers were treated with mm diisopropyl fluorophosphate (dfp; sigma-aldrich) for min at room temperature before scraping the cells. any remaining excess of dfp was removed after thoroughly washing the cells with ice-cold pbs. protein concentrations in the cell extracts were determined by bradford reaction (bio-rad protein assay) using known amounts of purified bovine serum albumin (bsa) as standards. specific acetyl-esterase activity of both recombinant he proteins (he . and he . ) was tested by an in situ staining assay as previously described [ ] . bsc cell monolayers were infected with the corresponding rvv-he and with rvv-haas the negative control. at hpi viral plaques expressing he were visualized after incubation with α-naphtyl acetate-fast blue bb solution (anae assay kit, sigma-aldrich) according to the manufacturer's instructions. cell monolayers were then stained with crystal violet solution ( . % in % methanol) to visualize all viral plaques. sialate-o-acetylesterase activity of rvv-infected bsc cell extracts in tne solution was determined with the synthetic substrate p-nitrophenyl acetate (pnpa; sigma-aldrich) as previously described [ ] . briefly, cell extracts were incubated with mm pnpa at °c, and the hydrolysis of pnpa was recorded by reading the absorbance at nm at different times. the enzymatic activity per μg of cell lysate, after subtracting the background activity present in the control cell extract infected with rvv-ha -, was calculated as previously described [ ] . these assays were performed in u-shaped -well plates (nunc, roskilde, denmark). mouse (mus musculus, strain swiss) blood obtained from the retro-orbital cavity of anesthetized animals was collected in two volumes of sterile alsever solution to prevent clotting. animals were handled following the guidelines of the animal experimentation committee of our institution, in strict accordance with the spanish law (rd - ). the red blood cells (rbc) obtained were washed with alsever solution, counted and diluted in pbs to obtain a % stock solution ( % solution corresponds to · rbcs per ml). hemmagglutination assays (ha) were set-up using two-fold dilutions of extracts from dfp-treated and untreated rvv-infected cells. fresh mouse rbc in pbs solution were added ( μl) to the he containing wells and incubated for two hours at °c. hemagglutination was scored and documented by photography, and plates were then placed at °c and a second image was taken after h incubation at this temperature. the hemagglutination units (hau) of he containing cell extracts were established as the reciprocal of the highest dilution causing hemagglutination. for the hemagglutination inhibition assays (hi), he cell extracts ( hau in μl pbs) were incubated ( h at °c) with two-fold serial dilutions of kaolin treated pig sera [ ] prior to adding μl per well of a mouse erythrocyte suspension (final concentration of . %). the hi titer was defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination by the viral antigen. serum samples with hi titers higher than log were considered positive. extracts from bsc cells infected with rvv-he . -myc or rvv-he . -myc, were prepared as described above in tne buffer containing % np . the soluble fractions were transferred to new tubes containing anti-c-myc coated beads previously equilibrated in tne buffer (mbl, naka-ku nagoya, japan). after h incubation at °c the mixtures of bead suspension and cell lysates were centrifuged in spin columns, washed thrice, and proteins were eluted with a c-myc tag peptide. the reagents and the protocol for the protein purification procedure were provided by the supplier. the purified proteins were analyzed by sds-page and western blot with the αhe polyclonal serum and anti-c-myc monoclonal antibodies to confirm their identity (clontech, takara bio inc., otsu, shiga, japan), and by coomasie blue staining of the gel to determine their purity and concentration using different amounts of bsa as the reference. an indirect elisa was set up to detect antibodies against ptov-he proteins in pig serum samples. the optimal protein concentration was established by checkerboard titration with positive and negative pig control sera. paired rows of a -well microtiter plate (maxisorp, nunc) were coated overnight ( °c) with purified he . -myc and he . -myc proteins diluted at a . μg/ml concentration. plates were thoroughly washed after each step by rinsing the plates thrice with pbs containing . % tween (pbst). after coating, plates were blocked for h at °c with pbst- % bsa. then, μl of each serum sample diluted : in pbst- % bsa was added in paired wells and incubated h at °c. a commercial goat anti-pig igg secondary antibody conjugated to horseradish peroxidase (sigma-aldrich) was used. the enzymatic reaction was developed using o-phenylenediamine dihydrochloride (opd-fast™, sigma-aldrich), and stopped after min by adding μl/well of n sulphuric acid. optical densities at nm (o.d. ) were recorded with a multichannel spectrophotometer (titertek multiscan mcc/ ). as negative control serum, a pool of sera from caesarianderived, colostrum-deprived (cd/cd) pigs kept under germ free conditions (spf) was used, and the positive control serum was a commercial porcine serum (abd serotec, kidlington, uk) previously determined to contain antibodies against ptov [ ] . the elisa cut-off was established for each antigen as the mean of the o.d. of negative control serum plus three times the standard deviation (he . -myc cut off = . ; and he . -myc cut off = . ). a previously described indirect elisa was used to detect antibodies to the highly conserved ptov nucleocapsid protein [ ] . statistical analyses of hi and elisa sera reactivities obtained from animals grouped by ages against both ptov-he . and ptov-he . were performed using two tailed t student's. means ± standard error of the mean (sem) for each group are shown. the detection of ptov strains belonging to the two defined he lineages, represented by markelo and p strains, in piglet fecal swabs collected on a spanish farm in the course of a ptov longitudinal survey was previously described [ ] . interestingly, two ptov strains, one from each of the described lineages were isolated from the same animal (pig , litter b) at different time-points of the piglet's life, at -and -weeks of age. the he from the virus isolated at week , named ptov-he . , was . % homologous to markelo he at the amino acid level, whereas the protein from the virus found at week , he . , was more closely related to p he ( . % homology). the direct comparison between both ptov-he . and ptov-he . gave a . % identity, a homology that is similar to those obtained when comparing previous reported he sequences from both he lineages [ , ] . to study the amino acid differences between both he lineages, a sequence alignment from he proteins corresponding to viruses identified in three different geographical regions was performed. the he sequences from the spanish ptov strains he . , he . , he . and he . [ ] were compared with those from other european strains markelo, p , p [ ] and bres [ ] , and from korean strains - and - and - and - [ ] . the alignment shows that several amino acid positions were conserved in a lineage specific manner ( figure a , grey shade), even in the geographically distant korean strains. these lineage specific amino acids were located in the markelo tridimensional structural model [ ] , and, as shown in figure b , these differential amino acids (blue balls) were found mainly placed on the surface of the receptor binding domain, suggesting that they could mark potential antigenic differences between both he lineages. to study the he proteins from ptov-he isolates . and . as the model for the markelo and p he lineages, and search for potential antigenic differences between them, both proteins were expressed by the recombinant vacv methodology. rvv carrying the corresponding he . or he . coding sequences inserted into the ha locus of the vacv genome, were generated (rvv-he . and rvv-he . ). in addition, a vacv lacking a functional ha protein, rvv-ha -, was produced to serve as the negative control. ptov-he proteins expressed upon infection of bsc cells with the rvv were specifically detected by western blot and immunofluorescence microscopy using the αhe rabbit antiserum. western blot analysis under denaturing conditions detected both he . and he . proteins in rvv-infected cell monolayers with the expected kda molecular weight (figure a ) corresponding to the glycosylated form of the protein [ ] . both he proteins were expressed at a similar extent. regarding the subcellular localization of recombinant he proteins analyzed by immunofluorescence, the ptov-he specific signal was widely distributed in the cytoplasm as well as associated to the nuclear and plasma membranes of bsc cells infected with either rvv-he . or rvv-he . but not in rvv-ha --infected cells ( figure b ). the acetyl-esterase activity of the recombinant ptov-he . and he . proteins was analyzed by in situ staining of infected cells by the anae assay [ ] . infected cells surrounding viral plaques produced by both rvv-he . and rvv-he . show a brownish staining whereas the rvv-haderived plaques remained unstained ( figure a ). ptov-he . and rvv-he . were also able to hydrolyze the acetyl group from the synthetic substrate p-nitrophenol acetate in the pnpa assay [ ] performed with extracts from infected cells. the graph in figure b shows the specific pnpa hydrolysis obtained with the he proteins. both he . and he . cell extracts show similar activities ( . and . mu/μg lysate respectively). ptov-he acetyl-esterase activity of the infected cell extracts was completely and irreversibly inhibited when the rvv-infected bsc cell monolayers were treated with the serine-esterase inhibitor dfp (see materials and methods) as shown in figure b . no esterase activity could be detected in extracts from cells infected with rvv-ha -, either untreated or treated with dfp. the lectin activity of he . and he . proteins was tested by hemagglutination assay using mouse erythrocytes. the hemagglutination assay was set up with two fold serial dilutions of dfp treated (dfp + ) and dfp untreated (dfp -) cell extracts, starting from μg per well of total protein. the lysate from cells infected with rvv-haserved as the negative control. at °c, both dfp + and dfp -rvv-he cell extracts were able to induce hemagglutination of mouse rbc at a similar ha titer, whereas the hacell extract did not hemagglutinate the mouse rbc at any of the amounts tested ( figure c ). when plates were placed at °c, the hemagglutination net induced by dfpcell extracts was disrupted due to their acetyl-esterase activity at that temperature, however hemagglutination was maintained in dfp-treated he containing wells, indicating that the acetyl-esterase activity was completely inhibited ( figure d ). the esterase activity and receptor binding results show that both recombinant ptov-he proteins were biologically active, indicating the acquisition of proper conformational folding. the hemagglutination caused by he proteins is due to the binding of their lectin or receptor binding domain to specific sialic acid determinants present on the surface of rbc. hence, in order to examine the potential existence of antigenic differences between the two lineages of ptov-he proteins, piglet serum samples were analyzed with an hi assay to detect the presence of specific antibodies against the receptor binding domain that prevents the rbc hemagglutination. in the hi test, dfp inactivated he . and he . cell lysates were used to avoid artifacts in the assay derived from the he esterase activity. using serum samples from the longitudinal survey, different hi antibody response dynamics against he . and he . proteins were observed. anti-he . antibodies were detected at week in % of the piglets ( / ) ( figure g ), even though all piglets from litter b were negative against that particular lineage at that time-point ( figure c ). with respect to the anti-he . inhibitory reaction in week sera, none of the animals in litter c was reactive against it ( figure f ), and only one piglet from litter b had specific anti-he . hi reactivity ( figure d ). however all piglets from litter a had high hi titers towards this lineage, in agreement with the high hi titer of their sow ( figure b ). independently of their lineage specificity, the hi titers decreased at week (coinciding with weaning) in all but one anti-he positive piglets, however, the percentages of positive animals against both he proteins remained constant until week ( figure g ). up to that week, the reactivity to he . was circumscribed to piglets within litter a ( figure b ). at week , just % of the piglets ( / ) were reactive against he . , and a bare % presented anti-he . antibodies ( figure g ). by week , % of the piglets ( / ) were positive against he . ( figure g ). although anti-he . reactivity was also observed in % ( / ) of the piglets, all those animals had higher hi titers against he . . to compare the reactivity of each serum sample against both he . and he . proteins the hi titers obtained against the two he proteins were plotted in figure h . while few samples had the same hi titers with both proteins (serum samples found over the diagonal), most of them showed preferential reactivity with one versus the other he lineage (serum samples found at both sides of the diagonal) and even some serum samples showed specific reactivity against only one of the he proteins ( serum samples were positive only for the he . and were specific for the he . ). these data clearly indicate that he lineage specific amino acid differences within the receptor domain were enough to determine that antibodies developed against one lineage do not interfere with the receptor recognition by the other he lineage. to further investigate the reactivity of antibodies in piglets' serum samples against he . and he . proteins and the dynamics of antibodies against each of them, an elisa method using purified myc-tagged he proteins (he . -myc and he . -myc) as antigens was used. the analysis by sds-page confirmed the purity of both preparations and their reactivities with both αhe polyclonal serum and anti-c-myc monoclonal antibodies were confirmed by western blot (see additional file ). all pig serum samples tested were positive against both he proteins by elisa ( figure ). one-week-old piglets had o.d. values akin to those of their corresponding sow against each he protein. piglets from litter a were highly reactive against both he . and he . , although the elisa values were slightly higher against the second one ( figure a and b) . piglets from litter b, had a low reactivity against both proteins ( figure c and d ). in contrast, piglets from litter c showed a higher reactivity against he . than against he . ( figure e and f). as it happened with hi titers, anti-he reactivity by elisa gradually diminished with piglet's age and by weeks of age, weeks after weaning, it reached the lowest level in most of the animals. at the next age analyzed ( weeks), most animals showed an increase in their anti-he reactivities against both ptov-he proteins, and these kept rising until weeks of age at least. to obtain an overview of the immune response against both ptov-he proteins, the mean elisa reactivities and hi titers of the serum samples from each litter grouped by ages were compared side-by-side. as shown in figure , both assays revealed statistically significant figure analysis by hi assay of the reactivity of sera obtained from pigs at different ages against ptov-he . and ptov-he . . extracts from bsc cells infected (moi ) for h with rvv-he . (panels a, c and e) or rvv-he . (panels b, d and f) and treated with dfp were diluted to contain hau in μl pbs and incubated ( h at °c) with two-fold serial dilutions of pig sera prior to adding the mouse rbc. serum samples were obtained from animals belonging to three litters (litter a, panels a and b; litter b, panels c and d; litter c; panels e and f) at different ages ( , , , antigenic differences between he . and he . . in animals from litters a and b, higher reactivity against he . was observed in the first weeks of the piglets' life, although low titers were observed in animals from litter b, which were barely detectable by the hi assay. on the contrary, in the first weeks, animals from litter c had higher antibody titers against he . by both assays. also, from these results it is clear that sera from -week old animals from the three litters reacted preferentially against he . , indicating a switch in antibody reactivity in animals from litter c. figure reactivity of sera obtained from pigs at different ages against ptov-he . and ptov-he . determined by elisa. purified he . -myc-and he . -myc proteins were used as coating antigen ( . ng/well). the same serum samples analyzed in figure were used in the elisa at a : dilution. igg elisa reactivities against he . -myc-(panels a, c and e) and he . -myc (panels b, d and f) of serum samples from pigs and their sows are represented. figure paired comparisons of the results of pig sera reactivities against ptov-he . and ptov-he . determined by hi and elisa. graphic representation of the mean hi (a) and elisa (b) titers of sera grouped by pig ages against both ptov-he . (black bars) and ptov-he . (grey bars). means ± standard error for each sample are shown. asterisks indicate statistically significant differences of sera reactivity against the two proteins (**p < . , *p < . ; student's t test). the dotted lines indicate the respective cut off values determined for each assay. the reactivity of the piglet's sera against the highly conserved n protein using a previously standardized elisa [ ] has already been analyzed [ ] . for comparison with the anti-he immune response here we provide the results of the analysis of sera from the individual piglets over time (see additional file ). although there are differences in the magnitude of the elisa titers among the different piglets, what is clear from these results is that once maternally acquired antibodies had vanished, which for the n protein occurs around weaning time (week of age), all animals developed their own anti-n antibodies that can readily be detected in most animals by week . these findings were in agreement with those obtained by hi and elisa using the he proteins as antigen, and indicate that all animals become infected by ptov soon after weaning. despite the great advances recently made on he knowledge, which include the elucidation of both btov-he and ptov-he tridimensional structures [ ] , the exact role of the he protein in the viral life cycle and the potential relevance of the differences between he lineages in the immunological response to the virus remain unclear. without an in vitro culture system and given the great difficulties and costs of in vivo research, the use of heterologous expression systems represents a useful and relatively inexpensive approach to study the ptov-he protein. here we used the recombinant vacv methodology to express two full ptov-he sequences, he . and he . , and their two corresponding soluble fractions attached to a c-myc tag. those he coding sequences had been previously identified during a thorough longitudinal study of ptov in a spanish farm [ ] , and each of them were found to belong to one of the two defined ptov-he lineages [ ] . these he sequences were obtained from the same animal in sequential collection points. this finding indicated that both lineages co-existed on the farm [ ] although with apparently different prevalences according to the age of the host. this result could lead to new possibilities to approach the ptov-he behavior in the virus' natural environment. both recombinant proteins show similar features regarding their molecular weight, glycosylation degree, and subcellular localization as those reported previously for ptov and btov [ , ] , indicating that the recombinant he proteins follow a correct biosynthetic pathway. in addition, the heterologous expressed he proteins were fully functional as receptor bindingreceptor destroying enzymes since both ptov-he proteins were able to hemagglutinate mouse erythrocytes, but also to hydrolyze the acetyl-ester linkage of glycan chains, as well as from acetylated synthetic compounds like pnpa and anae. the analysis of the amino acid changes found between both ptov-he lineages shows that there are amino acid residues that are conserved in a lineage specific manner even among strains identified in very distant geographic areas (different european countries and korea). this analysis also indicates that potential antigenic differences would be mainly determined by residues located at the receptor binding domain, and exposed on the surface of the protein according to the proposed structural model [ ] . hence, to elucidate if he lineage specific changes could determine antigenic differences on the receptor binding domain, the he . and . proteins were used as model proteins in hi assays with field serum samples from the same farm where both lineages were detected. significantly, by this assay most serum samples show preferential reactivity to one of the he proteins, or even specific reactivity against only one of them (see figure h ), indicating the existence of antigenic differences between the two he lineages. antigenic differences between he proteins from the two known btov lineages have also been described [ ] . in addition, to study the antibody response against the whole protein by a different approach, soluble c-myc tagged he . and he . proteins were generated by rvv methodology to obtain highly purified coating antigens that were used in elisa to test the same field serum samples. using both approaches, a high prevalence of antibodies against ptov-he was observed in both sows and piglets. these results were in agreement with those obtained using an elisa assay with the very immunogenic and highly conserved n protein as antigen [ ] . in the present study, similar anti-he response profiles over time were observed by both he-elisa and the more restricted lectin-specific hi test. a general decrease of antibody levels was seen from the first weeks of age until piglets' reached week , related to the extinction of maternally derived initial immunoglobulins. at -weeks of age, immune levels recovered due to the development of the pigs' own response to infections, and they increased at least until week . at this last sampled point, a general increase of reactivity against he . (p -like) was found in animal sera from the three litters, indicative of the prevalence of this lineage upon time. overall, similar antibody patterns were observed by elisa against the he proteins and the n protein, although a delay in both the extinction of maternally derived antibodies and the development of self-acquired antibodies against the he was observed in all animals analyzed (see figure and additional file ). the rising of antibodies to ptov antigens after weaning can be explained as a consequence of the animal grouping in livestock facilities for fattening purposes that provides encyclopedia of virology. rd edition etiology of community-acquired pediatric viral diarrhea: a prospective longitudinal study in hospitals, emergency departments, pediatric practices and child care centers during the winter rotavirus outbreak, to . the pediatric rotavirus epidemiology study for immunization study group emerging and re-emerging swine viruses bovine torovirus (breda virus) revisited identification and characterization of a porcine torovirus seroprevalence of porcine torovirus (ptov) in spanish farms a new family of vertebrate viruses: toroviridae. intervirology purification and partial characterization of a new enveloped rna virus (berne virus) new insights on the structure and morphogenesis of berne virus hemagglutinin-esterase, a novel structural protein of torovirus structure, function and evolution of the hemagglutininesterase proteins of corona-and toroviruses structural basis for ligand and substrate recognition by torovirus hemagglutinin esterases characterization of epidemic diarrhea outbreaks associated with bovine torovirus in adult cows genetic and antigenic characterization of newly isolated bovine toroviruses from japanese cattle the pathogenesis of torovirus infections in animals and humans hemagglutination mediated by the spike protein of cell-adapted bovine torovirus nidovirus sialate-o-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes the murine coronavirus hemagglutinin-esterase receptor-binding site: a major shift in ligand specificity through modest changes in architecture phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events longitudinal serological and virological study on porcine torovirus (ptov) in piglets from spanish farms detection and molecular characterization of porcine toroviruses in korea vaccinia virus: a suitable vehicle for recombinant vaccines? molecular characterization of a new ptov strain. evolutionary implications il- delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against hiv- env in a dose-dependent manner e. coli beta-glucuronidase (gus) as a marker for recombinant vaccinia viruses image processing and analysis in java identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza c virus and bovine coronavirus the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities comparison of macro and micro methods in kaolin and rbc treatment of sera used in the measles hemagglutination-inhibition test lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (ptov-he) protein we thank joquim segalés for providing porcine field serum samples. we also want to thank susana plazuelo for her excellent technical assistance in the analysis of sera by elisa. this work was supported by grants agl - and consolider-porcivir csd - from the spanish ministry of science and innovation. jaime pignatelli and julio alonso-padilla were both recipients of contracts financed with founding from the consolider-porcivir research project. the conditions for piglet infection and/or re-infection and the mixture of ptov strains in the same animal population. our results indicate that the immune response developed against one of the ptov lineages could not protect against the infection by other ptov isolates carrying an he protein belonging to a different lineage. hence, the specificity of the piglet's current immune response, its own or maternal, towards one or the other he lineage at a given time could determine the ptov strain that could infect or prevail in the animal. our hypothesis is that this ptov lineage alternation could explain the sustainment of both strains on the farm. in fact, even though the p -like (he . ) strain was not found by molecular detection in piglets at the earliest ages [ ] , the high reactivity observed by elisa in sows and young piglets and, more remarkably, by the hi in sow a and in -, -and -week old piglets from litter a meant that such a strain was already circulating in the herd from the very beginning of the sampling. particularly, in piglet the increasing reactivity against both he proteins from week to week indicates a temporal coexistence of both types of virus in the piglet at around weaning time. the lack of anti-he maternal antibodies with an hi capacity against either he protein might have facilitated the establishment of both ptov strains early on in the piglet's life. although we described that the two ptov-he lineages were detected within the same animal at two sequential sampling time points, the lack of detection of ptov-he . at week could have been due to the low abundance of this virus at the beginning of life, while at later times it became predominant, and therefore easier to detect. although the shift from ptov-he . to ptov-he . in the analyzed animals (markelo-like to p -like strains) seems to derive from immune pressure on the latter, the contribution of a potential better viral fitness provided by the former on older pigs' tissue environments cannot be discarded.the tendency to recombine modules of their genomes observed in nidovirales, together with the extensive mutation rates found, like in other rna viruses, would facilitate the evasion of immune responses and the rapid adaptation to new hosts and the new hosts' environments. from this study, where two ptov he genotypes were found co-existing on the same farm, we can speculate that the specificity of the immune response towards one or the other he lineage in piglets at a given time could determine the ptov strain that prevailed and spread. however, the potential additional contribution of the immune response to other viral antigens in virus selection also has to be considered. future studies with higher numbers of animals from different farms will be required to further support the proposed hypothesis.though the ptov-he protein in vivo function/s are still to be undoubtedly defined, its persistence in field strains, the tendency to undergo recombination events and the different antigenic characteristics of both he lineages indicate that he protein from torovirus plays an important role in virus-host interactions with implications in immune protection that could explain the broad spread of this virus in the pig population, causing chronic infections/re-infections of the animals. additional file : analysis of purified ptov-he . -myc and ptov-he . -myc proteins. (a) cell extract from bsc cells infected (moi ) with rvv-he . -myc or rvv-he . -myc (lysate), and affinity purified he . -myc and he . -myc proteins (protein) were fractionated by % sds-page, and the gel was stained with coomassie blue. (b) affinity purified he . -myc and he . -myc proteins were reacted in western blot with the αmyc and αhe antibodies. molecular size markers are given in kda.additional file : reactivity of sera obtained from pigs at different ages against the n protein. the same serum samples analyzed in figure were used in elisa at a : dilution using the ptov-n protein as antigen as previously described [ ] . the authors declare that they have no competing interests.authors' contributions jp and jap have contributed equally to this work. jp and jap participated in the design of the study, generated recombinant viruses to express recombinant he proteins, contributed to characterize the recombinant he proteins, carried out hi assays, participated in the analysis of the results and draft of the manuscript. dr conceived the study, participated in its design, coordination and draft of the manuscript. all authors read and approved the final manuscript.submit your next manuscript to biomed central and take full advantage of: key: cord- -frdsct authors: vogel, liesbeth; van der lubben, mariken; te lintelo, eddie g.; bekker, cornelis p.j.; geerts, tamara; schuijff, leontine s.; grinwis, guy c.m.; egberink, herman f.; rottier, peter j.m. title: pathogenic characteristics of persistent feline enteric coronavirus infection in cats date: - - journal: vet res doi: . /vetres/ sha: doc_id: cord_uid: frdsct feline coronaviruses (fcov) comprise two biotypes: feline enteric coronaviruses (fecv) and feline infectious peritonitis viruses (fipv). fecv is associated with asymptomatic persistent enteric infections, while fipv causes feline infectious peritonitis (fip), a usually fatal systemic disease in domestic cats and some wild felidae. fipv arises from fecv by mutation. fcov also occur in two serotypes, i and ii, of which the serotype i viruses are by far the most prevalent in the field. yet, most of our knowledge about fcov infections relates to serotype ii viruses, particularly about the fipv, mainly because type i viruses grow poorly in cell culture. hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype i viruses. kittens were inoculated oronasally with different doses of two independent fecv field strains, ucd and rm. persistent infection could be reproducibly established. the patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to weeks revealing subtle but reproducible differences between the two viruses. faecal virus, i.e. genomic rna, was detected during persistent fecv infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. the implications of our results, particularly our insights into the persistently infected state, are discussed. feline coronaviruses (fcov) present themselves in different forms. first of all there are two biotypes, the low-or avirulent viruses and the highly virulent ones. the former viruses cause intestinal infection with no or only mild clinical manifestations, i.e. diarrhoea in young kittens. these so-called feline enteric coronaviruses (fecv) are the predominant pathotype in the field. fecv infection often persists in apparently healthy animals, which shed the virus in their faeces. natural infection occurs via the faecal-oral route. fecv infection appears to be mainly restricted to the intestinal tract where the virus replicates in villous epithelial cells. the virulent pathotype, feline infectious peritonitis virus (fipv), causes severe systemic infection with lesions in many organs and tissues, most typically in the peritoneum, hence the disease's name feline infectious peritonitis (fip). unlike fecv, fipv does not spread readily among cats. instead, it originates de novo by spontaneous mutation from fecv in individual animals [ , , , ] often under conditions of immune suppression due to medication or coinfection [ ] . fip is a highly fatal disease that occurs in domestic cats and some wild felidae usually between and months of age. the disease is currently the leading infectious cause of death in domestic cats. in catteries to % of the animals are usually feline coronavirus seropositive, in single cat households seropositivity often varies between and % [ ] . from the seropositive cats, to % eventually develop fip [ ] . there is at present no effective way to prevent the infection or treat the disease. for recent reviews on fcov, see haijema et al. [ ] and pedersen [ ] . fcov also occur as two serotypes. based on in vitro neutralization tests, serotypes i and ii have been distinguished [ , ] , which can in turn be of either pathotype. in the field the serotype i viruses are largely predominant; they are responsible for to % of fcov morbidity and mortality. in europe and in the usa type ii viruses are quite rarely observed, in contrast to japan where they were reported to constitute some to % of all viruses [ ] . research on fcov infection has understandably focused mainly on fipv, its origin, its particular pathogenesis and disease. moreover, most of what we know about these viruses comes from work with serotype ii viruses, simply because efficient in vitro propagation of serotype i viruses has not yet been achieved. hence, in order to learn more about the characteristics of the most prevalent fcov infection in the field, we have in this study set up animal infection systems for serotype i fecv. using two independent field isolates we reproducibly observed persistent infection of which we evaluated the clinical, virological and serological features. faecal extracts containing an unknown titre of fecv strain ucd [ ] and fecv strain rm [ ] were kindly provided by n.c. pedersen and a. poland (both davis, ca, usa), respectively. they were used to prepare stocks of these viruses for our infection experiments as follows. specific pathogen-free (spf) kittens were inoculated oronasally with an aliquot of the faecal sample. faeces were collected daily and suspended in an equal volume of phosphate buffered saline (pbs). the suspensions were centrifuged at · g for min and the viral genome contents in the supernatants determined by quantitative rt-pcr (see below). extracts with the highest concentrations of viral rna were combined, aliquoted and stored frozen at À °c. the rna content of the fecv ucd and fecv rm stocks were · and · genome equivalents per ll, respectively. spf cats (harlan sprague dawley, inc., madison, usa), seronegative for fcov as checked by standard immunofluorescence assay, were used in all infection experiments. the animals were housed in groups in a closed animal facility. they were euthanized at the indicated end of each experiment by intravenous injection with an overdose of phenobarbital. for all experiments, approval of the university's ethical committee was obtained. with the aim to reproducibly establish persistence by experimental fecv infection, to elucidate possible differences in pathogenic characteristics among different strains, and to investigate possible dose-dependent differences in these characteristics, four experimental infections were performed, two with each of the fecv strains. in the first experiment with fecv ucd (experiment a , tab. i) two male spf cats of weeks old were inoculated oronasally each with . ml (i.e. · genome equivalents) of the fecv ucd faecal extract. the animals were monitored during days post inoculation for clinical signs, faecal virus shedding and serum antibody titres. in the second infection experiment with fecv ucd (experiment a , tab. i) eight female spf cats, weeks old and housed in four groups of two animals, were inoculated oronasally with . ml per cat of a series of -fold dilutions of the fecv ucd stock: undiluted and diluted À , À and À in pbs (two cats per dose). the cats were monitored for clinical signs, body weight, faecal virus shedding and serum antibody titres for days post inoculation. in the first infection experiment with fecv rm (experiment b , tab. i) four female spf cats of weeks old were inoculated oronasally with . ml (i.e. · genome equivalents) of the fecv rm faecal extract and monitored similarly for days post inoculation. in the second experiment with this virus (experiment b , tab. i) eight female spf cats, weeks old and housed in pairs, were inoculated oronasally with . ml per cat of several dilutions of the fecv rm stock: undiluted and diluted À , À and À in pbs (two cats per dose). they were again monitored for the standard parameters for days post inoculation. monitoring of clinical signs was done on a daily basis by scoring for such signs as depression, general activity, anorexia and faeces consistency. the body temperature and body weight were determined once or twice a week. body weight was expressed as the percentage of the body weight measured at the day of inoculation. to monitor faecal virus shedding, quantitative rt-pcr was performed using the taqman Ò procedure described by gut et al. [ ] , which targets the conserved b gene in the domain of the fcov genome. to extract template rna from faecal samples, faeces were weighed, suspended and diluted : (w/v) with pbs and centrifuged at · g for min to remove large particles. rna was extracted from the suspension using guanidinium thiocyanate with adsorption of viral rna onto silica as described by boom et al. [ ] . the eluted rna was diluted : in te buffer ( mm tris-hcl, mm edta, ph . ) before addition to the pcr mixture. the ll pcr mixture contained . ll master mix and . ll multiscribe and rnase inhibitor mix (applied biosystems, nieuwerkerk a/d ijssel, the netherlands), . lm forward primer fcov f, lm reverse primer fcov r, . lm probe fcov p and ll template rna or diluted standard (described in next paragraph). after a reverse transcription step of min at °c and a denaturation step of min at °c, cycles, each s at °c and min at °c, were performed. reverse transcription, amplification and real time monitoring of fluorescence were carried out in an abi prism Ò sequence detector. a synthetic rna standard for absolute quantification was produced using as a template the pst-blue plasmid (kindly provided by virbac laboratories inc., carros, france) containing a cdna copy of the rna fragment to be amplified in our assay behind the t rna promoter. the transcription reaction sample was dnase-treated, the rna purified by phenol/chloroform extraction and isopropanol precipitation, and the amount of rna determined by uv spectrophotometry. the rna stock was aliquoted and frozen at À °c. rna dilutions were tested by taqman Ò analysis, showing a linear relationship between the amount of input rna and the ct value over a range of at least log units, corresponding to · through · molecules (data not shown). twenty-five ll of s-methionine labelled fcov (strain - ) infected cell lysate was mixed with ml tesv buffer ( mm tris-hcl ph . , mm edta, mm nacl) containing % triton x- , . mg/ml bsa (sigma a- , sigma aldrich, zwijndrecht, the netherlands) and . % sds, to which ll cat serum was added. ascitis (# ; ll), obtained from an experimentally fipv - (serotype ii) infected cat [ ] , and anti-rm À · À · À b rm · undiluted f b rm · undiluted f · À · À · À the final pellets were resuspended in laemmli sample buffer and heated at °c for min. the precipitates were analysed by sds-page using a % polyacrylamide gel. after electrophoresis the gel was fixed in % methanol containing % acetic acid, dried and the radioactivity visualized by autoradiography. serum antibody titres were determined by a typespecific elisa. microtiter plates ( wells; microlon, greiner bio-one international b.v., alphen a/d rijn, the netherlands) were coated with ng whole virus particles of a cell culture adapted derivative of the serotype i fcov strain ucd- [ ] . the virus was grown on felis catus whole fetus (fcwf- ) cells [ ] , which were maintained as monolayer cultures in dulbecco's modified eagle's medium containing % foetal calf serum, iu of penicillin/ml, and lg of streptomycin/ml (all from life technologies ltd., paisley, uk). the virus was purified from the clarified cell culture supernatant by ultracentrifugation through a % (w/w) sucrose cushion. pellets were resuspended in tm buffer ( mm tris-hcl and mm mgcl adjusted to ph . ) and protein content determined with the bio-rad dc protein assay (bio-rad laboratories b.v., veenendaal, the netherlands). the plates were incubated with the virus, dissolved in . m carbonate buffer ph . , for . h at °c and overnight at °c. plates were blocked with % bsa in pbs-tween ( . %) for h at °c. the plates were washed three times with pbs-tween ( . %), -fold serial dilutions of cat serum in pbs-tween were added to the wells and the plates were incubated at room temperature for h. after washing the plates again with pbs-tween , peroxidase-conjugated goat anti-cat igg (cappel laboratories inc., downingtown, usa) was added to the wells and the plates were again incubated at room temperature for h. subsequently, the plates were rinsed and ll substrate solution containing . mg/ml tetramethylbenzidine-h o in . m sodium acetate was added to the wells. the substrate reaction was stopped by the addition of ll m h so . the absorbance at nm was determined on a el ultra microplate reader (biotek instruments, inc., winooski, usa). a titration curve was obtained by plotting the optical density as a function of the reciprocal serum dilution. the titre was defined as the highest dilution of serum corresponding to an optical density of . above the background, as extrapolated from the slope of the curve (background values were determined using a fcov negative serum). the significance of the differences in the serum antibody titres was analyzed using student's t-test. in order to detect the presence of viral rna in blood, edta blood was centrifuged for min at rpm. after separation of the plasma one volume of pbs was added to the blood cells and total rna was isolated using the total quick rna blood kit protocol (talent, trieste, italy). quantitative rt-pcr to detect viral genomic rna was performed as described above. for the detection of subgenomic mrna we used a rt-pcr protocol targeting the mrna specifying the viral membrane (m) protein as described earlier [ ] . to longitudinally screen the intestinal tract of persistently fecv infected cats for virus, the intestines of freshly euthanized animals were sectioned over their entire length into segments of cm (first part until appendix) or cm (remainder). the faecal content of each segment was collected in an eppendorf tube and subsequently the tissue segment was washed out thoroughly with cold pbs. the faecal material was suspended in an equal volume of pbs and centrifuged for min at rpm. the supernatant was collected, rna was extracted and the viral genome content was quantitated by real-time rt-pcr as described above. for rna isolation from the intestinal tissue, the washed segments were lysed overnight in lysis buffer l and subsequently the rna was purified and concentrated by the guanidinium thiocyanate-silica (sio ) protocol of boom et al. [ ] . pretreatment of the slides with citrate buffer ph . and heating using a microwave oven, endogenous peroxidase activity was blocked using hydrogen peroxide ( %) in methanol and subsequently sections were blocked with normal goat serum. after incubation with a primary antiserum (mouse anti-feline coronavirus mca , abd serotec, düsseldorf, germany) a goat anti mouse envision complex was added (dako, heverlee, belgium). subsequently sections were incubated with , '-diaminobenzidine tetrahydrochloride and counterstained with papanicolaou's hematoxylin. experiments a and b (tab. i) were performed to comparatively analyse the clinical, virological and serological course of an infection with two different avirulent serotype i fecv strains and to investigate whether persistent infection could be established. fecv ucd and fecv rm are independent virus isolates, maintained in cats only, and first described by pedersen et al. [ ] and hickman et al. [ ] , respectively. the viruses were applied by the natural route, i.e. by oronasal inoculation. two cats were inoculated with fecv ucd and four cats with fecv rm. all six cats were successfully infected and both infections persisted as indicated by faecal virus shedding which lasted for at least days (strain ucd) and days (strain rm), i.e. until the time the experiment was terminated and the animals euthanized. faecal virus shedding, as monitored by taqman Ò rt-pcr, became readily detectable from day to post inoculation and increased rapidly up to genomes/ll faeces in all animals. all cats seroconverted as determined both by ripa and by elisa (data not shown; detailed data are provided below for the dose-titration experiments). the two cats inoculated with fecv ucd had diminished appetite at several days in the period between day and day after inoculation and experienced weight loss during the first eight days after inoculation. the cats inoculated with fecv rm did not show any clinical signs of disease during the entire experimental period. they exhibited normal behaviour and the consistency of their faeces was also normal. to obtain a more detailed and comparative insight into fecv ucd and fecv rm infection and into the effect of virus dose on the outcome and persistence of infection, experiments a and b were performed. in both these experiments eight cats placed in groups of two were inoculated with different doses of the two viruses, ranging from · to · genome equivalents. all cats, even those inoculated with the lowest viral dose, became infected, as indicated by their faecal virus shedding and seroconversion. virus shedding became detectable already one day after inoculation with the two highest doses of fecv ucd and two days after inoculation of the cats with the two lowest doses of this virus. shedding of fecv rm was first detected at day in the animals that received the two highest doses and on day in the cats inoculated with the two lowest doses (fig. ) . in all animals, the amount of virus in the faeces subsequently increased within two days to peak levels of - genomes/ll faeces, after which shedding remained high for an extended period until day and day post inoculation in fecv ucd and fecv rm infected cats, respectively. thereafter the virus titres in the faeces declined. shedding seemed to decline more steeply in cats that had received the lowest viral doses. this was particularly the case for the fecv rm infected animals where virus was no longer detected from day after inoculation with the À or À diluted doses. all cats developed fcov-specific serum antibodies as determined by elisa and ripa (figs. and ) . the kinetics of appearance of the antibodies was comparable for both viruses. as illustrated for the fecv ucd infections in figure , the antibodies started to become detectable at days post inoculation. there was a clear tendency for the animals infected with the higher doses to develop somewhat higher, longer-lasting elisa titres. the elicited antibodies recognized the fcov structural proteins m, n and s as determined by ripa (fig. ) . antibodies to the n protein were predominant and the first to become detectable by this assay. all fecv ucd infected cats showed diminished appetite between days through post inoculation, resulting in loss of body weight gain (fig. ) . however, the animals produced normal stools and did not show any other signs of disease, with the exception of two cats. one cat, inoculated with undiluted faecal extract, developed a chronic fever at day , which subsided after day . the other cat, inoculated with À diluted faecal extract, developed a recurring fever, with temperatures ! °c on days , , , and post inoculation. the body temperature of the remaining cats stayed between . and . °c throughout the experiment. interestingly, two cats, inoculated with a À and a À dilution, respectively, which were apparently able to contain the persistent infection and to reduce viral titres in the faeces, showed a stronger increase in body weight than the other animals (fig. ) . the fecv rm infected cats grew normally, without weight loss or growth retardation, and showed no clinical signs of disease during the entire experimental period. the disease signs observed with the ucd strain are thus unlikely to be caused by experimental conditions such as manipulation-induced stress. the fecv infections had no effects on the number of peripheral blood lymphocytes. their numbers remained stable irrespective of the viral strain or dose when counted at various times post inoculation (data not shown). the presence of virus in blood was tested by performing a real-time rt-pcr on whole edta blood of fecv ucd infected cats. the results of these assays were variable. often no viral rna was detected and, if positive, no clear pattern was observed, as illustrated by figure . no sign of viral replication in the blood of these two animals could be detected as judged by an rt-pcr assay targeting subgenomic mrna for the viral m protein. in order to determine in which part(s) of the gut virus was present during viral persistence, we screened the contents of the entire intestines of two fecv ucd infected cats. these animals, from a similar infection experiment not described here, were still shedding virus at days and days post inoculation, the time they were euthanized. their intestines were sectioned over their entire length in -cm (from stomach up to the appendix) and -cm segments (remainder). viral rna in each segment was determined by real-time rt-pcr. as shown in figure , no virus was detectable in the first approximately cm long part comprising duodenum, jejunum and ileum. in both animals virus was found only in the approximately - cm final part of the gut, downstream of the appendix, i.e. in colon and rectum. while viral rna was found in the faeces part of essentially all segments of these compartments, no viral rna could be detected in several of the corresponding tissue specimens, suggesting that at this late stage of persistence the number or areas of virus-producing cells have become scarce. consistent with this interpretation, attempts to visualize infected cells in fixed sections of the epithelial lining of the intestines by immunoperoxidase staining were unsuccessful. while there is quite a number of reports describing natural infections caused by the prevalent serotype i fcov [ , , - , , , ] , studies investigating the course of fecv infection after controlled experimental infection are remarkably few [ , , , ] . yet, such studies are important as detailed knowledge about the biology of this infection should eventually allow us to better understand the conditions and requirements that give rise to the fatal pathotypic shift to fip. these insights in turn might provide leads to prevent this transition or treat the disease. here we describe the results of four infection experiments in which persistent infection was reproducibly established and in which we analysed a number of clinical, virological and serological parameters. inoculation through the natural, oronasal route invariably initiated an immediate infection as judged by the rapid onset of shedding of virus with the faeces. at the highest doses shedding started already at day (strain ucd) and (strain rm), at lower doses within three days after inoculation. titres rose rapidly, peaking about two days later at - genome equivalents/ll irrespective of the dose and staying high thereafter for the next to weeks. shedding continued during the subsequent two months at a gradually but slowly decreasing level except for the four animals inoculated with the lowest fecv rm doses, which all ceased shedding from about weeks onwards. these results are in part consistent with a study by pedersen et al. [ ] in which experimentally infected cats were systematically monitored over longer ( - months) periods. based on the shedding patterns of the cats that had been inoculated orally with (an unknown dose of) fecv strain rm, the authors of this study distinguished three excretion types: persistent, intermittent and self-limiting. though our monitoring period was too short to allow such classification, the shedding patterns we observed with this rm strain indicate that the inoculation dose can affect the type of excretion, lower doses giving rise to earlier clearance of the infection than higher doses. this is remarkable in view of the comparable excretion levels at the peak of shedding. for the ucd strain this distinction could not be made. within the range of inoculation doses that we used in our dose titration experiments, the threshold below which infection becomes ineffective was not reached. the observations in figure suggest that rather low doses are probably sufficient to establish fecv infection, consistent with the efficient spread of the virus in the field. for fecv ucd our lowest dose was · genome equivalents. the average amount of this virus shed during the first days of the infection was around · genomes/ ll. from this it is clear that only traces of the faeces from such infected cats will already suffice to initiate an infection in a new host. all fecv ucd and fecv rm infected cats seroconverted starting approximately days after inoculation. during the next weeks the serum igg titres increased but they never reached high levels. these systemic antibodies, which were directed against all major viral structural proteins, were obviously unable to efficiently clear the local fecv infection as shedding of virus with the faeces continued in their presence. our observations are consistent with the few reports regarding immune responses to experimental fecv infection, and in which low and often variable antibody titres were found in serum or plasma [ , , ] . little is known about mucosal immune responses to fecv infection in the gut. though probably the most important for the control and clearance of the infection, local immunity is evidently inadequate. it is quite conceivable that this failure is due to interference of viral factors with the immune system. fecv replicates in the gastrointestinal tract [ , ] . using rt-pcr, viral rna has however also been observed by us [ , , ] and others [ , , , ] in blood, some organs and haemolymphatic tissues of naturally infected cats; in experimentally infected animals viral rna appeared hard to detect in pbmc (this study) or blood monocytes [ ] . the significance of the apparent viraemia is still enigmatic. while cells of the monocyte lineage are the prime targets of fipv, these cells are poorly susceptibility to fecv and support its replication and spread very inefficiently, at least in vitro [ , ] . in one study using blood from cats, the monocytes from three cats were not susceptible to fecv while the cells from the other animals could be infected but did not sustain the infection [ ] . studies using an rt-pcr targeting viral mrna to detect fcov replication suggest this situation to be the case as well in vivo [ , ] ; actually, viral mrna or infected cells could not be observed in organs other than the intestinal tract. apparently, fecv is picked up by mononuclear cells in the gut and carried to organs and tissues with the blood. consistently, while studying haemolymphatic tissues, a major site for the accumulation of monocytes/macrophages, kipar et al. found significantly higher levels of viral rna in cats with fip than in healthy fcov positive cats [ ] . moreover, fcov antigen was detectable by immunohistology in these tissues only in fip cats [ , ] , confirming the low fecv replication activity in the mononuclear cells. fecv is tropic for cells of the mature apical epithelium of the intestinal villi, targeting cells from the caudal part of the duodenum to the cecum during acute infection [ ] . in chronically infected animals the lower part of the gastrointestinal tract was identified as a major site of viral replication, as indicated by rt-pcr detection of viral mrna and by immunohistochemical detection of infected cells [ ] . consistent with these findings, during experimentally established persistent infection we only observed fecv in the large intestine, downstream of the appendix, i.e. in colon and rectum. during the submission process of this manuscript a publication by kipar et al. [ ] appeared in which the colon was also identified as the major site of fcov persistence. fecv are known as low-or non-virulent pathogens. whether and to what extent virulence variation occurs is not really clear; no virulence variants have so far been described. here we studied two independent fecv strains isolated from cats many years apart and maintained by cat-to-cat passage only. interestingly, the two viruses behaved reproducibly different, the ucd strain being consistently more pathogenic than the rm strain, also when applied to older kittens (data not shown). in both infection experiments with fecv rm the cats grew normally, remained without fever, produced normal stools and did not show obvious signs of disease. in contrast, the fecv ucd infected cats had diminished appetite and exhibited weight loss early after inoculation. two of the cats developed chronic or recurrent fever, as observed also with another fecv isolate [ ] . the results indicate that variation in fecv 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virus infection two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein a mrna pcr for the diagnosis of feline infectious peritonitis intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses acknowledgements. we are very grateful to raoul de groot for stimulating discussions and advise. we also want to acknowledge the assistance and animal care provided by the employees of the central animal laboratory of the utrecht university. key: cord- -qea b i authors: eck, melanie; durán, margarita garcía; ricklin, meret e.; locher, samira; sarraseca, javier; rodríguez, maría josé; mccullough, kenneth c.; summerfield, artur; zimmer, gert; ruggli, nicolas title: virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: qea b i porcine reproductive and respiratory syndrome virus (prrsv) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. the vaccines currently available on the market elicit only limited protection. recombinant vesicular stomatitis virus (vsv) replicon particles (vrp) have been used successfully to induce protection against influenza a virus (iav) in chickens and bluetongue virus in sheep. in this study, vsv vrp expressing the prrsv envelope proteins gp , m, gp , gp , gp and the nucleocapsid protein n, individually or in combination, were generated and evaluated as a potential vector vaccine against prrsv infection. high level expression of the recombinant prrsv proteins was demonstrated in cell culture. however, none of the prrsv antigens expressed from vrp, with the exception of the n protein, did induce any detectable antibody response in pigs before challenge infection with prrsv. after challenge however, the antibody responses against gp , gp and gp appeared in average weeks earlier than in pigs vaccinated with the empty control vrp. no reduction of viremia was observed in the vaccinated group compared with the control group. when pigs were co-vaccinated with vrp expressing iav antigens and vrp expressing prrsv glycoproteins, only antibody responses to the iav antigens were detectable. these data show that the vsv replicon vector can induce immune responses to heterologous proteins in pigs, but that the prrsv envelope proteins expressed from vsv vrp are poorly immunogenic. nevertheless, they prime the immune system for significantly earlier b-cell responses following prrsv challenge infection. infections with porcine reproductive and respiratory syndrome virus (prrsv) causes reproductive failures in sows [ ] and respiratory disorders particularly in young pigs [ ] , which results in important economic losses worldwide [ , ] . recently, highly pathogenic prrsv strains have emerged in china [ ] and eastern europe [ ] . prrsv is an enveloped positive sense singlestranded rna virus belonging to the family arteriviridae within the order nidovirales [ ] . two prrsv genotypes can be distinguished, type prrsv of european origin and type prrsv originating from north america and china, both spreading worldwide with high genetic and antigenic diversity [ , ] . the prrsv genome consists of at least open reading frames (orf). orf a and b encode the non-structural proteins from two polyproteins pp a and pp ab that are further processed proteolytically, as well as two proteins nsp tf and nsp n resulting from ribosomal frameshifts within the nsp gene (for a detailed review see [ ] ). the remaining orfs encode the structural proteins on subgenomic messenger rnas. orf a, b and - , encode the glycoprotein (gp ) also termed gp a, the non-glycosylated protein b also termed e, the glycoproteins gp , gp , gp , the non-glycosylated membrane protein m (from orf ) and open access *correspondence: nicolas.ruggli@ivi.admin.ch institute of virology and immunology ivi, sensemattstrasse , mittelhäusern, switzerland full list of author information is available at the end of the article the nucleocapsid protein n (from orf ), respectively (reviewed in [ ] ). recently, an alternative orf a protein was identified as a minor component of the equine arteritis virus (eav) [ ] and the prrsv virions [ ] . gp and m form a disulphide-linked heterodimer that is essential for the formation of infectious particles [ , ] . for eav, the glycoproteins gp , gp and gp form a heterotrimeric complex that is stabilised by disulphide bonds, which has not been demonstrated for prrsv yet (reviewed in [ ] ). the prrsv gp -m and gp -gp -gp complexes are linked essentially through non-covalent interactions between gp and gp [ ] . the basic protein n associates with the viral rna genome to form the nucleocapsid. n is the most immunogenic prrsv structural protein. it elicits a strong antibody response a few days post infection (pi). these antibodies do however not neutralize the virus and are therefore not protective [ ] . the major neutralizing epitopes are found on gp [ ] [ ] [ ] [ ] and gp [ ] [ ] [ ] which are also the most diverse structural proteins between isolates [ ] . neutralizing epitopes were also found on m, gp and gp [ ] [ ] [ ] , but their contribution to protection is unclear. gp co-expressed with m elicits a better neutralizing ab response than gp alone [ , ] . however, neutralizing antibodies appear typically several weeks only after the onset of the first antibody response, simultaneously with clearance of the virus from the bloodstream [ , ] . the development of vaccines against prrsv has been only partially successful so far and remains a challenging task (for comprehensive reviews, see [ ] [ ] [ ] ). there are currently two types of prrsv vaccines on the market: modified live-virus vaccines (mlv) and inactivated vaccines [ ] [ ] [ ] . mlv are typically more efficacious than inactivated vaccines [ , ] . numerous alternative prrsv vaccine approaches have been explored with limited success so far (reviewed in [ , ] ). these efforts include for instance dna vaccines, subunit and peptide vaccines, viral vector vaccines and plant-derived vaccines [ , [ ] [ ] [ ] [ ] [ ] [ ] . propagation-incompetent recombinant vesicular stomatitis virus (vsv) represents yet another vector vaccine approach. recombinant vsv replicons lacking the vsv glycoprotein (g) gene and carrying genes of interest instead can be packaged in virus replicon particles (vrp) with high infectious titres using a complementing g-expressing cell line [ ] . such vrp were used successfully in the past to induce protection against sars coronavirus in a mouse model [ ] , influenza a virus (iav) in chickens and mice [ ] [ ] [ ] , and bluetongue virus (btv) in sheep [ ] . vsv vrp are safe due to the lack of glycoprotein g expression, preventing assembly and spread of virus particles. pigs have typically no pre-existing immunity against vsv. thus, vsv replicons represent an attractive novel vaccine platform for pigs. they have however not been evaluated in pigs yet. in the present study, vsv vrp were generated to express different combinations of the major and minor prrsv glycoproteins. the immunogenicity and protective potential of these vrp were assessed in pigs. marc- cells (atcc, lgc standards) were grown in dulbecco's modified eagle medium (dmem; life technologies) supplemented with % foetal bovine serum (fbs; biowest). bhk- cells were obtained from the german cell culture collection (dszm) and grown in earle's minimal essential medium eagle (mem; life technologies) supplemented with % fbs. bhk-g , a transgenic bhk- cell clone expressing the vsv g protein in a regulated manner, were maintained as described previously [ ] . the type prrsv strain olot/ was kindly provided by luis enjuanes (centro nacional de biotecnología, madrid, spain). this virus was a marc- -adapted variant of the original olot/ virus and was therefore propagated and titrated in marc- cells. for the detection of prrsv proteins, monoclonal antibody (mab) e directed against prrsv n, and mab vii d directed against amino acids - of prrsv gp were kindly provided by hans nauwynck (university ghent, belgium). a polyclonal rabbit serum against prrsv gp was obtained from luis enjuanes (centro nacional de biotecnología, madrid, spain). this serum was generated at biogenes (germany) with purified recombinant gp from prrsv olot/ expressed with the baculovirus system. the mab e c directed against prrsv m, and the mab ah against prrsv gp were a gift from ingenasa (madrid, spain). the rabbit anti-myc antiserum c , the mouse anti-flag m antibody and the anti-α-tubulin mab were purchased from sigma-aldrich. the mouse anti-ha antibody ca was from roche. the secondary antibodies goat anti-mouse igg alexa and goat anti-rabbit igg alexa were from molecular probes. the anti-swine igg antibody conjugated with rhodamine was purchased from rockland. the polyclonal rabbit anti-mouse igg coupled with horseradish peroxidase was from dako. the cdna of gp and m and the codon-optimized cdna of gp , gp and gp from the prrsv olot/ strain (genbank accession number kc ) were derived from psl-orf -orf and psl-gp - - , respectively, kindly provided by luis enjuanes (centro nacional de biotecnología, madrid, spain). the codonoptimized cdna of n from the olot/ strain (genbank accession number agw ) was synthesized by genscript (piscataway). for generation of recombinant vsv replicons, gp or n were inserted into the plasmid pvsv* using mlui and bsteii restriction sites upstream and downstream of the fourth transcription unit, replacing the g gene of vsv in analogy to a previous report [ ] . this replicon contained an additional transcription unit at position , expressing the green fluorescent protein (gfp) referred to by an asterisk (*) in the vector nomenclature. the resulting plasmids were designated pvsv*Δg(gp ) and pvsv*Δg(n), respectively. for generation of a dual antigen expression vector, the m cdna was inserted into pvsv*Δg(gp ) using xhoi and nhei restriction sites, thereby replacing the gfp gene in the fifth transcription unit. the resulting plasmid was designated pvsvΔg(gp /m). for generation of a triple antigen expression vector, the cdna of gp , gp and gp was inserted into a vsvΔg vector containing transcription units described recently [ ] using the mlui (in case of gp ), xhoi (in case of gp ) and nhei (in case of gp ) restriction sites. the resulting plasmid was designated pvsvΔg(gp /gp /gp ). for expression of a modified m and gp containing a short peptide epitope at the c terminus, the m and gp gene, respectively, were inserted without stop codon into the pcmv- tag- a plasmid vector (agilent technologies) upstream and in frame with a triple flag epitope (dykddddk)coding region followed by a stop codon. the m-flag and gp -flag open reading frames were amplified by pcr and inserted into the fourth transcription unit of pvsv*Δg, resulting in the plasmids pvsv*Δg(m-flag) and pvsv*Δg(gp -flag), respectively. the antigens gp and gp were modified by fusing a short ha epitope (ypydvpdya) to the c terminus, while gp was modified at the c terminus with a short myc epitope (eqkliseedl). the orfs were inserted into the fourth transcription unit of pvsv*Δg resulting in the plasmids pvsv*Δg(gp -ha), pvsv*Δg(gp -ha), and pvsv*Δg(gp -myc), respectively. for expression of the gp ectodomain (gp ecto) without the c-terminal transmembrane domain, a gene cassette encoding the amino acids - of gp (numbering according to genbank accession number agw ) was inserted in frame with the igκ leader sequence and an optimal signalase cleavage site in the mammalian expression vector psectag- a (invitrogen). in this way gp ecto was fused to a myc peptide epitope and a histidine tag ( xhis) at the c terminus, followed by a stop codon. this igκ-gp ecto-myc- xhis construct was then amplified by pcr and inserted into the fourth transcription unit of pvsv*Δg, resulting in the plasmid pvsv*Δg(gp ecto-myc). for expression of iav proteins, the cdna encoding ha, na, np and m of iav a/swine/belzig/ / (h n ) were kindly provided by jürgen and olga stech (friedrich-loeffler-institut, greifswald-insel riems, germany). the genes were inserted into the plasmid pvsv* using the mlui and bsteii restriction sites, replacing the g gene of vsv as described [ ] . all nucleotide sequences were confirmed by sanger sequencing. the recombinant replicons were propagated in the bhk-g helper cell line providing the vsv g protein in trans, yielding infectious vrp with titres of - infectious units (iu)/ ml as described previously [ ] . for the titrations, gfp expression was used as readout. for the detection of vrp that did not express gfp, infected cells were fixed with pbs containing % paraformaldehyde (pfa) for min, washed with pbs containing . m (w/v) glycine and then permeabilized with . % (v/v) triton x- . the cells were incubated with a rabbit anti-vsv serum and subsequently with a goat anti-rabbit horseradish peroxidase conjugate (dako) and stained with -amino- -ethylcarbazole (aec)/h o as substrate. an overview of all constructed vsv vrp is provided in table . marc- cells grown on -mm-diameter cover slips were inoculated for min at °c with recombinant vrp using a multiplicity of infection (moi) of iu/cell. at h post infection, the cells were fixed with % pfa for min and washed with pbs containing . m (w/v) glycine. the cells were permeabilized with . % (v/v) triton x- for - min and subsequently incubated with primary and secondary antibodies, diluted in pbs containing % bovine serum albumin (bsa). after each incubation period ( min, room temperature), the cells were washed three times with pbs. finally, the cells were washed with distilled water and embedded in mowiol - mounting medium (sigma-aldrich). all pigs were obtained from the specific pathogen free (spf) breeding facility of the institute of virology and immunology ivi. three experiments were performed. for each experiment the pigs were randomly assigned to treatment groups housed in separate stables. the groups were immunized by intramuscular injection of ml of cell culture supernatant containing - iu/ml recombinant vrp. in the first experiment, groups of pigs (n = ) were immunized three times at ½, ½ and ½ weeks of age with vsvΔg(gp /m) or with the control vsv*Δg vrp, respectively. in the second experiment, one group of pigs (n = ) was immunized twice at ½ and ½ weeks of age with a mixture of vsvΔg(gp /m) and vsvΔg(gp /gp /gp ) and the second group (n = ) received the control vsv*Δg vrp. in the third experiment, groups of pigs (n = ) were immunized three times at , and months of age with two different vsv vrp each injected at two different sites, i.e. vsv*Δg(n) and vsv*Δg(ha belz ), vsv*Δg(gp ecto-myc) and vsv*Δg(m belz ), vsv*Δg(gp -ha) and vsv*Δg(na belz ), vsv*Δg(control) and vsv*Δg(np belz ), respectively. in all experiments, the pigs were challenged via the intranasal route with tcid /animal of the prrsv strain olot/ - weeks after the last vaccination. the challenge virus was diluted in ml mem and administered dropwise intranasally. blood was taken before each vaccination and at regular intervals after vaccination and after the challenge infection. serum was stored at − °c. body temperature and clinical score were monitored daily according to a defined scoring system [ ] . the experiments in pigs were performed in compliance with the swiss animal protection law and approved by the animal welfare committee of the canton of berne, switzerland (authorization number be / ). titration by end point dilution was performed in marc- cells grown in -well plates. the cells were inoculated with tenfold serially diluted serum samples. at h pi, the cells were fixed and immunoperoxidase staining with the anti-n mab e was performed according to standard protocols. the % end point titre was expressed as tissue culture infectious dose % (tcid )/ ml, with a limit of detection of . log tcid /ml. total cellular rna was extracted from pig sera using the nucleospin multi virus kit (macherey-nagel) on a freedom evo robot (tecan) and stored at − °c. for detection of viral rna, a reverse transcriptase quantitative pcr (rt-qpcr) based on the amplification of the conserved region of orf was performed in duplicates employing gfp messenger rna as internal control [ ] . the results were expressed as the total number of cycles minus the quantification cycle (cq)-value. the elisa prrs x (idexx laboratories) was used for measuring anti-prrsv igg antibodies. samples to positive (s/p) ratios higher than . were considered positive. antibodies against prrsv gp , gp and gp were detected using in house competitive (gp ) and indirect (gp and gp ) elisas (ingenasa, madrid, spain). these elisas were performed with plates coated with recombinant gp , gp and gp antigens from prrsv olot/ . for the gp elisa, the percentage of competition was calculated using the formula [(odneg − odsample)/(odneg − odpos)] × . positive and negative controls were provided by ingenasa (spain). samples were considered positive if the percentage of competition was > %. for the indirect elisas, appropriate cut-offs were set. for the detection of c-reactive protein (crp; genway), haptoglobin (hp; genway) and ifn-γ (mabtech) in pig sera, commercially available elisas were used according to the manufacturer's protocols. porcine interferon-α (ifn-α) was determined by elisa as described previously [ ] . serum samples were heat inactivated at °c for min prior to performing the serum neutralization assay. µl of two-fold serially diluted sera were mixed with an equal volume of tcid of prrsv olot/ and incubated in -well tissue culture plates for h at °c. after h, marc- cells were added to each well. after days, the culture plate was fixed with % pfa. the presence of virus was detected by cpe and by staining with the anti-n mab and aec/h o . the virus neutralization titre was expressed as the reciprocal of the serum dilution leading to % reduction of infection. data were analysed with the graphpad prism . software. significant differences between groups were assessed by multiple t tests. p < . was considered significant. in previous work, a propagation-incompetent vsv replicon vector was generated by replacing the gene of the glycoprotein g in the fourth transcription unit of the viral genome with influenza virus genes. this replicon contained the gfp gene [referred to by an asterisk (*) in the vector nomenclature] in an additional transcription unit at position [ ] . based on this vector, we generated several vsv vrp expressing the individual structural antigens of prrsv olot/ or combinations thereof for evaluation as prrs vaccine candidates (see "materials and methods"; table ). the empty parental vsv*Δg vector served as control [ ] . the vsv vrp-mediated expression of the recombinant prrsv antigens was studied in marc- cells, taking advantage of the broad tropism of the vsv particles. this allowed direct comparison of the recombi- marc- cells infected with either vsv*Δg(gp -flag) or vsvΔg(gp /gp /gp ) reacted specifically with the anti-gp mab, while cells infected with either vsv*Δg(gp -ha) or vsvΔg(gp /gp /gp ) reacted with a polyclonal anti-gp immune serum (figure ). since a specific antibody against gp was not available, the expression of gp could not be demonstrated. any tag was omitted on purpose in the vsvΔg(gp /gp / gp ) to preserve the natural conformation of the three proteins. nevertheless, myc-tagged gp expression was detected from vsv*Δ(gp -myc) using the anti-myc serum (not shown). with the aim of evaluating whether protein secretion into the supernatant may enhance b-cell responses, the vsv*Δg(gp ecto-myc) replicon was constructed for the expression of the ectodomain of gp fused to a igκ leader sequence and an optimal signalase cleavage site. the myc-tagged ectodomain of gp reacted with the anti-gp mab and anti-myc serum as expected ( figure a ). western-blot analysis with the anti-myc serum demonstrated the presence of a kda protein in the cell lysate (ly) which was however missing in the supernatant (sn) of vsv*Δg(gp ecto-myc) infected marc- cells ( figure b ). this showed that the ectodomain of gp was retained in intracellular compartments despite the igκ signal sequence and the lack of the transmembrane domain. finally, expression of the prrsv n antigen by vsv*Δg(n) was demonstrated by immunofluorescence ( figure a ) and by western blot ( figure b ) using the anti-n mab. a kda protein was detected in the lysate of both, vsv*Δg(n)-and prrsv olot/ -infected cells. the gp and m complex constitutes the major protein component of the prrsv envelope against which neutralizing antibodies are formed [ , , ] . therefore, we first determined the immunogenicity of gp /mrecombinant vsv replicons in pigs. two groups of five pigs were immunized three times with vsvΔg(gp /m) or with the control vsv*Δg respectively, and were challenged weeks later with the homologous prrsv olot/ strain. following fever ( . - . °c) on day pi, whereas the control pigs had no fever despite slightly elevated body temperature ( figures a and b) . all animals developed a low viremia of short duration ( figures c and d) . a maximum mean virus titre of . log tcid /ml was reached in the serum at day post challenge. the virus was detectable up to day and viral rna up to day after challenge. no significant differences in viremia and viral rna in serum were observed between the vaccinated group and the control group at any time. before challenge infection, vaccination with vsvΔg(gp /m) did not induce any detectable antibody response against gp as measured by elisa (not shown). the first gp -specific antibody responses were detected by elisa in the vsvΔg(gp /m)-vaccinated group in out of five pigs on day after the challenge, and all pigs of this group were positive on day pi. all pigs of the vsv*Δg control group remained gp antibody negative at this time (table ). in the vsv*Δg control group, gp specific antibodies were detected for the first time on day pi in out of the pigs. seroconversion against n in response to the prrsv challenge infection was similar in the two groups, with the first n-specific seroconversion observed on day pi (table ) . virus neutralizing antibodies were not detected in any of the animals, neither before nor after the challenge (day and day pi). ifn-α and ifn-γ could not be detected in the serum at any time (data not shown). specific t-cell responses were not detected before challenge (not shown) and were therefore not further investigated. the acute phase proteins crp and hp which are early and sensitive markers of disease and inflammation including prrsv infection [ ] were increased in both groups between days and pi ( figures e and f) . however, no significant differences could be detected between vaccinated and control animals at any time. thus, apart from priming pigs for gp specific antibody responses after challenge infection, the vsvΔg(gp /m) vector could not induce any seroconversion against gp before challenge nor any sign of protection from viremia after infection. in order to determine whether the immune responses could be enhanced and induced before challenge by providing the three minor glycoproteins gp , gp and gp together with gp and m, pigs (n = ) were immunized twice with a mixture of vsvΔg(gp /m) and vsvΔg(gp /gp /gp ) or with vsv*Δg as control (n = ) followed by prrsv olot/ challenge. on day after challenge, two out of the four vaccinated pigs developed mild fever ( °c; figure a ) while all control animals remained free of fever (< °c; figure b ). all animals developed a short viremia following challenge infection with no significant differences in viral rna load and virus titres between the vaccinated and control groups (figures c and d) . as in the previous experiment, a maximum mean virus titre of . log tcid /ml was reached at day post challenge. again, before challenge infection, vaccination with vrp expressing the structural proteins of prrsv did not induce any detectable antibody response against gp , gp and gp as measured by elisa (not shown). following challenge infection, gp -specific antibody responses were detected on day pi in out of pigs, and all pigs vaccinated with vrp expressing the prrsv proteins seroconverted to gp at day pi (table ). all pigs of the vsv*Δg control group remained negative until day pi. seroconversion to gp (table ) vsv*Δg control group remained negative until day pi. a gp -specific immune response could be detected only in out of vaccinated animals on day pi. here also, the seroconversion against n following challenge was similar in the two groups ( table ) . none of the animals developed any neutralizing antibodies before nor after challenge (day and day pi). ifn-α and ifn-γ were not found in any of the sera in this experiment either (data not shown) whereas the serum crp and hp levels increased significantly in both groups between days and after challenge and declined subsequently. on day pi, the crp and hp levels were significantly different between the vaccinated and the control group ( figures e and f) , suggesting that the vaccinated group developed a slightly reduced inflammatory response after co-vaccination with vsvΔg(gp /m) and vsvΔg(gp / gp /gp ). together, these two vaccination trials show that vsv vrp cannot induce any detectable seroconversion against prrsv before challenge nor any protection against virus infection and viremia. nevertheless, these vectors can clearly prime pigs for earlier prrsv-specific antibody responses after challenge infection. the poor immunogenicity of vsv vrp expressing prrsv envelope proteins contrasts with previous reports showing protection against iav and btv infections in chickens and sheep, respectively [ , , ] . thus, the lack of antibody induction after immunization of pigs with vrp expressing the five prrsv envelope proteins raises the questions whether this vector vaccine is suitable for induction of antibody responses in pigs or whether the prrsv proteins are poorly immunogenic per se. in order to test this, the immunogenicity of prrsv and iav proteins expressed from vsv vrp was assessed in the same animal by co-vaccination with two different vsv vrp constructs expressing a prrsv protein (n, gp ecto, gp or empty vector) and a iav protein (ha, m , na, np), respectively, injected at two different sites (see "materials and methods"). after vaccinations, the pigs were challenged with the homologous prrsv olot/ strain. while, antibody responses were detected against all influenza virus proteins on day after vaccination (including neutralizing antibodies, not shown), no seroconversion against any of the prrsv proteins except n could be detected before challenge (table ). pigs vaccinated with vsv*∆g(n) seroconverted on day after vaccination ( days after the third vaccination). following challenge infection with prrsv olot/ , the control group seroconverted on day pi. there was no evidence of protection from virus infection after prrsv challenge as indicated by the kinetics of viremia (data not shown). nevertheless, a gp -specific immune response was induced earlier after challenge (day pi) in the vsv*Δg(gp ecto-myc) vaccinated pigs compared with the vsv*Δg-vaccinated pigs (no response on day pi). a gp -specific immune response was also induced on day pi whereas the vsv*Δg-vaccinated pigs stayed seronegative against gp . in line with the two previous immunization trials, these data show again a priming effect. more importantly however, simultaneous vaccination of the same pigs with two different vrp show clearly that the prrsv antigens are far less immunogenic than any of the iav antigens tested. the current prrsv vaccines on the market are of limited efficacy and come with several drawbacks [ ] . avoid the biological risks of virus spread and to circumvent potential immune modulating properties of modified live prrsv vaccines [ ] . therefore, we explored the immunogenic and protective potential of vsv replicon particles as a vectored vaccine approach against prrsv. such vrp were successfully used before as efficacious experimental vaccines against sars, iav and btv [ ] [ ] [ ] [ ] [ ] . in the present study, vsv replicons were engineered to express prrsv structural proteins, individually or in combination (table ) . emphasis was on gp , m, gp and gp , since these proteins are important targets for neutralizing and protective antibodies [ , , , ] , with the major neutralizing epitopes residing on gp [ ] . since several studies have shown that co-expression of prrsv antigens resulted in better humoral and cellular immune responses than expression of the individual proteins [ , ] , gp , m, gp , gp and gp proteins were co-expressed to partly mimic the formation of the gp /m and gp / / oligomers. vaccination of pigs with vrp expressing two or five prrsv envelope proteins in total did not result in any significant reduction of viremia compared with the control group. a tendency for reduced prrsv-related inflammatory responses was observed only when all five proteins were expressed. interestingly, vaccination with gp /m induced fever up to . °c for day following challenge infection with prrsv olot/ whereas mockvaccinated pigs remained asymptomatic. fever was less pronounced when all envelope proteins were included in the vaccine. enhanced disease following vaccination with recombinant gp and m was observed previously [ , ] . this was attributed to antibody-dependent enhancement of disease during natural infection, which is probably mediated by non-neutralizing antibodies [ , ] . such antibodies were shown to increase virus infection in porcine alveolar macrophage cultures and in vivo [ ] involving different fcγr isoforms [ , ] . neutralizing antibodies were not found at any time following vaccination and challenge infection. nevertheless, the vaccinated pigs seroconverted - days earlier than the mock-vaccinated animals after challenge virus infection, indicating that immunization with the recombinant vrp primed the immune system. in naïve pigs, the delayed seroconversion against the envelope proteins as opposed to the n protein after prrsv infection or after immunization with vectored vaccines is well table documented [ , , , ] . of note, with the marc- -adapted olot/ virus used in this study, seroconversion against n became detectable - days after challenge only (tables , , ) as opposed to the reported occurrence of anti-n antibodies as early as to days after prrsv infection [ , ] . this is probably related to the low level and short duration of replication of the marc- -adapted virus in pigs ( figures c, d, c, d) . despite the poor replication of this virus in vivo, vaccination of pigs with vrp did not result in any significant reduction of viremia. a better immunogenicity of prrsv proteins was reported when the antigens were modified, coupled to immunostimulatory molecules or administered as purified proteins along with adjuvants [ , ] . this raises the question whether the immunogenicity of the prrsv envelope proteins expressed from vrp is affected by glycosylation, subcellular localization, intracellular retention or low stability. in order to elaborate on this, gp was modified with the aim of obtaining secreted gp by replacing the leader sequence with a canonical igκ signal sequence followed by an optimal signalase cleavage site and by deleting the hydrophobic transmembrane anchor. however, the modified gp was retained in the cell, and no seroconversion against gp ecto was obtained in absence of challenge virus infection. the lack of gp secretion may be related to the fact that gp is naturally retained in intracellular compartments despite the n-terminal signal sequence [ ] . in order to address the question whether prrsv structural proteins are immunogenic at all when expressed by vsv vrp in pigs, a vector expressing prrsv n protein was included in one vaccination trial. n was chosen because it is the most immunogenic structural protein after prrsv infection [ , ] . indeed, vsv*Δg(n) was the only vrp capable of inducing a detectable antibody response in the absence of a viral challenge ( table ). the co-vaccination experiments with vrp expressing iav and prrsv envelope proteins corroborated these results. the iav proteins were strongly immunogenic while the prrsv envelope proteins were not, clearly demonstrating that the vsv vector per se is functional in pigs. although several vector vaccines failed to induce a protective immune response, immunizations essentially primed the immune system to a challenge infection [ , ] . these reports and the present study altogether suggest that the prrsv envelope proteins are expressed in a way to hide partially from the immune system. whether this immune evasion strategy is due solely to glycan shielding [ ] or to a particular intracellular topology remains to be investigated. future efforts in vectored vaccine development should consider these aspects to enhance the immunogenicity of the prrsv antigens. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states origin of highly pathogenic porcine reproductive and respiratory syndrome virus pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate nidovirales: evolving the largest rna virus genome the ever-expanding diversity of porcine reproductive and respiratory syndrome virus molecular evolution of prrsv in europe: current state of play arterivirus molecular biology and pathogenesis membrane proteins of arterivirus particles: structure, topology, processing and function discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd immunological responses of swine to porcine reproductive and respiratory syndrome virus infection the amino acid residues at and in gp of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody role of neutralizing antibodies in prrsv protective immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain the primary gp neutralization epitope of north american isolates of porcine reproductive and respiratory syndrome virus gp -specific neutralizing antibodies might be a driving force in prrsv evolution posttranslational processing and identification of a neutralization domain of the gp protein encoded by orf of lelystad virus a variable region in gp of european-type porcine reproductive and respiratory syndrome virus induces neutralizing antibodies against homologous but not heterologous virus strains heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection categorization of north american porcine reproductive and respiratory syndrome viruses: epitopic profiles of the n, m, gp and gp proteins and susceptibility to neutralization characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus co-expressing gp and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (prrsv) pathogenesis of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus vaccines: current status and strategies to a universal vaccine novel strategies and approaches to develop the next generation of vaccines against porcine reproductive and respiratory syndrome virus (prrsv) taming prrsv: revisiting the control strategies and vaccine design porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction impact of genetic diversity of european-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv vectored vaccines to protect against prrsv antigenic structures stably expressed by recombinant tgev-derived vectors immunization with dna vaccines containing porcine reproductive and respiratory syndrome virus open reading frames , , and may be related to the exacerbation of clinical disease after an experimental challenge vaccine production in plant systems: an aid to the control of viral diseases in domestic animals: a review baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain olot/ . involvement of orf and orf proteins in protection immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp /gp of porcine reproductive and respiratory syndrome virus and swine il- ctla mediated targeting enhances immunogenicity against prrsv in a dna prime/killed virus boost strategy use of influenza c virus glycoprotein hef for generation of vesicular stomatitis virus pseudotypes sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector a recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (h n ) vaccination with recombinant rna replicon particles protects chickens from h n highly pathogenic avian influenza virus vesicular stomatitis virus vectors expressing avian influenza h ha induce cross-neutralizing antibodies and long-term protection vesicular stomatitis virus replicon expressing the vp outer capsid protein of bluetongue we thank luis enjuanes, centro nacional de biotecnología, madrid, spain, for supplying the marc -adapted prrsv olot/ strain, the cdna for orf to and the rabbit anti-gp serum; we thank also hans nauwynck, university ghent, belgium for providing the mabs e and vii d, and ingenasa, madrid, spain for the mabs e c and ah . we thank markus gerber and nuria de la roja for excellent technical assistance, and daniel brechbühl and veronika ayala for animal care. the authors declare that they have no competing interests. authors' contributions nr, kcm, gz and as conceived the study. me, gz, nr, as, mgd and mjr designed the experiments. me performed most of the experiments. mgd, js and mjr performed the glycoprotein-specific elisas. sl and mer contributed with iav vrp. me, nr, gz and mgd wrote the manuscript. all authors read and approved the final manuscript. key: cord- - rehsd authors: opriessnig, tanja; gerber, priscilla f.; shen, huigang; de castro, alessandra marnie m. g.; zhang, jianqiang; chen, qi; halbur, patrick title: evaluation of the efficacy of a commercial inactivated genogroup b-based porcine epidemic diarrhea virus (pedv) vaccine and experimental live genogroup b exposure against b challenge date: - - journal: vet res doi: . /s - - -z sha: doc_id: cord_uid: rehsd porcine epidemic diarrhea virus strains from the g b cluster are considered less pathogenic compared to the g b cluster. the aim of this study was to compare the ability of g b-based live virus exposure against use of a commercial g b–based inactivated vaccine to protect growing pigs against g b challenge. thirty-nine pedv naïve pigs were randomly divided into five groups: exp-im- b (intramuscular g b exposure; g b challenge), exp-oral- b (oral g b exposure; g b challenge), vac-im- b (intramuscular commercial inactivated g b vaccination; g b challenge), pos-control (sham-vaccination; g b challenge) and neg-control (sham-vaccination; sham-challenge). pigs were vaccinated/exposed at weeks of age (day post-vaccination , dpv ), vac-im- b pigs were revaccinated at dpv , and the pigs were challenged at dpv . among all groups, vac-im- b pigs had significantly higher anti-pedv igg levels on dpv and while exp-oral- b pigs had significantly higher anti-pedv iga levels on dpv , , and . exp-oral- b also had detectable iga in feces. intramuscular pedv exposure did not result in a detectable antibody response in exp-im- b pigs. the fecal pedv rna levels in vac-im- b pigs were significantly lower – days after challenge compared to the pos-control group. under the study conditions a commercial inactivated g b-based vaccine protected pigs against g b challenge, as evidenced by reduction of pedv rna in feces for – logs during peak shedding and a shorter viral shedding duration. the oral, but not the intramuscular, experimental g b-based live virus exposure induced a high anti-pedv iga response prior to challenge, which apparently did not impact pedv shedding compared to pos-control pigs. clinical porcine epidemic diarrhea and its causative virus pedv were discovered in european pigs in the s [ , ] , spread to asia during the s and s [ ] , and became endemic in pigs on both continents [ , ] . approximately years ago pedv re-emerged as an important enteric disease of suckling and growing pigs [ ] . in , pedv was introduced for the first time to north america [ ] causing major disease and mortality [ ] . pedv can be differentiated into genogroups [ ] . on the basis of spike (s) gene sequences, pedv isolates can be divided into g a, g b, g a and g b [ , ] . g a includes historic pedv isolates such as cv and attenuated variants distributed in europe and asia, whilst g b includes the so called s-indel strains which can be found in europe, asia and north america. g a isolates are restricted to asia whereas g b isolates are present in asia and the ukraine [ ] , and since us introduction in are widespread in the us and considered the us prototype [ , ] . differences in pathogenicity between representative isolates of different genogroups have been demonstrated [ ] [ ] [ ] , with g b isolates usually being more pathogenic compared to g b isolates. partial crossprotection between pedv g b and g b isolates has been demonstrated experimentally [ ] . in january the first conditional licensed pedv vaccine was introduced to the north american pig market [ ] , and today an rna particle-based vaccine and an inactivated pedv vaccine are available in the us to immunize sows against pedv [ ] . while the use of these vaccines is often beneficial in previously exposed herds, they often fail in naïve herds [ ] . one reason for the variable vaccine efficacy observed under field conditions may be the usage of inactivated vaccines given intramuscularly rather than live virus vaccines given orally to induce a strong local enteric immunity. it would be risky to use a known pathogenic g b live vaccine virus in a pig population; however, using a less virulent variant such as g b instead may be safe and efficacious. the objectives of this study were to compare the efficacy of heterologous g b and homologous g b based vaccines in protecting growing pigs against g b challenge. specifically, an experimental g b-based live vaccine, administered orally or intramuscularly and a commercial g b-based inactivated vaccine administered intramuscularly were compared side by side. the experimental protocol was approved by the iowa state university institutional animal care and use committee (approval number: - - -s). thirty-nine, -week-old, colostrum-fed, arbitrarilyselected, crossbred, pedv naïve weaned pigs were randomly assigned to one of five rooms and groups, with - pigs in each group (table ) . all groups were fed ad libitum with a balanced, age-appropriate, pelleted feed ration. at weeks of age or dpv , exp-im- b, exp-oral- b and vac-im- b groups were vaccinated with different vaccines and routes as outlined in table , whereas pos-control and neg-control pigs were sham-vaccinated with saline. vac-im- b pigs were revaccinated at weeks of age (dpv ). at day postchallenge (dpc) or dpv , when the pigs were weeks old, they were challenged as shown in table . the pos-control group served as a challenge control group while the neg-control was sham-challenged and served as unvaccinated, unchallenged group. half of the pigs in each group were necropsied at dpv /dpc and the remainder at dpv /dpc . the experimental design and sample collection details are summarized in figure . blood was collected in serum separator tubes on a weekly basis (fisher scientific, pittsburgh, pa, usa), centrifuged at × g for min at °c, and the serum was stored at − °c until testing. rectal swabs were collected at dpv , , , and followed by daily collection until dpv /dpc using polyester swabs and stored in ml plastic tubes containing ml of sterile saline solution at − °c until testing. individual fecal samples were collected in ml plastic tubes and frozen immediately at − °c until testing. at weeks of age (dpv ), the exp-im- b and the exp-oral- b pigs were vaccinated with a g b (us s-indel-variant) live pedv isolate - at the th cell culture passage [ , ] as indicated in table . after cell culture adaption this virus was used to infect -day old pigs previously and had reduced pathogenicity compared to g b isolates [ ] . for the intramuscular vaccination, ml of the g b virus stock with a titer of × % tissue culture infectious dose (tcid ) per ml was mixed with . ml adjuplex ™ vaccine adjuvant (lot number slbp v; sigma-aldrich, st louis, mo, usa) prior to injection. the same g b cell culture adapted virus stock used in this study has been shown to have moderate to severe enteric pathogenicity in -day old pigs [ ] . each pig in the exp-im- b group received . ml intramuscularly into the neck, with a total pedv dose of × tcid . for the oral vaccination route, each exp-oral- b pig was administered ml of the g b virus stock with a titer of . × tcid per ml by slowly dripping the vaccine into the mouth of each pig with a total dose of . × tcid . adjuvant was not used for the oral vaccination route. pigs in the vac-im- b group were vaccinated intramuscularly with ml of a commercial conditionally-licensed inactivated pedv vaccine based on a g b strain (zoetis; serial number ) into the right neck. the vac-im- b group was revaccinated weeks later (dpv ) with another ml of the vaccine as recommended by the manufacturer. the pos-control group was sham-vaccinated intramuscularly in the neck with . ml saline and the neg-control group was sham-vaccinated orally with ml saline (table ) . the th passage of virulent pedv g b strain - e [ , ] was grown to a final titer of . × tcid per ml. at weeks of age, exp-im- b, exp-oral- b, vac-im- b and pos-control pigs (table ) the neg-control group was sham-vaccinated orally with saline and sham-inoculated with saline and served as unvaccinated and unchallenged control group. slowly dripping the inoculum into the mouth with a total dose of . × tcid . pigs in the neg-control group were sham-inoculated with ml saline orally. all pigs were weighed at dpv , at dpv /dpc and at dpv /dpc ( figure ). the average daily gain (adg) from dpv (vaccination ) to dpv /dpc (necropsy ) was calculated. after pedv challenge the fecal consistency was scored for each pig daily, ranging from to with = solid, = semisolid, = pasty, and = liquid. all pigs were examined daily for other signs of illness including lethargy, respiratory disease, inappetence and lameness. all serum samples were tested for the presence of pedv igg and iga antibodies by an in-house pedv g b s protein based indirect elisa [ , ] . for igg detection, a sample-to-positive (s/p) ratio of > . was considered positive, between . and . as suspect, and < . as negative. for the iga elisa an s/p ratio above or equal to . was considered positive. in addition, fecal samples collected at dpv , dpv , and at necropsy at dpv /dpc or dpv /dpc were also tested for presence of pedv iga antibodies [ ] . modifications for this assay included that samples were diluted : and the secondary antibody was diluted : . the positive cutoff for this assay was s/p ratio equal or greater than . . serum samples at dpv were titrated for anti-pedv virus neutralizing antibodies by an immunofluorescence assay as previously described [ ] . serum was diluted two-fold starting from : to : . titers were given as the reciprocal of the last dilution giving a positive result. total nucleic acids were extracted from all rectal swabs using the magmax ™ pathogen rna kit (applied biosystems, life technologies, carlsbad, ca, usa) on an automated nucleic acid extraction system (thermo scientific kingfisher ® flex, thermo fisher scientific, pittsburgh, pa, usa) according to the instructions of the manufacturer. all rna extracts were tested for the presence of pedv rna by a quantitative real-time pcr [ ] . samples were considered negative when no signal was observed within amplification cycles. half of the pigs in each group were necropsied at dpv /dpc and the remaining pigs were necropsied at dpv /dpc . the pigs were humanely euthanized by intravenous pentobarbital sodium overdose (fatal plus ® , vortech pharmaceuticals, ltd, dearborn, mi, usa). gross lesions were assessed by a veterinary pathologist and eight sections of small intestines, three sections of large intestines and one section of mesenteric lymph node were collected, fixed in % neutral-buffered formalin, and routinely processed for histological examination. microscopic lesions were evaluated by a veterinary pathologist blinded to the treatment groups. sections of small intestines were evaluated for the presence of villus atrophy and scored from (none) to (severe). pedvspecific antigen was detected by immunohistochemistry (ihc) using a monoclonal antibody specific for pedv (bionote, hwaseong-si, gyeonggi-do, korea) [ , ] . the amount of pedv antigen was scored by a pathologist blinded to treatment status. scores ranged from to with = no signal, = - % of villous enterocytes within the section showing a positive signal, = - % of villous enterocytes showing a positive signal, and = more than % of villous enterocytes showing a positive signal. for data analysis, jmp ® software version . . (sas institute, cary, nc, usa) was used. summary statistics were calculated for all the groups to assess the overall quality of the data set including normality. statistical analysis of the data was performed by one-way analysis of variance (anova) for continuous data. a p value of less than . was set as the statistically significant level. pairwise test using tukey's adjustment was subsequently performed to determine significant group differences. real-time pcr results (copies per ml of fecal swab suspension) were log transformed prior to statistical analysis. the area under the curve (auc) of viral shedding of each animal and the total auc for each group was calculated using the log transformed values of the viral loads from dpv to /dpc to . one-way anova and a bonferroni post hoc test were used to compare groups. non-repeated nominal data were assessed using a nonparametric kruskal-wallis one-way anova, and if significant, pairwise wilcoxon tests were used to evaluate differences among groups. clinical signs in the pedv-infected pigs were limited to diarrhea. three days after vaccination, / exp-oral- b pigs had semisolid feces and days later all pigs in this group had pasty feces. none of the pigs in the other groups had any fecal consistency changes and all pigs remained normal until pedv challenge. liquid fecal consistency was observed in / exp-im- b pigs by dpv /dpc and feces remained fluid in the majority of the pigs until dpv /dpc before becoming pasty-to-solid. in the remaining groups individual pigs had liquid feces for - days of duration (data now shown) with no differences among groups. the overall adg is summarized in table . there were no significant differences among groups. all pigs were negative for anti-pedv igg antibodies in serum samples by elisa at dpv and neg-control pigs remained seronegative for the duration of the study. anti-pedv igg antibodies were first detected in / vac-im-pedv pigs at dpv ( figure ). by dpv , / pigs in this group were seropositive and / were suspect. all vac-im- b pigs were anti-pedv igg positive by dpv . in the exp-oral- b group, / pigs were positive for anti-pedv igg antibodies by dpv , / were positive at dpv and at dpv , / pigs were positive by dpv /dpc , and at termination of the study / were positive and / was suspect ( figure ). the exp-im- b group remained anti-pedv igg negative until dpv / dpc at which time all three remaining pigs in this group were positive (figure ). one of four pos-control pigs seroconverted to pedv by dpc and all pigs in this group were seropositive by dpc . anti-pedv neutralizing antibodies were not detected in the exp-im- b, pos-control and neg-control groups at dpv . neutralizing antibody titers ranged from to with a geometric mean of . in the exp-oral- b group, and ranged - with a geometric mean of in the vac-im- b group. all pigs were negative for anti-pedv iga antibodies in serum samples by elisa at dpv and and neg-con-trol pigs remained seronegative for the duration of the study. anti-pedv iga antibodies in serum samples were first detected in / exp-oral- b pigs at dpv ( figure ). one week later at dpv , anti-pedv iga in sera were also detected in / vac-im- b pigs; however, in this group antibody levels decreased, and by dpv / dpc only / pigs were anti-pedv iga positive. iga antibodies against pedv in the exp-im- b group were detected by dpv /dpc in / pigs and by dpv /dpc in / pigs (figure ). one of four pos-control pigs had detectable anti-pedv iga antibodies by dpc and / pigs were seropositive by dpc (figure ). by dpv /dpc , one exp-oral- b pig had detectable iga levels in feces and by dpv /dpc one additional pig in this group was positive for pedv iga in feces (data not shown). by dpc, pedv iga antibodies in feces were present in / exp-im- b pigs, / exp-oral- b pigs, / pos-control pigs and / vac-im- b pigs (data not shown). all pigs were negative for pedv rna in fecal swabs on dpv and neg-control pigs remained negative for the duration of the study. after vaccination with a live g b strain, fecal shedding was detected in / exp-oral- b pigs by dpv and in / pigs by dpv (figure ). in addition, / exp-oral- b pigs had detectable amounts of pedv rna in serum by dpv (data not shown). on the day of challenge, / exp-oral- b pigs shed low amounts of pedv in feces. pedv rna was detected in rectal swabs of / exp-im- b pigs by dpv ; however, pedv rna was never detected in serum (data not shown). after challenge, / exp-im- b pigs shed virus by dpv /dpc . viral shedding in rectal swabs was first detected by dpv /dpc in / exp-oral- b pigs, in / vac-im- b pigs and in / pos-control pigs. group mean genomic copies of pedv rna in rectal swabs are summarized in figure . the average duration of pedv shedding was calculated by adding the number of consecutive pedv pcr positive days of each pig that remained in the study until dpv /dpc divided by all pigs in a group. the average duration of pedv shedding and the auc are summarized in table . at dpv /dpc , pedv-infected pigs regardless of vaccination status had hyperemic intestines that were fluidfilled. specifically, liquid intestinal content was noted in / exp-im- b pigs, in / exp-oral- b pigs, in / vac-im- b pigs and in / pos-control pigs. at dpv /dpc , / pos-control pigs had fluid filled intestines and a dilated colon without remarkable lesions in any of the other pigs. microscopic lesions were seen in / exp-im- b pigs, / exp-oral- b pigs, / vac-im- b pigs and / pos-control pigs which had mild to severe atrophic enteritis by dpv /dpc . there were no lesions in any of the other pigs. five of the six pigs with microscopic lesions also had moderate-to-high amounts of pedv figure group mean anti-pedv igg response in serum samples. the samples were collected at the day of initial vaccination (dpv ), and dpv , , , , and and tested by an in house igg pedv elisa. pigs were exposed to live g b pedv at weeks of age (dpv ) or were vaccinated at (dpv ) and (dpv ) weeks of age with a commercial inactivated pedv g b vaccine. pigs were challenged with pedv g b at weeks of age (dpv /day post challenge or dpc ). data presented as mean group elisa sample-to-positive (s/p) ratios ± sem. significantly different values for a dpc are indicated by different superscripts ( a,b,c ). the significance level was set to p < . . seropositive pigs/total number of pigs per group for groups that contained at least one seropositive pigs can be seen next to the group mean. antigen associated with the lesions (two exp-im-g b pigs, scores and ; a exp-oral-g b pig, score ; a vac-im-g b pig score ; and a pos-control pig, score ). there were no significant differences in antigen levels or severity of microscopic lesions among groups. no microscopic lesions nor pedv antigen were observed at dpv /dpc . vaccination strategies to protect against pedv are challenging, as the most vulnerable population is suckling pigs. vaccine efficacy studies using pregnant sows are difficult and costly. to select novel pedv vaccine candidates and to generate preliminary data, the growing pig model has been used [ ] . in this study growing pigs were used to test and compare the efficacy of live or inactivated vaccines to protect pigs against challenge with a highly virulent g b pedv isolate. pig veterinarians and producers often prefer intramuscular administration to assure each pig gets vaccinated with the appropriate dose. intramuscular administration is known to induce a systemic immune response [ ] . in this study, vac-im- b pigs had a strong anti-pedv igg response in serum which was significantly higher compared to exp-oral- b pigs. this could be due to the adjuvant amphigen ® used in the commercial product or due to the booster dose that the vac-im- b pigs received. in contrast to live virus exposure, inactivated vaccines are almost always given in dose regimens; hence in this study the vac-im-g b group received a booster dose whereas the exp-im-g b and exp-oral-g b pigs did not. in contrast to oral exposure to a live virus, pigs vaccinated with the commercial inactivated virus had a weak anti-iga response in serum and no anti-pedv iga response in feces. this is not surprising as inactivated vaccines often do not induce effective mucosal immunity in naïve pigs whereas oral exposure elicits better gut immunity [ ] . it has been shown that iga levels in serum correlates with iga measured in feces from experimentally infected piglets [ ] and in serum and colostrum and milk samples of sows orally immunized [ ] . these studies indicate that measuring iga levels in serum samples may be a marker of protection. for safety reasons, veterinarians and producers often prefer inactivated vaccines. however, for some viruses such as porcine reproductive and respiratory syndrome figure group mean anti-pedv iga response in serum samples. the samples were collected at the day of initial vaccination (dpv ), and dpv , , , , and and tested by an in house iga pedv elisa. pigs were exposed to live g b pedv at weeks of age (dpv ) or were vaccinated at (dpv ) and (dpv ) weeks of age with a commercial inactivated pedv g b vaccine. pigs were challenged with pedv g b at weeks of age (dpv /day post challenge or dpc ). data presented as mean group elisa sample-to-positive (s/p) ratios ± sem. significantly different values for a dpc are indicated by different superscripts ( a,b,c ). the significance level was set to p < . . seropositive pigs/total number of pigs per group for groups that contained at least one seropositive pigs can be seen next to the group mean. virus (prrsv), it has been shown that inactivated vaccines are largely ineffective [ ] . prrsv requires live virus to migrate to the lung and replicate at low levels to induce protection. similarly, pedv may also require local activation of the gut-associated mucosal system. in asia, where pedv vaccines have been available for decades, attenuated g a-based intramuscular vaccines are commonly used [ , ] . we attempted to inject a g b isolate intramuscularly with an adjuvant. under the study conditions, except for / exp-im- b pigs with low levels of pedv rna in rectal swabs at dpv, there was no sign of infection in this group based on lack of seroconversion and lack of detectable pedv rna in serum or feces. the two pedv rna positive exp-im- b samples were retested and results confirmed (data not shown). the pigs that were vaccinated intramuscularly with a commercial g b vaccine were protected against homologous g b challenge as evidenced by reduction of the amount of pedv rna in feces by - logs during peak shedding between dpc - (dpv - ) and shortening of the duration of viral shedding. viral titers to determine infectivity were not determined, but it has been shown previously that contact pigs can be infected for up to days after initial infection of a seeder pig group [ ] . in this study a homologous g b challenge for pigs vaccinated with the experimental g b live vaccine was not included due to space and cost reasons. in addition, g b isolates, considered to be more pathogenic compared to g b isolates [ , ] , appear to be the primary cause of clinical disease associated with pedv under field conditions and are more widely distributed compared to g b isolates. pigs orally vaccinated with an experimental heterologous g b live vaccine had a tendency for a shortened viral shedding duration; whereas pigs vaccinated intramuscularly with an experimental heterologous g b live vaccine were not protected. it has been shown that piglets orally inoculated with a virulent cv strain were fully protected after challenge, while protection was not complete in pigs orally inoculated with an attenuated cv strain [ ] . prior to usage the g b stock was passaged seven times which could have resulted in a low degree of attenuation. it is worth noting that pigs orally immunized with the g b live vaccine presented mild diarrhea and shed high levels of virus for at least weeks after immunization. this could pose risks of infection and potentially more serious clinical signs in younger piglets. results from studies on cross-protection between genogroups have been contradictory. a previous study showed that although g a-based vaccines (cv and dr strains) could provide protection against homologous challenge, they were not protective against contemporary chinese g b strain yc [ ] . it has been suggested that sows naturally-infected with a g b strain produce heterologous lactogenic protective immunity against g b strains months after initial infection [ ] . however, infection of - day old piglets with g b strain provided variable protection against a g b challenge - days later and the extent of protection was shown to be litter-dependent (mortality to %) [ ] . additionally, the antigen concentration in the commercial ( - tcid /dose) and experimental ( - tcid /dose) intramuscular vaccines may have contributed to differences in the protection observed in the current study. the dose of experimental vaccine was limited by the g b virus titer achieved after propagation. under the conditions of this study, a commercial inactivated g b-based pedv vaccine administered intramuscularly protected pigs against homologous challenge. in contrast, an experimental g b-based live virus vaccine given intramuscularly was not protective. the same virus given orally induced a high iga response but the virus shedding pattern after challenge mimicked that of the pos-control group suggesting limited protection. this could perhaps indicate that induction of a genotype specific humoral and/or cellular immune response may be important for pedv protection. . pigs were exposed to pedv at weeks of age (dpv ) with live pedv g b or vaccinated at (dpv ) and (dpv ) weeks of age with a commercial inactivated pedv g b vaccine. pigs were challenged with pedv g b at weeks of age (dpv /dpc ). a. mean group log pedv genomic copies per ml of fecal swab suspension ± sem. significant 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systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus epidemic strain yc of porcine epidemic diarrhea virus could provide piglets against homologous challenge previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe" strain the authors thank kelsey oakly and eve fontanella for assistance with the animal work and gustavo de-sousa-e-silva, marcelo nunes de almeida and will alberto lopez for assistance with the necropsies. the study was funded by the iowa livestock health advisory council (ilhac). the authors declare they have no competing interests. to performed the experiments, analysis of the data (including statistical analysis) and drafting of the manuscript. pfg performed analysis of samples and data. hs, ammgc, jz and qc performed the experiment. jz and qc provided the inoculum stocks. pgh performed the animal studies and necropsies, edited and finalized the manuscript. all authors read and approved the final manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.